Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Ogawa Yoshihiro Last modified date:2021.07.06

Professor / Department of Medical and Bioregulatory Science / Department of Clinical Medicine / Faculty of Medical Sciences


Papers
1. Kiyoshi Yoshioka, Hiroshi Nagahisa, Fumihito Miura, Hiromitsu Araki, Yasutomi Kamei, Yasuo Kitajima, Daiki Seko, Jumpei Nogami, Yoshifumi Tsuchiya, Narihiro Okazaki, Akihiko Yonekura, Seigo Ohba, Yoshinori Sumita, Ko Chiba, Kosei Ito, Izumi Asahina, Yoshihiro Ogawa, Takashi Ito, Yasuyuki Ohkawa, Yusuke Ono, Hoxa10 mediates positional memory to govern stem cell function in adult skeletal muscle, Science Advances, 10.1126/sciadv.abd7924, 7, 24, eabd7924-eabd7924, 2021.06.
2. Akifumi Kuwano, Miho Kurokawa, Motoyuki Kohjima, Koji Imoto, Shigeki Tashiro, Hideo Suzuki, Masatake Tanaka, Seiji Okada, Masaki Kato, Yoshihiro Ogawa, Microcirculatory disturbance in acute liver injury., Experimental and therapeutic medicine, 10.3892/etm.2021.10028, 21, 6, 596-596, 2021.06.
3. Yukina Takeichi, Takashi Miyazawa, Shohei Sakamoto, Yuki Hanada, Lixiang Wang, Kazuhito Gotoh, Keiichiro Uchida, Shunsuke Katsuhara, Ryuichi Sakamoto, Takaya Ishihara, Keiji Masuda, Naotada Ishihara, Masatoshi Nomura, Yoshihiro Ogawa, Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor., Diabetologia, 10.1007/s00125-021-05488-2, 2021.05.
4. Haruei Ogino, Keita Fukaura, Yoichiro Iboshi, Yousuke Nagamatsu, Hiroaki Okuno, Kei Nishioka, Yuichiro Nishihara, Yoshimasa Tanaka, Takatoshi Chinen, Eikich Ihara, Yoshihiro Ogawa, Role of the IL-23-T-bet/GATA3 Axis for the Pathogenesis of Ulcerative Colitis, Inflammation, 10.1007/s10753-020-01358-y, 44, 2, 592-603, 2021.04.
5. Hiromi Nagata, Jingya Lyu, Hitomi Imachi, Kensaku Fukunaga, Seisuke Sato, Toshihiro Kobayashi, Takanobu Saheki, Kayoko Seo, Japar B Salimah, Hisakazu Iwama, Ryuichi Sakamoto, Yoshihiro Ogawa, Koji Murao, AGEs inhibit scavenger receptor class B type I gene expression via Smad1 in HUVECs., Journal of molecular endocrinology, 10.1530/JME-20-0177, 66, 3, 223-231, 2021.03.
6. Maki Yokomoto-Umakoshi, Ryuichi Sakamoto, Hironobu Umakoshi, Yayoi Matsuda, Hiromi Nagata, Tazuru Fukumoto, Masatoshi Ogata, Yoshihiro Ogawa, Unilateral primary aldosteronism as an independent risk factor for vertebral fracture, Clinical Endocrinology, 10.1111/cen.14145, 92, 3, 206-213, 2020.03.
7. Mitsuru Esaki, Toshiki Horii, Ryoji Ichijima, Masafumi Wada, Seiichiro Sakisaka, Shuichi Abe, Naru Tomoeda, Yusuke Kitagawa, Kei Nishioka, Yosuke Minoda, Shinichi Tsuruta, Sho Suzuki, Hirotada Akiho, Eikichi Ihara, Yoshihiro Ogawa, Takuji Gotoda, Assistant skill in gastric endoscopic submucosal dissection using a clutch cutter., World journal of gastrointestinal surgery, 10.4240/wjgs.v13.i2.116, 13, 2, 116-126, 2021.02.
8. Hiroaki Okuno, Haruei Ogino, Eikichi Ihara, Kei Nishioka, Yoshimasa Tanaka, Takatoshi Chinen, Motoyuki Kohjima, Takamasa Oono, Masatake Tanaka, Takeshi Goya, Nao Fujimori, Yoichiro Iboshi, Takuji Gotoda, Yoshihiro Ogawa, Discriminant equation using mucosally expressed cytokines and transcription factor for making definite diagnosis of inflammatory bowel disease unclassified., BMC gastroenterology, 10.1186/s12876-021-01656-1, 21, 1, 73-73, 2021.02.
9. Akihisa Ohno, Nao Fujimori, Masami Miki, Takamasa Oono, Hisato Igarashi, Ryota Matsuda, Yutaka Koga, Yoshinao Oda, Takao Ohtsuka, Masafumi Nakamura, Tetsuhide Ito, Yoshihiro Ogawa, Collision of a pancreatic ductal adenocarcinoma and a pancreatic neuroendocrine tumor associated with multiple endocrine neoplasm type 1, Clinical Journal of Gastroenterology, 10.1007/s12328-020-01234-0, 2021.02.
10. Maki Yokomoto-Umakoshi, Hironobu Umakoshi, Takashi Miyazawa, Masatoshi Ogata, Ryuichi Sakamoto, Yoshihiro Ogawa, Investigating the causal effect of fibroblast growth factor 23 on osteoporosis and cardiometabolic disorders: A Mendelian randomization study., Bone, 10.1016/j.bone.2020.115777, 143, 115777-115777, 2021.02.
11. Masatoshi Murakami, Nao Fujimori, Yuta Suehiro, Tomonobu Hioki, Kazuhide Matsumoto, Takamasa Oono, Yoshihiro Ogawa, Successful endoscopic removal of a fully covered self-expandable metallic stent that fractured above a benign distal bile duct stricture., Endoscopy, 10.1055/a-1167-7861, 53, 1, E11-E12, 2021.01.
12. Tazuru Fukumoto, Hironobu Umakoshi, Masatoshi Ogata, Maki Yokomoto-Umakoshi, Yayoi Matsuda, Misato Motoya, Hiromi Nagata, Yui Nakano, Norifusa Iwahashi, Hiroki Kaneko, Norio Wada, Takashi Miyazawa, Ryuichi Sakamoto, Yoshihiro Ogawa, Significance of Discordant Results Between Confirmatory Tests in Diagnosis of Primary Aldosteronism., The Journal of clinical endocrinology and metabolism, 10.1210/clinem/dgaa812, 106, 2, e866-e874, 2021.01.
13. Maki Yokomoto-Umakoshi, Hironobu Umakoshi, Ryuichi Sakamoto, Tazuru Fukumoto, Masatoshi Ogata, Yui Nakano, Norifusa Iwahashi, Hiroki Kaneko, Noriko Mizoguchi, Akiko Hattori, Takashi Miyazawa, Yayoi Matsuda, Hisaya Kawate, Yoshihiro Ogawa, Role of deteriorated bone quality in the development of osteoporosis in pheochromocytoma and paraganglioma., Bone, 10.1016/j.bone.2020.115607, 142, 115607-115607, 2021.01.
14. Masatoshi Ogata, Hironobu Umakoshi, Tazuru Fukumoto, Yayoi Matsuda, Maki Yokomoto-Umakoshi, Hiromi Nagata, Norio Wada, Takashi Miyazawa, Ryuichi Sakamoto, Yoshihiro Ogawa, Significance of aldosterone gradient within left adrenal vein in diagnosing unilateral subtype of primary aldosteronism., Clinical endocrinology, 10.1111/cen.14320, 94, 1, 24-33, 2021.01.
15. Mitsuhiro Kawakubo, Miyako Tanaka, Kozue Ochi, Akiko Watanabe, Marie Saka-Tanaka, Yohei Kanamori, Naoki Yoshioka, Satoko Yamashita, Moritaka Goto, Michiko Itoh, Ibuki Shirakawa, Sayaka Kanai, Hiromi Suzuki, Makoto Sawada, Ayaka Ito, Masatoshi Ishigami, Mitsuhiro Fujishiro, Hiroshi Arima, Yoshihiro Ogawa, Takayoshi Suganami, Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis–associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects, Scientific reports, 10.1038/s41598-020-57935-6, 10, 1, 2020.12, Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor–deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism–independent effects of anagliptin on NASH and HCC development..
16. Nozomi Hanzawa, Koshi Hashimoto, Xunmei Yuan, Kenichi Kawahori, Kazutaka Tsujimoto, Miho Hamaguchi, Toshiya Tanaka, Yuya Nagaoka, Hiroshi Nishina, Sumiyo Morita, Izuho Hatada, Tetsuya Yamada, Yoshihiro Ogawa, Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing, Scientific reports, 10.1038/s41598-020-62035-6, 10, 1, 2020.12, Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues..
17. Akira Matsumoto, Hirohito Kuwata, Shinichiro Kimura, Hiroko Matsumoto, Kozue Ochi, Yuki Moro-oka, Akiko Watanabe, Hironori Yamada, Hitoshi Ishii, Taiki Miyazawa, Siyuan Chen, Toshiaki Baba, Hiroshi Yoshida, Taichi Nakamura, Hiroshi Inoue, Yoshihiro Ogawa, Miyako Tanaka, Yuji Miyahara, Takayoshi Suganami, Hollow fiber-combined glucose-responsive gel technology as an in vivo electronics-free insulin delivery system, Communications Biology, 10.1038/s42003-020-1026-x, 3, 1, 2020.12, Accumulating evidence demonstrates that not only sustained elevation of blood glucose levels but also the glucose fluctuation represents key determinants for diabetic complications and mortality. Current closed-loop insulin therapy option is limited to the use of electronics-based systems, although it poses some technical issues with high cost. Here we demonstrate an electronics-free, synthetic boronate gel-based insulin-diffusion-control device technology that can cope with glucose fluctuations and potentially address the electronics-derived issues. The gel was combined with hemodialysis hollow fibers and scaled suitable for rats, serving as a subcutaneously implantable, insulin-diffusion-active site in a manner dependent on the subcutaneous glucose. Continuous glucose monitoring tests revealed that our device not only normalizes average glucose level of rats, but also markedly ameliorates the fluctuations over timescale of a day without inducing hypoglycemia. With inherent stability, diffusion-dependent scalability, and week-long & acute glucose-responsiveness, our technology may offer a low-cost alternative to current electronics-based approaches..
18. Tomoaki Yoshida, Atsunori Tsuchiya, Masaru Kumagai, Suguru Takeuchi, Shunsuke Nojiri, Takayuki Watanabe, Masahiro Ogawa, Michiko Itoh, Masaaki Takamura, Takayoshi Suganami, Yoshihiro Ogawa, Shuji Terai, Blocking sphingosine 1-phosphate receptor 2 accelerates hepatocellular carcinoma progression in a mouse model of NASH, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2020.07.099, 530, 4, 665-672, 2020.10, The role of sphingosine 1-phosphate (S1P) and its sphingosine-1-phosphate receptors (S1PRs) in non-alcoholic steatohepatitis (NASH) is unclear. We aimed to analyze the role of S1P/S1PRs in a Melanocortin-4 receptor (Mc4r)-deficient NASH murine model using FTY720, the functional antagonist of S1PR1, S1PR3, S1PR4, and S1PR5, and JTE-013, the antagonist of S1PR2. We observed that, compared to that in the control, the mRNA of S1pr1 tended to decrease, whereas those of S1pr2 and S1pr3 significantly increased in Mc4r-knockout (KO) mice subjected to a Western diet (WD). While the fat area did not differ, fibrosis progression differed significantly between control mice and mice in which liver S1PRs were blocked. Lipidomic and metabolomic analysis of liver tissues showed that JTE-013-administered mice showed elevation of S-adenosyl-L-methionine level, which can induce aberrant methylation due to reduction in glycine N-methyltransferase (GNMT) and elevation in diacylglycerol (DG) and triacylglycerol (TG) levels, leading to increased susceptibility to hepatocellular carcinoma (HCC). These phenotypes are similar to those of Gnmt-KO mice, suggesting that blocking the S1P/S1PR2 axis triggers aberrant methylation, which may increase DG and TG, and hepatocarcinogenesis. Our observations that the S1P/S1PR2 axis averts HCC occurrence may assist in HCC prevention in NASH..
19. Yusuke Kobayashi, Tatsuya Haze, Yuichiro Yano, Kouichi Tamura, Isao Kurihara, Takamasa Ichijo, Takashi Yoneda, Takuyuki Katabami, Mika Tsuiki, Norio Wada, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Tetsuya Yamada, Ryuji Okamoto, Megumi Fujita, Kohei Kamemura, Koichi Yamamoto, Shoichiro Izawa, Akiyo Tanabe, Mitsuhide Naruse, Associations Between Changes in Plasma Renin Activity and Aldosterone Concentrations and Changes in Kidney Function After Treatment for Primary Aldosteronism, Kidney International Reports, 10.1016/j.ekir.2020.06.012, 5, 8, 1291-1297, 2020.08, Introduction: Greater reduction in estimated glomerular filtration rate (eGFR) after specific treatment for primary aldosteronism (PA) reflects improvement in glomerular hyperfiltration associated with PA and leads to better patient outcomes. However, little is known regarding the mechanisms underlying eGFR reduction after treatment for PA. Methods: We analyzed data from the nationwide PA registry in Japan. Patients were assigned to adrenalectomy (n = 438) and mineralocorticoid receptor (MR) antagonist (n = 746) groups. We assessed associations between changes in blood pressure (BP), plasma renin activity (PRA) and plasma aldosterone concentrations (PAC), and eGFR before and 6 months after treatment for both groups. Results: In a multivariable linear regression, the adjusted β values (95% confidence interval [CI]) for change in eGFR after treatment were −2.76 (−4.29, −1.22) ml/min per 1.73 m2 for PRA (per 3.2 ng/ml per hour), and 1.97 (1.08, 2.85) ml/min per 1.73 m2 for PAC (per 236.1 pg/ml) in the adrenalectomy group; and −0.45 (−0.89, −0.01) ml/min per 1.73 m2 for PRA and −0.72 (−1.62, 0.18) ml/min per 1.73 m2 for PAC in the MR antagonist group. Change in mean arterial pressure after treatment was not significantly associated with change in eGFR in either group. Changes in PRA and PAC but not BP before and 6 months after treatment for PA were associated with greater reductions in eGFR. Conclusion: Post-treatment improvements in glomerular hyperfiltration may be attributable to decreased MR activity in the kidneys, but not to reductions in systemic BP..
20. Miyako Tanaka, Marie Saka-Tanaka, Kozue Ochi, Kumiko Fujieda, Yuki Sugiura, Tomofumi Miyamoto, Hiro Kohda, Ayaka Ito, Taiki Miyazawa, Akira Matsumoto, Seiichiro Aoe, Yoshihiro Miyamoto, Naotake Tsuboi, Shoichi Maruyama, Makoto Suematsu, Sho Yamasaki, Yoshihiro Ogawa, Takayoshi Suganami, C-type lectin Mincle mediates cell death-triggered inflammation in acute kidney injury, Journal of Experimental Medicine, 10.1084/JEM.20192230, 217, 11, 2020.08, Accumulating evidence indicates that cell death triggers sterile inflammation and that impaired clearance of dead cells causes nonresolving inflammation; however, the underlying mechanisms are still unclear. Here, we show that macrophage-inducible C-type lectin (Mincle) senses renal tubular cell death to induce sustained inflammation after acute kidney injury in mice. Mincle-deficient mice were protected against tissue damage and subsequent atrophy of the kidney after ischemia-reperfusion injury. Using lipophilic extract from the injured kidney, we identified β-glucosylceramide as an endogenous Mincle ligand. Notably, free cholesterol markedly enhanced the agonistic effect of β-glucosylceramide on Mincle. Moreover, β-glucosylceramide and free cholesterol accumulated in dead renal tubules in proximity to Mincle-expressing macrophages, where Mincle was supposed to inhibit clearance of dead cells and increase proinflammatory cytokine production. This study demonstrates that β-glucosylceramide in combination with free cholesterol acts on Mincle as an endogenous ligand to induce cell death-triggered, sustained inflammation after acute kidney injury..
21. Mitsuru Esaki, Kazuhiro Haraguchi, Kazuya Akahoshi, Naru Tomoeda, Akira Aso, Soichi Itaba, Haruei Ogino, Yusuke Kitagawa, Hiroyuki Fujii, Kazuhiko Nakamura, Masaru Kubokawa, Naohiko Harada, Yosuke Minoda, Sho Suzuki, Eikichi Ihara, Yoshihiro Ogawa, Endoscopic mucosal resection vs endoscopic submucosal dissection for superficial non-ampullary duodenal tumors, World Journal of Gastrointestinal Oncology, 10.4251/wjgo.v12.i8.918, 12, 8, 918-930, 2020.08, BACKGROUND The selection of endoscopic treatments for superficial non-ampullary duodenal epithelial tumors (SNADETs) is controversial. AIM To compare the efficacy and safety of endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) for SNADETs. METHODS We retrospectively analyzed the data of patients with SNADETs from a database of endoscopic treatment for SNADETs, which included eight hospitals in Fukuoka, Japan, between April 2001 and October 2017. A total of 142 patients with SNADETs treated with EMR or ESD were analyzed. Propensity score matching was performed to adjust for the differences in the patient characteristics between the two groups. We analyzed the treatment outcomes, including the rates of en bloc/complete resection, procedure time, adverse event rate, hospital stay, and local or metastatic recurrence. RESULTS Twenty-eight pairs of patients were created. The characteristics of patients between the two groups were similar after matching. The EMR group had a significantly shorter procedure time and hospital stay than those of the ESD group [median procedure time (interquartile range): 6 (3-10.75) min vs 87.5 (68.5-136.5) min, P < 0.001, hospital stay: 8 (6-10.75) d vs 11 (8.25-14.75) d, P = 0.006]. Other outcomes were not significantly different between the two groups (en bloc resection rate: 82.1% vs 92.9%, P = 0.42; complete resection rate: 71.4% vs 89.3%, P = 0.18; and adverse event rate: 3.6% vs 17.9%, P = 0.19, local recurrence rate: 3.6% vs 0%, P = 1; metastatic recurrence rate: 0% in both). Only one patient in the ESD group underwent emergency surgery owing to intraoperative perforation. CONCLUSION EMR has significantly shorter procedure time and hospital stay than ESD, and provides acceptable curability and safety compared to ESD. Accordingly, EMR for SNADETs is associated with lower medical costs..
22. Yukihiko Okumura, Kenichi Kohashi, Yuki Tanaka, Masaki Kato, Yoshihiko Maehara, Yoshihiro Ogawa, Yoshinao Oda, Activation of the Akt/mammalian target of rapamycin pathway in combined hepatocellular carcinoma and cholangiocarcinoma
significant correlation between p-4E-BP1 expression in cholangiocarcinoma component and prognosis, Virchows Archiv, 10.1007/s00428-019-02741-3, 476, 6, 881-890, 2020.06, The Akt/mammalian target of rapamycin (mTOR) pathway, which plays an important role in regulating cellular functions including proliferation, motility, and invasion, is known to be activated in many cancers. Combined hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) (cHCC-CC) has wide histological diversity characterized by relatively poor prognosis. Because of a lack of investigation into its molecular mechanisms, no effective systemic therapy is currently available for unresectable cHCC-CC tumors. Here, we retrospectively examined the clinicopathological and activation statuses of the Akt/mTOR pathway in 89 cases of cHCC-CC. Expression levels of molecular markers associated with this signaling pathway, including phosphatase and tensin homologue deleted on chromosome 10 (PTEN), phosphorylated Akt (p-Akt), p-mTOR, p-ribosomal protein S6 (p-S6RP), and p-eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (p-4E-BP1), were measured by immunohistochemical staining. In addition, such activation in different cHCC-CC morphological categories was compared by dividing cases into those with HCC (n = 86), CC (n = 78), and intermediate components (n = 60). Comparison of prognosis among these groups revealed that p-4E-BP1 immunopositivity in cHCC-CC cases containing CC a component was a significant risk factor for poorer overall survival (P = 0.041). By evaluating factors in p-4E-BP1 expression in 78 cHCC-CC cases with a CC component, only lymph node metastasis was significantly correlated with positive immunostaining for p-4E-BP1 (P = 0.0222). In conclusion, p-4E-BP1 expression, especially in cHCC-CC cases with a CC component, was a notable Akt/mTOR pathway-related factor associated with poor prognosis. Assessing histological structure and p-4E-BP1 expression in cHCC-CC may be helpful for both predicting prognosis and using molecular targeted therapy..
23. Takayuki Watanabe, Atsunori Tsuchiya, Suguru Takeuchi, Shunsuke Nojiri, Tomoaki Yoshida, Masahiro Ogawa, Michiko Itoh, Masaaki Takamura, Takayoshi Suganami, Yoshihiro Ogawa, Shuji Terai, Development of a non-alcoholic steatohepatitis model with rapid accumulation of fibrosis, and its treatment using mesenchymal stem cells and their small extracellular vesicles, Regenerative Therapy, 10.1016/j.reth.2020.03.012, 14, 252-261, 2020.06, Introduction: Currently, there are no approved drugs for treating non-alcoholic steatohepatitis (NASH); however, mesenchymal stem cells (MSCs) and their small extracellular vesicles (sEVs), which possess immunomodulatory activities, are potential candidates. This study aimed to develop a mouse model of NASH with rapid accumulation of fibrosis using the pre-established melanocortin type-4 receptor knockout (Mc4r-KO) NASH mouse model and lipopolysaccharide (LPS), and to evaluate the therapeutic effect of MSCs and their sEVs. Methods: Mc4r-KO mice (8 weeks old, male) were fed a western diet (WD) for 8 weeks. Next, the mice were intraperitoneally injected with lipopolysaccharide (LPS) twice a week for 4 weeks while continuing the WD. To confirm the therapeutic effect of MSCs and sEVs, human adipose tissue-derived MSCs or their sEVs were administered 12 weeks after initiation of the WD, and serum testing, quantitative analysis of fibrosis, and quantitative reverse transcription-polymerase chain reaction qRT-PCR were performed. Results: By providing a WD combined with LPS treatment, we successfully developed a NASH model with rapid accumulation of fibrosis. Both human MSCs and their sEVs decreased serum alanine transaminase levels and inflammatory markers based on qRT-PCR. Histological analysis showed that MSC or sEV treatment did not affect fat accumulation. However, an improvement in fibrosis in the groups treated with MSCs and their sEVs was observed. Furthermore, after administering MSCs and sEVs, there was a significant increase in anti-inflammatory macrophages in the liver. Conclusion: We successfully developed a NASH model with rapid accumulation of fibrosis and confirmed the anti-inflammatory and anti-fibrotic effects of MSCs and their sEVs, which may be options for future therapy..
24. Seiichi Yano, Kenji Ashida, Ryuichi Sakamoto, Chihiro Sakaguchi, Masatoshi Ogata, Kengo Maruyama, Shohei Sakamoto, Munehiko Ikeda, Kenji Ohe, Shoko Akasu, Shimpei Iwata, Nobuhiko Wada, Yayoi Matsuda, Yoichi Nakanishi, Masatoshi Nomura, Yoshihiro Ogawa, Human leucocyte antigen DR15, a possible predictive marker for immune checkpoint inhibitor–induced secondary adrenal insufficiency, European Journal of Cancer, 10.1016/j.ejca.2020.02.049, 130, 198-203, 2020.05, Background: Immune checkpoint inhibitors (ICPis) induce various immune-related adverse events (irAEs), despite their beneficial effects in treating various advanced cancers. ICPi-induced secondary adrenal insufficiency is described as a prevalent and serious ‘pituitary irAE.’ However, its precise mechanism remains unclear, and no definitive predictive markers have been reported. Patients and methods: We enrolled and studied 11 patients with advanced cancer (aged 39–70 years; 6 male patients) receiving nivolumab, pembrolizumab or ipilimumab who developed pituitary irAEs. Their clinical data, including endocrine functions, were retrospectively assessed and human leucocyte antigen (HLA) genotypes were determined to compare the HLA allele frequencies in these patients and healthy controls. Results: Among 11 patients, 7, 3 and 1 patients exhibited malignant melanoma, non–small-cell lung cancer and gastric cancer, respectively. HLA type screening results revealed that HLA-DR15, B52 and Cw12 were observed in 9, 7, and 7 patients with pituitary irAE, respectively. DR15, B52 and Cw12 were significantly more prevalent in our group than in the healthy control group from the Japanese HLA-haplotype database (this study vs healthy control group); DR15: 81.8% vs 33.5% (n = 11, P = 0.0014), B52: 63.6% vs 21.0% (n = 11, P = 0.0026) and Cw12: 70% vs 21.3% (n = 10, P = 0.0013). Conclusions: HLA-DR15, B52 and Cw12 are possible predisposing factors for pituitary irAEs. HLA-DR15 is reportedly associated with autoimmune disease via interleukin-17 regulation, suggesting its involvement in pituitary irAE development. Using HLA haplotypes as pituitary irAE predictive markers, we could provide safe ICPi treatment and understand irAE pathogenesis..
25. Yuki Nakamura, Minato Yokoyama, Soichiro Yoshida, Hajime Tanaka, Toshiki Kijima, Junichiro Ishioka, Yoh Matsuoka, Kazutaka Saito, Isao Minami, Takanobu Yoshimoto, Shotaro Naito, Yoshihiro Ogawa, Tetsuya Yamada, Shinichi Uchida, Yasuhisa Fujii, Postoperative renal impairment and longitudinal change in renal function after adrenalectomy in patients with Cushing's syndrome, International Journal of Urology, 10.1111/iju.14205, 27, 5, 395-400, 2020.05, Objectives: To evaluate the renal function after adrenalectomy in patients with Cushing's syndrome in comparison with that in patients with primary aldosteronism. Methods: This retrospective study included 35 patients with Cushing's syndrome and 51 patients with primary aldosteronism who underwent unilateral adrenalectomy and were followed up for >6 months. The renal function was analyzed before and after adrenalectomy using the estimated glomerular filtration rate. Postoperative renal impairment was defined as a >25% reduction in the estimated glomerular filtration rate from baseline at 1 month after adrenalectomy. Multivariate logistic regression analyses were carried out to examine whether the differences between Cushing's syndrome and primary aldosteronism increased the risk of postoperative renal impairment. Longitudinal changes were calculated starting 1 month after adrenalectomy using the linear mixed model. Results: The mean estimated glomerular filtration rate in both groups significantly decreased at 1 month after adrenalectomy from baseline. Postoperative renal impairment was observed in four (11%) and 12 (24%) patients in the Cushing's syndrome and primary aldosteronism groups, respectively. Multivariate analysis showed that preoperative systolic blood pressure was independently associated with postoperative renal impairment, but not with the type of the disease. There was no significant increase or decrease in postoperative estimated glomerular filtration rate observed after the initial decrease after adrenalectomy in either group. Conclusions: Patients with Cushing's syndrome show the same persistent renal impairment after adrenalectomy as that reported in patients with primary aldosteronism. Attention should be given to possible masked renal damage in clinical practice for the management of Cushing's syndrome..
26. , Maki Yokomoto-Umakoshi, Hironobu Umakoshi, Tazuru Fukumoto, Yayoi Matsuda, Hiromi Nagata, Masatoshi Ogata, Hisaya Kawate, Takashi Miyazawa, Ryuichi Sakamoto, Yoshihiro Ogawa, Pheochromocytoma and paraganglioma
An emerging cause of secondary osteoporosis, Bone, 10.1016/j.bone.2020.115221, 133, 2020.04, Context: Pheochromocytoma (PHEO) and paraganglioma (PGL) (PHEO and PGL: PPGLs) are catecholamine-producing neuroendocrine tumors, which are known to be associated with low bone mineral density (BMD). However, it remains unknown whether PPGLs are associated with high prevalence of osteoporotic fracture and if so, whether their surgical resection improves BMD has been addressed. Objective: To evaluate the risk of vertebral fracture (VF) in PPGLs and the improvement of BMD after surgery. Design and settings: A retrospective cross-sectional study in a single referral center. Participants: This study included the following patients: 1) 49 patients with PPGLs and 61 patients with non-functional AT who were examined radiograph of the spine, 2) 23 patients with PPGLs who were examined BMD at follow-up. Intervention: 1) The prevalence of VF was evaluated between PPGLs and non-functional AT. 2) In PPGLs, BMD was evaluated at baseline and after surgery. Results: PPGLs had a higher prevalence of VF (43% [21/49]) than non-functional AT (16% [10/61]; p = 0.002). PPGLs were associated with VF after adjusting for age and sex (odds ratio, 4.47; 95% confidence interval, 1.76–11.3; p = 0.001). In PPGLs, BMD at the lumber spine was improved (before: 0.855 ± 0.198 g/cm2, after: 0.888 ± 0.169 g/cm2, mean of the difference: 0.032 g/cm2, p = 0.026), with 3.8% increase. Conclusion: This study demonstrates that PPGLs was associated with VF and that their surgical resection contributes to the improvement of BMD in the trabecular bone. These observations support the notion that PPGLs are an emerging cause of secondary osteoporosis..
27. Hironobu Umakoshi, Ryuichi Sakamoto, Yayoi Matsuda, Maki Yokomoto-Umakoshi, Hiromi Nagata, Tazuru Fukumoto, Masatoshi Ogata, Yoshihiro Ogawa, Role of Aldosterone and Potassium Levels in Sparing Confirmatory Tests in Primary Aldosteronism, The Journal of clinical endocrinology and metabolism, 10.1210/clinem/dgz148, 105, 4, 2020.04, CONTEXT: The current clinical guidelines suggest that confirmatory tests for primary aldosteronism (PA) may be excluded in some of patients who have elevated plasma aldosterone concentration (PAC) under plasma renin suppression. However, this has low-priority evidence and is under debate in use of serum potassium. OBJECTIVE: This study aimed to investigate an appropriate setting for sparing confirmatory tests in PA. DESIGN AND SETTING: A retrospective cross-sectional study in a single referral center. PARTICIPANTS: This study included 327 patients who had hypertension under plasma renin suppression and underwent the captopril challenge test (CCT) between January 2007 and April 2019. CCT results were used to diagnose PA. MAIN OUTCOME MEASURE: Diagnostic value of PAC and serum potassium in confirmation of PA. RESULTS: Of the studied patients, 252 of 327 (77%) were diagnosed with PA. All 61 patients with PAC > 30 ng/dL were diagnosed with PA. In patients with PAC between 20 and 30 ng/dL, 44 of 55 (80%) were diagnosed with PA, while all 26 with PAC between 20 to 30 ng/dL who had spontaneous hypokalemia were diagnosed with PA. The proportion of unilateral PA determined by adrenal vein sampling (AVS) was higher in patients who had PAC > 30 ng/dL or those with spontaneous hypokalemia who had PAC between 20 and 30 ng/dL than those who did not meet the criteria (76% vs. 17%, P < .001). CONCLUSION: Confirmatory tests in PA could be spared in patients who have typical features of PA and these patients had a high probability of unilateral PA on AVS..
28. Mitsuru Esaki, Yasuyo Hayashi, Hisatomo Ikehara, Eikichi Ihara, Toshiki Horii, Yu Tamura, Ryoji Ichijima, Shun Yamakawa, Akira Irie, Hitoshi Shibuya, Sho Suzuki, Chika Kusano, Yosuke Minoda, Hirotada Akiho, Yoshihiro Ogawa, Takuji Gotoda, The effect of scissor-type versus non-scissor-type knives on the technical outcomes in endoscopic submucosal dissection for superficial esophageal cancer
a multi-center retrospective study, Diseases of the esophagus : official journal of the International Society for Diseases of the Esophagus, 10.1093/dote/doz077, 33, 4, 2020.04, The Clutch Cutter was invented as a scissor-type knife for endoscopic submucosal dissection (ESD) of gastrointestinal neoplasms. ESD with the scissor-type knife (ESD-S) may be considered a technically easier procedure than ESD with non-scissor-type knives (ESD-NS). Therefore, this study aimed to compare the technical outcomes of ESD-S with those of ESD-NS for superficial esophageal cancer. This was a multicenter retrospective study. Patients with superficial esophageal cancer treated with ESD between October 2015 and March 2018 at three hospitals were retrospectively reviewed. The ESD-S group had 48 patients and the ESD-NS group had 114 patients. A propensity score matching analysis was performed to compensate for the confounding bias between both groups. Multivariate analyses and propensity score matching were used to adjust for age, sex, the tumor size, tumor location, tumor depth, degree of tumor circumference, operator level, usage of the traction method, and the sedation method. The primary outcome was the procedure time of the ESD. Secondary outcomes were the rate of en-bloc/complete resection and the rate of complications including perforation, delayed bleeding, and stricture. Propensity score matching analysis provided 36 matched pairs. Median procedure time in the ESD-S group was significantly shorter than that in the ESD-NS group (44.0 min vs. 66.5 min, P = 0.020). In addition, the treatment outcomes were similar in both groups (en-bloc resection: 100% vs. 97.2%, P = 1; complete resection: 88.9% vs. 86.1%, P = 1; curative resection: 80.6% vs. 77.8%, P = 1; perforation: 0% vs. 5.6%, P = 0.49; delayed bleeding: 0% in both groups; stricture: 2.8% vs. 8.3%, P = 0.61). ESD-S was associated with a shorter procedure time than ESD-NS, without an increase in the incidence of complications. Therefore, the scissor-type knife should be considered as an endo-knife for ESD of superficial esophageal cancers..
29. Shinichi Tsuruta, Kenichi Kohashi, Yuichi Yamada, Minako Fujiwara, Yutaka Koga, Eikichi Ihara, Yoshihiro Ogawa, Eiji Oki, Masafumi Nakamura, Yoshinao Oda, Solid-type poorly differentiated adenocarcinoma of the stomach
Deficiency of mismatch repair and SWI/SNF complex, Cancer Science, 10.1111/cas.14301, 111, 3, 1008-1019, 2020.03, ARID1A, one of the subunits in SWI/SNF chromatin remodeling complex, is frequently mutated in gastric cancers with microsatellite instability (MSI). The most frequent MSI in solid-type poorly differentiated adenocarcinoma (PDA) has been reported, but the SWI/SNF complex status in solid-type PDA is still largely unknown. We retrospectively analyzed 54 cases of solid-type PDA for the expressions of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6), SWI/SNF complex subunits (ARID1A, INI1, BRG1, BRM, BAF155, and BAF170) and EBER, and mutations in KRAS and BRAF. We analyzed 40 cases of another histological type of gastric cancer as a control group. The solid-type PDAs showed coexisting glandular components (76%), MMR deficiency (39%), and complete/partial loss of ARID1A (31%/7%), INI1 (4%/4%), BRG1 (48%/30%), BRM (33%/33%), BAF155 (13%/41%), and BAF170 (6%/2%), EBER positivity (4%), KRAS mutation (2%), and BRAF mutation (2%). Compared to the control group, MMR deficiency and losses of ARID1A, BRG1, BRM, and BAF155 were significantly frequent in solid-type PDAs. Mismatch repair deficiency was associated with the losses of ARID1A, BRG1, and BAF155 in solid-type PDAs. In the MMR-deficient group, solid components showed significantly more frequent losses of ARID1A, BRG1, BRM, and BAF155 compared to glandular components (P =.0268, P =.0181, P =.0224, and P =.0071, respectively). In the MMR-proficient group, solid components showed significantly more frequent loss of BRG1 compared to glandular components (P =.012). In conclusion, solid-type PDAs showed frequent losses of MMR proteins and the SWI/SNF complex. We suggest that loss of the SWI/SNF complex could induce a morphological shift from differentiated-type adenocarcinoma to solid-type PDA..
30. , Maki Yokomoto-Umakoshi, Ryuichi Sakamoto, Hironobu Umakoshi, Yayoi Matsuda, Hiromi Nagata, Tazuru Fukumoto, Masatoshi Ogata, Yoshihiro Ogawa, Unilateral primary aldosteronism as an independent risk factor for vertebral fracture, Clinical Endocrinology, 10.1111/cen.14145, 92, 3, 206-213, 2020.03, Context: Primary aldosteronism (PA) is known to increase vertebral fracture (VF), although the detailed mechanism remains to be elucidated. We hypothesized that the PA subtype is associated with VF. Objective: To evaluate whether unilateral PA is associated with the prevalence of VF. Design: This was a retrospective cross-sectional study in a single referral centre. Patients: We identified 210 hypertensive patients whose clinical data were available for case-detection results. One hundred and fifty-two patients were diagnosed with PA using captopril challenge tests. Measurements: We measured the prevalence of VF, according to PA subtype. Results: One hundred thirteen patients with PA were subtype classified by adrenal vein sampling. Of these, 37 patients had unilateral PA, 76 patients had bilateral PA, 58 patients had non-PA; 39 patients with PA were not subtype-classified. Patients with PA had a higher prevalence of VF (29%, 44/152) than those with non-PA (12%, 7/58; P =.011). Moreover, unilateral PA had a higher prevalence of VF (46%, 17/37) than bilateral PA (20%, 15/76; P =.021). There was no significant difference in the prevalence of VF between bilateral PA and non-PA. Unilateral PA was an independent risk factor for VF after adjusting for age and sex (OR: 3.16, 95% confidence interval: 1.12-8.92; P =.017). Among patients with unilateral PA, serum cortisol concentrations after 1-mg dexamethasone suppression test were higher in those with VF (1.32 ± 0.67 g/dL) than those without (0.96 ± 0.33 g/dL; P =.048). Conclusions: Unilateral PA is an independent risk factor for VF..
31. Taisuke Narazaki, Yasuhiro Nakashima, Yasuhiro Tsukamoto, Ruriko Nishida, Mariko Tsuda, Hiroki Muta, Daisaku Kimura, Toru Masuda, Akiko Takamatsu, Kenichi Kohashi, Daisuke Murakami, Motoaki Shiratsuchi, Yoshihiro Ogawa, Schizophyllum commune sinusitis after allogeneic bone marrow transplantation for myelodysplastic syndrome
A case report and literature review, Transplant Infectious Disease, 10.1111/tid.13205, 22, 1, 2020.02, Sinusitis is a serious infectious complication of allogeneic hematopoietic stem cell transplantation. Schizophyllum commune (S commune) is a common basidiomycete fungus that is rarely involved in human disease. We report herein a case of S commune sinusitis after allogeneic bone marrow transplantation. A 66-year-old man with myelodysplastic syndrome underwent allogeneic bone marrow transplantation and developed maxillary and ethmoid sinusitis. The sinusitis did not improve with liposomal amphotericin B after neutrophil engraftment, so we considered that surgical intervention was needed for the recovery of sinusitis. Endoscopic sinus surgery was performed. In the debridement tissue of paranasal mucosa, filamentous fungal elements were observed. Moreover, genetic analysis of the tissue revealed the presence of S commune. Schizophyllum commune should be recognized as a fungal pathogen that causes sinusitis after allogeneic hematopoietic stem cell transplantation. This case suggests the effectiveness of prompt surgical intervention with liposomal amphotericin B treatment for S commune sinusitis and the usefulness of genetic diagnosis for cases under antifungal treatment. (160 words)..
32. Yosuke Ikeda, Noriyuki Sonoda, Battsetseg Bachuluun, Shinichiro Kimura, Yoshihiro Ogawa, Toyoshi Inoguchi, Aberrant activation of bone marrow Ly6C high monocytes in diabetic mice contributes to impaired glucose tolerance, PloS one, 10.1371/journal.pone.0229401, 15, 2, 2020.01, Accumulating evidence indicates that diabetes and obesity are associated with chronic low-grade inflammation and multiple organ failure. Tissue-infiltrated inflammatory M1 macrophages are aberrantly activated in these conditions and contribute to hyperglycemia and insulin resistance. However, it is unclear at which stage these cells become aberrantly activated: as precursor monocytes in the bone marrow or as differentiated macrophages in tissues. We examined the abundance, activation state, and function of bone marrow-derived Ly6Chigh monocytes in mice with diabetes and/or obesity. Ly6Chigh monocytes were FACS-purified from six groups of male mice consisting of type 2 diabetes model db/db mice, streptozotocin (STZ) induced insulin depletion mice, high fat diet (HFD) induced obesity mice and each control mice. Ly6Chigh monocytes were then analyzed for the expression of inflammation markers by qRT-PCR. In addition, bone marrow-derived Ly6Chigh monocytes from db/+ and db/db mice were fluorescently labeled and injected into groups of db/db recipient mice. Cell trafficking to tissues and levels of markers were examined in the recipient mice. The expression of many inflammation-related genes was significantly increased in Ly6Chigh monocytes from db/db mice, compared with the control. Bone marrow-derived Ly6Chigh monocytes isolated from db/db mice, but not from db/+ mice, displayed prominent infiltration into peripheral tissues at 1 week after transfer into db/db mice. The recipients of db/db Ly6Chigh monocytes also exhibited significantly increased serum glucose levels and worsening tolerance compared with mice receiving db/+ Ly6Chigh monocytes. These novel observations suggest that activated Ly6Chigh monocytes may contribute to the glucose intolerance observed in diabetes..
33. Taisuke Narazaki, Motoaki Shiratsuchi, Takamitsu Matsushima, Mariko Tsuda, Yasuhiro Tsukamoto, Hiroki Muta, Toru Masuda, Daisaku Kimura, Akiko Takamatsu, Hidetaka Yamamoto, Yoshinao Oda, Hiroaki Miyoshi, Koichi Ohshima, Yayoi Matsuda, Ryuichi Sakamoto, Yasuhiro Nakashima, Yoshihiro Ogawa, Clinico-pathological characteristics of primary adrenal lymphomas–potential efficacy of autologous stem cell transplantation, Leukemia and Lymphoma, 10.1080/10428194.2020.1725507, 2020.01.
34. , Hiroki Kobayashi, Yoshihiro Nakamura, Masanori Abe, Isao Kurihara, Hiroshi Itoh, Takamasa Ichijo, Yoshiyu Takeda, Takashi Yoneda, Takuyuki Katabami, Mika Tsuiki, Norio Wada, Yoshihiro Ogawa, Ryuichi Sakamoto, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Takanobu Yoshimoto, Tetsuya Yamada, Ryuji Okamoto, Yuichi Matsuda, Megumi Fujita, Minemori Watanabe, Kouichi Tamura, Akiyo Tanabe, Mitsuhide Naruse, Effect of cosyntropin during adrenal venous sampling on subtype of primary aldosteronism
Analysis of surgical outcome, European journal of endocrinology, 10.1530/EJE-19-0860, 182, 3, 265-273, 2020.01, Objectives: We investigated the clinical significance of ACTH stimulation during adrenal venous sampling (AVS) by surgical outcome of primary aldosteronism (PA). Design: Multicenter retrospective study by Japan PA study. Method: We allocated 314 patients with both basal and ACTH-stimulated AVS data who underwent adrenalectomy to three groups: basal lateralization index (LI) ≥2 with ACTH-stimulated LI ≥4 on the ipsilateral side (Unilateral (U) to U group, n = 245); basal LI <2 with ACTH-stimulated LI ≥4 (Bilateral (B) to U group, n = 15); and basal LI ≥2 with ACTH-stimulated LI <4 (U to B group, n = 54). We compared surgical outcomes among the groups using the Primary Aldosteronism Surgical Outcome (PASO) criteria. Results: Compared with U to U group, U to B group had poor clinical and biochemical outcomes and low rates of adrenal adenoma as pathological findings (P = 0.044, 0.006, and 0.048, respectively), although there were no significant differences between U to U and B to U groups. All patients in U to B group with clinical and biochemical benefits, however, had adrenal adenoma as pathological findings and could be well differentiated from those with poor surgical outcomes via basal LI (>8.3), but not ACTH-stimulated LI. These results were similar even when we defined each group based on a cut-off value of 4 for basal LI. Conclusions: Although PA patients in U to B group had worse surgical outcomes than did those in U to U group, basal LI could discriminate among patients with better surgical outcomes in U to B group..
35. Mitsuru Esaki, Yosuke Minoda, Eikichi Ihara, Seiichiro Sakisaka, Shinichi Tsuruta, Taizo Hosokawa, Masafumi Wada, Yoshitaka Hata, Sho Suzuki, Aya Iwao, Shun Yamakawa, Akira Irie, Hirotada Akiho, Yoshihiro Ogawa, Efficacy of traction, using a clip-with-thread, for esophageal endoscopic submucosal dissection for esophageal lesions with fibrosis in an ex vivo pig training model, Turkish Journal of Gastroenterology, 10.5152/tjg.2020.19207, 31, 1, 58-64, 2020.01, Background/Aims: Endoscopic submucosal dissection (ESD) of recurrent esophageal carcinoma is technically difficult to perform due to submucosal fibrosis that develops after definitive chemoradiation therapy. Therefore, our aim was to evaluate the usefulness of clipwith- thread traction for ESD of esophageal lesions with submucosal fibrosis. Materials and Methods: Four endoscopists excised 16 lesions by ESD in an ex vivo pig training model. Mock lesions (30 mm in diameter) were created, including a 10-mm area of submucosal fibrosis in the center of each lesion. Each endoscopist performed two ESDs with traction (ESD-T) and two without traction (ESD-N). The primary outcome was the time required for submucosal dissection. Secondary outcomes were the rate of en bloc (complete) resection and perforation during the procedure, and the total amount of solution injected. Results: All esophageal ESDs were completed. The median dissection time was significantly shorter for the ESD-T group (median 12.5 min, interquartile range 10.2-14.5) when comparing to the ESD-N group (median 18.0 min, interquartile range 14.6-19.2) (P=0.040). The en bloc resection rate was 100% in both groups, with a rate of complete resection of 87.5% and a rate of perforation of 37.5% for both groups. The median amount of solution injected was not significantly different between the ESD-T (18.0 ml) and ESD-N (20.5 ml) groups (P=0.526). Conclusion: Clip-with-thread traction improved the performance of ESD for lesions with submucosal fibrosis. However, the method might not reduce the risk of perforation, which remains an important clinical issue to resolve..
36. Nao Fujimori, Yosuke Minoda, Masatoshi Murakami, Yuta Suehiro, Takamasa Oono, Eikichi Ihara, Yoshihiro Ogawa, Endoscopic removal of a lumen-apposing metal stent that migrated into the walled-off necrosis during the first drainage procedure, Endoscopy, 10.1055/a-0992-8900, 52, 2, E51-E52, 2020.01.
37. , Yuichi Fujii, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Koichi Yamamto, Atsushi Ogo, Toshihiko Yanase, Shintaro Okamura, Shozo Miyauchi, Megumi Fujita, Tomoko Suzuki, Hironobu Umakoshi, Tatsuki Ogasawara, Mika Tsuiki, Mitsuhide Naruse, Masanobu Yamada, Hiromi Rakugi, Takashi Kawamura, Osamu Ogawa, Akiyo Tanabe, Tomonobu Hasegawa, Masanori Abe, Ryuichi Sakamoto, Takuro Shinbo, Tatsuya Kai, Tomikazu Fukuoka, Masanori Murakami, Shigeatsu Hashimoto, Makito Tanabe, Mitsuhiro Kometani, Yuichirou Yoshikawa, Youichi Oono, Hisashi Fukuda, Takashi Yoneda, Historical changes and between-facility differences in adrenal venous sampling for primary aldosteronism in Japan, Journal of human hypertension, 10.1038/s41371-019-0229-4, 34, 1, 34-42, 2020.01, Primary aldosteronism (PA) is a common curable cause of hypertension. Adrenal venous sampling (AVS) is recommended for subtype diagnosis but is a difficult procedure. Recently, an increased prevalence of PA was reported, creating a greater demand for treatment of the condition in clinical facilities. The aim of the present study was to identify the historical changes over time and the differences between facilities in the success rate and subtype diagnosis of PA. The database of the PA registry developed by the Japan PA Study (JPAS) was used. A total of 2599 patients with PA who underwent AVS were evaluated. The overall success rate of AVS was 88%. The bilateral subtype was the dominant subtype, comprising 69% of cases. During the period 2004−2011 to 2011−2017, there were significant changes in the total number of AVS procedures (from 562 to 1732), ratio of ACTH administration with AVS (75 to 97%), success rate (79 to 90%), and proportion with bilateral subtype diagnosis (53 to 72%). There were also significant inter-facility differences in the number of AVS procedures (6 to 322), success rate (59 to 97%), and proportion with the bilateral subtype (44 to 86%). The principal enrolled department was Endocrinology (86%), and the ratio of unilateral PA was significantly higher in this department than in others (32% vs. 25%). In conclusion, the number of AVS procedures performed, the success rate, and the proportion with the bilateral subtype increased over time after normalizing the centre difference. Significant differences were observed between the centres..
38. Nao Fujimori, Masami Miki, Lingaku Lee, Kazuhide Matsumoto, Yu Takamatsu, Takehiro Takaoka, Katsuhito Teramatsu, Yuta Suehiro, Masatoshi Murakami, Hisato Igarashi, Takamasa Oono, Takao Ohtsuka, Masafumi Nakamura, Yutaka Koga, Yoshinao Oda, Tetsuhide Ito, Yoshihiro Ogawa, Natural history and clinical outcomes of pancreatic neuroendocrine neoplasms based on the WHO 2017 classification; a single-center experience of 30 years, Pancreatology, 10.1016/j.pan.2020.04.003, 2020.01, Background/Objectives: This single-center study aimed to evaluate treatment outcomes and long-term prognosis of patients with pancreatic neuroendocrine neoplasms (PanNENs) based on the World Health Organization (WHO) 2017 classification. Methods: We enrolled 245 patients with PanNENs treated at Kyushu University Hospital between January 1987 and March 2018. PanNENs were categorized according to the WHO 2017 classification or further subdivisions of Ki-67 index. Clinicopathological features, median survival time (MST), and prognostic factors were retrospectively analyzed. Results: The number of PanNENs, especially non-functioning PanNENs, has increased over the last decade. The mean MST of all patients was 202 months; which was longest in patients with NET G1 (n = 145, MST = 261 months) relative to NET G2 (n = 72, 132 months), NET G3 (n = 3, 34 months) and NEC G3 (n = 17, 9 months). Prognosis in patients with surgery as the first-line treatment was significantly better than in those with drug therapy. However, 26% of patients who underwent curative resection developed recurrence after a median time of 28.7 months. In unresectable PanNENs (n = 97), the MST and 5-year survival rate were 78 months and 55.8%, respectively. Poor differentiation, Ki-67 index of >10% and presence of liver metastasis were significant unfavorable predictors. Response to first-line therapy (stable disease/partial response) and three or more treatment regimens were significant favorable predictors for unresectable PanNENs according to multivariate analyses (p < 0.01). Conclusions: We demonstrated the utility of the WHO 2017 classification for PanNENs in the real clinical setting. For better prognosis in PanNENs, the use of three or more regimens should be considered..
39. , Ryo Nakamaru, Koichi Yamamoto, Hiromi Rakugi, Hiroshi Akasaka, Isao Kurihara, Takamasa Ichijo, Yoshiyu Takeda, Takuyuki Katabami, Mika Tsuiki, Norio Wada, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Takanobu Yoshimoto, Ryuji Okamoto, Megumi Fujita, Hiroki Kobayashi, Kouichi Tamura, Kohei Kamemura, Shintaro Okamura, Miki Kakutani, Akiyo Tanabe, Mitsuhide Naruse, Obesity predicts persistence of resistant hypertension after surgery in patients with primary aldosteronism, Clinical Endocrinology, 10.1111/cen.14203, 2020.01, Objective: Primary aldosteronism (PA) is considered a major cause of resistant hypertension (RHT). The prevalence of RHT has been recently reported to reach 18% in general hypertension. However, little is known about the prevalence and the outcomes after adrenalectomy of RHT in PA. Therefore, we aimed to clarify the prevalence and surgical outcomes in patients with both PA and RHT. Patients and Design: Among 550 patients who underwent adrenalectomy for unilateral PA in the Japan PA Study, RHT was defined as an uncontrolled blood pressure (≥140/90 mm Hg) despite treatment with at least any three antihypertensives or hypertension controlled with at least four drugs. Surgical outcome was assessed by the biochemical and clinical outcome. Results: Although 40 (7.3%) patients fulfilled the criteria for preoperative RHT, this should be underestimated because only 36% of patients with postoperative RHT were classified as having preoperative RHT. The prevalence of preoperative RHT was approximately 20% when estimated using the total number of patients with postoperative RHT and the ratio of postoperative RHT in patients with preoperative RHT. Although an improvement in hypertension was achieved in approximately 80% of patients with preoperative RHT, 20% of these exhibited persistent RHT. These patients were more obese than those for whom RHT improved after surgery. Notably, body mass index of ≥25 kg/m2 was an independent predictor of postoperative RHT. Conclusions: The prevalence of RHT in PA was lower than expected even with the adjustment for underestimation. Furthermore, obesity is an independent factor predicting the postoperative persistence of RHT..
40. T. Fukuda, R. Bouchi, M. Asakawa, T. Takeuchi, K. Shiba, K. Tsujimoto, C. Komiya, T. Yoshimoto, Y. Ogawa, T. Yamada, Sarcopenic obesity is associated with a faster decline in renal function in people with type 2 diabetes, Diabetic Medicine, 10.1111/dme.14153, 37, 1, 105-113, 2020.01, Aim: To evaluate the association between sarcopenic obesity and the decline in estimated GFR in people with type 2 diabetes. Methods: We enrolled 745 people with type 2 diabetes (mean age 64.6 years, 53.6% men). Body composition was evaluated using dual-energy X‑ray absorptiometry. Skeletal muscle index, calculated as appendicular non-fat mass (kg) divided by height squared (m2), was used to determine sarcopenia. Sarcopenic obesity was defined as the coexistence of sarcopenia and a ratio of android to gynoid fat mass greater than the median values in each gender. The association of sarcopenic obesity both with the annual rate of decline in estimated GFR and a >30% decline in estimated GFR was evaluated using multivariate linear regression models and Cox proportional hazard models, respectively. Results: Participants with sarcopenic obesity were at an increased risk of a high annual rate of decline in estimated GFR, even after adjustment for the confounding variables (standardized β = −0.228, P <0.001). Sarcopenic obesity was also significantly associated with risk of a >30% decline in estimated GFR (hazard ratio 4.52, 95% CI 2.16–9.47; P < 0.01) in multivariate model. Conclusions: Sarcopenic obesity evaluated by dual energy X‑ray absorptiometry is associated with a faster decline in renal function in people with type 2 diabetes..
41. Yosuke Minoda, Takatoshi Chinen, Takashi Osoegawa, Soichi Itaba, Kazuhiro Haraguchi, Hirotada Akiho, Akira Aso, Yorinobu Sumida, Keishi Komori, Haruei Ogino, Eikichi Ihara, Yoshihiro Ogawa, Superiority of mucosal incision-assisted biopsy over ultrasound-guided fine needle aspiration biopsy in diagnosing small gastric subepithelial lesions
A propensity score matching analysis, BMC Gastroenterology, 10.1186/s12876-020-1170-2, 20, 1, 2020.01, Background: Gastric subepithelial lesions, including gastrointestinal stromal tumors, are often found during routine gastroscopy. While endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) has been the gold standard for diagnosing gastric subepithelial lesions, alternative open biopsy procedures, such as mucosal incision-assisted biopsy (MIAB) has been reported useful. The aim of this study is to evaluate the efficacy of MIAB for the diagnosis of gastric SELs compared with EUS-FNAB. Methods: We retrospectively analyzed medical records of 177 consecutive patients with gastric SELs who underwent either MIAB or EUS-FNAB at five hospitals in Japan between January 2010 and January 2018. Diagnostic yield, procedural time, and adverse event rates for the two procedures were evaluated before and after propensity-score matching. Results: No major procedure-related adverse events were observed in either group. Both procedures yielded highly-accurate diagnoses once large enough samples were obtained; however, such successful sampling was more often accomplished by MIAB than by EUS-FNAB, especially for small SELs. As a result, MIAB provided better diagnostic yields for SELs smaller than 20-mm diameter. The diagnostic yields of both procedures were comparable for SELs larger than 20-mm diameter; however, MIAB required significantly longer procedural time (approximately 13 min) compared with EUS-FNAB. Conclusions: Although MIAB required longer procedural time, it outperformed EUS-FNAB when diagnosing gastric SELs smaller than 20-mm diameter..
42. Shunya Nakane, Akihiro Mukaino, Eikichi Ihara, Yoshihiro Ogawa, Autoimmune gastrointestinal dysmotility
the interface between clinical immunology and neurogastroenterology, Immunological Medicine, 10.1080/25785826.2020.1797319, 2020, Autoimmune gastrointestinal dysmotility (AGID), an idiopathic or paraneoplastic phenomenon, is a clinical form of limited autoimmune dysautonomia. The symptoms of AGID and gastrointestinal manifestations in patients with autoimmune rheumatic diseases are overlapping. Antineuronal autoantibodies are often detected in patients with AGID. Autoantibodies play a key role in GI dysmotility; however, whether they cause neuronal destruction is unknown. Hence, the connection between the presence of these autoantibodies and the specific interference in synaptic transmission in the plexus ganglia of the enteric nervous system has to be determined. The treatment options for AGID are not well-defined. However, theoretically, immunomodulatory therapies have been shown to be effective and are therefore used as the first line of treatment. Nonetheless, diverse combined immunomodulatory therapies should be considered for intractable cases of AGID. We recommend comprehensive autoimmune evaluation and cancer screening for clinical diagnosis of AGID. Univocal diagnostic criteria, treatment protocols, and outcome definitions for AGID are required for prompt diagnosis and treatment and appropriate management of immunotherapy, which will circumvent the need for surgeries and improve patient outcome. In conclusion, AGID, a disease at the interface of clinical immunology and neurogastroenterology, requires further investigations and warrants cooperation among specialists, especially clinical immunologists, gastroenterologists, and neurologists..
43. , M. Yokomoto-Umakoshi, H. Umakoshi, M. Ogata, T. Fukumoto, Y. Matsuda, T. Miyazawa, R. Sakamoto, Y. Ogawa, Coexistence of osteoporosis and atherosclerosis in pheochromocytoma
new insights into its long-term management, Osteoporosis International, 10.1007/s00198-020-05527-5, 2020, Summary: Osteoporosis and atherosclerosis frequently coexist in patients with pheochromocytoma. The presence of osteoporosis may predict that of atherosclerosis and vice versa in patients with PHEO. These findings have implications for the long-term management of the pheochromocytoma and its potential chronic complications. Introduction: Pheochromocytoma (PHEO), a catecholamine-producing tumor, is often found incidentally, and it may be present for years before it is diagnosed. However, long-term exposure to catecholamines excess may induce chronic complications, such as osteoporosis and atherosclerosis. We aimed to evaluate concomitant osteoporosis and atherosclerosis in patients with PHEO. Methods: Fifty-one patients with PHEO and 51 patients with a non-functional adrenal tumor were compared radiographically for the prevalence of vertebral fracture (VF), a typical osteoporotic fracture, and abdominal aortic calcification (AAC). Results: In patients with PHEO, the prevalence of AAC was higher in those with VF (58%) than in those without (6%, p < 0.001). AAC was associated with VF after adjusting for age and sex (odds ratio, 1.53; 95% confidence interval, 1.07–2.46; p = 0.003) in patients with PHEO. The degree of catecholamine excess correlated with the presence of VF and AAC (p = 0.007). The prevalence of VF was higher in patients with PHEO (37%) than those with non-functional AT (12%, p = 0.005), but the prevalence of AAC was comparable between the two groups (25% and 19%, p = 0.636). VF and AAC more frequently coexisted in patients with PHEO (22%) than in those with non-functional AT (2%, p = 0.003). Conclusion: This study represents the first demonstration that osteoporosis and atherosclerosis frequently coexist in patients with PHEO. The presence of osteoporosis may predict that of atherosclerosis and vice versa in patients with PHEO. These findings have implications for the long-term management of the PHEO and its potential chronic complications..
44. Ayumu Takeno, Ippei Kanazawa, Ken ichiro Tanaka, Masakazu Notsu, Keizo Kanasaki, Takamasa Oono, Yoshihiro Ogawa, Toshitsugu Sugimoto, High glucose promotes mineralization via bone morphogenetic protein 4-Smad signals in early stage of osteoblast differentiation, Diabetology International, 10.1007/s13340-020-00463-5, 2020, Diabetes mellitus is associated with bone fragility. Although osteoblast maturation is disturbed in patients with diabetes mellitus, the involvement of high glucose (HG) in different stages of osteoblast maturation is unclear. We used MC3T3-E1 cells, a murine osteoblastic cell line. The cells were incubated in high glucose medium (16.5 and 27.5 mM) with three different time courses: throughout 21 days, only first 7 days (early stage) and only last 7 days (late stage). Mineralization assay showed that HG throughout 21 days increased mineralization compared with control (5.5 mM). In the time course experiment, HG increased mRNA expression of Alp, osteocalcin (Ocn), runt-related transcription factor 2 and osterix on days 3 and 5. By contrast, long-term treatment with HG (14 and 21 days) decreased expression of these osteoblastic markers. HG only during early stage enhanced mineralization, while HG only during late stage had no effects. HG increased the expression of bone morphogenetic protein (BMP) 4 and enhanced phosphorylation of Smad1/5/8. Treatment with a BMP receptor antagonist LDN193189 prevented the HG-induced mineralization during early stage of osteoblast differentiation, indicating that HG in the early stage promotes mineralization by BMP4. In conclusion, the study demonstrates that continuous HG treatment might enhance early osteoblast differentiation but disturbs osteoblast maturation, and that BMP-4-Smad signal might be involved in the HG-induced differentiation and mineralization of osteoblasts..
45. Hiroaki Okuno, Haruei Ogino, Eikichi Ihara, Kei Nishioka, Yoichiro Iboshi, Takatoshi Chinen, Toshiaki Ochiai, Hirotada Akiho, Kazuhiko Nakamura, Takuji Gotoda, Yoshihiro Ogawa, Interleukin-1β as a predictor of glucocorticoid response in ulcerative colitis, Digestion, 10.1159/000507435, 2020, Background/Aim: Currently, there are no established biomarkers to differentiate between glucocorticoid (GC)-resistant and GC-sensitive ulcerative colitis (UC); however, interleukin (IL)-1β could be one such candidate biomarker. The aim of this study was to investigate whether mucosally expressed IL-1β could predict the response to GC in patients with UC. Methods: A total of 27 mucosal tissue samples from 10 patients with GC-resistant UC (GC-resistant group), 9 patients with GC-sensitive UC (GC-sensitive group), and 8 control patients (control group) were analyzed by qRT-PCR for the expression of IL-1β, GC receptor α (GRα), GRβ, and other inflammatory mediators. Rachmilewitz endoscopic index (REI) between the GC-resistant and GC-sensitive groups was matched to avoid any potential influence of inflammation. Results: The REI did not significantly differ between the GC-resistant and GC-sensitive groups. Mucosally expressed IL-1β levels in the GC-resistant group were significantly higher than those in the GC-sensitive group. However, there were no significant differences in the expression levels of GRα, GRβ, and other inflammatory mediators between the 2 groups. We could distinguish between the GC-resistant and GC-sensitive groups with a sensitivity of 90.0% and specificity of 77.8% based on mucosally expressed IL-1β. Conclusions: Mucosally expressed IL-1β can be used as a predictor of GC response in patients with UC..
46. Youichi Ohno, Masakatsu Sone, Nobuya Inagaki, Akiyuki Kawashima, Yoshiyu Takeda, Takashi Yoneda, Isao Kurihara, Hiroshi Itoh, Mika Tsuiki, Takamasa Ichijo, Takuyuki Katabami, Norio Wada, Ryuichi Sakamoto, Yoshihiro Ogawa, Takanobu Yoshimoto, Tetsuya Yamada, Junji Kawashima, Yuichi Matsuda, Hiroki Kobayashi, Kohei Kamemura, Koichi Yamamoto, Michio Otsuki, Shintaro Okamura, Shoichiro Izawa, Ryuji Okamoto, Kouichi Tamura, Akiyo Tanabe, Mitsuhide Naruse, Nadir Aldosterone Levels after Confirmatory Tests Are Correlated with Left Ventricular Hypertrophy in Primary Aldosteronism, Hypertension, 10.1161/HYPERTENSIONAHA.119.14601, 1475-1482, 2020, Left ventricular hypertrophy (LVH) is often seen in patients with primary aldosteronism (PA), and the prevalence of LVH is reportedly higher among patients with PA than patients with essential hypertension. However, the correlation between aldosterone levels and LVH is undefined, and how aldosterone affects LVH in patients with PA remains unclear. We, therefore, retrospectively assessed a large PA database established by the multicenter JPAS (Japan Primary Aldosteronism Study) to reveal the factors associated with LVH in patients with PA without suspected autonomous cortisol secretion. In the 1186 patients with PA studied, the basal plasma aldosterone concentration, plasma renin activity, and the aldosterone-to-renin ratio did not significantly correlate with left ventricular LV mass index (LVMI) in single or multiple regression analyses. However, the plasma aldosterone concentration after the captopril challenge test or saline-infusion test, which are associated with autonomous aldosterone secretion, correlated significantly with LVMI, even after adjusting for patients' backgrounds, including age and blood pressure. In addition, hypokalemia and the unilateral subtype also correlated with LVMI. Longitudinal subanalysis of medically or surgically treated patients with PA showed significant reductions in LVMI in both the surgery (63.0±18.1 to 55.3±19.5 g/m2.7, P<0.001) and drug treatment (56.8±14.1 to 52.1±13.5 g/m2.7, P<0.001) groups. Our results suggest the autonomous aldosterone secretion level, not the basal aldosterone level itself, is relevant to LVH in patients with PA. In addition, the elevated LVMI seen in patients with PA is at least partially reversible with surgical or medical treatment..
47. Maki Kawasaki, Naoko Arata, Naoko Sakamoto, Anna Osamura, Siori Sato, Yoshihiro Ogawa, Ichiro Yasuhi, Masako Waguri, Yuji Hiramatsu, Risk factors during the early postpartum period for type 2 diabetes mellitus in women with gestational diabetes, Endocrine Journal, 10.1507/endocrj.EJ19-0367, 67, 4, 427-437, 2020, For women with gestational diabetes mellitus (GDM), the evaluation of glucose tolerance (GT) in the early postpartum period is universally recommended. Nevertheless, few studies have evaluated the risk factors for T2DM on the basis of GT data obtained during the early postpartum period. We aimed to identify the risk factors for type 2 diabetes mellitus (T2DM) by evaluating GT in the first 12 weeks postpartum (12wPP) in women with GDM and to categorize the risk using a combination of the principal risk factors. This retrospective multicenter observational study included 399 East Asian women with GDM who underwent a 75-g oral glucose tolerance test (OGTT) within 12wPP, which was repeated annually or biennially and used to identify the postpartum development of T2DM. Forty-three women (10.8%) developed T2DM during a median follow-up period of 789 ± 477 days. The independent risk factors for T2DM were pre-pregnancy obesity (BMI ≥25 kg/m2), early postpartum impairment in glucose tolerance (IGT), and an early postpartum glycated hemoglobin (HbA1c) ≥5.7%. The odds ratios (95% confidence intervals) for T2DM were 3.2 (1.3–7.8) in women with either early postpartum IGT or pre-pregnancy obesity, 9.2 (3.0–28.3) in those with early postpartum IGT, pre-pregnancy obesity, and HbA1c <5.7%, and 51.4 (16.1–163.9) in those with early postpartum IGT, pre-pregnancy obesity, and HbA1c ≥5.7%, compared with those without obesity or IGT. T2DM risk in East Asian women with GDM should be stratified according to pre-pregnancy obesity and early postpartum IGT, and these patients should be followed up and receive appropriate care for their risk category..
48. Toshinobu Maki, Sayaka Maeno, Yasutaka Maeda, Mayumi Yamato, Noriyuki Sonoda, Yoshihiro Ogawa, Masanori Wakisaka, Toyoshi Inoguchi, Amelioration of diabetic nephropathy by SGLT2 inhibitors independent of its glucose-lowering effect
A possible role of SGLT2 in mesangial cells, Scientific reports, 10.1038/s41598-019-41253-7, 9, 1, 2019.12, Several clinical studies have shown the beneficial effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on diabetic nephropathy. The underlying mechanisms are not fully understood. We found that administration of canagliflozin at a low dose (0.01 mg/kg/day) did not affect either blood glucose levels or glycosuria, but it improved albuminuria and mesangial expansion in db/db mice to a similar extent as at a high dose (3.0 mg/kg/day) that lowered blood glucose levels. This indicated the existence of a tubular SGLT2-independent reno-protective mechanism. Here we focused on the potential role of SGLT2 in mesangial cells (MCs). Western blot analysis revealed the expression of SGLT2 in cultured mouse MCs. Exposure of MCs to high glucose levels for 72 h significantly increased the expression of SGLT2. Canagliflozin or ipragliflozin (both 100 nM) treatment inhibited glucose consumption in the medium under high-glucose conditions but not under normal-glucose conditions. Furthermore, canagliflozin inhibited high-glucose-induced activation of the protein kinase C (PKC)-NAD(P)H oxidase pathway and increases in reactive oxygen species (ROS) production. Thus, the inhibition of mesangial SGLT2 may cause an inhibition of PKC activation and ROS overproduction in diabetic nephropathy, and this may at least in part account for the reno-protective effect of SGLT2 inhibitors..
49. Jeenat Ferdous Urmi, Hiroaki Itoh, Keiko Muramatsu-Kato, Yukiko Kohmura-Kobayashi, Natsuyo Hariya, Divyanu Jain, Naoaki Tamura, Toshiyuki Uchida, Kazunao Suzuki, Yoshihiro Ogawa, Nobuaki Shiraki, Kazuki Mochizuki, Takeo Kubota, Naohiro Kanayama, Plasticity of histone modifications around Cidea and Cidec genes with secondary bile in the amelioration of developmentally-programmed hepatic steatosis, Scientific reports, 10.1038/s41598-019-52943-7, 9, 1, 2019.12, We recently reported that a treatment with tauroursodeoxycholic acid (TUDCA), a secondary bile acid, improved developmentally-deteriorated hepatic steatosis in an undernourishment (UN, 40% caloric restriction) in utero mouse model after a postnatal high-fat diet (HFD). We performed a microarray analysis and focused on two genes (Cidea and Cidec) because they are enhancers of lipid droplet (LD) sizes in hepatocytes and showed the greatest up-regulation in expression by UN that were completely recovered by TUDCA, concomitant with parallel changes in LD sizes. TUDCA remodeled developmentally-induced histone modifications (dimethylation of H3K4, H3K27, or H3K36), but not DNA methylation, around the Cidea and Cidec genes in UN pups only. Changes in these histone modifications may contribute to the markedly down-regulated expression of Cidea and Cidec genes in UN pups, which was observed in the alleviation of hepatic fat deposition, even under HFD. These results provide an insight into the future of precision medicine for developmentally-programmed hepatic steatosis by targeting histone modifications..
50. Yukihiro Inoguchi, Kenji Ichiyanagi, Hiroaki Ohishi, Yasutaka Maeda, Noriyuki Sonoda, Yoshihiro Ogawa, Toyoshi Inoguchi, Hiroyuki Sasaki, Poorly controlled diabetes during pregnancy and lactation activates the Foxo1 pathway and causes glucose intolerance in adult offspring, Scientific reports, 10.1038/s41598-019-46638-2, 9, 1, 2019.12, Exposure to maternal diabetes during pregnancy results in diabetes in offspring, but its underlying mechanisms are unclear. Here, we investigated the phenotype and molecular defects of the offspring of poorly controlled diabetic female mice generated by streptozotocin (STZ) administration. Offspring was exposed to maternal diabetes during pregnancy and lactation. The body weight of STZ offspring was lower than that of control offspring at birth and in adulthood, and glucose tolerance was impaired in adult STZ offspring. Interestingly, the phenotype was more pronounced in male offspring. We next investigated the morphology of islets and expression of β cell-related genes, but no significant changes were observed. However, transcriptome analysis of the liver revealed activation of the fork head box protein O1 (Foxo1) pathway in STZ male offspring. Notably, two key gluconeogenesis enzyme genes, glucose 6 phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), were upregulated. Consistent with this finding, phosphorylation of Foxo1 was decreased in the liver of STZ male offspring. These changes were not obvious in female offspring. The activation of Foxo1 and gluconeogenesis in the liver may have contributed to the impaired glucose tolerance of STZ male offspring..
51. Masahiro Asakawa, Michiko Itoh, Takayoshi Suganami, Takeru Sakai, Sayaka Kanai, Ibuki Shirakawa, Xunmei Yuan, Tomomi Hatayama, Shu Shimada, Yoshimitsu Akiyama, Katsuhito Fujiu, Yutaka Inagaki, Ichiro Manabe, Shoji Yamaoka, Tetsuya Yamada, Shinji Tanaka, Yoshihiro Ogawa, Upregulation of cancer-associated gene expression in activated fibroblasts in a mouse model of non-alcoholic steatohepatitis, Scientific reports, 10.1038/s41598-019-56039-0, 9, 1, 2019.12, Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride–induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor–deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the ‘pathways in cancer’ were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC..
52. Taisuke Narazaki, Motoaki Shiratsuchi, Mariko Tsuda, Yasuhiro Tsukamoto, Hiroki Muta, Toru Masuda, Daisaku Kimura, Akiko Takamatsu, Ryota Nakanishi, Eiji Oki, Minako Fujiwara, Yoshinao Oda, Yasuhiro Nakashima, Yoshihiro Ogawa, Intestinal Behçet's Disease with Primary Myelofibrosis Involving Trisomy 8, Acta Haematologica, 10.1159/000501019, 142, 4, 253-256, 2019.11, Behçet's disease (BD) is a disorder characterized by systemic inflammation of multiple organs, including the intestines. Several studies have reported a relationship between myelodysplastic syndrome and BD, and trisomy 8 was frequently seen, especially in intestinal BD. However, the association of BD with primary myelofibrosis (PMF) has not been well documented. A 58-year-old Japanese female was diagnosed with PMF in 2014. The symptoms of PMF resolved with ruxolitinib. However, she developed fever and intestinal perforation due to multiple ulcers in the terminal ileum in 2017. Intestinal perforation recurred 1 month later, and the dose of ruxolitinib was tapered. After discontinuation of ruxolitinib, she presented with recurrent oral aphthous ulcers and uveitis. Subsequently, intestinal perforation recurred, and she was diagnosed with intestinal BD. Trisomy 8 was identified in her peripheral blood. She underwent steroid therapy, azathioprine, and infliximab. This case suggests relationships between PMF, trisomy 8, and BD..
53. Keishi Komori, Eikichi Ihara, Yosuke Minoda, Haruei Ogino, Taisuke Sasaki, Minako Fujiwara, Yoshinao Oda, Yoshihiro Ogawa, The Altered Mucosal Barrier Function in the Duodenum Plays a Role in the Pathogenesis of Functional Dyspepsia, Digestive Diseases and Sciences, 10.1007/s10620-019-5470-8, 64, 11, 3228-3239, 2019.11, Background: An altered gastrointestinal barrier function is reportedly associated with the pathogenesis of functional dyspepsia (FD); however, the pathogenesis of FD has not yet been fully elucidated. Aims: The objective of the present study was to determine whether the mucosal barrier function is impaired in patients with FD and to investigate the mechanisms underlying FD. Methods: The present study included patients with FD (FD group, n = 24), non-FD patients with abdominal symptoms (symptomatic control group, n = 14), and patients with no abdominal symptoms (asymptomatic control group, n = 20). The groups were compared regarding the mucosal electrical impedance (MI) values of the stomach and duodenum, which were measured using a tissue conductance meter during esophagogastroduodenoscopy. Results: There were no significant differences between the three groups in the MI of the stomach. In contrast, the duodenal MI of the FD group (17.8 ± 4.3 Ω) was significantly lower than those of the symptomatic control group (27.2 ± 6.4 Ω, p < 0.0001) and asymptomatic control group (23.0 ± 7.4 Ω, p = 0.016). The expression of zonula occludens-1 (ZO-1) was significantly lower in the FD group than in the symptomatic control group (p = 0.011), where ZO-1 was positively correlated with the duodenal MI (β = 0.513, p = 0.017). The interleukin (IL)-1β expression was significantly higher in the FD group than in the symptomatic control group (p = 0.041), where IL-1β was inversely correlated with the duodenal MI (β = − 0.600, p = 0.004). Conclusions: The mucosal barrier function of the duodenum was altered in patients with FD. Both a decreased ZO-1 and increased IL-1β may play a role in the pathogenesis of FD..
54. Ryoko Takei, Tomoaki Inoue, Noriyuki Sonoda, Motoyuki Kohjima, Misato Okamoto, Ryuichi Sakamoto, Toyoshi Inoguchi, Yoshihiro Ogawa, Bilirubin reduces visceral obesity and insulin resistance by suppression of inflammatory cytokines, PloS one, 10.1371/journal.pone.0223302, 14, 10, 2019.10, Objective Although previous studies have reported a negative relationship between serum bilirubin concentration and the development of diabetes mellitus (DM), the relationship between bilirubin and insulin resistance has not been thoroughly assessed. This study was designed to determine the relationships between bilirubin, body fat distribution, and adipose tissue inflammation in patients with type 2 DM and the effect of bilirubin in an obese animal model. Method Body fat distribution was measured using an abdominal dual bioelectrical impedance analyzer in patients with type 2 DM. We also measured glycemic control, lipid profile, serum bilirubin concentration and other clinical characteristics, and determined their relationships with body fat distribution. In the animal study, biliverdin (20 mg/kg daily) was orally administered to high-fat diet (HFD)-induced obese (DIO) mice for 2 weeks, after which intraperitoneal insulin tolerance testing was performed. Then, adipocyte area, adipocytokine expression, and macrophage polarization were evaluated in epididymal adipose tissues. Results In the clinical study, univariate analysis showed that a lower bilirubin concentration was significantly correlated with higher body mass index, waist circumference, triglyceride, uric acid, creatinine, visceral fat area and lower HDL-C. In multivariate analyses, bilirubin concentration significantly correlated with diastolic blood pressure, creatinine, and visceral fat area. However, there was no association between bilirubin concentration and subcutaneous fat area. In the animal study, DIO mice treated with biliverdin had smaller adipocytes than untreated DIO mice and biliverdin improved HFD-induced insulin resistance. Biliverdin treatment reversed the higher gene expression of Cd11c, encoding an M1 macrophage marker, and Tnfa, encoding the proinflammatory cytokine tumor necrosis factor-α, in the adipose tissues of DIO mice. These data suggest biliverdin administration alleviates insulin resistance by ameliorating inflammation and the dysregulation of adipocytokine expression in adipose tissues of DIO mice. Conclusions Bilirubin may protect against insulin resistance by ameliorating visceral obesity and adipose tissue inflammation..
55. Youichi Ohno, Masakatsu Sone, Nobuya Inagaki, Yoshiyu Takeda, Isao Kurihara, Mika Tsuiki, Takamasa Ichijo, Norio Wada, Takuyuki Katabami, Yoshihiro Ogawa, Shintaro Okamura, Tomikazu Fukuoka, Tatsuya Kai, Shoichiro Izawa, Yuichiro Yoshikawa, Shigeatsu Hashimoto, Masanobu Yamada, Yoshiro Chiba, Mitsuhide Naruse, Latent Autonomous Cortisol Secretion from Apparently Nonfunctioning Adrenal Tumor in Nonlateralized Hyperaldosteronism, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2018-02790, 104, 10, 4382-4389, 2019.10, Context: Adrenal tumors (ATs), even those diagnosed as nonfunctioning, may cause metabolic disorders. Some primary aldosteronism (PA) patients with ATs are diagnosed with bilateral PA based on adrenal venous sampling (AVS), and their ATs are apparently nonfunctioning. Objective: To clarify the influence of apparently nonfunctioning ATs, we compared hormone levels and clinical complications between bilateral PA cases with and without ATs. Design, setting, and participants: After retrospectively assessing 2814 patients with PA in the multicenter Japan PA study, bilateral PA cases on AVS were divided into cases with and without ATs by computed tomography findings. Importantly, patients with cortisol levels >1.8 μg/dL after the 1-mg dexamethasone suppression test (DST) were excluded. Clinical characteristics and biochemical data were compared between them. The correlation between AT size and hormone levels was also analyzed. Main outcome measures: Analyzed were 196 bilateral PA patients with ATs and 331 those without ATs. Although basal cortisol and aldosterone levels were similar between them, cortisol levels after the 1-mg DST and the prevalences of diabetes mellitus and proteinuria were significantly higher and ACTH levels and plasma renin activity were significantly lower in cases with ATs than in those without. After adjusting for patients' backgrounds, cortisol levels after the 1-mg DST and plasma renin activity remained significantly different between them. Moreover, cortisol levels after the 1-mg DST and ACTH levels correlated with AT size. Conclusions: Apparently nonfunctioning ATs in bilateral PA cases may cause latent autonomous cortisol secretion, inducing diabetes and proteinuria..
56. Hiroki Kobayashi, Masanori Abe, Yoshihiro Nakamura, Katsutoshi Takahashi, Megumi Fujita, Yoshiyu Takeda, Takashi Yoneda, Isao Kurihara, Hiroshi Itoh, Mika Tsuiki, Norio Wada, Takamasa Ichijo, Takuyuki Katabami, Yoshihiro Ogawa, Junji Kawashima, Takanobu Yoshimoto, Masakatsu Sone, Nobuya Inagaki, Minemori Watanabe, Kohei Kamemura, Yuichi Matsuda, Shoichiro Izawa, Makito Tanabe, Akiyo Tanabe, Tomoko Suzuki, Mitsuhide Naruse, Association between acute fall in estimated glomerular filtration rate after treatment for primary aldosteronism and long-term decline in renal function, Hypertension, 10.1161/HYPERTENSIONAHA.119.13131, 74, 3, 630-638, 2019.09, Primary aldosteronism causes renal structural damage after glomerular hyperfiltration, and primary aldosteronism-specific treatment leads to an acute fall in estimated glomerular filtration rate (eGFR). We investigated whether this change affected the long-term eGFR slope in a retrospective cohort from the multicenter Japan Primary Aldosteronism Study. We allocated patients with primary aldosteronism to the adrenalectomy (n=202) and MR (mineralocorticoid receptor) antagonist (n=303) groups based on their treatment history and analyzed the association between the initial eGFR fall and long-term eGFR slope. The increased age, low serum potassium levels, high eGFR, and high plasma aldosterone levels were independent predictors for a large initial eGFR fall in both groups. Our analysis of tertiles based on the initial eGFR fall revealed that in the MR antagonist group, patients with a small initial eGFR fall had a significantly steeper long-term eGFR slope than those with a large initial fall (tertile 1 versus 2, P=0.025; tertile 1 versus 3, P=0.017). These associations were not identified in the adrenalectomy group. Thus, the smaller the acute fall in eGFR by initiation of MR antagonists, the greater was the rate of long-term eGFR decline. While the acute fall in eGFR induced by primary aldosteronism-specific treatment is occasionally a clinical concern, our findings highlight the favorable implications of the acute fall with respect to long-term renal outcomes..
57. Yoshimi Kodama, Fuminori Hyodo, Mayumi Yamato, K. Yasukawa, Y. Minami, Noriyuki Sonoda, Yoshihiro Ogawa, Kazuhiro Ichikawa, Toyoshi Inoguchi, Dynamic nuclear polarization magnetic resonance imaging and the oxygen-sensitive paramagnetic agent OX63 provide a noninvasive quantitative evaluation of kidney hypoxia in diabetic mice, Kidney International, 10.1016/j.kint.2019.04.034, 96, 3, 787-792, 2019.09, Renal hypoxia may play an important role in the progression of diabetic nephropathy. However, tools that noninvasively and quantitatively measure oxygen tension in the kidney are lacking. Here, we evaluated the feasibility of a noninvasive and quantitative imaging technique using dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) in combination with the oxygen-sensitive paramagnetic agent OX63 for measuring oxygen tension in the kidney. Our results demonstrate that the DNP-MRI technique can yield quantitative maps of oxygen tension in the mouse renal cortex. Using this procedure, we also showed that oxygen tension was less elevated in the renal cortex of both streptozotocin-induced type 1 diabetic mice and db/db mice, a model of type 2 diabetes, than in the renal cortex of age-matched control mice of each respective model. Oxygen tension in streptozotocin-exposed mice was significantly improved by insulin treatment. Thus, the noninvasive and quantitative DNP-MRI technique appears to be useful for studying the pathophysiological role of hypoxia..
58. Yosuke Minoda, Haruei Ogino, Takatoshi Chinen, Eikichi Ihara, Kazuhiro Haraguchi, Hirotada Akiho, Nobuyoshi Takizawa, Akira Aso, Yosuke Tomita, Mitsuru Esaki, Keishi Komori, Yoshihiro Otsuka, Tsutomu Iwasa, Yoshihiro Ogawa, Objective validity of the Japan Narrow-Band Imaging Expert Team classification system for the differential diagnosis of colorectal polyps, Digestive Endoscopy, 10.1111/den.13393, 31, 5, 544-551, 2019.09, Background and Aim: The Japan Narrow-Band Imaging (NBI) Expert Team (JNET) classification is a recently proposed NBI magnifying endoscopy-based classification system for colorectal tumors. Although the usefulness of this system has been reported by JNET experts, its objective validity remains unclear. We tested its validity and usefulness for the diagnosis of colorectal polyps by including colonoscopy experts and non-experts as test participants. Methods: Forty NBI images of polyps of various JNET types were shown to 22 doctors (11 experts and 11 non-gastrointestinal [GI] trainees) who had not examined the patients. The doctors diagnosed the polyps based solely on the surface and vessel patterns in the magnified images and the JNET classification system. Concordance rates of their diagnoses with the pathological findings of the polyps were determined, and the results for experts and non-GI trainees were compared. Results: Both for colonoscopy experts and non-GI trainees, the JNET classification system was particularly useful for classifying polyps as benign or malignant. Although the accuracy rates for classifying polyps into each JNET type varied among colonoscopy experts, those who were familiar with the JNET classification system were able to diagnose polyps with approximately 90% accuracy. Common mistakes were attributable to misunderstandings of the wording in the JNET classification chart and lack of proper training. Conclusion: The JNET classification system is a practical approach for the diagnosis of colorectal polyps. Training is required even for experienced colonoscopists to adopt the system properly. Common pitfalls must be shared among colonoscopists to improve the accuracy of the diagnosis..
59. Taisuke Narazaki, Yasuhiro Nakashima, Yasuhiro Tsukamoto, Mariko Tsuda, Toru Masuda, Daisaku Kimura, Akiko Takamatsu, Koichi Ohshima, Motoaki Shiratsuchi, Yoshihiro Ogawa, Transformation of follicular lymphoma to double-hit lymphoma during adjuvant chemotherapy for concurrent ovarian carcinoma, International journal of hematology, 10.1007/s12185-019-02656-4, 110, 3, 375-380, 2019.09, The frequency of multiple primary malignant neoplasms (MPMN) is increasing due to population aging. Since consensus guidelines for the treatment of MPMN are lacking, treatment strategies are determined by disease status on a per-patient basis. In this report, we describe a case of MPMN with follicular lymphoma (FL) grade 1 that transformed to double-hit lymphoma during adjuvant chemotherapy for concurrent ovarian carcinoma. A 64-year-old woman was diagnosed with MPMN of FL and endometrioid carcinoma by staging laparotomy and lymph node biopsy. She received four cycles of adjuvant chemotherapy (carboplatin and paclitaxel) for endometrioid carcinoma, but during chemotherapy, the FL grade 1 transformed to double-hit lymphoma. We speculate that adjuvant chemotherapy for endometrioid carcinoma may have triggered the transformation of FL in the present case..
60. Xiaopeng Bai, Eikichi Ihara, Yoshihihro Otsuka, Shinichi Tsuruta, Katsuya Hirano, Yoshimasa Tanaka, Haruei Ogino, Mayumi Hirano, Takatoshi Chinen, Hirotada Akiho, Kazuhiko Nakamura, Yoshinao Oda, Yoshihiro Ogawa, Involvement of different receptor subtypes in prostaglandin E2-induced contraction and relaxation in the lower esophageal sphincter and esophageal body, European Journal of Pharmacology, 10.1016/j.ejphar.2019.172405, 857, 2019.08, Prostaglandin E2 (PGE2) plays a role in the pathogenesis of gastro-esophageal reflux disease (GERD). There are 4 subtypes of PGE2, PGE2 receptor 1, 2, 3 and 4 (EP 1–4). In GERD patents, PGE2, EP2 and EP4 are upregulated. However, the effects of PGE2 on esophageal motility remain elusive. We examined how PGE2 regulates motility in the porcine circular smooth muscle of the lower esophageal sphincter (LES), and the circular and longitudinal smooth muscle of the esophagus body in organ bath. PGE2 induced tonic relaxation in the LES and circular smooth muscle, but transient contraction in longitudinal smooth muscle. The relaxation of the LES and circular smooth muscle was similar in pattern and mechanism, but was much larger in the LES. The relaxation was completely blocked by a voltage-gated K+ channel blocker or 40 mM K+ depolarization, indicating the involvement of K+ channel. Longitudinal smooth muscle contraction was completely blocked by an L-type Ca2+ channel blocker, showing the contribution of Ca2+ movement. The involvement of the EP receptor in motility was examined with selective receptor agonists and antagonists. Activation of EP2 and EP4 caused relaxation in the LES and circular smooth muscle. Compatible with PGE2, EP2 and EP4 agonists caused more significant relaxation in the LES than in circular smooth muscle. EP1 contributed to the longitudinal smooth muscle contraction. The different effects of PGE2 in the LES, circular and longitudinal smooth muscle contributes to esophageal motility, their impairment might increase the amount and frequency of esophageal reflux..
61. Mitsuha Morisaki, Isao Kurihara, Hiroshi Itoh, Mitsuhide Naruse, Yoshiyu Takeda, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Takanobu Yoshimoto, Yoshihiro Ogawa, Masakatsu Sone, Mika Tsuiki, Hirotaka Shibata, Junji Kawashima, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Tomoko Suzuki, Predictors of Clinical Success after Surgery for Primary Aldosteronism in the Japanese Nationwide Cohort, Journal of the Endocrine Society, 10.1210/js.2019-00295, 3, 11, 2012-2022, 2019.08, Context: Aldosterone-producing adenomas are a curable subtype of primary aldosteronism (PA); however, hypertension persists in some patients after adrenalectomy. Objective: To identify factors associated with, and develop prediction models for, blood pressure (BP) normalization or improvement after adrenalectomy. Design: Retrospective analysis of patients treated between 2006 and 2018, with a 6-month follow-up. Setting: A nationwide, 29-center Japanese registry encompassing 15 university hospitals and 14 city hospitals. Patients: We categorized 574 participants in the Japan Primary Aldosteronism Study, who were diagnosed with PA and underwent adrenalectomy, as BP normalized or improved, on the basis of their presentations at 6 months postsurgery. Main Outcome Measure: The rate of complete, partial, and absent clinical success. Predictive factors related to BP outcomes after PA surgery were also evaluated. Results: Complete clinical success was achieved in 32.6% and partial clinical success was achieved in 53.0% of the patients at 6 months postsurgery. The following five variables were independent predictors for BP normalization: ≤7 years of hypertension, body mass index ≤25 kg/m2, no more than one antihypertensive medication, absence of medical history of diabetes, and female sex. The area under the receiver operator characteristic curve was 0.797 in the BP normalization model. Conclusion: We established models that predicted postoperative BP normalization in patients with PA. These should be useful for shared decision-making regarding adrenalectomy for PA..
62. Hiroshi Akasaka, Koichi Yamamoto, Hiromi Rakugi, Motonori Nagasawa, Ryo Nakamaru, Takamasa Ichijo, Yoshiyu Takeda, Isao Kurihara, Takuyuki Katabami, Mika Tsuiki, Norio Wada, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Kohei Kamemura, Takanobu Yoshimoto, Yuichi Matsuda, Megumi Fujita, Hiroki Kobayashi, Minemori Watanabe, Kouichi Tamura, Shintaro Okamura, Shozo Miyauchi, Shoichiro Izawa, Yoshiro Chiba, Akiyo Tanabe, Mitsuhide Naruse, Sex Difference in the Association between Subtype Distribution and Age at Diagnosis in Patients with Primary Aldosteronism, Hypertension, 10.1161/HYPERTENSIONAHA.119.13006, 74, 2, 368-374, 2019.08, Primary aldosteronism (PA) is the most frequent cause of secondary hypertension. Adrenal vein sampling (AVS) is an established method for finding patients with the unilateral subtype of PA, for which adrenalectomy is an applicable treatment. In this study, we analyzed a large database of patients with PA who underwent adrenal vein sampling, to investigate the sex differences in the impact of age at diagnosis on the subtype and cause of PA. In 2122 patients, women with the unilateral subtype were younger than men with the same subtype and women with the bilateral subtype. Younger age and older age were associated with unilateral PA in women and men, respectively. After stratification by tertiles of age, there was a trend of decreased and increased incidence of unilateral PA with aging in women and men, respectively. Male sex was a predictor of unilateral PA in middle-aged and older patients but not in younger patients. We also found that obesity, a known factor associated with idiopathic hyperaldosteronism, was positively associated with bilateral PA in younger patients but not in older patients. These findings suggest that the proportion of operable patients with unilateral PA differs depending on the combination of sex and age, and that other than obesity, the cause of PA is also associated with the bilateral subtype in older patients..
63. Masami Miki, Takamasa Oono, Nao Fujimori, Takehiro Takaoka, Ken Kawabe, Yoshihiro Miyasaka, Takao Ohtsuka, Daisuke Saito, Masafumi Nakamura, Yasuyuki Ohkawa, Yoshinao Oda, Mikita Suyama, Tetsuhide Ito, Yoshihiro Ogawa, CLEC3A, MMP7, and LCN2 as novel markers for predicting recurrence in resected G1 and G2 pancreatic neuroendocrine tumors, Cancer Medicine, 10.1002/cam4.2232, 8, 8, 3748-3760, 2019.07, Although the postoperative recurrence rate for pancreatic neuroendocrine tumors (PNETs) is reported to be 13.5%-30%, the paucity of valuable biomarkers to predict recurrence poses a problem for the early detection of relapse. Hence, this study aimed to identify new biomarkers to predict the recurrence of PNETs. We performed RNA sequencing (RNA-Seq) on RNA isolated from frozen primary tumors sampled from all localized G1/G2 PNETs resected curatively from 1998 to 2015 in our institution. We calculated differentially expressed genes (DEGs) in tumor with and without recurrence (≥3 years) for the propensity-matched cohort. Gene ontology analysis for the identified DEGs was also performed. Furthermore, we evaluated the expression levels of candidate genes as recurrence predictors via immunostaining. Comparison of transcriptional levels in tumors with and without recurrence identified 166 DEGs. Up- and downregulated genes with high significance in these tumors were mainly related to extracellular organization and cell adhesion, respectively. We observed the top three upregulated genes, C-type lectin domain family 3 member A (CLEC3A), matrix metalloproteinase-7 (MMP7), and lipocalin2 (LCN2) immunohistochemically and compared their levels in recurrent and nonrecurrent tumors. Significantly higher recurrence rate was shown in patients with positive expression of CLEC3A (P = 0.028), MMP7 (P = 0.003), and LCN2 (P = 0.040) than that with negative expression. We identified CLEC3A, MMP7, and LCN2 known to be associated with the phosphatidylinositol-3-kinase/Akt pathway, as potential novel markers to predict the postoperative recurrence of PNETs..
64. Takuyuki Katabami, Hisashi Fukuda, Hidekazu Tsukiyama, Yasushi Tanaka, Yoshiyu Takeda, Isao Kurihara, Hiroshi Ito, Mika Tsuiki, Takamasa Ichijo, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamoto, Atsushi Ogo, Toshihiko Yanase, Tomoko Suzuki, Mitsuhide Naruse, Clinical and biochemical outcomes after adrenalectomy and medical treatment in patients with unilateral primary aldosteronism, Journal of hypertension, 10.1097/HJH.0000000000002070, 37, 7, 1513-1520, 2019.07, Objectives:Current clinical guidelines of primary aldosteronism recommend adrenalectomy (AdX) for unilateral primary aldosteronism based on the studies showing the potential superiority of AdX over the medical treatment. However, since most medically treated cases consisted of bilateral primary aldosteronism and all surgically treated cases consisted of unilateral primary aldosteronism, the different subtype of primary aldosteronism could be a bias for their effects. This study compared the effects of AdX and medical therapy in patients with unilateral primary aldosteronism confirmed by adrenal vein sampling.Methods:Of the 339 patients with unilateral primary aldosteronism in the Japan Primary Pldosteronism Study data base, unilateral AdX and treatment with mineral corticoid receptor antagonists (MRAs) was done in 276 patients (AdX group) and in 63 patients (MRAs group), respectively. The effects were compared by the clinical (improvement of blood pressure) and biochemical outcomes (improvement of hypokalemia).Results:At baseline, use of potassium replacement, plasma aldosterone concentration, aldosterone-to-renin ratio, estimated glomerular filtration rate, and prevalence of adrenal mass on imaging were higher in the AdX group than in the MRAs group. At 6 months after commencement of specific treatment for primary aldosteronism, clinical outcome and biochemical outcome in the AdX group were superior than those in the MRAs group. The difference of the outcome between the two groups were the case even after adjusting for the different clinical backgrounds in the two groups before the specific treatment.Conclusion:Our study provides evidence that AdX is the first choice of treatment in the patients with unilateral primary aldosteronism in terms of clinical and biochemical outcome..
65. Motonori Nagasawa, Koichi Yamamoto, Hiromi Rakugi, Masao Takeda, Hiroshi Akasaka, Hironobu Umakoshi, Mika Tsuiki, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Takamasa Ichijo, Takuyuki Katabami, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Atsushi Ogo, Shintaro Okamura, Shozo Miyauchi, Toshihiko Yanase, Tomoko Suzuki, Takashi Kawamura, Mitsuhide Naruse, Influence of antihypertensive drugs in the subtype diagnosis of primary aldosteronism by adrenal venous sampling, Journal of hypertension, 10.1097/HJH.0000000000002047, 37, 7, 1493-1499, 2019.07, Objectives:Because of the influence on the renin-angiotensin-aldosterone system, it is recommended to avoid, if possible, the use of angiotensin-converting-enzyme inhibitors, angiotensin II type 1 receptor blockers, diuretics, β-blockers, and mineralocorticoid receptor antagonists during the diagnostic period of primary aldosteronism. A laterality index more than 4 in adrenocorticotropic hormone (ACTH)-stimulated adrenal venous sampling (ACTH-AVS) is a widely used classification of the unilateral subtype that can benefit from adrenalectomy. Here, we revealed clinical features of patients taking drugs that could affect the primary aldosteronism diagnosis (DAPD) and investigated whether the classification with laterality index more than 4 in ACTH-AVS is applicable to these patients.Patients and methods:Using a large database of primary aldosteronism patients in Japan, we analyzed 2122 patients with successful ACTH-AVS.Results:Patients who received any DAPD (n = 209) showed higher prevalence of comorbidity burdens and took more antihypertensive drugs compared with patients without DAPD. In patients taking DAPD, those with laterality index more than 4 had a higher prevalence of hypokalemia, a higher aldosterone-to-renin ratio and a higher prevalence of adrenal mass than those with laterality index of 4 or less. Adrenalectomy was performed in 76% patients with laterality index more than 4 and 20% with laterality index of 4 or less. Patients who underwent adrenalectomy showed biochemical cure in 89% with laterality index more than 4 and 50% with laterality index of 4 or less (P = 0.001). Multivariate regression analysis showed that laterality index more than 4 was an independent predictor of a biochemical cure. Biochemical cure rate in patients with laterality index more than 4 was consistently high, irrespective of the potential effect of individual DAPD on laterality index.Conclusion:Our findings suggest that in primary aldosteronism patients to whom DAPD were administrated due to severe clinical features, laterality index more than 4 in ACTH-AVS could accurately predict a biochemical cure after adrenalectomy..
66. Takashi Osoegawa, Yosuke Minoda, Eikichi Ihara, Keishi Komori, Akira Aso, Ayako Goto, Soichi Itaba, Haruei Ogino, Kazuhiko Nakamura, Naohiko Harada, Kosuke Makihara, Shinichi Tsuruta, Hidetaka Yamamoto, Yoshihiro Ogawa, Mucosal incision-assisted biopsy versus endoscopic ultrasound-guided fine-needle aspiration with a rapid on-site evaluation for gastric subepithelial lesions
A randomized cross-over study, Digestive Endoscopy, 10.1111/den.13367, 31, 4, 413-421, 2019.07, Objectives: This study aimed to compare the diagnostic yield of mucosal incision-assisted biopsy (MIAB) and endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) with a rapid on-site evaluation (ROSE) for gastric subepithelial lesions (SEL) suspected of being gastrointestinal stromal tumors (GIST) with an intraluminal growth pattern. Methods: This was a prospective randomized, cross-over multicenter study. The primary outcome was the diagnostic yield of EUS-FNA and MIAB. The secondary outcomes were the technical success rate, complication rate, procedure time and biopsy frequency. Results: A total of 47 patients were randomized to the MIAB group (n = 23) and EUS-FNA group (n = 24). There was no significant difference in the diagnostic yield of MIAB and EUS-FNA (91.3% vs 70.8%, P = 0.0746). The complication rates of MIAB and EUS-FNA did not differ to a statistically significant extent. The mean procedure time in the MIAB group was significantly longer than that in the EUS-FNA group (34 vs 26 min, P = 0.0011). Conclusions: The diagnostic yield of MIAB was satisfactorily as high as EUS-FNA with ROSE for gastric SEL with an intraluminal growth pattern..
67. K. Fukaura, E. Ihara, H. Ogino, Y. Iboshi, K. Muta, X. Bai, S. Hamada, Y. Hata, T. Iwasa, A. Aso, K. Nakamura, Y. Ogawa., Mucosally expressed cytokines are associated with the esophageal motility function., Digestion, 10.1159/000487708. , 98, 2, 95-103, 2019.06.
68. Kanako Toda, Koji Mizutani, Isao Minami, Ming Ye, Takahiro Arakawa, Kohji Mitsubayashi, Yoshihiro Ogawa, Kouji Araki, Kayoko Shinada, Effects of oral health instructions on glycemic control and oral health status of periodontitis patients with type 2 diabetes mellitus
A preliminary observation, Journal of Dental Sciences, 10.1016/j.jds.2019.01.009, 14, 2, 171-177, 2019.06.
69. Shinichi Tsuruta, Yoshihiro Ohishi, Minako Fujiwara, Eikichi Ihara, Yoshihiro Ogawa, Eiji Oki, Masafumi Nakamura, Yoshinao Oda, Gastric hepatoid adenocarcinomas are a genetically heterogenous group; most tumors show chromosomal instability, but MSI tumors do exist, Human Pathology, 10.1016/j.humpath.2019.03.006, 88, 27-38, 2019.06, The Cancer Genome Atlas Research Network classified gastric adenocarcinoma into four molecular subtypes: (1) Epstein-Barr virus–positive (EBV), (2) microsatellite-instable (MSI), (3) chromosomal instable (CIN), and (4) genomically stable (GS). The molecular subtypes of gastric hepatoid adenocarcinomas are still largely unknown. We analyzed 52 hepatoid adenocarcinomas for the expression of surrogate markers of molecular subtypes (MLH1, p53, and EBER in situ hybridization) and some biomarkers (p21, p16, Rb, cyclin D1, cyclin E, β-catenin, Bcl-2, IMP3, ARID1A and HER2), and mutations of TP53, CTNNB1, KRAS, and BRAF. We analyzed 36 solid-type poorly differentiated adenocarcinomas as a control group. Hepatoid adenocarcinomas were categorized as follows: EBV group (EBER-positive), no cases (0%); MSI group (MLH1 loss), three cases (6%); “CIN or GS” (CIN/GS) group (EBER-negative, MLH1 retained), 49 cases (94%). In the CIN/GS group, most of the tumors (59%) had either p53 overexpression or TP53 mutation and a coexisting tubular intestinal-type adenocarcinoma component (90%), suggesting that most hepatoid adenocarcinomas should be categorized as a true CIN group. Hepatoid adenocarcinomas showed relatively frequent expressions of HER2 (score 3+/2+: 21%/19%). Hepatoid adenocarcinomas showed shorter survival, more frequent overexpressions of p16 (67%) and IMP3 (98%) than the control group. None of hepatoid adenocarcinomas had KRAS or CTNNB1 mutations except for one case each, and no hepatoid adenocarcinomas had BRAF mutation. In conclusion, gastric hepatoid adenocarcinomas are a genetically heterogenous group. Most hepatoid adenocarcinomas are “CIN,” but a small number of hepatoid adenocarcinomas with MSI do exist. Hepatoid adenocarcinomas are characterized by overexpressions of p16 and IMP3..
70. Kentaro Mori, Kyoichiro Tsuchiya, Suguru Nakamura, Yasutaka Miyachi, Kumiko Shiba, Yoshihiro Ogawa, Kenichiro Kitamura, Ipragliflozin-induced adipose expansion inhibits cuff-induced vascular remodeling in mice, Cardiovascular Diabetology, 10.1186/s12933-019-0886-1, 18, 1, 2019.06, Background: Perivascular adipose tissue (PVAT) plays a critical role in the pathogenesis of cardiovascular disease. It is unclear whether inhibition of sodium glucose cotransporter 2 (SGLT2) in subjects with type 2 diabetes (T2DM) could affect PVAT characters, and whether the SGLT2 inhibitors-induced changes of adipose tissue, especially the alternation of adipose tissue-derived secretory factors, affect vascular pathophysiology. Methods: Western-type diet (WD) fed wild-type mice were treated with or without an SGLT2 inhibitor ipragliflozin (Ipra) for 10 weeks. WEHI 274.1 and primary vascular smooth muscle cells were incubated with conditioned media (CM) of epididymal adipose tissue (Epi) or abdominal PVAT of Ipra- or vehicle-treated mice fed a WD. Epi of Ipra- or vehicle-treated mice fed a WD was implanted onto cuff-placed femoral arteries of apoE-deficient mice. Results: Ipra increased adipocyte size associated with decreased expression of pro-inflammatory and fibrosis-related genes in abdominal PVAT of WD-fed mice. Ipra also suppressed WD-induced macrophages accumulation, fibrosis, and adipocyte death in abdominal PVAT. In CM of abdominal PVAT from Ipra-treated mice, concentration of leptin was significantly lower than that from vehicle-treated mice. In vitro, migration of WEHI 274.1 and primary vascular smooth muscle cells were more enhanced by CM of Epi or abdominal PVAT from vehicle-treated mice than that from Ipra-treated mice. Perivascular implantation of Epi from Ipra-treated mice to apolipoprotein E-deficient mice attenuated cuff-induced neointimal hyperplasia and vascular remodeling compared to that from vehicle-treated mice. Conclusions: The Ipra-induced changes of abdominal PVAT will lead to a better understanding of unveiled mechanisms by which SGLT2 inhibitors prevent cardiovascular complications in T2DM, and the development of new therapeutic strategies targeting PVAT..
71. Keiichiro Uchida, Masatoshi Nomura, Tadashi Yamamoto, Yoshihiro Ogawa, Noriyoshi Teramoto, Rab8a is involved in membrane trafficking of Kir6.2 in the MIN6 insulinoma cell line, Pflugers Archiv European Journal of Physiology, 10.1007/s00424-018-02252-1, 471, 6, 877-887, 2019.06, Although ATP-sensitive K+ (KATP) channels play an important role in the secretion of insulin by pancreatic beta cells, the mechanisms that regulate the intracellular transport of KATP channel subunit proteins (i.e., Kir6.2 and sulfonylurea receptor 1 (SUR1)) to the plasma membrane remain uncharacterized. We investigated the possibility that an interaction between KATP channel subunit proteins and Rab8a protein, a member of the RAS superfamily, may be involved in the membrane trafficking of KATP channels. Co-immunoprecipitation and immunostaining experiments using co-expression systems with fluorescent protein-tagged Kir6.2 were carried out to identify the coupling of KATP channels and Rab8a proteins in the insulin-secreting cell line, MIN6. Rab8a protein co-localized with Kir6.2 protein, a channel pore subunit (in a granular pattern), and with insulin. Knockdown of the Rab8a gene with RNA interference using small interfering RNA systems caused reductions in the amount of total KATP and plasma membrane surface KATP channels without decreasing the messenger RNA transcription of the KATP channel subunits. Rab8a gene knockdown also enhanced glucose-induced insulin secretion. These results suggest that Rab8a may be involved in membrane trafficking of KATP channels and the maintenance of normal insulin secretion in the MIN6 pancreatic beta cell line..
72. , Yuko Akehi, Toshihiko Yanase, Ryoko Motonaga, Hironobu Umakoshi, Mika Tsuiki, Yoshiyu Takeda, Takashi Yoneda, Isao Kurihara, Hiroshi Itoh, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Kenji Ashida, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamoto, Atsushi Ogo, Shintaro Okamura, Shozo Miyauchi, Tomikazu Fukuoka, Shoichiro Izawa, Shigeatsu Hashimoto, Masanobu Yamada, Yuichiro Yoshikawa, Tatsuya Kai, Tomoko Suzuki, Takashi Kawamura, Mitsuhide Naruse, High prevalence of diabetes in patients with primary aldosteronism (PA) associated with subclinical hypercortisolism and prediabetes more prevalent in bilateral than unilateral PA
A large, multicenter cohort study in Japan, Diabetes care, 10.2337/dc18-1293, 42, 5, 938-945, 2019.05, OBJECTIVE To investigate the prevalence and causes of diabetes in patients with primary aldosteronism (PA) in a multi-institutional cohort study in Japan. RESEARCH DESIGN AND METHODS The prevalence of diabetes was determined in 2,210 patients with PA (diagnosed or glycated hemoglobin [HbA1c] ‡6.5% [‡48 mmol/mol]; NGSP) and compared with that of the Japanese general population according to age and sex. In 1,386 patients with PA and clear laterality (unilateral or bilateral), the effects of plasma aldosterone concentration (PAC), hypokalemia (<3.5 mEq/L), suspected subclinical hypercortisolism (SH; serum cortisol ‡1.8 mg/dL after 1-mg dexamethasone suppression test), and PA laterality on the prevalence of diabetes or prediabetes (5.7% £ HbA1c <6.5% [39 mmol/mol £ HbA1c <48 mmol/mol]) were examined. RESULTS Of the 2,210 patients with PA, 477 (21.6%) had diabetes. This prevalence is higher than that in the general population (12.1%) or in 10-year cohorts aged 30–69 years. Logistic regression or x2 test revealed a significant contribution of suspected SH to diabetes. Despite more active PA profiles (e.g., higher PAC and lower potassium concentrations) in unilateral than bilateral PA, BMI and HbA1c values were significantly higher in bilateral PA. PA laterality had no effect on the prevalence of diabetes; however, the prevalence of prediabetes was significantly higher in bilateral than unilateral PA. CONCLUSIONS Individuals with PA have a high prevalence of diabetes, which is associated mainly with SH. The prevalence of prediabetes is greater for bilateral than unilateral PA, suggesting a unique metabolic cause of bilateral PA..
73. Yoshiyu Takeda, Hironobu Umakoshi, Yoshimichi Takeda, Takashi Yoneda, Isao Kurihara, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamto, Atsushi Ogo, Toshihiko Yanase, Tomoko Suzuki, Mitsuhide Naruse, Impact of adrenocorticotropic hormone stimulation during adrenal venous sampling on outcomes of primary aldosteronism, Journal of hypertension, 10.1097/HJH.0000000000001964, 37, 5, 1077-1082, 2019.05, Background: Adrenal venous sampling (AVS) is essential for identifying a surgically curable form of primary aldosteronism. Adrenocorticotropic hormone (ACTH) infusion or bolus has been reported to improve the success rate of AVS, although the effects on lateralization and its outcomes in unilateral primary aldosteronism are unclear. Methods: The success rate and lateralization indices were examined in a cohort of 2197 Japanese patients with primary aldosteronism from 28 centres who underwent AVS. Outcomes were analysed in 267 patients with aldosterone-producing adenomas (APAs). Results: ACTH loading during AVS improved the success rate from 67 to 89%, while lateralization indices decreased from 62 to 28%. Bolus, bolus along with continuous infusion or continuous infusion of ACTH did not affect both indices. The absence of clinical success (i.e. unchanged or increased blood pressure) was 33% and absence of biochemical success (persistent hypokalaemia or persistently raised aldosterone-to-renin ratio, or both) was 15%. The clinical and biochemical success rates did not differ between the three groups [lateralization index >2 in basal condition (LIb) and lateralization index >4 after ACTH loading (lateralization indices), and LIb >2 R lateralization indices<4, LIb<2Rlateralization indices>4]. The three groups (LIb>4Rlateralization indices>4, LIb>4Rlateralization indices<4 and LIb<4Rlateralization indices>4) did not show any significant differences of clinical and biochemical outcome. Conclusion: ACTH loading during AVS improved the success rate but decreased laterality. ACTH did not affect the clinical and biochemical outcomes in APA patients. These data showed that the use of ACTH during AVS was helpful for improving the success rate, but did not contribute to better outcomes..
74. Tomomi Yamada, Yuto Kashiwagi, Takemi Rokugawa, Hideaki Kato, Haruyo Konishi, Tadateru Hamada, Ryohei Nagai, Yusaku Masago, Michiko Itoh, Takayoshi Suganami, Yoshihiro Ogawa, Kohji Abe, Evaluation of hepatic function using dynamic contrast-enhanced magnetic resonance imaging in melanocortin 4 receptor-deficient mice as a model of nonalcoholic steatohepatitis, Magnetic Resonance Imaging, 10.1016/j.mri.2018.11.013, 57, 210-217, 2019.04, Introduction: Melanocortin 4 receptor-deficient (MC4R-KO) mice fed a high-fat diet (HFD) develop liver pathology similar to human nonalcoholic steatohepatitis (NASH). However, although liver histology and blood biochemistry have been reported, hepatic function has not been evaluated. In the present study, we evaluated hepatic function in MC4R-KO mice fed an HFD using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium‑ethoxybenzyl‑diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). Materials and methods: Wild type (WT) mice and MC4R-KO mice were fed a standard diet (SD) or an HFD for 20 weeks. The hepatic signal intensity was obtained from DCE-MRI images, and relative enhancement (RE), the time to maximum RE (Tmax), and the half-life of RE elimination (T1/2) were calculated. Histopathological analysis was then performed. Results: Histological analysis with nonalcoholic fatty liver disease activity score (NAS) revealed that MC4R-KO mice fed an HFD achieved the NAS of 5. There was moderate fibrosis in MC4R-KO mice fed an HFD. DCE-MRI with Gd-EOB-DTPA showed that Tmax and T1/2 were significantly longer in MC4R-KO mice fed an HFD compared with wild type (WT) mice (Tmax, WT, 3.9 ± 0.4 min; MC4R-KO, 7.4 ± 1.5 min; T1/2, WT, 23.7 ± 1.9 min; MC4R-KO, 62.5 ± 18.5 min). Tmax and T1/2 were significantly correlated with histopathologic score (steatosis vs. Tmax, rho = 0.48, P = 0.04; steatosis vs. T1/2, rho = 0.50, P = 0.03; inflammation vs. Tmax, rho = 0.55, P = 0.02; inflammation vs. T1/2, rho = 0.61, P < 0.01; ballooning vs. T1/2, rho = 0.51, P = 0.03;fibrosis vs Tmax, rho = 0.72, P < 0.01; fibrosis vs T1/2, rho = 0.75, P < 0.01). Conclusions: MC4R-KO mice fed an HFD developed obesity and NASH. The liver kinetics of Gd-EOB-DTPA were significantly different in MC4R-KO mice fed an HFD from WT mice, and correlated with the histopathologic score. These results suggest that MC4R-KO mice fed an HFD mimic the hepatic pathology and liver function of human NASH, and therefore might be useful for the study of hepatic dysfunction during the fibrotic stage of NASH..
75. Masami Miki, Nao Fujimori, Takamasa Oono, Ken Kawabe, Akihisa Ohno, Kazuhide Matsumoto, Katsuhito Teramatsu, Yuichi Tachibana, Yoshihiro Ogawa, Relapse patterns and predictors of IgG4-related diseases involved with autoimmune pancreatitis
A single-center retrospective study of 115 patients, Journal of Digestive Diseases, 10.1111/1751-2980.12708, 20, 3, 152-158, 2019.03, Objective: Autoimmune pancreatitis is an autoimmune disorder accompanied by clinicopathological manifestations that have been established as immunoglobulin (IgG)4-related diseases (IgG4-RD). Other IgG4-RD are often involved with autoimmune pancreatitis. They sometimes relapse despite a favorable response to steroid therapy. This study aimed to clarify the patterns and risk factors for extrapancreatic relapse. Methods: We reviewed the data of 115 patients diagnosed with definite autoimmune pancreatitis type 1 and followed up for > 1 year. We analyzed two items: the timing and pattern of extrapancreatic relapse, and risk factors for relapse with three common manifestations: IgG4-related sclerosing cholangitis (SC), IgG4-related dacryoadenitis and sialadenitis (DS), and IgG4-related retroperitoneal fibrosis (RF). Results: Remission was achieved in all patients, except one. The extrapancreatic relapse rates were 11.0%, 19.7%, and 40% within 3, 5, and 10 years, respectively. Of 26 patients with extrapancreatic relapse, nine (34.6%) relapsed with a new IgG4-RD. Based on multivariate analysis, the interval between symptom onset and steroid initiation, and the presence of RF at onset were significant risk factors for relapse with SC and RF, respectively. Conclusions: Our results indicate that they may be various extrapancreatic relapse patterns especially in autoimmune pancreatitis with other organ involvement. Patients with a delayed initiation of steroids or RF at onset should be carefully followed up as high-risk groups for SC and RF relapse..
76. C. Sakaguchi, K. Ashida, S. Yano, K. Ohe, N. Wada, N. Hasuzawa, Y. Matsuda, S. Sakamoto, R. Sakamoto, H. Uchi, M. Furue, M. Nomura, Y. Ogawa, A case of nivolumab-induced acute-onset type 1 diabetes mellitus in melanoma, Current oncology (Toronto, Ont.), 10.3747/co.26.4130, 26, 1, e115-e118, 2019.02, Nivolumab, an anti-PD-1 antibody, is now considered an important therapeutic agent in several advanced malignancies. However, immune-related adverse events such as endocrinopathies have been reported with its use. Thyroid disorder and isolated adrenocorticotropic hormone deficiency have frequently been reported as nivolumab-induced immune-related adverse events. Another endocrinopathy is nivolumab-induced type 1 diabetes mellitus (t1dm), described as diabetes mellitus with rapid onset and complete insulin insufficiency, at times leading to fulminant t1dm. We report the case of a 68-year-old woman who developed pancreatic islet-related autoantibody-negative t1dm, possibly induced by nivolumab, under continuous glucocorticoid administration. She was treated with nivolumab for advanced malignant melanoma, concomitant with 10 mg prednisolone daily for thrombophlebitis tapered to 5 mg after 13 courses of nivolumab therapy. At approximately the 27th course of nivolumab therapy, she showed elevated plasma glucose levels despite preserved insulin secretion. A month later, she developed diabetic ketoacidosis. Her insulin secretion decreased and finally was exhausted. She was diagnosed with acute-onset rather than fulminant t1dm because of a rapidly progressive course to diabetic ketoacidosis during just more than 1 week. She is currently receiving insulin replacement. There has been no recurrence of the melanoma. Thus, nivolumab might induce autoimmune diabetes mellitus, with patients having t1dm-sensitive human leucocyte antigen being more susceptible even when receiving glucocorticoids. Physicians should be aware that nivolumab could potentially induce t1dm as a critical immune-related adverse event..
77. Takahiro Yamazaki, Yumie Morimoto-Kobayashi, Kumiko Koizumi, Chika Takahashi, Shiori Nakajima, Sayoko Kitao, Yoshimasa Taniguchi, Mikio Katayama, Yoshihiro Ogawa, Secretion of a gastrointestinal hormone, cholecystokinin, by hop-derived bitter components activates sympathetic nerves in brown adipose tissue, Journal of Nutritional Biochemistry, 10.1016/j.jnutbio.2018.10.009, 64, 80-87, 2019.02, Matured hop bitter acids (MHBA) are oxidation products from bitter components in hops, which are used widely as food materials to add flavor and bitterness in beer production. Our previous study has shown that MHBA induces thermogenesis in brown adipose tissue (BAT) via sympathetic nerves in rodents and reduces body fat in healthy adults. However, it is unclear how MHBA affects the sympathetic nervous system. In this study, we demonstrate that MHBA treatment of enteroendocrine cells increases Ca2+ levels and induces the secretion of the gastrointestinal hormone, cholecystokinin (CCK), in a dose-dependent manner. These effects were eliminated by Ca2+ depletion from the medium or blockers of L-type voltage-sensitive Ca2+ channels during pretreatment. Induction of CCK secretion by MHBA was also confirmed using isolated rat small intestines. Elevation of the sympathetic nerve activity innervating BAT (BAT-SNA) and BAT temperature by MHBA administration in rats was blocked by pretreatment with a CCK receptor 1 (CCK1R) antagonist. Moreover, the intraperitoneal injection of CCK fragment elevated BAT-SNA, and this increase was blocked by subdiaphragmatic vagotomy. These results demonstrate that MHBA induces CCK secretion in the gastrointestinal tracts and elevates BAT-SNA via CCK1R and vagal afferent nerves. In addition, MHBA increases BAT temperature via CCK1R. Our findings reveal a novel mechanism of the beneficial metabolic effects of food ingredients..
78. K. Komori, E. Ihara, Y. Minoda, H.Ogino, T. Sasaki, M. Fujiwara, Y. Oda, Y. Ogawa., The altered mucosal barrier function in the duodenum plays a role in the pathogenesis of functional dyspepsia., Dig Dis Sci., 10.1007/s10620-019-5470-8., 2019.01.
79. Chihiro Sakaguchi, Kenji Ashida, Kenichi Kohashi, Kenji Ohe, Yoichi Fujii, Seiichi Yano, Yayoi Matsuda, Shohei Sakamoto, Ryuichi Sakamoto, Yoshinao Oda, Masatoshi Nomura, Yoshihiro Ogawa, A case of autonomous cortisol secretion in a patient with subclinical Cushing's syndrome, GNAS mutation, and paradoxical cortisol response to dexamethasone, BMC Endocrine Disorders, 10.1186/s12902-019-0345-8, 19, 1, 2019.01, Background: Increased urinary free cortisol in response to the oral administration of dexamethasone is a paradoxical reaction mainly reported in patients with primary pigmented nodular adrenocortical disease. Here, we describe the first case of subclinical Cushing's syndrome represented by autonomous cortisol secretion and paradoxical response to oral dexamethasone administration, harboring an activating mutation in the α subunit of the stimulatory G protein (GNAS). Case presentation: A 65-year-old woman was diagnosed with subclinical Cushing's syndrome during an evaluation for bilateral adrenal masses. Tumors of unknown origin were found in the heart, brain, thyroid gland, colon, pancreas, and both adrenal glands. Adenocarcinoma of the sigmoid colon and systemic brown-patchy skin pigmentation were also present. Her urinary cortisol levels increased in response to oral dexamethasone, while serum dehydroepiandrosterone-sulfate was not suppressed. After right adrenalectomy, genetic analysis of the resected tumor revealed the somatic GNAS activating mutation, p.R201H. Paradoxical urinary cortisol response persisted even after unilateral adrenal resection, although serum and urinary cortisol levels were attenuated. Conclusions: This patient harbored a GNAS activating mutation, and presented with a mild cortisol- and androgen-producing adrenal adenoma. Administration of oral dexamethasone paradoxically increased cortisol levels, possibly via the stimulation of the cyclic adenosine monophosphate-dependent protein kinase A signaling pathway, which is seen in patients with pigmented nodular adrenocortical disease or Carney complex. GNAS mutations may provide clues to the mechanisms of hyper-function and tumorigenesis in the adrenal cortex, especially in bilateral adrenal masses accompanied by multiple systemic tumors. Examining GNAS mutations could help physicians detect extra-adrenal malignancies, which may contribute to an improved prognosis for patients with this type of Cushing's syndrome..
80. , Norio Wada, Yui Shibayama, Takashi Yoneda, Takuyuki Katabami, Isao Kurihara, Mika Tsuiki, Takamasa Ichijo, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Takanobu Yoshimoto, Yuichi Matsuda, Megumi Fujita, Hiroki Kobayashi, Kouichi Tamura, Kohei Kamemura, Michio Otsuki, Shintaro Okamura, Mitsuhide Naruse, Lateralizing asymmetry of adrenal imaging and adrenal vein sampling in patients with primary aldosteronism, Journal of the Endocrine Society, 10.1210/js.2019-00131, 3, 7, 1393-1402, 2019.01, Context: In patients with primary aldosteronism (PA), it remains unclear whether aldosteroneproducing adenomas are likely to develop in the left or right adrenal gland. Objective: To investigate left-right differences of PA laterality diagnoses via CT imaging and adrenal vein sampling (AVS). Design: Retrospective, observational study. Patients: From the Japan Primary Aldosteronism Study, 1493 patients with PA were enrolled who underwent CT and ACTH-stimulated AVS. Measurements: Left or right adrenal nodular lesion distribution and laterality observed on CT scans and from AVS were noted. Results: Both on CT scans and AVS, unilateral results were observed more frequently on the left side than on the right side (25.1% vs 15.4% and 17.3% vs 13.5%, respectively; P < 0.01 for both diagnostic techniques). There was no significant difference in the concordance rate for CT and AVS between patients with left and right unilateral nodular lesions observed on CT scans (44.1% and 50.9%, respectively; P 5 0.15). In patients with nodules <20 mm, the concordance rate was significantly greater on the right side than the left side (45.8% vs 56.4%; P 5 0.03). In patients with bilateral results of AVS, unilateral nodular lesions were detected more frequently on the left side than the right side (17.8% vs 9.4%; P < 0.01). Conclusion: These results suggest aldosterone-producing adenomas and nonfunctioning tumors are more likely to develop on the left side in patients with PA and that misdiagnosis of CT-based lateralization may occur more frequently on the left side..
81. Chihiro Sakaguchi, Seiichi Yano, Kenji Ashida, Naoko Wada, Kenji Ohe, Hiromi Nagata, Yayoi Matsuda, Shohei Sakamoto, Ryuichi Sakamoto, Keizo Ohnaka, Hiroshi Uchi, Masutaka Furue, Masatoshi Nomura, Yoshihiro Ogawa, A case of acute exacerbation of chronic adrenal insufficiency due to ipilimumab treatment for advanced melanoma, American Journal of Case Reports, 10.12659/AJCR.913021, 20, 106-110, 2019, Objective: Unusual clinical course Background: Ipilimumab is a therapeutic human monoclonal antibody that targets the T-cell inhibitory molecule, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and is classified as an immune checkpoint inhibitor that has been shown to improve prognosis in patients with advanced melanoma. However, several immune-related adverse events have been reported to be associated with ipilimumab Treatment. A case of acute exacerbation of chronic adrenal insufficiency is presented that highlights that glucocorticoid dosage for patients undergoing steroid treatment at the time of ipilimumab treatment has yet to be established. Case Report: A 50-year-old Japanese woman was diagnosed with malignant melanoma on the sole of her right foot. During her second course of ipilimumab treatment, she developed acute adrenal insufficiency caused by isolated adrenocorticotropic hormone (ACTH) deficiency, which required treatment with oral hydrocortisone. However, the symptoms of her adrenal insufficiency worsened, and she commenced treatment with 12 courses of nivolumab, a therapeutic human monoclonal antibody that blocks programmed cell death protein 1 (PD-1) on the surface of T-cells. She did not require corticosteroid support during nivolumab treatment. Conclusions: This case report highlights the risk of exacerbating adrenal insufficiency during treatment with ipilimumab. The differences in clinical outcome in this patient between ipilimumab and nivolumab treatment might be explained by the different mechanisms between ipilimumab and nivolumab on immune function..
82. Shohei Hamada, Eikichi Ihara, Hiroko Ikeda, Kazumasa Muta, Haruei Ogino, Takatoshi Chinen, Yoshimasa Tanaka, Yoshihiro Ogawa, Clinical Characterization of Vonoprazan-Refractory Gastroesophageal Reflux Disease, Digestion, 10.1159/000503340, 2019, Introduction: The newly developed vonoprazan (a potassium-competitive acid blocker) has a greater ability to suppress gastric acid production than convention proton pump inhibitors (PPIs). The objective of the present study was to determine how vonoprazan influences the pathogenesis of refractory gastroesophageal reflux disease (GERD) in clinical practice. Methods: Between March 2013 and November 2018, a total of 73 refractory GERD patients (34 in the conventional PPI group versus 39 in the vonoprazan group) were enrolled in this retrospective study. We then compared the underlying disease conditions between the 2 groups, examined by high-resolution manometry and multichannel intraluminal impedance/pH (MII-pH) monitoring. Results: There was a significant difference in the proportion of underlying disease conditions, including erosive esophagitis, non-erosive reflux disease, reflux hypersensitivity, functional heartburn and oesophageal motility disorder (EMD), between the conventional PPI (6, 14, 23, 40 and 17% respectively) and vonoprazan groups (0, 0, 10, 49, and 41% respectively; p < 0.01). No cases of acid-related GERD were observed in the vonoprazan group. When the EMD patients were excluded, the lower oesophageal acid exposure time of the vonoprazan group (0.1% [0.0-0.5%], n = 23) was significantly lower than that of the conventional PPI group (0.35% [0.1-3.9%], n = 28; p < 0.05), and the gastric pH <4 holding time of the vonoprazan group (7.7% [0.7-34.5%]) was also significantly lower than that of the conventional PPI group (61.6% [49.4-74.3%], p < 0.01). Conclusions: Vonoprazan serves as a diagnostic tool to exclude acid-related GERD..
83. Koji Imoto, Motoyuki Kohjima, Tomonobu Hioki, Tomoyuki Kurashige, Miho Kurokawa, Shigeki Tashiro, Hideo Suzuki, Akifumi Kuwano, Masatake Tanaka, Seiji Okada, Masaki Kato, Yoshihiro Ogawa, Clinical Features of Liver Injury Induced by Immune Checkpoint Inhibitors in Japanese Patients, Canadian Journal of Gastroenterology and Hepatology, 10.1155/2019/6391712, 2019, 2019, Aim. Immune checkpoint inhibitors (ICIs) have improved the survival rate of patients carrying various malignant neoplasms. Despite their efficacy, ICIs occasionally induce liver injury as an immune-related adverse event (irAE). This study aimed to reveal the clinical features of the hepatic irAE in Japanese patients. Methods. Among 387 patients treated with ICIs, those who developed drug-induced liver injury were investigated. We also describe the histological findings and clinical courses of four patients with hepatic irAE who underwent liver biopsy. Results. Among the 56 patients with all-grade liver injury, only 11 (19.6%) showed hepatocellular-type liver injury, which resembled autoimmune hepatitis. Thirty-four patients (60.7%) developed cholestatic or mixed-type liver injury, although only one patient showed abnormal image findings in the bile duct. Most patients with grade ≤2 liver injury improved spontaneously, while two patients with biliary dysfunction required ursodeoxycholic acid or prednisolone. Among eight patients with grade ≥3 liver injury, three required no immunosuppressants and five were treated with prednisolone (three of five patients required other types of immunosuppressants). Four patients in the case series showed diverse clinical features in terms of hepatotoxic pattern, symptoms, and the interval time between the initiation of immunotherapy and the onset of the hepatic irAE. Conclusions. Our findings suggest that ICIs could cause microscopic biliary disorder without any abnormal image finding. Because the hepatic irAE presents diverse clinical features, liver biopsy is recommended to provide appropriate treatments..
84. Yoshihiro Niitsu, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Fuminori Satou, Motoyoshi Tsujino, Kazuki Ota, Atsushi Kudo, Minoru Tanabe, Tetsuya Yamada, Yoshihiro Ogawa, Clinical outcomes of 20 Japanese patients with insulinoma treated with diazoxide, Endocrine Journal, 10.1507/endocrj.EJ18-0353, 66, 2, 149-155, 2019, Diazoxide is recognized as an effective medical treatment for insulinoma. However, due to its adverse effects, such as fluid retention, it is sometimes difficult to employ diazoxide at an effective dose in clinical practice. This study aimed to clarify the clinical factors, which may affect efficacy and safety of the diazoxide treatment. We retrospectively evaluated the medical records of 20 patients with insulinoma including 4 malignant cases. The patients were divided into two groups according to the presence or absence of favorable outcomes or adverse effects, and the clinical features of both groups were compared. Diazoxide was effective and ineffective in each 9 patients, respectively. In other 2 cases, the efficacy could not be determined. In the effective group, all patients had benign insulinoma. Additionally, the tumor size determined by imaging test was tended to smaller than the ineffective group but not statistically significant when malignant cases were excluded (p = 0.065). Fluid retention was observed more frequently in females than in males (p = 0.025). Five patients displayed unacceptable thrombocytopenia within a few weeks after the administration of diazoxide. In these patients, the diazoxide dose was significantly higher than that in the other patients [400 mg/day (250–500 mg/day) vs. 225 mg/day (50–425 mg/day), p = 0.027]. These findings may be informative in determining the indication and dose of diazoxide against insulinoma. In addition, a careful evaluation of platelet count would be required for a few weeks after the initiation of diazoxide treatment..
85. Takaaki Fujimoto, Yasuhisa Mori, Yohei Nakashima, Takao Ohtsuka, So Nakamura, Yoshitaka Gotoh, Kenjiro Date, Yoshihiko Sadakari, Kohei Nakata, Yoshihiro Miyasaka, Takashi Osoegawa, Akira Aso, Eikichi Ihara, Kazuhiko Nakamura, Yoshihiro Ogawa, Shuji Shimizu, Masafumi Nakamura, Endoscopic retrograde cholangiopancreatography in patients with surgically altered gastrointestinal anatomy
A retrospective study, International Surgery, 10.9738/INTSURG-D-17-00137.1, 103, 3-4, 184-190, 2019, Objective: The aim of this study was to evaluate the difficulty of endoscopic retrograde cholangiopancreatography (ERCP) procedures when performed in patients with different types of surgically altered gastrointestinal (GI) anatomies. Summary of background data: Clinical data of 102 consecutive patients with surgically altered GI anatomy who underwent ERCP using a double-balloon enteroscope or a regular gastroendoscope between January 2008 and March 2015 were retrospectively reviewed. Methods: The success rate of reaching the destination, the time until reaching the destination, the success rate of the procedures, and complications were assessed for each type of altered GI anatomy using a double-balloon enteroscope and a regular gastroendoscope. Results: A total of 180 ERCP procedures were performed. The total success rate of reaching the destination was 91% (164 of 180), and that of treatment was 88% (144 of 164). The success rate of reaching the destination in patients with Roux-en-Y hepaticojejunostomy (HJ þ R-Y) was significantly lower than that of the other types of reconstruction. The time until reaching the destination was significantly longer in patients after R-Y reconstruction (gastrectomy or HJ) than that after Billroth-II gastrectomy or pancreatoduodenectomy. GI perforation occurred in 2 patients after R-Y reconstruction (1 patient after gastrectomy, and 1 patient after HJ). However, no other complications, such as severe pancreatitis, bleeding, or air embolism, were observed. Conclusions: ERCP for patients with surgically altered GI anatomy is feasible. Improvement of the success rate of reaching the destination in patients after HJ þ R-Y and prevention of perforation in those with R-Y reconstruction are necessary..
86. Tsukasa Miyagahara, Nao Fujimori, Takamasa Oono, Misato Okamoto, Naoichi Sato, Noriyuki Sonoda, Kenichi Kohashi, Kousei Ishigami, Yoshihiro Ogawa, Fulminant type 1 diabetes mellitus following acute pancreatitis and hypoglycemia with sequential imaging of the pancreas using computed tomography
A case report, Journal of Japanese Society of Gastroenterology, 10.11405/nisshoshi.116.161, 116, 2, 161-167, 2019, Wc herein report a rare case of fulminant type 1 diabetes mellitus (FT1DM) following acute pancreatitis and hypoglycemia, in which the pancreas was evaluated by serial computed tomography (CT). A 30-year-old male presented to a local hospital with a two-day history of abdominal pain and was diagnosed with acute pancreatitis based on elevated serum amylase and peripancreatic fluid collection on CT images. The patient developed sudden hypoglycemia (plasma glucose, 45mg/dL: serum C-peptide, 3.4ng/mL) the next day and hyperglycemia (plasma glucose, 250-480mg/dL) on admission day four. CT revealed a low attenuation area extending from the pancreatic head to the pancreatic tail. On admission day eight, he was referred to our hospital and diagnosed with FT1DM after he developed ketoacidosis immediately after hospitalization, with a plasma glucose level of 442mg/dL, hemoglobin Ale concentration of 5.7% and undetectable urinary C- peptide with a serum C-peptide level of O.lng/mL before and after intravenous glucagon loading. CT imaging revealed dramatic improvement at the time, and no pancreatic islets were detected in the pancreatic biopsy specimens..
87. Yujiro Nakano, Takanobu Yoshimoto, Ryo Watanabe, Masanori Murakami, Tatsuya Fukuda, Kazutaka Saito, Yasuhisa Fujii, Takumi Akashi, Toshihiro Tanaka, Tetsuya Yamada, Mitsuhide Naruse, Yoshihiro Ogawa, MiRNA299 involvement in CYP11B2 expression in aldosterone-producing adenoma, European journal of endocrinology, 10.1530/EJE-18-0882, 181, 1, 69-78, 2019, Objective: The pathophysiology of aldosterone-producing adenomas (APAs) has been intensively investigated using genetic and epigenetic approaches. However, the role of miRNAs in APA is not fully understood. The present study profiled miRNAs in APAs as an exploratory approach to elucidate their pathophysiological roles in APAs. Design: Tissues of APAs and other adrenocortical adenomas were obtained from patients who underwent adrenalectomy. Methods: Candidate miRNAs differentially detected from samples were exam ined by whole miRNA sequencing. The expression of candidate miRNAs in APA tissues were further vali dated by real-time quantitative polymerase chain reaction (qPCR). Further, differential miRNA expression between APAs with and without KCNJ5 somatic mutations was examined. Prediction of miRNA target genes was performed by bioinformatics analysis. For specific miRNAs, correlation analysis between the levels of their target genes and CYP11B2 was analyzed in APA tissues. Results: Our study determined differential expression of six miRNAs in A PA or APA with KCNJ5 mutations. We further demonstrated that miR299 levels were negatively correlated with mRNA levels of CACNB2, which encodes the beta-subunit of the L-type calcium channel. Additionally, we found s ignificant correlations among miR299, CACNB2, and CYP11B2 levels in APA tissues. Conclusions: Our study suggests the possible pathophysiological involvement of specific miRNAs in calcium signaling and aldosterone hypersecretion in APAs. Further studies, including in vitro analyses, are required to clarify these findings..
88. Keita Fukaura, Yoichiro Iboshi, Haruei Ogino, Eikichi Ihara, Kazuhiko Nakamura, Yuichiro Nishihara, Kei Nishioka, Takatoshi Chinen, Tsutomu Iwasa, Akira Aso, Ayako Goto, Kazuhiro Haraguchi, Hirotada Akiho, Naohiko Harada, Yoshihiro Ogawa, Mucosal Profiles of Immune Molecules Related to T Helper and Regulatory T Cells Predict Future Relapse in Patients with Quiescent Ulcerative Colitis, Inflammatory bowel diseases, 10.1093/ibd/izy395, 25, 6, 1019-1027, 2019, Background: T helper (Th)- and regulatory T (Treg) cell-related immune molecules are implicated in ulcerative colitis (UC). However, the association between their mucosal expression during remission and the subsequent clinical course of UC is unknown. Methods: The expression of cytokines and transcription factors related to Th1, Th2, Th17, and Treg in endoscopic mucosal biopsy specimens from 40 UC patients in clinical remission and 9 controls was measured by quantitative polymerase chain reaction. The relationship between their expression patterns, as stratified by Mayo Endoscopic Subscore (MES), and any future relapse was evaluated by univariate and multivariate analyses. Results: Six of 40 patients (baseline MES 0/1/2, 22/14/4) experienced a relapse during the study period (median, 37 months). At baseline, even in the MES0 patients, the interleukin (IL)-17A of the patients was significantly upregulated in comparison with controls (P = 0.0351). Future relapse was associated with a higher baseline expression of IL-17A, IL-17F, and IL-21 in MES0/1, and the upregulation of IL-17F and IL-21 remained statistically significant when limited to MES0 patients. Kaplan-Meier analysis revealed that as a single marker, a higher IL-21 level best grouped patients with an increased risk of relapse (P = 0.0042). Furthermore, a multivariate model that consisted of IL-21 and T-bet showed an even greater value (P = 0.0001). Conclusions: The profiles of Th/Treg-related gene expression in the colonic mucosa are altered, even during clinical and endoscopic remission of UC, with a detectable Th17-predominant profile predicting future relapse. This association might represent latent immune dysregulation during disease quiescence and has the potential to be utilized to improve patient care..
89. , Akiyuki Kawashima, Masakatsu Sone, Nobuya Inagaki, Yoshiyu Takeda, Hiroshi Itoh, Isao Kurihara, Hironobu Umakoshi, Takamasa Ichijo, Takuyuki Katabami, Norio Wada, Yoshihiro Ogawa, Junji Kawashima, Megumi Fujita, Shozo Miyauchi, Shintaro Okamura, Tomikazu Fukuoka, Toshihiko Yanase, Shoichiro Izawa, Yuichiro Yoshikawa, Shigeatsu Hashimoto, Masanobu Yamada, Tatsuya Kai, Tomoko Suzuki, Mitsuhide Naruse, Renal impairment is closely associated with plasma aldosterone concentration in patients with primary aldosteronism, European journal of endocrinology, 10.1530/EJE-19-0047, 181, 3, 339-350, 2019, Objective: Several clinical studies have reported that renal impairments are sometimes observed in patients with primary aldosteronism (PA). We analyzed the prevalence of renal impairments in PA patients and identified parameters that increase the risk for them. Design: This is a retrospective cross-sectional study. We assessed the PA database established by the multicenter Japan PA study (JPAS). Data were also collected from patients with essential hypertension (EHT). Methods: We compared the prevalences of proteinuria and lowered estimated glomerular filtration rate (eGFR) between patients with PA and age, sex, blood pressure and duration of hypertension-matched patients with EHT. We also performed logistic regression analysis to identify parameters that increase the risk for these renal impairments. Results: Among 2366 PA patients, the prevalences of proteinuria and lowered eGFR were 10.3 and 11.6%, respectively. The prevalence of proteinuria was significantly higher in PA patients than matched-EHT patients (16.8 vs 4.4%), whereas there was no significant difference in the prevalence of lowered eGFR (17.2 vs 15.0%). The logistic regression analysis also showed that the plasma aldosterone concentration (PAC) significantly increases the risk of proteinuria and lowered eGFR, independent of other known risk factors. Conclusion: Plasma aldosterone levels are closely associated with renal impairment in patients with PA. This is contrast to our earlier finding that the PAC was not itself linearly associated with cardiovascular events such as stroke or ischemic heart disease. The mechanism underlying the kidney damage in patients with PA may differ from that affecting the cardiovascular system..
90. Mitsuru Esaki, Yosuke Minoda, Masafumi Wada, Seiichiro Sakisaka, Shinichi Tsuruta, Taizo Hosokawa, Takahiro Matsuguchi, Ryoji Ichijima, Sho Suzuki, Yu Tamura, Aya Iwao, Shun Yamakawa, Akira Irie, Eikichi Ihara, Yoshihiro Ogawa, Self-Completion Method of Endoscopic Submucosal Dissection Using Endosaber without Any Other Device or Assistance
An ex vivo Porcine Model Study, Digestion, 10.1159/000502771, 2019, Background: Endoscopic submucosal dissection (ESD) is a standard treatment for tumors of the gastrointestinal tract. We developed a self-completion method of ESD using Endosaber to eliminate the need for an additional device or human assistance during the procedure. The aim of this study was to evaluate the technical feasibility and outcomes of this method in an ex vivo porcine training model. Methods: This was a pilot study, and the procedures were performed by 4 experts. Mock lesions measuring 15 mm in diameter were prepared at the posterior wall in the middle or lower esophagus obtained from domestic pigs. Each operator performed ESD on the mock lesions in 3 models. The primary outcome was ESD completion rate using the self-completion method. The secondary outcomes were procedure time, en bloc resection rate, perforation rate, and number of injections during the procedure. Results: All 12 ESDs were completed using the self-completion method. The median procedure time (interquartile range) was 483.5 (399-619.3) s (median incision time: 240.4 [168.3-332.5] s; median dissection time: 222 [182.8-257] s). En bloc resection rate was 100%. No perforation was noted during any of the procedures. The median number of injections was 10.5 (9-14.3). The procedure time decreased significantly with increase in experience (p = 0.020). Conclusions: The self-completion ESD method using one Endosaber without any assistance achieved a 100% en bloc resection rate without any perforation. The need for an additional device or assistance was successfully eliminated. This method may prove to be a simple and cost-effective ESD procedure for lesions in humans..
91. Koji Imoto, Motoyuki Kohjima, Tomoyuki Kurashige, Taiji Mutsuki, Shigeki Tashiro, Hideo Suzuki, Akifumi Kuwano, Masaki Kato, Yoshihiro Ogawa, Successful endoscopic treatment of hepatoduodenal fistula formed during sorafenib treatment for hepatocellular carcinoma with duodenal invasion, Acta Hepatologica Japonica, 10.2957/kanzo.60.91, 60, 3, 91-98, 2019, Here, we have reported a case of hepatocellular carcinoma (HCC) with a duodenal fistula that was treated with endoscopic procedure in a 79-year-old female patient who was admitted to our hospital with melena. She was diagnosed with recurrent HCC with duodenal bulb invasion and hemorrhage. After 2 months of sorafenib treatment, an improvement in HCC was noted, while a hepatoduodenal fistula was detected at the invasion site. Hepatoduodenal fistula occurring in duodenal invasion of HCC often produces intractable infection and worsens prognosis. We successfully closed the fistula using argon plasma coagulation (APC), following endoscopic injection with fibrin glue and polyglycolic acid sheet coverage. Recent developments in chemotherapy for advanced HCC may provide similar cases with hepatoduodenal fistula occurring in duodenal invasion of HCC and endoscopic treatment could be one of the therapeutic options in such cases..
92. Yasutaka Miyachi, Kyoichiro Tsuchiya, Kumiko Shiba, Kentaro Mori, Chikara Komiya, Naomi Ogasawara, Yoshihiro Ogawa, A reduced M1-like/M2-like ratio of macrophages in healthy adipose tissue expansion during SGLT2 inhibition, Scientific reports, 10.1038/s41598-018-34305-x, 8, 1, 2018.12, The adipose tissue includes various stromal cells, such as preadipocytes, endothelial cells, fibroblasts, and immune cells, which are involved in adipose tissue functions. We previously reported that, in obese mice, the sodium–glucose cotransporter 2 inhibitor ipragliflozin (Ipra) promoted the expansion of the epididymal adipose tissue (Epi) with increase of serum ketone body concentration. The Ipra-induced adipose tissue expansion did not deteriorate adipose inflammation, or systemic glucose/lipid metabolism, referred to as “healthy adipose tissue expansion.” Here we found that Ipra promoted healthy adipose tissue expansion with a reduced ratio of pro-inflammatory M1-like adipose tissue macrophages (ATMs) to anti-inflammatory M2-like ATMs. Ipra downregulated the gene expression of interleukin (IL)−15 (Il15) in stromal cells of Epi. IL-15 inhibited lipogenesis in 3T3-L1 cells associated with downregulation of the lipogenic gene. Ketone body β-hydroxybutyrate suppressed Il15 gene induction in M1-polarized cultured macrophages, and a ketogenic diet reproduced the adipose tissue expansion without deteriorating systemic glucose metabolism in mice. Our data indicate that the phenotypic switch of ATMs could mediate healthy adipose tissue expansion by treatment with Ipra, and it may offer new insights into the pathophysiological mechanisms of adipose tissue expansion..
93. Akihiro Mukaino, Hitomi Minami, Hajime Isomoto, Hitomi Hamamoto, Eikichi Ihara, Yasuhiro Maeda, Osamu Higuchi, Tohru Okanishi, Yohei Kokudo, Kazushi Deguchi, Fumisato Sasaki, Toshihito Ueki, Ken ya Murata, Takeshi Yoshida, Mistuyo Kinjo, Yoshihiro Ogawa, Akio Ido, Hidenori Matsuo, Kazuhiko Nakao, Shunya Nakane, Anti-ganglionic AChR antibodies in Japanese patients with motility disorders, Journal of gastroenterology, 10.1007/s00535-018-1477-8, 53, 12, 1227-1240, 2018.12, Background: The existence of several autoantibodies suggests an autoimmune basis for gastrointestinal (GI) dysmotility. Whether GI motility disorders are features of autoimmune autonomic ganglionopathy (AAG) or are related to circulating anti-ganglionic acetylcholine receptor (gAChR) antibodies (Abs) is not known. The aim of this study was to determine the associations between autonomic dysfunction, anti-gAChR Abs, and clinical features in patients with GI motility disorders including achalasia and chronic intestinal pseudo-obstruction (CIPO). Methods: First study: retrospective cohort study and laboratory investigation. Samples from 123 patients with seropositive AAG were obtained between 2012 and 2017. Second study: prospective study. Samples from 28 patients with achalasia and 14 patients with CIPO were obtained between 2014 and 2016, and 2013 and 2017, respectively. In the first study, we analyzed clinical profiles of seropositive AAG patients. In the second study, we compared clinical profiles, autonomic symptoms, and results of antibody screening between seropositive, seronegative achalasia, and CIPO groups. Results: In the first study, we identified 10 patients (8.1%) who presented with achalasia, or gastroparesis, or paralytic ileus. In the second study, we detected anti-gAChR Abs in 21.4% of the achalasia patients, and in 50.0% of the CIPO patients. Although patients with achalasia and CIPO demonstrated widespread autonomic dysfunction, bladder dysfunction was observed in the seropositive patients with CIPO as a prominent clinical characteristic of dysautonomia. Conclusions: These results demonstrate a significant prevalence of anti-gAChR antibodies in patients with achalasia and CIPO. Anti-gAChR Abs might mediate autonomic dysfunction, contributing to autoimmune mechanisms underlying these GI motility disorders..
94. Kumiko Shiba, Kyoichiro Tsuchiya, Chikara Komiya, Yasutaka Miyachi, Kentaro Mori, Noriko Shimazu, Shinobu Yamaguchi, Naomi Ogasawara, Makoto Katoh, Michiko Itoh, Takayoshi Suganami, Yoshihiro Ogawa, Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH, Scientific reports, 10.1038/s41598-018-19658-7, 8, 1, 2018.12, Sodium glucose cotransporter 2 (SGLT2) inhibitors, an antidiabetic drug, promotes urinary excretion of glucose by blocking its reabsorption in the renal proximal tubules. It is unclear whether SGLT2 inhibition could attenuate nonalcoholic steatohepatitis (NASH) and NASH-associated hepatocellular carcinoma. We examined the preventive effects of an SGLT2 inhibitor canagliflozin (CANA) in Western diet (WD)-fed melanocortin 4 receptor-deficient (MC4R-KO) mice, a mouse model of human NASH. An eight-week CANA treatment attenuated hepatic steatosis in WD-fed MC4R-KO mice, with increased epididymal fat mass without inflammatory changes. CANA treatment for 20 weeks inhibited the development of hepatic fibrosis in WD-fed MC4R-KO mice. After one year of CANA treatment, the number of liver tumors was significantly reduced in WD-fed MC4R-KO mice. In adipose tissue, CANA suppressed the ratio of oxidative to reduced forms of glutathiones (GSSG/GSH) in WD-fed MC4R-KO mice. Treatment with GSH significantly attenuated the H2O2-induced upregulation of genes related to NADPH oxidase in 3T3-L1 adipocytes, and that of Il6, Tgfb, and Pdgfb in RAW264.7 cells. This study provides evidence that SGLT2 inhibitors represent the unique class of drugs that can attenuate or delay the onset of NASH and eventually hepatocellular carcinoma, at least partly, through "healthy adipose expansion"..
95. Yasuyo Hayashi, Mitsuru Esaki, Sho Suzuki, Eikichi Ihara, Azusa Yokoyama, Seiichiro Sakisaka, Taizo Hosokawa, Yoshimasa Tanaka, Takahiro Mizutani, Shinichi Tsuruta, Aya Iwao, Shun Yamakawa, Akira Irie, Yosuke Minoda, Yoshitaka Hata, Haruei Ogino, Hirotada Akiho, Yoshihiro Ogawa, Clutch Cutter knife efficacy in endoscopic submucosal dissection for early gastric neoplasms, World Journal of Gastrointestinal Oncology, 10.4251/wjgo.v10.i12.487, 10, 12, 487-495, 2018.12, AIM To compare the outcomes of endoscopic submucosal dissection (ESD) for gastric neoplasms using Clutch Cutter (ESD-C) or other knives (ESD-O). METHODS This was a single-center retrospective study. Gastric neoplasms treated by ESD between April 2016 and October 2017 at Kitakyushu Municipal Medical Center were reviewed. Multivariate analyses and propensity score matching were used to reduce biases. Covariates included factors that might affect outcomes of ESD, including age, sex, underlying disease, anti-thrombotic drugs use, tumor location, tumor position, tumor size, tumor depth, tumor morphology, tumor histology, ulcer (scar), and operator skill. The treatment outcomes were compared among two groups. The primary outcome was ESD procedure time. Secondary outcomes were en bloc, complete, and curative resection rates, and adverse events rates including perforation and delayed bleeding. RESULTS A total of 155 patients were included in this study; 44 pairs were created by propensity score matching. Background characteristics were quite similar among two groups after matching. Procedure time was significantly shorter for ESD-C (median; 49 min) than for ESD-O (median; 88.5 min) (P < 0.01). However, there was no significant difference in treatment outcomes between ESD-C and ESD-O including en bloc resection rate (100% in both groups), complete resection rate (100% in both groups), curative resection rate (86.4% vs 88.6%, P = 0.730), delayed bleeding (2.3% vs 6.8%, P = 0.62) and perforation (0% in both groups). CONCLUSION ESD-C achieved shorter procedure time without an increase in complication risk. Therefore, ESD-C could become an effective ESD option for gastric neoplasms..
96. Xunmei Yuan, Kazutaka Tsujimoto, Koshi Hashimoto, Kenichi Kawahori, Nozomi Hanzawa, Miho Hamaguchi, Takami Seki, Makiko Nawa, Tatsuya Ehara, Yohei Kitamura, Izuho Hatada, Morichika Konishi, Nobuyuki Itoh, Yoshimi Nakagawa, Hitoshi Shimano, Takako Takai-Igarashi, Yasutomi Kamei, Yoshihiro Ogawa, Epigenetic modulation of Fgf21 in the perinatal mouse liver ameliorates diet-induced obesity in adulthood, Nature communications, 10.1038/s41467-018-03038-w, 9, 1, 2018.12, The nutritional environment to which animals are exposed in early life can lead to epigenetic changes in the genome that influence the risk of obesity in later life. Here, we demonstrate that the fibroblast growth factor-21 gene (Fgf21) is subject to peroxisome proliferator-activated receptor (PPAR) α-dependent DNA demethylation in the liver during the postnatal period. Reductions in Fgf21 methylation can be enhanced via pharmacologic activation of PPARα during the suckling period. We also reveal that the DNA methylation status of Fgf21, once established in early life, is relatively stable and persists into adulthood. Reduced DNA methylation is associated with enhanced induction of hepatic FGF21 expression after PPARα activation, which may partly explain the attenuation of diet-induced obesity in adulthood. We propose that Fgf21 methylation represents a form of epigenetic memory that persists into adulthood, and it may have a role in the developmental programming of obesity..
97. Toshihiro Goto, Michiko Itoh, Takayoshi Suganami, Sayaka Kanai, Ibuki Shirakawa, Takeru Sakai, Masahiro Asakawa, Toshihiro Yoneyama, Toshihiro Kai, Yoshihiro Ogawa, Obeticholic acid protects against hepatocyte death and liver fibrosis in a murine model of nonalcoholic steatohepatitis, Scientific reports, 10.1038/s41598-018-26383-8, 8, 1, 2018.12, Accumulating evidence has suggested that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) are therapeutically useful for non-Alcoholic steatohepatitis (NASH). However, it is still unclear how FXR agonists protect against NASH and which cell type is the main target of FXR agonists. In this study, we examined the effects of OCA on the development of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice that progressively developed hepatic steatosis and NASH on Western diet (WD). Treatment with OCA effectively prevented chronic inflammation and liver fibrosis in WD-fed MC4R-KO mice with only marginal effect on body weight and hepatic steatosis. Hepatic crown-like structure (hCLS) is a unique histological structure characteristic of NASH, which triggers hepatocyte death-induced interstitial fibrosis. Intriguingly, treatment with OCA markedly reduced hCLS formation even after MC4R-KO mice developed NASH, thereby inhibiting the progression of liver fibrosis. As its mechanism of action, OCA suppressed metabolic stress-induced p53 activation and cell death in hepatocytes. Our findings in this study highlight the role of FXR in hepatocytes in the pathogenesis of NASH. Collectively, this study demonstrates the anti-fibrotic effect of OCA in a murine model of NASH with obesity and insulin resistance, which suggests the clinical implication for human NASH..
98. Yohei Minami, Noriyuki Sonoda, Eiichi Hayashida, Hiroaki Makimura, Makoto Ide, Noriko Ikeda, Masahiro Ohgidani, Takahiro A. Kato, Yoshihiro Seki, Yasutaka Maeda, Shigenobu Kanba, Ryoichi Takayanagi, Yoshihiro Ogawa, Toyoshi Inoguchi, P66Shc Signaling Mediates Diabetes-Related Cognitive Decline, Scientific reports, 10.1038/s41598-018-21426-6, 8, 1, 2018.12, Accumlating evidence have suggested that diabetes mellitus links dementia, notably of Alzheimer's disease (AD). However, the underlying mechanism remains unclear. Several studies have shown oxidative stress (OS) to be one of the major factors in the pathogenesis of diabetic complications. Here we show OS involvement in brain damage in a diabetic animal model that is at least partially mediated through an AD-pathology-independent mechanism apart from amyloid-β accumulation. We investigated the contribution of the p66Shc signaling pathway to diabetes-related cognitive decline using p66Shc knockout (-/-) mice. p66Shc (-/-) mice have less OS in the brain and are resistant to diabetes-induced brain damage. Moreover, p66Shc (-/-) diabetic mice show significantly less cognitive dysfunction and decreased levels of OS and the numbers of microglia. This study postulates a p66Shc-mediated inflammatory cascade leading to OS as a causative pathogenic mechanism in diabetes-associated cognitive impairment that is at least partially mediated through an AD-pathology-independent mechanism..
99. Mariko Tsuda, Motoaki Shiratsuchi, Yasuhiro Nakashima, Motohiko Ikeda, Hiroki Muta, Taisuke Narazaki, Toru Masuda, Daisaku Kimura, Akiko Takamatsu, Masanori Matsumoto, Yoshihiro Fujimura, Koichi Kokame, Takamitsu Matsushima, Yoshihiro Ogawa, Upshaw-Schulman syndrome diagnosed during pregnancy complicated by reversible cerebral vasoconstriction syndrome, Transfusion and Apheresis Science, 10.1016/j.transci.2018.10.023, 57, 6, 790-792, 2018.12, Upshaw-Schulman syndrome (USS) is an inherited type of thrombotic thrombocytopenic purpura (TTP) that is extremely rare, but often diagnosed during pregnancy. Reversible cerebral vasoconstriction syndrome (RCVS) is the transient stenosis of several cerebral arteries that is frequently diagnosed post-partum. We describe a 28-year-old woman with USS complicated by RCVS after delivery that was treated by plasma exchange with a good outcome. She was referred to our hospital with thunderclap headache, anemia and thrombocytopenia that occurred immediately postpartum. She was diagnosed with TTP and multiple cerebral infarctions. Plasma exchange promptly improved her symptoms on hospital day 3. Moreover, multiple stenoses of cerebral arteries indicating RCVS were resolved. Since her sister also had an episode of thrombocytopenia during pregnancy, inherited TTP was suspected and genetic analyses confirmed USS. Pregnancy is a risk for not only TTP, but also RCVS. Endothelial damage might be an underlining cause and vasospasm after delivery is a trigger of RCVS. Plasma exchange was effective against both TTP and RCVS..
100. Masao Takeda, Koichi Yamamoto, Hiroshi Akasaka, Hiromi Rakugi, Mitsuhide Naruse, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Hironobu Umakoshi, Mika Tsuiki, Takamasa Ichijo, Takuyuki Katabami, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Atsushi Ogo, Shintaro Okamura, Shozo Miyauchi, Toshihiko Yanase, Tomoko Suzuki, Takashi Kawamura, Clinical characteristics and postoperative outcomes of primary aldosteronism in the elderly, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2018-00059, 103, 10, 3620-3629, 2018.10, Context: Primary aldosteronism (PA) in the elderly has increased in importance in association with population aging. Objective: To investigate the characteristics and outcomes of elderly patients with PA undergoing adrenalectomy. Patients and Methods: Using a database of patients with PA who underwent adrenal venous sampling (AVS),wecompared elderly patients (65 years old)with nonelderly patients (,65 years old) in terms of characteristics, subtype classification in ACTH-stimulated AVS, and outcomes after adrenalectomy. Results: The elderly group had a higher prevalence of comorbidities than the nonelderly group. The proportion of the unilateral subtype [defined as a lateralization index (LI) .4] was comparable between the age groups. In patients who received adrenalectomy, biochemical cure was comparable between the groups, whereas persistent hypertension was more common in the elderly group. The prevalences of hyperkalemia and renal impairment (chronic kidney disease stage 3b or higher) were higher in the elderly group. Multiple regression analysis showed that the duration of hypertension predicted persistent hypertension and hyperkalemia and that preoperative estimated glomerular filtration rate predicted renal impairment in the elderly group. LI .4 in AVS was an independent predictor of biochemical cure after adrenalectomy in the elderly group but not in the nonelderly group. Age was negatively associated with biochemical cure in patients with LI #4. Conclusion: Adrenalectomy contributes to biochemical improvement in elderly patients if determined in accordance with AVS. The treatment strategy should be determined considering the high postoperative incidence of persistent hypertension and hyperkalemia in elderly patients with a long history of hypertension or renal impairment in those with reduced renal function..
101. Taisuke Narazaki, Shojiro Haji, Yasuhiro Nakashima, Yasuhiro Tsukamoto, Mariko Tsuda, Akiko Takamatsu, Hirofumi Ohno, Takamitsu Matsushima, Tomoko Matsumoto, Keiji Nogami, Midori Shima, Motoaki Shiratsuchi, Yoshihiro Ogawa, Acquired hemophilia A associated with autoimmune pancreatitis with serum IgG4 elevation, International journal of hematology, 10.1007/s12185-018-2441-3, 108, 3, 335-338, 2018.09, A case of acquired hemophilia A (AHA) that developed in a patient with autoimmune pancreatitis (AIP) is presented. A 64-year-old woman was diagnosed with AIP in 2007. The symptoms resolved with prednisolone (PSL). Although the dose of PSL was tapered to 7.5 mg/day for maintenance, serum IgG4 levels remained high. She suddenly presented with subcutaneous bleeding in 2015. Her activated partial thromboplastin time was prolonged (80.0 s). A mixing test showed an inhibitor pattern, factor VIII (FVIII) activity was less than 1%, and FVIII inhibitor was 290 BU/mL. She was diagnosed with AHA. Her serum IgG4 was elevated to 133 mg/dL. She was treated first with PSL alone, but she developed bladder tamponade. Cyclophosphamide and activated prothrombin complex concentrate were combined with PSL. She then achieved hemostasis, and FVIII inhibitor disappeared. FVIII inhibitor had been detected since PSL was tapered and AHA recurred two months later. An enzyme-linked immunosorbent assay showed that the inhibitor was mainly IgG4 and IgG1. This case suggests that elevation of IgG4 may be associated with the development of both AHA and AIP..
102. T. Ohashi, M. Kato, A. Yamasaki, A. Kuwano, H. Suzuki, M. Kohjima, and Y. Ogawa., Effects of high fructose intake on liver injury progression in high fat diet induced fatty liver disease in ovariectomized female mice., Food Chem Toxicol , 10.1016/j.fct.2018.05.006., 118, 190-197, 2018.08.
103. M. Esaki, S. Suzuki, Y. Hayashi, A. Yokoyama, S. Abe, T. Hosokawa, S. Tsuruta, Y. Minoda, Y. Hata, H. Ogino, H. Akiho, E. Ihara and Y. Ogawa. , Propensity score-matching analysis to compare clinical outcomes of endoscopic submucosal dissection for early gastric cancer in the postoperative and non-operative stomachs., BMC Gastroenterol, 10.1186/s12876-018-0855-2., 18, 1, 125, 2018.08.
104. Hironobu Umakoshi, Mika Tsuiki, Maki Yokomoto-Umakoshi, Yoshiyu Takeda, Yoneda Takashi, Isao Kurihara, Hiroshi Itoh, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Kenji Ashida, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamto, Atsushi Ogo, Shintaro Okamura, Shozo Miyauchi, Tomikazu Fukuoka, Shoichiro Izawa, Toshihiko Yanase, Shigeatsu Hashimoto, Masanobu Yamada, Yuichiro Yoshikawa, Tatsuya Kai, Tomoko Suzuki, Takashi Kawamura, Mitsuhide Naruse, Correlation between lateralization index of adrenalvenoussamplingandstandardized outcome in primary aldosteronism, Journal of the Endocrine Society, 10.1210/JS.2018-00055, 2, 8, 893-902, 2018.08, Objectives: The aim of this study was to investigate the impact of adrenal venous sampling (AVS) lateralization cutoffs on surgical outcomes. Patients and Methods: Cosyntropin-stimulated AVS was used to guide surgical management of 377 patients with primary aldosteronism (PA) who were evaluated 6 months after surgery. Main Outcome Measures: The proportion of patients that achieved clinical benefit and complete biochemical success based on the AVS aldosterone lateralization index (LI) was determined. Results: Clinical benefit was achieved in 29 of 47 patients with an LI between 2 and 4, in 66 of 101 with an LI between 4 and 10, and in 158 of 203 with an LI > 10 (P, 0.01 for trend). Complete biochemical success was achieved in 27 of 42 with an LI between 2 and 4, in 60 of 76 with an LI between 4 and 10, and in 127 of 155 with an LI > 10 (P = 0.024 for trend). After adjustment for confounders and using those patients with an LI between 2 and 4 as a reference, a clinical benefit was associated only with those with an LI > 10 (OR, 2.30; 95% CI, 1.03 to 5.16), whereas complete biochemical success was associated with those with an LI between 4 and 10 (OR, 2.83; 95% CI, 1.14 to 7.01) or LI > 10 (OR, 3.55; 95% CI, 1.47 to 8.55). Conclusions: Difference of clinical outcome was relatively small when strict LI diagnostic threshold was used; biochemical cure was sufficiently achieved when an LI > 4 was used. Our study by standardized outcome measures validated that an LI > 4 may be appropriate for determining unilateral disease in PA..
105. Tomoko Ohashi, Masaki Kato, Akihiro Yamasaki, Akifumi Kuwano, Hideo Suzuki, Motoyuki Kohjima, Yoshihiro Ogawa, Effects of high fructose intake on liver injury progression in high fat diet induced fatty liver disease in ovariectomized female mice, Food and Chemical Toxicology, 10.1016/j.fct.2018.05.006, 118, 190-197, 2018.08, Epidemiology shows that the morbidity of nonalcoholic fatty liver disease (NAFLD) is increased in postmenopausal women and chronic high fructose intake induces NAFLD progression. To analyze the effects of high fructose intake on estrogen deficiency, we evaluated liver disease progression using ovariectomized mice fed with a high fat diet (HFD) for 12 weeks. Hepatic steatosis developed in all HFD groups. Fructose intake significantly increased the liver weight and serum alanine aminotransferase, which was not exacerbated by ovariectomy alone. Ovariectomy enhanced the hepatic inflammatory activity shown by tumor necrosis factor α upregulation in the groups with or without fructose intake. Both fructose intake and ovariectomy increased the hepatocytes with ballooning degeneration and hepatic macrophage infiltration and activated hepatic stellate cells. Coexistence of fructose intake and ovariectomy markedly enhanced liver cell destruction, macrophage accumulation, and progression of fibrosis. Liver damage was ameliorated by 17β-estradiol supplementation. These findings suggest that high fructose intake enhanced the progression of NAFLD in ovariectomized female mice..
106. Junichi Kiyasu, Fumiko Arakawa, Shojiro Haji, Yoshimichi Tachikawa, Mariko Tsuda, Yasuhiro Tsukamoto, Motohiko Ikeda, Hiroki Muta, Takamitsu Matsushima, Hiroaki Miyoshi, Motoaki Shiratsuchi, Yoshihiro Ogawa, Kouichi Ohshima, Yuji Yufu, Methotrexate-associated lymphoproliferative disorders with angioimmunoblastic T-cell lymphoma-like features accompanied by gamma-heavy chain disease in a patient with rheumatoid arthritis, Pathology International, 10.1111/pin.12703, 68, 8, 485-490, 2018.08, Although gamma heavy chain disease (γ-HCD) lesions occasionally morphologically resemble angioimmunoblastic T-cell lymphoma (AITL), no association has been described in detail due to the rarity of the disease. In this report, we present a rare manifestation of methotrexate (MTX)-associated lymphoproliferative disorders (LPDs) with AITL-like features accompanied by γ-HCD in a 75-year-old man with rheumatoid arthritis (RA). A biopsy specimen was evaluated using immunohistochemistry, clonal analyses of immunoglobulin VH and T-cell receptor γ gene rearrangements by polymerase chain reaction, and Sanger sequencing for confirmation of the structure of deleted γ-HCD clones. The histological features characterized by proliferation of CD4- and PD-1-positive medium-sized T cells and arborizing high endothelial venules together with numbers of small lymphocytes, eosinophils, plasma cells, and histiocytes in the background mimicked those of AITL, but did not completely fulfill the diagnostic criteria. Clonal analysis demonstrated that the specimen contained multiple LPDs of both B-cell and T-cell lineages. Sequence analysis confirmed the co-existence of a clone responsible for production of the abnormal heavy chain. This report provides new insights into the pathology of γ-HCD. Multiple host-derived factors (e.g., RA and/or use of MTX) may be responsible for the occurrence of LPDs of multiple lineages within a single lymph node..
107. Keita Fukaura, Eikichi Ihara, Haruei Ogino, Yoichiro Iboshi, Kazumasa Muta, Bai Xiaopeng, Shohei Hamada, Yoshitaka Hata, Tsutomu Iwasa, Akira Aso, Kazuhiko Nakamura, Yoshihiro Ogawa, Mucosally Expressed Cytokines are Associated with the Esophageal Motility Function, Digestion, 10.1159/000487708, 98, 2, 95-103, 2018.08, Background and Aim: Although basic research has shown that certain cytokines affect gastrointestinal motility, the clinical evidence is lacking. The objective of this study was to explore the association between mucosally expressed cytokines and the esophageal motility function in humans. Methods: We enrolled a total of 57 patients with suspected esophageal motility disorders (EMDs) who underwent high-resolution manometry. Results: The diagnoses of the patients were as follows: normal esophageal motility (n = 25), ineffective esophageal motility (n = 5), esophagogastric junction outflow obstruction (EGJOO; n = 10), distal esophageal spasm (n = 5), achalasia (n = 10), absent contractility (n = 1), and jackhammer esophagus (n = 1). The expression of tumor necrosis factor (TNF)-α in the esophagogastric junction (EGJ) was significantly higher in EGJOO (14.6, 14.0-15.8, n = 10) than in normal esophageal motility (13.3, 12.8-14.1, n = 25); however, there was no difference in the expression of TNF-α between achalasia (13.4, 13.0-14.1, n = 10) and normal esophageal motility (13.3, 12.8-14.1, n = 25). EGJOO was discriminated from achalasia/normal by a linear discriminant analysis (AUC = 0.917). A multivariable regression analysis revealed that interleukin (IL)-13 and IL-23A were predictive of the distal contractile integral, whereas TNF-α and IL-6 were predictive of the basal EGJ pressure. Conclusions: The esophageal motility was associated with mucosally expressed cytokines in humans; these cytokines could be useful targets for the diagnosis and treatment of EMDs..
108. Mitsuru Esaki, Sho Suzuki, Yasuyo Hayashi, Azusa Yokoyama, Shuichi Abe, Taizo Hosokawa, Shinichi Tsuruta, Yosuke Minoda, Yoshitaka Hata, Haruei Ogino, Hirotada Akiho, Eikichi Ihara, Yoshihiro Ogawa, Propensity score-matching analysis to compare clinical outcomes of endoscopic submucosal dissection for early gastric cancer in the postoperative and non-operative stomachs, BMC Gastroenterology, 10.1186/s12876-018-0855-2, 18, 1, 2018.08, Background: Endoscopic submucosal dissection (ESD) of the postoperative stomach (ESD-P) for early gastric cancer (EGC) is considered a technically difficult procedure. However, it is difficult to compare the outcomes of ESD-P and ESD of the non-operative stomach (ESD-N) because their baseline characteristics are different. Therefore, we aimed to compare the technical outcomes of ESD-P with those of ESD-N using a propensity score-matching analysis to compensate for the differences. Methods: The chart records of 1046 patients with EGC who were treated with ESD between January 2004 and July 2016 at Kitakyushu Municipal Medical Center in Japan were reviewed in this retrospective study. Multivariate analyses and propensity score-matching were performed for age, sex, lesion location, lesion size, tumor invasion, tumor size, ulcer (scar), and operator skill. The primary outcome was procedure time. Secondary outcomes were percentages of en bloc, complete, and curative resections, and percentages of adverse events, which were evaluated between the two groups. Results: Forty-one patients were in the ESD-P group and 1005 patients were in the ESD-N group. Propensity score-matching created 41 matched pairs. According to the adjusted comparisons, ESD-P required a significantly longer procedure time (85 min vs 51 min, p < 0.001). Other treatment outcomes showed an en bloc resection rate of 100% for both groups (p = 1) and complete resection rates of 95.1 and 97.6% (p = 1), curative resection rates of 90.2 and 90.2% (p = 1), perforation during ESD rates of 2.4 and 0% (p = 1), and postprocedure bleeding rates of 2.4 and 2.4% (p = 1) for the ESD-P and ESD-N groups, respectively. For the ESD-P group, lesions on the suture line or anastomotic site were significantly associated with longer procedure times (p = 0.038). Conclusions: ESD-P was a more time-consuming procedure than ESD-N. However, ESD-P and ESD-N achieved high rates of curative resection with a low rate of adverse events for the treatment of EGC. ESD could be selected as the treatment for EGC even in the postoperative stomach provided that careful attention is given to lesions on the suture line or anastomotic site..
109. T. Iwasa, R. Nakadate, S. Onogi, Y. Okamoto, J. Arata, S. Oguri, H. Ogino H, E. Ihara, K. Ohuchida, T. Akahoshi, T. Ikeda, Y. Ogawa, M. Hashizume., A new robotic-assisted flexible endoscope with single-hand control: endoscopic submucosal dissection in the ex vivo porcine stomach., Surgical Endoscopy, 10.1007/s00464-018-6188-y., 32, 7, 3386-3392, 2018.07.
110. Tsutomu Iwasa, Ryu Nakadate, Shinya Onogi, Yasuharu Okamoto, Jumpei Arata, Susumu Oguri, Haruei Ogino, Eikichi Ihara, Kenoki Ohuchida, Tomohiko Akahoshi, Tetsuo Ikeda, Yoshihiro Ogawa, Makoto Hashizume, A new robotic-assisted flexible endoscope with single-hand control
endoscopic submucosal dissection in the ex vivo porcine stomach, Surgical endoscopy, 10.1007/s00464-018-6188-y, 32, 7, 3386-3392, 2018.07, Background: Difficulties in endoscopic operations and therapeutic procedures seem to occur due to the complexity of operating the endoscope dial as well as difficulty in performing synchronized movements with both hands. We developed a prototype robotic-assisted flexible endoscope that can be controlled with a single hand in order to simplify the operation of the endoscope. The aim of this study was to confirm the operability of the robotic-assisted flexible endoscope (RAFE) by performing endoscopic submucosal dissection (ESD). Methods: Study 1: ESD was performed manually or with RAFE by an expert endoscopist in ex vivo porcine stomachs; six operations manually and six were performed with RAFE. The procedure time per unit circumferential length/area was calculated, and the results were statistically analyzed. Study 2: We evaluated how smoothly a non-endoscopist can move a RAFE compared to a manual endoscope by assessing the designated movement of the endoscope. Results: Study 1: En bloc resection was achieved by ESD using the RAFE. The procedure time was gradually shortened with increasing experience, and the procedure time of ESD performed with the RAFE was not significantly different from that of ESD performed with a manual endoscope. Study 2: The time for the designated movement of the endoscope was significantly shorter with a RAFE than that with a manual endoscope as for a non-endoscopist. Conclusions: The RAFE that we developed enabled an expert endoscopist to perform the ESD procedure without any problems and allowed a non-endoscopist to control the endoscope more easily and quickly than a manual endoscope. The RAFE is expected to undergo further development..
111. Seiichi Yano, Kenji Ashida, Hiromi Nagata, Kenji Ohe, Naoko Wada, Yukina Takeichi, Yuki Hanada, Yuta Ibayashi, Lixiang Wang, Shohei Sakamoto, Ryuichi Sakamoto, Hiroshi Uchi, Motoaki Shiratsuchi, Masutaka Furue, Masatoshi Nomura, Yoshihiro Ogawa, Nivolumab-induced thyroid dysfunction lacking antithyroid antibody is frequently evoked in Japanese patients with malignant melanoma, BMC Endocrine Disorders, 10.1186/s12902-018-0267-x, 18, 1, 2018.06, Background: Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs. Methods: After excluding 10 patients, data of 24 patients with malignant melanoma (aged 17-85years; 54% female) were retrospectively analyzed. Results: Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (P<0.01). There were no significant differences between patients with and without thyroid irAEs regarding age, sex, tumor stage, response to nivolumab therapy, baseline thyroid function, antithyroid peroxidase antibody (anti-TPO Ab) and antithyroglobulin antibody (anti-Tg Ab). Conclusions: Thyroid dysfunction was a common irAE of nivolumab in malignant melanoma. Neither anti-TPO Ab nor anti-Tg Ab was associated with the risk for nivolumab-induced thyroid dysfunction. A conventional ATA-independent mechanism might be involved in thyroid irAEs. Further studies are required to clarify the mechanism and identify the predictive factors of thyroid irAEs..
112. Yosuke Minoda, Keishi Komori, Ryoko Naruo, Tsutomu Iwasa, Haruei Ogino, Eikichi Ihara, Yoshihiro Ogawa, Recovery technique using a double scope to rescue failed primary endoscopic ligation, Endoscopy, 10.1055/a-0631-7760, 50, 9, E244-E245, 2018.06.
113. K. Yasunaga, T. Ito, M. Miki, K. Ueda, T. Fujiyama, Y. Tachibana, N. Fujimori, K. Kawabe and Y. Ogawa., Using CRISPR/Cas9 to knock out amylase in acinar cells decreases pancreatitis-induced autophagy., Biomed Res Int., 10.1155/2018/8719397. , 17, 8719397, 2018.05.
114. M. Miki, N. Fujimori, T. Oono, K. Kawabe, A. Ohno, K. Matsumoto, K. Teramatsu, Y. Tachibana and Y. Ogawa., Relapse patterns and predictors of IgG4-related diseases involved with autoimmune pancreatitis: A single-center retrospective study of 115 patients. , J Dig Dis., 10.1111/1751-2980.12708., 30, 3, 152-158, 2018.05.
115. A. Mukaino, H. Minami, H. Isomoto, H. Hamamoto, E. Ihara. Y. Maeda, O. Higuchi, T. Okanishi, Y. Kokudo, K. Deguchi, F. Sasaki, T. Ueki, K. Murata, T. Yoshida, M. Kinjo, Y. Ogawa, A. Ido, H. Matsuo, K. Nakao, S. Nakane., Anti-ganglionic acetylcholine receptor antibodies in Japanese patients with motility disorders. , J Gastroenterol, 10.1007/s00535-018-1477-8., 53, 12, 1227-1240, 2018.05.
116. T. Goto, M. Itoh, T. Suganami, S. Kanai, I. Shirakawa, T. Sakai, M. Asakawa, T. Yoneyama, T. Kai, and Y. Ogawa., Clinicopathogenic and molecular characteristics of synchronous colorectal carcinoma with mismatch repair deficiency., Sci Rep. , 10.1038/s41598-018-26383-8., 8, 1, 8157, 2018.05.
117. , Hironobu Umakoshi, Tatsuki Ogasawara, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamto, Atsushi Ogo, Toshihiko Yanase, Shintaro Okamura, Shozo Miyauchi, Tomoko Suzuki, Mika Tsuiki, Mitsuhide Naruse, Accuracy of adrenal computed tomography in predicting the unilateral subtype in young patients with hypokalaemia and elevation of aldosterone in primary aldosteronism, Clinical Endocrinology, 10.1111/cen.13582, 88, 5, 645-651, 2018.05, Context: The current Endocrine Society Guideline suggests that patients aged <35 years with marked primary aldosteronism (PA) and unilateral adrenal lesions on adrenal computed tomography (CT) scan may not need adrenal vein sampling (AVS) before proceeding to unilateral adrenalectomy. This suggestion is, however, based on the data from only one report in the literature. Objective: We sought to determine the accuracy of CT findings in young PA patients who had unilateral adrenal disease on CT with hypokalaemia and elevation of aldosterone. Design and patients: We retrospectively studied 358 PA patients (n = 30, aged <35 years; n = 39, aged 35-40 years; n = 289, aged ≥40 years) with hypokalaemia and elevation of aldosterone and unilateral disease on CT who had successful AVS. Main outcome measure: Accuracy of CT findings is determined by AVS findings and/or surgical outcomes in patients aged <35 years. Results: Concordance of the diagnosis between CT and AVS was 90% (27/30) in patients aged <35 years, 79% (31/39) in patients aged 35-40 years and 69% (198/289) in those aged ≥40 years (trend for P <.01). Surgical benefit was confirmed in three patients aged <35 years and in three patients aged 35-40 years with the available surgical data who had discordance between CT and AVS findings. Collectively, the diagnostic accuracy of CT findings was 100% (30/30) if aged <35 years and 87% (34/39) if aged 35-40 years. Conclusion: Primary aldosteronism patients aged <35 years with hypokalaemia and elevation of aldosterone and unilateral disease on adrenal CT could be spared AVS..
118. Makiko Abe, Nitin Shivappa, Hidemi Ito, Isao Oze, Tetsuya Abe, Yasuhiro Shimizu, Yasuhisa Hasegawa, Chikako Kiyohara, Masatoshi Nomura, Yoshihiro Ogawa, James R. Hebert, Keitaro Matsuo, Dietary inflammatory index and risk of upper aerodigestive tract cancer in Japanese adults, Oncotarget, 10.18632/oncotarget.25288, 9, 35, 24028-24040, 2018.05, Background: The inflammatory potential of diet that has been shown to be associated with cancer risk. We examined the association between dietary inflammatory potential as measured by the dietary inflammatory index (DII®) and risk of upper aerodigestive tract cancers in a Japanese case-control study. Results: A positive association was observed between increasing DII scores and overall upper aerodigestive tract cancers, and across anatomic subsites. For upper aerodigestive tract cancers, the ORQ4vsQ1 = 1.73 (95% CI: 1.37-2.20); head and neck cancer, the ORQ4vsQ1 was 1.92 (95% CI: 1.42-2.59); and for esophageal cancer, the ORQ4vsQ1 was1.71 (95% CI: 1.54-1.90). Risks for hypopharyngeal and nasopharyngeal cancers were greatly elevated: (ORQ4vsQ1 = 4.05 (95% CI: 1.24-13.25) for hypopharyngeal cancer and ORQ4vsQ1 = 4.99 (95% CI: 1.14-21.79) for nasopharyngeal cancer. Conclusion: A more pro-inflammatory diet was associated with an elevated risk of upper aerodigestive tract cancers after accounting for important confounders. All anatomic subsites, except larynx, showed the consistently elevated risk with increasing DII score. Those subsites with known etiological associations with persistent infection showed the largest elevation in risk. These results warrant further evaluation in future studies. Materials and Methods: This is a case-control study of 1,028 cases and 3,081 age- and sex-matched non-cancer controls recruited at Aichi Cancer Center. DII scores were computed based on estimates of macro- and micro-nutrients from a self-administered food frequency questionnaire. Scores were further categorized into quartiles (based on the distribution in controls). Conditional logistic regression models were fit to estimate odds ratio (OR) and 95% confidence intervals (CIs) adjusted for smoking, ethanol consumption, alcohol flushing, number of teeth, and occupation group..
119. Norimichi Chiyonobu, Shu Shimada, Yoshimitsu Akiyama, Kaoru Mogushi, Michiko Itoh, Keiichi Akahoshi, Satoshi Matsumura, Kosuke Ogawa, Hiroaki Ono, Yusuke Mitsunori, Daisuke Ban, Atsushi Kudo, Shigeki Arii, Takayoshi Suganami, Shoji Yamaoka, Yoshihiro Ogawa, Minoru Tanabe, Shinji Tanaka, Fatty Acid Binding Protein 4 (FABP4) Overexpression in Intratumoral Hepatic Stellate Cells within Hepatocellular Carcinoma with Metabolic Risk Factors, American Journal of Pathology, 10.1016/j.ajpath.2018.01.012, 188, 5, 1213-1224, 2018.05, Metabolic syndrome is a newly identified risk factor for hepatocellular carcinoma (HCC); however, tumor-specific biomarkers still remain unclear. We performed cross-species analysis to compare gene signatures of HCC from human patients and melanocortin 4 receptor-knockout mice, which develop HCC with obesity, insulin resistance, and dyslipidemia. Unsupervised hierarchical clustering and principle component analysis of 746 differentially expressed orthologous genes classified HCC of 152 human patients and melanocortin 4 receptor-knockout mice into two distinct subgroups, one of which included mouse HCC and was causatively associated with metabolic risk factors. Nine genes commonly overexpressed in human and mouse metabolic disease-associated HCC were identified; fatty acid binding protein 4 (FABP4) was remarkably enriched in intratumoral activated hepatic stellate cells (HSCs). Subclones constitutively expressing FABP4 were established from a human HSC cell line in which expression levels of inflammatory chemokines, including IL-1A and IL-6, were up-regulated through NF-κB nuclear translocation, resulting in recruitment of macrophages. An immunohistochemical validation study of 106 additional human HCC samples indicated that FABP4-positive HSCs were distributed in tumors of 38 cases, and the FABP4-high group consisted of patients with nonviral and nonalcoholic HCC (P = 0.027) and with multiple metabolic risk factors (P < 0.001) compared with the FABP4-low group. Thus, FABP4 overexpression in HSCs may contribute to hepatocarcinogenesis in patients with metabolic risk factors by modulation of inflammatory pathways..
120. Yui Shibayama, Norio Wada, Mitsuhide Naruse, Isao Kurihara, Hiroshi Ito, Takashi Yoneda, Yoshiyu Takeda, Hironobu Umakoshi, Mika Tsuiki, Takamasa Ichijo, Hisashi Fukuda, Takuyuki Katabami, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Yuichi Ohno, Masakatsu Sone, Megumi Fujita, Katsutoshi Takahashi, Hirotaka Shibata, Kohei Kamemura, Yuichi Fujii, Koichi Yamamoto, Tomoko Suzuki, The occurrence of apparent bilateral aldosterone suppression in adrenal vein sampling for primary aldosteronism, Journal of the Endocrine Society, 10.1210/js.2017-00481, 2, 5, 398-407, 2018.05, Context: In adrenal venous sampling (AVS) for patients with primary aldosteronism (PA), apparent bilateral aldosterone suppression (ABAS), defined as lower aldosterone/cortisol ratios in the bilateral adrenal veins than that in the inferior vena cava, is occasionally experienced. ABAS is uninterpretable with respect to lateralization of excess aldosterone production.We previously reported that ABAS was not a rare phenomenon and was significantly reduced after adrenocorticotropic hormone (ACTH) administration. Objective: To validate the effects of ACTH administration and adding sampling positions in the left adrenal vein on the prevalence of ABAS in the larger Japan Primary Aldosteronism Study. Patients: The data from 1689 patients with PAwho underwent AVS between January 2006 and October 2016 were studied. All patients in the previous study, the West Japan Adrenal Vein Sampling study, were excluded. Outcome Measurements: The prevalence of ABAS was investigated at two sampling positions in the left adrenal vein, the central vein and the common trunk, without and with ACTH administration. Results: The prevalence of ABAS with ACTH administration was significantly lower than that without ACTH administration [without ACTH vs with ACTH: 79/440 (18.0%) vs 45/591 (7.6%); P < 0.001]. With ACTH administration, the prevalence of ABAS was not different between the sampling position, at the central vein and at the common trunk [33/591 (5.6%) vs 32/591 (5.4%); P = 1.00]. Conclusions: The effectiveness of ACTH administration for the reduction of ABAS in AVS regardless of the sampling position in the left adrenal vein was confirmed in the larger cohort..
121. Tatsuya Fukuda, Ryotaro Bouchi, Takato Takeuchi, Kazutaka Tsujimoto, Isao Minami, Takanobu Yoshimoto, Yoshihiro Ogawa, Sarcopenic obesity assessed using dual energy X-ray absorptiometry (DXA) can predict cardiovascular disease in patients with type 2 diabetes
A retrospective observational study, Cardiovascular Diabetology, 10.1186/s12933-018-0700-5, 17, 1, 2018.04, Background: Sarcopenic obesity, defined as reduced skeletal muscle mass and power with increased adiposity, was reported to be associated with cardiovascular disease risks in previous cross-sectional studies. Whole body dual-energy X-ray absorptiometry (DXA) can simultaneously evaluate both fat and muscle mass, therefore, whole body DXA may be suitable for the diagnosis of sarcopenic obesity. However, little is known regarding whether sarcopenic obesity determined using whole body DXA could predict incident cardiovascular disease (CVD). The aim of this study was to investigate the impact of sarcopenic obesity on incident CVD in patients with type 2 diabetes. Methods: A total of 716 Japanese patients (mean age 65±13 years; 47.0% female) were enrolled. Android fat mass (kg), gynoid fat mass (kg), and skeletal muscle index (SMI) calculated as appendicular non-fat mass (kg) divided by height squared (m2), were measured using whole body DXA. Sarcopenic obesity was defined as the coexistence of low SMI and obesity determined by four patterns of obesity as follows: android to gynoid ratio (A/G ratio), android fat mass or percentage of body fat (%BF) was higher than the sex-specific median, or body mass index (BMI) was equal to or greater than 25 kg/m2. The study endpoint was the first occurrence or recurrence of CVD. Results: Over a median follow up of 2.6 years (IQR 2.1-3.2 years), 53 patients reached the endpoint. Sarcopenic obesity was significantly associated with incident CVD even after adjustment for the confounding variables, when using A/G ratio [hazard ratio (HR) 2.63, 95% CI 1.10-6.28, p=0.030] and android fat mass (HR 2.57, 95% CI 1.01-6.54, p=0.048) to define obesity, but not %BF (HR 1.67, 95% CI 0.69-4.02, p=0.252), and BMI (HR 1.55, 95% CI 0.44-5.49, p=0.496). Conclusions: The present data suggest that the whole body DXA is valuable in the diagnosis of sarcopenic obesity (high A/G ratio or android fat mass with low SMI) to determine the risk of CVD events in patients with type 2 diabetes. Meanwhile, sarcopenic obesity classified with low SMI, and high %BF or BMI was not associated with incident CVD..
122. K. Kawahori, K. Hashimoto, X. Yuan, K. Tsujimoto, N. Hanzawa, M. Hamaguchi, S. Kase, K. Fujita, K. Tagawa, H. Okazawa, Y. Nakajima, N. Shibusawa, M. Yamada, and Y. Ogawa. , Mild maternal hypothyroxinemia during pregnancy induces persistent DNA hypermethylation in the hippocampal brain-derived neurotrophic factor gene in mouse offspring. , Thyroid, 10.1089/thy.2017.0331., 28, 3, 395-406, 2018.03.
123. H. Umakoshi, M. Tsuiki, Y. Takeda, I. Kurihara, H. Itoh, T. Katabami, T. Ichijo, N. Wada, T. Yoshimoto, Y. Ogawa, J. Kawashima, M. Sone, N. Inagaki, K. Takahashi, M. Watanabe, Y. Matsuda, H. Kobayashi, H. Shibata, K. Kamemura, M. Otsuki, Y. Fujii, K. Yamamto, A. Ogo, T. Yanase, T. Suzuki, M. Naruse,, JPAS Study Group. Significance of computed tomography and serum potassium in predicting subtype diagnosis of primary aldosteronism., J. Clin. Endocrinol. Metab., 10.1210/jc.2017-01774., 103, 3, 900-908, 2018.03.
124. Xiaopeng Bai, Eikichi Ihara, Katsuya Hirano, Yoshimasa Tanaka, Kayoko Nakano, Satomi Kita, Takahiro Iwamoto, Haruei Ogino, Mayumi Hirano, Yoshinao Oda, Kazuhiko Nakamura, Yoshihiro Ogawa, Endogenous Hydrogen Sulfide Contributes to Tone Generation in Porcine Lower Esophageal Sphincter Via Na+/Ca2+ Exchanger, CMGH, 10.1016/j.jcmgh.2017.11.004, 5, 3, 209-221, 2018.03, Background and Aims: Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. Methods: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. Results: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. Conclusions: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia..
125. Kenichi Kawahori, Koshi Hashimoto, Xunmei Yuan, Kazutaka Tsujimoto, Nozomi Hanzawa, Miho Hamaguchi, Saori Kase, Kyota Fujita, Kazuhiko Tagawa, Hitoshi Okazawa, Yasuyo Nakajima, Nobuyuki Shibusawa, Masanobu Yamada, Yoshihiro Ogawa, Mild Maternal Hypothyroxinemia during Pregnancy Induces Persistent DNA Hypermethylation in the Hippocampal Brain-Derived Neurotrophic Factor Gene in Mouse Offspring, Thyroid, 10.1089/thy.2017.0331, 28, 3, 395-406, 2018.03, Background: Thyroid hormones are essential for normal development of the central nervous system (CNS). Experimental rodents have shown that even a subtle thyroid hormone insufficiency in circulating maternal thyroid hormones during pregnancy may adversely affect neurodevelopment in offspring, resulting in irreversible cognitive deficits. This may be due to the persistent reduced expression of the hippocampal brain-derived neurotrophic factor gene Bdnf, which plays a crucial role in CNS development. However, the underlying molecular mechanisms remain unclear. Methods: Thiamazole (MMI; 0.025% [w/v]) was administered to dams from two weeks prior to conception until delivery, which succeeded in inducing mild maternal hypothyroxinemia during pregnancy. Serum thyroid hormone and thyrotropin levels of the offspring derived from dams with mild maternal hypothyroxinemia (M offspring) and the control offspring (C offspring) were measured. At 70 days after birth, several behavior tests were performed on the offspring. Gene expression and DNA methylation status were also evaluated in the promoter region of Bdnf exon IV, which is largely responsible for neural activity-dependent Bdnf gene expression, in the hippocampus of the offspring at day 28 and day 70. Results: No significant differences in serum thyroid hormone or thyrotropin levels were found between M and C offspring at day 28 and day 70. M offspring showed an impaired learning capacity in the behavior tests. Hippocampal steady-state Bdnf exon IV expression was significantly weaker in M offspring than it was in C offspring at day 28. At day 70, hippocampal Bdnf exon IV expression at the basal level was comparable between M and C offspring. However, it was significantly weaker in M offspring than in C offspring after the behavior tests. Persistent DNA hypermethylation was also found in the promoter region of Bdnf exon IV in the hippocampus of M offspring compared to that of C offspring, which may cause the attenuation of Bdnf exon IV expression in M offspring. Conclusions: Mild maternal hypothyroxinemia induces persistent DNA hypermethylation in Bdnf exon IV in offspring as epigenetic memory, which may result in long-term cognitive disorders..
126. Miyako Tanaka, Michiko Itoh, Yoshihiro Ogawa, Takayoshi Suganami, Molecular mechanism of obesity-induced ‘metabolic’ tissue remodeling, Journal of Diabetes Investigation, 10.1111/jdi.12769, 9, 2, 256-261, 2018.03, Chronic inflammation is a common molecular basis underlying a variety of chronic diseases. Accumulating evidence has also suggested that chronic inflammation contributes to the pathogenesis of obesity and diabetes, which have been considered as metabolic diseases. For the past several decades, there has been dramatic progress in understanding the underlying mechanism of adipose tissue dysfunction induced by obesity. Tissue remodeling is one of the histological features of chronic inflammation, in which stromal cells dramatically change in number and cell type. Indeed, adipose tissue remodeling is induced by various stromal cells, and results in the impairment of adipose tissue function, such as adipocytokine production and lipid storage, which leads to systemic metabolic disorder. In addition to adipose tissue, the liver is another example of obesity-induced tissue remodeling. In the present review, we discuss how obesity induces interstitial fibrosis in adipose tissue and the liver, particularly focusing on the role of macrophages..
127. Tatsuya Fukuda, Ryotaro Bouchi, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Ratio of visceral-to-subcutaneous fat area predicts cardiovascular events in patients with type 2 diabetes, Journal of Diabetes Investigation, 10.1111/jdi.12713, 9, 2, 396-402, 2018.03, Aims/Introduction: To investigate whether the ratio of visceral fat area (VFA) to subcutaneous fat area (SFA; V/S ratio) could be predictive of cardiovascular disease (CVD) as compared with VFA or SFA in patients with diabetes. Materials and Methods: A total of 682 patients with type 2 diabetes (mean age 64 ± 13 years; 41% women) were enrolled. VFA (cm2) and SFA (cm2) were assessed by a dual bioelectrical impedance analyzer. The patients were divided into four groups according to the quartiles of the V/S ratio. The study end-point was the first occurrence or recurrence of CVD. Results: Over a median follow up of 2.5 years, 21 patients reached the end-point. The number of patients who reached the end-point was increased along with the increasing of the V/S ratio quartiles. The V/S ratio was significantly associated with incident or recurrent CVD (hazard ratio [HR] 1.82, 95% CI: 1.09–3.04, P = 0.021) after adjusting for estimated glomerular filtration rate (HR 0.98, 95% CI: 0.96–1.00), brain-type natriuretic peptide (HR 1.00, 95% CI: 1.00–1.01), use of antiplatelet agents (HR 4.26, 95% CI: 1.63–11.13), coefficient of variation of R-R intervals (HR 0.85, 95% CI: 0.69–1.10) and glycated hemoglobin (HR 1.37, 95% CI: 1.05–1.79). The addition of the V/S ratio to age, estimated glomerular filtration rate, brain-type natriuretic peptide, antiplatelet agents and glycated hemoglobin significantly improved classification performance for CVD using net reclassification improvement (0.60, 95% CI: 0.21–1.00) and the integrated discrimination improvement (0.02, 95% CI: 0.00–0.05). Conclusions: The V/S ratio measured by dual bioelectrical impedance analyzer is an independent predictor of CVD in patients with type 2 diabetes..
128. Yukino Hatazawa, Yusuke Ono, Yuma Hirose, Sayaka Kanai, Nobuharu L. Fujii, Shuichi Machida, Ichizo Nishino, Takahiko Shimizu, Masaki Okano, Yasutomi Kamei, Yoshihiro Ogawa, Reduced Dnmt3a increases Gdf5 expression with suppressed satellite cell differentiation and impaired skeletal muscle regeneration, FASEB Journal, 10.1096/fj.201700573R, 32, 3, 1452-1467, 2018.03, DNAmethylation is an epigenetic mechanism regulating gene expression. In this study, we observed that DNAmethyltransferase 3a (Dnmt3a) expression is decreased after muscle atrophy.Wemade skeletalmuscle-specific Dnmt3a-knockout (Dnmt3a-KO) mice. The regeneration capacity after muscle injury was markedly decreased in Dnmt3a-KOmice.DiminishedmRNAand protein expression of Dnmt3awere observed in skeletalmuscles aswell as in satellite cells, which are important formuscle regeneration, in Dnmt3a-KOmice. Dnmt3a-KO satellite cell showed smaller in size (length/area), suggesting suppressed myotube differentiation. Microarray analysis of satellite cells showed that expression of growth differentiation factor 5 (Gdf5)mRNAwasmarkedly increased in Dnmt3a-KOmice. TheDNA methylation level of the Gdf5 promoter wasmarkedly decreased in Dnmt3a-KO satellite cells. In addition, DNA methylation inhibitor azacytidine treatment increased Gdf5 expression in wild-type satellite cells, suggesting Gdf5 expression is regulated byDNAmethylation. Also, we observed increased inhibitor of differentiation (a target of Gdf5)mRNA expression in Dnmt3a-KO satellite cells. Thus, Dnmt3a appears to regulate satellite cell differentiation viaDNAmethylation. Thismechanism may play a role in the decreased regeneration capacity during atrophy such as in aged sarcopenia..
129. Y. Minami, N. Sonoda, E. Hayashida, H. Makimura, M. Ide, N. Ikeda, M. Ohgidani, T.A. Kato, Y. Seki, Y. Maeda, S. Kanba, R. Takayanagi, Y. Ogawa, and T. Inoguchi., p66Shc signaling mediates diabetes-related cognitive decline., Sci. Rep., 10.1038/s41598-018-21426-6., 8, e3213, 2018.02.
130. X. Yuan, K. Tsujimoto, K. Hashimoto, K. Kawahori, N. Hanzawa, M. Hamaguchi, T. Seki, M. Nawa, T. Ehara, Y. Kitamura, I. Hatada, M. Konishi, N. Itoh, Y. Nakagawa, H. Shimano, T. Takai-Igarashi, Y. Kamei, and Y. Ogawa., Epigenetic modulation of Fgf21 in the perinatal mouse liver ameliorates diet-induced obesity in adulthood., Nat. Commun., 10.1038/s41467-018-03038-w., 9, e636, 2018.02.
131. N. Chiyonobu, S. Shimada, Y. Akiyama, K. Mogushi, M. Itoh, K. Akahoshi, S. Matsumura, K. Ogawa, H. Ono, Y. Mitsunori, D. Ban, A. Kudo, S. Arii, T. Suganami, S. Yamaoka, Y. Ogawa, M. Tanabe, and S. Tanaka., FABP4 overexpressed in intratumoral hepatic stellate cells within hepatocellular carcinoma with metabolic risk factors., Am. J. Pathol., 10.1016/j.ajpath., 188, 1213-1224, 2018.02.
132. Ryotaro Bouchi, Tatsuya Fukuda, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Dipeptidyl peptidase 4 inhibitors attenuates the decline of skeletal muscle mass in patients with type 2 diabetes, Diabetes/Metabolism Research and Reviews, 10.1002/dmrr.2957, 34, 2, 2018.02, Background: Activation of dipeptidyl peptidase 4 has been reported to be associated with impairment of insulin signalling in skeletal muscle, presumably leading to loss of muscle function. This study was aimed to investigate whether the use of dipeptidyl peptidase 4 inhibitors (DPP4i) could attenuate the progressive loss of muscle mass in patients with type 2 diabetes. Methods: A total 105 patients with type 2 diabetes (mean age 62 ± 12 years; 39% female) were studied in this retrospective observational study. To reduce the bias due to confounding variables, propensity-score matching analysis was performed. Change in skeletal muscle index measured by the whole body dual-energy X-ray absorptiometry at 1-year follow-up was evaluated. One-year changes in visceral and subcutaneous fat area and liver attenuation index were also determined by abdominal computed tomography. Results: Overall, 37 of 105 (35.2%) patients were treated with DPP4i. The estimated change in skeletal muscle index in patients with DPP4i was significantly higher than that in patients without (0.05 ± 0.06 vs −0.10 ± 0.04 kg, P =.046). In a propensity-matched population (N = 48), the same finding was observed (0.04 ± 0.03 in DPP4i versus −0.12 ± 0.03 kg in non-DPP4i, P =.033). There were no significant differences in changes of visceral and subcutaneous fat area and liver attenuation index between patients with DPP4i and those without. Conclusions: Our data suggest the potential of DPP4i to prevent the progressive loss of muscle mass with ageing in patients with type 2 diabetes..
133. Tomoaki Inoue, Noriyuki Sonoda, Shinsuke Hiramatsu, Shinichiro Kimura, Yoshihiro Ogawa, Toyoshi Inoguchi, Serum Bilirubin Concentration is Associated with Left Ventricular Remodeling in Patients with Type 2 Diabetes Mellitus
A Cohort Study, Diabetes Therapy, 10.1007/s13300-018-0368-6, 9, 1, 331-338, 2018.02, Introduction: Previous studies have shown that serum bilirubin concentration is inversely associated with the risk of cardiovascular disease. The relationship between serum bilirubin concentration and left ventricular geometry, however, has not been investigated in patients with diabetes mellitus. Methods: In this cohort study, 158 asymptomatic patients with type 2 diabetes mellitus without overt heart disease were enrolled. Left ventricular structure and function were assessed using echocardiography. Serum bilirubin concentration, glycemic control, lipid profile, and other clinical characteristics were evaluated, and their association with left ventricular geometry was determined. Patients with New York Heart Association Functional Classification greater than I, left ventricular ejection fraction less than 50%, history of coronary artery disease, severe valvulopathy, chronic atrial fibrillation, or creatinine clearance less than 30 ml/min, and those receiving insulin treatment, were excluded. Results: Univariate analyses showed that relative wall thickness (RWT) was significantly correlated with diastolic blood pressure (P = 0.003), HbA1c (P = 0.024), total cholesterol (P = 0.043), urinary albumin (P = 0.023), and serum bilirubin concentration (P = 0.009). There was no association between left ventricular mass index and serum bilirubin concentration. Multivariate linear regression analysis showed that log RWT was positively correlated with diastolic blood pressure (P = 0.010) and that log RWT was inversely correlated with log bilirubin (P = 0.003). In addition, the patients with bilirubin less than 0.8 mg/dl had a higher prevalence of concentric left ventricular remodeling compared with those with bilirubin 0.8 mg/dl or more. Conclusion: Our study shows that the serum bilirubin concentration may be associated with the progression of concentric left ventricular remodeling in patients with type 2 diabetes mellitus..
134. Mitsuru Esaki, Sho Suzuki, Yasuyo Hayashi, Azusa Yokoyama, Shuichi Abe, Taizo Hosokawa, Haruei Ogino, Hirotada Akiho, Eikichi Ihara, Yoshihiro Ogawa, Splash M-knife versus Flush Knife BT in the technical outcomes of endoscopic submucosal dissection for early gastric cancer
A propensity score matching analysis, BMC Gastroenterology, 10.1186/s12876-018-0763-5, 18, 1, 2018.02, Background: Endoscopic submucosal dissection (ESD) is a standard treatment for early gastric cancer. A new multi-functional ESD device was developed to achieve complete ESD with a single device. A metal plate attached to its distal sheath achieves better hemostasis during the procedure than the other needle-knife device, Flush Knife BT®, that has been conventionally used. The aim of this study was to compare the technical outcomes of ESD for early gastric cancer using the Splash M-Knife® with those using the Flush Knife BT. Methods: We conducted a retrospective review of the case records of 149 patients with early gastric cancer treated with ESD using the needle-type ESD knives between January 2012 and August 2016 at Kitakyushu Municipal Medical Center. Lesions treated with ESD using the Splash M-knife (ESD-M) and the Flush Knife BT (ESD-F) were compared. Multivariate analyses and propensity score matching were used to compensate for the differences in age, gender, underlying disease, antithrombotic drug use, lesion location, lesion position, macroscopic type, tumor size, presence of ulceration, operator level and types of electrosurgical unit used. The primary endpoint was the requirement to use hemostatic forceps in the two groups. The secondary endpoints of procedure time, en bloc and complete resection rates, and adverse events rates were evaluated for the two groups. Results: There were 73 patients in the ESD-M group, and 76 patients in the ESD-F group. Propensity score matching analysis created 45 matched pairs. Adjusted comparisons between the two groups showed a significantly lower usage rate of hemostatic forceps in the ESD-M group than in the ESD-F group (6.7% vs 84.4%, p<0.001). Treatment outcomes showed an en bloc resection rate of 100% in both groups; complete resection rate of 95.6% vs 100%, p=0.49; median procedure time of 74.0 min vs 71.0 min, p=0.90; post-procedure bleeding of 2.2% vs 2.2%, p=1, in the ESD-M and ESD-F groups, respectively. There were no perforations in either group. Conclusions: ESD-M appeared to reduce the usage of hemostatic forceps during ESD for early gastric cancer without increasing the adverse effects. Thus, it may contribute to a reduction in the total ESD cost..
135. K. Shiba, K. Tsuchiya, C. Komiya, Y. Miyachi, K. Mori, N. Shimazu, S. Yamaguchi, N. Ogasawara, M. Katoh, M. Itoh, T. Suganami, and Y. Ogawa., Canagliflozin, an SGLT2 inhibitor, attenuates the development of hepatocellular carcinoma in a mouse model of human NASH. , Sci. Rep. , 8, e2362, 2018.01.
136. K. Hashimoto, E. Nishihara, M. Matsumoto, S. Matsumoto, Y. Nakajima, K. Tsujimoto, H. Yamakage, N. Asahara-Satoh, J. Noh, K. Ito, A. Miyauchi, M. Mori, M. Yamada, and Y. Ogawa., Sialic acid-binding immunoglobulin-like lectin1 as a novel predictive biomarker for relapse in Graves’ disease: a multicenter study., Thyroid, 10.1089/thy, 28, 1, 50-59, 2018.01.
137. L. Lee , T. Ito, H. Igarashi, M. Miki, N. Fujimori, K. Kawabe, RT Jensen, Y. Ogawa., Dose and schedule modification are required for long-term continuation of sunitinib in Japanese patients with advanced pancreatic neuroendocrine tumors., Cancer Chemother Pharmacol., 10.1007/s00280-017-3482-7., 81, 1, 163-169, 2018.01.
138. Kazumasa Muta, Eikichi Ihara, Yusuke Kitagawa, Shohei Hamada, Keita Fukaura, Tsutomu Iwasa, Akira Aso, Haruei Ogino, Kazuhiko Nakamura, Yoshihiro Ogawa, A case of non-cardiac chest pain caused by esophageal motility disorder observed on esophageal high-resolution manometry, Journal of Japanese Society of Gastroenterology, 10.11405/nisshoshi.115.401, 115, 4, 401-408, 2018.01, Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University A 20-year-old man was referred to our hospital with dysphagia and chest pain. Heart disease was denied. No abnormality was observed in upper esophagogastroduodenoscopy and fluoroscopy; furthermore, no gastric acid-related symptoms were observed on combined esophageal multichannel intraluminal impedance and pH monitoring. Esophageal high-resolution manometry (HRM) performed by liquid swallow revealed normal peristalsis; however, HRM performed while the patient was taking solid meals showed abnormal contraction, and the patient simultaneously complained of chest pain. Therefore, we diagnosed this case as non-cardiac chest pain due to esophageal motility disorder..
139. Lingaku Lee, Tetsuhide Ito, Hisato Igarashi, Masami Miki, Nao Fujimori, Ken Kawabe, Robert T. Jensen, Yoshihiro Ogawa, Dose and schedule modification are required for long-term continuation of sunitinib in Japanese patients with advanced pancreatic neuroendocrine tumors, Cancer chemotherapy and pharmacology, 10.1007/s00280-017-3482-7, 81, 1, 163-169, 2018.01, Purpose: This study aimed to clarify the efficacy and safety of sunitinib in Japanese patients with pancreatic neuroendocrine tumors (PNET), especially by focusing on dose and schedule modification. Methods: Sixteen patients with advanced PNET treated with sunitinib were reviewed retrospectively. Efficacy was evaluated by progression-free survival (PFS) and objective tumor response. Toxicity profile was assessed regularly. Correlation between relative dose intensity (RDI) and treatment period was also evaluated. Results: The median PFS was 25.8 months, and the probability of PFS at 1-year was 92%. The objective response rate and clinical benefit rate were 44% and 69%, respectively. The common adverse drug reactions (ADRs) were hand-foot syndrome (88%), neutropenia (75%), leucopenia (75%), and diarrhea (63%). Due to the development of severe ADRs, 81% required dose reduction and 31% discontinued sunitinib treatment, respectively. Prolonged treatment period was significantly correlated with decreased RDI (Spearman r = − 0.57, P = 0.022). The median RDI among 9 patients whom continued sunitinib more than 1 year was 49%. Conclusions: Sunitinib showed significant clinical benefit in Japanese patients with advanced PNET in the real-world clinical setting. Successful management of ADRs with appropriate dose reduction and interruption can enable long-term continuation of sunitinib..
140. Yoko M. Nakao, Yoshihiro Miyamoto, Kenji Ueshima, Kazuhiro Nakao, Michikazu Nakai, Kunihiro Nishimura, Shinji Yasuno, Kiminori Hosoda, Yoshihiro Ogawa, Hiroshi Itoh, Hisao Ogawa, Kenji Kangawa, Kazuwa Nakao, Effectiveness of nationwide screening and lifestyle intervention for abdominal obesity and cardiometabolic risks in Japan
The metabolic syndrome and comprehensive lifestyle intervention study on nationwide database in Japan (MetS ACTION-J study), PloS one, 10.1371/journal.pone.0190862, 13, 1, 2018.01, Background Lifestyle interventions can substantially improve obesity and cardiometabolic risks. However, evidence of long-term benefits of national intervention is sparse. We aimed to evaluate the long-term effectiveness of a nationwide program for abdominal obesity. Methods A retrospective cohort study was performed using a longitudinal nationwide individual data in subjects aged 40–74 years who underwent checkups in fiscal year (FY) 2008. Lifestyle interventions were provided via interview in subjects with abdominal obesity and at least one cardiometabolic risk factor. Subjects who attended the lifestyle intervention (participants) were compared to those who did not attend (non-participants). Outcomes were waist circumferences (WC) and body mass index (BMI) reduction, reversal of metabolic syndrome (MetS), and changes in cardiometabolic risks. We used a three-step process with robust analytic approaches to account for selection bias that included traditional multivariate analysis, propensity-score matching and instrumental variable (IV) analyses. Results Of 19,969,722 subjects, 4,370,042 were eligible for analyses; 111,779 participants and 907,909 non-participants. A higher percentage of participants had 5% reductions in obesity profiles at year 3, compared to non-participants (WC, 21.4% vs 16.1%; BMI, 17.6% vs 13.6%; p<0.001 each). Participants also had higher reversal for MetS (adjusted odds ratio 1.31; 95% confidence interval: 1.29–1.33; p<0.001). Greater reductions in cardiometabolic risks were observed in participants. Those results were confirmed in analyses using a propensity score-matched cohort (n = 75,777, each) and IV analyses. Limitations of this work include the use of non-randomized national data in Japan to assess the effectiveness of the nationwide preventive program. Conclusions In the nationwide lifestyle intervention for abdominal obesity, the at-risk population achieved significant reductions in WC, BMI, and cardiometabolic risks in 3 years. This study provides evidence that the nationwide program effectively achieved long-term improvement in abdominal obesity and cardiometabolic risks..
141. Maki Kawasaki, Naoko Arata, Celine Miyazaki, Rintaro Mori, Toru Kikuchi, Yoshihiro Ogawa, Erika Ota, Obesity and abnormal glucose tolerance in offspring of diabetic mothers
A systematic review and meta-analysis, PloS one, 10.1371/journal.pone.0190676, 13, 1, 2018.01, Background Rising prevalence of childhood obesity and type 2 diabetes mellitus (T2DM) is an emerging public health issue. Objectives To investigate the association of maternal hyperglycemia exposure during pregnancy with obesity and abnormal glucose tolerance in offspring, and the age at occurrence. Methods We searched MEDLINE and EMBASE for observational studies on obesity and diabetes in offspring of diabetic mothers (gestational diabetes mellitus (GDM), type 1 diabetes mellitus (T1DM) and T2DM), and those on non-diabetic mothers. We performed fixed effect meta-analysis for all studies except when heterogeneity was detected. The quality of studies was evaluated using the Risk of Bias Assessment Tool for Nonrandomized Studies (RoBANS) Results Twenty observational studies were included involving a total of 26,509 children. Offspring of GDM mother had higher BMI z-score in childhood (pooled MD: 0.14, 95%CI: 0.04–0.24, seven studies, 21,691children, low quality of evidence). Offspring of T1DM mothers had higher BMI z-score from prepubertal to adolescent (pooled MD: 0.35, 95% CI: 0.13–0.58, three studies, 844 children, low quality of evidence) compared with control. After adjustment for maternal pre-pregnancy BMI, this association remained in offspring of T1DM, but disappeared in those of GDM mothers. Offspring of GDM mother had higher 2-hour plasma glucose from prepubertal to early adulthood (pooled MD: 0.43 mmol/L, 95% CI: 0.18–0.69, five studies, 890 children), while those of T1DM mothers had higher rate of T2DM in 2–5 years old to early adulthood (pooled odds ratio [OR], 6.10: 95% CI: 1.23–30.37, two studies, 448 children, very low quality of evidence) compared with control. As there was only one study with offspring of T2DM mothers, evidence is sparse. Limitations Only observational studies were included, with a few adequately adjusted for covariables. Conclusions Exposure to maternal hyperglycemia was associated with offspring obesity and abnormal glucose tolerance especially in offspring of T1DM mothers, but the evidence relies on observational studies with low quality of evidence only..
142. Koshi Hashimoto, Eijun Nishihara, Masako Matsumoto, Shunichi Matsumoto, Yasuyo Nakajima, Kazutaka Tsujimoto, Hajime Yamakage, Noriko Satoh-Asahara, Jaeduk Yoshimura Noh, Koichi Ito, Akira Miyauchi, Masatomo Mori, Masanobu Yamada, Yoshihiro Ogawa, Sialic Acid-Binding Immunoglobulin-Like Lectin1 as a Novel Predictive Biomarker for Relapse in Graves' Disease
A Multicenter Study, Thyroid : official journal of the American Thyroid Association, 10.1089/thy.2017.0244, 28, 1, 50-59, 2018.01, BACKGROUND: There are currently no reliable biomarkers to predict relapse in Graves' disease (GD). In the present study, we investigated novel diagnostic biomarkers to predict the long-term remission of or relapse in GD. METHODS: A DNA microarray analysis was performed to examine gene expression in the peripheral leukocytes of a frequently relapsing patient with GD and a patient in long-term remission after the discontinuation of antithyroid drugs (ATDs). Based on the DNA microarray analysis, we focused on Sialic acid-binding immunoglobulin-like lectin1 (SIGLEC1) as a candidate novel biomarker to predict GD relapse. Three hundred and fifty-eight patients with GD in the thyroid clinics of four different hospitals in Japan were included in a cross-sectional study to establish whether SIGLEC1 mRNA levels distinguish GD relapse experience from long-term remission. An additional 55 patients with GD were enrolled in a prospective study to clarify whether SIGLEC1 mRNA levels at ATD discontinuation predict GD relapse. RESULTS: SIGLEC1 mRNA levels were significantly higher in patients with GD relapse experience than in those in long-term remission. Based on the receiver operating characteristic analysis, we found that high SIGLEC1 mRNA levels (≥258.9 copies) significantly distinguished GD relapse experience from long-term remission (p < 0.0001; sensitivity 66.7%, specificity 70.1%). In the prospective study, when the optimal cutoff value from the receiver operating characteristic curve analysis was applied to SIGLEC1 mRNA positivity at ATD discontinuation, SIGLEC1-positive patients (≥258.9 copies) showed a significantly higher cumulative risk of relapse than SIGLEC1-negative patients (<258.9 copies) (p = 0.022, the log-rank test). CONCLUSIONS: SIGLEC1 mRNA levels have potential as a novel predictive biomarker for GD relapse..
143. Masami Miki, Ken Kawabe, Hisato Igarashi, Tatsuro Abe, Yoshihiro Ohishi, Risa Hashimoto, Takashi Karashima, Ichiro Yamasaki, Keiji Inoue, Tetsuhide Ito, Yoshihiro Ogawa, An advanced well-differentiated pancreatic neuroendocrine carcinoma (NET-G3) associated with von hippel-lindau disease, Internal Medicine, 10.2169/internalmedicine.0416-17, 57, 14, 2007-2011, 2018, A 45-year old woman who underwent several surgeries for tumors associated with von Hippel-Lindau disease (VHL) was referred to our hospital due to a pancreatic tumor and liver tumors. She was diagnosed with pancreatic neuroendocrine tumor (NET) with a Ki67 index of 40% based on the examination of a biopsy specimen of the liver tumors. She was treated with everolimus for 6 months and sunitinib for 6 weeks as first-and second-line therapies. She survived for 13 months. At autopsy the diagnosis of pancreatic neuroendocrine tumor (NET)-G3 was confirmed. We herein report an aggressive clinical course of VHL-related NET G3. The further accumulation of cases is required to reach a consensus on treatment for this disease..
144. Kayoko Nakano, Hidetaka Yamamoto, Minako Fujiwara, Yutaka Koga, Shinichi Tsuruta, Eikichi Ihara, Eiji Oki, Masafumi Nakamura, Yoshihiro Ogawa, Yoshinao Oda, Clinicopathologic and molecular characteristics of synchronous colorectal carcinoma with mismatch repair deficiency, American Journal of Surgical Pathology, 10.1097/PAS.0000000000000947, 42, 2, 172-182, 2018, Synchronous colorectal carcinoma (CRC) is a unique disease associated with a high prevalence (∼35%) of microsatellite instability and occasionally with Lynch syndrome. The clinicopathologic and molecular features of synchronous CRC are poorly understood, particularly in Japanese patients. We examined 118 Japanese patients (236 tumors) with synchronous CRC and 117 Japanese patients (117 tumors) with solitary CRC with immunohistochemical staining for TP53 and mismatch repair (MMR) protein (MLH1, MSH2, PMS2, and MSH6) and mutation analyses of KRAS and BRAF genes. The results revealed no significant differences in clinicopathologic, histologic, and molecular findings between the synchronous and solitary CRC groups. Among the 118 synchronous CRC patients, 15 (12.7%) showed loss of MMR protein(s) expression in at least 1 tumor, whereas 103 (87.3%) showed intact expression of all 4 MMR proteins in both tumors. Of note, all patients with MMR deficiency had excellent prognoses. The 15 patients were further subdivided into 2 groups: the Concordant group, with concordant MMR loss (n=9, 7.6%) and the Discordant group, with discordant MMR loss (n=6, 5.1%). The Concordant patients showed concurrent MLH1/PMS2 loss (n=3), concurrent MSH2/ MSH6 loss (n=4) and isolated MSH6 loss (n=2) in both tumors, whereas the Discordant patients showed concurrent MLH1/PMS2 loss (n=2), isolated PMS2 loss (n=2) and isolated MSH6 loss (n=2) in a single tumor. On the basis of the MMR expression pattern and BRAF mutation, the Concordant and Discordant groups were suspected to include Lynch syndrome, Lynch-like syndrome and sporadic MLH1 promoter hypermethylated CRC. In addition, KRAS mutation was present in only 1 tumor in a single patient in each group. In conclusion, the frequency of MMR protein deficiency in synchronous CRC in the Japanese population may be lower compared with the reported data from Western populations. MMR protein loss and KRAS and BRAF mutations in synchronous CRCs were heterogenous even in an individual patient..
145. , Hiroki Kobayashi, Masanori Abe, Masayoshi Soma, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Hironobu Umakoshi, Mika Tsuiki, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hirotaka Shibata, Kohei Kamemura, Toshihiko Yanase, Michio Otsuki, Yuichi Fujii, Koichi Yamamoto, Atsushi Ogo, Kazutaka Nanba, Akiyo Tanabe, Tomoko Suzuki, Mitsuhide Naruse, Development and validation of subtype prediction scores for the workup of primary aldosteronism, Journal of hypertension, 10.1097/HJH.0000000000001855, 36, 11, 2269-2276, 2018, Objectives: A subtype prediction score for primary aldosteronism has not yet been developed and validated using a large dataset. This study aimed to develop and validate a new subtype prediction score and to compare it with existing scores using a large multicenter database. Methods: In total, 1936 patients with primary aldosteronism were randomly assigned to the development and validation datasets, constituting 1290 and 646 patients, respectively. Three prediction scores were generated with or without confirmatory tests, using logistic regression analysis. In the validation dataset, new and existing prediction scores were compared using receiver operating characteristic curve, net reclassification improvement, and integrated discrimination improvement analyses. Results: The new prediction score is simply calculated using serum potassium levels [>3.9 mmol/l (four points); 3.5-3.9 mmol/l (three points)], the absence of adrenal nodules during computed tomography (three points), a baseline plasma aldosterone concentration of <210.0 pg/ ml (two points), a baseline aldosterone/renin ratio of less than 620 (two points), and female sex (one point). Using the validation dataset, we found that a new subtype prediction score of at least 8 had a positive predictive value of 93.5% for bilateral hyperaldosteronism. The new prediction score for bilateral hyperaldosteronism was better than the existing prediction scores in the receiver operating characteristic curve and net reclassification improvement analyses. Conclusion: The new prediction score has clear advantages over the existing prediction scores in terms of diagnostic accuracy, feasibility, and the potential for generalization in a large population. These data will help healthcare professionals to better select patients who require adrenal venous sampling..
146. Yujiro Nakano, Takanobu Yoshimoto, Tatsuya Fukuda, Masanori Murakami, Ryotaro Bouchi, Isao Minami, Koshi Hashimoto, Yasuhisa Fujii, Kazunori Kihara, Yoshihiro Ogawa, Effect of eplerenone on the glomerular filtration rate (GFR) in primary aldosteronism
Sequential changes in the GFR during preoperative eplerenone treatment to subsequent adrenalectomy, Internal Medicine, 10.2169/internalmedicine.0438-17, 57, 17, 2459-2466, 2018, Objective Eplerenone (EPL) is a mineralo-corticoid receptor antagonist that is highly selective and has few side effects. This study was conducted to examine whether or not EPL treatment was able to reverse glomerular hyperfiltration, as an indicator of aldosterone renal action, in primary aldosteronism (PA) patients. Methods Changes in the estimated glomerular filtration rate (ΔGFR) were examined in 102 PA patients with EPL treatment. Furthermore, the sequential ΔGFR in 40 patients initially treated with EPL followed by adrenalectomy was examined in order to evaluate the extent of the remaining glomerular hyperfiltration in the patients treated with EPL. Results EPL decreased the GFR at 1 month after treatment. The GFR at baseline was the sole significant predictor for the ΔGFR. Patients initially treated by EPL followed by adrenalectomy showed three different ΔGFR patterns during the treatment, despite having comparable doses of EPL and comparable control of blood pressure and serum potassium levels. The urinary aldosterone excretion was significantly different among these three groups, and the group with no decrease in the GFR after EPL treatment showed greater urinary aldosterone excretion. Glomerular hyperfiltration was completely restored only in 17.5% of our unilateral PA patients after EPL treatment. Conclusion The present study revealed that blockade of aldosterone action by EPL could, at least partially, reverse glomerular hyperfiltration in PA. Whether or not these differential effects on the GFR affect the long-term outcome needs to be investigated, especially in patients with unilateral PA who do not want adrenalectomy and choose the EPL treatment option..
147. Koshi Hashimoto, Yoshihiro Ogawa, Epigenetic switching and neonatal nutritional environment, Advances in Experimental Medicine and Biology, 10.1007/978-981-10-5526-3_3, 19-25, 2018, The hepatic metabolic function changes sequentially during early life in mammals to adapt to the drastic changes in the nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. De novo lipogenesis is induced upon the onset of oral intake, when the major nutritional source switches to carbohydrate. However, how a particular metabolic pathway is activated during the liver maturation is poorly understood. We found that the expression of glycerol-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme of de novo hepatic lipogenesis, is epigenetically regulated in the mouse liver by DNA methylation. In the neonatal liver, DNA methylation of the GPAT1 gene (Gpam) promoter, which is likely to be induced by DNA methyltransferase (Dnmt) 3b, inhibited the recruitment of sterol regulatory element-binding protein-1c (SREBP-1c), whereas in the adult, decreased DNA methylation resulted in active chromatin conformation, allowing the recruitment of SREBP-1c. Maternal nutritional environment affects the DNA methylation status in the Gpam promoter, GPAT1 expression, and triglyceride content in the liver of the offspring. We also found DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation is specifically induced in the lactation period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveals that the DNA demethylation is PPARα-dependent. These findings indicate the gene- and lifestage-specific DNA demethylation of a particular metabolic pathway in the neonatal liver to adapt the marked changes in nutritional environment in early life..
148. Yasuhiro Tsukamoto, Junichi Kiyasu, Mariko Tsuda, Motohiko Ikeda, Motoaki Shiratsuchi, Yoshihiro Ogawa, Yuji Yufu, Fatal disseminated tuberculosis during treatment with ruxolitinib plus prednisolone in a patient with primary myelofibrosis
A case report and review of the literature, Internal Medicine, 10.2169/internalmedicine.9165-17, 57, 9, 1297-1300, 2018, A 73-year-old man with primary myelofibrosis (PMF) was being treated with hydroxyurea, which was changed to ruxolitinib treatment because of worsening constitutional symptoms. Although ruxolitinib rapidly induced relief, he developed a high-grade fever. A comprehensive fever work-up found no apparent cause of the fever, except for PMF. Therefore, we increased the dose of ruxolitinib and added prednisolone, which was gradually withdrawn with resolution of the fever. However, the patient subsequently developed disseminated tuberculosis and died eight months after initiation of ruxolitinib. Our case highlights the importance of assessing and monitoring the immune status of patients receiving ruxolitinib..
149. Maki Kawasaki, Naoko Arata, Yoshihiro Ogawa, Obesity and abnormal glucose tolerance in the offspring of mothers with diabetes, Current Opinion in Obstetrics and Gynecology, 10.1097/GCO.0000000000000479, 30, 6, 361-368, 2018, Purpose of review Type 2 diabetes and obesity during childhood, puberty, and adulthood have become more common. This trend presents a global problem in terms of public health and health economics. Associations between intrauterine exposure to hyperglycemia, obesity, and abnormal glucose tolerance (AGT) in offspring have been reported in populations at high risk of diabetes such as Pima Indians, but these associations have not been established in other groups. In this review, we summarize the evidence on obesity and AGT in the offspring of mothers with diabetes. Recent findings Although there are many reports indicating that the incidence of obesity or overweight is higher in the offspring of mothers with gestational diabetes, there is no consensus on whether maternal prepregnancy obesity has a larger impact than intrauterine exposure to hyperglycemia. While the risk of AGT or type 2 diabetes in the offspring of mothers with gestational diabetes is thought to increase after puberty, the incidence of AGT is elevated by the age of 7 years in the offspring of mothers with untreated gestational diabetes. Maternal gestational diabetes is a risk factor for AGT or type 2 diabetes independent of maternal prepregnancy BMI. When the offspring of women who had gestational diabetes and received therapeutic intervention in two randomized controlled studies were followed, the prevalence of obesity and impaired fasting glucose was lower in some 7-year-old girls, but the effect of maternal intervention was limited. The risk of obesity or overweight is higher in the offspring of mothers with type 1 diabetes, even after adjustment for maternal prepregnancy BMI. The risk of type 2 diabetes in such offspring is also higher. Although the offspring of mothers with type 2 diabetes are likely to be at high risk for type 2 diabetes, there are only limited reports supporting this hypothesis. Summary Intrauterine exposure to hyperglycemia is associated with obesity and AGT among offspring. The present review suggests that these associations might depend on the type of maternal diabetes, that is, the timing and degree of exposure to hyperglycemia. There are only a small number of studies on the effect of therapeutic interventions for maternal diabetes on metabolism in the offspring..
150. , Youichi Ohno, Masakatsu Sone, Nobuya Inagaki, Toshinari Yamasaki, Osamu Ogawa, Yoshiyu Takeda, Isao Kurihara, Hironobu Umakoshi, Takamasa Ichijo, Takuyuki Katabami, Norio Wada, Yoshihiro Ogawa, Takanobu Yoshimoto, Junji Kawashima, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Shozo Miyauchi, Kohei Kamemura, Tomikazu Fukuoka, Koichi Yamamoto, Michio Otsuki, Tomoko Suzuki, Mitsuhide Naruse, Obesity as a key factor underlying idiopathic hyperaldosteronism, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2018-00866, 103, 12, 4456-4464, 2018, Context: Recently, the relationship between primary aldosteronism (PA) and various metabolic disorders, including obesity, diabetes mellitus, and dyslipidemia, has been discussed. However, in PA, aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA) have different etiologies. Objective: Our objectives were to clarify differences in obesity and metabolic disorders between APA and IHA and to gain insight in the pathogenesis of IHA. Design, Setting, and Participants: This is a retrospective cross-sectional study. We assessed the PA database established by the multicenter Japan Primary Aldosteronism Study. For comparative analysis, data were also collected from 274 patients with essential hypertension (EHT). Main Outcome Measures: We compared prevalences of obesity and metabolic disorders between patients with APA and patients with IHA. Comparisons with sex-, age-, and blood pressure-matched patients with EHT were also performed. Correlations between metabolic parameters and plasma aldosterone concentrations (PACs) in each subtype were analyzed. Results: Analysis of 516 patients with APA and 1015 patients with IHA revealed PACs were significantly higher in patients with APA than patients with IHA. By contrast, after we adjusted for clinical backgrounds, the prevalence of obesity was significantly higher in patients with IHA than in patients with APA or EHT. Although the prevalences of diabetes mellitus and dyslipidemia did not significantly differ between patients with IHA and patients with APA, triglyceride and HbA1c were significantly higher in patients with IHA than in patients with APA. There was no significant correlation between metabolic parameters and PACs in either subtype. Conclusions: Patients with IHA tend to be obese despite lower PACs than in patients with APA. The present results suggest that obesity-related factors contribute to the pathogenesis of IHA..
151. , Youichi Ohno, Masakatsu Sone, Nobuya Inagaki, Toshinari Yamasaki, Osamu Ogawa, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Hironobu Umakoshi, Mika Tsuiki, Takamasa Ichijo, Takuyuki Katabami, Yasushi Tanaka, Norio Wada, Yui Shibayama, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Katsutoshi Takahashi, Megumi Fujita, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamoto, Atsushi Ogo, Shintaro Okamura, Shozo Miyauchi, Tomikazu Fukuoka, Shoichiro Izawa, Takashi Yoneda, Shigeatsu Hashimoto, Toshihiko Yanase, Tomoko Suzuki, Takashi Kawamura, Yasuharu Tabara, Fumihiko Matsuda, Mitsuhide Naruse, Prevalence of Cardiovascular Disease and Its Risk Factors in Primary Aldosteronism A Multicenter Study in Japan, Hypertension, 10.1161/HYPERTENSIONAHA.117.10263, 71, 3, 530-537, 2018, There have been several clinical studies examining the factors associated with cardiovascular disease (CVD) in patients with primary aldosteronism (PA); however, their results have left it unclear whether CVD is affected by the plasma aldosterone concentration or hypokalemia. We assessed the PA database established by the multicenter JPAS (Japan Primary Aldosteronism Study) and compared the prevalence of CVD among patients with PA with that among age-, sex-, and blood pressure-matched essential hypertension patients and participants with hypertension in a general population cohort. We also performed binary logistic regression analysis to determine which parameters significantly increased the odds ratio for CVD. Of the 2582 patients with PA studied, the prevalence of CVD, including stroke (cerebral infarction, cerebral hemorrhage, or subarachnoid hemorrhage), ischemic heart disease (myocardial infarction or angina pectoris), and heart failure, was 9.4% (stroke, 7.4%; ischemic heart disease, 2.1%; and heart failure, 0.6%). The prevalence of CVD, especially stroke, was higher among the patients with PA than those with essential hypertension/ hypertension. Hypokalemia (K+ ≤3.5 mEq/L) and the unilateral subtype significantly increased adjusted odds ratios for CVD. Although aldosterone levels were not linearly related to the adjusted odds ratio for CVD, patients with plasma aldosterone concentrations ≥125 pg/mL had significantly higher adjusted odds ratios for CVD than those with plasma aldosterone concentrations <125 pg/mL. Thus, patients with PA seem to be at a higher risk of developing CVD than patients with essential hypertension. Moreover, patients with PA presenting with hypokalemia, the unilateral subtype, or plasma aldosterone concentration ≥125 pg/mL are at a greater risk of CVD and have a greater need for PA-specific treatments than others..
152. Seizaburo Masuda, Masanori Murakami, Ryotaro Bouchi, Isao Minami, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Repeated hypoglycemic episodes with postprandial hyperinsulinemia after the recovery from acute weight loss revealed by continuous glucose monitoring and the oral glucose tolerance test, Internal Medicine, 10.2169/internalmedicine.9452-17, 57, 5, 697-700, 2018, We herein report a case of a 20-year-old woman who experienced hypoglycemia in parallel with acute weight loss confirmed by continuous glucose motoring (CGM). When she recovered from the acute weight loss, CGM revealed nocturnal and postprandial hypoglycemia. Six months were required to resolve the hypoglycemia and hyperinsulinemia after the recovery of her weight. Our case suggests that the adaption of insulin secretion to the rapid loss of weight and to the recovery of weight may require a long period of time, leading to the excessive secretion of insulin relative to the glucose level and repeated hypoglycemic episodes with postprandial hyperinsulinemia..
153. Hironobu Umakoshi, Mika Tsuiki, Yoshiyu Takeda, Isao Kurihara, Hiroshi Itoh, Takuyuki Katabami, Takamasa Ichijo, Norio Wada, Takanobu Yoshimoto, Yoshihiro Ogawa, Junji Kawashima, Masakatsu Sone, Nobuya Inagaki, Katsutoshi Takahashi, Minemori Watanabe, Yuichi Matsuda, Hiroki Kobayashi, Hirotaka Shibata, Kohei Kamemura, Michio Otsuki, Yuichi Fujii, Koichi Yamamto, Atsushi Ogo, Toshihiko Yanase, Tomoko Suzuki, Mitsuhide Naruse, Study Group JPAS Study Group, Significance of computed tomography and serum potassium in predicting subtype diagnosis of primary aldosteronism, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2017-01774, 103, 3, 900-908, 2018, Context: The number of centers with established adrenal venous sampling (AVS) programs for the subtype diagnosis of primary aldosteronism (PA) is limited. Objective: Aim was to develop an algorithm for AVS based on subtype prediction by computed tomography (CT) and serum potassium. Design: A multi-institutional retrospective cohort study in Japan. Patients: A total of 1591 patients with PA were classified into four groups according to CT findings and potassium status. Subtype diagnosis of PA was determined by AVS. Main Outcome Measure: Prediction value of the combination of CT findings and potassium status for subtype diagnosis. Results: The percentages of unilateral hyperaldosteronism on AVS were higher in patients with unilateral disease on CT than those with bilateral normal results on CT (50.8% vs 14.6%, P, 0.01), and these percentages were higher in those with hypokalemia than those with normokalemia (58.4% vs 11.5%, P, 0.01). The prevalence and odds ratio for unilateral hyperaldosteronism on AVS were as follows: bilateral normal on CT with normokalemia, 6.2% (reference); unilateral disease on CT with normokalemia, 23.8% and 4.8 [95% confidence interval (CI), 3.1 to 7.2]; bilateral normal on CT with hypokalemia, 38.1% and 9.4 (95% CI, 6.2 to 14.1), and unilateral disease on CT with hypokalemia, 70.6% and 36.4 (95% CI, 24.7 to 53.5). Conclusions: Patients with PA with bilateral normal results on CT and normokalemia likely have a low prior probability of a lateralized form of AVS and could be treated medically, whereas those with unilateral disease on CT and hypokalemia have a high probability of a lateralized form of AVS..
154. Kohei Yasunaga, Tetsuhide Ito, Masami Miki, Keijiro Ueda, Takashi Fujiyama, Yuichi Tachibana, Nao Fujimori, Ken Kawabe, Yoshihiro Ogawa, Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy, BioMed Research International, 10.1155/2018/8719397, 2018, 2018, Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to "impaired autophagy flux". Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles..
155. Kohjiro Ueki, Takayoshi Sasako, Yukiko Okazaki, Masayuki Kato, Sumie Okahata, Hisayuki Katsuyama, Mikiko Haraguchi, Ai Morita, Ken Ohashi, Kazuo Hara, Atsushi Morise, Kazuo Izumi, Naoki Ishizuka, Yasuo Ohashi, Mitsuhiko Noda, Takashi Kadowaki, Masakazu Haneda, Yasunori Iwashima, Toshihiro Suda, Naoki Tamasawa, Makoto Daimon, Jo Satoh, Noriko Takebe, Yasushi Ishigaki, Tsuyoshi Watanabe, Hiroaki Satoh, Kikuo Kasai, Yoshimasa Aso, Shun Ishibashi, Shigehiro Katayama, San e. Ishikawa, Masafumi Kakei, Kazuyuki Namai, Naotake Hashimoto, Yoshifumi Suzuki, Shunichiro Onishi, Koutaro Yokote, Masafumi Matsuda, Masahiro Masuzawa, Yoichi Hayashi, Satoshi Saito, Norikazu Ogihara, Hisamitsu Ishihara, Naoko Tajima, Kazunori Utsunomiya, Akira Shimada, Hiroshi Itoh, Ryuzo Kawamori, Hirotaka Watada, Michio Hayashi, Yasumichi Mori, Teruo Shiba, Akihiro Isogawa, Hiroshi Sakura, Masato Odawara, Kazuyuki Tobe, Kazuhisa Tsukamoto, Toshimasa Yamauchi, Tamio Teramoto, Yukio Hirata, Isao Uchimura, Yoshihiro Ogawa, Gen Yoshino, Takahisa Hirose, Hiroshi Kajio, Yoshihito Atsumi, Akira Shimada, Yoichi Oikawa, Atsushi Araki, Akio Ueki, Atsushi Ohno, Masafumi Kitaoka, Yoshikuni Fujita, Tatsumi Moriya, Taiki Tojo, Masayoshi Shichiri, Daisuke Suzuki, Masao Toyoda, Kumiko Hamano, Rieko Komi, Yasuo Terauchi, Nobuaki Kuzuya, Masayo Yamada, Toshinari Takamura, Mitsuo Imura, Hiroshi Tanaka, Masayuki Hayashi, Yasuhisa Kato, Mitsuyasu Itoh, Atsushi Suzuki, Mikihiro Nakayama, Takahisa Sano, Eitaro Nakashima, Yasuhiro Sumida, Yutaka Yano, Tsuyoshi Tanaka, Kazuya Murata, Atsunori Kashiwagi, Hiroshi Maegawa, Shigeo Kono, Nobuya Inagaki, Keisuke Kosugi, Tetsuyuki Yasuda, Yasunao Yoshimasa, Ichiro Kishimoto, Toshihiko Sato, Masayuki Hosoi, Tomoyuki Yamasaki, Munehide Matsuhisa, Iichiro Shimomura, Ataru Taniguchi, Akira Kuroe, Takeshi Kurose, Takeshi Ohara, Kazuhiko Sakaguchi, Mitsuyoshi Namba, Kohei Kaku, Masazumi Fujiwara, Ikki Shimizu, Keizo Ono, Osamu Ebisui, Yukio Tanizawa, Yosuke Okada, Shoichi Natori, Takehiko Kodera, Naoichi Sato, Makoto Ide, Kentaro Yamada, Fumio Umeda, Shoichi Natori, Tomoaki Eto, Kazuo Mimura, Shinsuke Hiramatsu, Tomoaki Inoue, Ryoko Takei, Atsushi Ogo, Katsumi Eguchi, Eiji Kawasaki, Yuji Koide, Eiichi Araki, Hideaki Jinnouchi, Hiroaki Yamamoto, Mitsutaka Motoyoshi, Toru Hiyoshi, Yasushi Tanaka, Tadahisa Momoki, Koichiro Sato, Akihiko Yoneyama, Kenichi Ito, Hiroshi Sobajima, Hiroshi Ikegami, Masaki Ikeda, Hiroki Ikeda, Kenji Takahashi, Hirofumi Makino, Yasuo Ueda, Masamitsu Nakazato, Effect of an intensified multifactorial intervention on cardiovascular outcomes and mortality in type 2 diabetes (J-DOIT3)
an open-label, randomised controlled trial, The Lancet Diabetes and Endocrinology, 10.1016/S2213-8587(17)30327-3, 5, 12, 951-964, 2017.12, Background Limited evidence suggests that multifactorial interventions for control of glucose, blood pressure, and lipids reduce macrovascular complications and mortality in patients with type 2 diabetes. However, safe and effective treatment targets for these risk factors have not been determined for such interventions. Methods In this multicentre, open-label, randomised, parallel-group trial, undertaken at 81 clinical sites in Japan, we randomly assigned (1:1) patients with type 2 diabetes aged 45–69 years with hypertension, dyslipidaemia, or both, and an HbA1c of 6·9% (52·0 mmol/mol) or higher, to receive conventional therapy for glucose, blood pressure, and lipid control (targets: HbA1c <6·9% [52·0 mmol/mol], blood pressure <130/80 mm Hg, LDL cholesterol <120 mg/dL [or 100 mg/dL in patients with a history of coronary artery disease]) or intensive therapy (HbA1c <6·2% [44·3 mmol/mol], blood pressure <120/75 mm Hg, LDL cholesterol <80 mg/dL [or 70 mg/dL in patients with a history of coronary artery disease]). Randomisation was done using a computer-generated, dynamic balancing method, stratified by sex, age, HbA1c, and history of cardiovascular disease. Neither patients nor investigators were masked to group assignment. The primary outcome was occurrence of any of a composite of myocardial infarction, stroke, revascularisation (coronary artery bypass surgery, percutaneous transluminal coronary angioplasty, carotid endarterectomy, percutaneous transluminal cerebral angioplasty, and carotid artery stenting), and all-cause mortality. The primary analysis was done in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00300976. Findings Between June 16, 2006, and March 31, 2009, 2542 eligible patients were randomly assigned to intensive therapy or conventional therapy (1271 in each group) and followed up for a median of 8·5 years (IQR 7·3–9·0). Two patients in the intensive therapy group were found to be ineligible after randomisation and were excluded from the analyses. During the intervention period, mean HbA1c, systolic blood pressure, diastolic blood pressure, and LDL cholesterol concentrations were significantly lower in the intensive therapy group than in the conventional therapy group (6·8% [51·0 mmol/mol] vs 7·2% [55·2 mmol/mol]; 123 mm Hg vs 129 mm Hg; 71 mm Hg vs 74 mm Hg; and 85 mg/dL vs 104 mg/dL, respectively; all p<0·0001). The primary outcome occurred in 109 patients in the intensive therapy group and in 133 patients in the conventional therapy group (hazard ratio [HR] 0·81, 95% CI 0·63–1·04; p=0·094). In a post-hoc breakdown of the composite outcome, frequencies of all-cause mortality (HR 1·01, 95% CI 0·68–1·51; p=0·95) and coronary events (myocardial infarction, coronary artery bypass surgery, and percutaneous transluminal coronary angioplasty; HR 0·86, 0·58–1·27; p=0·44) did not differ between groups, but cerebrovascular events (stroke, carotid endarterectomy, percutaneous transluminal cerebral angioplasty, and carotid artery stenting) were significantly less frequent in the intensive therapy group (HR 0·42, 0·24–0·74; p=0·002). Apart from non-severe hypoglycaemia (521 [41%] patients in the intensive therapy group vs 283 [22%] in the conventional therapy group, p<0·0001) and oedema (193 [15%] vs 129 [10%], p=0·0001), the frequencies of major adverse events did not differ between groups. Interpretation Our results do not fully support the efficacy of further intensified multifactorial intervention compared with current standard care for the prevention of a composite of coronary events, cerebrovascular events, and all-cause mortality. Nevertheless, our findings suggest a potential benefit of an intensified intervention for the prevention of cerebrovascular events in patients with type 2 diabetes. Funding Ministry of Health, Labour and Welfare of Japan, Asahi Kasei Pharma, Astellas Pharma, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Eli Lilly, GlaxoSmithKline, Kissei Pharmaceutical, Kowa Pharmaceutical, Mitsubishi Tanabe Pharma, Mochida Pharmaceutical, MSD, Novartis Pharma, Novo Nordisk, Ono Pharmaceutical, Pfizer, Sanwa Kagaku Kenkyusho, Shionogi, Sumitomo Dainippon Pharma, Taisho Toyama Pharmaceutical, and Takeda..
156. Atsumi Tsuji-Hosokawa, Kei Takasawa, Risa Nomura, Yuichi Miyakawa, Chikahiko Numakura, Atsushi Hijikata, Tsuyoshi Shirai, Yoshihiro Ogawa, Kenichi Kashimada, Tomohiro Morio, Molecular mechanisms of insulin resistance in 2 cases of primary insulin receptor defect-associated diseases, Pediatric Diabetes, 10.1111/pedi.12508, 18, 8, 917-924, 2017.12, Background: Defects of the insulin receptor gene (INSR) cause wide spectra of congenital insulin resistance. Monoallelic defects result in milder insulin-resistant diabetes mellitus with acanthosis nigricans (IRAN, type A). Whereas, leprechaunism (Donahue syndrome), the most severe condition with lethality during the infantile period is caused by biallelic defects of INSR. Materials and Methods: We detected 2 missense mutations in 2 cases of leprechaunism and IRAN, type A, and reduced mRNA expression in the leprechaunism case. We performed an in vitro analysis to confirm that the 2 missense mutations are causative. Results: The heterozygote mutations c.3436G>A (p.Gly1146Arg) and c.294C>A (p.Ser98Arg) were identified in a male patient with IRAN, type A and a female patient with leprechaunism, respectively. Gly1146Arg was previously reported in a diabetic case without precise functional analyses, and Ser98Arg is a novel mutation. Gly1146 and Ser98 are located on the tyrosine kinase domain and ligand-binding domain of INSR, respectively, and in vitro analyses (assay for insulin binding and phosphorylation) revealed that each mutation disrupted protein functions and properties. In the leprechaunism case, mutations in INSR other than Ser98Arg were not identified, and qRT-PCR analysis revealed that mRNA expression of INSR in lymphocytes was reduced in the leprechaunism case. Conclusion: Our study indicates that the 2 missense mutations of INSR, Gly1146Arg, and Ser98Arg, are responsible for insulin resistance, and, suggests that mutations not contained within INSR, but leading to decreased INSR expression should be considered for the patients who show insulin resistance without any mutations in the coding sequence of INSR..
157. Po Jen Chien, Takuma Suzuki, Masato Tsujii, Ming Ye, Isao Minami, Kanako Toda, Hiromi Otsuka, Koji Toma, Takahiro Arakawa, Kouji Araki, Yasuhiko Iwasaki, Kayoko Shinada, Yoshihiro Ogawa, Kohji Mitsubayashi, Biochemical Gas Sensors (Biosniffers) Using Forward and Reverse Reactions of Secondary Alcohol Dehydrogenase for Breath Isopropanol and Acetone as Potential Volatile Biomarkers of Diabetes Mellitus, Analytical chemistry, 10.1021/acs.analchem.7b03191, 89, 22, 12261-12268, 2017.11, This study describes two biosniffers to determine breath acetone and isopropanol (IPA) levels and applies them for breath measurement in healthy subjects and diabetic patients. Secondary alcohol dehydrogenase (S-ADH) can reduce acetone and oxidize nicotinamide adenine dinucleotide (NADH to NAD+) in a weak acid environment. NADH can be excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. S-ADH can also oxidize IPA and reduce NAD+ to NADH when it is in an alkaline environment. Thus, IPA can be detected by the increase of fluorescence. The developed biosniffers show rapid response, high sensitivity and high selectivity. The breath acetone and IPA analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb and 15.4 ± 11.3 ppb. Both acetone and IPA did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (p < 1 × 10-6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (p < 0.01). The breath IPA also had a higher concentration in diabetic patients (23.1 ± 20.1 ppb, p < 0.01), but only T2D patients presented a statistical difference (23.9 ± 21.3 ppb, p < 0.01). These findings are worthwhile in the study of breath biomarkers for diabetes mellitus diagnosis. Additionally, the developed biosniffers provide a new technique for volatolomics research..
158. Michiko Itoh, Takayoshi Suganami, Hideaki Kato, Sayaka Kanai, Ibuki Shirakawa, Takeru Sakai, Toshihiro Goto, Masahiro Asakawa, Isao Hidaka, Hiroshi Sakugawa, Koji Ohnishi, Yoshihiro Komohara, Kenichi Asano, Isao Sakaida, Masato Tanaka, Yoshihiro Ogawa, CD11c+ resident macrophages drive hepatocyte death-triggered liver fibrosis in a murine model of nonalcoholic steatohepatitis, JCI Insight, 10.1172/jci.insight.92902, 2, 22, 2017.11, Although recent evidence has pointed to the role of organ- and pathogenesis-specific macrophage subsets, it is still unclear which subsets are critically involved in the pathogenesis of nonalcoholic steatohepatitis (NASH). Using melanocortin-4 receptor-deficient (MC4R-KO) mice fed Western diet (WD), which exhibit liver phenotypes similar to those of human NASH, we found a histological structure, termed hepatic crown-like structure (hCLS), in which CD11c+ macrophages surround dead/dying hepatocytes, a prominent feature of NASH. Here, we demonstrate that hCLS-constituting macrophages could be a novel macrophage subset that drives hepatocyte death-triggered liver fibrosis. In an "inducible NASH model," hepatocyte death induces hCLS formation and liver fibrosis sequentially in the short term. In combination with the long-term WD feeding model, we also showed that resident macrophages are a major cellular source of CD11c+ macrophages constituting hCLS, which exhibited gene expression profiles distinct from CD11c- macrophages scattered in the liver. Moreover, depletion of CD11c+ macrophages abolished hCLS formation and fibrogenesis in NASH. Our clinical data suggest the role of CD11c+ macrophages in the disease progression from simple steatosis to NASH. This study sheds light on the role of resident macrophages, in addition to recruited macrophages, in the pathogenesis of NASH..
159. Kyoichiro Tsuchiya, Yoshihiro Ogawa, Forkhead box class O family member proteins
The biology and pathophysiological roles in diabetes, Journal of Diabetes Investigation, 10.1111/jdi.12651, 8, 6, 726-734, 2017.11, Forkhead box class O family member proteins (FoxOs) of transcription factors are essential regulators of cellular homeostasis, including glucose and lipid metabolism, oxidative stress response and redox signaling, cell cycle progression, and apoptosis. Altered FoxO1 expression and activity have been associated with glucose intolerance, dyslipidemia and complications of diabetes. In the liver, they direct carbons to glucose or lipid utilization, thus providing a unifying mechanism for the two abnormalities of the diabetic liver: excessive glucose production, and increased lipid synthesis and secretion. In the pancreas, FoxO1 is necessary to maintain β-cell differentiation, and could be promising targets for β-cell regeneration. In endothelial cells, FoxOs strongly promote atherosclerosis through suppressing nitric oxide production and enhancing inflammatory responses. In the present review, we summarize the basic biology and pathophysiological significance of FoxOs in diabetes..
160. Norihiko Ohara, Isao Minami, Ryotaro Bouchi, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Loss of skeletal muscle mass and its predictors in type 2 diabetes patients under a multifaceted treatment approach, Diabetology International, 10.1007/s13340-017-0325-z, 8, 4, 366-374, 2017.11, Background: Weight loss, which is an effective method for reducing visceral fat, may cause a concomitant loss of skeletal muscle mass. The aim of this study was to elucidate the changes in visceral fat and skeletal muscle mass in response to diabetes treatment including weight control. Methods: For 6 months we observed the changes in the body compositions of 72 Japanese patients with type 2 diabetes who underwent multifaceted treatment including educational hospitalization. Visceral fat area (VFA) and appendicular skeletal muscle mass (ASM) were measured using a bioelectrical impedance method and dual-energy X-ray absorptiometry, respectively. Results: During the follow-up period, VFA reduced significantly whereas the average ASM did not change. Changes in ASM were strongly positively associated with changes in body weight (r = 0.50). Additionally, in an analysis of covariance, an above-median BMI (27 kg/m2) and above-median VFA (110 cm2) at baseline were found to be independent predictors of ASM reduction prevention. Of the 55 patients who lost weight, those who had a baseline VFA of ≥110 cm2 had significantly greater reductions in VFA than those with a baseline VFA of <110 cm2 (p < 0.01). ASM reduced significantly in patients with a VFA of <110 cm2 (p < 0.01), but not in those with a VFA of ≥110 cm2 (p = 0.98). Conclusions: Baseline accumulation of visceral fat may predict a preferential reduction of visceral fat rather than skeletal muscle during weight control programs in type 2 diabetes patients..
161. Ryotaro Bouchi, Tatsuya Fukuda, Takato Takeuchi, Isao Minami, Takanobu Yoshimoto, Yoshihiro Ogawa, Sarcopenia is associated with incident albuminuria in patients with type 2 diabetes
A retrospective observational study, Journal of Diabetes Investigation, 10.1111/jdi.12636, 8, 6, 783-787, 2017.11, Sarcopenia, defined as age-related loss of skeletal muscle mass and function, increases the risk of albuminuria. However, it has still unknown whether sarcopenia could increase the risk for the progression of albuminuria. A total 238 patients with type 2 diabetes (mean age 64 ± 12 years; 39.2% women) were studied in the present retrospective observational study. The prevalence of sarcopenia was 17.6%. During the median follow-up period of 2.6 years, albuminuria was measured 5.8 ± 1.8 times, and progression of albuminuria was observed in 14.9% of patients with normoalbuminuria, as was 11.5% in those with microalbuminuria. Sarcopenia was significantly associated with both progression (hazard ratio 2.61, 95% confidence interval 1.08–6.31, P = 0.034) and regression (hazard ratio 0.23, 95% confidence interval 0.05–0.98, P = 0.048) of albuminuria by multivariate Cox regression analysis. The present data suggest that sarcopenia is an important determinant of both progression and regression of albuminuria in patients with type 2 diabetes..
162. Akira Matsumoto, Miyako Tanaka, Hiroko Matsumoto, Kozue Ochi, Yuki Moro-Oka, Hirohito Kuwata, Hironori Yamada, Ibuki Shirakawa, Taiki Miyazawa, Hitoshi Ishii, Kazunori Kataoka, Yoshihiro Ogawa, Yuji Miyahara, Takayoshi Suganami, Synthetic "smart gel" provides glucose-responsive insulin delivery in diabetic mice, Science Advances, 10.1126/sciadv.aaq0723, 3, 11, 2017.11, Although previous studies have attempted to create "electronics-free" insulin delivery systemsusing glucose oxidase and sugar-binding lectins as a glucose-sensingmechanism, no successful clinical translation has hitherto beenmade. These protein-based materials are intolerant of long-term use and storage because of their denaturing and/or cytotoxic properties. We provide a solution by designing a protein-free and totally synthetic material-based approach. Capitalizing on the sugar-responsive properties of boronic acid, we have established a synthetic polymer gel-based insulin delivery device confined within a single catheter, which exhibits an artificial pancreas-like function in vivo. Subcutaneous implantation of the device in healthy and diabetic mice establishes a closed-loop systemcomposed of "continuous glucose sensing" and "skin layer"-regulated insulin release. As a result, glucose metabolism was controlled in response to interstitial glucose fluctuation under both insulin-deficient and insulin-resistant conditions with at least 3-week durability. Our "smart gel" technology could offer a user-friendly and remarkably economic (disposable) alternative to the current state of the art, thereby facilitating availability of effective insulin treatment not only to diabetic patients in developing countries but also to those patients who otherwise may not be strongly motivated, such as the elderly, infants, and patients in need of nursing care..
163. Yukihiko Okumura, Kenichi Kohashi, Huanlin Wang, Masaki Kato, Yoshihiko Maehara, Yoshihiro Ogawa, Yoshinao Oda, Combined primary hepatic neuroendocrine carcinoma and hepatocellular carcinoma with aggressive biological behavior (adverse clinical course)
A case report, Pathology Research and Practice, 10.1016/j.prp.2017.06.001, 213, 10, 1322-1326, 2017.10, Combined primary hepatic neuroendocrine carcinoma (PHNEC) and hepatocellular carcinoma (HCC) is a very rare malignant hepatic tumor. Its prognosis and histological features are uncertain. Here we report the case of such a tumor in a 70-year-old male Japanese patient with adverse prognosis. The patient underwent a right hepatic lobectomy for a tumor mass that measured 11 × 10 cm in diameter located in the right lobe of the liver, treated with transcatheter arterial chemoembolization (TACE) and percutaneous transhepatic portal vein embolization (PTPE) therapy five weeks before the operation. Histologically, the hepatic tumor was composed of predominantly HCC and admixed with a small part of neuroendocrine carcinoma (NEC). The NEC component was distributed as a collision-type tumor separated by fibrous bands from HCC and the combined-type tumor, focally intermingling with HCC. One month after the surgery, metastasis to abdominal lymph nodes and the lumbar vertebra was detected. Although the additional treatments of systematic chemotherapy and radiation therapy were performed, the patient died 3 months after the initial surgery..
164. Masanori Murakami, Takanobu Yoshimoto, Kazuhiko Nakabayashi, Yujiro Nakano, Takahiro Fukaishi, Kyoichiro Tsuchiya, Isao Minami, Ryotaro Bouchi, Kohji Okamura, Yasuhisa Fujii, Koshi Hashimoto, Ken Ichiro Hata, Kazunori Kihara, Yoshihiro Ogawa, Molecular characteristics of the KCNJ5 mutated aldosterone-producing adenomas, Endocrine-Related Cancer, 10.1530/ERC-17-0117, 24, 10, 531-541, 2017.10, The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5, has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations..
165. Peng Xiong, Motoaki Shiratsuchi, Takamitsu Matsushima, Jiyuan Liao, Emi Tanaka, Yasuhiro Nakashima, Ryoichi Takayanagi, Yoshihiro Ogawa, Regulation of expression and trafficking of perforin-2 by LPS and TNF-α, Cellular Immunology, 10.1016/j.cellimm.2017.07.001, 320, 1-10, 2017.10, Perforin-2 is constitutively expressed in macrophages that are required for bacterial control. In this study, we found that perforin-2 is expressed in human macrophages with two isoforms: full-length perforin-2a and a splice variant, perforin-2b. Two isoforms show different subcellular distributions. Perforin-2a was predominantly localized to the membrane of endosome-like vesicles by a C-terminal transmembrane domain. In contrast, the short isoform perforin-2b lacking the transmembrane domain failed to localize to the membrane of vesicles. Furthermore, we determined that the pro-inflammatory stimuli LPS and TNF-α induced perforin-2a expression via the NF-κB pathway and triggered perforin-2a vesicles fusion with lysosomes. On the other hand, we detected the secretion of perforin-2b in response to LPS stimulation. Taken together, our data provide the evidence that membrane-bound and secretory isoforms of perforin-2 are present in human macrophages and may play important roles in immune defense..
166. Eulalia A. Coutinho, Shiki Okamoto, Ayako Wendy Ishikawa, Shigefumi Yokota, Nobuhiro Wada, Takahiro Hirabayashi, Kumiko Saito, Tatsuya Sato, Kazuyo Takagi, Chen Chi Wang, Kenta Kobayashi, Yoshihiro Ogawa, Seiji Shioda, Yumiko Yoshimura, Yasuhiko Minokoshi, Activation of SF1 neurons in the ventromedial hypothalamus by DREADD technology increases insulin sensitivity in peripheral tissues, Diabetes, 10.2337/db16-1344, 66, 9, 2372-2386, 2017.09, The ventromedial hypothalamus (VMH) regulates glucose and energy metabolism in mammals. Optogenetic stimulation of VMH neurons that express steroidogenic factor 1 (SF1) induces hyperglycemia. However, leptin acting via the VMH stimulates whole-body glucose utilization and insulin sensitivity in some peripheral tissues, and this effect of leptin appears to be mediated by SF1 neurons. We examined the effects of activation of SF1 neurons with DREADD (designer receptors exclusively activated by designer drugs) technology. Activation of SF1 neurons by an intraperitoneal injection of clozapine-N-oxide (CNO), a specific hM3Dq ligand, reduced food intake and increased energy expenditure in mice expressing hM3Dq in SF1 neurons. It also increased whole-body glucose utilization and glucose uptake in red-type skeletal muscle, heart, and interscapular brown adipose tissue, as well as glucose production and glycogen phosphorylase a activity in the liver, thereby maintaining blood glucose levels. During hyperinsulinemic-euglycemic clamp, such activation of SF1 neurons increased insulin-induced glucose uptake in the same peripheral tissues and tended to enhance insulin-induced suppression of glucose production by suppressing gluconeogenic gene expression and glycogen phosphorylase a activity in the liver. DREADD technology is thus an important tool for studies of the role of the brain in the regulation of insulin sensitivity in peripheral tissues..
167. Norio Miyamura, Shoji Hata, Tohru Itoh, Minoru Tanaka, Miki Nishio, Michiko Itoh, Yoshihiro Ogawa, Shuji Terai, Isao Sakaida, Akira Suzuki, Atsushi Miyajima, Hiroshi Nishina, Erratum
YAP determines the cell fate of injured mouse hepatocytes in vivo, Nature communications, 10.1038/ncomms16146, 8, 16146, 2017.08, This corrects the article DOI: 10.1038/ncomms16017..
168. Tatsuya Fukuda, Ryotaro Bouchi, Masahiro Terashima, Yuriko Sasahara, Masahiro Asakawa, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Ipragliflozin Reduces Epicardial Fat Accumulation in Non-Obese Type 2 Diabetic Patients with Visceral Obesity
A Pilot Study, Diabetes Therapy, 10.1007/s13300-017-0279-y, 8, 4, 851-861, 2017.08, Introduction: Epicardial fat (EF) was reported to be independently associated with cardiovascular disease regardless of obesity. We have previously reported that a sodium-glucose co-transporter-2 (SGLT2) inhibitor, luseogliflozin, reduces the EF volume (EFV) in parallel with the reduction of body weight in obese patients (BMI ≥25 kg/m2) with type 2 diabetes. However, it is unknown whether SGLT2 inhibitors could reduce EFV in non-obese patients (BMI <25 kg/m2) with type 2 diabetes. Therefore, we evaluated the effect of SGLT2 inhibitors on the EFV in non-obese type 2 diabetic patients with visceral obesity in this pilot study. Methods: Nine of type 2 diabetic patients (mean age 66 ± 8 years; 33% female) with HbA1c 6.5–9.0%, body mass index (BMI, kg/m2) <25.0, and visceral fat area (VFA, cm2) ≥100 were enrolled. Participants were administered ipragliflozin 50 mg daily. EFV [median (interquartile range), cm3] was measured by magnetic resonance imaging. Primary endpoint was the change in EFV at 12 weeks. VFA and liver attenuation index (LAI), skeletal muscle index (SMI), and body fat (%) were also assessed at baseline and at 12 weeks. Results: The EFV was significantly reduced from 102 (79–126) cm3 to 89 (66–109) cm3 by ipraglifrozin (p = 0.008). The body weight, BMI, HbA1c, fasting plasma glucose, insulin, homeostasis model assessment-insulin resistance, triglycerides, leptin, body fat, android, gynoid, and VFA were significantly reduced and high-density lipoprotein cholesterol was significantly increased by ipraglifrozin at 12 weeks, whereas SFA and LAI were unchanged. The change in EFV was significantly correlated with the change in BMI. Conclusions: A12-week intervention of ipragliflozin reduced the EFV in non-obese type 2 diabetic patients with visceral adiposity. Clinical Trial Registration: UMIN Clinical Trial Registry: UMIN000019071. Funding: Astellas Pharma Inc. and the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan..
169. Ryotaro Bouchi, Tatsuya Fukuda, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Insulin Treatment Attenuates Decline of Muscle Mass in Japanese Patients with Type 2 Diabetes, Calcified Tissue International, 10.1007/s00223-017-0251-x, 101, 1, 2017.07, Sarcopenia is defined as an age-related loss of skeletal muscle mass and strength, and is a major cause of disability and mobility limitations. Recent studies have demonstrated that type 2 diabetes and insulin signaling deficiencies contribute to the progression of sarcopenia, suggesting that a sufficient supply of insulin to the skeletal muscles may be important for the maintenance of muscle function; however, little has been reported regarding whether insulin treatment can protect against sarcopenia. We conducted a retrospective observational study to examine the impact of insulin treatment on the muscle mass of patients with type 2 diabetes. A total of 312 patients (mean age: 64 ± 11 years; 40.8% female; 27.6% treated with insulin) were studied in this retrospective observational study. Skeletal muscle index (SMI) and grip strength (kg) were used to assess sarcopenia. The prevalence of sarcopenia was 18.0%. Insulin treatment was shown to be protective against the annual decline of SMI (standardized β 0.195; p = 0.025) even after adjusting for covariates, including age, gender, duration of diabetes, and body mass index. In a cohort matched by propensity scores, insulin treatment significantly increased the 1-year change in SMI (mean ± SE) compared with non-insulin-treated group (2.40 ± 0.98% vs. −0.43 ± 0.98%; p = 0.050). Our data suggest that insulin treatment could attenuate the progression of sarcopenia in patients with type 2 diabetes..
170. Norio Miyamura, Shoji Hata, Tohru Itoh, Minoru Tanaka, Miki Nishio, Michiko Itoh, Yoshihiro Ogawa, Shuji Terai, Isao Sakaida, Akira Suzuki, Atsushi Miyajima, Hiroshi Nishina, YAP determines the cell fate of injured mouse hepatocytes in vivo, Nature communications, 10.1038/ncomms16017, 8, 2017.07, The presence of senescent, transformed or damaged cells can impair tissue function or lead to tumorigenesis; therefore, organisms have evolved quality control mechanisms to eliminate them. Here, we show that YAP activation induced by inactivation of the Hippo pathway specifically in damaged hepatocytes promotes their selective elimination by using in vivo mosaic analysis in mouse liver. These damaged hepatocytes migrate into the hepatic sinusoids, undergo apoptosis and are engulfed by Kupffer cells. In contrast, YAP activation in undamaged hepatocytes leads to proliferation. Cellular stresses such as ethanol that damage both liver sinusoidal endothelial cells and hepatocytes switch cell fate from proliferation to migration/apoptosis in the presence of activated YAP. This involves the activation of CDC42 and Rac that regulate cell migration. Thus, we suggest that YAP acts as a stress sensor that induces elimination of injured cells to maintain tissue and organ homeostasis..
171. Ryotaro Bouchi, Tatsuya Fukuda, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Association of sarcopenia with both latent autoimmune diabetes in adults and type 2 diabetes
A cross-sectional study, Journal of Diabetes and Its Complications, 10.1016/j.jdiacomp.2017.02.021, 31, 6, 992-996, 2017.06, Background To investigate the association of both latent autoimmune diabetes in adults (LADA) and type 2 diabetes (T2DM) with muscle mass and function (sarcopenia). Methods Japanese patients with LADA (N = 20), T2DM (N = 208), and control subjects (N = 41) were included in this cross-sectional study. The definition of LADA was based on age of onset (≥ 30), positive glutamic acid decarboxylase autoantibodies, and insulin requirement within the first 6 months after diagnosis. Sarcopenia was diagnosed by the criteria for Asians, using skeletal muscle index (male < 7.0 and female < 5.4) and grip strength (male < 26.0 kg and female < 18.0 kg). The odds ratio (OR) with a 95% confidence interval (CI) was estimated using logistic regression. Results The prevalence of sarcopenia was higher in LADA (35.0%) than in either T2DM (13.3%) or control subjects (9.8%). LADA was significantly associated with an increased risk for sarcopenia in a multivariate model in which age and body mass index were incorporated (OR: 9.57, 95% CI: 1.86–49.27). In contrast, T2DM tended to be associated with an increased risk for sarcopenia (OR: 2.99, 95% CI: 0.83–10.80). Conclusions This study provides evidence that patients with LADA are at a high risk for sarcopenia compared to those with T2DM or to control subjects..
172. Masami Miki, Tetsuhide Ito, Masayuki Hijioka, Lingaku Lee, Kohei Yasunaga, Keijiro Ueda, Takashi Fujiyama, Yuichi Tachibana, Ken Kawabe, Robert T. Jensen, Yoshihiro Ogawa, Utility of chromogranin B compared with chromogranin A as a biomarker in Japanese patients with pancreatic neuroendocrine tumors, Japanese journal of clinical oncology, 10.1093/jjco/hyx032, 47, 6, 520-528, 2017.06, Objective: Currently, serum chromogranin A is a well-established biomarker for pancreatic neuroendocrine tumors; however, other pancreatic diseases, oral use of a proton pump inhibitor and renal impairment can affect chromogranin A. Meanwhile, chromogranin B, belonging to the same granin family as chromogranin A, is not fully examined in these conditions. The present study aimed to evaluate the utility of chromogranin B as a pancreatic neuroendocrine tumor biomarker. Methods: Serum chromogranin B levels were determined by radioimmunoassay and serum chromogranin A levels by enzyme-linked immunosorbent assay in pancreatic neuroendocrine tumor (n = 91) and other pancreatic conditions, and in healthy people (n = 104), to assess the relationships with clinical features. Results: The diagnostic ability of chromogranin B was as good as chromogranin A. The area under the curve was 0.79 for chromogranin B (sensitivity/specificity: 72%/77%), and 0.78 for chromogranin A (sensitivity/specificity: 79%/64%). Chromogranin B was not affected by proton pump inhibitor use and age, which affected chromogranin A. The number of cases without liver metastases was larger in pancreatic neuroendocrine tumor patients with positive chromogranin B and negative chromogranin A. Though chromogranin A significantly elevated cases with proton pump inhibitor treatment and had positive correlation with age, chromogranin B did not have the tendencies. However, both chromogranin B and chromogranin A elevated in the case with renal impairment. In addition, the logistic regression analysis showed that chromogranin B was superior to chromogranin A in differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases. Conclusions: Compared with chromogranin A, chromogranin B may be more useful during proton pump inhibitor treatment and can detect tumors without liver metastases. In addition, chromogranin B may be an excellent biomarker when differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases is required..
173. Tatsuya Fukuda, Ryotaro Bouchi, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Association of diabetic retinopathy with both sarcopenia and muscle quality in patients with type 2 diabetes
A crosssectional study, BMJ Open Diabetes Research and Care, 10.1136/bmjdrc-2017-000404, 5, 1, 2017.05, Objective To examine whether the existence and severity of diabetic retinopathy (DR) could be associated with the prevalent sarcopenia and muscle quality in patients with type 2 diabetes. Research design and methods This is a cross-sectional study of 316 patients with type 2 diabetes (mean age 65±12 years; 38% female). Body compositions were measured by the dual-energy X-ray absorptiometry. Patients were divided into three groups: patients without DR (NDR), with non-proliferative DR (NPDR) and proliferative DR (PDR). Sarcopenia was diagnosed according to the criteria for Asians, using both skeletal muscle index (SMI) and grip strength (kg). Muscle quality was also determined by the grip strength divided by SMI. Logistic regression analyses were carried out to assess the cross-sectional association of the severity of DR with sarcopenia. In addition, linear regression analyses were performed to determine the associations between DR and muscle quality. Selection of covariates in the multivariate logistic and linear regression analyses was done by a stepwise procedure. Results Among the patients examined, NDR, NPDR and PDR were diagnosed in 261, 38 and 17 patients, respectively. The prevalence of sarcopenia significantly increased along with the progression of DR. Multivariate logistic regression analysis showed that PDR is significantly associated with sarcopenia (OR 7.78, 95% CI 1.52 to 39.81, p=0.014) and low muscle strength (OR 6.25, 95% CI 1.15 to 33.96, p=0.034). Multivariate linear regression analysis additionally showed that the existence of DR was significantly associated with the muscle quality (standardized β −0.136, p=0.005 for NPDR, standardized β −0.146, p=0.003 for PDR). Conclusions This study provides evidence that PDR is significantly associated with sarcopenia, and the existence of DR increases the risk for low muscle quality in patients with type 2 diabetes..
174. Takashi Fujiyama, Tetsuhide Ito, Keijiro Ueda, Yuichi Tachibana, Kohei Yasunaga, Masami Miki, Takehiro Takaoka, Lingaku Lee, Ken Kawabe, Yoshihiro Ogawa, Serum levels of Wisteria floribunda agglutinin-positive Mac-2 binding protein reflect the severity of chronic pancreatitis, Journal of Digestive Diseases, 10.1111/1751-2980.12475, 18, 5, 302-308, 2017.05, OBJECTIVES: To evaluate the utility of serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA +-M2BP) level as a marker for chronic pancreatitis (CP). METHODS: We measured the serum WFA+-M2BP level of 74 patients with CP who had undergone endoscopic retrograde cholangiopancreatography and 30 normal controls (NC) using a glycan sugar chain-based immunoassay and investigated the relationship between serum WFA+-M2BP levels and the Cambridge classification of CP. RESULTS: Serum WFA+-M2BP level was significantly higher in patients with CP than in NC (0.64 ± 0.28 vs 0.34 ± 0.25, P < 0.001). The levels (expressed as cut-off index) of WFA+-M2BP for the classification of mild, moderate and marked CP were 0.44, 0.63 and 0.87, respectively. Thus, serum WFA+-M2BP levels increased with increasing CP severity. With a cut-off value of 0.34, 0.59 and 0.61, the area under the receiver operating characteristic curve, sensitivity and specificity were 0.829, 91.9% and 63.3% for mild CP; 0.891, 81.8% and 85.0% for moderate CP; and 0.888, 92.0% and 74.7% for marked CP, respectively. Multivariate analysis revealed that elevated serum WFA+-M2BP was independently associated with moderate and marked CP, respectively. CONCLUSION: Serum WFA+-M2BP level is a useful marker for grading CP severity..
175. Shojiro Haji, Motoaki Shiratsuchi, Takamitsu Matsushima, Akiko Takamatsu, Mariko Tsuda, Yasuhiro Tsukamoto, Emi Tanaka, Hirofumi Ohno, Eriko Fujioka, Yuriko Ishikawa, Ken Ichi Imadome, Yoshihiro Ogawa, Achievement of disease control with donor-derived EB virus-specific cytotoxic T cells after allogeneic peripheral blood stem cell transplantation for aggressive NK-cell leukemia, International journal of hematology, 10.1007/s12185-016-2131-y, 105, 4, 540-544, 2017.04, Aggressive NK-cell leukemia (ANKL) is characterized by systemic infiltration of Epstein-Barr virus (EBV)-associated natural killer cells and poor prognosis. We report a case of ANKL in which EBV-specific cytotoxic T lymphocytes (CTLs) were induced. A 41-year-old male suffered from fever, pancytopenia, and hepatosplenomegaly. The number of abnormal large granular lymphocytes in the bone marrow was increased and the cells were positive for CD56 and EBV-encoded small nuclear RNAs. The patient was diagnosed with ANKL and achieved a complete response following intensive chemotherapy. He then underwent allogeneic peripheral blood stem cell transplantation from his sister. Conditioning therapy consisted of total body irradiation and cyclophosphamide. Graft-versus-host disease prophylaxis consisted of cyclosporine and methotrexate. On day 31, complete donor chimerism was achieved and no acute graft-versus-host disease developed. The ANKL relapsed on day 80, and cyclosporine was rapidly tapered and chemotherapy was started. During hematopoietic recovery, the number of atypical lymphocytes increased, but they were donor-derived EBV-specific CTLs. The patient achieved a partial response and EBV viral load decreased to normal range. Unfortunately, ANKL worsen again when the CTLs disappeared from his blood. This is the first case report of ANKL in which induced EBV-specific CTLs may have contributed to disease control..
176. Kyoko Shirakabe, Takuya Omura, Yoshio Shibagaki, Emiko Mihara, Keiichi Homma, Yukinari Kato, Akihiko Yoshimura, Yoshinori Murakami, Junichi Takagi, Seisuke Hattori, Yoshihiro Ogawa, Mechanistic insights into ectodomain shedding
Susceptibility of CADM1 adhesion molecule is determined by alternative splicing and O-glycosylation, Scientific reports, 10.1038/srep46174, 7, 2017.04, Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation..
177. Ogawa Yoshihiro, Reduction of visceral fat by liraglutide is associated with ameliorations of hepatic steatosis, albuminuria, and micro-inflammation in type 2 diabetic patients with insulin treatment: a randomized control trial., Endocr. J. , 64, 3, 269-281, 2017.03.
178. Ogawa Yoshihiro, Dopamine-secreting paraganglioma in the retroperitoneum., Endocr. Pathol., 28, 1, 36-40, 2017.03.
179. Chikara Komiya, Miyako Tanaka, Kyoichiro Tsuchiya, Noriko Shimazu, Kentaro Mori, Shunsaku Furuke, Yasutaka Miyachi, Kumiko Shiba, Shinobu Yamaguchi, Kenji Ikeda, Kozue Ochi, Kazuhiko Nakabayashi, Ken Ichiro Hata, Michiko Itoh, Takayoshi Suganami, Yoshihiro Ogawa, Antifibrotic effect of pirfenidone in a mouse model of human nonalcoholic steatohepatitis, Scientific reports, 10.1038/srep44754, 7, 2017.03, Non-alcoholic steatohepatitis (NASH) is characterized by steatosis with lobular inflammation and hepatocyte injury. Pirfenidone (PFD) is an orally bioavailable pyridone derivative that has been clinically used for the treatment of idiopathic pulmonary fibrosis. However, it remains unknown whether PFD improves liver fibrosis in a mouse model with human NASH-like phenotypes. In this study, we employed melanocortin 4 receptor-deficient (MC4R-KO) mice as a mouse model with human NASH-like phenotypes to elucidate the effect and action mechanisms of PFD on the development of NASH. PFD markedly attenuated liver fibrosis in western diet (WD)-fed MC4R-KO mice without affecting metabolic profiles or steatosis. PFD prevented liver injury and fibrosis associated with decreased apoptosis of liver cells in WD-fed MC4R-KO mice. Pretreatment of PFD inhibited the tumor necrosis factor-α (TNF-α)-induced liver injury and fibrogenic responses associated with decreased apoptosis of liver cells in wild-type mice. PFD also prevented TNF-α-induced hepatocyte apoptosis in vitro with reduced activation of caspase-8 and -3. This study provides evidence for the antifibrotic effect of PFD in a mouse model of human NASH. The data of this study highlight hepatocyte apoptosis as a potential therapeutic target, and suggest that PFD can be repositioned as an antifibrotic drug for human NASH..
180. Yusuke Matsuda, Noriko Kimura, Takanobu Yoshimoto, Yoshihiro Sekiguchi, Junzo Tomoishi, Ichiro Kasahara, Yoshihito Hara, Yoshihiro Ogawa, Dopamine-Secreting Paraganglioma in the Retroperitoneum, Endocrine Pathology, 10.1007/s12022-016-9457-0, 28, 1, 36-40, 2017.03, Pheochromocytomas and paragangliomas, which exclusively produce dopamine, are very rare. Herein, we report for the first time a Japanese case of an exclusively dopamine-producing paraganglioma accompanied by detailed immunohistochemical analyses. A 70-year-old Japanese woman was referred to our hospital for functional examination of her left retroperitoneal mass. Her adrenal functions were normal, except for excessive dopamine secretion. After the tumorectomy, her dopamine level normalized. The histopathological diagnosis of the tumor was paraganglioma; this was confirmed by positive immunostaining of chromogranin A (CgA), tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and succinate dehydrogenase gene subunit B (SDHB). However, the immunostaining of CgA in the tumor cells showed peculiar dot-like staining located corresponding to Golgi complex in the perinuclear area, rather than the diffuse cytoplasmic staining usually observed in epinephrine- or norepinephrine-producing functional pheochromocytomas and paragangliomas. The immunohistochemical results suggested that the tumor cells had sparse neuroendocrine granules in the cytoplasm, resulting in inhibition of catecholamine synthesis from dopamine to norepinephrine in neurosecretory granules. This may be the mechanism responsible for exclusive dopamine secretion in the present case..
181. Ryotaro Bouchi, Masahiro Terashima, Yuriko Sasahara, Masahiro Asakawa, Tatsuya Fukuda, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Luseogliflozin reduces epicardial fat accumulation in patients with type 2 diabetes
A pilot study, Cardiovascular Diabetology, 10.1186/s12933-017-0516-8, 16, 1, 2017.03, Background: Accumulation of epicardial fat (EF) is associated with increased cardio-metabolic risks and coronary events, independently of traditional cardiovascular risk factors. Therefore, the reduction of EF volume (EFV) may be associated with reduced cardio-metabolic risks and future cardiovascular events. Sodium-glucose co-transporter-2 (SGLT2) inhibitors reduce body fat including visceral fat and cardiovascular events in patients with type 2 diabetes. However, it has still been unknown whether SGLT2 inhibitors can reduce EFV. Methods: Type 2 diabetic patients with HbA1c 6.5-9.0% and body mass index (BMI, kg/m2)≥25.0 were enrolled in this single arm pilot study. Participants were administered luseogliflozin 2.5mg daily and the dosage was tolerated to be increased up to 5.0mg daily. EFV [median (interquartile range), cm3] was measured by magnetic resonance imaging. Primary endpoint was the decrease in EFV at 12weeks. Visceral fat area (VFA, cm2) and liver attenuation index (LAI) measured by the abdominal computed tomography, and skeletal muscle index (SMI) and body fat (%) measured by the whole body dual-energy X-ray absorptiometry were also determined at baseline and at 12weeks. Results: Nineteen patients (mean age: 55±12years; 26% female) completed this study. Luseogliflozin treatment significantly reduced EFV at 12weeks [117 (96-136) to 111 (88-134), p=0.048]. The body weight, BMI, systolic and diastolic blood pressure, HbA1c, fasting plasma glucose, insulin, homeostasis model assessment-insulin resistance (HOMA-IR), triglycerides, SMI, and body fat were significantly reduced by luseogliflozin at 12weeks. The reduction of EFV was significantly correlated with the reduction of C-reactive protein (r=0.493, p=0.019). Neither VFA nor LAI were significantly reduced by the luseogliflozin treatment. No severe adverse events were observed. Conclusions: Our data suggest that luseogliflozin could reduce the EFV in parallel with the improvement of systemic micro-inflammation and the reduction of body weight in Japanese patients with type 2 diabetes. The reduction of muscle mass after the administration of SGLT2 inhibitors may require a particular attention. Trial registration umin.ac.jp, UMIN000019072.
182. Yasutaka Miyachi, Kyoichiro Tsuchiya, Chikara Komiya, Kumiko Shiba, Noriko Shimazu, Shinobu Yamaguchi, Michiyo Deushi, Mizuko Osaka, Kouji Inoue, Yuta Sato, Sayaka Matsumoto, Junichi Kikuta, Kenjiro Wake, Masayuki Yoshida, Masaru Ishii, Yoshihiro Ogawa, Roles for Cell-Cell Adhesion and Contact in Obesity-Induced Hepatic Myeloid Cell Accumulation and Glucose Intolerance, Cell Reports, 10.1016/j.celrep.2017.02.039, 18, 11, 2766-2779, 2017.03, Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance..
183. Ogawa Yoshihiro, Abdominal paraganglioma in a young woman with 1p36 deletion syndrome., Am. J. Med. Genet. Part A. , 173, 2, 495-500, 2017.02.
184. Miki Murakoshi, Kei Takasawa, Masato Nishioka, Masahiro Asakawa, Kenichi Kashimada, Takanobu Yoshimoto, Toshiyuki Yamamoto, Kazuhiro Takekoshi, Yoshihiro Ogawa, Masayuki Shimohira, Abdominal paraganglioma in a young woman with 1p36 deletion syndrome, American Journal of Medical Genetics, Part A, 10.1002/ajmg.a.38020, 173, 2, 495-500, 2017.02, 1p36 deletion syndrome is the most common terminal deletion syndrome, and the genomic regions that contribute to specific 1p36 deletion syndrome-related phenotypes were recently identified. Deletions in the 1p36 region have been documented in various tumor tissues, which indicates correlation between loss of heterozygosity of 1p36 and tumor development, and the existence of tumor suppressors in this region. Therefore, it was suspected that patients with 1p36 deletion syndrome have a higher risk of tumor development; however, only a few child cases of neuroblastoma with 1p36 deletion syndrome have been reported. We report the first case of 1p36 deletion syndrome with paraganglioma (PGL) and include genetic investigation. The 24-year-old woman with 1p36 deletion syndrome had severe intellectual disability, dilated cardiomyopathy, and distinct dysmorphic features, and presented with persistent vomiting accompanied by hypertension (178/115 mmHg). Abdominal CT revealed a 40 × 50 mm retroperitoneal mass and substantial elevations of plasma and urine norepinephrine (15.4 nmol/L and 1022 µmol/mol creatinine, respectively); abnormal uptake of 123I-MIBG in the tumor led to PGL diagnosis. The patient was not able to have surgery because of substantial surgical risks; however, a combination of α- and β-blockade was effective for blood pressure control. Array CGH revealed a deletion over 4.5 Mb, from the 1p telomere but excluding the SDHB region. Comprehensive mutational analysis of PGL-associated genes (RET, VHL, TMEM127, MAX, and SDHA/B/C/D) was negative. These results indicate that the germline 1p36 deletion might be “1st hit” of tumor development, and PGL might be a novel complication of 1p36 deletion syndrome..
185. Lingaku Lee, Tetsuhide Ito, Hisato Igarashi, Keijiro Ueda, Takashi Fujiyama, Ken Kawabe, Yoshihiro Ogawa, Impact of everolimus on Japanese patients with advanced pancreatic neuroendocrine neoplasms, Journal of Hepato-Biliary-Pancreatic Sciences, 10.1002/jhbp.418, 24, 2, 95-102, 2017.02, Background: Although everolimus has become a key therapeutic agent in patients with advanced pancreatic neuroendocrine neoplasms (PNEN), its efficacy and safety in clinical practice remains unclear. Methods: Forty-seven patients with advanced PNEN treated with everolimus were reviewed retrospectively. To evaluate the safety of everolimus as a long-term treatment, the patients were divided into two groups according to treatment duration: group A, ≤1 year (n = 21); group B, >1 year (n = 26). Results: Among 42 patients with pancreatic neuroendocrine tumors (PNET), the median progression-free survival, overall survival, and objective response rate were 27.5 months, 60.8 months, and 19.0%, respectively. Two patients with pancreatic neuroendocrine carcinomas (PNEC) with lower Ki-67 index and well-differentiated tumors showed favorable responses. More patients in group A discontinued everolimus owing to adverse drug reactions (ADRs) than in group B. The median relative dose intensity was significantly lower in group B than group A (P = 0.045), whereas the drug interruption rate was significantly higher in group B than group A (P < 0.001). Conclusions: Everolimus showed significant clinical benefit in Japanese patients with advanced PNEN. Prevention of severe ADRs by appropriate dose reduction and interruption is necessary for a long-term continuation of everolimus..
186. Masaki Tanaka, Ansa Kobayashi, Masahiro Asakawa, Momoko Akihisa, Atsuko Miyake, Mitsunobu Kawamura, Yoshihiro Ogawa, A case of an elderly woman with concurrent pancreatic cancer and slowly progressive type 1 diabetes mellitus, Journal of the Japan Diabetes Society, 10.11213/tonyobyo.60.806, 60, 12, 806-812, 2017.01, The patient was a 76-year-old woman who had been diagnosed with diabetes when she was 75 years of age. Her blood glucose was initially well controlled by oral antidiabetic therapy. However, she was admitted to our hospital at approximately 6 months after her diagnosis because her casual blood glucose and HbAlc suddenly increased to 675 mg/dL and 11.9 %, respectively, and she also developed ketosis. An investigation to determine the cause of her blood glucose deterioration revealed pancreatic cancer. However, her anti-GAD antibody titer was also extremely high (124,000 U/mL), and she was diagnosed as simultaneously having slowly progressive insulin-dependent diabetes mellitus (SPIDDM). The development of SPIDDM in elderly patients is rare and there have only been a few reports about SPIDDM being diagnosed concurrently with pancreatic cancer. HLA genes were detected in the present patient. These genes are potentially related to disease resistance. It is possible that disease resistance HLA led to the simultaneous development of SPIDDM and pancreatic cancer in this elderly patient. Although pancreatic cancer and SPIDDM rarely occur simultaneously in elderly patients, the possible combination of a malignant tumor and type 1 diabetes mellitus (such as SPIDDM) should be considered if a patient's blood glucose control suddenly deteriorates during diabetes treatment..
187. Rie Nishitani, Keiko Ashidate, Mitsunobu Kawamura, Yoshihiro Ogawa, A case of diabetes mellitus accompanied by tumor-forming pancreatitis that was difficult to distinguish from pancreatic cancer, Journal of the Japan Diabetes Society, 60, 1, 37-41, 2017.01, A 44-year-old-man was diagnosed with diabetes in a medical examination and was admitted to our hospital due to hyperglycemia (FPG 285 mg/dL, HbAlc 10.8 %). He had experienced acute pancreatitis on 3 occasions. The laboratory data on admission revealed that the patient's CEA and DUPAN-2 levels were elevated. Abdominal enhanced CT and MRI demonstrated a tumor of 3 cm in diameter in the pancreatic head. Although pancreatic cancer was strongly suspected, a PET scan was negative. A percutaneous biopsy of the pancreas and a cytological analysis of the pancreatic juice were performed and the pathological diagnosis was tumor-forming pancreatitis. The acute onset of diabetes is considered to be a predictive marker of pancreatic cancer. It is very difficult to differentiate tumor-forming pancreatitis from pancreatic cancer. We should therefore make a comprehensive diagnosis using imaging, an analysis of the tumor marker levels, and a pathological examination..
188. Satoru Uchida, Mina Yamaguchi, Hiromi Hasegawa, Noriaki Ohkiba, Ryotaro Bouchi, Isao Minami, Yoshihiro Ogawa, A case of type B insulin resistance syndrome with postprandial hyperglycemia successfully treated with liraglutide, Journal of the Japan Diabetes Society, 60, 3, 244-252, 2017.01, We herein report a patient with type B insulin resistance in whom liraglutide significantly improved postprandial hyperglycemia that had been highly resistant to other treatments, including insulin therapy. The patient was a 73-year-old Japanese male whose hyperglycemia had worsened rapidly after being diagnosed as diabetes. His fasting plasma glucose was 77 mg/dL, HbA1c was 8.6 %, and serum insulin level was 366 /μU/mL. As anti-insulin receptor antibody was detected, we diagnosed the patient with type B insulin resistance. CGM (Continuous Glucose Monitoring) showed that liraglutide reduced the average blood glucose level from 124 mg/dL (standard deviation [SD] 59 mg/dL) to 80 mg/dL (SD 16 mg/dL) by improving the postprandial hyperglycemia and did not increase the time in a hypoglycemic state (27 % to 23 %). The meal tolerance test showed that liraglutide significantly enhanced the reactivity of the plasma insulin responses after food intake. Liraglutide was also effective as a treatment when the patient's postprandial hyperglycemia worsened again, in parallel with increasing inhibited insulin receptor rates. Our findings in this case imply the specific effect of liraglutide in improving type B insulin resistance complicated with postprandial hyperglycemia..
189. Masanori Murakami, Takanobu Yoshimoto, Yoshihiro Ogawa, Molecular analysis of aldosterone-producing adenomas, Japanese Journal of Clinical Chemistry, 46, 1, 28-33, 2017.01.
190. Ryotaro Bouchi, Yujiro Nakano, Tatsuya Fukuda, Takato Takeuchi, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Reduction of visceral fat by liraglutide is associated with ameliorations of hepatic steatosis, albuminuria, and micro-inflammation in type 2 diabetic patients with insulin treatment
A randomized control trial, Endocrine Journal, 10.1507/endocrj.EJ16-0449, 64, 3, 269-281, 2017.01, Liraglutide, an analogue of human glucagon-like peptide 1, reduces cardiovascular events in patients with type 2 diabetes; however, it has still been unknown by which mechanisms liraglutide could reduce cardiovascular events. Type 2 diabetic patients with insulin treatment were enrolled in this randomized, open-label, comparative study. Participants were randomly assigned to liraglutide plus insulin (liraglutide group) and insulin treatment (control group) at 1:1 allocation. Primary endpoint was the change in viscera fat are (VFA, cm2) at 24 weeks. Liver attenuation index (LAI) measured by abdominal computed tomography, urinary albumin-to-creatinine ratio (ACR, mg/g), and C-reactive protein (CRP) levels, skeletal muscle index (SMI), and quality of life (QOL) related to diabetes treatment were also determined. Seventeen patients (8; liraglutide group, 9; control group, mean age 59 ± 13 years; 53% female) completed this study. Liraglutide treatment significantly reduced VFA at 24 weeks; whereas, SFA was unchanged. ACR, LAI, and CRP levels were significantly reduced by liraglutide at 24 weeks and there was no difference in SMI between the two groups. Changes in VFA from baseline to 24 weeks were significantly associated with those in LAI, albuminuria, and HbA1c. Liraglutide treatment significantly improved QOL scores associated with anxiety and dissatisfaction with treatment and satisfaction with treatment. No severe adverse events were observed in both groups. Our data suggest that liraglutide could reduce visceral adiposity in parallel with attenuation of hepatic fat accumulation, albuminuria and micro-inflammation and improve QOL related to diabetes care in insulin-treated patients with type 2 diabetes..
191. Yoshihiro Ogawa, Symposium, Therapeutic Research, 38, 1, 21-24, 2017.01.
192. Ryotaro Bouchi, Tatsuya Fukuda, Takato Takeuchi, Yujiro Nakano, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Gender difference in the impact of gynoid and android fat masses on the progression of hepatic steatosis in Japanese patients with type 2 diabetes, BMC Obesity, 10.1186/s40608-017-0163-3, 4, 1, 2017, Background: Increased visceral adiposity is strongly associated with non-alcoholic fatty liver disease (NAFLD). However, little attention has been paid to the association between the change in subcutaneous adipose mass and the progression of non-alcoholic fatty liver disease (NAFLD). We aimed to investigate whether increased subcutaneous adipose tissue (gynoid fat mass) could be protective against the progression of NAFLD in Japanese patients with type 2 diabetes. Methods: This is a retrospective observational study of 294 Japanese patients with type 2 diabetes (65 ± 10 years old, 40% female). Liver attenuation index (LAI) measured by abdominal computed tomography was used for the assessment of hepatic steatosis. Both gynoid (kg) and android (kg) fat masses were measured by the whole body dual-energy X-ray absorptiometry. One-year changes in LAI, gynoid, and android fat masses were evaluated in both male and female patients. Linear regression analysis with a stepwise procedure was used for the statistical analyses to investigate the association of the changes in gynoid and android fat masses with the change in LAI. Results: LAI levels at baseline were 1.15 ± 0.31 and 1.10 ± 0.34 in female and male patients (p = 0.455). The change in gynoid fat mass was significantly and positively associated with the change in LAI in both univariate (standardized β 0.331, p = 0.049) and multivariate (standardized β 0.360, p = 0.016) models in the female patients. However, no significant association was observed in males. In contrast, the increase in android fat mass was significantly associated with the reduced LAI in both genders in the multivariate models (standardized β -0.651, p < 0.001 in females and standardized β -0.519, p = 0.042 in males). Conclusions: This study provides evidence that increased gynoid fat mass may be protective against the progression of NAFLD in female Japanese patients with type 2 diabetes..
193. Motoki Kawasaki, Motoyoshi Tsujino, Fuminori Sato, Maya Sakurada, Kenji Nishida, Takayasu Kise, Yuko Hijioka, Mitsugu Ishizawa, Kazuaki Enatsu, Yoshihiro Ogawa, IgG4-related hypophysitis with subtle hypopituitarism in an elderly diabetic patient
Is treatment or observation preferable?, Internal Medicine, 10.2169/internalmedicine.8851-17, 56, 20, 2733-2738, 2017, A 70-year-old man with diabetes mellitus presented with an enlarged pituitary stalk in 2014. IgG4-related parotitis and submandibular sialoadenitis were diagnosed in 2012. He denied any symptoms related to a pituitary mass. His visual field was intact, and his hypopituitarism was subtle. The serum IgG4 level was elevated. A lip biopsy revealed strong fibrosis and hyper-infiltration of IgG4-positive plasma cells. Based on these findings, IgG4-related hypophysitis was diagnosed. The patient was carefully followed without specific intervention. His clinical condition showed no change until December 2016, suggesting a stable, natural course. Care should be taken when considering glucocorticoid therapy, especially for elderly diabetic patients, given possible side effects..
194. Yoshihiro Ogawa, Michiko Itoh, Metabolic syndrome and NAFLD/NASH, Journal of Japanese Society of Gastroenterology, 114, 5, 834-838, 2017.
195. Mitsuhiro Kishino, Takanobu Yoshimoto, Masashi Nakadate, Yoshiaki Katada, Eiichiro Kanda, Shuichiro Nakaminato, Yukihisa Saida, Yoshihiro Ogawa, Ukihide Tateishi, Optimization of left adrenal vein sampling in primary aldosteronism
Coping with asymmetrical cortisol secretion, Endocrine Journal, 10.1507/endocrj.EJ16-0433, 64, 3, 347-355, 2017, We evaluated the influence of catheter sampling position and size on left adrenal venous sampling (AVS) in patients with primary aldosteronism (PA) and analyzed their relationship to cortisol secretion. This retrospective study included 111 patients with a diagnosis of primary aldosteronism who underwent tetracosactide-stimulated AVS. Left AVS was obtained from two catheter positions — the central adrenal vein (CAV) and the common trunk. For common trunk sampling, 5-French catheters were used in 51 patients, and microcatheters were used in 60 patients. Autonomous cortisol secretion was evaluated with a low-dose dexamethasone suppression test in 87 patients. The adrenal/inferior vena cava cortisol concentration ratio [selectivity index (SI)] was significantly lower in samples from the left common trunk than those of the left CAV and right adrenal veins, but this difference was reduced when a microcatheter was used for common trunk sampling. Sample dilution in the common trunk of the left adrenal vein can be decreased by limiting sampling speed with the use of a microcatheter. Meanwhile, there was no significant difference in SI between the left CAV and right adrenal veins. Laterality, determined according to aldosterone/cortisol ratio (A/C ratio) based criteria, showed good reproducibility regardless of sampling position, unlike the absolute aldosterone value based criteria. However, in 11 cases with autonomous cortisol co-secretion, the cortisol hypersecreting side tended to be underestimated when using A/C ratio based criteria. Left CAV sampling enables symmetrical sampling, and may be essential when using absolute aldosterone value based criteria in cases where symmetrical cortisol secretion is uncertain..
196. Keijiro Ueda, Tetsuhide Ito, Ken Kawabe, Lingaku Lee, Takashi Fujiyama, Yuichi Tachibana, Masami Miki, Kohei Yasunaga, Takehiro Takaoka, Akihiro Nishie, Yoshiki Asayama, Robert T. Jensen, Yoshihiro Ogawa, Should the selective arterial secretagogue injection test for insulinoma localization be evaluated at 60 or 120 seconds?, Internal Medicine, 10.2169/internalmedicine.9107-17, 56, 22, 2985-2991, 2017, Objective The selective arterial secretagogue injection (SASI) test is considered indispensable for the accurate localization of insulinoma. However, the optimum timing of the post-injection evaluation is controversial, as some studies recommend 60 seconds [SASI (60 seconds)] while others support 120 seconds [SASI (120 seconds)]. The aim of this study was to determine the optimum timing for the SASI test evaluation for insulinoma localization. Methods Thirteen patients with surgically proven insulinoma were studied retrospectively. For the SASI test, immunoreactive insulin (IRI) was determined at baseline and at 30, 60, 90, and 120 seconds after calcium gluconate injection. A two-fold or greater increase in IRI over the baseline value was considered positive. The localization abilities of SASI (60 seconds) and SASI (120 seconds) were then compared. Results In 13 patients, a secretagogue was injected into 40 arteries supplying the pancreas. In the SASI (60 seconds) and SASI (120 seconds), the respective findings were as follows: positive predictive value, 72.2% and 68.2%; false positive rate, 25.0% and 35.0%; and rate of positivity in the head and body/tail, 38.5% and 46.2%. When the artery with the largest change was taken as the dominant artery, the localization detection sensitivity was 76.9% for SASI (60 seconds) and 92.3% for SASI (120 seconds). The sensitivity of morphological imaging techniques for localization ranged from 61.5-91.7%. Conclusion Compared with SASI (60 seconds) or morphological imaging, the insulinoma localization ability of SASI (120 seconds) was superior. Given these findings, we believe that the IRI level should be measured at 120 seconds in the SASI test..
197. Hideyuki Okuma, Ryotaro Bouchi, Seizaburo Masuda, Takato Takeuchi, Masanori Murakami, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Suppression of extrapancreatic glucagon by octreotide may reduce the fasting and postprandial glucose levels in a diabetic patient after total pancreatectomy, Internal Medicine, 10.2169/internalmedicine.8963-17, 56, 22, 3061-3066, 2017, A 52-year-old woman was treated with sensor augmented pump therapy after undergoing total pancreatectomy for a nonfunctional pancreatic neuroendocrine tumor (NET). The secretion of both endogenous insulin and pancreatic glucagon were completely depleted. Octreotide long acting repeatable (Oct-LAR) was administered for the treatment of liver metastasis of NET. Both the fasting and postprandial glucagon levels decreased immediately after the administration of Oct-LAR. In a continuous glucose monitoring analysis, episodes of nocturnal hypoglycemia was found to increase and an improvement of postprandial hyperglycemia was observed. This case suggests that octreotide may reduce the glucose level in both the fasting and postprandial states, in part by the suppression of extrapancreatic glucagon..
198. Hideyuki Okuma, Koshi Hashimoto, Xin Wang, Noriaki Ohkiba, Nozomi Murooka, Norikazu Akizuki, Takeshi Inazawa, Yoshihiro Ogawa, Systemic sarcoidosis with thyroid involvement, Internal Medicine, 10.2169/internalmedicine.8324-16, 56, 16, 2181-2186, 2017, A 66-year-old woman, who was diagnosed with iritis, visited our hospital due to general malaise. A blood analysis revealed hypercalcemia. Computed tomography revealed mediastinal and hilar lymph node hyperpla-sia. Moreover,67Gallium scintigraphy demonstrated strong accumulation in the lesions, suggesting sarcoidosis. A core needle biopsy (CNB) of the hypoechoic areas of the thyroid was performed because the patient refused to undergo a bronchoscopic examination. The scattering of slightly acidophilic epithelioid cell granulo-mas was observed in the pathological examination of the biopsy specimen. Based on this finding, the patient was diagnosed with sarcoidosis. Although sarcoidosis rarely involves the thyroid gland, in the present case, thyroid CNB was an alternative diagnostic method that allowed a pathological diagnosis to be obtained..
199. Makiko Abe, Hidemi Ito, Isao Oze, Masatoshi Nomura, Yoshihiro Ogawa, Keitaro Matsuo, The more from East-Asian, the better
Risk prediction of colorectal cancer risk by GWAS-identified SNPs among Japanese, Journal of Cancer Research and Clinical Oncology, 10.1007/s00432-017-2505-4, 143, 12, 2481-2492, 2017, Background Little is known about the difference of genetic predisposition for CRC between ethnicities; however, many genetic traits common to colorectal cancer have been identified. This study investigated whether more SNPs identified in GWAS in East Asian population could improve the risk prediction of Japanese and explored possible application of genetic risk groups as an instrument of the risk communication. Methods 558 Patients histologically verified colorectal cancer and 1116 first-visit outpatients were included for derivation study, and 547 cases and 547 controls were for replication study. Among each population, we evaluated prediction models for the risk of CRC that combined the genetic risk group based on SNPs from GWASs in Euro-pean-population and a similarly developed model adding SNPs from GWASs in East Asian-population. We examined whether adding East Asian-specific SNPs would improve the discrimination. Results Six SNPs (rs6983267, rs4779584, rs4444235, rs9929218, rs10936599, rs16969681) from 23 SNPs by European-based GWAS and five SNPs (rs704017, rs11196172, rs10774214, rs647161, rs2423279) among ten SNPs by Asian-based GWAS were selected in CRC risk prediction model. Compared with a 6-SNP-based model, an 11-SNP model including Asian GWAS-SNPs showed improved discrimination capacity in Receiver operator characteristic analysis. A model with 11 SNPs resulted in statistically significant improvement in both derivation (P = 0.0039) and replication studies (P = 0.0018) compared with six SNP model. We estimated cumulative risk of CRC by using genetic risk group based on 11 SNPs and found that the cumulative risk at age 80 is approximately 13% in the high-risk group while 6% in the low-risk group. Conclusion We constructed a more efficient CRC risk prediction model with 11 SNPs including newly identified East Asian-based GWAS SNPs (rs704017, rs11196172, rs10774214, rs647161, rs2423279). Risk grouping based on 11 SNPs depicted lifetime difference of CRC risk. This might be useful for effective individualized prevention for East Asian..
200. Ogawa Yoshihiro, SF-1 deficiency causes lipid accumulation in Leydig cells via suppression of STAR and CYP11A1., Endocrine , 54, 2, 484-496, 2016.11.
201. Yoshihiro Ogawa, Molecular Mechanism of Lifestyle-related Diseases
See Both the Wood and Trees!, Fukuoka igaku zasshi = Hukuoka acta medica, 107, 11, 191-198, 2016.11, Energy homeostasis is maintained locally through parenchymal-stromal cell interaction and systemically through metabolic organ network. In obese adipose tissue, saturated fatty acids, which are released as a danger signal from hypertrophied adipocytes, stimulates a pathogen sensor TLR4 in the infiltrating macrophages, thus establishing a vicious cycle between adipocytes and macrophages to stimulate adipose tissue inflammation. Histologically, macrophages aggregate to constitute crown-like structures (CLS), where they are thought to scavenge the residual lipid droplets of dead adipocytes. Free fatty acids, when released from obese visceral fat depots, are transported in large quantities to the liver via the portal vein, where they are accumulated as ectopic fat, thus developing non-alcoholic fatty liver disease (NAFLD). There is a unique histological feature termed÷hepatic CLS (hCLS)øin the non-alcoholic steatohepatitis (NASH) liver, where macrophages aggregate to surround dead hepatocytes with large lipid droplets. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. Our data suggest that hCLS serves as an origin of hepatic inflammation and fibrosis during the progression from simple steatosis to NASH. Sodium glucose cotransporter 2 (SGLT2) inhibitors, an oral antidiabetic drug, promotes the urinaryexcretion of glucose by blocking its reabsorption in renal proximal tubules. Inhibition of SGLT2 lowers is expected to reduce body weight because of urinary calorie loss. Interestingly, SGLT2 inhibition improves hepatic steatosis in obese mice irrespective of body weight reduction. There is an inverse correlation between liver weight and adipose tissue weight in obese mice with SGLT2 inhibition, suggesting that SGLT2 inhibition induces the÷healthyøadipose tissue expansion and prevents ectopic fat accumulation in the liver. Our data suggest that seeing both the wood and trees is Required to understand the molecular mechanism of lifestyle-related diseases..
202. Megumi Hatano, Toshiro Migita, Tomokazu Ohishi, Yuichi Shima, Yoshihiro Ogawa, Ken Ichirou Morohashi, Yukihiro Hasegawa, Futoshi Shibasaki, SF-1 deficiency causes lipid accumulation in Leydig cells via suppression of STAR and CYP11A1, Endocrine, 10.1007/s12020-016-1043-1, 54, 2, 484-496, 2016.11, Genetic mutations of steroidogenic factor 1 (also known as Ad4BP or Nr5a1) have increasingly been reported in patients with 46,XY disorders of sex development (46,XY disorders of sex development). However, because the phenotype of 46,XY disorders of sex development with a steroidogenic factor 1 mutation is wide-ranging, its precise diagnosis remains a clinical problem. We previously reported the frequent occurrence of lipid accumulation in Leydig cells among patients with 46,XY disorders of sex development with a steroidogenic factor 1 mutation, an observation also reported by other authors. To address the mechanism of lipid accumulation in this disease, we examined the effects of steroidogenic factor 1 deficiency on downstream targets of steroidogenic factor 1 in in vitro and in vivo. We found that lipid accumulation in Leydig cells was enhanced after puberty in heterozygous steroidogenic factor 1 knockout mice compared with wild-type mice, and was accompanied by a significant decrease in steroidogenic acute regulatory protein and CYP11A1 expression. In mouse Leydig cell lines, steroidogenic factor 1 knockdown induced a remarkable accumulation of neutral lipids and cholesterol with reduced androgen levels. Steroidogenic factor 1 knockdown reduced the expression of steroidogenic acute regulatory protein and CYP11A1, both of which are transcriptional targets of steroidogenic factor 1 and key molecules for steroidogenesis from cholesterol in the mitochondria. Knockdown of either steroidogenic acute regulatory protein or CYP11A1 also induced lipid accumulation, and knockdown of both had an additive effect. Our data suggested that lipid accumulation in the Leydig cells of the 46,XY disorders of sex development phenotype with a steroidogenic factor 1 mutation is due, at least in part, to the suppression of steroidogenic acute regulatory protein and CYP11A1, and a resulting increase in unmetabolized cholesterol..
203. Ogawa Yoshihiro, Inflammatory responses increase secretion of MD-1 protein. , Int. Immunol., 28, 10, 503-512, 2016.10.
204. Richard Thomas Jennings, Erdenezaya Odkhuu, Akina Nakashima, Naoko Morita, Toshihiko Kobayashi, Ikuko Yamai, Miyako Tanaka, Takayoshi Suganami, Sanae Haga, Michitaka Ozaki, Yasuharu Watanabe, Yoshinori Nagai, Kiyoshi Takatsu, Takane Kikuchi-Ueda, Isao Ichimonji, Yoshihiro Ogawa, Hidekazu Takagi, Tatsuya Yamazaki, Kensuke Miyake, Sachiko Akashi-Takamura, Inflammatory responses increase secretion of MD-1 protein, International immunology, 10.1093/intimm/dxw031, 28, 10, 503-512, 2016.10, Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo. To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases..
205. Ogawa Yoshihiro, Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation. , Biochem. Biophys. Res. Commun., 476, 4, 614-619, 2016.08.
206. Masanori Murakami, Takanobu Yoshimoto, Yujiro Nakano, Kyoichiro Tsuchiya, Isao Minami, Ryotaro Bouchi, Yasuhisa Fujii, Kazuhiko Nakabayashi, Koshi Hashimoto, Ken ichiro Hata, Kazunori Kihara, Yoshihiro Ogawa, Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2016.06.007, 476, 4, 614-619, 2016.08, Background The adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. Methods We evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. Results We revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. Conclusion The present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs..
207. Ogawa Yoshihiro, Increased visceral adiposity with normal weight is associated with prevalence of non-alcoholic fatty liver disease in Japanese patients with type 2 diabetes., J. Diabetes. Investig., 7, 607-614, 2016.07.
208. Ryotaro Bouchi, Takato Takeuchi, Momoko Akihisa, Norihiko Ohara, Yujiro Nakano, Rie Nishitani, Masanori Murakami, Tatsuya Fukuda, Masamichi Fujita, Isao Minami, Masatomo Mihara, Takanobu Yoshimoto, Yoshihiro Ogawa, Increased visceral adiposity with normal weight is associated with the prevalence of non-alcoholic fatty liver disease in Japanese patients with type 2 diabetes, Journal of Diabetes Investigation, 10.1111/jdi.12443, 7, 4, 607-614, 2016.07, Aims/Introduction: To investigate the impact of increased visceral adiposity with normal weight (OB[−]VA[+]) on the prevalence of non-alcoholic fatty liver disease in patients with type 2 diabetes. Materials and Methods: This was a cross-sectional study of 140 Japanese patients with type 2 diabetes (mean age 65 ± 11 year; 44.6% women). Visceral fat area (VFA; cm2) and liver attenuation index (LAI) were assessed by abdominal computed tomography. The patients were divided into four groups by VFA and body mass index (BMI; kg/m2) as follows: BMI <25 kg/m2 and VFA <100 cm2 (OB[−]VA[−]), BMI ≥25 kg/m2 and VFA <100 cm2 (OB[+]VA[−]), BMI <25 kg/m2 and VFA ≥100 cm2 (OB[−]VA[+]), and BMI ≥25 kg/m2 and VFA ≥100 cm2 (OB[+]VA[+]). Multivariate linear regression and logistic regression analysis were carried out to determine the impact of OB(−)VA(+) on LAI. Results: In the present study, 25.0% were OB(−)VA(+) patients, where the LAI levels were lower (1.09 ± 0.22) than those in OB(−)VA(−) patients (1.23 ± 0.15), and were equivalent to those in OB(+)VA(+) patients (1.03 ± 0.26). In multivariate linear regression analysis, OB(−)VA(+) was independently associated with LAI (standardized β−0.212, P = 0.014). In multivariate logistic regression analysis, OB(−)VA(+) was a significant predictor of LAI <0.9 (odds ratio 5.88, 95% confidence interval 1.03−33.52, P = 0.046). Conclusions: The present study provides evidence that increased visceral adiposity with normal weight is a strong predictor for the prevalence of non-alcoholic fatty liver disease in Japanese patients with type 2 diabetes..
209. Ogawa Yoshihiro, A case of Cushing's syndrome with multiple adrenocortical adenomas composed of compact cells and clear cells. , Endocr. Pathol., 27, 2, 136-141, 2016.06.
210. Masahiro Asakawa, Takanobu Yoshimoto, Mitsutane Ota, Mitsuyuki Numasawa, Yuriko Sasahara, Takato Takeuchi, Yujiro Nakano, Norihiko Oohara, Masanori Murakami, Ryotaro Bouchi, Isao Minami, Kyoichiro Tsuchiya, Koshi Hashimoto, Hajime Izumiyama, Naoko Kawamura, Kazunori Kihara, Mariko Negi, Takumi Akashi, Yoshinobu Eishi, Hironobu Sasano, Yoshihiro Ogawa, A Case of Cushing’s Syndrome with Multiple Adrenocortical Adenomas Composed of Compact Cells and Clear Cells, Endocrine Pathology, 10.1007/s12022-016-9423-x, 27, 2, 136-141, 2016.06, A 58-year-old woman was referred to our hospital for Cushingoid features and diagnosed as adrenal Cushing’s syndrome due to a right adrenocortical mass (60 × 55 mm). The mass was composed of three different tumors; the first one was homogeneously lipid-poor neoplasm measuring 20 × 13 mm located at the most dorsal region, the second one was heterogeneous and lipid-rich tumor containing multiple foci of calcification measuring 50 × 32 mm located at the central region, and the last one was heterogeneous harboring dilated and tortuous vessels and lipid-poor one measuring 35 × 18 mm at the most ventral region of the adrenal gland. A right adrenalectomy was subsequently performed by open surgery. Macroscopic and microscopic analyses revealed that all three tumors were adrenocortical adenomas; the first one represents a pigmented adrenocortical adenoma, the second one adrenocortical adenoma associated with degeneration, and the third one adrenocortical adenoma harboring extensive degeneration. Immunohistochemical analysis of the steroidogenic enzymes also revealed that all of the tumors had the capacity of synthesizing cortisol. This is a very rare case of Cushing’s syndrome caused by multiple adrenocortical adenomas including a pigmented adenoma. Immunohistochemical analysis of steroidogenic enzymes contributed to understanding of steroidogenesis in each of these three different adrenocortical adenomas in this case..
211. Takanori Chiba, Erika Ishihara, Norio Miyamura, Rika Narumi, Mihoko Kajita, Yasuyuki Fujita, Akira Suzuki, Yoshihiro Ogawa, Hiroshi Nishina, MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status, Scientific reports, 10.1038/srep28383, 6, 2016.06, Cell competition is a cell-cell interaction by which a cell compares its fitness to that of neighboring cells. The cell with the relatively lower fitness level is the "loser" and actively eliminated, while the cell with the relatively higher fitness level is the "winner" and survives. Recent studies have shown that cells with high Yes-associated protein (YAP) activity win cell competitions but the mechanism is unknown. Here, we report the unexpected finding that cells overexpressing constitutively active YAP undergo apical extrusion and are losers, rather than winners, in competitions with normal mammalian epithelial cells. Inhibitors of metabolism-related proteins such as phosphoinositide-3-kinase (PI3K), mammalian target of rapamycin (mTOR), or p70S6 kinase (p70S6K) suppressed this apical extrusion, as did knockdown of vimentin or filamin in neighboring cells. Interestingly, YAP-overexpressing cells switched from losers to winners when co-cultured with cells expressing K-Ras (G12V) or v-Src. Thus, the role of YAP in deciding cell competitions depends on metabolic factors and the status of neighboring cells..
212. Rumi Hachiya, Takuya Shiihashi, Ibuki Shirakawa, Yorihiro Iwasaki, Yoshihiro Matsumura, Yumiko Oishi, Yukiteru Nakayama, Yoshihiro Miyamoto, Ichiro Manabe, Kozue Ochi, Miyako Tanaka, Nobuhito Goda, Juro Sakai, Takayoshi Suganami, Yoshihiro Ogawa, The H3K9 methyltransferase Setdb1 regulates TLR4-mediated inflammatory responses in macrophages, Scientific reports, 10.1038/srep28845, 6, 2016.06, Proinflammatory cytokine production in macrophages involves multiple regulatory mechanisms, which are affected by environmental and intrinsic stress. In particular, accumulating evidence has suggested epigenetic control of macrophage differentiation and function mainly in vitro. SET domain, bifurcated 1 (Setdb1, also known as Eset) is a histone 3 lysine 9 (H3K9)-specific methyltransferase and is essential for early development of embryos. Here we demonstrate that Setdb1 in macrophages potently suppresses Toll-like receptor 4 (TLR4)-mediated expression of proinflammatory cytokines including interleukin-6 through its methyltransferase activity. As a molecular mechanism, Setdb1-deficiency decreases the basal H3K9 methylation levels and augments TLR4-mediated NF-κB recruitment on the proximal promoter region of interleukin-6, thereby accelerating interleukin-6 promoter activity. Moreover, macrophage-specific Setdb1-knockout mice exhibit higher serum interleukin-6 concentrations in response to lipopolysaccharide challenge and are more susceptible to endotoxin shock than wildtype mice. This study provides evidence that the H3K9 methyltransferase Setdb1 is a novel epigenetic regulator of proinflammatory cytokine expression in macrophages in vitro and in vivo. Our data will shed insight into the better understanding of how the immune system reacts to a variety of conditions..
213. Ogawa Yoshihiro, Retrograde pyelonephritis and lumbar spondylitis due to Salmonella typhi in a type 2 diabetic patient with neurogenic bladder., J. Diabetes. Investig., 7, 436-439, 2016.05.
214. Tatsuya Fukuda, Ryotaro Bouchi, Isao Minami, Norihiko Ohara, Yujiro Nakano, Rie Nishitani, Masanori Murakami, Takato Takeuchi, Momoko Akihisa, Masamichi Fujita, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Retrograde pyelonephritis and lumbar spondylitis as a result of Salmonella typhi in a type 2 diabetes patient with neurogenic bladder, Journal of Diabetes Investigation, 10.1111/jdi.12375, 7, 3, 436-439, 2016.05, We present a case of a 62-year-old diabetic woman with acute pyelonephritis and spondylitis caused by Salmonella typhi. She was admitted to Tokyo Medical Dental University Hospital, Tokyo, Japan, because of unconsciousness and was diagnosed with sepsis by retrograde pyelonephritis as a result of Salmonella typhi. Antibiotics treatment was immediately started; however, she subsequently developed lumbar spondylitis, and long-term conservative treatment with antibiotics and a fixing device were required. This is the first report of a diabetic patient who developed retrograde urinary tract infection with Salmonella typhi, followed by sepsis and spondylitis. The infection could be a result of diabetic neuropathy, presenting neurogenic bladder and hydronephrosis. The patient was successfully treated with antibiotics and became asymptomatic with normal inflammatory marker levels, and no clinical sign of recurrence was observed in the kidney and spine at 4 months..
215. Ogawa Yoshihiro, Clinical relevance of dual-Energy X-ray absorptiometry (DXA) as a simultaneous evaluation of fatty liver disease and atherosclerosis in patients with type 2 diabetes., Cardiovasc. Diabetol. , 15, e64, 2016.04.
216. Ryotaro Bouchi, Yujiro Nakano, Norihiko Ohara, Takato Takeuchi, Masanori Murakami, Masahiro Asakawa, Yuriko Sasahara, Mitsuyuki Numasawa, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Clinical relevance of dual-energy X-ray absorptiometry (DXA) as a simultaneous evaluation of fatty liver disease and atherosclerosis in patients with type 2 diabetes, Cardiovascular Diabetology, 10.1186/s12933-016-0384-7, 15, 1, 2016.04, Background: Whole body dual-energy X-ray absorptiometry (DXA) can simultaneously measure both regional fat and non-fat mass. Android-to-gynoid (A/G) ratio measured by DXA has been reported to be associated with cardiovascular risks and visceral adiposity; however, little is known regarding its relationship with fatty liver disease and atherosclerosis among patients with diabetes. This study was designed to investigate the association of android and gynoid fat mass measured by DXA with fatty liver disease and atherosclerosis in patients with type 2 diabetes. Methods: This is a cross-sectional study of 259 patients with type 2 diabetes (mean age 64 ± 13 years; 40.2 % female). Android and gynoid fat mass (kg) were measured by DXA. Skeletal muscle index (SMI) was calculated as appendicular non-fat mass (kg) divided by height (m2). Visceral fat area (VFA, cm2), subcutaneous fat area (SFA, cm2), and liver attenuation index (LAI) were assessed by abdominal computed tomography. Intima media thickness (IMT, mm) in common carotid arteries was determined by carotid ultrasonography. Results: A/G ratio was significantly correlated with VFA (r = 0.72, p < 0.001), SFA (r = 0.32, p < 0.001) and LAI (r = -0.26, p < 0.001). A/G ratio (standardized β -0.223, p = 0.002) as well as VFA (standardized β -0.226, p = 0.001) were significantly associated with LAI in the univariate model. A/G ratio remained to be significantly associated with LAI (standardized β -0.224, p = 0.005) after adjusting for covariates including body mass index and transaminases. Among patients with low SMI (SMI < 7.0 in male and < 5.4 in female), A/G ratio was significantly associated with carotid IMT in the multivariate model (standardized β 0.408, p = 0.014). Conclusions: DXA can be used to simultaneously estimate the risks for both fatty liver disease and atherosclerosis in patients with type 2 diabetes..
217. Ogawa Yoshihiro, Indirect measure of visceral adiposity "a body shape index" (ABSI) is associated with arterial stiffness in patients with type 2 diabetes. , BMJ Open Diab. Res. Care, 4, 1, e000188, 2016.03.
218. Ogawa Yoshihiro, Ipragliflozin improves hepatic steatosis in obese mice and liver dysfunction in type 2 diabetic patients irrespective of body weight reduction. , PLoS ONE , 11, 3, e0151511, 2016.03.
219. Chikara Komiya, Kyoichiro Tsuchiya, Kumiko Shiba, Yasutaka Miyachi, Shunsaku Furuke, Noriko Shimazu, Shinobu Yamaguchi, Kazuo Kanno, Yoshihiro Ogawa, Ipragliflozin improves hepatic steatosis in obese mice and liver dysfunction in type 2 diabetic patients irrespective of body weight reduction, PloS one, 10.1371/journal.pone.0151511, 11, 3, 2016.03, Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis..
220. Yasutaka Miyachi, Kyoichiro Tsuchiya, Yoshihiro Ogawa, Lifestyle-related diseases and an inter-organ metabolic network, Clinical calcium, 26, 3, 392-398, 2016.03, Lifestyle-related diseases such as type 2 diabetes, hypertension and dyslipidemia are a prominent cause of mortality in Japan, and there is a strong requirement for elucidation of detailed molecular mechanisms and effective therapeutic strategies. Obesity-induced adipose tissue inflammation leads to dysregulation of adipokine production, which can cause lifestyle-related diseases. The interaction of organ systems via endocrine or neural networks is recognized as an important factor in the pathogenesis and promotion of lifestyle-related diseases. Therefore, further investigation for the interaction between adipose tissues and bones can provide new treatment strategies of metabolic bone disorders..
221. Yoshihiro Ogawa, Takanobu Yoshimoto, Programs for Continuing Medical Education
A session; 3. Primary aldosteronism as a cause of secondary hypertension, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.105.434, 105, 3, 434-438, 2016.03.
222. Koshi Hashimoto, Tetsuya Tagami, Hajime Yamakage, Kazuya Muranaka, Masashi Tanaka, Shinji Odori, Shigeo Kono, Akira Shimatsu, Yoshihiro Ogawa, Noriko Satoh-Asahara, Serum free thyroxine levels are associated with the efficacy of weight reduction therapy in obese female patients, Endocrine Journal, 10.1507/endocrj.EJ15-0498, 63, 3, 221-229, 2016.03, Thyroid function is strongly associated with obesity. The aim of this study is to investigate whether serum free thyroxine (FT4) and/or thyrotropin (TSH) levels are associated with the efficacy of weight reduction therapy in obese patients. We enrolled a total of 283 obese patients and cross-sectionally investigated the association of serum FT4 and/or TSH levels with metabolic features. Furthermore, in 97 obese patients who received 6-month weight reduction therapy, we assessed the relationship of serum FT4 and/or TSH levels to the efficacy of weight reduction therapy. Neither baseline serum FT4 nor TSH levels showed any correlations with body weight (BW) and body mass index (BMI) in these obese patients. However, in 57 obese female patients who underwent weight reduction therapy for six months, serum FT4 levels prior to the therapy was negatively correlated with the degrees of reduction of BW (r = -0.354, p = 0.007) and BMI (r = -0.373, p = 0.004). The correlation between baseline serum FT4 levels with the efficacy of weight reduction therapy was not observed in obese male or postmenopausal female patients. This study demonstrates that baseline serum FT4 levels are associated with weight reduction in obese female premenopausal patients. Therefore, baseline FT4 levels can be used as a clinical, noninvasive, hormonal predictor of weight reduction efficacy in obese patients..
223. Yoshihiro Ogawa, Koshi Hashimoto, The 43rd Scientific Meeting
Perspectives of Internal Medicine; Genetic predisposition and related life-style underlying metabolic disorders; 2. Genetic and Environmental Susceptibility : 2) Genome and epigenome in obesity, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 105, 3, 376-382, 2016.03.
224. Ogawa Yoshihiro, Serum free thyroxine levels is associated with the efficacy of weight reduction therapy in obese female patients., Endocr. J., 63, 3, 221-229, 2016.02.
225. Ogawa Yoshihiro, Gene expression profiling of white adipose tissue reveals paternal transmission of proneness to obesity., Sci. Rep., 6, e21693, 2016.02.
226. Sumiyo Morita, Kazuhiko Nakabayashi, Tomoko Kawai, Keiko Hayashi, Takuro Horii, Mika Kimura, Yasutomi Kamei, Yoshihiro Ogawa, Kenichiro Hata, Izuho Hatada, Gene expression profiling of white adipose tissue reveals paternal transmission of proneness to obesity, Scientific reports, 10.1038/srep21693, 6, 2016.02, Previously, we found that C57BL/6J (B6) mice are more prone to develop obesity than PWK mice. In addition, we analyzed reciprocal crosses between these mice and found that (PWK x B6) F1 mice, which have B6 fathers, are more likely to develop dietary obesity than (B6 x PWK) F1 mice, which have B6 mothers. These results suggested that diet-induced obesity is paternally transmitted. In this study, we performed transcriptome analysis of adipose tissues of B6, PWK, (PWK x B6) F1, and (B6 x PWK) F1 mice using next-generation sequencing. We found that paternal transmission of diet-induced obesity was correlated with genes involved in adipose tissue inflammation, metal ion transport, and cilia. Furthermore, we analyzed the imprinted genes expressed in white adipose tissue (WAT) and obesity. Expression of paternally expressed imprinted genes (PEGs) was negatively correlated with body weight, whereas expression of maternally expressed imprinted genes (MEGs) was positively correlated. In the obesity-prone B6 mice, expression of PEGs was down-regulated by a high-fat diet, suggesting that abnormally low expression of PEGs contributes to high-fat diet-induced obesity in B6 mice. In addition, using single-nucleotide polymorphisms that differ between B6 and PWK, we identified candidate imprinted genes in WAT..
227. Ogawa Yoshihiro, MDCK cells expressing constitutively active Yes-associated protein (YAP) undergo apical extrusion depending on neighboring cell status., Sci. Rep., 6, e28383, 2016.01.
228. Ogawa Yoshihiro, The H3K9 methyltransferase Setdb1 regulates TLR4-mediated inflammatory response in macrophages. , Sci. Rep. , 6, e28845, 2016.01.
229. Ryotaro Bouchi, Norihiko Ohara, Masahiro Asakawa, Yujiro Nakano, Takato Takeuchi, Masanori Murakami, Yuriko Sasahara, Mitsuyuki Numasawa, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Is visceral adiposity a modifier for the impact of blood pressure on arterial stiffness and albuminuria in patients with type 2 diabetes?, Cardiovascular Diabetology, 10.1186/s12933-016-0335-3, 15, 1, 2016.01, Background: We aimed to investigate whether visceral adiposity could modify the impact of blood pressure on arterial stiffness and albuminuria in patients with type 2 diabetes. Methods: This cross-sectional study examines the interaction of visceral adiposity with increased blood pressure on arterial stiffness and albuminuria. 638 patients with type 2 diabetes (mean age 64 ± 12 years; 40 % female) were enrolled. Visceral fat area (VFA, cm2) was assessed by a dual-impedance analyzer, whereby patients were divided into those with VFA < 100 (N = 341) and those with VFA ≥ 100 (N = 297). Albuminuria was measured in a single 24-h urine collection (UAE, mg/day) and brachial-ankle pulse wave velocity (ba-PWV, cm/s) was used for the assessment of arterial stiffening. Linear regression analyses were used to investigate the association of systolic blood pressure (SBP) and VFA with UAE and baPWV. Results: Patients with VFA ≥ 100 were significantly younger, had higher SBP, HbA1c, triglycerides, UAE, alanine aminotransferase, C-reactive protein and lower high-density lipoprotein and shorter duration of diabetes than those with VFA < 100. SBP was significantly and almost equivalently associated with ba-PWV both in VFA < 100 (standardized β 0.224, p = 0.001) and VFA ≥ 100 (standardized β 0.196, p = 0.004) patients in the multivariate regression analysis adjusting for covariates including age, gender, HbA1c, diabetic complications and the use of insulin and anti-hypertensive agents. By contrast, the association of SBP with UAE was stronger in patients with VFA ≥ 100 (standardized β 0.263, p = 0.001) than that in patients with VFA < 100 (standardized β 0.140, p = 0.080) in the multivariate regression model. In the whole cohort, the significant interaction between SBP and VFA on UAE (standardized β 0.172, p = 0.040) but not on ba-PWV (standardized β -0.008, p = 0.916) was observed. Conclusions: The effect of increased blood pressure on arterial stiffness is almost similar in type 2 diabetic patients with both low and high visceral adiposity, while its association with albuminuria is stronger in the latter..
230. Ryotaro Bouchi, Masahiro Asakawa, Norihiko Ohara, Yujiro Nakano, Takato Takeuchi, Masanori Murakami, Yuriko Sasahara, Mitsuyuki Numasawa, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, Indirect measure of visceral adiposity ‘A body shape index’ (ABSI) is associated with arterial stiffness in patients with type 2 diabetes, BMJ Open Diabetes Research and Care, 10.1136/bmjdrc-2015-000188, 4, 1, 2016, Objective: Among indirect measures of visceral adiposity, A Body Shape Index (ABSI), which is defined as waist circumference (WC)/(body mass index (BMI)2/3×height1/2), is unique in that ABSI is positively correlated with visceral adiposity and is supposed to be independent of BMI. ABSI has been also shown to be linearly and positively associated with visceral fat mass and all-cause and cardiovascular disease (CVD) in the general population. It is, however, uncertain whether ABSI could be associated with arterial stiffness in patients with diabetes. Methods: This is a cross-sectional study of 607 patients with type 2 diabetes (mean age 64±12 years; 40.0% female). Visceral fat area (VFA, cm2) and subcutaneous fat area (SFA, cm2) were assessed with a dual-impedance analyzer. In order to estimate the risk for CVD, brachial-ankle pulse wave velocity (baPWV, cm) was used for the assessment of arterial stiffness. Results: ABSI was significantly and positively correlated with VFA (r=0.138, p=0.001) and negatively associated with BMI (r=−0.085, p=0.037). The correlation of z-score for ABSI with VFA remained significant (r=0.170, p<0.001) but not with BMI (r=0.009, p=0.820). ABSI (standardized β 0.095, p=0.043) but not WC (standardized β −0.060, p=0.200) was significantly and positively correlated with baPWV in the multivariate model including BMI as a covariate. Conclusions: ABSI appears to reflect visceral adiposity independently of BMI and to be a substantial marker of arterial stiffening in patients with type 2 diabetes..
231. Yoshihiro Ogawa, Obesity, diabetes, and gut microbiome, Therapeutic Research, 37, 4, 338-340, 2016.
232. Masanori Murakami, Takanobu Yoshimoto, Isao Minami, Ryotaro Bouchi, Kyoichiro Tsuchiya, Koshi Hashimoto, Hajime Izumiyama, Yasuhisa Fujii, Takashi Endo, Takumi Akashi, Koshiro Nishimoto, Kuniaki Mukai, Kazunori Kihara, Yoshihiro Ogawa, A Novel Somatic Deletion Mutation of ATP2B3 in Aldosterone-Producing Adenoma, Endocrine Pathology, 10.1007/s12022-015-9400-9, 26, 4, 328-333, 2015.12, Aldosterone-producing adenoma (APA) is a form of primary aldosteronism (PA). Recent studies suggested that somatic mutations in the KCNJ5, ATP1A1, ATP2B3, and CACNA1D genes are involved in the pathogenesis of APA. We report a case of a 62-year-old man diagnosed as PA with left adrenal mass. He underwent adrenalectomy for treatment. We identified a novel somatic deletion mutation in ATP2B3 in the adrenal tumor: c.1269_1274delTGTGCT which spans three codons (423–425) resulting in p.Val424_Leu425del. Immunohistochemical analysis revealed strong expression of aldosterone synthase (CYP11B2) in the tumor tissue, which is consistent with APA. Here, we identified a novel somatic deletion mutation in ATP2B3, which results in the amino acid sequences increasing intracellular calcium concentrations as reported previously, leading to increased aldosterone synthase (CYP11B2) expression and following excess aldosterone production in the APA cells. The novel ATP2B3 mutation detected in our case supports the pathogenic significance of the locus spanning the codon 424–426 of ATP2B3..
233. Masahiro Asakawa, Hiroshi Mitsui, Momoko Akihisa, Tetsuo Sekine, Yoshihiro Niitsu, Arisa Kobayashi, Atsuko Miyake, Naoaki Hashimoto, Mitsunobu Kawamura, Yoshihiro Ogawa, Efficacy and safety of sitagliptin for the treatment of diabetes mellitus complicated by chronic liver injury, SpringerPlus, 10.1186/s40064-015-1135-z, 4, 1, 2015.12, Aim: To investigate the efficacy and safety of a dipeptidyl peptidase-4 inhibitor, sitagliptin, for treating diabetes mellitus complicated by chronic liver injury. Methods: Sitagliptin was administered for 13.7 ± 10.1 months to 122 patients with DM complicated by chronic liver injury (including 19 patients with liver cirrhosis), and changes in hemoglobin A1c (HbA1c) and liver enzymes (transaminases, etc.) were evaluated. Results: HbA1c was reduced from 8.48 ± 1.43% to 7.87 ± 1.35% (P < 0.001). Among liver enzymes, alanine aminotransferase (ALT) levels improved from 75.1 ± 45.2 to 65.8 ± 35.8 IU/L (P = 0.012) and gamma-glut amyl-trans peptidase from 155.2 ± 161.1 to 133.2 ± 127.4 IU/L (P = 0.044). Among the causes of liver injury, non-alcoholic fatty liver disease and alcoholic liver disease both showed the reductions in HbA1c with no deterioration of liver enzymes. An analysis of 19 patients with liver cirrhosis also showed reductions in HbA1c with no deterioration of liver enzymes. Conclusion: It is suggested that sitagliptin can be administered effectively and safely to patients with diabetes mellitus complicated by chronic liver injury, including liver cirrhosis..
234. Nanami Senoo, Noriyuki Miyoshi, Naoko Goto-Inoue, Kimiko Minami, Ryoji Yoshimura, Akihito Morita, Naoki Sawada, Junichiro Matsuda, Yoshihiro Ogawa, Mitsutoshi Setou, Yasutomi Kamei, Shinji Miura, PGC-1 α -mediated changes in phospholipid profiles of exercise-trained skeletal muscle, Journal of Lipid Research, 10.1194/jlr.M060533, 56, 12, 2286-2296, 2015.12, Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α ), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α . We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle..
235. Keiko Muramatsu-Kato, Hiroaki Itoh, Yukiko Kohmura-Kobayashi, Urmi J. Ferdous, Naoaki Tamura, Chizuko Yaguchi, Toshiyuki Uchida, Kazunao Suzuki, Koshi Hashimoto, Takayoshi Suganami, Yoshihiro Ogawa, Naohiro Kanayama, Undernourishment in utero Primes Hepatic Steatosis in Adult Mice Offspring on an Obesogenic Diet; Involvement of Endoplasmic Reticulum Stress, Scientific reports, 10.1038/srep16867, 5, 2015.11, In order to investigate the possible involvement of endoplasmic reticulum (ER) stress in the developmental origins of hepatic steatosis associated with undernourishment in utero, we herein employed a fetal undernourishment mouse model by maternal caloric restriction in three cohorts; cohort 1) assessment of hepatic steatosis and the ER stress response at 9 weeks of age (wks) before a high fat diet (HFD), cohort 2) assessment of hepatic steatosis and the ER stress response on a HFD at 17 wks, cohort 3) assessment of hepatic steatosis and the ER stress response at 22 wks on a HFD after the alleviation of ER stress with a chemical chaperone, tauroursodeoxycholic acid (TUDCA), from 17 wks to 22 wks. Undernourishment in utero significantly deteriorated hepatic steatosis and led to the significant integration of the ER stress response on a HFD at 17 wks. The alleviation of ER stress by the TUDCA treatment significantly improved the parameters of hepatic steatosis in pups with undernourishment in utero, but not in those with normal nourishment in utero at 22 wks. These results suggest the pivotal involvement of the integration of ER stress in the developmental origins of hepatic steatosis in association with undernourishment in utero..
236. Ryotaro Bouchi, Takato Takeuchi, Momoko Akihisa, Norihiko Ohara, Yujiro Nakano, Rie Nishitani, Masanori Murakami, Tatsuya Fukuda, Masamichi Fujita, Isao Minami, Hajime Izumiyama, Koshi Hashimoto, Takanobu Yoshimoto, Yoshihiro Ogawa, High visceral fat with low subcutaneous fat accumulation as a determinant of atherosclerosis in patients with type 2 diabetes, Cardiovascular Diabetology, 10.1186/s12933-015-0302-4, 14, 1, 2015.10, Background: Abdominal visceral obesity has been reported to be associated with cardiovascular risks than body mass index, waist circumference, and abdominal subcutaneous fat. On the other hand, there is evidence that subcutaneous fat has a beneficial role against cardio-metabolic risks such as diabetes or dyslipidemia. However, little is known regarding the association between high visceral fat with low subcutaneous fat accumulation and the risk for atherosclerosis. Methods: This study was designed to elucidate whether high visceral fat with low subcutaneous fat accumulation enhances the risk for atherosclerosis in patients with type 2 diabetes. This is a cross-sectional study of 148 patients with type 2 diabetes (mean age 65 ± 12 years; 44.5 % female). Visceral fat area (VFA, cm2) and subcutaneous fat area (SFA, cm2) were assessed by abdominal computed tomography. Carotid intima media thickness (CIMT, mm) measured by ultrasonography was used for the assessment of atherosclerosis. Patients were divided into four groups: SFA < 100 cm2 and VFA < 100 cm2 [S(-)V(-)], SFA ≥ 100 cm2 and VFA < 100 cm2 [S(+)V(-)], SFA < 100 cm2 and VFA ≥ 100 cm2 [S(-)V(+)], and SFA ≥ 100 cm2 and VFA ≥ 100 cm2 [S(+)V(+)]. Linear regression analysis with a stepwise procedure was used for the statistical analyses. Results: Among the patients examined, 16.3 % were S(-)V(+). Mean (95 % confidence interval) of CIMT adjusting for age and gender were 0.80 (0.69-0.91), 0.86 (0.72-1.01), 1.28 (1.11-1.44) and 0.83 (0.77-0.88) in patients with S(-)V(-), S(+)V(-), S(-)V(+) and S(+)V(+), respectively (p < 0.001). The S(-)V(+) patients exhibited significantly older than S(-)V(-) patients and those with S(+)V(+) and had a highest VFA-SFA ratio (V/S ratio) among the four groups. S(-)V(+) patients were male predominant (100 % male), and S(+)V(-) patients showed female predominance (82 % female). In multivariate linear regression analysis (Adjusted R2 = 0.549), S(-)V(+) was significantly associated with CIMT (Standardized β 0.423, p < 0.001). Notably, S(+)V(+) was inversely associated with CIMT in the multivariate model. Conclusions: This study provides evidence that high visceral fat with low subcutaneous fat accumulation is an important determinant of carotid atherosclerosis and high subcutaneous fat could be protective against atherosclerosis in patients with type 2 diabetes..
237. Mari Hotta, Reiko Horikawa, Hiroyo Mabe, Shin Yokoyama, Eiko Sugiyama, Tadato Yonekawa, Masamitsu Nakazato, Yuri Okamoto, Chisato Ohara, Yoshihiro Ogawa, Epidemiology of anorexia nervosa in Japanese adolescents, BioPsychoSocial Medicine, 10.1186/s13030-015-0044-2, 9, 1, 2015.08, Background: No epidemiologic survey examining eating disorders in Japan has been done at a national level since 1992. The prevalence of anorexia nervosa, as assessed by questionnaires to hospitals, is thought to be underestimated because patients with anorexia nervosa tend to avoid consultations. In conformity with the School Health and Safety Act of Japan, schools are required to have physicians perform a medical examination of students every year. The teachers in charge of health education and school physicians determine the height, weight, and health condition, and examine the medical records of each student. Therefore, we as members of the Survey Committee for Eating Disorders of the Japanese Ministry of Health, Labour, and Welfare conducted an epidemiologic survey using questionnaires sent to schools in seven prefectures to determine the current prevalence of anorexia nervosa among adolescents. Methods: We sent a questionnaire to elementary, junior high, and senior high schools. Questionnaires contained items on the number of students, patients with anorexia nervosa in each grade who were diagnosed by specialists, and students who the school physician strongly suspected to have anorexia nervosa but who did not undergo a clinical examination in a medical institution. Results: We found patients of both sexes with anorexia nervosa aged 9-10 years in elementary schools. The point prevalence of anorexia nervosa for girls, including strongly suspected cases, in the three grades of junior high school and three grades of senior high school were 0-0.17 %, 0-0.21 %, 0.17-0.40 %, 0.05-0.56 %, 0.17-0.42 % and 0.09-0.43 %, respectively. We also confirmed a prominent sex difference in the prevalence of anorexia nervosa. The prevalence of boys was one third that of girls in some prefectures. One third to one half of diagnosed and strongly suspected students with anorexia nervosa had not received medical consultation or treatment. Conclusions: Although the prevalence of anorexia nervosa had regional differences in Japan, it has reached levels comparable to those in Western societies. Because no eating disorder center exists and the treatment environment is poor, national action to address this disease is a pressing need in Japan..
238. Masanori Murakami, Takanobu Yoshimoto, Kazuhiko Nakabayashi, Kyoichiro Tsuchiya, Isao Minami, Ryotaro Bouchi, Hajime Izumiyama, Yasuhisa Fujii, Kosei Abe, Chiharu Tayama, Koshi Hashimoto, Takayoshi Suganami, Ken Ichiro Hata, Kazunori Kihara, Yoshihiro Ogawa, Integration of transcriptome and methylome analysis of aldosterone-producing adenomas, European Journal of Endocrinology, 10.1530/EJE-15-0148, 173, 2, 185-195, 2015.08, Objective: The pathophysiology of aldosterone-producing adenomas (APA) has been investigated intensively through genetic and genomic approaches. However, the role of epigenetics in APA is not fully understood. In the present study, we explored the relationship between gene expression and DNA methylation status in APA. Methods: We conducted an integrated analysis of transcriptome and methylome data of paired APA-adjacent adrenal gland (AAG) samples from the same patient. The adrenal specimens were obtained from seven Japanese patients with APA who underwent adrenalectomy. Gene expression and genome-wide CpG methylation profiles were obtained from RNA and DNA samples that were extracted from those seven paired tissues. Results: Methylome analysis showed global CpG hypomethylation in APA relative to AAG. The integration of gene expression and methylation status showed that 34 genes were up-regulated with CpG hypomethylation in APA. Of these, three genes (CYP11B2, MC2R, and HPX) may be related to aldosterone production, and five genes (PRRX1, RAB38, FAP, GCNT2, and ASB4) are potentially involved in tumorigenesis. Conclusion: The present study is the first methylome analysis to compare APA with AAG in the same patients. Our integrated analysis of transcriptome and methylome revealed DNA hypomethylation in APA and identified several up-regulated genes with DNA hypomethylation that may be involved in aldosterone production and tumorigenesis..
239. Yukino Hatazawa, Nanami Senoo, Miki Tadaishi, Yoshihiro Ogawa, Osamu Ezaki, Yasutomi Kamei, Shinji Miura, Metabolomic analysis of the skeletal muscle mice overexpressing PGC-1α, PloS one, 10.1371/journal.pone.0129084, 10, 6, 2015.06, Peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors whose expression increases in the skeletal muscle during exercise. We have previously made transgenic mice overexpressing PGC-1α in the skeletal muscle (PGC-1α-Tg mice). PGC-1α upregulates the expression of genes associated with red fibers, mitochondrial function, fatty acid oxidation, and branched chain amino acid (BCAA) degradation. However, global analyses of the actual metabolic products have not been investigated. In this study, we conducted metabolomic analysis of the skeletal muscle in PGC-1α-Tg mice by capillary electrophoresis with electrospray ionization time-of-flight mass spectrometry. Principal component analysis and hierarchical cluster analysis showed clearly distinguishable changes in the metabolites between PGC-1α-Tg and wild-type control mice. Changes were observed in metabolite levels of various metabolic pathways such as the TCA cycle, pentose phosphate pathway, nucleotide synthesis, purine nucleotide cycle, and amino acid metabolism, including BCAA and β-alanine. Namely, metabolic products of the TCA cycle increased in PGC-1α-Tg mice, with increased levels of citrate (2.3-fold), succinate (2.2-fold), fumarate (2.8-fold), and malate (2.3-fold) observed. Metabolic products associated with the pentose phosphate pathway and nucleotide biosynthesis also increased in PGC-1α-Tg mice. Meanwhile, BCAA levels decreased (Val, 0.7-fold; Leu, 0.8-fold; and Ile, 0.7-fold), and Glu (3.1-fold) and Asp (2.2-fold) levels increased. Levels of β-alanine and related metabolites were markedly decreased in PGC-1α-Tg mice. Coordinated regulation of the TCA cycle and amino acid metabolism, including BCAA, suggests that PGC-1α plays important roles in energy metabolism. Moreover, our metabolomics data showing the activation of the purine nucleotide pathway, malate-aspartate shuttle, as well as creatine metabolism, which are known to be active during exercise, further suggests that PGC-1α regulates metabolism in exercise. Thus, we demonstrated the roles of PGC-1α in the skeletal muscle at the metabolite level..
240. Koshi Hashimoto, Yoshihiro Ogawa, The Update of Obesity Syndrome
Molecular Mechanism, Pathophysiology and Therapies. Topics: I. Recent Topics on Diagnosis and Pathophysiology of the Obesity Syndrome; 2. Progress in genomic and epigenetic medicine against obesity, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.104.697, 104, 4, 697-702, 2015.04.
241. Kuniha Konuma, Michiko Itoh, Takayoshi Suganami, Sayaka Kanai, Nobutaka Nakagawa, Takeru Sakai, Hiroyuki Kawano, Mitsuko Hara, Soichi Kojima, Yuichi Izumi, Yoshihiro Ogawa, Eicosapentaenoic acid ameliorates non-alcoholic steatohepatitis in a novel mouse model using melanocortin 4 receptor-deficient mice, PloS one, 10.1371/journal.pone.0121528, 10, 3, 2015.03, Many attempts have been made to find novel therapeutic strategies for non-alcoholic steatohepatitis (NASH), while their clinical efficacy is unclear. We have recently reported a novel rodent model of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice, which exhibit the sequence of events that comprise hepatic steatosis, liver fibrosis, and hepatocellular carcinoma with obesity-related phenotypes. In the liver of MC4R-KO mice, there is a unique histological feature termed hepatic crown-like structures (hCLS), where macrophages interact with dead hepatocytes and fibrogenic cells, thereby accelerating inflammation and fibrosis. In this study, we employed MC4R-KO mice to examine the effect of highly purified eicosapentaenoic acid (EPA), a clinically available n-3 polyunsaturated fatty acid, on the development of NASH. EPA treatment markedly prevented the development of hepatocyte injury, hCLS formation and liver fibrosis along with lipid accumulation. EPA treatment was also effective even after MC4R-KO mice developed NASH. Intriguingly, improvement of liver fibrosis was accompanied by the reduction of hCLS formation and plasma kallikrein-mediated transforming growth factor-β activation. Moreover, EPA treatment increased the otherwise reduced serum concentrations of adiponectin, an adipocytokine with anti-inflammatory and anti-fibrotic properties. Collectively, EPA treatment effectively prevents the development and progression of NASH in MC4R-KO mice along with amelioration of hepatic steatosis. This study unravels a novel anti-fibrotic mechanism of EPA, thereby suggesting a clinical implication for the treatment of NASH..
242. Sachiko Kobayashi, Atsunori Fukuhara, Michio Otsuki, Takayoshi Suganami, Yoshihiro Ogawa, Eiichi Morii, Iichiro Shimomura, Fat/vessel-derived secretory protein (Favine)/CCDC3 is involved in lipid accumulation, Journal of Biological Chemistry, 10.1074/jbc.M114.592493, 290, 12, 7443-7451, 2015.03, We previously identified a novel gene encoding Favine/CCDC3 (NCBI protein entry NP-083080), a possible secretory factor, the mRNA of which is highly expressed in adipose tissue and the aorta. The Favine mRNA levels are increased in the course of differentiation of rat primary adipocytes and are more elevated in the adipose tissue of genetically obese and diet-induced obese mice than in lean mice. However, its biological function has not yet been elucidated until now. Here, we tested the hypothesis that Favine is involved in lipid metabolism in adipocytes. We found that overexpression of Favine promoted 3T3-L1 adipocyte differentiation. To further investigate the function of Favine in vivo, we generated Favine knock-out (KO) mice. Favine KO mice exhibited a lean phenotype as they aged. The weights of white adipose tissue and liver were less, and adipocyte size was smaller in Favine KO mice compared with wild-type littermates (WT). Expression levels of lipogenic genes, such as fatty-acid synthase (FAS), acetyl-CoA carboxylase α (ACC1), and diacylglycerol O-acyltransferase-2 (Dgat2), were decreased in adipose tissue of Favine KO mice. In 1-year-old mice, Favine deficiency decreased the number of inflammatory cells in white adipose tissue and diminished hepatic steatosis. In vitro, deficiency of Favine attenuated differentiation of primary adipocytes. Taken together, these data demonstrate that Favine has adipogenic and lipogenic effects on adipocytes..
243. Tatsuya Ehara, Yasutomi Kamei, Xunmei Yuan, Mayumi Takahashi, Sayaka Kanai, Erina Tamura, Kazutaka Tsujimoto, Takashi Tamiya, Yoshimi Nakagawa, Hitoshi Shimano, Takako Takai-Igarashi, Izuho Hatada, Takayoshi Suganami, Koshi Hashimoto, Yoshihiro Ogawa, Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver, Diabetes, 10.2337/db14-0158, 64, 3, 775-784, 2015.03, The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor a (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and lifestage- specific DNA demethylation of a particular metabolic pathway..
244. Masatoshi Takagi, Hatsume Uno, Rina Nishi, Masataka Sugimoto, Setsuko Hasegawa, Jinhua Piao, Norimasa Ihara, Sayaka Kanai, Saori Kakei, Yoshifumi Tamura, Takayoshi Suganami, Yasutomi Kamei, Toshiaki Shimizu, Akio Yasuda, Yoshihiro Ogawa, Shuki Mizutani, ATM Regulates Adipocyte Differentiation and Contributes to Glucose Homeostasis, Cell Reports, 10.1016/j.celrep.2015.01.027, 10, 6, 957-967, 2015.02, Ataxia-telangiectasia (A-T) patients occasionally develop diabetes mellitus. However, only limited attempts have been made to gain insight into the molecular mechanism of diabetes mellitus development in A-T patients. We found that Atm-/- mice were insulin resistant and possessed less subcutaneous adipose tissue as well as a lower level of serum adiponectin than Atm+/+ mice. Furthermore, invitro studies revealed impaired adipocyte differentiation in Atm-/- cells caused by the lack of induction of C/EBPα and PPARγ, crucial transcription factors involved in adipocyte differentiation. Interestingly, ATM was activated by stimuli that induced differentiation, and the binding of ATM to C/EBPβ and p300 was involved in the transcriptional regulation of C/EBPα and adipocyte differentiation. Thus, our study sheds light on the poorly understood role of ATM in the pathogenesis of glucose intolerance in A-T patients and providesinsight into the role of ATM in glucose metabolism..
245. Masahiro Asakawa, Tetsuo Sekine, Yoshihiro Niitsu, Arisa Kobayashi, Atsuko Miyake, Mitsunobu Kawamura, Yoshihiro Ogawa, Difficult medical management in elderly diabetic patients with polypharmacy
An analysis of cases accompanied by frequent consciousness loss and asymptomatic hypoglycemia, Journal of the Japan Diabetes Society, 58, 9, 688-694, 2015.01, An 80-year-old woman was admitted because of a frequent loss of consciousness and hypoglycemic episodes. She had developed diabetes mellitus in 1978 and was started on insulin treatment in 1990 and multiple daily injections in 2005. Her glycemic control during these periods was moderate (HbA1c: 7 % to 9 %). Bisoprolol at a dose of 2.5 mg for hypertension was administered starting in 2006. In 2014, she suffered from frequent episodes of loss of consciousness and vomiting (two to three times per month) within three months before admission, in addition to frequent hypoglycemic episodes. On the second hospital day, she experienced loss of consciousness and vomiting, and severe bradycardia (heart rate: 30 beats per minute) was observed. The blood glucose level at that time was not low (221 mg/dl). An electrocardiogram showed sinus arrest, considered to be caused by bisoprolol, and temporary cardiac pacing was needed. Pacing became unnecessary the next day, and no further loss of consciousness or vomiting dependent on severe bradycardia occurred. Since polypharmacy is common in the treatment of diabetes mellitus among elderly patients, it is important to check medications thoroughly when a patient suffers from new symptoms..
246. Ryotaro Bouchi, Yoshihiro Ogawa, Gut Microbiota and Internal Diseases
Update Information. Topics: IV. Obesity, diabetes and gut microbiota, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.104.57, 104, 1, 57-65, 2015.01.
247. Naoto Tsuda, Shin Kumadaki, Chika Higashi, Makoto Ozawa, Mikihiko Shinozaki, Yutaka Kato, Koutarou Hoshida, Satomi Kikuchi, Yoshihisa Nakano, Yoshihiro Ogawa, Shoji Furusako, Intestine-targeted DGAT1 inhibition improves obesity and insulin resistance without skin aberrations in mice, PloS one, 10.1371/journal.pone.0112027, 9, 11, 2014.11, Objective:Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice.
Design and Methods:We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500), reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO) mice.
Results:The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice.
Conclusions: Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance. Copyright:.
248. Jinyoung Park, Young Sil Yoon, Hye Sook Han, Yong Hoon Kim, Yoshihiro Ogawa, Keun Gyu Park, Chul Ho Lee, Seong Tae Kim, Seung Hoi Koo, SIK2 is critical in the regulation of lipid homeostasis and adipogenesis in vivo, Diabetes, 10.2337/db13-1423, 63, 11, 3659-3673, 2014.11, Cyclic AMP promotes chronic expression of target genes mainly by protein kinase A-dependent activation of CREB transcription factor machineries in the metabolic tissues. Here, we wanted to elaborate whether CREB-regulated transcription factor (CRTC)2 and its negative regulator salt-inducible kinase (SIK)2 are involved in the transcriptional control of the metabolic pathway in adipocytes. SIK2 knockout (SIK2 KO) mice exhibited higher blood glucose levels that were associated with impaired glucose and insulin tolerance. Hypertriglyceridemia was apparent in SIK2 KO mice, mainly due to the increased lipolysis from white adipocytes and the decreased fatty acid uptake in the peripheral tissues. Investigation of white adipocytes revealed the increases in fat cell size and macrophage infiltration, which could be linked to the metabolic anomaly that is associated in these mice. Interestingly, SIK2 KO promoted the enhancement in the CRTC2-CREB transcriptional pathway in white adipocytes. SIK2 KO mice displayed increased expression of activating transcription factor (ATF)3 and subsequent downregulation of GLUT4 expression and reduction in high-molecular weight adiponectin levels in the plasma, leading to the reduced glucose uptake in the muscle and white adipocytes. The effect of SIK2-dependent regulation of adipocyte metabolism was further confirmed by in vitro cell cultures of 3T3 L1 adipocytes and the differentiated preadipocytes from the SIK2 or CRTC2 KO mice. Collectively, these data suggest that SIK2 is critical in regulating whole-body glucose metabolism primarily by controlling the CRTC2-CREB function of the white adipocytes..
249. Yasutomi Kamei, Maki Hattori, Yukino Hatazawa, Tomomi Kasahara, Masanobu Kanou, Sayaka Kanai, Xunmei Yuan, Takayoshi Suganami, Wouter H. Lamers, Tadahiro Kitamura, Yoshihiro Ogawa, FOXO1 activates glutamine synthetase gene in mouse skeletal muscles through a region downstream of 3’-UTR
Possible contribution to ammonia detoxification, American Journal of Physiology - Endocrinology and Metabolism, 10.1152/ajpendo.00177.2014, 307, 6, E485-E493, 2014.09, Skeletal mus- cle is a reservoir of energy in the form of protein, which is degraded under catabolic conditions, resulting in the formation of amino acids and ammonia as a byproduct. The expression of FOXO1, a forkhead- type transcription factor, increases during starvation and exercise. In agreement, transgenic FOXO1-Tg mice that overexpress FOXO1 in skeletal muscle exhibit muscle atrophy. The aim of this study was to examine the role of FOXO1 in amino acid metabolism. The mRNA and protein expressions of glutamine synthetase (GS) were increased in skeletal muscle of FOXO1-Tg mice. Fasting induced FOXO1 and GS expression in wild-type mice but hardly increased GS expression in muscle-specific FOXO1 knockout (FOXO1-KO) mice. Activation of FOXO1 also increased GS mRNA and protein expression in C2C12 myoblasts. Using a transient transfection reporter assay, we observed that FOXO1 activated the GS reporter construct. Mutation of a putative FOXO1-binding consensus sequence in the downstream genomic region of GS decreased basal and FOXO1-dependent re- porter activity significantly. A chromatin immunoprecipitation assay showed that FOXO1 was recruited to the 3= region of GS in C2C12 myoblasts. These results suggest that FOXO1 directly upregulates GS expression. GS is considered to mediate ammonia clearance in skel- etal muscle. In agreement, an intravenous ammonia challenge in- creased blood ammonia concentrations to a twofold higher level in FOXO1-KO than in wild-type mice, demonstrating that the capacity for ammonia disposal correlated inversely with the expression of GS in muscle. These data indicate that FOXO1 plays a role in amino acid metabolism during protein degradation in skeletal muscle..
250. Yukino Hatazawa, Miki Tadaishi, Yuta Nagaike, Akihito Morita, Yoshihiro Ogawa, Osamu Ezaki, Takako Takai-Igarashi, Yasuyuki Kitaura, Yoshiharu Shimomura, Yasutomi Kamei, Shinji Miura, PGC-1α-mediated branched-chain amino acid metabolism in the skeletal muscle, PloS one, 10.1371/journal.pone.0091006, 9, 3, 2014.03, Peroxisome proliferator-activated receptor (PPAR) c coactivator 1a (PGC-1α) is a coactivator of various nuclear receptors and other transcription factors, which is involved in the regulation of energy metabolism, thermogenesis, and other biological processes that control phenotypic characteristics of various organ systems including skeletal muscle. PGC-1α in skeletal muscle is considered to be involved in contractile protein function, mitochondrial function, metabolic regulation, intracellular signaling, and transcriptional responses. Branched-chain amino acid (BCAA) metabolism mainly occurs in skeletal muscle mitochondria, and enzymes related to BCAA metabolism are increased by exercise. Using murine skeletal muscle overexpressing PGC-1α and cultured cells, we investigated whether PGC-1α stimulates BCAA metabolism by increasing the expression of enzymes involved in BCAA metabolism. Transgenic mice overexpressing PGC-1α specifically in the skeletal muscle had increased the expression of branched-chain aminotransferase (BCAT) 2, branched-chain a-keto acid dehydrogenase (BCKDH), which catabolize BCAA. The expression of BCKDH kinase (BCKDK), which phosphorylates BCKDH and suppresses its enzymatic activity, was unchanged. The amount of BCAA in the skeletal muscle was significantly decreased in the transgenic mice compared with that in the wild-type mice. The amount of glutamic acid, a metabolite of BCAA catabolism, was increased in the transgenic mice, suggesting the activation of muscle BCAA metabolism by PGC-1α. In C2C12 cells, the overexpression of PGC-1α significantly increased the expression of BCAT2 and BCKDH but not BCKDK. Thus, PGC-1α in the skeletal muscle is considered to significantly contribute to BCAA metabolism. Copyright:.
251. Naoki Sawada, Aihua Jiang, Fumihiko Takizawa, Adeel Safdar, Andre Manika, Yevgenia Tesmenitsky, Kyu Tae Kang, Joyce Bischoff, Hermann Kalwa, Juliano L. Sartoretto, Yasutomi Kamei, Laura E. Benjamin, Hirotaka Watada, Yoshihiro Ogawa, Yasutomi Higashikuni, Chase W. Kessinger, Farouc A. Jaffer, Thomas Michel, Masataka Sata, Kevin Croce, Rica Tanaka, Zolt Arany, Endothelial PGC-1α mediates vascular dysfunction in diabetes, Cell metabolism, 10.1016/j.cmet.2013.12.014, 19, 2, 246-258, 2014.02, Endothelial dysfunction is a central hallmark of diabetes. The transcriptional coactivator PGC-1α is a powerful regulator of metabolism, but its role in endothelial cells remains poorly understood. We show here that endothelial PGC-1α expression is high in diabetic rodents and humans and that PGC-1α powerfully blocks endothelial migration in cell culture and vasculogenesis in vivo. Mechanistically, PGC-1α induces Notch signaling, blunts activation of Rac/Akt/eNOS signaling, and renders endothelial cells unresponsive to established angiogenic factors. Transgenic overexpression of PGC-1α in the endothelium mimics multiple diabetic phenotypes, including aberrant re-endothelialization after carotid injury, blunted wound healing, and reduced blood flow recovery after hindlimb ischemia. Conversely, deletion of endothelial PGC-1α rescues the blunted wound healing and recovery from hindlimb ischemia seen in type 1 and type 2 diabetes. Endothelial PGC-1α thus potently inhibits endothelial function and angiogenesis, and induction of endothelial PGC-1α contributes to multiple aspects of vascular dysfunction in diabetes..
252. Kana Inoue, Norikazu Maeda, Takuya Mori, Ryohei Sekimoto, Yu Tsushima, Keisuke Matsuda, Masaya Yamaoka, Takayoshi Suganami, Hitoshi Nishizawa, Yoshihiro Ogawa, Tohru Funahashi, Iichiro Shimomura, Possible involvement of opa-interacting protein 5 in adipose proliferation and obesity, PloS one, 10.1371/journal.pone.0087661, 9, 2, 2014.02, Obesity is an epidemic matter increasing risk for cardiovascular diseases and metabolic disorders such as type 2 diabetes. We recently examined the association between visceral fat adiposity and gene expression profile of peripheral blood cells in human subjects. In a series of studies, Opa (Neisseria gonorrhoeae opacity-associated)-interacting protein 5 (OIP5) was nominated as a molecule of unknown function in adipocytes and thus the present study was performed to investigate the role of OIP5 in obesity. Adenovirus overexpressing Oip5 (Ad-Oip5) was generated and infected to 3T3-L1 cells stably expressing Coxsackie-Adenovirus Receptor (CAR-3T3-L1) and to mouse subcutaneous fat. For a knockdown experiment, siRNA against Oip5 (Oip5-siRNA) was introduced into 3T3-L1 cells. Proliferation of adipose cells was measured by BrdU uptake, EdU-staining, and cell count. Significant increase of Oip5 mRNA level was observed in obese white adipose tissues and such increase was detected in both mature adipocytes fraction and stromal vascular cell fraction. Ad-Oip5-infected CAR-3T3-L1 preadipocytes and adipocytes proliferated rapidly, while a significant reduction of proliferation was observed in Oip5-siRNA-introduced 3T3-L1 preadipocytes. Fat weight and number of adipocytes were significantly increased in Ad-Oip5-administered fat tissues. Oip5 promotes proliferation of pre- and mature-adipocytes and contributes adipose hyperplasia. Increase of Oip5 may associate with development of obesity..
253. Yorihiro Iwasaki, Takayoshi Suganami, Rumi Hachiya, Ibuki Shirakawa, Misa Kim-Saijo, Miyako Tanaka, Miho Hamaguchi, Takako Takai-Igarashi, Michikazu Nakai, Yoshihiro Miyamoto, Yoshihiro Ogawa, Activating transcription factor 4 links metabolic stress to interleukin-6 expression in macrophages, Diabetes, 10.2337/db13-0757, 63, 1, 152-161, 2014.01, Chronic inflammation is a molecular element of the metabolic syndrome and type 2 diabetes. Saturated fatty acids (SFAs) are considered to be an important proinflammatory factor. However, it is still incompletely understood how SFAs induce proinflammatory cytokine expression. Hereby we report that activating transcription factor (ATF) 4, a transcription factor that is induced downstream of metabolic stresses including endoplasmic reticulum (ER) stress, plays critical roles in SFA-induced interleukin-6 (Il6) expression. DNA microarray analysis using primary macrophages revealed that the ATF4 pathway is activated by SFAs. Haploinsufficiency and short hairpin RNA-based knockdown of ATF4 in macrophages markedly inhibited SFA- and metabolic stress-induced Il6 expression. Conversely, pharmacological activation of the ATF4 pathway and overexpression of ATF4 resulted in enhanced Il6 expression. Moreover, ATF4 acts in synergy with the Toll-like receptor-4 signaling pathway, which is known to be activated by SFAs. At a molecular level, we found that ATF4 exerts its proinflammatory effects through at least two different mechanisms: ATF4 is involved in SFAinduced nuclear factor-kB activation; and ATF4 directly activates the Il6 promoter. These findings provide evidence suggesting that ATF4 links metabolic stress and Il6 expression in macrophages..
254. Sumiyo Morita, Takuro Horii, Mika Kimura, Yuji Arai, Yasutomi Kamei, Yoshihiro Ogawa, Izuho Hatada, Paternal allele influences high fat diet-induced obesity, PloS one, 10.1371/journal.pone.0085477, 9, 1, 2014.01, C57BL/6J (B6) mice are susceptible to high-fat diet (HFD)-induced obesity and have been used in metabolism research for many decades. However, the genetic component of HFD-induced obesity has not yet been elucidated. This study reports evidence for a paternal transmission of HFD-induced obesity and a correlated expression of Igf2 and Peg3 (paternal expressed gene 3) imprinted genes. We found that PWK mice are resistant to HFD-induced obesity compared to C57BL/6J mice. Therefore, we generated and analyzed reciprocal crosses between these mice, namely; (PWKxB6) F1 progeny with B6 father and (B6xPWK) F1 progeny with PWK father. The (PWKxB6) F1 mice were more sensitive to diet-induced obesity compared to (B6xPWK) F1 mice, suggesting a paternal transmission of diet-induced obesity. Expression analysis of imprinted genes in adipocytes revealed that HFD influences the expression of some of the imprinted genes in adipose tissue in B6 and PWK mice. Interestingly, Igf2 and Peg3, which are paternally expressed imprinted genes involved in the regulation of body fat accumulation, were down-regulated in B6 and (PWKxB6) F1 mice, which are susceptible to HFD-induced obesity, but not in PWK and (B6xPWK) F1 mice, which are resistant. Furthermore, in vitro analysis showed that Igf2, but not Peg3, had an anti-inflammatory effect on TNF-α induced MCP-1 expression in adipocytes. Taken together, our findings suggest that the down-regulation of Igf2 and Peg3 imprinted genes in adipocytes may be involved in the paternal transmission of HFD-induced obesity..
255. Masahiro Asakawa, Arisa Niwa, Momoko Akihisa, Mizuho Sawada, Atsuko Miyake, Mitsunobu Kawamura, Yoshihiro Ogawa, A case of type 1 diabetes with frequent hypoglycemic episodes induced by primary hypothyroidism, Journal of the Japan Diabetes Society, 57, 4, 242-248, 2014, A 46-year-old male was admitted due to recurrent hypoglycemic episodes. He developed diabetes mellitus in 2000 and became insulin-dependent in 2006. Following the initiation of insulin treatment, his glycemic control stabilized (HbAlc: 6 % to 7 %). However, in 2013 (at 46 years of age), the frequency of the hypoglycemic episodes increased starting three months prior to hospitalization, which occurred after the patient experienced a low-grade fever for approximately three weeks. Symptoms such as easy fatigability, coldness, hypohidrosis and weight gain were noted concomitantly with the onset of frequent hypoglycemic episodes. He was diagnosed with primary hypothyroidism, and thyroid hormone replacement therapy was started. As a result of switching the patient's insulin therapy from multiple injections to an insulin pump in the hospital, the hypoglycemic episodes became infrequent The patient's frequent hypoglycemic episodes were thought to be induced by primary hypothyroidism due to destructive thyroiditis complicating type 1 diabetes. Thyroid hormone deficiency delays the intestinal absorption of glucose, decreases gluconeogenesis in the liver and impairs glucagon secretion as the counterregulatory response to hypoglycemia Accordingly, hypothyroidism can cause hypoglycemia This case serves as a reminder that hypothyroidism should be taken into consideration as a possible cause of hypoglycemia..
256. Ryo Ito, Noriko Satoh-Asahara, Hajime Yamakage, Yousuke Sasaki, Shinji Odori, Shigeo Kono, Hiromichi Wada, Takayoshi Suganami, Yoshihiro Ogawa, Koji Hasegawa, Akira Shimatsu, An increase in the EPA/AA ratio is associated with improved arterial stiffness in obese patients with dyslipidemia, Journal of atherosclerosis and thrombosis, 10.5551/jat.19976, 21, 3, 248-260, 2014, Aim: Previous epidemiological studies demonstrated that the ratio of n-6 to n-3 polyunsaturated fatty acids is associated with cardiovascular diseases. We herein investigated whether the beneficial effect of highly purified eicosapentaenoic acid(EPA) on arterial stiffness is associated with changes in the ratio of polyunsaturated fatty acids, such as EPA, docosahexaenoic acid(DHA) and dihomo-γ-linolenic acid (DGLA), relative to arachidonic acid(AA), in obese Japanese patients with dyslipidemia. Methods: The EPA/AA, DHA/AA and DGLA/AA ratios were compared between obese patients with(n=94) and without (n=31) dyslipidemia. Among the former group, 88 patients received either highly purified EPA treatment(1.8g daily, n=45) or treatment without EPA(control, n=43). Results: At baseline, the ratios of DHA/AA and DGLA/AA were significantly(P<0.05) higher in obese patients with dyslipidemia than in those without, while the EPA/AA ratio was similar between patients with and without dyslipidemia. EPA significantly reduced the hemoglobin A1c, total cholesterol, triglycerides, CRP, cardio-ankle vascular index(CAVI) (an index of arterial stiffness) and the DGLA/AA ratio relative to the control at three months after the treatment. On the other hand, EPA significantly increased the adiponectin level and EPA/AA ratio(P<0.05). A multivariate regression analysis revealed that only age, an increase in the EPA/AA ratio and a decrease in the CRP level were significant determinants of a reduction of the CAVI by EPA. Conclusion: These findings suggest that EPA improves the arterial stiffness in association with an increase in the EPA/AA ratio and a decrease in inflammation in obese patients with dyslipidemia..
257. Miyako Tanaka, Kenji Ikeda, Takayoshi Suganami, Chikara Komiya, Kozue Ochi, Ibuki Shirakawa, Miho Hamaguchi, Satoshi Nishimura, Ichiro Manabe, Takahisa Matsuda, Kumi Kimura, Hiroshi Inoue, Yutaka Inagaki, Seiichiro Aoe, Sho Yamasaki, Yoshihiro Ogawa, Macrophage-inducible C-type lectin underlies obesity-induced adipose tissue fibrosis, Nature communications, 10.1038/ncomms5982, 5, 2014, In obesity, a paracrine loop between adipocytes and macrophages augments chronic inflammation of adipose tissue, thereby inducing systemic insulin resistance and ectopic lipid accumulation. Obese adipose tissue contains a unique histological structure termed crown-like structure (CLS), where adipocyte-macrophage crosstalk is known to occur in close proximity. Here we show that Macrophage-inducible C-type lectin (Mincle), a pathogen sensor for Mycobacterium tuberculosis, is localized to macrophages in CLS, the number of which correlates with the extent of interstitial fibrosis. Mincle induces obesity-induced adipose tissue fibrosis, thereby leading to steatosis and insulin resistance in liver. We further show that Mincle in macrophages is crucial for CLS formation, expression of fibrosis-related genes and myofibroblast activation. This study indicates that Mincle, when activated by an endogenous ligand released from dying adipocytes, is involved in adipose tissue remodelling, thereby suggesting that sustained interactions between adipocytes and macrophages within CLS could be a therapeutic target for obesity-induced ectopic lipid accumulation..
258. Chikako Aoyama-Mani, Shoji Kawachi, Yoshihiro Ogawa, Norihiro Kato, Vascular complications and coagulation-related changes in the perioperative period in japanese patients undergoing non-cardiac surgery, Journal of atherosclerosis and thrombosis, 10.5551/jat.21139, 21, 5, 414-434, 2014, Aim: To properly assess the guidance for perioperative management, we undertook a clinical epidemiology study with the primary aim of evaluating the incidence of perioperative vascular complications and their associated factors in a cohort of Japanese patients who underwent non-cardiac surgery in a tertiary medical care center. Methods: This observational study comprised two parts. In the first part, thrombotic and bleeding events and their risk factors in the perioperative period were evaluated in a total of 2,654 consecutive patients. In the second part, perioperative changes in coagulation-related factors, including the thrombin-antithrombin complex (TAT) and platelet aggregation activity, were serially characterized in 82 individuals randomly chosen from the consecutive patients. Results: The incidence of perioperative vascular complications was as follows: 1.0% for major bleeding, 0.21% for stroke and 0.21% for venous thromboembolism. No episodes of symptomatic myocardial infarction were identified in the studied population. Perioperative changes in coagulationrelated factors were found to be complex and correlated in the mixed direction of pro- and anticoagulation. The TAT values showed prolonged (across postoperative days 1-5) and prominent (>116% increase) perioperative activation of coagulation, whereas global coagulation parameters, such as the prothrombin time, showed a tendency of anticoagulation in the immediate postoperative period. Conclusions: Our data confirm the relatively low incidence of perioperative vascular complications in the general Japanese non-cardiac surgical population. Given the delicate balance between thrombotic and bleeding events, it is important to comprehensively understand the associations between the patient's baseline risk factors and vascular complications for effective clinical management..
259. Michiko Itoh, Hideaki Kato, Takayoshi Suganami, Kuniha Konuma, Yoshio Marumoto, Shuji Terai, Hiroshi Sakugawa, Sayaka Kanai, Miho Hamaguchi, Takahiro Fukaishi, Seiichiro Aoe, Kazunari Akiyoshi, Yoshihiro Komohara, Motohiro Takeya, Isao Sakaida, Yoshihiro Ogawa, Hepatic crown-like structure
A unique histological feature in non-alcoholic steatohepatitis in mice and humans, PloS one, 10.1371/journal.pone.0082163, 8, 12, 2013.12, Although macrophages are thought to be crucial for the pathogenesis of chronic inflammatory diseases, how they are involved in disease progression from simple steatosis to non-alcoholic steatohepatitis (NASH) is poorly understood. Here we report the unique histological structure termed "hepatic crown-like structures (hCLS)" in the mouse model of human NASH; melanocortin-4 receptor deficient mice fed a Western diet. In hCLS, CD11c-positive macrophages aggregate to surround hepatocytes with large lipid droplets, which is similar to those described in obese adipose tissue. Histological analysis revealed that hCLS is closely associated with activated fibroblasts and collagen deposition. When treatment with clodronate liposomes effectively depletes macrophages scattered in the liver, with those in hCLS intact, hepatic expression of inflammatory and fibrogenic genes is unaffected, suggesting that hCLS is an important source of inflammation and fibrosis during the progression of NASH. Notably, the number of hCLS is positively correlated with the extent of liver fibrosis. We also observed increased number of hCLS in the liver of non-alcoholic fatty liver disease/NASH patients. Collectively, our data provide evidence that hCLS is involved in the development of hepatic inflammation and fibrosis, thereby suggesting its pathophysiologic role in disease progression from simple steatosis to NASH. Copyright:.
260. Takuya Mori, Norikazu Maeda, Kana Inoue, Ryohei Sekimoto, Yu Tsushima, Keisuke Matsuda, Masaya Yamaoka, Takayoshi Suganami, Hitoshi Nishizawa, Yoshihiro Ogawa, Tohru Funahashi, Iichiro Shimomura, A Novel Role for Adipose Ephrin-B1 in Inflammatory Response, PloS one, 10.1371/journal.pone.0076199, 8, 10, 2013.10, Aims:Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.Methods and Results:EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.Conclusions:EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation..
261. Rachael A. Potter, Alissa D. DeLong, Sierra M. Smith, Benjamin M. Erb, Bryon Renwand, Yasutomi Kamei, Yoshihiro Ogawa, Thomas J. McLoughlin, FoxO1 inhibits skeletal muscle hypertrophy through mTOR-independent mechanisms, Journal of Exercise Physiology Online, 16, 4, 32-50, 2013.09, The canonical Akt/mTOR signaling pathway plays a strong role in promoting skeletal muscle hypertrophy through regulating anabolic and catabolic signaling cascades. The transcription factor, FoxO1, a downstream molecular target of Akt signaling, may play a negative role in skeletal muscle hypertrophy through suppression of growth signaling and/or upregulation of atrophy gene expression. Using a transgenic mouse model in which FoxO1 is specifically expressed within skeletal muscle, we tested the hypotheses: (a) that FoxO1 inhibits skeletal muscle hypertrophy in vivo; and (b) that inhibition of skeletal muscle hypertrophy conferred through FoxO1 expression is associated with suppression of Akt/mTOR signaling and upregulation of muscle atrophy F-box (MAFbx/atrogin-1) gene expression. The findings confirm that FoxO1 inhibits skeletal muscle hypertrophy associated with 2 wks of mechanical overload (synergist ablation), evidenced through dampened increases in muscle mass, protein content, and muscle cross sectional area. We conclude that FoxO1 overexpression hampers the ability of skeletal muscle to hypertrophy, and that this suppression involves mechanisms independent of mTOR signaling..
262. Yukiko Kobayashi Kohmura, Naohiro Kanayama, Keiko Muramatsu, Naoaki Tamura, Chizuko Yaguchi, Toshiyuki Uchida, Kazunao Suzuki, Kazuhiro Sugihara, Seiichiro Aoe, Takeshi Sasaki, Takayoshi Suganami, Yoshihiro Ogawa, Hiroaki Itoh, Association between body weight at weaning and remodeling in the subcutaneous adipose tissue of obese adult mice with undernourishment in utero, Reproductive Sciences, 10.1177/1933719112466300, 20, 7, 813-827, 2013.07, Rapid growth in infancy considerably increases the risk of obesity and metabolic disorders in adulthood especially among neonates born small. To investigate the mechanism involved, we developed an animal model of undernourishment in utero by maternal caloric restriction, in which the Z scores of body weight at weaning (19.5 days) positively correlated with parameters of obesity, metabolic disorders, and remodeling of subcutaneous adipose tissue, such as numbers of macrophages in adipose tissue, the ratio of inflammatory M1 to anti-inflammatory M2 macrophages, estimated by gene expression of specific antigens, and the relative ratio of small adipocytes less than 30 μm in diameter, on a high-fat diet at 17 weeks of age. To our knowledge, this is the first report of a possible connection between infantile body weight and adipose tissue remodeling in obesity after undernourishment in utero..
263. Fuminori Sato, Motoyoshi Tsujino, Yasutaka Miyachi, Ikue Ishimoto, Yuka Nagata, Chikara Komiya, Takuya Ohashi, Yukiko Kurosawa, Maya Sakurada, Kenji Nishida, Yoshihiro Ogawa, Efficacy of liraglutide in Japanese patients with type 2 diabetes assessed using continuous glucose monitoring, Journal of the Japan Diabetes Society, 56, 7, 430-435, 2013.07, We evaluated the efficacy of a GLP-1 analogue, liraglutide, using continuous glucose monitoring (CGM). The subjects included 13 patients with type 2 diabetes for which oral antidiabetic agents had been used. The daily mean blood glucose level, SD of the daily blood glucose level, duration of hyperglycemia (blood glucose > 180 mg/d/), mean amplitude of glycemic excursions (MAGE) and J-index were compared before and after treatment with liraglutide. The BMI, duration of diabetes, CPR index and postprandial CPR level were examined in relation to the recovery rate of the mean blood glucose level and the SD. The daily mean blood glucose level significantly improved from 151.0 mg/d/ to 121.0 mg/d/ (median, p = 0.002). The SD of the daily blood glucose level (43.0 mg/d/ to 14.0 mg/d/, p = 0.002), duration of hyperglycemia (23.0 % to 0.0 %, p = 0.002), MAGE (109.0 mg/d/ to 33.0 mg/d/, p = 0.001) and J-index (36.9 to 18.5, p = 0.002) significantly improved. A significantly negative correlation was observed between the recovery rate of the SD and the duration of diabetes. Liraglutide improves the average blood glucose level and also has a positive effect on flattening the blood glucose levels..
264. Mayumi Takahashi, Yasutomi Kamei, Tatsuya Ehara, Xunmei Yuan, Takayoshi Suganami, Takako Takai-Igarashi, Izuho Hatada, Yoshihiro Ogawa, Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.04.041, 434, 4, 873-878, 2013.05, DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo..
265. Fumihiko Takizawa, Shuki Mizutani, Yoshihiro Ogawa, Naoki Sawada, Glucose-independent persistence of PAI-1 gene expression and H3K4 tri-methylation in type 1 diabetic mouse endothelium
Implication in metabolic memory, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.02.064, 433, 1, 66-72, 2013.03, Clinical trials with type 1 and type 2 diabetes have identified a phenomenon known as " metabolic memory" in which previous periods of hyperglycemia result in the long-lasting deleterious impact on cardiovascular events. Emerging evidence shows that transient hyperglycemic exposure of human endothelial cells induces histone 3 lysine 4 mono-methylation (H3K4me1) on the promoter and persistent mRNA expression of RelA and IL-8 genes, suggesting that epigenetic histone modification and chromatin structure remodeling is a key event underlying metabolic memory. This burgeoning hypothesis, however, critically remains to be tested for relevance in the disease process of diabetes in vivo, and for broader applicability to an array of genes involved in endothelial dysfunction. To address this, we used type 1 diabetes mouse model induced by streptozocin to be hyperglycemic for 8. weeks, and isolated endothelial cells that were used either freshly after isolation or after 2 to 3-week cell culture in normoglycemic conditions. mRNA expression profiling in diabetic mouse endothelial cells revealed significant and persistent up-regulation of Serpine1 encoding PAI-1, the hypo-fibrinolytic mediator leading to thrombotic diseases in diabetes, along with Rock2, Fn1 and Ccl2, whereas only Serpine 1 was persistently elevated in high glucose-treated mouse endothelial cells. Chromosome immunoprecipitation assay in type 1 diabetic mouse endothelial cells showed predominant enrichment of H3K4 tri-methylation on Serpine1 promoter, suggesting a unique epigenetic regulation in diabetic mice as opposed to high glucose-treated human ECs. Our study demonstrates the importance of combining in vivo models of diabetes with high glucose-treated cell culture to better assess the epigenetic mechanisms relevant to disease..
266. Satomi Yogosawa, Shin Mizutani, Yoshihiro Ogawa, Tetsuro Izumi, Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor γ and C/EBPα, Diabetes, 10.2337/db12-0295, 62, 1, 115-123, 2013.01, We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-β receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator-activated receptor γ ( PPAR γ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention..
267. Natsuko Yamakawa, Umeharu Ohto, Sachiko Akashi-Takamura, Koichiro Takahashi, Shin Ichiroh Saitoh, Natsuko Tanimura, Takayoshi Suganami, Yoshihiro Ogawa, Takuma Shibata, Toshiyuki Shimizu, Kensuke Miyake, Human TLR4 polymorphism D299G/T399I alters TLR4/MD-2 conformation and response to a weak ligand monophosphoryl lipid A, International immunology, 10.1093/intimm/dxs084, 25, 1, 45-52, 2013.01, A cell surface heterodimer Toll-like receptor 4 (TLR4)/MD-2 senses lipopolysaccharide (LPS), a principal membrane component of Gram-negative bacteria. LPS binds to MD-2 and induces dimerization of TLR4/MD-2. Dimerized TLR4 activates downstream signaling. TLR4 polymorphism replacing Asp299 with Gly and Thr399 with Ile (D299G/T399I) causes LPS hyporesponsiveness, and is associated with a variety of infectious and noninfectious diseases. However, a molecular mechanism underlying the LPS hyporesponsiveness remains controversial. We here asked whether the TLR4 polymorphism influenced cell surface expression of TLR4/MD-2, ligand-dependent TLR4/MD-2 dimerization or TLR4/ MD-2 responses to a weak agonist monophosphoryl lipid A (MPL). A newly established anti-TLR4 mAb detected D299G/T399I TLR4/MD-2 on Ba/F3 cells whereas a previous anti-TLR4 mAb did not, suggesting that the D299G/T399I polymorphism caused a conformational change in TLR4. Hyporesponsiveness of D299G/T399I TLR4/MD-2 was much more apparent when cells were stimulated with MPL than with lipid A. MPL-dependent TLR4/MD-2 dimerization was impaired by the D299G/T399I polymorphism. The D299G/T399I polymorphism did not alter LPS-binding to soluble TLR4/MD-2, but impaired its dimerization. These results suggest that the D299G/T399I TLR4 polymorphism impairs TLR4/MD-2 responses by altering ligand-dependent dimerization..
268. Michiya Kida, Toru Sugiyama, Takanobu Yoshimoto, Yoshihiro Ogawa, Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells, European Journal of Pharmaceutical Sciences, 10.1016/j.ejps.2012.11.001, 48, 1-2, 211-215, 2013.01, Hydrogen sulfide (H2S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H2S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H 2S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H2S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H 2S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H2S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function..
269. Takayoshi Suganami, Yoshihiro Ogawa, [Role of chronic inflammation in adipose tissue in the pathophysiology of obesity]., Unknown Journal, 71, 2, 225-230, 2013.01, Obesity may be viewed as a chronic low-grade inflammatory disease as well as a metabolic disease. Evidence has accumulated suggesting that chronic inflammation in adipose tissue leads to dramatic changes in number and cell type of stromal cells during the course of obesity, which is referred to as"adipose tissue remodeling". Among stromal cells, macrophages in obese adipose tissue are considered to be crucial for adipose tissue inflammation, which results in dysregulated adipocytokine production and ectopic fat accumulation. Understanding the molecular mechanism underlying adipose tissue inflammation would contribute to the identification of novel therapeutic strategies to prevent or treat obesity-induced metabolic derangements..
270. Noriko Satoh-Asahara, Akira Shimatsu, Hiromichi Wada, Takayoshi Suganami, Koji Hasegawa, Yoshihiro Ogawa, Response to Comment on
Satoh-Asahara et al. Highly purified eicosapentaenoic acid increases interleukin-10 levels of peripheral blood monocytes in obese patients with dyslipidemia. Diabetes Care 2012; 35:2631-2639, Diabetes care, 10.2337/dc13-0376, 36, 7, e110, 2013.
271. Noriko Satoh-Asahara, Akira Shimatsu, Yousuke Sasaki, Hidenori Nakaoka, Akihiro Himeno, Mayu Tochiya, Shigeo Kono, Tomohide Takaya, Koh Ono, Hiromichi Wada, Takayoshi Suganami, Koji Hasegawa, Yoshihiro Ogawa, Highly purified eicosapentaenoic acid increases interleukin-10 levels of peripheral blood monocytes in obese patients with dyslipidemia, Diabetes care, 10.2337/dc12-0269, 35, 12, 2631-2639, 2012.12, OBJECTIVE - It has recently been highlighted that proinflammatory (M1) macrophages predominate over anti-inflammatory (M2) macrophages in obesity, thereby contributing to obesityinduced adipose inflammation and insulin resistance. A recent clinical trial revealed that highly purified eicosapentaenoic acid (EPA) reduces the incidence of major coronary events. In this study, we examined the effect of EPA on M1/M2-like phenotypes of peripheral blood monocytes in obese dyslipidemic patients. RESEARCH DESIGN AND METHODS - Peripheral bloodmonocyteswere prepared from 26 obese patients without and 90 obese patients with dyslipidemia. Of the latter 90 obese patients with dyslipidemia, 82 patientswere treated with orwithout EPA treatment (1.8 g daily) for 3months. RESULTS - Monocytes in obese patients with dyslipidemia showed a significantly lower expression of interleukin-10 (IL-10), an M2 marker, than those without dyslipidemia. EPA significantly increased serum IL-10 and EPA levels, the EPA/arachidonic acid (AA) ratio, andmonocyte IL-10 expression and decreased the pulse wave velocity (PWV), an index of arterial stiffness, compared with the control group. After EPA treatment, the serum EPA/AA ratio was significantly correlated with monocyte IL-10 expression. Only increases in monocyte IL-10 expression and serum adiponectin were independent determinants of a decreased PWV by EPA. Furthermore, EPA significantly increased the expression and secretion of IL-10 in human monocytic THP-1 cells through a peroxisome proliferator-activated receptor (PPAR)g-dependent pathway. CONCLUSIONS - This study is the first to show that EPA increases the monocyte IL-10 expression in parallel with decrease of arterial stiffness, which may contribute to the antiatherogenic effect of EPA in obese dyslipidemic patients..
272. Tatsuya Ehara, Yasutomi Kamei, Mayumi Takahashi, Xunmei Yuan, Sayaka Kanai, Erina Tamura, Miyako Tanaka, Tomomi Yamazaki, Shinji Miura, Osamu Ezaki, Takayoshi Suganami, Masaki Okano, Yoshihiro Ogawa, Role of DNA methylation in the regulation of lipogenic glycerol-3-phosphate acyltransferase 1 gene expression in the mouse neonatal liver, Diabetes, 10.2337/db11-1834, 61, 10, 2442-2450, 2012.10, The liver is a major organ of lipid metabolism, which is markedly changed in response to physiological nutritional demand; however, the regulation of hepatic lipogenic gene expression in early life is largely unknown. In this study, we show that expression of glycerol- 3-phosphate acyltransferase 1 (GPAT1; Gpam), a rate-limiting enzyme of triglyceride biosynthesis, is regulated in the mouse liver by DNA methylation, an epigenetic modification involved in the regulation of a diverse range of biological processes in mammals. In the neonatal liver, DNA methylation of the Gpam promoter, which is likely to be induced by Dnmt3b, inhibited recruitment of the lipogenic transcription factor sterol regulatory element-binding protein- 1c (SREBP-1c), whereas in the adult, decreased DNA methylation resulted in active chromatin conformation, allowing recruitment of SREBP-1c. Maternal overnutrition causes decreased Gpam promoter methylation with increased GPAT1 expression and triglyceride content in the pup liver, suggesting that environmental factors such as nutritional conditions can affect DNA methylation in the liver. This study is the first detailed analysis of the DNA-methylation-dependent regulation of the triglyceride biosynthesis gene Gpam, thereby providing new insight into the molecular mechanism underlying the epigenetic regulation of metabolic genes and thus metabolic diseases..
273. Fumihiko Maekawa, Shigeki Shimba, Shota Takumi, Tomoharu Sano, Takehiro Suzuki, Jinhua Bao, Mika Ohwada, Tatsuya Ehara, Yoshihiro Ogawa, Keiko Nohara, Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork, Epigenetics, 10.4161/epi.21539, 7, 9, 1046-1056, 2012.09, DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNa methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNa expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNa expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNa expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNa methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNa methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNa expression and the consequent dynamic changes in global DNa methylation..
274. T. Kuwabara, K. Mori, M. Mukoyama, M. Kasahara, H. Yokoi, Y. Saito, Y. Ogawa, H. Imamaki, T. Kawanishi, A. Ishii, K. Koga, K. P. Mori, Y. Kato, A. Sugawara, K. Nakao, Exacerbation of diabetic nephropathy by Hyperlipidaemia is mediated by Toll-like receptor 4 in mice, Diabetologia, 10.1007/s00125-012-2578-1, 55, 8, 2256-2266, 2012.08, Aims/hypothesis: Hyperlipidaemia is an independent risk factor for the progression of diabetic nephropathy, but its molecular mechanism remains elusive. We investigated in mice how diabetes and hyperlipidaemia cause renal lesions separately and in combination, and the involvement of Toll-like receptor 4 (TLR4) in the process. Methods: Diabetes was induced in wild-type (WT) and Tlr4 knockout (KO) mice by intraperitoneal injection of streptozotocin (STZ). At 2 weeks after STZ injection, normal diet was substituted with a high-fat diet (HFD). Functional and histological analyses were carried out 6 weeks later. Results: Compared with treatment with STZ or HFD alone, treatment of WT mice with both STZ and HFD markedly aggravated nephropathy, as indicated by an increase in albuminuria, mesangial expansion, infiltration of macrophages and upregulation of pro-inflammatory and extracellularmatrix-associated gene expression in glomeruli. In Tlr4 KO mice, the addition of an HFD to STZ had almost no effects on the variables measured. Production of protein S100 calcium binding protein A8 (calgranulin A; S100A8), a potent ligand for TLR4, was observed in abundance in macrophages infiltrating STZ-HFD WT glomeruli and in glomeruli of diabetic nephropathy patients. High-glucose and fatty acid treatment synergistically upregulated S100a8 gene expression in macrophages from WT mice, but not from KO mice. As putative downstream targets of TLR4, phosphorylation of interferon regulatory factor 3 (IRF3) was enhanced in kidneys of WT mice co-treated with STZ and HFD. Conclusions/interpretation: Activation of S100A8/TLR4 signalling was elucidated in an animal model of diabetic glomerular injury accompanied with hyperlipidaemia, which may provide novel therapeutic targets in progressive diabetic nephropathy..
275. Yasuharu Watanabe, Tomoya Nakamura, Sho Ishikawa, Shiho Fujisaka, Isao Usui, Koichi Tsuneyama, Yoshinori Ichihara, Tsutomu Wada, Yoichiro Hirata, Takayoshi Suganami, Hirofumi Izaki, Shizuo Akira, Kensuke Miyake, Hiro Omi Kanayama, Michio Shimabukuro, Masataka Sata, Toshiyasu Sasaoka, Yoshihiro Ogawa, Kazuyuki Tobe, Kiyoshi Takatsu, Yoshinori Nagai, The radioprotective 105/MD-1 complex contributes to diet-induced obesity and adipose tissue inflammation, Diabetes, 10.2337/db11-1182, 61, 5, 1199-1209, 2012.05, Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesityassociated metabolic disorders..
276. Takayoshi Suganami, Miyako Tanaka, Yoshihiro Ogawa, Adipose tissue inflammation and ectopic lipid accumulation, Endocrine Journal, 10.1507/endocrj.EJ12-0271, 59, 10, 849-857, 2012, Obesity may be viewed as a chronic low-grade inflammatory disease as well as a metabolic disease. Indeed, unbalanced production of pro- and anti-inflammatory adipocytokines critically contributes to the obesity-induced insulin resistance. In addition to lipid-laden mature adipocytes, adipose tissue is composed of various stromal cells such as preadipocytes, endothelial cells, fibroblasts, and immune cells that may be involved in adipose tissue functions. Accumulating evidence has suggested that adipocytes and stromal cells in adipose tissue change dramatically in number and cell type during the course of obesity, which is referred to as "adipose tissue remodeling." Among stromal cells, infiltration of macrophages in obese adipose tissue precedes the development of insulin resistance in animal models, suggesting that they are crucial for adipose tissue inflammation. We have provided evidence suggesting that a paracrine loop involving saturated fatty acids and tumor necrosis factor-α derived from adipocytes and macrophages, respectively, aggravates obesity-induced adipose tissue inflammation. On the other hand, storing excessive energy as triglyceride is also a fundamental function of adipose tissue. Recent evidence suggests that reduced lipid storage in obese adipose tissue contributes to ectopic lipid accumulation in non-adipose tissues such as the liver, skeletal muscle, and pancreas, where lipotoxicity impairs their metabolic functions. Notably, chronic inflammation is capable of inducing insulin resistance, lipolysis, and interstitial fibrosis in adipose tissue, all of which may reduce the lipid-storing function. Understanding the molecular mechanism underlying adipose tissue remodeling may lead to the identification of novel therapeutic strategies to prevent or treat obesity-induced adipose tissue inflammation..
277. Hirohide Nambu, Miyuki Fukushima, Hirohiko Hikichi, Takao Inoue, Norihiro Nagano, Yoshio Tahara, Tadahiro Nambu, Junko Ito, Yoshihiro Ogawa, Satoshi Ozaki, Hisashi Ohta, Characterization of metabolic phenotypes of mice lacking GPR61, an orphan G-protein coupled receptor, Life Sciences, 10.1016/j.lfs.2011.09.002, 89, 21-22, 765-772, 2011.11, Aims: GPR61 is an orphan G protein-coupled receptor whose function remains unknown. The purpose of the present study is to elucidate the importance of GPR61 in metabolism by characterization of GPR61-deficient mice. Main methods: Male GPR61-deficient mice were characterized regarding various metabolic parameters, including food intake, body weight, oxygen consumption, body temperature, locomotor activity, and in a pair feeding study. Hypothalamic gene expression was analyzed using real-time quantitative RT-PCR. Key findings: GPR61-deficient mice exhibited marked hyperphagia and heavier body weight than wild-type mice. Hyperphagia of GPR61-deficient mice was observed before the differences in body weight became apparent between the genotypes. When body weight difference did become apparent between genotypes, increases in visceral fat pad weight, liver weight, liver triglyceride (TG) content, plasma leptin, and plasma insulin were observed in GPR61-deficient mice, suggesting that GPR61 deficiency caused obesity associated with hyperphagia. Oxygen consumption, body temperature, and locomotor activity were not significantly different between GPR61-deficient and wild-type mice. Pair-fed GPR61-deficient mice had a greater fat mass than wild-type mice despite comparable body weight in both genotypes. The mRNA levels of proopiomelanocortin (POMC) and brain-derived neurotropic factor (BDNF) in the hypothalamus of GPR61-deficient mice were significantly lower than those of wild-type mice. Significance: GPR61-deficient mice exhibited obesity associated with hyperphagia. These findings suggest that GPR61 is involved in the regulation of food intake and body weight, and may be of importance when considering GPR61 as a therapeutic target for obesity or eating disorders..
278. Michiko Itoh, Takayoshi Suganami, Nobutaka Nakagawa, Miyako Tanaka, Yukio Yamamoto, Yasutomi Kamei, Shuji Terai, Isao Sakaida, Yoshihiro Ogawa, Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis, American Journal of Pathology, 10.1016/j.ajpath.2011.07.014, 179, 5, 2454-2463, 2011.11, Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the "two hit" hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptordeficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies..
279. Lingyun Zhang, Takashi Sugiyama, Nao Murabayashi, Takashi Umekawa, Ning Ma, Yuki Kamimoto, Yoshihiro Ogawa, Norimasa Sagawa, The inflammatory changes of adipose tissue in late pregnant mice, Journal of Molecular Endocrinology, 10.1530/JME-11-0030, 47, 2, 157-165, 2011.10, The infiltration of classically activated macrophages (M1) and alternatively activated macrophages (M2) in subcutaneous adipose tissue (SAT) and parametrial adipose tissue (PAT) was analyzed to investigate whether local inflammatory change in adipose tissue occurs in late pregnancy. C57BL/6N female mice at 6 weeks of age were fed a normal chow diet for 4 weeks prior to mating at 10 weeks of age and were sampled on day 17 of pregnancy. The serum levels of adipokines and biochemical markers were measured using ELISA and enzymatic methods. The identification of M1 and M2 was analyzed by double immunofluorescence with anti-F4/80 and anti-CD11c antibodies. The gene expression of adipokines in adipose tissues was analyzed by quantitative RT-PCR. The pregnant group showed adipocyte hypertrophy, higher macrophage infiltration, and higher M1/M2 in both SAT and PAT compared with the non-pregnant (NP) group. Serum levels of free fatty acids, tumor necrosis factor α (TNFα), interleukin 6 (IL6), and IL10 were higher, and serum levels of adiponectin were lower in the pregnant group than those in the NP group. The gene expressions of CD68, Itgax, CCR2, TNFa, and PAI1 in SAT during pregnancy were significantly higher than those in the NP group, as were the gene expressions of CD68, Emrl, Itgax, MCP1, TNFα, IL6, PAI1, adiponectin, and IL10 in PAT. These results suggest that the low-grade inflammation of adipose tissue indicated by increased macrophage infiltration occurs in late normal pregnancy..
280. R. Hachiya, T. Suganami, Y. Ogawa, Adipose tissue and chronic inflammation, Journal of the Japan Diabetes Society, 54, 7, 480-482, 2011.07.
281. Miyako Tanaka, Takayoshi Suganami, Misa Kim-Saijo, Chitoku Toda, Makoto Tsuiji, Kozue Ochi, Yasutomi Kamei, Yasuhiko Minokoshi, Yoshihiro Ogawa, Role of central Leptin signaling in the starvation-induced alteration of B-cell development, Journal of Neuroscience, 10.1523/JNEUROSCI.6562-10.2011, 31, 23, 8373-8380, 2011.06, Nutritional deprivation or malnutrition suppresses immune function in humans and animals, thereby conferring higher susceptibility to infectious diseases. Indeed, nutritional deprivation induces atrophy of lymphoid tissues such as thymus and spleen and decreases the number of circulating lymphocytes. Leptin, a major adipocytokine, is exclusively produced in the adipose tissue in response to the nutritional status and acts on the hypothalamus, thereby regulating energy homeostasis. Although leptin plays a critical role in the starvation-induced T-cell-mediated immunosuppression, little is known about its role in B-cell homeostasis under starvation conditions. Here we show the alteration of B-cell development in the bone marrow of fasted mice, characterized by decrease in pro-B, pre-B, and immature B cells and increase in mature B cells. Interestingly, intracerebroventricular leptin injection was sufficient to prevent the alteration of B-cell development of fasted mice. The alteration of B lineage cells in the bone marrow of fasted mice was markedly prevented by oral administration of glucocorticoid receptor antagonist RU486 (11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one). It was also effectively prevented by intracerebroventricular injection of neuropeptide Y Y1 receptor antagonist BIBP3226 [(2R)-5-(diaminomethylideneamino)-2-[(2,2-diphenylacetyl)amino]-N-[(4-hydroxyphenyl)methyl]pentanamide], along with suppression of the otherwise increased serum corticosterone concentrations. This study provides the first in vivo evidence for the role of central leptin signaling in the starvation-induced alteration of B-cell development. The data of this study suggest that the CNS, which is inherent to integrate information from throughout the organism, is able to control immune function..
282. Takayoshi Suganami, Yoshihiro Ogawa, [Obesity
Progress in diagnosis and treatment; Topics, IV. Recent topics; 2. Obesity and inflammation]., Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.100.989, 100, 4, 989-995, 2011.04.
283. Masayuki Ichioka, Takayoshi Suganami, Naoto Tsuda, Ibuki Shirakawa, Yoichiro Hirata, Noriko Satoh-Asahara, Yuri Shimoda, Miyako Tanaka, Misa Kim-Saijo, Yoshihiro Miyamoto, Yasutomi Kamei, Masataka Sata, Yoshihiro Ogawa, Increased expression of macrophage-inducible C-type lectin in adipose tissue of obese mice and humans, Diabetes, 10.2337/db10-0864, 60, 3, 819-826, 2011.03, OBJECTIVE - We have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity. RESEARCH DESIGN AND METHODS - We performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression. RESULTS - We found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro. CONCLUSIONS - Our data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation..
284. Noriko Satoh-Asahara, Takayoshi Suganami, Takafumi Majima, Kazuhiko Kotani, Yasuhisa Kato, Rika Araki, Kazunori Koyama, Taiichiro Okajima, Makito Tanabe, Mariko Oishi, Akihiro Himeno, Shigeo Kono, Akira Sugawara, Masakazu Hattori, Yoshihiro Ogawa, Akira Shimatsu, Urinary cystatin C as a potential risk marker for cardiovascular disease and chronic kidney disease in patients with obesity and metabolic syndrome, Clinical Journal of the American Society of Nephrology, 10.2215/CJN.04830610, 6, 2, 265-273, 2011.02, Background and Objectives: Obesity and metabolic syndrome (MS) increase the risk of cardiovascular disease (CVD), chronic kidney disease (CKD), and all-cause mortality. Serum cystatin C (S-CysC), a marker of GFR, has been shown to be associated with CVD and CKD. This study was designed to elucidate the association of urinary CysC (U-CysC), a marker of renal tubular dysfunction, with CVD and CKD risk factors in patients with obesity and MS. Design, setting, participants, & measurements: The U-CysC-creatinine ratio (UCCR) was examined in 343 Japanese obese outpatients enrolled in the multi-centered Japan Obesity and Metabolic Syndrome Study. Results: UCCR was positively correlated with urine albumin-creatinine ratio (UACR) and S-CysC and negatively correlated with estimated GFR (eGFR). Among obese patients, UCCR was significantly higher in MS patients than in non-MS patients. UCCR had significant correlations with the number of components of MS and arterial stiffness, all of which are CVD predictors, similarly to UACR (P < 0.05). Interestingly, diet- and exercise-induced weight reduction for 3 months significantly decreased only UCCR among all of the renal markers examined (P < 0.01), in parallel with the decrease in BMI, HbA1c, and arterial stiffness, suggesting the beneficial effect of weight reduction on renal tubular dysfunction. Conclusions: This study demonstrates that UCCR is significantly associated with renal dysfunction, the severity of MS, arterial stiffness, and weight change in obese patients. The data of this study suggest that UCysC could serve as a CVD and CKD risk factor in patients with obesity and MS..
285. Naoto Tsuda, Takayoshi Suganami, Yoshihiro Ogawa, [Molecular mechanism of adipose tissue remodeling]., Nihon rinsho. Japanese journal of clinical medicine, 69 Suppl 1, 279-284, 2011.01.
286. Satoshi Sugita, Yasutomi Kamei, Fumiko Akaike, Takayoshi Suganami, Sayaka Kanai, Maki Hattori, Yasuko Manabe, Nobuharu Fujii, Takako Takai-Igarashi, Miki Tadaishi, Jun Ichiro Oka, Hiroyuki Aburatani, Tetsuya Yamada, Hideki Katagiri, Saori Kakehi, Yoshifumi Tamura, Hideo Kubo, Kenichi Nishida, Shinji Miura, Osamu Ezaki, Yoshihiro Ogawa, Increased systemic glucose tolerance with increased muscle glucose uptake in transgenic mice overexpressing RXRγ in skeletal muscle, PloS one, 10.1371/journal.pone.0020467, 6, 5, 2011, Background: Retinoid X receptor (RXR) γ is a nuclear receptor-type transcription factor expressed mostly in skeletal muscle, and regulated by nutritional conditions. Previously, we established transgenic mice overexpressing RXRγ in skeletal muscle (RXRγ mice), which showed lower blood glucose than the control mice. Here we investigated their glucose metabolism. Methodology/Principal Findings: RXRγ mice were subjected to glucose and insulin tolerance tests, and glucose transporter expression levels, hyperinsulinemic-euglycemic clamp and glucose uptake were analyzed. Microarray and bioinformatics analyses were done. The glucose tolerance test revealed higher glucose disposal in RXRγ mice than in control mice, but insulin tolerance test revealed no difference in the insulin-induced hypoglycemic response. In the hyperinsulinemic-euglycemic clamp study, the basal glucose disposal rate was higher in RXRγ mice than in control mice, indicating an insulin-independent increase in glucose uptake. There was no difference in the rate of glucose infusion needed to maintain euglycemia (glucose infusion rate) between the RXRγ and control mice, which is consistent with the result of the insulin tolerance test. Skeletal muscle from RXRγ mice showed increased Glut1 expression, with increased glucose uptake, in an insulin-independent manner. Moreover, we performed in vivo luciferase reporter analysis using Glut1 promoter (Glut1-Luc). Combination of RXRγ and PPARδ resulted in an increase in Glut1-Luc activity in skeletal muscle in vivo. Microarray data showed that RXRγ overexpression increased a diverse set of genes, including glucose metabolism genes, whose promoter contained putative PPAR-binding motifs. Conclusions/Significance: Systemic glucose metabolism was increased in transgenic mice overexpressing RXRγ. The enhanced glucose tolerance in RXRγ mice may be mediated at least in part by increased Glut1 in skeletal muscle. These results show the importance of skeletal muscle gene regulation in systemic glucose metabolism. Increasing RXRγ expression may be a novel therapeutic strategy against type 2 diabetes..
287. Ayumi Sato, Hiroyuki Kawano, Tatsuto Notsu, Masahiko Ohta, Masanori Nakakuki, Kiyoshi Mizuguchi, Michiko Itoh, Takayoshi Suganami, Yoshihiro Ogawa, Antiobesity effect of eicosapentaenoic acid in high-fat/high-sucrose diet-induced obesity
Importance of hepatic lipogenesis, Diabetes, 10.2337/db09-1554, 59, 10, 2495-2504, 2010.10, OBJECTIVE - Given the pleiotropic effect of eicosapentaenoic acid (EPA), it is interesting to know whether EPA is capable of improving obesity. Here we examined the anti-obesity effect of EPA in mice with two distinct models of obesity. RESEARCH DESIGN AND METHODS - Male C57BL/6J mice were fed a high-fat/high-sucrose diet (25.0% [w/w] fat, 32.5% [w/w] sucrose) (HF/HS group) or a high-fat diet (38.1% [w/w] fat, 8.5% [w/w] sucrose) (HF group) for 4-20 weeks. A total of 5% EPA was administered by partially substituting EPA for fat in the HF/HS + EPA and HF + EPA groups. RESULTS - Both the HF/HS and HF groups similarly developed obesity. EPA treatment strongly suppresses body weight gain and obesity-related hyperglycemia and hyperinsulinemia in HF/HS-fed mice (HF/HS + EPA group), where hepatic triglyceride content and lipogenic enzymes are increased. There is no appreciable effect of EPA on body weight in HF-fed mice (HF + EPA group) without enhanced expression of hepatic lipogenic enzymes. Moreover, EPA is capable of reducing hepatic triglyceride secretion and changing VLDL fatty acid composition in the HF/HS group. By indirect calorimetry analysis, we also found that EPA is capable of increasing energy consumption in the HF/HS + EPA group. CONCLUSIONS - This study is the first demonstration that the anti-obesity effect of EPA in HF/HS-induced obesity is associated with the suppression of hepatic lipogenesis and steatosis. Because the metabolic syndrome is often associated with hepatic lipogenesis and steatosis, the data suggest that EPA is suited for treatment of the metabolic syndrome..
288. Yoshihiro Ogawa, Homeostatic inflammation, an emerging concept, Endocrine Journal, 10.1507/endocrj.EDT10-08, 57, 8, 657-658, 2010.10.
289. Takanobu Yamamoto, Takayoshi Suganami, Minako Kiso-Narita, Peggy A. Scherle, Yasutomi Kamei, Mitsuaki Isobe, Shigeki Higashiyama, Yoshihiro Ogawa, Insulin-induced ectodomain shedding of heparin-binding epidermal growth factor-like growth factor in adipocytes in vitro, Obesity, 10.1038/oby.2010.2, 18, 10, 1888-1894, 2010.10, Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein, which is proteolytically cleaved to release a soluble form via members of the a disintegrin and metalloproteinase (ADAM) family of proteolytic enzymes. This study was designed to elucidate the molecular mechanism underlying insulin-induced HB-EGF shedding in adipocytes in vitro. The 3T3-L1 adipocytes with stable expression of alkaline phosphatase (AP)-tagged proHB-EGF (3T3-L1/HB-EGF-AP adipocytes) were developed and AP activities of conditioned media were determined. Using 3T3-L1/HB-EGF-AP adipocytes, we demonstrated that insulin induces HB-EGF shedding in differentiated 3T3-L1 adipocytes in a dose- and time-dependent manner. There is no significant increase in insulin-induced HB-EGF shedding in undifferentiated 3T3-L1 preadipocytes. Studies with metalloprotease inhibitors suggested that insulin-induced HB-EGF shedding in adipocytes is mediated at least in part via ADAM17. Treatment with recombinant HB-EGF results in a dose- and time-dependent increase in HB-EGF shedding in adipocytes, which is significantly suppressed by pharmacologic blockade of ADAM17 (P < 0.01). Moreover, insulin-induced HB-EGF shedding in adipocytes is significantly inhibited by AG1478, an EGF receptor antagonist (P < 0.01). This study provides in vitro evidence that insulin induces HB-EGF shedding in 3T3-L1 adipocytes. Our data also suggest the role of ADAM17 in insulin-induced HB-EGF shedding in adipocytes..
290. Yuri Okazaki, Noriyasu Ohshima, Ikumi Yoshizawa, Yasutomi Kamei, Stefania Mariggiò, Keiko Okamoto, Masahiro Maeda, Yoshihito Nogusa, Yuichiro Fujioka, Takashi Izumi, Yoshihiro Ogawa, Yoshitsugu Shiro, Masanobu Wada, Norihisa Kato, Daniela Corda, Noriyuki Yanaka, A novel glycerophosphodiester phosphodiesterase, GDE5, controls skeletal muscle development via a non-enzymatic mechanism, Journal of Biological Chemistry, 10.1074/jbc.M110.106708, 285, 36, 27652-27663, 2010.09, Mammalian glycerophosphodiester phosphodiesterases (GP-PDEs) have been identified recently and shown to be implicated in several physiological functions. This study isolated a novel GP-PDE, GDE5, and showed that GDE5 selectively hydrolyzes glycerophosphocholine (GroPCho) and controls skeletal muscle development. We show that GDE5 expression was reduced in atrophied skeletal muscles in mice and that decreasing GDE5 abundance promoted myoblastic differentiation, suggesting that decreased GDE5 expression has a counter-regulatory effect on the progression of skeletal muscle atrophy. Forced expression of full-length GDE5 in cultured myoblasts suppressed myogenic differentiation. Unexpectedly, a truncated GDE5 construct (GDE5 Δ C471), which contained a GP-PDE sequence identified in other GP-PDEs but lacked GroPCho phosphodiesterase activity, showed a similar inhibitory effect. Furthermore, transgenic mice specifically expressing GDE5 Δ C471 in skeletal muscle showed less skeletal muscle mass, especially type II fiber-rich muscle. These results indicate that GDE5 negatively regulates skeletal muscle development even without GroPCho phosphodiesterase activity, providing novel insight into the biological significance of mammalian GP-PDE function in a non-enzymatic mechanism..
291. Kenji Yamashiro, Tetsuo Sasano, Katsuyoshi Tojo, Iyuki Namekata, Junko Kurokawa, Naoki Sawada, Takayoshi Suganami, Yasutomi Kamei, Hikaru Tanaka, Naoko Tajima, Kazunori Utsunomiya, Yoshihiro Ogawa, Tetsushi Furukawa, Erratum to Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages [Biochem. Biophys. Res. Commun., 398, (2010), 284-289], Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2010.08.076, 400, 3, 2010.09.
292. Takayoshi Suganami, Yoshihiro Ogawa, Adipose tissue macrophages
Their role in adipose tissue remodeling, Journal of Leukocyte Biology, 10.1189/jlb.0210072, 88, 1, 33-39, 2010.07, The adipose tissue secretes a large number of bioactive substances, adipocytokines, which may be involved in a variety of physiologic and pathologic processes. Unbalanced production of pro- and anti-inflammatory adipocytokines seen in visceral fat obesity contributes critically to the development of the metabolic syndrome. Evidence has accumulated indicating that obesity is associated with a state of chronic, low-grade inflammation, suggesting that inflammation may be a potential mechanism, whereby obesity leads to insulin resistance. Indeed, obese adipose tissue is characterized by adipocyte hypertrophy, followed by increased angiogenesis, immune cell infiltration, extracellular matrix overproduction, and thus, increased production of proinflammatory adipocytokines during the progression of chronic inflammation. The dynamic change found in the adipose tissue can be referred to as "adipose tissue remodeling," in which stromal cells change dramatically in number and cell type during the course of obesity. Among stromal cells, infiltration of macrophages in the adipose tissue precedes the development of insulin resistance in animal models, suggesting that they are crucial for obesity-related adipose tissue inflammation. We have demonstrated that a paracrine loop involving saturated fatty acids and TNF-α derived from adipocytes and macrophages, respectively, aggravates obesity-induced adipose tissue inflammation. Notably, saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced lipolysis, serve as a naturally occurring ligand for TLR4 complex, thereby activating macrophages. Understanding the molecular mechanism underlying adipose tissue remodeling may lead to the identification of novel, therapeutic strategies to prevent or treat obesity-induced adipose tissue inflammation..
293. Kenji Yamashiro, Tetsuo Sasano, Katsuyoshi Tojo, Iyuki Namekata, Junko Kurokawa, Naoki Sawada, Takayoshi Suganami, Yasutomi Kamei, Hikaru Tanaka, Naoko Tajima, Kazunori Utsunomiya, Yoshihiro Ogawa, Tetsushi Furukawa, Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2010.06.082, 398, 2, 284-289, 2010.07, There is considerable evidence indicating that intracellular Ca2+ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca2+ concentrations ([Ca2+]i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca2+ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca2+ imaging suggested that the LPS-induced increase in [Ca2+]i involves both the TRPV2-mediated intracellular and extracellular Ca2+ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca2+ mobilization from intracellular Ca2+ store and extracellular Ca2+. In addition to Ca2+ mobilization through the IP3-receptor, TRPV2-mediated intracellular Ca2+ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca2+ entry is involved in NFκB-independent IL-6 production..
294. Yoshihiro Yamazaki, Yasutomi Kamei, Satoshi Sugita, Fumiko Akaike, Sayaka Kanai, Shinji Miura, Yukio Hirata, Bruce R. Troen, Tadahiro Kitamura, Ichizo Nishino, Takayoshi Suganami, Osamu Ezaki, Yoshihiro Ogawa, The cathepsin L gene is a direct target of FOXO1 in skeletal muscle, Biochemical Journal, 10.1042/BJ20091346, 427, 1, 171-178, 2010.04, FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominantnegative form of FOXO1 or in skeletal-muscle- specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy..
295. Yasutomi Kamei, Takayoshi Suganami, Tatsuya Ehara, Sayaka Kanai, Koji Hayashi, Yuji Yamamoto, Shinji Miura, Osamu Ezaki, Masaki Okano, Yoshihiro Ogawa, Increased expression of DNA methyltransferase 3a in obese adipose tissue
Studies with transgenic mice, Obesity, 10.1038/oby.2009.246, 18, 2, 314-321, 2010.02, Epigenetic mechanisms are likely to be involved in the development of obesity. This study was designed to examine the role of a DNA methyltransferase (Dnmt3a), in obese adipose tissue. The gene expression of Dnmts was examined by quantitative real-time PCR analysis. Transgenic mice overexpressing Dnmt3a in the adipose tissue driven by the aP2 promoter were created (Dnmt3a mice). DNA methylation of downregulated genes was examined using bisulfite DNA methylation analysis. Dnmt3a mice were fed a methyl-supplemented or high-fat diet, and subjected to body weight measurement and gene expression analysis of the adipose tissue. Expression of Dnmt3a was markedly upregulated in the adipose tissue of obese mice. The complementary DNA (cDNA) microarray analysis of Dnmt3a mice revealed a slight decrease in the gene expression of secreted frizzled-related protein 1 (SFRP1) and marked increase in that of interferon responsive factor 9 (IRF9). In the SFRP1 promoter, DNA methylation was not markedly increased in Dnmt3a mice relative to wild-type mice. In experiments with a high-fat diet or methyl-supplemented diet, body weight did not differ significantly with the genotypes. Gene expression levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were higher in Dnmt3a mice than in wild-type mice on a high-fat diet. This study suggests that increased expression of Dnmt3a in the adipose tissue may contribute to obesity-related inflammation. The data highlight the potential role of Dnmt3a in the adult tissue as well as in the developing embryo and cancer..
296. Miyako Tanaka, Takayoshi Suganami, Satoshi Sugita, Yuri Shimoda, Masato Kasahara, Seiichiro Aoe, Motohiro Takeya, Shu Takeda, Yasutomi Kamei, Yoshihiro Ogawa, Role of central leptin signaling in renal macrophage infiltration, Endocrine Journal, 10.1507/endocrj.K09E-296, 57, 1, 61-72, 2010.02, Monocytes/macrophages are key mediators of wound repair, tissue remodeling, and inflammation. However, the molecular mechanisms underlying macrophage recruitment to the site of inflammation is not fully understood. Leptin acts directly on the hypothalamus, thereby regulating food intake and energy expenditure. The leptin receptor, a single transmembrane protein that belongs to the gp130 family of cytokine receptor superfamily, is expressed not only in the hypothalamus but in a variety of peripheral tissues, suggesting the role of leptin as a pro-inflammatory adipocytokine in peripheral tissues. Here, we show that deficiency of leptin signaling reduces renal macrophage infiltration after unilateral ureteral obstruction (UUO). Bone marrow transplantation studies using leptin signaling-deficient db/db mice revealed that leptin signaling in bone marrow cells may not play a major role in the UUO-induced renal macrophage infiltration. Interestingly, central leptin administration reverses the otherwise reduced UUO-induced renal macrophage infiltration in leptin-deficient ob/ob mice. This is effectively abolished by central co-administration of SHU9119, a melanocortin-3 receptor/melanocortin-4 receptor antagonist. This study demonstrates that central leptin administration in ob/ob mice accelerates renal macrophage infiltration through the melanocortin system, thereby suggesting that the central nervous system, which is inherent to integrate information from throughout the organism, is able to control peripheral inflammation..
297. Hiroyuki Koshiyama, Yoshihiro Ogawa, Kiyoshi Tanaka, Issei Tanaka, Integrated network systems and evolutionary developmental endocrinology, Medical Hypotheses, 10.1016/j.mehy.2009.07.028, 74, 1, 132-138, 2010.01, Endocrine system has been considered to be a linear one, but the 'real world endocrine system' is a complex system, which is difficult to investigate using conventional strategies, such as single nucleotide polymorphism, genome-wide analysis, or gene targeting in animals. Here we propose a new strategy to comprehend the endocrine system as a complex network system. We introduced several novel concepts, such as complex system, network analysis, systems biology and evolutionary medicine, into the comprehension of endocrine system as a whole complex network system. This system is considered to be a scale-free network with key molecules such as acetyl CoA, NAD or ATP as 'hubs'. This system is robust against simple mutations, but various complex diseases may attack hubs. The system is also 'fractals', since there exist similar network systems among cells, proteins, and transcription factors in the lower levels, and there are similar ones among disease and social network in the higher levels. We propose to call this model 'Integrated Network Systems and Evolutionary DEvelopmental ENdocrinology (INS-EDEN)'. This novel framework will facilitate us to develop a new approach for understanding and treatment of various complex diseases related to endocrinology, and identify a unified theory of complex diseases..
298. Noriko Satoh, Akira Shimatsu, Akihiro Himeno, Yousuke Sasaki, Hajime Yamakage, Kazunori Yamada, Takayoshi Suganami, Yoshihiro Ogawa, Unbalanced M1/M2 phenotype of peripheral blood monocytes in obese diabetic patients
Effect of pioglitazone, Diabetes care, 10.2337/dc09-1315, 33, 1, e7, 2010.01.
299. Haruta Mogami, Shigeo Yura, Hiroaki Itoh, Makoto Kawamura, Tsuyoshi Fujii, Ayako Suzuki, Seiichiro Aoe, Yoshihiro Ogawa, Norimasa Sagawa, Ikuo Konishi, Shingo Fujii, Isocaloric high-protein diet as well as branched-chain amino acids supplemented diet partially alleviates adverse consequences of maternal undernutrition on fetal growth, Growth Hormone and IGF Research, 10.1016/j.ghir.2009.03.002, 19, 6, 478-485, 2009.12, Maternal undernutrition causes fetal growth restriction. Protein is a vital dietary nutrient for fetal growth, and branched-chain amino acids (BCAA) are noted to have anabolic actions. In this study, we investigated the effects of maternal high-protein diet or BCAA-supplemented diet upon fetal growth under the condition of maternal calorie restriction. Pregnant mice were calorie-restricted (undernutrition: UN), using either a standard diet (S-UN group), high-protein diet (HP-UN group), or BCAA-supplemented diet (BCAA-UN group) to 70% of the control; dams fed ad libitum with a standard diet (S-NN group) from 10.5 days post coitum (dpc) to 18.5 dpc. The fetal weights of UN groups were significantly decreased compared to that of S-NN. However, the fetal weights of HP-UN and BCAA-UN were significantly higher by 5% and 4%, respectively, than those of S-UN, concomitant with augmentation of the gene and protein expressions of IGF-I and IGF-II in fetal liver. A high-protein diet as well as BCAA-supplemented diet partially improved fetal growth restriction caused by maternal calorie-restriction, suggesting a pivotal role of them in the amelioration of fetal growth restriction..
300. Takayoshi Suganami, Xunmei Yuan, Yuri Shimoda, Kozue Uchio-Yamada, Nobutaka Nakagawa, Ibuki Shirakawa, Takako Usami, Takamitsu Tsukahara, Keizo Nakayama, Yoshihiro Miyamoto, Kazuki Yasuda, Junichiro Matsuda, Yasutomi Kamei, Shigetaka Kitajima, Yoshihiro Ogawa, Activating transcription factor 3 constitutes a negative feedback mechanism that attenuates saturated fatty Acid/Toll-like receptor 4 signaling and macrophage activation in obese adipose tissue, Circulation research, 10.1161/CIRCRESAHA.109.196261, 105, 1, 25-32, 2009.07, Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-α production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-α promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKA mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation..
301. Tsuyoshi Chiba, Yasutomi Kamei, Takahiko Shimizu, Takuji Shirasawa, Aki Katsumata, Lisa Shiraishi, Satoshi Sugita, Yoshihiro Ogawa, Shinji Miura, Osamu Ezaki, Overexpression of FOXO1 in skeletal muscle does not alter longevity in mice, Mechanisms of Ageing and Development, 10.1016/j.mad.2009.04.004, 130, 7, 420-428, 2009.07, Caloric restriction (CR) is the most robust and reproducible intervention that can extend lifespan in rodents. Studies in invertebrates have led to the identification of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signaling pathway, including DAF-16 (C. elegans) and dFOXO (Drosophila). Mice subjected to CR for 8 weeks showed an increase in FOXO1 mRNA and other longevity-related genes: Gadd 45α, glutamine synthase, and catalase in skeletal muscle. To investigate whether FOXO1 expression affects longevity in mammals, transgenic mice were studied that over-express FOXO1 in their skeletal muscle (FOXO1 mice), and in which muscle atrophy occurs. FOXO1 mice showed increases in Gadd 45α, and glutamine synthase proteins in skeletal muscle. In FOXO1 mice, the phosphorylation/dephosphorylation state of the p70 S6K and 4E-BP1 proteins were not altered, suggesting that translation initiation of protein synthesis might not be suppressed. The lifespan of FOXO1 mice was similar to their wild-type littermates. FOXO1 overexpression could not prevent aging-induced reduction in catalase, CuZu-SOD, and Mn-SOD mRNA in skeletal muscle. These data suggest that an increase in FOXO1 protein and its activation in skeletal muscle does not extend lifespan in mice..
302. Ayaka Ito, Takayoshi Suganami, Yoshihiro Ogawa, Inflammatory changes in obese adipose tissue, Nippon rinsho. Japanese journal of clinical medicine, 67, 2, 270-276, 2009.01, Evidence has accumulated suggesting that obesity is a state of chronic, low grade inflammation; it may represent a potential mechanism whereby obesity leads to the metabolic derangements. To understand the molecular mechanism underlying the development of obesity and obesity-related metabolic syndrome, it is of great use to divide the obesity-related 'adipose tissue remodeling' into three phases; 1) adipocyte proliferation and hypertrophy, 2) macrophage infiltration, and 3) adipocyte-macrophage interaction. Elucidation of the molecular mechanism underlying the inflammatory changes in each phase of the obesity-related 'adipose tissue remodeling' may lead to the identification of new therapeutic targets that may reduce obesity-induced inflammatory changes and thus the metabolic syndrome..
303. Noriko Satoh, Akira Shimatsu, Kazuhiko Kotani, Akihiro Himeno, Takafumi Majima, Kazunori Yamada, Takayoshi Suganami, Yoshihiro Ogawa, Highly purified eicosapentaenoic acid reduces cardio-ankle vascular index in association with decreased serum amyloid A-LDL in metabolic syndrome, Hypertension Research, 10.1038/hr.2009.145, 32, 11, 1004-1008, 2009, A recent clinical trial revealed that highly purified eicosapentaenoic acid (EPA), an n-3 polyunsaturated fatty acid, reduces the incidence of cardiovascular diseases. However, the detailed mechanism underlying the anti-atherogenic effect of EPA is still poorly understood. In this study, we examined the effect of EPA on cardio-ankle vascular index (CAVI), a new index of arterial stiffness that is less influenced by blood pressure (BP), as well as on serum amyloid A-low-density lipoprotein (SAA-LDL), an oxidized LDL (oxLDL), in the metabolic syndrome. Ninety-two obese Japanese subjects with metabolic syndromes were randomly divided into two groups (n=46): the EPA-treated group (1.8 g administered daily for 3 months) and the control group. Measurements were taken to assess the changes in glucose-lipid metabolism, SAA-LDL, C-reactive protein (CRP), leptin, adiponectin and pulse wave velocity (PWV), and CAVI. EPA treatment significantly reduced the levels of immunoreactive insulin, triglycerides, SAA-LDL, CRP, PWV and CAVI and increased the levels of adiponectin relative to the control group for 3 months (P<0.05). Stepwise multivariate linear regression analysis revealed that the only significant determinant for a decrease in CAVI by EPA is a reduction in SAA-LDL (P<0.05). Moreover, the EPA-induced reduction of SAA-LDL was only significantly correlated with a decrease in total cholesterol and an increase in adiponectin (P<0.05). This study is the first demonstration that EPA improves arterial stiffness and is less influenced by BP, possibly through the suppression of SAA-LDL, thereby leading to a reduction in the frequency of cardiovascular disease development in metabolic syndrome..
304. Makoto Kawamura, Hiroaki Itoh, Shigeo Yura, Haruta Mogami, Tsuyoshi Fujii, Hisashi Makino, Yoshihiro Miyamoto, Yasunao Yoshimasa, Seiichiro Aoe, Yoshihiro Ogawa, Norimasa Sagawa, Norio Kanayama, Ikuo Konishi, Isocaloric high-protein diet ameliorates systolic blood pressure increase and cardiac remodeling caused by maternal caloric restriction in adult mouse offspring, Endocrine Journal, 10.1507/endocrj.K08E-286, 56, 5, 679-689, 2009, Epidemiologic studies have shown that in utero malnutrition is a risk factor for adult cardiovascular disease (cVd). recently, we reported a mouse animal model of 30% maternal caloric reduction, in which offspring showed a significant increase in systolic blood pressure (SBP) as well as in cardiac remodeling-associated morphological parameters such as cardiac enlargement and coronary perivascular fibrosis in adulthood. Using a similar animal model, we here demonstrated that an increased level of protein consumption during an undernourished pregnancy (high-protein diet; HPD) corrected for the development of cVd risk factors found in fetal undernourishment with less protein consumption (standardprotein diet; SPD). In contrast, maternal ad libitum feeding with HPD resulted in significantly elevated SBP and cardiac enlargement in offspring at 16 wks. appropriate maternal protein ingestion might partly protect against the development of cVd risk factors in offspring..
305. Noriko Satoh, Akira Shimatsu, Yasuhisa Kato, Rika Araki, Kazunori Koyama, Taiichiro Okajima, Makito Tanabe, Mariko Ooishi, Kazuhiko Kotani, Yoshihiro Ogawa, Evaluation of the cardio-ankle vascular index, a new indicator of arterial stiffness independent of blood pressure, in obesity and metabolic syndrome, Hypertension Research, 10.1291/hypres.31.1921, 31, 10, 1921-1930, 2008.12, Aortic stiffness is predictive of cardiovascular diseases (CVD) and mortality in lifestyle-related diseases. The cardio-ankle vascular index (CAVI), a new index of arterial stiffness, was recently developed by measuring of pulse wave velocity (PWV) and blood pressure (BP). CAVI is adjusted for BP based on stiffness parameter β and is less influenced by BP, suggesting its superiority over brachial-ankle PWV (baPWV). However, there are currently no reports on the usefulness of CAVI as an atherogenic index in obesity and metabolic syndrome (MS). Among the 325 obese Japanese outpatients enrolled in the multi-centered Japan Obesity and Metabolic Syndrome Study, 216 patients (67%) met the criteria of MS according to the modified National Cholesterol Education Program-Adult Treatment Panel III. CAVI values were significantly higher in MS than in non-MS patients, whereas there was no significant difference in body mass index, total cholesterol, and low-density lipoprotein-cholesterol between both groups. CAVI values were weakly correlated with BP but closely correlated with the severity of MS and MS-related parameters such as hypoadiponectinemia, relative to baPWV. Furthermore, weight-reduction therapy through diet and exercise over a 3-month period significantly decreased CAVI values in parallel with increasing adiponectin. This study demonstrates for the first time that CAVI is a good indicator of arterial stiffness. It is closely correlated with the severity of MS and CVD risks in obesity and independent of BP, and is thus superior to baPWV. Therefore, the determination of arterial stiffness by CAVI may be useful for evaluating and managing the CVD risks of MS patients..
306. Hideto Yamada, Masayuki Yoshida, Yasutaka Nakano, Takayoshi Suganami, Noriko Satoh, Tomoya Mita, Kosuke Azuma, Michiko Itoh, Yukio Yamamoto, Yasutomi Kamei, Minoru Horie, Hirotaka Watada, Yoshihiro Ogawa, In vivo and in vitro inhibition of monocyte adhesion to endothelial cells and endothelial adhesion molecules by eicosapentaenoic acid, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/ATVBAHA.108.171736, 28, 12, 2173-2179, 2008.12, Objective - A large-scale, prospective, randomized clinical trial has recently revealed that the addition of highly purified eicosapentaenoic acid (EPA) to low-dose statin therapy significantly reduces the incidence of major coronary events. Here we investigated in vivo and in vitro effect of EPA on monocyte adhesion to endothelial cells and adhesion molecules. Methods and Results - A new en face immunohistochemistry of endothelial surface in combination with confocal microscopy revealed marked reduction of lipopolysaccharide (LPS)-induced monocyte adhesion to the aortic endothelium in parallel with the suppression of vascular cell adhesion molecule 1 (VCAM-1) and nuclear translocation of nuclear factor-κB p65 in EPA-treated mice relative to vehicle-treated groups. In an in vitro adhesion assay system under physiological flow conditions, EPA inhibited LPS-induced monocyte adhesion and endothelial adhesion molecules. We found significant decrease in plasma concentrations of soluble intercellular adhesion molecule 1 (sICAM-1) and sVCAM-1 in patients with the metabolic syndrome after a 3-month administration of highly purified EPA (1.8 g daily). Multivariate regression analysis revealed that EPA administration is the only independent determinant of sICAM-1 and sVCAM-1. Conclusions - This study provides evidence that EPA inhibits monocyte adhesion to endothelial cells in parallel with the suppression of endothelial adhesion molecules in vivo and in vitro..
307. Ayaka Ito, Takayoshi Suganami, Akira Yamauchi, Mikako Degawa-Yamauchi, Miyako Tanaka, Ryuji Kouyama, Yuko Kobayashi, Nao Nitta, Kazuki Yasuda, Yukio Hirata, William A. Kuziel, Motohiro Takeya, Shiro Kanegasaki, Yasutomi Kamei, Yoshihiro Ogawa, Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue, Journal of Biological Chemistry, 10.1074/jbc.M804220200, 283, 51, 35715-35723, 2008.12, The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2-/- BMCs (ob/ob + CCR2-/- BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2-/- BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue..
308. Yasutomi Kamei, Yoshihiro Ogawa, Skeltal muscle function and FOXO, Seikagaku, 80, 11, 1026-1029, 2008.12.
309. T. Nagae, K. Mori, M. Mukoyama, M. Kasahara, H. Yokoi, T. Suganami, K. Sawai, T. Yoshioka, M. Koshikawa, Y. Saito, Y. Ogawa, T. Kuwabara, I. Tanaka, A. Sugawara, T. Kuwahara, K. Nakao, Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis, Kidney International, 10.1038/ki.2008.98, 74, 1, 70-80, 2008.07, Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-β-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation..
310. Takuya Toyoda, Yasutomi Kamei, Hirotsugu Kato, Satoshi Sugita, Motohiro Takeya, Takayoshi Suganami, Yoshihiro Ogawa, Effect of peroxisome proliferator-activated receptor-α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, Obesity, 10.1038/oby.2008.62, 16, 6, 1199-1207, 2008.06, Objective: This study was designed to examine the effect of peroxisome proliferator-activated receptor-α (PPAR-α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR-α ligands (Wy-14,643 and fenofibrate) were added to 3T3-L1 adipocytes, RAW264 macrophages, or co-culture of 3T3-L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) mRNA expression and secretion were examined. PPAR-α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR-α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR-α-deficient mice. Results: In the co-culture of 3T3-L1 adipocytes and RAW264 macrophages, PPAR-α ligands reduced MCP-1 and TNF-α mRNA expression and secretion in vitro relative to vehicle-treated group. The anti-inflammatory effect of Wy-14,643 was observed in adipocytes treated with macrophage-conditioned media or mouse recombinant TNF-α and in macrophages treated with adipocyte-conditioned media or palmitate. Systemic administration of PPAR-α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy-14,643 also exerted an anti-inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR-α-deficient mice. Discussion: This study provides evidence for the anti-inflammatory effect of PPAR-α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity-related metabolic syndrome..
311. Shigeo Yura, Hiroaki Itoh, Norimasa Sagawa, Hiroshi Yamamoto, Hiroaki Masuzaki, Kazuwa Nakao, Makoto Kawamura, Haruta Mogami, Yoshihiro Ogawa, Shingo Fujii, Neonatal exposure to leptin augments diet-induced obesity in leptin-deficient ob/ob mice, Obesity, 10.1038/oby.2008.57, 16, 6, 1289-1295, 2008.06, Objective: Epidemiological evidence has revealed that undernutrition in utero is closely associated with obesity and related detrimental metabolic sequelae in adulthood. Recently, using a wild-type (wt) mouse model in which offspring were exposed to intrauterine undernutrition (UN offspring), we reported that the premature leptin surge during neonatal growth promotes lifelong changes in energy regulating circuitry in the hypothalamus, thus playing an important role in the development of pronounced obesity on a high-fat diet (HFD) in adulthood. Here, we further evaluate the essential involvement of leptin in the developmental origins of obesity using leptin-deficient ob/ob mice. Methods and Procedures: We assessed the progression of obesity on an HFD in adult leptin-deficient ob/ob male mice that were exposed to intrauterine undernutrition by maternal food restriction (ob/ob UN offspring) or to leptin treatment during the neonatal period; this treatment is comparable to the premature leptin surge observed in the wt-UN offspring. Results: On an HFD, the body weight of the male ob/ob UN offspring paralleled that of the ob/ob offspring exposed to normal intrauterine nutrition (ob/ob NN offspring). In contrast, early exposure to leptin in the ob/ob NN offspring during early neonatal growth reproduced the development of pronounced obesity on an HFD in adulthood. Discussion: The presence of leptin and associated energy regulation are indispensable in the acceleration of obesity on an HFD caused by undernutrition in utero. The premature leptin surge plays an essential role in the developmental origins of obesity as a programming signal during the early neonatal period..
312. Yasutomi Kamei, Shinji Miura, Takayoshi Suganami, Fumiko Akaike, Sayaka Kanai, Satoshi Sugita, Aki Katsumata, Hiroyuki Aburatani, Terry G. Unterman, Osamu Ezaki, Yoshihiro Ogawa, Regulation of SREBP1c gene expression in skeletal muscle
Role of retinoid x receptor/liver x receptor and forkhead-O1 transcription factor, Endocrinology, 10.1210/en.2007-1461, 149, 5, 2293-2305, 2008.05, Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-α (RXRα) and liver X receptor-α (LXRα). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Herewereport that gene expression of RXRγ is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXRγ or RXRα, together with LXRα, activate the SREBP1c promoter in vitro. Moreover, transgenic mice over-expressing RXRγ specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXRγ showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXRγ and SREBP1c. In addition, FOXO1 suppressed RXRα/LXRα-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle..
313. H. Yokoi, M. Mukoyama, K. Mori, M. Kasahara, T. Suganami, K. Sawai, T. Yoshioka, Y. Saito, Y. Ogawa, T. Kuwabara, A. Sugawara, K. Nakao, Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice, Kidney International, 10.1038/sj.ki.5002722, 73, 4, 446-455, 2008.01, Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes..
314. Atsutaka Yasui, Hitoshi Nishizawa, Yosuke Okuno, Kentaro Morita, Hironori Kobayashi, Kenichiro Kawai, Morihiro Matsuda, Ken Kishida, Shinji Kihara, Yasutomi Kamei, Yoshihiro Ogawa, Tohru Funahashi, Iichiro Shimomura, Foxo1 represses expression of musclin, a skeletal muscle-derived secretory factor, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2007.10.013, 364, 2, 358-365, 2007.12, Musclin is a novel skeletal muscle-derived secretory factor, whose mRNA level is markedly regulated by nutritional status. In the present study, we investigated the mechanism of musclin mRNA regulation by insulin. In C2C12 myocytes, insulin-induced upregulation of musclin mRNA was significantly decreased by treatment of phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and was abolished in C2C12 myocytes stably expressing a constitutively active Foxo1 (Foxo1-3A), suggesting the involvement of Foxo1 in the regulation of musclin mRNA. Promoter deletion analysis of musclin promoter revealed that the region of -303/-123 is important for the repression of promoter activity by Foxo1. Chromatin immunoprecipitation assay showed that Foxo1 bound to musclin promoter. Musclin mRNA level was markedly downregulated in gastrocnemius muscle of Foxo1 transgenic mice. Our results demonstrated that Foxo1 downregulates musclin mRNA expression both in vitro and in vivo, which should explain insulin-mediated upregulation of this gene in muscle cells..
315. Rumi Hachiya, Yuko Ohashi, Yasutomi Kamei, Takayoshi Suganami, Hiroshi Mochizuki, Norimasa Mitsui, Masaaki Saitoh, Masako Sakuragi, Gen Nishimura, Hirofumi Ohashi, Tomonobu Hasegawa, Yoshihiro Ogawa, Intact kinase homology domain of natriuretic peptide receptor-B is essential for skeletal development, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2007-1101, 92, 10, 4009-4014, 2007.10, Context: Natriuretic peptide receptor-B (NPR-B, GC-B in rodents; gene name NPR2) is a guanylyl cyclase-coupled receptor that mediates the effect of C-type natriuretic peptide. Homozygous mutations in human NPR-B cause acromesomelic dysplasia, type Maroteaux (OMIM 602875), an autosomal recessive skeletal dysplasia. NPR-B has an intracellular kinase homology domain (KHD), which has no kinase activity, and its functional significance in vivo is currently unknown. Objective: We examined the functional significance of a novel NPR-B KHD mutation in humans. Patients and Methods: A 28-yr-old Japanese male presented with marked short stature (118.5 cm, -9.3 SD). His limbs showed marked shortening in the middle and distal segments. His parents had relatively short stature with height z-scores of -2.75 and -0.98 (his father and mother, respectively). Direct sequencing of coding region of the NPR2 gene of the family was performed. The mutant receptor activity was investigated by saturation binding assay and cGMP measurement. Additionally, interaction between the mutant and wild type allele was investigated by the titration experiments. Results: We identified a novel missense mutation L658F in KHD of NPR-B in homozygous and heterozygous states in the patient and his parents, respectively. The mutation conferred normal binding affinity for C-type natriuretic peptide but no discernible ligand-induced cGMP production. Furthermore, L658F mutant impaired wild-type NPR-B-mediated cGMP production in a dose-dependent manner, suggesting that short stature found in L658F heterozygote can be caused by its dominant-negative effect. Conclusions: This study provides the first evidence that intact KHD of NPR-B is essential for skeletal development..
316. Michiko Itoh, Takayoshi Suganami, Noriko Satoh, Kanami Tanimoto-Koyama, Xunmei Yuan, Miyako Tanaka, Hiroyuki Kawano, Takashi Yano, Seiichiro Aoe, Motohiro Takeya, Akira Shimatsu, Hideshi Kuzuya, Yasutomi Kamei, Yoshihiro Ogawa, Increased adiponectin secretion by highly purified eicosapentaenoic acid in rodent models of obesity and human obese subjects, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/ATVBAHA.106.136853, 27, 9, 1918-1925, 2007.09, OBJECTIVES - Fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) or n-3 PUFAs have been shown to reduce the incidence of coronary heart disease. Here we investigated the effect of highly purified eicosapentaenoic acid (EPA) on production of adiponectin, the only established antiatherogenic and antiinflammatory adipocytokine, in rodent models of obesity and human obese subjects. METHODS AND RESULTS - We demonstrated that EPA increases adiponectin secretion in genetically obese ob/ob mice and high-fat diet-induced obese mice. In the in vitro coculture of adipocytes and macrophages, EPA reversed the coculture-induced decrease in adiponectin secretion at least in part through downregulation of tumor necrosis factor-α in macrophages. We also showed significant increase in plasma adiponectin concentrations in human obese subjects after a 3-month treatment with EPA (1.8 g daily). Multivariate regression analysis revealed that EPA treatment is the only independent determinant of plasma adiponectin concentrations. CONCLUSION - This study demonstrates that EPA increases adiponectin secretion in rodent models of obesity and human obese subjects, possibly through the improvement of the inflammatory changes in obese adipose tissue. Because EPA has reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial, this study provides important insight into its therapeutic implication in obesity-related metabolic sequelae..
317. Satoshi Sugita, Yasutomi Kamei, Jun Ichiro Oka, Takayoshi Suganami, Yoshihiro Ogawa, Macrophage-colony stimulating factor in obese adipose tissue
Studies with heterozygous op/+ mice, Obesity, 10.1038/oby.2007.237, 15, 8, 1988-1995, 2007.08, Objective: We examined the gene expression of macrophage-colony stimulating factor (M-CSF) in mice with diet-induced obesity and in genetically obese mice. We also examined the effect of decreased M-CSF signaling on the susceptibility to obesity and macrophage recruitment into the adipose tissue of mice. Research Methods and Procedures: The adipose tissue from mice with diet-induced obesity, obese KKAy mice, and ob/ob obese mice was used for RNA preparation. Production of M-CSF and monocyte chemoattractant protein-1 (MCP-1) was examined by quantitative real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. The op/+ heterozygous mice, with decreased functional M-CSF expression, were placed on a high-fat diet or crossed with KKAy mice to study the susceptibility to obesity. The gene expression of macrophage markers in adipose tissue was examined.Results: The expression of M-CSF was not significantly changed in mice on a high-fat diet or in either type of genetic obesity (KKAy or ob/ob mice). No change in the degree of obesity or macrophage-related gene expression (F4/80, CD68, and MCP-1) in the adipose tissue was observed in op/+ mice compared with +/+ control mice, which were either treated with a high-fat diet or crossed with KKAy mice. Discussion: This study demonstrated that there was no significant change in the expression of M-CSF in the adipose tissue from obese mice and only a minor phenotypic change, such as macrophage infiltration, in the adipose tissue from op/+ mice, suggesting that M-CSF does not play a major role in macrophage recruitment in the adipose tissue of obese mice..
318. Ayaka Ito, Takayoshi Suganami, Yoshihiro Miyamoto, Yasunao Yoshimasa, Motohiro Takeya, Yasutomi Kamei, Yoshihiro Ogawa, Role of MAPK phosphatase-1 in the induction of monocyte chemoattractant protein-1 during the course of adipocyte hypertrophy, Journal of Biological Chemistry, 10.1074/jbc.M701549200, 282, 35, 25445-25452, 2007.08, Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy..
319. H. Kobayashi, M. Kasahara, M. Hino, S. Takahara, K. Ikeda, C. Son, T. Iwakura, N. Matsuoka, A. Yoshimoto, N. Ohgo, R. Kasai, T. Ishihara, Y. Ogawa, A novel heterozygous splice-site mutation of LEM domain-containing 3 in a Japanese kindred with Buschke-Ollendorff syndrome, Journal of Endocrinological Investigation, 10.1007/BF03347437, 30, 3, 263-265, 2007.03.
320. Takayoshi Suganami, Tae Mieda, Michiko Itoh, Yuri Shimoda, Yasutomi Kamei, Yoshihiro Ogawa, Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a Toll-like receptor 4 mutation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2006.12.190, 354, 1, 45-49, 2007.03, Obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. We have provided in vitro evidence that saturated fatty acids, which are released from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for Toll-like receptor 4 (TLR4) to induce the inflammatory changes in macrophages. Here we show the attenuation of adipose tissue inflammation in C3H/HeJ mice carrying a functional mutation in the TLR4 gene relative to control C3H/HeN mice during a 16-week high-fat diet. We also find that adiponectin mRNA expression is significantly reduced by co-culture of hypertrophied 3T3-L1 adipocytes and C3H/HeN peritoneal macrophages, which is reversed, when co-cultured with C3H/HeJ peritoneal macrophages. This study provides in vivo evidence that TLR4 plays a role in obesity-related adipose tissue inflammation and thus helps to identify the therapeutic targets that may reduce obesity-induced inflammation and the metabolic syndrome..
321. Licht Miyamoto, Taro Toyoda, Tatsuya Hayashi, Shin Yonemitsu, Masako Nakano, Satsuki Tanaka, Ken Ebihara, Hiroaki Masuzaki, Kiminori Hosoda, Yoshihiro Ogawa, Gen Inoue, Tohru Fushiki, Kazuwa Nakao, Effect of acute activation of 5′-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle, Journal of Applied Physiology, 10.1152/japplphysiol.01034.2006, 102, 3, 1007-1013, 2007.03, 5′-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4- carboxamide-1-β-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the α1- and α2-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis..
322. Ken Ebihara, Toru Kusakabe, Masakazu Hirata, Hiroaki Masuzaki, Fumiko Miyanaga, Nozomi Kobayashi, Tomohiro Tanaka, Hideki Chusho, Takashi Miyazawa, Tatsuya Hayashi, Kiminori Hosoda, Yoshihiro Ogawa, Alex M. DePaoli, Masanori Fukushima, Kazuwa Nakao, Efficacy and safety of leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2006-1546, 92, 2, 532-541, 2007.02, Background: Lack of leptin is implicated in insulin resistance and other metabolic abnormalities in generalized lipodystrophy; however, the efficacy, safety, and underlying mechanisms of leptin-replacement therapy in patients with generalized lipodystrophy remain unclear. Methods: Seven Japanese patients with generalized lipodystrophy, two acquired and five congenital type, were treated with the physiological replacement dose of recombinant leptin during an initial 4-month hospitalization followed by outpatient follow-up for up to 36 months. Results: The leptin-replacement therapy with the twice-daily injection dramatically improved fasting glucose (mean ± SE, 172 ± 20 to 120 ± 12 mg/dl, P < 0.05) and triglyceride levels (mean ± SE, 700 ± 272 to 260 ± 98 mg/dl, P < 0.05) within 1 wk. The leptin-replacement therapy reduced insulin resistance evaluated by euglycemic clamp method and augmented insulin secretion at glucose tolerance test with different responses between acquired and congenital types. Improvement of the fatty liver was also observed. The efficacy and safety of the once-daily injection were comparable to those of the twice-daily injection. The leptin-replacement therapy ameliorated macro- and microalbuminuria and showed no deterioration of neuropathy and retinopathy of these patients. The leptin-replacement therapy is beneficial to diabetic complications and lipodystrophic ones. Two patients developed antileptin antibodies but not neutralizing antibodies. The therapy was well tolerated, and its effects were maintained for up to 36 months without any notable adverse effects such as hypoglycemia, high blood pressure, or reduction of bone mineral density. Conclusions: The present study demonstrates the efficacy and safety of the long-term leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy..
323. Yasutomi Kamei, Takayoshi Suganami, Takashi Kohda, Fumitoshi Ishino, Kazuki Yasuda, Shinji Miura, Osamu Ezaki, Yoshihiro Ogawa, Peg1/Mest in obese adipose tissue is expressed from the paternal allele in an isoform-specific manner, FEBS Letters, 10.1016/j.febslet.2006.12.002, 581, 1, 91-96, 2007.01, Paternally expressed 1 (Peg1)/mesoderm specific transcript (Mest) is an imprinted gene, which is only transcribed from the paternal (father's) allele. In some human cancer tissues, an alternatively spliced variant of PEG1/MEST mRNA using a different promoter of a distinct first exon is expressed from both paternal and maternal alleles. We previously reported that Peg1/Mest expression was markedly up-regulated in obese adipose tissue in mice. Moreover, transgenic overexpression of Peg1/Mest in the adipose tissue resulted in the enlargement of adipocytes in size. Given the potential pathophysiologic relevance in obesity, we examined the nature of increased expression of Peg1/Mest in obese adipose tissue. In obese adipose tissue, expression of Peg1/Mest was increased, but not that of other imprinted genes tested. The transcription rate of Peg1/Mest was increased in obese adipose tissue. We found at least four isoforms of mouse Peg1/Mest generated by use of the alternative first exons. We also demonstrated that the abundantly expressed Peg1/Mest in obese adipose tissue retained monoallelic expression. This is the first report of monoallelic induction of Peg1/Mest in adult tissues..
324. Noriko Satoh, Akira Shimatsu, Kazuhiko Kotani, Naoki Sakane, Kazunori Yamada, Takayoshi Suganami, Hideshi Kuzuya, Yoshihiro Ogawa, Purified eicosapentaenoic acid reduces small dense LDL, remnant lipoprotein particles, and C-reactive protein in metabolic syndrome, Diabetes care, 10.2337/dc06-1179, 30, 1, 144-146, 2007.01.
325. Takayoshi Suganami, Kanami Tanimoto-Koyama, Junko Nishida, Michiko Itoh, Xunmei Yuan, Shinji Mizuarai, Hidehito Kotani, Shoji Yamaoka, Kensuke Miyake, Seiichiro Aoe, Yasutomi Kamei, Yoshihiro Ogawa, Role of the Toll-like receptor 4/NF-κB pathway in saturated fatty acid-induced inflammatory changes in the interaction between adipocytes and macrophages, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/01.ATV.0000251608.09329.9a, 27, 1, 84-91, 2007.01, OBJECTIVE - Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-α derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. METHODS AND RESULTS - Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-κB (NF-κB), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-κB markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFα production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the β3-adrenergic stimulation, resulted in the activation of the TLR4/NF-κB pathway. CONCLUSION - These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-κB activation..
326. Jiyoung Park, Sik Choe Sung, A. Hyun Choi, Ho Kim Kang, Jin Yoon Myeong, Takayoshi Suganami, Yoshihiro Ogawa, Bum Kim Jae, Increase in glucose-6-phosphate dehydrogenase in adipocytes stimulates oxidative stress and inflammatory signals, Diabetes, 10.2337/db05-1570, 55, 11, 2939-2949, 2006.11, In adipocytes, oxidative stress and chronic inflammation are closely associated with metabolic disorders, including insulin resistance, obesity, cardiovascular disease, and type 2 diabetes. However, the molecular mechanisms underlying these metabolic disorders have not been thoroughly elucidated. In this report, we demonstrate that overexpression of glucose-6-phosphate dehydrogenase (G6PD) in adipocytes stimulates oxidative stress and in-flammatory responses, thus affecting the neighboring macrophages. Adipogenic G6PD overexpression promotes the expression of pro-oxidative enzymes, including inducible nitric oxide synthase and NADPH oxidase, and the activation of nuclear factor-κB (NF-κB) signaling, which eventually leads to the dysregulation of adipocytokines and inflammatory signals. Furthermore, secretory factors from G6PD-overexpressing adipocytes stimulate macrophages to express more proinflammatory cytokines and to be recruited to the adipocytes; this would cause chronic inflammatory conditions in the adipose tissue of obesity. These effects of G6PD overexpression in adipocytes were abolished by pretreatment with NF-κB inhibitors or antioxidant drugs. Thus, we propose that a high level of G6PD in adipocytes may mediate the onset of metabolic disorders in obesity by increasing the oxidative stress and inflammatory signals..
327. H. Kobayashi, M. Kasahara, M. Hino, H. Yoshimura, S. Takahara, K. Ikeda, C. Son, T. Iwakura, A. Yoshimoto, T. Ishihara, Y. Ogawa, A novel heterozygous deletion frameshift mutation of GATA3 in a Japanese kindred with the hypoparathyroidism, deafness and renal dysplasia syndrome [2], Journal of Endocrinological Investigation, 10.1007/BF03347383, 29, 9, 851-853, 2006.10.
328. H. Makino, M. Mukoyama, K. Mori, T. Suganami, M. Kasahara, K. Yahata, T. Nagae, H. Yokoi, K. Sawai, Y. Ogawa, S. Suga, Y. Yoshimasa, A. Sugawara, I. Tanaka, K. Nakao, Transgenic overexpression of brain natriuretic peptide prevents the progression of diabetic nephropathy in mice, Diabetologia, 10.1007/s00125-006-0352-y, 49, 10, 2514-2524, 2006.10, Aims/hypothesis: Brain natriuretic peptide (BNP) is a potent vasorelaxing and natriuretic peptide that is secreted from the heart and has cardioprotective properties. We have previously generated hypotensive transgenic mice (BNP-Tg mice) that overproduce BNP in the liver, which is released into the circulation. Using this animal model, we successfully demonstrated the amelioration of renal injury after renal ablation and in proliferative glomerulonephritis. Glomerular hyperfiltration is an early haemodynamic derangement, representing one of the key mechanisms of the pathogenesis of diabetic nephropathy. Based on the suggested involvement of increased endogenous natriuretic peptides, the aim of this study was to investigate their role in the development and progression of diabetic nephropathy. Materials and methods: We evaluated the progression of renal injury and fibrogenesis in BNP-Tg mice with diabetes induced by streptozotocin. We also investigated the effect of BNP on high glucose-induced signalling abnormalities in mesangial cells. Results: After induction of diabetes, control mice exhibited progressively increased urinary albumin excretion with impaired renal function, whereas these changes were significantly ameliorated in BNP-Tg mice. Notably, diabetic BNP-Tg mice revealed minimal mesangial fibrogenesis with virtually no glomerular hypertrophy. Glomerular upregulation of extracellular signal-regulated kinase, TGF-β and extracellular matrix proteins was also significantly inhibited in diabetic BNP-Tg mice. In cultured mesangial cells, activation of the above cascade under high glucose was abrogated by the addition of BNP. Conclusions/interpretation: Chronic excess of BNP prevents glomerular injury in the setting of diabetes, suggesting that renoprotective effects of natriuretic peptides may be therapeutically applicable in preventing the progression of diabetic nephropathy..
329. Noriko Sato, Yoshihiro Ogawa, Prohormone convertase 1 (PC1) gene mutation, Nippon rinsho. Japanese journal of clinical medicine, Suppl 3, 249-253, 2006.09.
330. Noriko Satoh, Akira Shimatsu, Kazunori Yamada, Megumi Aizawa-Abe, Takayoshi Suganami, Hideshi Kuzuya, Yoshihiro Ogawa, An α-glucosidase inhibitor, voglibose, reduces oxidative stress markers and soluble intercellular adhesion molecule 1 in obese type 2 diabetic patients, Metabolism: Clinical and Experimental, 10.1016/j.metabol.2006.01.016, 55, 6, 786-793, 2006.06, Postprandial hyperglycemia and hyperlipidemia are considered risk factors for cardiovascular disease. This study was designed to elucidate whether improving the postprandial state by voglibose, an α-glucosidase inhibitor, leads to the reduction of oxidative stress markers and soluble adhesion molecules in obese type 2 diabetic patients. A total of 30 Japanese obese type 2 diabetic patients were randomly assigned and treated for 3 weeks with either diet alone (the control group) or diet plus voglibose (0.9 mg daily) (the voglibose group) (n = 15 each). Analysis of the diurnal metabolic profiles revealed a significant reduction of postprandial hyperglycemia and hyperlipidemia in the voglibose group relative to the control group (P < .05), despite the similar improvement in body mass index and hemoglobin A1c in both groups. Voglibose also decreased significantly the plasma levels of soluble intercellular adhesion molecule 1 and urinary excretion of 8-iso-prostaglandin F2α and 8-hydroxydeoxyguanosine (P < .01) and C-reactive protein (P < .05) relative to the control group. In conclusion, this study represents the first demonstration that voglibose reduces oxidative stress generation and soluble intercellular adhesion molecule 1 in parallel with the reduction of postprandial hyperglycemia and hyperlipidemia in obese type 2 diabetic patients..
331. Hiromasa Kobayashi, I. Fujisawa, K. Ikeda, C. Son, T. Iwakura, A. Yoshimoto, M. Kasahara, T. Ishihara, Y. Ogawa, A novel heterozygous missense mutation in the vasopressin moiety is identified in a Japanese person with neurohypophyseal diabetes insipidus, Journal of Endocrinological Investigation, 10.1007/BF03345549, 29, 3, 252-256, 2006.03, The autosomal dominant familial neuronal diabetes insipidus (adFNDI) is caused by diverse mutations in one allele of the gene that encodes the arginine vasopressin (AVP) precursor protein, AVP-neurophysin II (AVP-NP II). Most of the mutations identified so far are located in either the signal peptide or NP II moiety. Two recently published mutations in the AVP gene identified in kindreds with adFNDI predict a substitution of histidine for tyrosine at position 2 and a deletion of phenylalanine at position 3 in AVP. They are unique among adFNDI mutations in that they are the only adFNDI mutations that affect amino acid residues in the AVP moiety of the pro-hormone. Here, we report a novel heterozygous missense mutation in the AVP moiety of the AVP-NP II gene in a Japanese person with neurohypophyseal diabetes insipidus (DI). This mutation occurs at position 2 in AVP and predicts a substitution of serine for tyrosine (Y21S). It is expected to interfere with normal binding of AVP with NP II, and thus result in misfolding of the precursor proteins. The data of this study support the notion that mutations affecting the AVP moiety can result in the initiation of the pathological processes..
332. Masako Nakano, Taku Hamada, Tatsuya Hayashi, Shin Yonemitsu, Licht Miyamoto, Taro Toyoda, Satsuki Tanaka, Hiroaki Masuzaki, Ken Ebihara, Yoshihiro Ogawa, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Akira Otaka, Toru Fushiki, Kazuwa Nakao, α2 Isoform-specific activation of 5′ adenosine monophosphate-activated protein kinase by 5-aminoimidazole-4-carboxamide-1- β-D-ribonucleoside at a physiological level activates glucose transport and increases glucose transporter 4 in mouse skeletal muscle, Metabolism: Clinical and Experimental, 10.1016/j.metabol.2005.09.003, 55, 3, 300-308, 2006.03, 5′Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the α1 and α2 isoforms of AMPK, the α2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of α2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the α2 isoform, we used 5-aminoimidazole-4- carboxamide-1-β-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the α2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the α2 isoform for 90 minutes, but not the α1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the α2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in α2 activity paralleled the phosphorylation of Thr172, the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase β, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the α2 isoform of AMPK..
333. Noriko Sato, Yoshihiro Ogawa, Clinical features of the metabolic syndrome and its molecular mechanism, Nippon rinsho. Japanese journal of clinical medicine, 64 Suppl 9, 349-355, 2006.01.
334. Noriko Satoh, Yoshihiro Ogawa, Leptin, Nippon rinsho. Japanese journal of clinical medicine, 64 Suppl 5, 168-172, 2006.01.
335. Kazumichi Onigata, Yoshihiro Ogawa, POMC gene mutations in human, Nippon rinsho. Japanese journal of clinical medicine, Suppl 3, 255-259, 2006.01.
336. Takayoshi Suganami, Yoshihiro Ogawa, Pathophysiologic role of macrophages infiltrated into obese adipose tissue, Nippon rinsho. Japanese journal of clinical medicine, 64 Suppl 9, 198-202, 2006.01.
337. Hiroyuki Koshiyama, Yoshihiro Ogawa, Kiyoshi Tanaka, Issei Tanaka, The unified hypothesis of interactions among the bone, adipose and vascular systems
'Osteo-lipo-vascular interactions', Medical Hypotheses, 10.1016/j.mehy.2005.11.024, 66, 5, 960-963, 2006, Epidemiological evidence has established a link among hyperlipidemia, visceral obesity, osteoporosis, and cardiovascular diseases (CVD). We here propose the hypothesis that the associations of those disorders are based on interaction of the three organs, i.e. the bone, adipose, and vascular tissues, possibly through multiple interactions among several humoral factors and/or transcription factors. The unified hypothesis of three organs, which we call 'osteo-lipo-vascular interactions', may be explained by the common origin of the cells in each organ. The mesenchymal stem cells are capable of differentiating into osteoblasts, vascular smooth muscle cells, and adipocytes. Alternatively, macrophages may evolve into osteoclasts or infiltrate both the vascular and adipose tissues, thereby leading to chronic inflammation. This unified concept of three organs may provide insights into the development of a new drug for the treatment of osteoporosis, obesity, hyperlipidemia or CVD..
338. Tomohiro Tanaka, Hiroaki Masuzaki, Ken Ebihara, Yoshihiro Ogawa, Shintaro Yasue, Hideo Yukioka, Hideki Chusho, Fumiko Miyanaga, Takashi Miyazawa, Muneya Fujimoto, Toru Kusakabe, Nozomi Kobayashi, Tatsuya Hayashi, Kiminori Hosoda, Kazuwa Nakao, Transgenic expression of mutant peroxisome proliferator-activated receptor γ in liver precipitates fasting-induced steatosis but protects against high-fat diet-induced steatosis in mice, Metabolism: Clinical and Experimental, 10.1016/j.metabol.2005.05.015, 54, 11, 1490-1498, 2005.11, Steatosis is one of the most common liver diseases and is associated with the metabolic syndrome. A line of evidence suggests that peroxisome proliferator-activated receptor (PPAR) α and PPARγ are involved in its pathogenesis. Hepatic overexpression of PPARγ1 in mice provokes steatosis, whereas liver-specific PPARγ disruption ameliorates steatosis in ob/ob mice, suggesting that hepatic PPARγ functions as an aggravator of steatosis. In contrast, PPARα-null mice are susceptible to steatosis because of reduced hepatic fatty acid oxidation. PPARγ with mutations in its C-terminal ligand-binding domain (L468A/E471A mutant PPARγ1) have been reported as a constitutive repressor of both PPARα and PPARγ activities in vitro. To elucidate the effect of cosuppression of PPARα and PPARγ on steatosis, we generated mutant PPARγ transgenic mice (Liver mt PPARγ Tg) under the control of liver-specific human serum amyloid P component promoter. In the liver of transgenic mice, PPARα and PPARγ agonist-induced augmentation of the expression of downstream target genes of PPARα and PPARγ, respectively, was significantly attenuated, suggesting PPARα and PPARγ cosuppression in vivo. Suppression of PPARα and PPARγ target genes was also observed in the fasted and high-fat-fed conditions. Liver mt PPARγ Tg were susceptible to fasting-induced steatosis while being protected against high-fat diet-induced steatosis. The opposite hepatic outcomes in Liver mt PPARγ Tg as a result of fasting and high-fat feeding may indicate distinct roles of PPARα and PPARγ in 2 different types of nutritionally provoked steatosis..
339. Takayoshi Suganami, Junko Nishida, Yoshihiro Ogawa, A paracrine loop between adipocytes and macrophages aggravates inflammatory changes
Role of free fatty acids and tumor necrosis factor α, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/01.ATV.0000183883.72263.13, 25, 10, 2062-2068, 2005.10, Objective - Weight gain is associated with infiltration of fat by macrophages, suggesting that they are an important source of inflammation in obese adipose tissue. Here we developed an in vitro coculture system composed of adipocytes and macrophages and examined the molecular mechanism whereby these cells communicate. Methods and Results - Coculture of differentiated 3T3-L1 adipocytes and macrophage cell line RAW264 results in the marked upregulation of proinflammatory cytokines, such as tumor necrosis factor α(TNF-α), and the downregulation of the antiinflammatory cytokine adiponectin. Such inflammatory changes are induced by the coculture without direct contact, suggesting the role of soluble factors. A neutralizing antibody to TNF-α, which occurs mostly in macrophages, inhibits the inflammatory changes in 3T3-L1, suggesting that TNF-α is a major macrophage-derived mediator of inflammation in adipocytes. Conversely, free fatty acids (FFAs) may be important adipocyte-derived mediators of inflammation in macrophages, because the production of TNF-α in RAW264 is markedly increased by palmitate, a major FFA released from 3T3-L1. The inflammatory changes in the coculture are augmented by use of either, hypertrophied 3T3-L1 or adipose stromal vascular fraction obtained from obese ob/ob mice. Conclusions - We postulate that a paracrine loop involving FFAs and TNF-α between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue..
340. Ryuji Kouyama, Takayoshi Suganami, Junko Nishida, Miyako Tanaka, Takuya Toyoda, Minako Kiso, Tsuyoshi Chiwata, Yoshihiro Miyamoto, Yasunao Yoshimasa, Akiyoshi Fukamizu, Masatsugu Horiuchi, Yukio Hirata, Yoshihiro Ogawa, Attenuation of diet-induced weight gain and adiposity through increased energy expenditure in mice lacking angiotensin II type 1a receptor, Endocrinology, 10.1210/en.2005-0003, 146, 8, 3481-3489, 2005.08, Given that angiotensin II (AII) type 1 and 2 receptors (Agtr1 and Agtr2) are expressed in adipose tissue, AII may act directly on adipose tissue. However, regardless of whether AII directly modulates adipose tissue growth and metabolism in vivo and, if so, whether it is mediated via Agtr1 are still matters of debate. To understand the functional role of Agtr1 in adipose tissue growth and metabolism in vivo, we examined the metabolic phenotypes of mice lacking Agtr1a (Agtr1a -/- mice) during a high-fat diet. The Agtr1a -/- mice exhibited the attenuation of diet-induced body weight gain and adiposity, and insulin resistance relative to wild-type littermates (Agtr1a +/+ mice). They also showed increased energy expenditure accompanied by sympathetic activation, as revealed by increased rectal temperature and oxygen consumption, increased expression of uncoupling protein-1 mRNA in brown adipose tissue, and increased urinary catecholamine excretion. The heterozygous Agtr1a-deficient mice (Agtr1a +/- mice) also exhibited metabolic phenotypes similar to those of Agtr1a -/- mice. Using mouse embryonic fibroblasts derived from Agtr1a +/+ and Agtr1a -/- mice, we found no significant difference between genotypes in the ability to differentiate into lipid-laden mature adipocytes. In primary cultures of mouse mature adipocytes, AII increased the expression of mRNAs for some adipocytokines, which was abolished by pharmacological blockade of Agtr1. This study demonstrates that Agtr1a -/- mice exhibit attenuation of diet-induced weight gain and adiposity through increased energy expenditure. The data also suggest that AII does not affect directly adipocyte differentiation, but can modulate adipocytokine production via Agtr1..
341. Tomohiro Tanaka, Shuji Hidaka, Hiroaki Masuzaki, Shintaro Yasue, Yasuhiko Minokoshi, Ken Ebihara, Hideki Chusho, Yoshihiro Ogawa, Taro Toyoda, Kenji Sato, Fumiko Miyanaga, Muneya Fujimoto, Tsutomu Tomita, Toru Kusakabe, Nozomi Kobayashi, Hideki Tanioka, Tatsuya Hayashi, Kiminori Hosoda, Hironobu Yoshimatsu, Toshiie Sakata, Kazuwa Nakao, Skeletal muscle AMP-activated protein kinase phosphorylation parallels metabolic phenotype in leptin transgenic mice under dietary modification, Diabetes, 10.2337/diabetes.54.8.2365, 54, 8, 2365-2374, 2005.08, Leptin augments glucose and lipid metabolism independent of its effect on satiety. Administration of leptin in rodents increases skeletal muscle β-oxidation by activating AMP-activated protein kinase (AMPK). We previously reported that, as hyperleptinemic as obese human subjects, transgenic skinny mice overexpressing leptin in liver (LepTg) exhibit enhanced insulin sensitivity and lipid clearance. To assess skeletal muscle AMPK activity in leptin-sensitive and -insensitive states, we examined phosphorylation of AMPK and its target, acetyl CoA carboxylase (ACC), in muscles from LepTg under dietary modification. Here we show that phosphorylation of AMPK and ACC are chronically augmented in LepTg soleus muscle, with a concomitant increase in the AMP-to-ATP ratio and a significant decrease in tissue triglyceride content. Despite preexisting hyperleptinemia, high-fat diet (HFD)-fed LepTg develop obesity, insulin-resistance, and hyperlipidemia. In parallel, elevated soleus AMPK and ACC phosphorylation in regular diet-fed LepTg is attenuated, and tissue triglyceride content is increased in those given HFD. Of note, substitution of HFD with regular diet causes a robust recovery of soleus AMPK and ACC phosphorylation in LepTg, with a higher rate of body weight reduction and a regain of insulin sensitivity. In conclusion, soleus AMPK and ACC phosphorylation in LepTg changes in parallel with its insulin sensitivity under dietary modification, suggesting a close association between skeletal muscle AMPK activity and sensitivity to leptin..
342. Shigeo Yura, Hiroaki Itoh, Norimasa Sagawa, Hiroshi Yamamoto, Hiroaki Masuzaki, Kazuwa Nakao, Makoto Kawamura, Maki Takemura, Kazuyo Kakui, Yoshihiro Ogawa, Shingo Fujii, Role of premature leptin surge in obesity resulting from intrauterine undernutrition, Cell metabolism, 10.1016/j.cmet.2005.05.005, 1, 6, 371-378, 2005.06, Intrauterine undernutrition is closely associated with obesity related to detrimental metabolic sequelae in adulthood. We report a mouse model in which offspring with fetal undernutrition (UN offspring), when fed a high-fat diet (HFD), develop pronounced weight gain and adiposity. In the neonatal period, UN offspring exhibited a premature onset of neonatal leptin surge compared to offspring with intrauterine normal nutrition (NN offspring). Unexpectedly, premature leptin surge generated in NN offspring by exogenous leptin administration led to accelerated weight gain with an HFD. Both UN offspring and neonatally leptin-treated NN offspring exhibited an impaired response to acute peripheral leptin administration on a regular chow diet (RCD) with impaired leptin transport to the brain as well as an increased density of hypothalamic nerve terminals. The present study suggests that the premature leptin surge alters energy regulation by the hypothalamus and contributes to "developmental origins of health and disease."..
343. Hiroshi Iwakura, Kiminori Hosoda, Choel Son, Junji Fujikura, Tsutomu Tomita, Michio Noguchi, Hiroyuki Ariyasu, Kazuhiko Takaya, Hiroaki Masuzaki, Yoshihiro Ogawa, Tatsuya Hayashi, Gen Inoue, Takashi Akamizu, Hiroshi Hosoda, Masayasu Kojima, Hiroshi Itoh, Shinya Toyokuni, Kenji Kangawa, Kazuwa Nakao, Analysis of rat insulin II promoter-ghrelin transgenic mice and rat glucagon promoter-ghrelin transgenic mice, Journal of Biological Chemistry, 10.1074/jbc.M411358200, 280, 15, 15247-15256, 2005.04, We developed and analyzed two types of transgenic mice: rat insulin II promoter-ghrelin transgenic (RIP-G Tg) and rat glucagon promoter-ghrelin transgenic mice (RGP-G Tg). The pancreatic tissue ghrelin concentration measured by C-terminal radioimmunoassay (RIA) and plasma desacyl ghrelin concentration of RIP-G Tg were about 1000 and 3.4 times higher than those of nontransgenic littermates, respectively. The pancreatic tissue n-octanoylated ghrelin concentration measured by N-terminal RIA and plasma n-octanoylated ghrelin concentration of RIP-G Tg were not distinguishable from those of nontransgenic littermates. RIP-G Tg showed suppression of glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion, pancreatic insulin mRNA and peptide levels, β cell mass, islet architecture, and GLUT2 and PDX-1 immunoreactivity in RIP-G Tg pancreas were not significantly different from those of nontransgenic littermates. Islet batch incubation study did not show suppression of insulin secretion of RIP-G Tg in vitro. The insulin tolerance test showed lower tendency of blood glucose levels in RIP-G Tg. Taking lower tendency of triglyceride level of RIP-G Tg into consideration, these results may indicate that the suppression of insulin secretion is likely due to the effect of desacyl ghrelin on insulin sensitivity. RGP-G Tg, in which the pancreatic tissue ghrelin concentration measured by C-RIA was about 50 times higher than that of nontransgenic littermates, showed no significant changes in insulin secretion, glucose metabolism, islet mass, and islet architecture. The present study raises the possibility that desacyl ghrelin may have influence on glucose metabolism..
344. Yuichi Oike, Masaki Akao, Kunio Yasunaga, Toshimasa Yamauchi, Tohru Morisada, Yasuhiro Ito, Takashi Urano, Yoshishige Kimura, Yoshiaki Kubota, Hiromitsu Maekawa, Takeshi Miyamoto, Keishi Miyata, Shun Ichiro Matsumoto, Jura Sakai, Naomi Nakagata, Motohiro Takeya, Haruhiko Koseki, Yoshihiro Ogawa, Takashi Kadowaki, Toshio Suda, Angiopoietin-related growth factor antagonizes obesity and insulin resistance, Nature medicine, 10.1038/nm1214, 11, 4, 400-408, 2005.04, Angiopoietin-related growth factor (AGF), a member of the angiopoietin-like protein (Angptl) family, is secreted predominantly from the liver into the systemic circulation. Here, we show that most (>80%) of the AGF-deficient mice die at about embryonic day 13, whereas the surviving AGF-deficient mice develop marked obesity, lipid accumulation in skeletal muscle and liver, and insulin resistance accompanied by reduced energy expenditure relative to controls. In parallel, mice with targeted activation of AGF show leanness and increased insulin sensitivity resulting from increased energy expenditure. They are also protected from high-fat diet-induced obesity, insulin resistance and nonadipose tissue steatosis. Hepatic overexpression of AGF by adenoviral transduction, which leads to an approximately 2.5-fold increase in serum AGF concentrations, results in a significant (P < 0.01) body weight loss and increases insulin sensitivity in mice fed a high-fat diet. This study establishes AGF as a new hepatocyte-derived circulating factor that counteracts obesity and related insulin resistance..
345. Hiromasa Kobayashi, Kaori Ikeda, Cheol Son, Toshio Iwakura, Takashi Ishihara, Megumu Hino, Tatsuhide Inoue, Eiji Nii, Yoshihiro Ogawa, GC79/TRPS1 and tumorigenesis in humans [3], American Journal of Medical Genetics, 10.1002/ajmg.a.30596, 134 A, 3, 341-343, 2005.04.
346. Yoshihiro Ogawa, New prospects for the treatment of obesity - Leptin and the discovery of anti-obesity drugs -, Japan Medical Association Journal, 48, 2, 64-67, 2005.02, Obesity is a typical multifactorial disease that results from complex interactions between hereditary predisposition and environmental factors, making it extremely difficult to approach from a molecular level. At the end of 1994, an obese gene product, leptin, was discovered, and, since then, obesity research has produced a variety of new findings. Leptin is secreted from the adipose tissue and to act directly on the hypothalamus, causing appetite suppression and accelerated energy metabolism, thereby denoting a relationship to obesity and weight gain. A number of hypothalamic appetite regulators have been found, and it has recently become apparent that many of these regulators are controlled by leptin. In contrast, many genes that are known to cause human obesity and to develop from single-gene mutations regulate energy metabolism by leptin, and they have attracted attention as possible anti-obesity drugs. This paper outlines new anti-obesity drug research and development that have emerged since the discovery of leptin..
347. Takayoshi Suganami, Masashi Mukoyama, Kiyoshi Mori, Hideki Yokoi, Masao Koshikawa, Kazutomo Sawai, Shuji Hidaka, Ken Ebihara, Tomohiro Tanaka, Akira Sugawara, Hiroshi Kawachi, Charles Viason, Yoshihiro Ogawa, Kazuwa Nakao, Prevention and reversal of renal injury by leptin in a new mouse model of diabetic nephropathy, FASEB Journal, 10.1096/fj.04-2183fje, 19, 1, 127-129, 2005.01.
348. Rika Kawakami, Yoshihiko Saito, Ichiro Kishimoto, Masaki Harada, Koichiro Kuwahara, Nobuki Takahashi, Yasuaki Nakagawa, Michio Nakanishi, Keiji Tanimoto, Satoru Usami, Shinji Yasuno, Hideyuki Kinoshita, Hideki Chusho, Naohisa Tamura, Yoshihiro Ogawa, Kazuwa Nakao, Overexpression of brain natriuretic peptide facilitates neutrophil infiltration and cardiac matrix metalloproteinase-9 expression after acute myocardial infarction, Circulation, 10.1161/01.CIR.0000147829.78357.C5, 110, 21, 3306-3312, 2004.11, Background - Recent clinical trials have shown that systemic infusion of nesiritide, a recombinant human brain natriuretic peptide (BNP), improves hemodynamic parameters in acutely decompensated hearts. This suggests that BNP exerts a direct cardioprotective effect and might thus be a useful therapeutic agent with which to treat acute myocardial infarction (MI). In the present study, we used BNP-transgenic (BNP-Tg) mice with elevated plasma BNP to determine whether and how BNP contributes to left ventricular remodeling and healing after MI. Methods and Results - We examined the accumulation of neutrophils and the expression and activation of matrix metalloproteinase (MMP)-9 in the ventricles of male BNP-Tg mice and their nontransgenic (non-Tg) littermates during the early phase after acute MI. The numbers of neutrophils infiltrating the infarcted area were significantly increased in BNP-Tg mice 3 days after MI. In addition, both the gene expression and zymographic activity of MMP-9, but not MMP-2, were significantly higher in BNP-Tg than non-Tg mice. Double immunostaining revealed that neutrophils are the main source of the MMP-9, although doxycycline, an MMP inhibitor, had no effect on neutrophil infiltration of the infarcted area in BNP-Tg mice. Conclusions - These results demonstrate that elevated plasma BNP facilitates neutrophil infiltration of the infarcted area after MI and increases the activity of the MMP-9 they produce. This suggests that BNP plays a key role in the processes of extracellular matrix remodeling and wound-healing during the early phase after acute MI..
349. Noriko Satoh, Mitsuhide Naruse, Takeshi Usui, Tetsuya Tagami, Takayoshi Suganami, Kazunori Yamada, Hideshi Kuzuya, Akira Shimatsu, Yoshihiro Ogawa, Leptin-to-adiponectin ratio as a potential atherogenic index in obese type 2 diabetic patients, Diabetes care, 10.2337/diacare.27.10.2488, 27, 10, 2488-2490, 2004.10.
350. Eri Suganami, Hitoshi Takagi, Hirokazu Ohashi, Kiyoshi Suzuma, Izumi Suzuma, Hideyasu Oh, Daisuke Watanabe, Tomonari Ojima, Takayoshi Suganami, Yasushi Fujio, Kazuwa Nakao, Yoshihiro Ogawa, Nagahisa Yoshimura, Leptin stimulates ischemia-induced retinal neovascularization
Possible role of vascular endothelial growth factor expressed in retinal endothelial cells, Diabetes, 10.2337/diabetes.53.9.2443, 53, 9, 2443-2448, 2004.09, Diabetic retinopathy is the leading cause of new blindness in adults in developed countries. Leptin, an adipocyte-derived hormone, stimulates endothelial proliferation and angiogenesis. This study was designed to elucidate the pathophysiologic role of leptin in the progression of retinal neovascularization. Using the retinopathy of prematurity model, a mouse model of ischemia-induced retinal neovascularization, we have demonstrated more pronounced retinal neovascularization in 17-day-old transgenic mice overexpressing leptin than in age-matched wild-type littermates. Ischemia-induced retinal neovascularization was markedly suppressed in 17-day-old leptin-deficient ob/ob mice. Western blot analysis revealed that a biologically active leptin receptor isoform is expressed in mouse retinal endothelial cells. Leptin receptor expression was also detected in primary cultures of porcine retinal endothelial cells, where it upregulated vascular endothelial growth factor (VEGF) mRNA expression. This effect was thought to be mediated at least partly through the activation of signal transducers and activators of transcription (STAT)3, because adenoviral transfection of the dominant-negative form of STAT3 abolished the leptin-induced upregulation of VEGF mRNA expression in retinal endothelial cells. This study provides evidence that leptin stimulates the ischemia-induced retinal neovasucularization possibly through the upregulation of endothelial VEGF, thereby suggesting that leptin antagonism may offer a novel therapeutic strategy to prevent or treat diabetic retinopathy..
351. Akiko Takahashi-Yasuno, Hiroaki Masuzaki, Takashi Miyawaki, Naoki Matsuoka, Yoshihiro Ogawa, Tatsuya Hayashi, Kiminori Hosoda, Yasunao Yoshimasa, Gen Inoue, Kazuwa Nakao, Association of Ob-R Gene Polymorphism and Insulin Resistance in Japanese Men, Metabolism: Clinical and Experimental, 10.1016/j.metabol.2003.12.012, 53, 5, 650-654, 2004.05, Leptin and its receptors are known to play a role in glucose metabolism. We succeeded in cloning human Ob-R cDNA and revealed 7 single nucleotide polymorphisms (SNPs) (Lys109Arg, Arg223Gln, Ser343Ser, Ser492Thr, Lys656Asn, Ala976Asp, and Pro1019Pro) in the coding region of Ob-Rb. Although these 7 SNPs were not associated with an obese phenotype, several studies have reported that some of them were associated with impaired glucose metabolism. To clarify whether the Arg223Gln and A3057G (Pro1019Pro) polymorphisms influence glucose metabolism in Japanese, 696 Japanese men were genotyped. Individually, the Arg223Gln and the A3057G polymorphisms were not associated with the glucose metabolic parameters. No associations were found between haplotype and clinical parameters. However, in 327 subjects with normal glucose tolerance (NGT), the subjects with Arg/Gln or Gln/Gln + A/A haplotype showed significantly higher serum insulin levels and homeostasis model assessment (HOMA) index than those with Arg/Arg + A/A haplotype and Arg/Gln or Gln/Gln + A/G or G/G haplotype. The subjects with Arg/Gln or Gln/Gln + A/A haplotype showed a significantly lower fasting glucose to insulin (GI) ratio than those with Arg/Arg + A/A haplotype. These results suggest that the Ob-R gene may serve as a modifier gene for insulin resistance in Japanese men..
352. Ken Ebihara, Toru Kusakabe, Hiroaki Masuzaki, Nozomi Kobayashi, Tomohiro Tanaka, Hideki Chusho, Fumiko Miyanaga, Takashi Miyazawa, Tatsuya Hayashi, Kiminori Hosoda, Yoshihiro Ogawa, Kazuwa Nakao, Gene and Phenotype Analysis of Congenital Generalized Lipodystrophy in Japanese
A Novel Homozygous Nonsense Mutation in Seipin Gene, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2003-031211, 89, 5, 2360-2364, 2004.05, Congenital generalized lipodystrophy (CGL), Berardinelli-Seip syndrome, is a rare metabolic disorder characterized by a near total lack of adipose tissue from birth or early infancy. Recently, seipin, encoding a 398-amino acid protein of unknown function, and AGPAT2, encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase 2, were identified as causative genes for CGL. Seipin mutations were found in patients from families originating from Europe and the Middle East. AGPAT2 mutations were found predominantly in African ancestry. However, no information is available on these genes in the pathogenesis of CGL in Asian ancestry. We examined the sequences of the entire coding region of seipin and AGPAT2 in four Japanese CGL patients from independent families. Their average body fat content was 4.7 ± 0.5%, and the plasma leptin level was 1.15 ± 0.14 ng/ml. We identified a novel nonsense mutation of seipin at codon 275 (R275X). Of four CGL patients, three were homozygous for R275X. No seipin mutation was found in any exon in one patient. We did not find any AGPAT2 mutations in our Japanese patients, suggesting that AGPAT2 is a minor causative gene, if any, for CGL in Japanese. This is the first report on gene and phenotype analysis of CGL in Japanese..
353. Mercy A. Nuamah, Shigeo Yura, Norimasa Sagawa, Hiroaki Itoh, Hiroko Mise, Daizo Korita, Kazuyo Kakui, Maki Takemura, Yoshihiro Ogawa, Kazuwa Nakao, Shingo Fujii, Significant increase in maternal plasma leptin concentration in induced delivery
A possible contribution of pro-inflammatory cytokines to placental leptin secretion, Endocrine Journal, 10.1507/endocrj.51.177, 51, 2, 177-187, 2004.04, Maternal plasma leptin concentration is significantly increased during pregnancy. However, its roles in pregnancy, especially in labor, have not been fully clarified. We measured plasma leptin concentrations in pregnant women during the course of induced labor, just after spontaneous vaginal delivery and Cesarean section at term. We also studied the regulation of leptin secretion from term placental tissue and BeWo cells, a trophoblastic cell-line. Plasma leptin concentrations increased significantly during labor (58.9 ± 9.2 ng/ml) compared to those before labor induction (37.5 ± 5.8 ng/ml, P<0.05), then decreased 3-6 days postpartum (14 ± 3 ng/ml, n = 6, P<0.0001) to the levels of normal nonpregnant women. Leptin concentrations within an hour and 24 hours after spontaneous vaginal delivery were significantly higher than those after Cesarean section (P<0.05 for both comparisons). Similarly, leptin mRNA expression in placental tissues obtained after spontaneous vaginal delivery was significantly greater than that in those obtained after Cesarean section without labor (P<0.05). IL-1α and TNF-α treatment significantly stimulated leptin secretion and leptin mRNA expression in explant culture of human term placental tissue and in BeWo cells as compared with those in vehicle controls (P<0.05, for all comparisons). By contrast, oxytocin and prostaglandin F treatment had no effects on leptin secretion from explant culture of human term placental tissue or from BeWo cells. These data indicate that pro-inflammatory cytokines might stimulate placental leptin secretion, thus finally contributing to the increase in plasma leptin concentration during labor..
354. F. Elefteriou, S. Takeda, K. Ebihara, J. Magre, N. Patano, C. Ae Kim, Y. Ogawa, X. Liu, S. M. Ware, W. J. Craigen, J. J. Robert, C. Vinson, K. Nakao, J. Capeau, G. Karsenty, Serum leptin level is a regulator of bone mass, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0308744101, 101, 9, 3258-3263, 2004.03, Leptin is a powerful inhibitor of bone formation in vivo. This antiosteogenic function involves leptin binding to its receptors on ventromedial hypothalamic neurons, the autonomous nervous system and β-adrenergic receptors on osteoblasts. However, the mechanisms whereby leptin controls the function of ventromedial hypothalamic antiosteogenic neurons remain unclear. In this study, we compared the ability of leptin to regulate body weight and bone mass and show that leptin antiosteogenic and anorexigenic functions are affected by similar amounts of leptin. Using a knock-in of LacZ in the leptin locus, we failed to detect any leptin synthesis in the central nervous system. However, increasing serum leptin level, even dramatically, reduced bone mass. Conversely, reducing serum-free leptin level by overexpressing a soluble receptor for leptin increased bone mass. Congruent with these results, the high bone mass of lipodystrophic mice could be corrected by restoring serum leptin level, suggesting that leptin is an adipocyte product both necessary and sufficient to control bone mass. Consistent with the high bone mass phenotype of lipodystrophic mice, we observed an advanced bone age, an indirect reflection of premature bone formation, in lipodystrophic patients. Taken together, these results indicate that adipocyte-derived circulating leptin is a determinant of bone formation and suggests that leptin antiosteogenic function is conserved in vertebrates..
355. Yoshihiro Ogawa, Biological function of leptin and obesity-related diseases, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.93.2435, 93, 11, 2435-2441, 2004.01.
356. Akihiro Yasoda, Yasato Komatsu, Kazuwa Nakao, Yoshihiro Ogawa, C-type natriuretic peptide(CNP)--a novel stimulator of bone growth formed through endochondral ossification, Nippon rinsho. Japanese journal of clinical medicine, 62 Suppl 2, 77-81, 2004.01.
357. Akihiro Yasoda, Yasato Komatsu, Kazuwa Nakao, Yoshihiro Ogawa, Growth promoting effect of natriuretic peptides on bones formed through endochondral ossification, Nippon rinsho. Japanese journal of clinical medicine, 62 Suppl 9, 60-64, 2004.01.
358. Yoshihiro Ogawa, Kazuwa Nakao, Involvement of leptin in the pathogenesis of hypertension and hypertensive target-organ damage, Nippon rinsho. Japanese journal of clinical medicine, 62 Suppl 3, 216-221, 2004.01.
359. Akihiro Yasoda, Yasato Komatsu, Hideki Chusho, Takashi Miyazawa, Ami Ozasa, Masako Miura, Tatsuya Kurihara, Tomohiro Rogi, Shoji Tanaka, Michio Suda, Naohisa Tamura, Yoshihiro Ogawa, Kazuwa Nakao, Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway, Nature medicine, 10.1038/nm971, 10, 1, 80-86, 2004.01, Achondroplasia is the most common genetic form of human dwarfism, for which there is presently no effective therapy. C-type natriuretic peptide (CNP) is a newly identified molecule that regulates endochondral bone growth through GC-B, a subtype of particulate guanylyl cyclase. Here we show that targeted overexpression of CNP in chondrocytes counteracts dwarfism in a mouse model of achondroplasia with activated fibroblast growth factor receptor 3 (FGFR-3) in the cartilage. CNP prevented the shortening of achondroplastic bones by correcting the decreased extracellular matrix synthesis in the growth plate through inhibition of the MAPK pathway of FGF signaling. CNP had no effect on the STAT-1 pathway of FGF signaling that mediates the decreased proliferation and the delayed differentiation of achondroplastic chondrocytes. These results demonstrate that activation of the CNP-GC-B system in endochondral bone formation constitutes a new therapeutic strategy for human achondroplasia..
360. Akiko Takahashi-Yasuno, Hiroaki Masuzaki, Takashi Miyawaki, Yoshihiro Ogawa, Naoki Matsuoka, Tatsuya Hayashi, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Kazuwa Nakao, Leptin receptor polymorphism is associated with serum lipid levels and impairment of cholesterol lowering effect by simvastatin in Japanese men, Diabetes Research and Clinical Practice, 10.1016/S0168-8227(03)00163-3, 62, 3, 169-175, 2003.12, Objective: To investigate whether leptin receptor (Ob-R) Arg223Gln polymorphism influences serum lipid levels and whether this polymorphism affects the efficiency of the cholesterol lowering HMG-CoA reductase inhibitor, simvastatin [Clin. Cardiol. 16 (1993) 317]. Design: Case-control association study. Subjects: We studied 201 Japanese men without medical care, and 78 Japanese who took simvastatin. Methods: Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Serum lipid and leptin levels were determined. Results: Subjects with the Arg/Arg homozygotes had significantly higher serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels than those with the Arg/Gln heterozygotes and Gln/Gln homozygotes (TC: Arg/Arg: 213±3, Arg/Gln: 196±6, Gln/Gln: 184±5, P=0.004 for comparison among three genotypes, P=0.008 for difference between Arg/Arg and Arg/Gln, and P=0.025 for difference between Arg/Arg and Gln/Gln, LDL-C: Arg/Arg: 127±3, Arg/Gln: 112±6, Gln/Gln: 114±8, P=0.027) for comparison among three genotypes and P=0.011 for difference between Arg/Arg and Arg/Gln. Subjects with the Arg/Arg homozygotes had significantly lower serum high density lipoprotein cholesterol (HDL-C) levels than those with the Arg/Gln heterozygotes and Gln/ Gln homozygotes (Arg/Arg: 55±1, Arg/Gln: 62±3, Gln/Gln: 57±7, P=0.046) for comparison among three genotypes and P=0.013 for difference between Arg/Arg and Arg/Gln. In addition, in 78 patients with hypercholesterolemia who took 5 mg simvastatin, the TC lowering effect by simvastatin in subjects with the Arg/Arg homozygotes was significantly lower than in those with the Arg/Gln heterozygotes and Gln/Gln homozygotes (the reduction in serum TC levels; 62±4 vs. 79±6, P=0.044). Conclusions: We demonstrate that Ob-R Arg223Gln polymorphism in Japanese men is associated with significant elevation of serum TC and LDL-C levels. Our data also show that the Arg/Arg homozygotes tend to show lowered level of serum HDL-C. Furthermore, this polymorphism tends to show an attenuated response to an HMG-CoA reductase inhibitor in terms of the cholesterol lowering effect. These results suggest that the Ob-R gene may serve as a novel modifier gene for hypercholesterolemia in Japanese men..
361. Nobuki Takahashi, Yoshihiko Saito, Koichiro Kuwahara, Masaki Harada, Ichiro Kishimoto, Yoshihiro Ogawa, Rika Kawakami, Yasuaki Nakagawa, Micio Nakanishi, Kazuwa Nakao, Angiotensin II-induced ventricular hypertrophy and extracellular signal-regulated kinase activation are suppressed in mice overexpressing brain natriuretic peptide in circulation, Hypertension Research, 10.1291/hypres.26.847, 26, 10, 847-853, 2003.10, Atrial and brain (B-type) natriuretic peptides (ANP and BNP, respectively) are known to exert various cardioprotective effects. For instance, knocking out the expression of ANP, BNP, or their receptor, guanylyl cyclase-A, induces cardiac hypertrophy and/or fibrosis. The cardiac effects of elevated circulating natriuretic peptides are less well understood, however. We therefore compared angiotensin (Ang) II-induced cardiac hypertrophy and fibrosis in BNP-transgenic (Tg) mice, in which circulating BNP levels were elevated by increased secretion from the liver, and their non-Tg littermates. Left ventricular expression of Ang II type 1a receptor was similar in BNP-Tg and non-Tg mice, and there was no significant difference in the elevation of blood pressure elicited by chronic infusion or acute injection of Ang II. Nevertheless, cardiac hypertrophy and fibrosis were significantly diminished in BNP-Tg mice chronically infused with Ang II. In addition, ventricular activation of extracellular signal-regulated kinase (ERK) induced by acute injection of Ang II was also diminished in BNP-Tg mice, as was activation of ERK kinase (MEK). Conversely, expression of mitogen-activated protein kinase phosphatase (MKP) was significantly increased in the ventricles of BNP-Tg mice. Based on these findings, we conclude that elevated circulating BNP exerts cardioprotective effects via inhibition of a ventricular ERK pathway. The mechanism responsible for this inhibition likely involves 1) increased ventricular MKP expression and 2) inhibition of transduction mediators situated upstream of ERK..
362. Yoshihiro Ogawa, K. Nakao, Leptin, Journal of the Japan Diabetes Society, 46, 9, 730-732, 2003.10.
363. F. Miyanaga, Y. Ogawa, K. Ebihara, S. Hidaka, T. Tanaka, S. Hayashi, H. Masuzaki, K. Nakao, Leptin as an adjunct of insulin therapy in insulin-deficient diabetes, Diabetologia, 10.1007/s00125-003-1193-6, 46, 10, 1329-1337, 2003.10, Aims/hypothesis. The purpose of this study was to assess the therapeutic implication of leptin in insulin-deficient diabetes. Methods. Insulin-deficient diabetes was induced by streptozotocin (STZ) in transgenic skinny mice overexpressing leptin. Plasma concentrations of glucose, insulin, and leptin were measured. The effects on body weight, food intake, and hypothalamic gene expressions were analyzed. After diabetes was induced, graded doses of insulin ranging from 0.4 to 51.2 mU·g-1·day-1 were injected. Co-administration of leptin and insulin was also carried out using osmotic pumps. Results. After STZ injection, both transgenic and nontransgenic littermates developed marked hyperglycaemia as a result of severe hypoinsulinaemia [termed diabetic transgenic skinny mice overexpressing leptin (diabetic TGM) and diabetic non-transgenic littermates (diabetic WT) respectively], although diabetic TGM were more sensitive to exogenously administered insulin than diabetic WT. Diabetic WT were hypoleptinaemic and hyperphagic relative to non-diabetic WT, whereas diabetic TGM, which remained hyperleptinaemic, were less hyperphagic than diabetic WT. After STZ injection, hypothalamic expressions of orexigenic and anorexigenic peptide mRNAs were upregulated and down-regulated, respectively, in diabetic WT, whereas they were unchanged in diabetic TGM. Diabetic TGM became normoglycaemic, when treated with insulin at such doses that did not improve hyperglycaemia in diabetic WT. We found that a subthreshold dose of insulin that does not affect glucose homeostasis is effective in improving the diabetes in normal mice rendered diabetic by STZ injection, when combined with leptin. Conclusions/ interpretation. This study suggests that leptin could be used as an adjunct of insulin therapy in insulin-deficient diabetes, thereby providing an insight into the therapeutic implication of leptin as an anti-diabetic agent..
364. Noriko Satoh, Yoshihiro Ogawa, Takeshi Usui, Tetsuya Tagami, Shigeo Kono, Hiroko Uesugi, Hiroyuki Sugiyama, Akira Sugawara, Kazunori Yamada, Akira Shimatsu, Hideshi Kuzuya, Kazuwa Nakao, Antiatherogenic effect of pioglitazone in type 2 diabetic patients irrespective of the responsiveness to its antidiabetic effect, Diabetes care, 10.2337/diacare.26.9.2493, 26, 9, 2493-2499, 2003.09, OBJECTIVE - Thiazolidinediones (TZDs), a class of insulin-sensitizing agents used clinically to treat type 2 diabetes, are also antiatherogenic. This study was designed to elucidate the relationship between the antiatherogenic and antidiabetic effects of pioglitazone, a TZD, in type 2 diabetic patients. RESEARCH DESIGN AND METHODS - A total of 136 Japanese type 2 diabetic patients were included and divided into two groups: the pioglitazone-treated group (30 mg daily for 3 months) (n = 70) and the untreated control group (n = 66). The changes in glycolipid metabolism as well as plasma high-sensitivity C-reactive protein (CRP), leptin, adiponectin, and pulse wave velocity (PWV) were monitored to analyze the relationship between the antiatherogenic and antidiabetic effects of pioglitazone. RESULTS - The pioglitazone treatment significantly reduced hyperglycemia, hyperinsulinemia, and HbA1c levels and increased plasma adiponectin concentrations relative to the control group (P < 0.01). It also significantly decreased CRP and PWV (P < 0.01). The antiatherogenic effect was observed in both the nonresponders showing < 1% of reduction in HbA1c (n = 30) and responders showing > 1% of reduction (n = 40). ANCOVA revealed that treatment with pioglitazone was associated with a low CRP and PWV, independent of the changes in parameters related to glucose metabolism. CONCLUSIONS - This study represents the first demonstration of the antiatherogenic effect of pioglitazone in both nonresponders and responders with respect to its antidiabetic effect and suggests that pioglitazone can exert its antiatherogenic effect independently of its antidiabetic effect..
365. Mercy A. Nuamah, Norimasa Sagawa, Shigeo Yura, Hiroko Mise, Hiroaki Itoh, Yoshihiro Ogawa, Kazuwa Nakao, Shingo Fujii, Free-to-total leptin ratio in maternal plasma is constant throughout human pregnancy, Endocrine Journal, 10.1507/endocrj.50.421, 50, 4, 421-428, 2003.08, To clarify the mechanism of leptin resistance during pregnancy, we measured plasma leptin concentrations, free to total leptin ratio (percent free leptin) and soluble leptin receptor concentrations in pregnant women, and compared the results with those in non-pregnant women. We collected plasma samples from 23 non-pregnant and 31 pregnant women in the third trimester. Plasma samples from 5 pregnant women were collected longitudinally in each trimester. Plasma leptin concentrations in pregnant women in the second trimester (17.4 ± 3.2 ng/ml) were higher than those in the first trimester of pregnancy (11.0 ± 2.8 ng/ml, n = 5), as previously reported. However, percent free leptin did not change significantly throughout pregnancy. Percent free leptin correlated with total leptin concentrations (ng/ml) in non-pregnant women (r = 0.727, P<0.0001), but not in women in the third trimester of pregnancy (r = 0.006). Constant percent free leptin during pregnancy despite increased leptin concentrations indicates increased leptin binding capacity in pregnant women, that might partly contribute to the establishment of leptin resistance. On the other hand, soluble leptin receptor concentrations showed significant negative correlation with BMI and plasma leptin concentrations in pregnant women (r = -0.470, P<0.01 and r = -0.493, P<0.01, respectively) but not in non-pregnant women. These data suggest the possibility that soluble leptin receptor is a minor component of leptin binding capacity in the plasma of pregnant women..
366. T. Nakagawa, Y. Ogawa, K. Ebihara, M. Yamanaka, A. Tsuchida, M. Taiji, H. Noguchi, K. Nakao, Antiobesity and antidiabetic effects of brain-derived neurotrophic factor in rodent models of leptin resistance, International Journal of Obesity, 10.1038/sj.ijo.0802265, 27, 5, 557-565, 2003.05, OBJECTIVE: Obesity in rodents and humans is mostly associated with elevated plasma leptin concentrations, suggesting a new pathological concept of 'leptin resistance'. We have demonstrated that brain-derived neurotrophic factor (BDNF) can improve obesity and diabetes of C57BL/KsJ db/db (db/db) mice. In this study, we investigated whether or not BDNF is effective in two different models of leptin resistance, an acquired model and a genetic model. DESIGN: C57BL/6J mice rendered obese by consumption of a high-fat diet (diet-induced obesity (DIO) mice) were used as an acquired model and lethal yellow ogouti mice (KKAy mice) as a genetic model of leptin resistance. Food intake and glucose metabolism were studied after acute or repetitive administration of BDNF. RESULTS: Intraperitoneal administration of BDNF (10 mg/kg, twice/day) significantly reduced cumulative food intake of DIO and KKAy mice, whereas they were unresponsive to leptin administration. Repetitive subcutaneous administration of BDNF (10 mg/kg daily for 6 days) reduced food intake and improved impaired glucose tolerance in DIO mice. Pair feeding of vehicle-treated DIO mice with the same amount of chow consumed by the BDNF-treated group did not improve the impaired glucose homeostasis, indicating that the antidiabetic effect is not due to decreased food intake. We also observed that BDNF is effective in improving obesity and diabetes of KKAy mice. CONCLUSION: This study demonstrated antiobesity and antidiabetic effects of BDNF in two different models of leptin resistance, thereby suggesting the therapeutic potential of BDNF in the treatment of leptin-resistant obesity and diabetes..
367. Kazuo Chin, Takaya Nakamura, Kenichi Takahashi, Kensuke Sumi, Yoshihiro Ogawa, Hiroaki Masuzaki, Shigeo Muro, Noboru Hattori, Hisako Matsumoto, Akio Niimi, Tsutomu Chiba, Kazuwa Nakao, Michiaki Mishima, Motoharu Ohi, Takashi Nakamura, Effects of obstructive sleep apnea syndrome on serum aminotransferase levels in obese patients, American Journal of Medicine, 10.1016/S0002-9343(02)01570-X, 114, 5, 370-376, 2003.04, PURPOSE: Obesity has been associated with obstructive sleep apnea and hepatic steatosis. We investigated the effects of obstructive sleep apnea and treatment with nasal continuous positive airway pressure (CPAP) on serum aminotransferase levels in obese patients. METHODS: We studied 40 obese men with obstructive sleep apnea syndrome. None had hepatitis B antigen or C antibody, autoimmune disease, or an excessive intake of alcohol. Serum levels of aspartate aminotransferase, alanine aminotransferase, triglyceride, glucose, insulin, and leptin were determined in the afternoon and in the morning immediately after sleep, before and after nasal CPAP treatment. RESULTS: Aminotransferase levels were abnormal in 35% (n = 14) of patients. Before treatment, mean (± SD) aspartate aminotransferase levels were higher in the morning than in the previous afternoon (presleep, 34 ± 20 IU/L; postsleep, 39 ± 28 IU/L; P = 0.006). The overnight mean increases in aminotransferase levels were less marked after the first night of nasal CPAP treatment (aspartate aminotransferase: from 6 ± 11 IU/L to 2 ± 6 IU/L, P = 0.0003; alanine aminotransferase: from 5 ± 9 IU/L to 2 ± 6 IU/L, P = 0.006). Leptin levels (n = 23) decreased significantly after treatment (P = 0.0002), whereas insulin resistance (calculated by the homeostasis model assessment method) and triglyceride levels were unchanged. Improvements in aspartate and alanine aminotransferase levels were maintained after 1 and 6 months of nasal CPAP treatment. CONCLUSION: Nasal CPAP therapy may have beneficial effects on serum aminotransferase abnormalities in obese patients who have obstructive sleep apnea..
368. Koji Takeuchi, Shinichi Kato, Masanori Takeeda, Yoshihiro Ogawa, Masato Nakashima, Masahiro Matsumoto, Facilitation by endogenous prostaglandins of capsaicin-induced gastric protection in rodents through EP2 and IP receptors, Journal of Pharmacology and Experimental Therapeutics, 10.1124/jpet.102.044156, 304, 3, 1055-1062, 2003.03, We investigated the role that prostaglandins (PGs) and EP receptors play in facilitating the gastroprotective action of capsaicin against HCl/ethanol in rats and mice. Male Sprague-Dawley rats and C57BL/6 mice were used after 18 h of fasting. The animals were given HCl/ethanol (60% in 150 mM HCl) p.o. and killed 1 h later. Capsaicin or various EP agonists were given p.o. 30 min or i.v. 10 min before HCl/ethanol. In some cases, indomethacin or various EP agonists were given s.c. 30 min or i.v 10 min before capsaicin, respectively. Gastric lesions induced by HCl/ethanol were significantly inhibited by PGE2 as well as capsaicin. The effect of PGE2 was antagonized by ONO-AE-829 (EP1 antagonist), whereas the capsaicin action was mitigated by indomethacin as well as sensory deafferentation but not by ONO-AE-829. The generation of mucosal PGE2, was not affected by either capsaicin or sensory deafferentation, but was significantly inhibited by indomethacin. Although neither butaprost (EP2), ONO-NT-012 (EP3), nor 11-deoxy PGE1 (EP4) alone had any effect on HCl/ethanol-induced gastric lesions, only butaprost restored the protective action of capsaicin in the presence of indomethacin. Capsaicin provided a protective action against HCl/ethanol-induced gastric lesions in wild-type (+/+) mice in an indomethacin-sensitive manner, and this action was similarly observed in EP1 (-/-) and EP3 (-/-) mice but not in the animals lacking IP receptors. These results suggest that capsaicin exhibits gastric cytoprotection, essentially by stimulating sensory neurons, and this action is facilitated by endogenous PGs through EP2/IP receptors, probably sensitizing the sensory neurons to capsaicin..
369. Kenichi Yamahara, Hiroshi Itoh, Tae Hwa Chun, Yoshihiro Ogawa, Jun Yamashita, Naoki Sawada, Yasutomo Fukunaga, Masakatsu Sone, Takami Yurugi-Kobayashi, Kazutoshi Miyashita, Hirokazu Tsujimoto, Hyun Kook, Robert Feil, David L. Garbers, Franz Hofmann, Kazuwa Nakao, Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0538059100, 100, 6, 3404-3409, 2003.03, Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMP/cGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPs/cGMP/cGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk1/2 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPs/cGMP/cGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia..
370. Y. Ogawa, H. Kobayashi, K. Ebihara, T. Hayashi, K. Hosoda, H. Inoue, T. Ishihara, H. Kurahachi, K. Nakao, Congenital obesity caused by a novel mutation of human melanocortin receptor 4 (MC 4 R), Journal of the Japan Diabetes Society, 46, 1, 29-33, 2003.01.
371. Yasato Komatsu, Hideki Chusho, Naohisa Tamura, Akihiro Yasoda, Takashi Miyazawa, Michio Suda, Masako Miura, Yoshihiro Ogawa, Kazuwa Nakao, Significance of C-type natriuretic peptide (CNP) in endochondral ossification
Analysis of CNP knockout mice, Journal of Bone and Mineral Metabolism, 10.1007/s007740200048, 20, 6, 331-336, 2002.12.
372. Hiroshi Iwakura, Kiminori Hosoda, Ryuichiro Doi, Izumi Komoto, Haruo Nishimura, Choel Son, Junji Fujikura, Tsutomu Tomita, Kazuhiko Takaya, Yoshihiro Ogawa, Tatsuya Hayashi, Gen Inoue, Takashi Akamizu, Hiroshi Hosoda, Masayasu Kojima, Kenji Kangawa, Masayuki Imamura, Kazuwa Nakao, Ghrelin expression in islet cell tumors
Augmented expression of ghrelin in a case of glucagonoma with multiple endocrine neoplasm type I, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.2002-020882, 87, 11, 4885-4888, 2002.11, Ghrelin is a 28-amino acid peptide that regulates GH release together with GHRH and somatostatin. The expression of ghrelin has been detected in the stomach, small intestine, hypothalamus, pituitary gland, kidney, placenta, and testis. Recently it was reported that ghrelin is present in pancreatic α-cells and that it stimulates insulin secretion. In this study, we examined the ghrelin expression in two cases of glucagonoma and two cases of insulinoma by Northern blot analysis and immunohistochemistry. Ghrelin expression was identified in a case of glucagonoma associated with multiple endocrine neoplasm type I both by Northern blot analysis using total RNA and by immunohistochemistry, although the plasma ghrelin level was not elevated. This is the first case of tumor in which ghrelin gene expression was detected by Northern blot analysis using total RNA..
373. Shinichi Kato, Yoshihiro Ogawa, Kenji Kanatsu, Mitsuaki Okayama, Toshio Watanabe, Tetsuo Arakawa, Koji Takeuchi, Ulcerogenic influence of selective cyclooxygenase-2 inhibitors in the rat stomach with adjuvant-induced arthritis, Journal of Pharmacology and Experimental Therapeutics, 10.1124/jpet.102.040659, 303, 2, 503-509, 2002.11, Cyclooxygenase (COX)-2 inhibitors have been developed as new gastric sparing anti-inflammatory drugs. We previously reported that the ulcerogenic response to conventional nonselective COX inhibitors, such as indomethacin and aspirin, was markedly increased in arthritic rats. The ulcerogenic effect of selective COX-2 inhibitors in arthritic animals, however, remains unknown. The present study was designed to examine the influence of selective COX-2 inhibitors, such as rofecoxib and celecoxib, on gastric mucosal integrity in rats with adjuvant-induced arthritis. Arthritis was induced in male dark Agouti rats by injection of Freund's complete adjuvant into the right hind paw. Two weeks after the injection, the animals were fasted for 18 h, various COX inhibitors were administered orally, and the mucosa was examined for lesions 4 h later. Oral administration of indomethacin caused hemorrhagic gastric lesions in both normal and arthritic rats, although the severity of lesions was significantly greater in the latter group. In contrast, neither rofecoxib nor celecoxib caused any gastric damage in normal rats, but both drugs provoked hemorrhagic gastric lesions in arthritic rats. The expression of COX-2 mRNA and immuno-positive cells was observed in the gastric mucosa of arthritic but not normal rats. The gastric mucosal prostaglandin (PG) E2 content was significantly elevated in arthritic rats in a rofecoxib-sensitive manner. In conclusion, COX-2 inhibitors produce gastric lesions in arthritic rats, similar to the nonselective COX-inhibitors. COX-2 is up-regulated in the stomach of arthritic rats, and PGs produced by COX-2 play a role in maintaining the integrity of the gastric mucosa..
374. Norimasa Sagawa, Shigeo Yura, Hiroaki Itoh, Kazuyo Kakui, Maki Takemura, Mercy A. Nuamah, Yoshihiro Ogawa, Hiroaki Masuzaki, Kazuwa Nakao, Shingo Fujii, Possible role of placental leptin in pregnancy
A review, Endocrine, 10.1385/ENDO:19:1:65, 19, 1, 65-71, 2002.10, Leptin was initially identified as an adipocyte-derived hormone that decreases food intake and body weight via its receptor in the hypothalamus. Subsequent animal studies revealed various physiologic functions of leptin. Leptin plays an essential role in reproduction by regulating gonadotropin-releasing hormone secretion from the hypothalamus. It also modulates glucose metabolism by increasing insulin sensitivity and activates the sympathetic nervous system. In humans, leptin is also produced by placental trophoblasts and is secreted into both the maternal and fetal circulation. Leptin production in the placenta is increased in pregnancies complicated with several pathologic conditions. Leptin gene expression in the placenta is augmented in severe preeclampsia, and maternal plasma leptin levels in severe preeclampsia are significantly higher than those in normotensive pregnant women. Leptin production in the placenta is also increased in diabetic pregnancy with insulin treatment. Furthermore, leptin is proposed to play a functional role in implantation by virtue of its stimulatory effect on matrix metalloproteinase expression in cytotrophoblast. Dysregulation of leptin metabolism and/or function in the placenta may be implicated in the pathogenesis of various disorders during pregnancy, such as recurrent miscarriage, gestational diabetes, intrauterine growth retardation, and preeclampsia. In this review, possible roles of placental leptin are discussed..
375. Takashi Miyazawa, Yoshihiro Ogawa, Hideki Chusho, Akihiro Yasoda, Naohisa Tamura, Yasato Komatsu, Alexander Pfeifer, Franz Hofmann, Kazuwa Nakao, Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification, Endocrinology, 10.1210/en.2002-220307, 143, 9, 3604-3610, 2002.09, Longitudinal bone growth is determined by endochondral ossification at the growth plate, which is located at both ends of long bones and vertebrae, and involves many systemic hormones and local regulators. C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, occurs at the growth plate and acts locally as a positive regulator of endochondral ossification through the intracellular accumulation of cyclic GMP (cGMP). The increase in cGMP concentrations is known to activate different signaling mediators, such as cyclic nucleotide phosphodiesterases, cGMP-regulated ion channels, and cGMP-dependent protein kinases (cGKs). The type II cGK (cGKII)-deficient mice (Prkg2-/- mice) develop dwarfism as a result of impaired endochondral ossification, suggesting that cGKII is important for the CNP-mediated endochondral ossification. However, given that Prkg2-/- mice differ from CNP-deficient mice (Nppc-/- mice) in the growth plate histology, which downstream mediator(s) of cGMP play key roles in the process is still an enigma. Here we show that targeted expression of CNP in the growth plate chondrocytes fails to rescue the skeletal defect of Prkg2-/- mice. Using cultured fetal mouse tibias, an in vitro model system of endochondral ossification, we also demonstrated that CNP cannot increase the longitudinal bone growth, and chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis in Prkg2-/- mice. This study provides in vivo and in vitro genetic evidence that cGKII plays a critical role in CNP-mediated endochondral ossification..
376. Hiroyuki Ariyasu, Kazuhiko Takaya, Hiroshi Hosoda, Hiroshi Iwakura, Ken Ebihara, Kiyoshi Mori, Yoshihiro Ogawa, Kiminori Hosoda, Takashi Akamizu, Masayasu Kojima, Kenji Kangawa, Kazuwa Nakao, Delayed short-term secretory regulation of ghrelin in obese animals
Evidenced by a specific RIA for the active form of ghrelin, Endocrinology, 10.1210/en.2002-220225, 143, 9, 3341-3350, 2002.09, Ghrelin is an acylated peptide, whose lipid modification is essential for its biological activities. Previous studies demonstrated that it strongly stimulates GH release and has a potent orexigenic action. Meanwhile, there is enough evidence showing that feeding states influence plasma ghrelin levels. Fasting stimulates ghrelin secretion, and feeding reduces plasma ghrelin levels. In this study we examined the regulation of plasma ghrelin by fasting in genetically obese animals considering its molecular forms. Plasma levels of active form of ghrelin as well as those of total ghrelin were reduced in ob/ob and db/db mice compared with those in their control mice. Zucker fatty (fa/fa) rats also showed lower plasma ghrelin levels by fasting than the control rats. Insulin-induced hypoglycemia, however, stimulated ghrelin secretion in the fasted fatty rats. Moreover, glucose injection was revealed to reduce plasma ghrelin levels in rats. The effect of the severity of obesity on secretory regulation of ghrelin was also studied. Older fatty rats showed low plasma ghrelin levels even after 48-h fasting. These data suggest that the short-term secretory regulation of total ghrelin and the active form of ghrelin is delayed in obese animals and that blood glucose levels may be involved in the delayed regulation..
377. Y. Ogawa, K. Kanatsu, T. Iino, S. Kato, Y. I. Jeong, N. Shibata, K. Takada, K. Takeuchi, Protection against dextran sulfate sodium-induced colitis by microspheres of ellagic acid in rats, Life Sciences, 10.1016/S0024-3205(02)01737-X, 71, 7, 827-839, 2002.07, Ellagic acid (EA), a naturally occurring plant phenol, has the antioxidant and anti-inflammatory activities. In the present study, we examined the effect of EA contained in microspheres on the ulcerative colitis induced experimentally in rats by dextran sulfate sodium (DSS). Experimental colitis was induced in male Fisher 344 rats by daily treatment with 3% DSS solution in drinking water for 7 days. EA of microspheres (mcEA: 1∼10 mg/kg as EA contents) was administered p.o. twice daily for 6 days. In a preliminary study, we found that these microsphere capsules, when administered p.o., are effectively dissolved in the proximal to the ileo-cecal junction and distributed to the terminal ileum and the colon. The ulceration area, colon length, and mucosal myeloperoxidase (MPO) activity as well as thiobarbituric acid-reactive substances (TBARS) were measured on 7th day after the onset of DSS treatment. The DSS treatment for 7 days caused severe mucosal lesions in the colon, accompanied with the increases of MPO activity and TBARS as well as the decreases of body weight gain and colon length. Administration of mcEA reduced the severity of DSS-induced colitis in a dose-dependent manner, and a significant effect was observed at 10 mg/kg, the ED50 being 2.3 mg/kg. This mcEA treatment also significantly mitigated changes in various biochemical parameters in the colonic mucosa induced by DSS. Although plain EA (without using microspheres) was also effective in reducing the severity of DSS-induced colitis, this effect was much less potent as compared with that of mcEA; the ED50 was about 15 times higher than that of mcEA. In addition, a significant effect on DSS-induced colitis was also obtained by intra-rectal administration of superoxide dismutase, an anti-oxidative agent. These results suggest that EA prevents the ulcerative colitis induced by DSS, probably by radical scavenging and/or anti-oxidative actions. The microspheres used in this study may be useful for delivering an orally administered drug specifically to the colon..
378. K. Takeuchi, Y. Ogawa, S. Kagawa, H. Ukawa, Gastric ulcerogenic responses following barrier disruption in knockout mice lacking prostaglandin EP1 receptors, Alimentary Pharmacology and Therapeutics, Supplement, 10.1046/j.1365-2036.16.s2.21.x, 16, 2, 74-82, 2002.04, Background/aims: Endogenous prostaglandins (PGs) arc considered to play a pivotal role in maintaining the mucosal integrity of the stomach after injury. In the present study, we evaluated the mucosal ulcerogenic and mucosal blood flow (GMBF) responses in the stomach after damage by taurocholate (TC) in knockout mice lacking EP1 or EP3 receptors. Methods: Under urethane anaesthesia, a mouse stomach was mounted in an ex vivo chamber, exposed to 20 mmol/L TC for 20 min and treated with 20 mmol/L HCl before and after TC. GMBF was measured with a laser Doppler flowmeter. Results: Mucosal exposure to TC in wild-type mice caused a marked decrease in potential difference (PD), followed by an increase in H+ loss and GMBF. The decreased PD was gradually normalized after removal of TC from the chamber, with minimal damage in the mucosa 1 h after TC treatment. This hyperaemic response was inhibited by indomethacin, resulting in severe lesions in the mucosa without any change in PD or H+ loss. None of these responses induced by TC were altered in EP3-/- mice. However, in mice lacking EP1 receptors, TC treatment did not increase GMBF, despite causing PD reduction and acid loss, and resulted in severe damage in the mucosa. These responses were closely similar to those observed in animals pretreated with ONO-8711, a EP1 receptor antagonist. Mucosal PGE2 content was significantly increased after TC, similarly in all groups of mice. Conclusion: These results confirm a mediator role for PGE 2 in gastric hyperaemic response following mucosal exposure to TC and suggest that endogenous PGs may contribute to maintaining mucosal integrity after barrier disruption, mainly through activation of the EP1 receptor subtype..
379. N. Sagawa, S. Yura, H. Itoh, H. Mise, K. Kakui, D. Korita, M. Takemura, M. A. Nuamah, Y. Ogawa, H. Masuzaki, K. Nakao, S. Fujii, Role of leptin in pregnancy - A review, Placenta, 10.1053/plac.2002.0814, 23, SUPPL. 1, S80-S86, 2002.04, Leptin is an adipocyte-derived hormone that decreases food intake and body weight via its receptor in the hypothalamus. In rodents, it also modulates glucose metabolism by increasing insulin sensitivity. We previously reported that leptin is produced by human placental trophoblasts. We also revealed that leptin gene expression in the placenta was augmented in severe pre-eclampsia, and suggested that placental hypoxia may play a role in this augmentation. Maternal plasma leptin levels correlated well with mean blood pressure, but not with body mass index. Plasma leptin levels in pre-eclamptic women with IUGR were higher than those without IUGR (P<0.05). We further examined the effects of hyperleptinemia on the course of pregnancy by using transgenic mice (Tg) overexpressing leptin. In pregnant Tg mice, food intake was significantly less than non-Tg, and the fetal body weights were reduced to approximately 70 per cent of those of non-Tg. Resistin is a novel adipocyte-derived hormone that decreases insulin sensitivity and increases plasma glucose concentration, thus contributing the development of obesity-related type II diabetes mellitus. We recently found that resistin gene is expressed in the human placenta as well as adipose tissue. In this review, possible roles of placental leptin and resistin are discussed..
380. Yoshihiro Ogawa, Hiroaki Masuzaki, Ken Ebihara, Mitsuyo Shintani, Megumi Aizawa-Abe, Fumiko Miyanaga, Kazuwa Nakao, Pathophysiogical role of leptin in lifestyle-related diseases
Studies with transgenic skinny mice overexpressing leptin, Journal of Diabetes and Its Complications, 10.1016/S1056-8727(01)00204-5, 16, 1, 119-122, 2002.03, Leptin is a major adipocyte-derived hormone that is involved in the regulation of food intake and energy expenditure. Plasma leptin concentrations are elevated in obese subjects, suggesting its pathophysiological role in obesity-related lifestyle-related diseases. We have recently succeeded in the generation of transgenic skinny mice overexpressing leptin. They exhibit increased glucose metabolism and insulin sensitivity accompanied by a significant increase in insulin signaling for glucose utilization in the skeletal muscle and liver. They also show blood pressure elevation through the sympathetic activation. Introduction of the lethal yellow agouti (Ay) allele into transgenic skinny mice results in late-onset obesity and diabetes with blood pressure elevation similar to those found in nontransgenic agouti mice (Ay/+ mice). After caloric restriction, blood pressure elevation is reversed but insulin resistance still remains in Ay/+ mice in parallel with a reduction of plasma leptin concentrations. By contrast, blood pressure elevation is sustained but insulin resistance is reversed in transgenic mice overexpressing leptin with the Ay allele (Tg/+:Ay/+ mice), which remain hyperleptinemic. Collectively, our data suggest the pathophysiologic and therapeutic implication of leptin in obesity-related insulin resistance and hypertension..
381. Hiromasa Kobayashi, Yoshihiro Ogawa, Mitsuyo Shintani, Ken Ebihara, Makiko Shimodahira, Toshio Iwakura, Megumu Hino, Takashi Ishihara, Katsuji Ikekubo, Hiroyuki Kurahachi, Kazuwa Nakao, A novel homozygous missence mutation of melanocortin-4 receptor (MC4R) in a Japanese woman with obesity, Diabetes, 10.2337/diabetes.51.1.243, 51, 1, 243-246, 2002.01, The melanocortin-4 receptor (MC4R) is a member of the seven membrane-spanning G protein-coupled receptor superfamily and signals through the activation of adenylyl cyclase. The MC4R mutations are the most common known monogenic cause of human obesity. However, no such mutations have been found in Japanese obese subjects. Here we report a novel homozygous missense mutation of MC4R (G98R) in a nondiabetic Japanese woman with severe early-onset obesity, which is located in its second transmembrane domain. Her birth weight was 3,360 g, and she gained weight progressively from 10 months of age. At 40 years of age, her weight reached 160 kg and a BMI of 62 kg/m2. Her parents, who are heterozygous for the mutation, have BMIs of 26 and 27 kg/m2. In vitro transient transfection assays revealed no discernable agonist ligand binding and cAMP production in HEK293 cells expressing the mutant receptor, indicating a severe loss-of-function mutation. This study represents the first demonstration of a pathogenic mutation of MC4R in Japan and will provide further insight into the pathophysiologic role of the hypothalamic melanocortin system in human obesity..
382. T. Miyawaki, H. Masuzaki, Y. Ogawa, K. Hosoda, H. Nishimura, N. Azuma, A. Sugawara, I. Masuda, M. Murata, T. Matsuo, T. Hayashi, G. Inoue, Y. Yoshimasa, K. Nakao, Clinical implications of leptin and its potential humoral regulators in long-term low-calorie diet therapy for obese humans, European Journal of Clinical Nutrition, 10.1038/sj.ejcn.1601363, 56, 7, 593-600, 2002.01, Objective: To address the clinical implications of leptin and to re-examine the relationship between leptin and its potential humoral regulators such as insulin, nonesterified fatty acids (NEFA) and triiodothyronine (T3) in low-calorie diet (LCD) for obese humans. Design: Longitudinal study. Setting: University and foundation hospitals. Subjects: Ten obese men and 10 premenopausal obese women. Interventions: Five men and five women took 800 kcal/day LCD and another five men and five women took 1400 kcal/day balanced deficit diet (BDD) during 4 weeks. Results: Plasma leptin levels in the LCD group decreased more markedly (46.2±14.6 to 13.2±3.6 ng/ml) than that expected for the decrement in percentage fat (39.0±1.7 to 35.9±1.7%) and body mass index (BMI; 35.4±2.4 to 33.1±2.2 kg/m2), while that in the BDD group did not decrease significantly (14.9±3.5 to 13.4±2.8 ng/ml). The ratio of the decrease in leptin levels to that of BMI during the first week was significantly greater than that during the following 3 weeks (39.5±2.7 vs 29.3±2.1%, P=0.017). The plasma insulin and T3 levels also fell substantially in the first week and continued to decrease during the entire course. Plasma leptin levels measured weekly in each subject were correlated well with insulin (r= 0.586, P=0.0003) and T3 (r=0.785, P=0.0004). Multiple regression analyses after adjustment for the time course and BMI revealed that serum levels of T3 were independently correlated with plasma leptin levels (r=0.928, P<0.0001). The plasma NEFA level was markedly elevated during the first 2 weeks and decreased thereafter. Conclusions: A rapid fall in leptin during the first week of LCD, coordinated by insulin, T3 and NEFA, should be beneficial for responding to decreased energy intake. Inversely, in view of the powerful effect of leptin on energy dissipation, the present findings suggest the potential usefulness of leptin in combination with caloric restriction for the treatment of obesity. Sponsorship: The Ministry of Education, Culture, Sports, Science and Technology of Japan and the Ministry of Health, Labour and Welfare of Japan..
383. Taeko Iino, Kimihito Tashima, Masakazu Umeda, Yoshihiro Ogawa, Masanori Takeeda, Kanji Takata, Koji Takeuchi, Effect of ellagic acid on gastric damage induced in ischemic rat stomachs following ammonia or reperfusion, Life Sciences, 10.1016/S0024-3205(01)01493-X, 70, 10, 1139-1150, 2002.01, We examined the effect of ellagic acid (EA), one of the polyphenols that are abundantly contained in whisky as a nonalcoholic component, on gastric lesions induced by ammonia plus ischemia or ischemia/reperfusion in rats, in relation to the antioxidative system. Under urethane anesthesia, a rat stomach was mounted in an ex vivo chamber, and the following two experiments were performed; 1) a stomach was made ischemic (1.5 ml/100 g body weight) for 20 min, followed by reperfusion for 15 min in the presence of 100 mM HCl; 2) a stomach was made ischemic by bleeding from the carotid artery (1 ml/100 g body weight), followed by intragastric application of ammonia (NH4OH: 120 mM). EA (0.1-10 mg/ml) was applied in the chamber 30 min before the onset of ischemia. Gastric potential difference (PD) and mucosal blood flow (GMBF) were measured before, during and after 20 min of ischemia. Ischemia/reperfusion caused a profound drop in GMBF followed by a return, and resulted in hemorrhagic lesions in the stomach in the presence of 100 mM HCl. These lesions were dose-dependently prevented by EA with suppression of lipid peroxidation but no effect on GMBF, and the effect at 6 mg/ml was almost equivalent to that of superoxide dismutase (SOD: 15000 unit/kg/hr) infused i.v. during a test-period. On the other hand, application of NH4OH to the ischemic stomach produced a marked reduction in PD, resulting in severe hemorrhagic lesions. These changes were prevented with both EA and SOD. In addition, EA had a potent scavenging action against monochloramine in vitro. These results suggest that EA exhibits gastric protective action against gastric lesions induced by NH4OH or reperfusion in the ischemic stomach, probably due to its anti-oxidative activity. This property of EA partly explains the less damaging effect of whisky in the stomach and may be useful as the prophylactic for Helicobacter pylori-associated gastritis..
384. Ken Ebihara, Yoshihiro Ogawa, Kazuwa Nakao, Insulin resistant transgenic mice, Nippon rinsho. Japanese journal of clinical medicine, 60 Suppl 8, 79-84, 2002.01.
385. Yoshihiro Ogawa, Kazuwa Nakao, Leptin and leptin receptor, Nippon rinsho. Japanese journal of clinical medicine, 60 Suppl 7, 571-576, 2002.01.
386. Hiromasa Kobayashi, Megumu Hino, Makiko Shimodahira, Toshio Iwakura, Takashi Ishihara, Katsuji Ikekubo, Yoshihiro Ogawa, Kazuwa Nakao, Hiroyuki Kurahachi, Missense mutation of TRPS1 in a family of tricho-rhino-phalangeal syndrome type III, American Journal of Medical Genetics, 10.1002/ajmg.10081, 107, 1, 26-29, 2002.01, We report a new Japanese family with tricho-rhino-phalangeal syndrome type III (TRPS III) who have a missense mutation (Arg908Gn) of the TRPS1 gene (TRPS1) in affected individuals of the family. This study supports the notion that TRPS III results from missense mutations in exon 6 of TRPS1..
387. Mitsuyo Shintani, Yoshihiro Ogawa, Kazuwa Nakao, Obesity induced by abnormality in leptin receptor and melanocortin-4 receptor, Nippon rinsho. Japanese journal of clinical medicine, 60, 2, 404-409, 2002.01, Obesity is a multifactorial disease that arises from complex interactions between genetic predisposition and environmental factors. It increases a risk of cardiovascular and metabolic diseases such as diabetes, hypertension, and hyperlipidemia. Recent molecular genetic studies have disclosed some monogenic forms of obesity in humans. Leptin directly exerts its anorexigenic effects on hypothalamic arcuate nucleus. alpha-melanocyte stimulating hormone (alpha-MSH) derived from proopiomelanocortin (POMC) and melanocortin-4 receptor (MC4-R) have been reported to be involved in the downstream of leptin actions. In this paper, we summarize the clinical characteristics and the mechanisms of obesity caused by genetic abnormalities in leptin receptor and melanocortin-4 receptor..
388. Hiroaki Masuzaki, Yoshihiro Ogawa, Megumi Aizawa-Abe, Kazuwa Nakao, Transgenic approach toward leptin biology
The clinical implications of leptin for the treatment of obesity-associated diabetes and obesity-related hypertension, Endocrine Journal, 10.1507/endocrj.49.109, 49, 2, 109-119, 2002.01.
389. Michio Suda, Kiyoshi Tanaka, Akihiro Yasoda, Yasato Komatsu, Hideki Chusho, Masako Miura, Naohisa Tamura, Yoshihiro Ogawa, Kazuwa Nakao, C-type natriuretic peptide/guanylate cyclase B system in ATDC5 cells, a chondrogenic cell line, Journal of Bone and Mineral Metabolism, 10.1007/s007740200019, 20, 3, 136-141, 2002, Natriuretic peptides constitute a family of three structurally related peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Particulate guanylate cyclases, GC-A, and GC-B, are the receptors for these peptides to mediate their action. ANP and BNP possess high affinities for GC-A, and CNP is the preferred ligand for GC-B. In this article, we report our study of the expression and possible role(s) of natriuretic peptides in ATDC5 cells, which represent a chondrogenic cell line. ATDC5 cells produced cyclic guanosine monophosphate (cGMP) in response to natriuretic peptides. CNP was far more potent than ANP in terms of cGMP production. The messages for GC-A and GC-B were demonstrated by means of Northern blot analysis, and the presence of CNP was shown by Southern blotting coupled with reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that the CNP/GC-B system is preferentially expressed in ATDC5 cells. GC-B mRNA expression was higher at 14 days after confluency than that at confluency. CNP or 8-bromo cGMP reduced [3H] thymidine uptake and slightly increased the message for collagen type X, which is a marker of hypertrophic chondrocytes. These data suggest that the CNP/GC-B system is likely to be an autocrine/paracrine regulator of ATDC5 cells, thus affecting both their growth and differentiation..
390. Y. Fujita, M. Murakami, Y. Ogawa, H. Masuzaki, M. Tanaka, S. Ozaki, K. Nakao, T. Mimori, Leptin inhibits stress-induced apoptosis of T lymphocytes, Clinical and Experimental Immunology, 10.1046/j.1365-2249.2002.01797.x, 128, 1, 21-26, 2002, Leptin, which is secreted by adipocytes, the placenta and the stomach, not only controls appetite through leptin receptors in the hypothalamus but also regulates cell-mediated immunity. In this study we have demonstrated that continuous injection of leptin prevents the reduction in lymphocyte numbers normally observed in fasted and steroid-injected mice. Consistent with leptin-induced protection, we observed up-regulation of the bcl-xL gene as a result of signal transduction via leptin receptors on lymphocytes. We suggest that leptin might contribute to the recovery of immune suppression in malnourished mice by inhibiting lymphocyte apoptosis..
391. K. Shimizu, T. Nakamura, A. Niimi, N. Hattori, M. Mishima, K. Chin, T. Nakamura, R. Nohara, R. Hosokawa, S. Sasayama, H. Masuzaki, Y. Ogawa, K. Nakao, M. Ohi, Plasma leptin levels and cardiac sympathetic function in patients with obstructive sleep apnoea-hypopnoea syndrome, Thorax, 10.1136/thorax.57.5.429, 57, 5, 429-434, 2002, Background: The control of body weight and cardiac sympathetic function in patients with obstructive sleep apnoea-hypopnoea syndrome (OSAHS) are important because both factors have significant effects on the mortality of these patients. It has recently been reported that OSAHS has a significant effect on the secretion of leptin, a hormone involved in the control of body weight and sympathetic nerve activity. In addition to the circadian rhythm of leptin secretion, the effects of one night of treatibility with nasal continuous positive airway pressure (nCPAP) and the mechanism of the effects of nCPAP on nocturnal leptin secretion in patients with OSAHS has not yet been elucidated. Methods: Blood samples were obtained at 21.00 hours, 00.00 hours, 03.00 hours, and 06.30 hours from 21 subjects with OSAHS (mean apnoea and hypopnoea index 52.4/h), with and without nCPAP treatment. Iodine-123 (1123)-meta-iodobenzylguanidine (MIBG) imaging was used to evaluate myocardial sympathetic function before nCPAP treatment. Results: Plasma leptin reached a peak level at 00:00 hours (p<0.01) in patients with OSAHS, both with and without nCPAP treatment. The first night of nCPAP treatment significantly decreased the plasma leptin levels at 03.00 hours (without nCPAP: mean (SE) 21.6 (4.7) ng/ml; with nCPAP: 19.3 (4.1) ng/ml, p<0.02) and at 06.30 hours (without nCPAP: 17.6 (3.8) ng/ml; with nCPAP: 15.2 (3.2) ng/ml, p<0.01). The magnitude of the decrease in leptin levels after nCPAP treatment was significantly correlated with cardiac sympathetic function measured before nCPAP treatment (p<0.03). Conclusions: Patients with OSAHS undergo nocturnal increases in leptin levels in spite of interruption of sleep due to apnoea and hypopnoea, a trend seen in normal subjects. Plasma leptin levels in patients with OSAHS decreased significantly after the first night of nCPAP treatment. Enhanced cardiac sympathetic function in these patients may contribute to the leptin levels before nCPAP treatment and vice versa..
392. S. Hidaka, Y. Ogawa, K. Nakao, Potential molecular targets for anti-obesity drugs - After the discovery of leptin, Folia Pharmacologica Japonica, 10.1254/fpj.118.309, 118, 5, 309-314, 2001.12, The discovery of the adipose-derived hormone leptin has generated interest in the interaction between peripheral signals and brain targets involved in the regulation of feedings and energy balance. Potential anti-obesity drugs can be based on any intervention between the neuropeptide and its receptor that would alter the biological responses mediated by the neuronal network, in particular, food intake, metabolism and energy expenditure. Modulation of neurons in the arcuate nucleus by leptin results in reduced expression of neuropeptide Y and agouti-related protein, and increased expression of pro-opiomelanocortin (the precursor of a-melanocyte-stimulating hormone) and cocaine- and amphetamine- regulated transcript. Whether leptin finds its way into general usage as an anti-obesity drug, the use of modern methods to identify and target the components of leptin signaling pathway will form the basis for new pharmacological approaches to the treatment of obesity..
393. Mitsuyo Shintani, Haruo Nishimura, Shin Yonemitsu, Yoshihiro Ogawa, Tatsuya Hayashi, Kiminori Hosoda, Gen Inoue, Kazuwa Nakao, Troglitazone Not only Increases GLUT4 but Also Induces Its Translocation in Rat Adipocytes, Diabetes, 10.2337/diabetes.50.10.2296, 50, 10, 2296-2300, 2001.10, Thiazolidinediones, insulin-sensitizing agents, have been reported to increase glucose uptake along with the expression of glucose transporters in adipocytes and cardiomyocytes. Recently, we have further suggested that the translocation of GLUT4 is stimulated by thiazolidinediones in L6 myocytes. However, the direct effects of thiazolidinediones on translocation of glucose transporters have not yet been determined. In this study, using hemagglutinin epitope-tagged GLUT4 (GLUT4-HA), we provide direct evidence of the effect of troglitazone on the translocation of GLUT4 in rat epididymal adipocytes. Primary cultures of rat adipocytes were transiently transfected with GLUT4-HA and over-expressed eightfold compared with endogenous GLUT4 in transfected cells. A total of 24 h of treatment with troglitazone (10-4 mol/l) increased the cell surface level of GLUT4-HA by 1.5 ± 0.03-fold (P < 0.01) without changing the total amount of GLUT4-HA, whereas it increased the protein level of endogenous GLUT4 (1.4-fold) without changing that of GLUT1. Thus, the direct effect on the translocation can be detected apart from the increase in endogenous GLUT4 content using GLUT4-HA. Troglitazone not only increased the translocation of GLUT4-HA on the cell surface in the basal state but also caused a leftward shift in the dose-response relations between GLUT4-HA translocation and insulin concentration in the medium (ED50: from ∼0.1 to 0.03 nmol/l). These effects may partly contribute to the antidiabetic activity of troglitazone in patients with obesity and type 2 diabetes..
394. Y. Ogawa, M. Abe, K. Nakao, Leptin and cardiovascular lesions, Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.90.705, 90, 4, 705-710, 2001.04.
395. Hideki Chusho, Naohisa Tamura, Yoshihiro Ogawa, Akihiro Yasoda, Michio Suda, Takashi Miyazawa, Kenji Nakamura, Kazuki Nakao, Tatsuya Kurihara, Yasato Komatsu, Hiroshi Itoh, Kiyoshi Tanaka, Yoshihiko Saito, Motoya Katsuki, Kazuwa Nakao, Dwarfism and early death in mice lacking C-type natriuretic peptide, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.071389098, 98, 7, 4016-4021, 2001.03, Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc-/- mice). The Nppc-/- mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc-/- mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia..
396. H. Nishimura, Y. Ogawa, K. Nakao, Human obesity and point mutations of leptin and leptin receptor, Nippon rinsho. Japanese journal of clinical medicine, 59, 3, 571-577, 2001.03, Obesity-related genes were isolated and identified in these several years. Development of transgenic mice has clarified the molecular mechanisms of obesity. There have been reported point mutations of the genes correspond to leptin(ob-gene) and leptin receptor(db-gene). Thereby leptin is considered to be one of the most important regulators of energy metabolism also in human..
397. Naoki Matsuoka, Yoshihiro Ogawa, Hiroaki Masuzaki, Ken Ebihara, Megumi Aizawa-Abe, Noriko Satoh, Eiichiro Ishikawa, Yukio Fujisawa, Atsushi Kosaki, Kazunori Yamada, Hideshi Kuzuya, Kazuwa Nakao, Decreased triglyceride-rich lipoproteins in transgenic skinny mice overexpressing leptin, American Journal of Physiology - Endocrinology and Metabolism, 10.1152/ajpendo.2001.280.2.e334, 280, 2 43-2, E334-E339, 2001.02, Leptin is an adipocyte-derived circulating satiety factor with a variety of biological effects. Evidence has accumulated suggesting that leptin may modulate glucose and lipid metabolism. In the present study, we examined lipid metabolism in transgenic skinny mice with elevated plasma leptin concentrations. The plasma concentrations of triglycerides and free fatty acids in transgenic skinny mice were 71.5 (P < 0.01) and 89.1% (P < 0.05) of those in their nontransgenic littermates, respectively. Separation of plasma into lipoprotein classes by ultracentrifugation revealed that very low density lipoprotein-triglyceride concentrations were markedly reduced in transgenic skinny mice relative to the controls. The clearance of triglycerides estimated by a fat-loading test was enhanced in transgenic skinny mice; the triglyceride concentration in transgenic skinny mice 3 h after fat loading was 39.7% (P < 0.05) of that of their nontransgenic littermates. Postheparin plasma lipoprotein lipase activity increased 1.4-fold (P < 0.05) in transgenic skinny mice. Our data demonstrated a significant reduction in plasma triglyceride concentrations, accompanied by increased lipoprotein lipase activity in transgenic skinny mice overexpressing leptin, suggesting that leptin plays a role in long-term triglyceride metabolism..
398. H. Nishimura, Y. Ogawa, K. Nakao, Structures and functions of leptin and leptin receptor, Nippon rinsho. Japanese journal of clinical medicine, 59 Suppl 2, 509-514, 2001.02.
399. Kazuki Yasuda, Naoya Koda, Hiroko Kadowaki, Yoshihiro Ogawa, Satoshi Kimura, Takashi Kadowaki, Yasuo Akanuma, A Japanese case of congenital hyperinsulinism with hyperammonemia due to a mutation in glutamate dehydrogenase (GLUD1) gene, Internal Medicine, 10.2169/internalmedicine.40.32, 40, 1, 32-37, 2001.01, We describe a Japanese case of neonatal hyperinsulinism due to a de novo mutation (Gly446Asp) in glutamate dehydrogenase gene (GLUD1). A boy suffered from hypoglycemic coma with relative hyperinsulinemia on day 1 after birth, and received subtotal pancreatectomy. Examination of the resected pancreas revealed a diffuse increase in endocrine cells, consistent with 'nesidioblastosis'. He is now 15 years old and has exhibited mild but persistent hyperammonemia, which is a very unique feature of the disorder caused by GLUD1 activating mutations. He has also been suffering from seizures and mental retardation. Thus, GLUD1 mutations can be a cause of congenital hyperinsulinism in Japanese..
400. Takashi Kuramoto, Kazuhiro Kitada, Toshihide Inui, Yoshifumi Sasaki, Kazumi Ito, Takao Hase, Saburo Kawagachi, Yoshihiro Ogawa, Kazuwa Nakao, Gregory S. Barsh, Minako Nagao, Toshikazu Ushijima, Tadao Serikawa, Attractin/mahogany/zitter plays a critical role in myelination of the central nervous system, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.98.2.559, 98, 2, 559-564, 2001.01, The rat zitter (zi) mutation induces hypomyelination and vacuolation in the central nervous system (CNS), which result in early-onset tremor and progressive flaccid paresis. By positional cloning, we found a marked decrease in Attractin (Atrn) mRNA in the brain of the zi/zi rat and identified zi as an 8-bp deletion at a splice donor site of Atrn. Atrn has been known to play multiple roles in regulating physiological processes that are involved in monocyte-T cell interaction, agouti-related hair pigmentation, and control of energy homeostasis. Rat Atrn gene encoded two isoforms, a secreted and a membrane form, as a result of alternative splicing. The zi mutation at the Atrn locus darkened coat color when introduced into agouti rats, as also described in mahogany (mg) mice, carrying the homozygous mutation at the Atrn locus. Transgenic rescue experiments showed that the membrane-type Atrn complemented both neurological alteration and abnormal pigmentation in zi/zi rats, but that the secreted-type Atrn complemented neither mutant phenotype. Furthermore, we discovered that mg mice exhibited hypomyelination and vacuolation in the CNS associated with body tremor. We conclude from these results that the membrane Atrn has a critical role in normal myelination in the CNS and would provide insights into the physiology of myelination as well as the etiology of myelin diseases..
401. Y. Ogawa, N. Tamura, H. Chusho, K. Nakao, Brain natriuretic peptide appears to act locally as an antifibrotic factor in the heart, Canadian Journal of Physiology and Pharmacology, 10.1139/y01-052, 79, 8, 723-729, 2001.01, In addition to cardiac myocyte hypertrophy, proliferation and increased extracellular matrix production of cardiac fibroblasts occur in response to cardiac overload. This remodeling of the cardiac interstitium is a major determinant of pathologic hypertrophy leading to ventricular dysfunction and heart failure. Atrial and brain natriuretic peptides (ANP and BNP) are cardiac hormones produced primarily by the atrium and ventricle, respectively. Plasma ANP and BNP concentrations are elevated in patients with hypertension, cardiac hypertrophy, and acute myocardial infarction, suggesting their pathophysiologic roles in these disorders. ANP and BNP exhibit diuretic, natriuretic, and vasodilatory activities via a guanylyl cyclase-coupled natriuretic peptide receptor subtype (guanylyl cyclase-A or GC-A). Here we report the generation of mice with targeted disruption of BNP (BNP-/- mice). We observed focal fibrotic lesions in ventricles from BNP-/- mice with a remarkable increase in ventricular mRNA expression of ANP, angiotensin converting enzyme (ACE), transforming growth factor (TGF)-β3, and pro-α1(I) collagen [Col α1(I)], which are implicated in the generation and progression of ventricular fibrosis. Electron microscopic examination revealed supercontraction of sarcomeres and disorganized myofibrils in some ventricular myocytes from BNP-/- mice. No signs of cardiac hypertrophy and systemic hypertension were noted in BNP-/- mice. In response to acute cardiac pressure overload induced by aortic constriction, massive fibrotic lesions were found in all the BNP-/- mice examined, accompanied by further increase of mRNA expression of TGF-β3 and Col α1(I). We postulate that BNP acts as a cardiocyte-derived antifibrotic factor in the ventricle..
402. Kentaro Doi, Tadashi Ikeda, Hiroshi Itoh, Koji Ueyama, Kiminori Hosoda, Yoshihiro Ogawa, Jun Yamashita, Tae Hwa Chun, Mayumi Inoue, Ken Masatsugu, Naoki Sawada, Yasutomo Fukunaga, Takatoshi Saito, Masakatsu Sone, Kenichi Yamahara, Hyun Kook, Masashi Komeda, Makiko Ueda, Kazuwa Nakao, C-type natriuretic peptide induces redifferentiation of vascular smooth muscle cells with accelerated reendothelialization, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/01.ATV.21.6.930, 21, 6, 930-936, 2001.01, We recently reported that C-type natriuretic peptide (CNP) occurs in vascular endothelial cells and acts as a vascular-type natriuretic peptide. In the present study, we stimulated the cGMP cascade in proliferating smooth muscle cells (SMCs), in which particulate guanylate cyclase-B, the specific receptor for CNP, is predominantly expressed, by use of an adenovirus encoding rat CNP cDNA (Ad.CNP). In the Ad.CNP-treated cultured SMCs, CNP caused the growth inhibition of SMCs at G1, phase with an early increase of p21CIP1/WAF1 expression and subsequent upregulation of p16INK4a. The expression of smooth muscle myosin heavy chain-2, which is the molecular marker of highly differentiated SMCs, was reinduced in the Ad.CNP-treated SMCs. The Ad.CNP-treated SMCs also reexpressed particulate guanylate cyclase-A, which shows high affinity to atrial and brain natriuretic peptide and is exclusively expressed in well-differentiated SMCs. CNP, which was overexpressed in rabbit femoral arteries in vivo at the time of balloon injury, significantly suppressed neointimal formation. Furthermore, an enhancement of the expression of smooth muscle myosin heavy chain-2 occurred in the residual neointima. In addition, early regeneration of endothelial cells was observed in the Ad.CNP-infected group. Thus, stimulation of cGMP cascade in proliferating dedifferentiated SMCs can induce growth inhibition and redifferentiation of SMCs with accelerated reendothelialization..
403. Masayo Nakagawa, Issei Tanaka, Masashi Mukoyama, Shin Ichi Suga, Yoshihiro Ogawa, Naohisa Tamura, Rieko Ishibashi, Masahisa Goto, Osamu Nakagawa, Akira Sugawara, Kazuwa Nakao, Monoclonal antibody against brain natriuretic peptide and characterization of brain natriuretic peptide-transgenic mice, Journal of hypertension, 10.1097/00004872-200103000-00016, 19, 3, 475-483, 2001.01, Objective: Brain natriuretic peptide (BNP) is a ventricular hormone with natriuretic, diuretic and vasodilatory actions. Acute infusion of BNP reduces cardiac pre- and after-load in healthy and diseased subjects, but its long-term therapeutic usefulness remains unclear. Design: We prepared a monoclonal antibody specific to mouse BNP, and characterized transgenic mice overexpressing BNP in the liver (BNP-Tg mice) as a model of its chronic overproduction. Methods: Radioimmunoassay and neutralization experiments using the monoclonal antibody, KY-mBNP-I, were performed in BNP-Tg mice in conjunction with examinations of blood pressure (BP) and other markers for body fluid homeostasis. Results: We developed highly sensitive radioimmunoassay to mouse BNP. In BNP-Tg mice, the plasma BNP concentration increased more than 100-fold, while ventricular BNP concentration did not alter, suggesting that ventricular BNP production was not down-regulated in BNP-Tg mice. The BNP concentration in the kidneys was 10-fold higher than nontransgenic (nonTg) littermates, accompanied with marked reduction in the atrial natriuretic peptide (ANP) concentration, that may be due to binding of circulating BNP to the natriuretic peptide receptors. BNP-Tg mice showed significantly low arterial BP, and a bolus intraperitoneal administration of KY-mBNP-I completely abolished enhanced cGMP excretion in the urine and significantly increased the systolic BP. Conclusion: These results suggested that biological actions of BNP last and reduce cardiac overload in its long-term overproduction in the transgenic mouse model..
404. S. Hidaka, Y. Ogawa, K. Ebihara, M. Shintani, M. Abe, F. Miyanaga, K. Nakao, Pathology and significance of leptin resistance in obesity, Nippon rinsho. Japanese journal of clinical medicine, 59, 3, 472-480, 2001.01, Leptin, the protein product of the ob gene, is predominantly secreted from white adipose tissue, and acts on the brain to regulate food intake, energy expenditure, and neuroendocrine function. Obese rodent and humans are mostly associated with high circulating leptin levels. These findings have led to the conclusion that obese individuals are relatively insensitive to endogenous leptin termed 'leptin resistance'. The potential sites for leptin resistance include the blood-brain-barrier transport system and the leptin signaling mechanism in leptin-responsive neurons in the hypothalamus. In this review, we describe leptin, leptin receptor, and potential hypothesis of leptin resistance..
405. Mitsuyo Shintani, Yoshihiro Ogawa, Ken Ebihara, Megumi Aizawa-Abe, Fumiko Miyanaga, Kazuhiko Takaya, Tatsuya Hayashi, Gen Inoue, Kiminori Hosoda, Masayasu Kojima, Kenji Kangawa, Kazuwa Nakao, Rapid publication ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway, Diabetes, 10.2337/diabetes.50.2.227, 50, 2, 227-232, 2001.01, Ghrelin, an endogenous ligand for growth hormone secretagogue (GHS) receptor originally isolated from the stomach, occurs in the hypothalamic arcuate nucleus and may play a role in energy homeostasis. Synthetic GHSs have activated the hypothalamic arcuate neurons containing neuropeptide Y (NPY), suggesting the involvement of NPY in some of ghrelin actions. This study was designed to elucidate the role of ghrelin in the regulation of food intake. A single intracerebroventricular (ICV) injection of ghrelin (5-5,000 ng/rat) caused a significant and dose-related increase in cumulative food intake in rats. Ghrelin (500 ng/rat) was also effective in growth hormone-deficient spontaneous dwarf rats. Hypothalamic NPY mRNA expression was increased in rats that received a single ICV injection of ghrelin (500 ng/rat)( ∼ 6 160% of that in vehicle-treated groups, P < 0.05). The ghrelin's orexigenic effect was abolished dose-dependently by ICV co-injection of NPY Y1 receptor antagonist (10-30 μg/rat). The leptin-induced inhibition of food intake was reversed by ICV co-injection of ghrelin in a dose-dependent manner (5-500 ng/rat). Leptin reduced hypothalamic NPY mRNA expression by 35% (P < 0.05), which was abolished by ICV co-injection of ghrelin (500 ng/rat). This study provides evidence that ghrelin is an orexigenic peptide that antagonizes leptin action through the activation of hypothalamic NPY/Y1 receptor pathway..
406. Shin Yonemitsu, Haruo Nishimura, Mitsuyo Shintani, Ryou Inoue, Yuji Yamamoto, Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Gen Inoue, Tatsuya Hayashi, Kazuwa Nakao, Troglitazone induces GLUT4 translocation in L6 myotubes, Diabetes, 10.2337/diabetes.50.5.1093, 50, 5, 1093-1101, 2001.01, A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10-5 mol/l) caused a substantial increase in the 2-deoxy-[3H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 μmol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 × 10-6 mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-λ, PKC-β2, and PKC-ξ; or 5′ AMP-activated protein kinase activity. α-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle..
407. C. Son, K. Hosoda, J. Matsuda, J. Fujikura, S. Yonemitsu, H. Iwakura, H. Masuzaki, Y. Ogawa, T. Hayashi, H. Itoh, H. Nishimura, G. Inoue, Y. Yoshimasa, Y. Yamori, K. Nakao, Up-regulation of uncoupling protein 3 gene expression by fatty acids and agonists for PPARs in L6 myotubes, Endocrinology, 10.1210/endo.142.10.8446, 142, 10, 4189-4194, 2001.01, Uncoupling protein 3 (UCP3), which uncouples electron transport from ATP synthesis, is expressed at high levels in the skeletal muscle, an important organ in glucose and lipid metabolism. Because several reports proposed that fatty acids induced UCP3 gene expression in skeletal muscle in vivo, in the present study we examined the regulation of UCP3 gene expression by various fatty acids using L6 myotubes. UCP3 gene expression was increased in L6 myotubes by various fatty acids or by α-bromopalmitate, a nonmetabolized derivative of palmitic acid. Because fatty acids are also known as agonists for PPARs, we examined the involvement of PPARs in the regulation of the UCP3 gene expression. L-165041, a PPARδ agonist, increased UCP3 gene expression in L6 myotubes, whereas neither Wy 14,643, a PPARα agonist, nor Pioglitazone, a PPARγ agonist, increased it. Therefore, we conclude that UCP3 gene expression is increased by the activation of PPARδ in L6 myotubes and postulate that PPARδ mediates at least some part of the increased UCP3 gene expression by fatty acids in skeletal muscle in vivo..
408. Shinichi Kato, Yoshihiro Ogawa, Akiko Tanaka, Tomonori Kunikata, Koji Takeuchi, Delayed healing of gastric ulcers in adjuvant arthritis rats
Role of acid secretion and basic fibroblast growth factor, Digestion, 10.1159/000051886, 63, 3, 171-179, 2001, Background/Aim: We examined the healing process of chronic gastric ulcers in adjuvant-induced arthritic rats and investigated the mechanism for delayed ulcer healing in arthritic rats, in relation to acid secretion and basic fibroblast growth factor (bFGF). Methods: Arthritis was induced in male dark Agouti rats by a single injection of Freund's complete adjuvant (FCA), while gastric ulcers were induced by thermal cauterization (70°C for 30 s) 7 days after FCA injection. Results: Injection of FCA induced severe arthritis in all animals with a marked acid hypersecretion. The healing of gastric ulcers was significantly delayed in arthritic rats as compared with normal rats. Daily administration of indomethacin delayed ulcer healing in both normal and arthritic rats, but this effect was more pronounced in the latter. In contrast, the healing of gastric ulcers was significantly promoted in both normal and arthritic rats by omeprazole at a dose that inhibited acid secretion completely. The delayed healing of gastric ulcers was not influenced by twice daily administration of NG-nitro-L-arginine methyl ester, aminoguanidine or FR167653 (IL-1/TNF-α synthesis inhibitor), but was significantly accelerated by CS-23 (recombinant human bFGF) in a dose-dependent manner, without effect on the acid secretion. The expression of bFGF was markedly increased after ulceration, but this response was decreased in arthritic rats. Conclusion: The healing of gastric ulcers was delayed in arthritic rats, and this mechanism may be partly attributable to both acid hypersecretion and less expression of bFGF..
409. Shinichi Kato, Akiko Tanaka, Yoshihiro Ogawa, Kenji Kanatsu, Koichi Seto, Tomoyuki Yoneda, Koji Takeuchi, Effect of polaprezinc on impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats - Role of insulin-like growth factors (IGF)-1, Medical Science Monitor, 7, 1, 20-25, 2001, Polaprezinc, N-(3-aminopropionyl)-L-histidinatozinc, has been shown to stimulate the production of insulin-like growth factor-1 (IGF-1) in mesenchymal cells, the polypeptide playing a role in the gastric epithelial wound repair. The present study was performed to examine the effect of polaprezinc on the impaired healing of chronic gastric ulcers in adjuvant-induced arthritic rats, in relation to IGF-1. Arthritis was induced in male Dark Agouti (DA) rats by a single injection of Freund's complete adjuvant (FCA), and the gastric ulcers were induced by thermal cauterization (70°C for 30 sec) 7 days after FCA injection. Omeprazole (30 mg/kg) was administered p.o. once daily, while recombinant human IGF-1 (rhIGF-1) (30 μg/kg, s.c.) or polaprezinc (3-10 mg/kg, p.o.) was administered twice daily, starting from 3 days after ulceration for 14 days. The healing of gastric ulcers was significantly delayed in arthritic rats as compared to normal rats on day 10 and 17 following ulceration. The expression of IGF-1 mRNA was markedly increased in the ulcerated mucosa, but this response was apparently attenuated in arthritic rats. Repeated administration of polaprezinc accelerated the healing of gastric ulcers in both normal and arthritic rats, in a dose-dependent manner, and this effect was more pronounced in arthritic rats. Likewise, treatment with omeprazole also significantly promoted the healing of gastric ulcers in both normal and arthritic rats. On the other hand, rhIGF-1 significantly promoted the gastric ulcer healing in arthritic rats Without any effect on that in normal rats. These results suggest that the impaired healing of chronic gastric ulcers in arthritic rats is, at least partly, accounted for by less expression of IGF-1, and the polaprezinc improves the delayed healing of gastric ulcers in arthritic rats, probably through an increase in IGF-1 production..
410. Taeko Iino, Koichi Nakahara, Wataru Miki, Yoshinobu Kiso, Yoshihiro Ogawa, Shinichi Kato, Koji Takeuchi, Less damaging effect of whisky in rat stomachs in comparison with pure ethanol, Digestion, 10.1159/000048864, 64, 4, 214-221, 2001, Background/Aim: Ellagic acid (EA), one of the polyphenols that are abundantly contained in whisky as a nonalcoholic component, has antioxidant and anti-inflammatory activities. In the present study, we compared the action of whisky and pure ethanol on the rat gastric mucosa, and examined the role of EA in the less-damaging effect of whisky in the stomach. Methods: Under urethane anesthesia, a rat stomach was mounted in an ex vivo chamber, perfused with saline, and the transmucosal potential difference (PD) was measured before and after exposure to whisky (Yamazaki, Suntory) and ethanol (43%). In a separate study, the animals were given whisky or ethanol (1 ml, 43%) p.o. under unanesthetized conditions, killed 1 h later, and the gastric mucosa was examined for hemorrhagic lesions. Results: Both whisky and ethanol caused a PD reduction, resulting in damage in the stomach, but these responses were less marked in the case of whisky. Although the reduced PD recovered gradually after removal of ethanol, this process was significantly expedited by co-application of EA (80 μg/ml), the recovery rate being much the same as that observed after exposure to whisky. The less-damaging effect of whisky was confirmed in unanesthetized rats after p.o. administration of these agents. In addition, EA (1-30 mg/kg), administered p.o. together with absolute ethanol (99.9%), reduced the severity of gastric lesions induced by ethanol, in a dose-dependent manner, and the effect at 30 mg/kg was equivalent to that obtained by the whisky component containing several low- and high-molecular-weight polyphenols. EA had a scavenging action against both oxygen and hydroxyl radicals in vitro, the effect being equivalent to that of catechol or α-tocopherol. Conclusion: These results suggest that whisky is less irritating to the gastric mucosa, as compared with pure ethanol, and this property of whisky may be explained by EA, one of polyphenols contained in whisky, and its radical scavenging action..
411. T. Suganami, M. Mukoyama, A. Sugawara, K. Mori, T. Nagae, M. Kasahara, K. Yahata, H. Makino, Y. Fujinaga, Yoshihiro Ogawa, I. Tanaka, K. Nakao, Overexpression of brain natriuretic peptide in mice ameliorates immune-mediated renal injury, Journal of the American Society of Nephrology, 12, 12, 2652-2663, 2001, One of major causes of end-stage renal disease is glomerulonephritis, the treatment of which remains difficult clinically. It has already been shown that transgenic mice that overexpress brain natriuretic peptide (BNP), with a potent vasorelaxing and natriuretic property, have ameliorated glomerular injury after subtotal nephrectomy. However, the role of natriuretic peptides in immune-mediated renal injury still remains unknown. Therefore, the effects of chronic excess of BNP on anti-glomerular basement membrane nephritis induced in BNP-transgenic mice (BNP-Tg) were investigated and the mechanisms how natriuretic peptides act on mesangial cells in vitro were explored. After induction of nephritis, severe albuminuria (∼21-fold above baseline), tissue damage, including mesangial expansion and cell proliferation, and functional deterioration developed in nontransgenic littermates. In contrast, BNP-Tg exhibited much milder albuminuria (approximately fourfold above baseline), observed only at the initial phase, and with markedly ameliorated histologic and functional changes. Up-regulation of transforming growth factor-β (TGF-β and monocyte chemoattractant protein-1 (MCP-1), as well as increased phosphorylation of extracellular signal-regulated kinase (ERK), were also significantly inhibited in the kidney of BNP-Tg. In cultured mesangial cells, natriuretic peptides counteracted the effects of angiotensin II with regard to ERK phosphorylation and fibrotic action. Because angiotensin II has been shown to play a pivotal role in the progression of nephritis through induction of TGF-β and MCP-1 that may be ERK-dependent, the protective effects of BNP are likely to be exerted, at least partly, by antagonizing the renin-angiotensin system locally. The present study opens a possibility of a novel therapeutic potential of natriuretic peptides for treating immune-mediated renal injury..
412. Hiroyuki Mizoguchi, Yoshihiro Ogawa, Kenji Kanatsu, Akiko Tanaka, Shinichi Kato, Koji Takeuchi, Protective effect of rebamipide on indomethacin-induced intestinal damage in rats, Journal of Gastroenterology and Hepatology (Australia), 10.1046/j.1440-1746.2001.02592.x, 16, 10, 1112-1119, 2001, Background and Aim: We evaluated the effect of rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl] propionic acid), a novel anti-ulcer drug, on indomethacin-induced small intestinal lesions in rats. Methods: The animals were administered indomethacin (10 mg/kg, s.c.), and they were killed 24 h later. Rebamipide (30-300 mg/kg) was administered p.o. twice, 30 min before, and 6 h after indomethacin. Results: Indomethacin caused hemorrhagic lesions in the rat small intestine, accompanied by an increase in enterobacterial translocation, inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) activities, as well as thiobarbituric acid (TBA) reactants, and these changes were significantly prevented by the supplementation with 16,16-dimethyl prostaglandin E2 (dmPGE2; 10 μg/kg, i.v.) or the pretreatment of animals with the antibiotic ampicillin. Treatment of the animals with rebamipide dose-dependently prevented the development of intestinal lesions, and this effect was mimicked by i.v. administration of superoxide dismutase (SOD: 3000 U/kg) + catalase (CAT: 5000 U/kg). The protection by rebamipide was accompanied by a significant suppression of the increase in both MPO and iNOS activities, and a complete inhibition of the increase in TBA reactants, while SOD + CAT significantly inhibited the increase of MPO activity and TBA reactants, but not iNOS activity. The bacterial translocation following indomethacin was also significantly decreased by either rebamipide or SOD + CAT. Conclusion: These results confirmed the importance of enterobacteria and iNOS/NO in the pathogenesis of indomethacin-induced small intestinal lesions, and suggested that rebamipide prevents the development of these lesions, probably by its radical scavenging action..
413. Koji Takeuchi, Shinichi Kato, Yoshihiro Ogawa, Kenji Kanatsu, Masakazu Umeda, Role of endogenous prostacyclin in gastric ulcerogenic and healing responses - A study using IP-receptor knockout mice, Journal of Physiology Paris, 10.1016/S0928-4257(01)00011-0, 95, 1-6, 75-80, 2001, Endogenous prostaglandins (PGs) play an important role in the cytoprotective and healing responses in the stomach, by altering various functions, i.e., an increase of the mucosal blood flow, yet the role of prostacyclin (PGI2) and its receptor (IP-receptor) in these responses remains unclarified. In the present study, we used IP-receptor knockout mice [IP (-/-)] and examined the importance of IP-receptors in gastric ulcerogenic, cytoprotective and healing responses in these animals. The studies included the ulcerogenic response to cold-restraint stress, the cytoprotective response to a mild irritant (20 mM taurocholate: TC) and capsaicin, and the healing response of chronic gastric ulcers induced by thermo-cauterization. We first checked the absence of IP-receptors by examining the effect of cicaprost (a PGI2 agonist, topical mucosal application) on gastric mucosal blood flow and found that this agent increased the mucosal blood flow in wild-type [WT (+/+)] mice but not in IP (+/-) mice. Cold-restraint stress (4 h) induced gastric lesions in both groups of mice, but the severity of damage was significantly greater in IP (-/-) mice. Prior p.o. administration of both TC and capsaicin exhibited a marked cytoprotection against HCl/ethanol-induced gastric damage in WT (+/+) mice, both responses being significantly mitigated in the presence of indomethacin. The adaptive cytoprotection induced by TC was similarly observed in IP (-/-) mice, while the capsaicin protection was totally attenuated in the animals lacking IP receptors. On the other hand, the healing of gastric ulcers was significantly delayed by daily administration of indomethacin in WT (+/+) mice. However, this process was not altered in IP (-/-) mice. These results suggest that endogenous PGI2 is involved in the gastric ulcerogenic response to stress, but not in the healing of pre-existing gastric ulcers. In addition, PGI2 and its receptors may play a crucial role in capsaicin-induced gastric protection but not in the adaptive cytoprotection-induced by mild irritants..
414. Hiroyuki Ariyasu, Kazuhiko Takaya, Tetsuya Tagami, Yoshihiro Ogawa, Kiminori Hosoda, Takashi Akamizu, Michio Suda, Toshikiyo Koh, Koshi Natsui, Shigetake Toyooka, Gotaro Shirakami, Takeshi Usui, Akira Shimatsu, Kentaro Doi, Hiroshi Hosoda, Masayasu Kojima, Kenji Kangawa, Kazuwa Nakao, Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans, Journal of Clinical Endocrinology and Metabolism, 10.1210/jcem.86.10.7885, 86, 10, 4753-4758, 2001, Ghrelin, an endogenous ligand for the GH secretagogue receptor, was isolated from rat stomach and is involved in a novel system for regulating GH release. Although previous studies in rodents suggest that ghrelin is also involved in energy homeostasis and that ghrelin secretion is influenced by feeding, little is known about plasma ghrelin in humans. To address this issue, we studied plasma ghrelin-like immunoreactivity levels and elucidated the source of circulating ghrelin and the effects of feeding state on plasma ghrelin-like immunoreactivity levels in humans. The plasma ghrelin-like immunoreactivity concentration in normal humans measured by a specific RIA was 166.0 ± 10.1 fmol/ml. Northern blot analysis of various human tissues identified ghrelin mRNA found most abundantly in the stomach and plasma ghrelin-like immunoreactivity levels in totally gastrectomized patients were reduced to 35% of those in normal controls. Plasma ghrelin-like immunoreactivity levels were increased by 31% after 12-h fasting and reduced by 22% immediately after habitual feeding. In patients with anorexia nervosa, plasma ghrelin-like immunoreactivity levels were markedly elevated compared with those in normal controls (401.2 ± 58.4 vs. 192.8 ± 19.4 fmol/ml) and were negatively correlated with body mass indexes. We conclude that the stomach is a major source of circulating ghrelin and that plasma ghrelin-like immunoreactivity levels reflect acute and chronic feeding states in humans..
415. Ken Ebihara, Yoshihiro Ogawa, Hiroaki Masuzaki, Mitsuyo Shintani, Fumiko Miyanaga, Megumi Aizawa-Abe, Tatsuya Hayashi, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Oksana Gavrilova, Marc L. Reitman, Kazuwa Nakao, Transgenic overexpression of leptin rescues insulin resistance and diabetes in a mouse model of lipoatrophic diabetes, Diabetes, 10.2337/diabetes.50.6.1440, 50, 6, 1440-1448, 2001, Lipoatrophic diabetes is caused by a deficiency of adipose tissue and is characterized by severe insulin resistance, hypoleptinemia, and hyperphagia. The A-ZIP/F-1 mouse (A-ZIPTg/+) is a model of severe lipoatrophic diabetes and is insulin resistant, hypoleptinemic, hyperphagic, and shows severe hepatic steatosis. We have also produced transgenic "skinny" mice that have hepatic overexpression of leptin (LepTg/+) and no adipocyte triglyceride stores, and are hypophagic and show increased insulin sensitivity. To explore the pathophysiological and therapeutic roles of leptin in lipoatrophic diabetes, we crossed LepTg/+ and A-ZIPTg/+ mice, producing doubly transgenic mice (LepTg/+:A-ZIPTg/+) virtually lacking adipose tissue but having greatly elevated leptin levels. The LepTg/+:A-ZIPTg/+ mice were hypophagic and showed improved hepatic steatosis. Glucose and insulin tolerance tests revealed increased insulin sensitivity, comparable to LepTg/+ mice. These effects were stable over at least 6 months of age. Pair-feeding the A-ZIPTg/+ mice to the amount of food consumed by LepTg/+:A-ZIPTg/+ mice did not improve their insulin resistance, diabetes, or hepatic steatosis, demonstrating that the beneficial effects of leptin were not due to the decreased food intake. Continuous leptin administration that elevates plasma leptin concentrations to those of LepTg/+:A-ZIPTg/+ mice also effectively improved hepatic steatosis and the disorder of glucose and lipid metabolism in A-ZIP/F-1 mice. These data demonstrate that leptin can improve the insulin resistance and diabetes of a mouse model of severe lipoatrophic diabetes, suggesting that leptin may be therapeutically useful in the long-term treatment of lipoatrophic diabetes..
416. Yoshihiro Ogawa, Kazuwa Nakao, Transgenic skinny mice overexpressing leptin, Seikagaku, 72, 7, 554-558, 2000.12.
417. Kiyoshi Mori, Kensei Yahata, Masashi Mukoyama, Takayoshi Suganami, Hisashi Makino, Tetsuya Nagae, Hiroaki Masuzaki, Yoshihiro Ogawa, Akira Sugawara, Yo ichi Nabeshima, Kazuwa Nakao, Disruption of klotho gene causes an abnormal energy homeostasis in mice, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2000.3864, 278, 3, 665-670, 2000.11, klotho mice, which genetically lack klotho gene expression, are characterized with various systemic phenotypes resembling human aging, and also with growth retardation. Here we show that klotho mice have a barely detectable amount of the white adipose tissue but their brown adipose tissue (BAT) is comparably preserved. Glucose tolerance and insulin sensitivity in klotho mice are increased compared to those in wild-type mice as revealed by intraperitoneal glucose and insulin tolerance tests. Uncoupling protein-1 gene expression of BAT and body temperature in klotho mice are lower than those in wild-type mice, suggesting that klotho mice have less energy expenditure than wild-type mice. Histological examination suggests that klotho mice possess less energy storage than wild-type mice with respect to glycogen in the liver and lipid in BAT. All these changes of parameters for energy homeostasis in klotho mice are very similar to those reported under food-restricted conditions. However, the amount of food intake is not different between klotho and wild-type mice when normalized for body weight. The present study elucidates the importance of klotho gene expression for the maintenance of normal energy homeostasis. (C) 2000 Academic Press..
418. Masato Kasahara, Masashi Mukoyama, Akira Sugawara, Hisashi Makino, Takayoshi Suganami, Yoshihiro Ogawa, Masayo Nakagawa, Kensei Yahata, Masahisa Goto, Rieko Ishibashi, Naohisa Tamura, Issei Tanaka, Kazuwa Nakao, Ameliorated glomerular injury in mice overexpressing brain natriuretic peptide with renal ablation, Journal of the American Society of Nephrology, 11, 9, 1691-1701, 2000.09, Brain natriuretic peptide (BNP) is a cardiac hormone produced by the ventricle, and its secretion is markedly increased in heart failure, hypertension, and renal failure. Transgenic mice that overexpress BNP in the liver (BNP-Tg) were recently generated, resulting in low BP. To elucidate the role of BNP in renal pathophysiology, the effect of chronic excess of BNP in transgenic mice on glomerular injury after subtotal nephrectomy induced by resection of the renal poles was examined. After nephrectomy, glomerular cross-sectional areas in control nontransgenic mice markedly increased as compared with those in sham-operated mice (+81 ± 7%), whereas there was only a modest increase in BNP-Tg (+10 ± 6%). Expansion of the mesangial area and increase in the intraglomerular cell number were also inhibited in BNP-Tg. Glomerular expressions of transforming growth factor-β and fibronectin were increased with hypertrophy and were significantly suppressed in BNP-Tg. Furthermore, increases in the urinary albumin excretion and BP were significantly ameliorated in BNP-Tg. Chronic hydralazine treatment in nephrectomized nontransgenic mice failed to inhibit glomerular hypertrophy. These findings indicate that the chronic excess of BNP in mice ameliorates glomerular hypertrophy and mesangial expansion after renal ablation. The results also suggest that the observed effects of natriuretic peptides under reduced renal mass are not due merely to systemic BP reduction and may be therapeutically applicable in various renal diseases..
419. Masafumi Nakayama, Hirofumi Yasue, Michihiro Yoshimura, Yukio Shimasaki, Hisao Ogawa, Kiyotaka Kugiyama, Yuji Mizuno, Eisaku Harada, Shota Nakamura, Teruhiko Ito, Yoshihiko Saito, Yoshihiro Miyamoto, Yoshihiro Ogawa, Kazuwa Nakao, T-786 → C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with myocardial infarction, especially without coronary organic stenosis, American Journal of Cardiology, 10.1016/S0002-9149(00)01041-9, 86, 6, 628-634, 2000.09, Recently, we discovered a T-786 → C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene that is associated with coronary spasm. The precise mechanism(s) of myocardial infarction (MI), especially without coronary organic stenosis, has not been elucidated, but it seems possible that coronary spasm plays a key role in the mechanism. In this study, we examined the frequency with which the T-786 → C mutation occurred in 359 patients with MI who were compared with 195 controls. In the MI group, the frequency of C/C, C/T, and T/T genotypes was 1%, 22%, and 77%, respectively. In the control group, the frequency of C/C, C/T, and T/T genotypes were 0%, 8%, and 92%, respectively. The frequency of the C allele was significantly higher in the MI group than in the control group (p < 0.001). In the MI group, 30 of 359 patients (8%) with MI had no stenosed vessels angiographically, 158 (44%) had 1 stenosed vessel, 80 (22%) had 2 stenosed vessels, and 91 (25%) exhibited 3 stenosed vessels. Total and low- density lipoprotein cholesterol levels and the incidence of diabetes mellitus increased as the number of stenosed vessels increased (p < 0.01, respectively). The frequency of the T-786 → C mutation was significantly higher in MI patients with no stenosed vessels (50%) than in those with stenosed vessels (p < 0.003). In conclusion, the T-786 → C mutation was strongly associated with MI, especially without coronary arterial stenosis, in Japanese patients. The association may be due to the impaired effects of nitric oxide in the cardiovascular system. (C) 2000 by Excerpta Medica, Inc..
420. Megumi Aizawa-Abe, Yoshihiro Ogawa, Hiroaki Masuzaki, Ken Ebihara, Noriko Satoh, Hidenori Iwai, Naoki Matsuoka, Tatsuya Hayashi, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Kazuwa Nakao, Pathophysiological role of leptin in obesity-related hypertension, Journal of Clinical Investigation, 10.1172/JCI8341, 105, 9, 1243-1252, 2000.05, To explore the pathophysiological role of leptin in obesity-related hypertension, we examined cardiovascular phenotypes of transgenic skinny mice whose elevated plasma leptin concentrations are comparable to those seen in obese subjects. We also studied genetically obese KKA(y) mice with hyperleptinemia, in which hypothalamic melanocortin system is antagonized by ectopic expression of the agouti protein. Systolic blood pressure (BP) and urinary catecholamine excretion are elevated in transgenic skinny mice relative to nontransgenic littermates. The BP elevation in transgenic skinny mice is abolished by α1-adrenergic, β-adrenergic, or ganglionic blockers at doses that do not affect BP in nontransgenic littermates. Central administration of an α-melanocyte-stimulating hormone antagonist causes a marked increase in cumulative food intake but no significant changes in BP. The obese KKA(y) mice develop BP elevation with increased urinary catecholamine excretion relative to control KK mice. After a 2-week caloric restriction, BP elevation is reversed in nontransgenic littermates with the A(y) allele, in parallel with a reduction in plasma leptin concentrations, but is sustained in transgenic mice overexpressing leptin with the A(y) allele, which remain hyperleptinemic. This study demonstrates BP elevation in transgenic skinny mice and obese KKA(y) mice that are both hyperleptinemic, thereby suggesting the pathophysiological role of leptin in some forms of obesity-related hypertension..
421. Naohisa Tamura, Yoshihiro Ogawa, Hideki Chusho, Kenji Nakamura, Kazuki Nakao, Michio Suda, Masato Kasahara, Ryuju Hashimoto, Goro Katsuura, Masashi Mukoyama, Hiroshi Itoh, Yoshihiko Saito, Issei Tanaka, Hiroki Otani, Motoya Katsuki, Kazuwa Nakao, Cardiac fibrosis in mice lacking brain natriuretic peptide, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.070371497, 97, 8, 4239-4244, 2000.04, Cardiac fibrosis, defined as a proliferation of interstitial fibroblasts and biosynthesis of extracellular matrix components in the ventricles of the heart, is a consequence of remodeling processes initiated by pathologic events associated with a variety of cardiovascular disorders, which leads to abnormal myocardial stiffness and, ultimately, ventricular dysfunction. Brain natriuretic peptide (BNP) is a cardiac hormone produced primarily by ventricular myocytes, and its plasma concentrations are markedly elevated in patients with congestive heart failure and acute myocardial infarction. However, its precise functional significance has been undefined. In this paper, we report the generation of mice with targeted disruption of BNP (Nppb(-/-) mice). We observed multifocal fibrotic lesions in the ventricles from Nppb(-/-) mice. No signs of systemic hypertension and ventricular hypertrophy are noted in Nppb(-/-) mice. In response to ventricular pressure overload, focal fibrotic lesions are increased in size and number in Nppb(-/- ) mice, whereas no focal fibrotic changes are found in wild-type littermates (Nppb(+/+) mice). This study establishes BNP as a cardiomyocyte-derived antifibrotic factor in vivo and provides evidence for its role as a local regulator of ventricular remodeling..
422. Shigeo Yura, Yoshihiro Ogawa, Norimasa Sagawa, Hiroaki Masuzaki, Hiroaki Itoh, Ken Ebihara, Megumi Aizawa-Abe, Shingo Fujii, Kazuwa Nakao, Accelerated puberty and late-onset hypothalamic hypogonadism in female transgenic skinny mice overexpressing leptin, Journal of Clinical Investigation, 10.1172/JCI8353, 105, 6, 749-755, 2000.03, Excess or loss of body fat can be associated with infertility, suggesting that adequate fat mass is essential for proper reproductive function. Leptin is an adipocyte-derived hormone that is involved in the regulation of food intake and energy expenditure, and its synthesis and secretion are markedly increased in obesity. Short-term administration of leptin accelerates the onset of puberty in normal mice and corrects the sterility of leptin-deficient ob/ob mice. These findings suggest a role for leptin as an endocrine signal between fat depots and the reproductive axis, but the effect of hyperleptinemia on the initiation and maintenance of reproductive function has not been elucidated. To address this issue, we examined the reproductive phenotypes of female transgenic skinny mice with elevated plasma leptin concentrations comparable to those in obese subjects. With no apparent adipose tissue, female transgenic skinny mice exhibit accelerated puberty and intact fertility at younger ages followed by successful delivery of healthy pups. However, at older ages, they develop hypothalamic hypogonadism characterized by prolonged menstrual cycles, atrophic ovary, reduced hypothalamic gonadotropin releasing hormone contents, and poor pituitary luteinizing hormone secretion. This study has demonstrated for the first time to our knowledge that accelerated puberty and late-onset hypothalamic hypogonadism are associated with chronic hyperleptinemia, thereby leading to a better understanding of the pathophysiological and therapeutic implication of leptin..
423. Yuji Yamamoto, Yasunao Yoshimasa, Makoto Koh, Junko Suga, Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Haruo Nishimura, Yoshihiko Watanabe, Gen Inoue, Kazuwa Nakao, Constitutively active mitogen-activated protein kinase kinase increases GLUT1 expression and recruits both GLUT1 and GLUT4 at the cell surface in 3T3-L1 adipocytes, Diabetes, 10.2337/diabetes.49.3.332, 49, 3, 332-339, 2000.03, To address a role of mitogen-activated protein kinase (MAPK) in the regulation of glucose transport, we made a constitutively active mutant of MAPK kinase (MAPKK) and introduced it into 3T3-L1 preadipocytes by using a retrovirus-mediated transfection procedure. The deletion of 20 amino acids (those between and including 32 and 51) in the amino terminal region of Xenopus MAPKK and the replacement of serine residues on the 218 and 222 positions by glutamic acid (dSESE-MAPKK) let Xenopus MAPKK constitutively active. The isolated cell clones differently expressing dSESE-MAPKK (clone 219 higher expression, clone 233 lower expression) efficiently differentiated to adipocytes by a standard differentiation cocktail. Accordingly, the increased expression of dSESE-MAPKK protein during differentiation resulted in the increased basal MAPK activity in clone 219 adipocytes and, to a lesser extent, in clone 233 adipocytes. In contrast to clone 233 and parental adipocytes, basal 2-deoxyglucose uptake was enhanced fourfold in clone 219 adipocytes, in accordance with increased expression of GLUT1 mRNA and protein. Whereas GLUT4 mRNA was similarly expressed in all of the adipocytes, GLUT4 protein appeared to decrease in clone 219 adipocytes. More importantly, subcellular fractionation studies showed that the localization of both GLUT1 and GLUT4 in the plasma membranes (PMs) was markedly increased in the basal state in clone 219 adipocytes compared with that in clone 233 and parental adipocytes, in which both glucose transporters were preferentially located in intracellular compartments. Consequently, insulin-induced translocation of GLUT1 was abolished in clone 219 adipocytes, although the remaining intracellular GLUT4 was still responsive to insulin stimulation, which led to the movement to the PM. As combined effects on the situation of GLUT1 and GLUT4, the foldness of insulin stimulation of glucose transport based on the basal activity was reduced in cells expressing constitutively active MAPKK. These results imply that chronic activation of MAPK could be one of the mechanisms for insulin resistance..
424. Mitsuyo Shintani, Haruo Nishimura, Shin Yonemitsu, Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Kazuwa Nakao, Downregulation of leptin by free fatty acids in rat adipocytes
Effects of triacsin C, palmitate, and 2-bromopalmitate, Metabolism: Clinical and Experimental, 10.1016/S0026-0495(00)90154-9, 49, 3, 326-330, 2000.01, Free fatty acid (FFA) has been reported to decrease leptin mRNA levels in 3T3-L1 adipocytes. When using this cell line, it is difficult to determine the protein levels because a very small amount of leptin is secreted into the medium. The effect of FFA on leptin secretion from adipocytes has not yet been determined. In addition, in vivo studies have failed to demonstrate a FFA-induced decrease in plasma leptin levels. To clarify the effect of FFA on leptin production, we investigated the leptin protein level in the medium and the mRNA level in primary cultured rat adipocytes treated with triacsin C, which is a potent inhibitor of acyl-coenzyme A (CoA) synthetase, palmitate, and 2-bromopalmitate. Triacsin C (0 to 5 x 10-5 mol/L) decreased leptin concentrations in the culture medium in a close-dependent manner. Leptin mRNA levels were decreased to 10% of the control in the presence of triacsin C. The concentration of triacsin C needed to suppress leptin production was similar to the K(i) value (≃ 10-5 mol/L) for inhibition of acyl-CoA synthetase. Both palmitate and 2-bromopalmitate decreased leptin concentrations but did not affect the triacsin C-induced decrease in leptin additively. In conclusion, both protein and mRNA levels of leptin were decreased by triacsin C and FFA in primary cultured rat adipocytes. Our findings suggest that FFA is involved in the regulation of leptin production in adipocytes. (C) 2000 by W.B. Saunders Company..
425. M. Yoshimura, H. Yasue, M. Nakayama, Y. Shimasaki, H. Ogawa, K. Kugiyama, Y. Saito, Y. Miyamoto, Y. Ogawa, T. Kaneshige, H. Hiramatsu, T. Yoshioka, S. Kamitani, H. Teraoka, K. Nakao, Genetic risk factors for coronary artery spasm
Significance of endothelial nitric oxide synthase gene T-786→C and missense Glu298Asp variants, Journal of Investigative Medicine, 48, 5, 367-374, 2000.01, Background: We recently identified two endothelial nitric oxide synthase (eNOS) gene polymorphisms, Glu298Asp and T-786→C, which are independently associated with coronary spasm. eNOS gene intron 4b/a polymorphism is also reported to be involved in smoking-dependent coronary artery disease. The genetic linkage among these polymorphisms remains unknown. Also, it is unclear which variant is most responsible for coronary spasm. In the present study, we first examined the genetic linkage among these three variants. Next, we studied the risk factors of coronary spasm by using all significant genetic and conventional risk factors in a large-scale study. Methods: The genotype and allele frequencies for the T-786→C, intron 4b/a, and Glu298Asp variants were assessed in 423 randomly selected DNA samples to examine their genetic linkages. The relative capacities of all risk factors to predict coronary spasm were then analyzed using multiple logistic regression in 201 patients with coronary spasm and 345 volunteers. Results: Comparison of allele frequencies revealed that the eNOS intron 4a allele was significantly linked to the T-786→C mutation (P<0.00001), whereas there was not a linkage between the intron 4a allele and the Glu298Asp variant (P=0.1437) or between the Glu298Asp variant and the T-786→C mutation (P=0.1996). Multiple logistic regression revealed that the most predictive independent risk factor for coronary spasm was the T-786→C mutation (P<0.001), followed by cigarette smoking (P<0.001), hypertension (P=0.004), and the Glu298Asp variant (P=0.028). Conclusions: We found that the T-786→C mutation and the intron 4a allele are in linkage disequilibrium. We previously showed that the T-786→C mutation reduced eNOS gene promoter activity. In that context, our results strongly suggest that the T-786→C mutation underlies the functional characteristics of the intron 4a allele. Further, multiple logistic regression analysis revealed that the T-786→C mutation is the most predictive risk factor for coronary spasm, followed by cigarette smoking. Given that those effects are potentially additive, patients carrying the eNOS gene variants should be strongly cautioned against smoking..
426. Kazuhiko Takaya, Hiroyuki Ariyasu, Naotetsu Kanamoto, Hiroshi Iwakura, Akihiro Yoshimoto, Masaki Harada, Kiyoshi Mori, Yasato Komatsu, Takeshi Usui, Akira Shimatsu, Yoshihiro Ogawa, Kiminori Hosoda, Takashi Akamizu, Masayasu Kojima, Kenji Kangawa, Kazuwa Nakao, Ghrelin strongly stimulates growth hormone (GH) release in humans, Journal of Clinical Endocrinology and Metabolism, 10.1210/jcem.85.12.7167, 85, 12, 4908-4911, 2000.01, Ghrelin is a recently identified endogenous ligand for the GH secretagogue receptor and is involved in a novel system for regulating GH release. However, little is known about its GH-releasing activity and other endocrine effects in humans. To address this issue, we studied the GH, ACTH, cortisol, PRL, LH, FSH, and TSH responses to synthetic human ghrelin. In four normal male adults (28-37 yr), iv ghrelin administration released GH in a dose-dependent manner and 0.2, 1.0, and 5.0 μg/kg ghrelin produced 43.3 ± 6.0, 81.5 ± 12.7, and 107.0 ± 10.7 ng/mL of the GH peak values at 30 min, respectively. ACTH, cortisol, and PRL levels were also elevated after ghrelin injection, while the lowest dose (0.2 μg/kg) resulted in only minimum peak values of these hormones (22.8 ± 3.0 pg/mL, 9.4 ± 1.9 μg/dL, and 4.6 ± 0.6 ng/mL, respectively). There were no significant changes in LH, FSH, or TSH levels. This is the first study showing evidence that ghrelin strongly stimulates GH release in humans..
427. M. Shintani, Y. Ogawa, K. Nakao, Insulin resistance, role of leptin and leptin receptor, Nippon rinsho. Japanese journal of clinical medicine, 58, 2, 327-332, 2000.01, Leptin, the obese gene product, is an adipocyte-derived satiety factor which is involved in the regulation of food intake and energy expenditure. Obesity often accompanies insulin resistance and high levels of leptin. In in vitro studies, leptin has been reported to increase fatty acid oxidation and decrease fatty acid synthesis in adipocytes and hepatocytes. The direct effects of leptin on glucose metabolism and insulin signaling have not been clarified yet. In in vivo studies, however, leptin has been reported to improve insulin sensitivity and glucose metabolism in normal and obese rodents acting mainly through hypothalamus. Moreover leptin has been reported to have antidiabetic effects in insulin-deficient diabetes rats and lipoatrophic diabetes mice. It is suggested that leptin modulates insulin sensitivity and glucose disposal and that leptin may have a pathophysiological and therapeutic implications in diabetes..
428. Kensei Yahata, Kiyoshi Mori, Hiroshi Arai, Susumu Koide, Yoshihiro Ogawa, Masashi Mukoyama, Akira Sugawara, Shoichi Ozaki, Issei Tanaka, Yo Ichi Nabeshima, Kazuwa Nakao, Molecular cloning and expression of a novel klotho-related protein, Journal of Molecular Medicine, 10.1007/s001090000131, 78, 7, 389-394, 2000.01, Klotho protein is a novel β-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one β-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic β-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein..
429. H. Masuzaki, Y. Ogawa, K. Ebihara, K. Nakao, Molecular medicine in leptin biology, Nippon rinsho. Japanese journal of clinical medicine, 58 Suppl 1, 157-162, 2000.01.
430. M. Aizawa-Abe, Y. Ogawa, H. Masuzaki, K. Nakao, Ob gene, Nippon rinsho. Japanese journal of clinical medicine, 58 Suppl 1, 551-555, 2000.01.
431. Tae Hwa Chun, Hiroshi Itoh, Takatoshi Saito, Ken Ichi Yamahara, Kentaro Doi, Yuko Mori, Yoshihiro Ogawa, Jun Yamashita, Tokuji Tanaka, Mayumi Inoue, Ken Masatsugu, Naoki Sawada, Yasutomo Fukunaga, Kazuwa Nakao, Oxidative stress augments secretion of endothelium-derived relaxing peptides, C-type natriuretic peptide and adrenomedullin, Journal of hypertension, 10.1097/00004872-200018050-00010, 18, 5, 575-580, 2000.01, Objective. Excess oxidative stress is one of the major metabolic abnormalities on vascular walls in hypertension and atherosclerosis. In order to further elucidate the endothelial function under oxidative stress, the effect of hydrogen peroxide (H2O2) on expression of two novel endothelium-derived vasorelaxing peptides, C-type natriuretic peptide (CNP) and adrenomedullin (AM) from bovine carotid artery endothelial cells (BCAECs) was examined. Methods. BCAECs were treated with H2O2 (0.1-1.0 mmol/l) and/or an antioxidant, N-acetylcysteine (NAC) (5-10 mmol/l), and incubated for 48 h. The concentrations of CNP and AM were measured with the specific radioimmunassays that we originally developed. CNP and AM mRNA expressions were also examined by reverse transcription-polymerase chain reaction (RT-PCR). Results. Treatment of BCAECs with 0.5 and 1 mmol/l H2O2 induced 9- and 10-fold increases of CNP concentration in the media. Addition of 10 mmol/l NAC significantly suppressed the effect of H2O2 by 52%. RT-PCR analysis showed that CNP mRNA expression in BCAECs was also rapidly augmented within 1 h with H2O2 (1 mmol/l) treatment, and reached a peak at 3 h to show a 10-fold increase. AM secretion from BCAECs also increased to two-fold with exposure to 0.5 mmol/l H2O2, accompanied with the augmented level of AM mRNA. NAC 10 mmol/l completely suppressed the effect of H2O2 on AM secretion. Conclusions. In this study, it has been demonstrated that H2O2 augments endothelial secretion of the two endothelium-derived relaxing peptides, CNP and AM. Our findings suggest the increased secretion of CNP and AM from endothelium under oxidative stress may function to compensate the impaired nitric oxide-dependent vasorelaxation in hypertension and atherosclerosis. (C) Lippincott Williams and Wilkins..
432. H. Masuzaki, Y. Ogawa, K. Nakao, The role of leptin in human obesity and related diseases--recent progress and future directions, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 45, 6 Suppl, 1125-1132, 2000.01.
433. H. Chusho, Y. Ogawa, N. Tamura, M. Suda, A. Yasoda, T. Miyazawa, I. Kishimoto, Y. Komatsu, H. Itoh, K. Tanaka, Y. Saito, D. L. Garbers, K. Nakao, Genetic models reveal that brain natriuretic peptide can signal through different tissue-specific receptor-mediated pathways, Endocrinology, 10.1210/endo.141.10.7692, 141, 10, 3807-3813, 2000, Brain natriuretic peptide (BNP), a hormone produced primarily by the cardiac ventricle, is thought to be involved in a variety of homeostatic processes through its cognate receptor, guanylyl cyclase A (GC-A). We previously created transgenic mice overexpressing BNP under the control of the liver-specific human serum amyloid P component promoter (BNP-transgenic mice) and demonstrated that they exhibit reduced blood pressure and cardiac weight accompanied by an elevation of plasma cGMP concentrations and marked skeletal overgrowth through the activation of endochondral ossification. To address whether BNP exerts its biological effects solely through GC-A, we produced BNP-transgenic mice lacking GC-A (BNP-Tg/GC-A(-/-) mice) and examined their cardiovascular and skeletal phenotypes. The GC-A(-/-) mice are hypertensive with cardiac hypertrophy relative to wild-type littermates, which is not alleviated by overexpression of BNP in BNP-Tg/GC-A(-/-) mice. The BNP-Tg/GC-A(-/-) mice, however, continue to exhibit marked longitudinal growth of vertebrae and long bones comparably to BNP-Tg mice. This study provides genetic evidence that BNP reduces blood pressure and cardiac weight through GC-A, whereas it dramatically alters endochondral ossification in the absence of this receptor. Therefore, the BNP-Tg/GC-A(-/-) mice provide the first experimental model demonstrating that this natriuretic peptide can signal in a tissue-specific manner through a receptor other than GC-A..
434. M. Kotani, I. Tanaka, Y. Ogawa, T. Suganami, T. Matsumoto, S. Muro, Y. Yamamoto, A. Sugawara, Y. Yoshimasa, N. Sagawa, S. Narumiya, K. Nakao, Multiple signal transduction pathways through two prostaglandin E receptor EP3 subtype isoforms expressed in human uterus, Journal of Clinical Endocrinology and Metabolism, 10.1210/jcem.85.11.6989, 85, 11, 4315-4322, 2000, PGE2 is known to induce uterine contraction by increasing intra-cellular Ca2+. In the present study, to investigate other functions of PGE2 in human uterus, two EP3 isoforms were isolated by the RT-PCR method using human uterus polyadenylated ribonucleic acid (RNA). These EP3 isoforms, named EP3-V and EP3-VI, are composed of 402 and 393 amino acid residues, respectively, which are unique compared with EP3 isoforms of other species. Their N-terminal 359 amino acid residues are identical to those of previously reported human EP3 isoforms, whereas the two isoforms contained a novel amino acid sequence in their C-terminal tails. The dissociation constant values of EP3-V and EP3-VI for PGE2 were 3.9 and 1.4 nmol/L, respectively, which were consistent with those of previously reported EPa isoforms. Signaling experiments revealed that M&B28767, an EP3 agonist, not only inhibited forskolin-induced cAMP concentrations, but also activated mitogen-activated protein kinase in Chinese hamster ovary cells stably expressing EP3-V and EP3-VI. These responses were abolished by treatment with pertussis toxin. In addition, M&B28767 increased cAMP concentrations in EP3-VI-expressing cells, whereas it did not in EP3-V-expressing cells. M&B28767 did not stimulate phosphoinositide turnover in EP3-V- or EP3-VI-expressing cells. EP3-V and EP3-VI messenger RNAs (mRNAs) were detected abundantly in human uterus, whereas weak, but substantial, bands were detected in the lung and kidney in RT-PCR specific for each mRNA. In situ hybridization revealed EP3-V and EP3-VI mRNAs in the human myometrium, but not in the endometrium. The present study suggests that EP3-V and EP3-VI are possibly involved in the proliferation of cells in human myometrium..
435. Ken Ebihara, Yoshihiro Ogawa, Goro Katsuura, Yoshito Numata, Hiroaki Masuzaki, Noriko Satoh, Mikio Tamaki, Takeshi Yoshioka, Minoru Hayase, Naoki Matsuoka, Megumi Aizawa-Abe, Yasunao Yoshimasa, Kazuwa Nakao, Involvement of agouti-related protein, an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action, Diabetes, 10.2337/diabetes.48.10.2028, 48, 10, 2028-2033, 1999.10, To understand the role of agouti-related protein (AGRP), an endogenous antagonist of hypothalamic melanocortin receptor, in leptin action, we produced a full-length recombinant AGRP and examined its effect on the satiety effect of leptin. We also studied leptin's regulation of hypothalamic AGRP mRNA expression. A single intracerebroventricular (ICV) injection of AGRP significantly increased cumulative food intake and body weight in a dose-dependent manner in rats. The leptin-induced inhibition of food intake and body weight was reversed by co-injection of AGRP in a dose-dependent manner. Hypothalamic AGRP mRNA expression was upregulated in leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice and downregulated in lethal yellow agouti mice (KKA(y) mice) with hyperleptinemia. A single ICV injection of leptin reversed the increased AGRP mRNA levels in ob/ob mice but not in db/db mice. In control mice and KKA(y) mice, AGRP mRNA expression was upregulated during fasting, when plasma leptin concentrations were decreased. No significant increase in AGRP mRNA expression was noted during fasting in control mice and KKA(y) mice treated with leptin. This study provides the first direct evidence that AGRP is a negative regulator of leptin action, and leptin downregulates hypothalamic AGRP production. Because leptin is shown to increase hypothalamic α-melanocyte stimulating hormone (e-MSH) production, our data suggest that its action via the hypothalamic melanocortin system is determined by the balance between the levels of its agonist and antagonist, α-MSH and AGRP..
436. Yoshihiro Ogawa, Hiroaki Masuzaki, Kiminori Hosoda, Megumi Aizawa-Abe, Junko Suga, Michio Suda, Ken Ebihara, Hidenori Iwai, Naoki Matsuoka, Noriko Satoh, Hiroyuki Odaka, Hisao Kasuga, Yukio Fujisawa, Gen Inoue, Haruo Nishimura, Yasunao Yoshimasa, Kazuwa Nakao, Increased glucose metabolism and insulin sensitivity in transgenic skinny mice overexpressing leptin, Diabetes, 10.2337/diabetes.48.9.1822, 48, 9, 1822-1829, 1999.09, Excess of body fat, or obesity, is a major health problem and confers a higher risk of cardiovascular and metabolic disorders such as diabetes, hypertension, and coronary heart disease. Leptin is an adipocyte-derived satiety factor that plays an important role in the regulation of energy homeostasis, and its synthesis and secretion are markedly increased in obese subjects. To explore the metabolic consequences of an increased amount of leptin on a long-term basis in vivo, we generated transgenic skinny mice with elevated plasma leptin concentrations comparable to those in obese subjects. Overexpression of leptin in the liver has resulted in complete disappearance of white and brown adipose tissue for a long period of time in mice. Transgenic skinny mice exhibit increased glucose metabolism accompanied by the activation of insulin signaling in the skeletal muscle and liver. They also show small-sized livers with a marked decrease in glycogen and lipid storage. The phenotypes are in striking contrast to those of recently reported animal models of lipoatrophic diabetes and patients with lipoatrophic diabetes with reduced amount of leptin. The present study provides evidence that leptin is an adipocyte-derived antidiabetic hormone in vivo and suggests its pathophysiologic and therapeutic implications in diabetes..
437. Noriko Satoh, Yoshihiro Ogawa, Goro Katsuura, Yoshito Numata, Tetsuo Tsuji, Minoru Hayase, Ken Ebihara, Hiroaki Masuzaki, Kiminori Hosoda, Yasunao Yoshimasa, Kazuwa Nakao, Sympathetic activation of leptin via the ventromedial hypothalamus
Leptin-induced increase in catecholamine secretion, Diabetes, 10.2337/diabetes.48.9.1787, 48, 9, 1787-1793, 1999.09, Leptin is an adipocyte-derived blood-borne satiety factor that acts directly on the hypothalamus, thereby regulating food intake and energy expenditure. We have demonstrated that the hypothalamic arcuate nucleus (Arc) is a primary site of the satiety effect of leptin (Neurosci Lett 224:149-152, 1997). To explore the hypothalamic pathway of sympathetic activation of leptin, we examined the effects of a single intravenous or intracerebroventricular injection of recombinant human leptin on catecholamine secretion in rats. We also examined the effects of direct microinjection of leptin into the ventromedial hypothalamus (VMH), Arc, paraventricular nucleus (PVN), and dorsomedial hypothalamus (DMH) in rats. To further assess whether sympathetic activation of leptin is mediated via the VMH, we also examined the effects of a single intravenous injection of leptin in VMH-lesioned rats. A single injection of leptin (0.25-1.0 mg i.v./rat or 0.5-2.0 pg i.c.v./rat) increased plasma norepinephrine (NE) and epinephrine (EPI) concentrations in a dose-dependent manner. Plasma NE and EPI concentrations were increased significantly when leptin was injected directly into the VMH but were unchanged when injected into the Arc, PVN, and DMH. Plasma NE and EPI concentrations were unchanged in VMH-lesioned rats that received a single intravenous injection of leptin. The present study provides evidence that a leptin-induced increase in catecholamine secretion is mediated primarily via the VMH and suggests the presence of distinct hypothalamic pathways mediating the satiety effect and sympathetic activation of leptin..
438. Kazuo Chin, Kouichi Shimizu, Takaya Nakamura, Noboru Narai, Hiroaki Masuzaki, Yoshihiro Ogawa, Michiaki Mishima, Takashi Nakamura, Kazuwa Nakao, Motoharu Ohi, Changes in intra-abdominal visceral fat and serum leptin levels in patients with obstructive sleep apnea syndrome following nasal continuous positive airway pressure therapy, Circulation, 10.1161/01.CIR.100.7.706, 100, 7, 706-712, 1999.08, Background-Obstructive sleep apnea syndrome (OSAS) is a common disorder in obese subjects. Visceral fat accumulation (VFA) is a better predictor of coronary heart disease than body mass index. Leptin is a hormone involved in the control of body weight and fat distribution. The effect of nasal continuous positive airway pressure (NCPAP) treatment on VFA and serum leptin levels in OSAS patients has not been known. Methods and Results-VFA and subcutaneous fat accumulation (SFA) were assessed by CT before and after NCPAP treatment in 22 OSAS patients (mean apnea and hypopnea index >50 episodes/h). Serum leptin levels of another 21 OSAS patients were measured before and after 3 to 4 days of NCPAP to gain insight into the mechanism by which NCPAP affects fat distribution. VFA and SFA decreased significantly after 6 months of NCPAP treatment (236 ± 16 to 182±14cm2, P=0.0003 and 215±21 to 189±18 cm2 P=0.003, respectively). VFA decreased significantly in the body weight reduction group (n=9, P<0.01) and the no body weight reduction group (n=13, P<0.03). In contrast, SFA changed significantly in the body weight reduction group only (P<0.01). Leptin levels decreased significantly following 3 to 4 days of NCPAP (P<0.01), whereas body weight, fasting insulin, and cortisol levels did not change significantly. Conclusions-Correction of sleep disordered breathing by NCPAP may be used to reduce VFA in OSAS patients. OSAS may have significant effects on the serum leptin levels..
439. Hiroaki Masuzaki, Yoshihiro Ogawa, Megumi Aizawa-Abe, Kiminori Hosoda, Junko Suga, Ken Ebihara, Noriko Satoh, Hidenori Iwai, Gen Inoue, Haruo Nishimura, Yasunao Yoshimasa, Kazuwa Nakao, Glucose metabolism and insulin sensitivity in transgenic mice overexpressing leptin with lethal yellow agouti mutation
Usefulness of leptin for the treatment of obesity-associated diabetes, Diabetes, 10.2337/diabetes.48.8.1615, 48, 8, 1615-1622, 1999.08, Leptin acts as an adipocyte-derived blood-borne satiety factor that can increase glucose metabolism. To elucidate the therapeutic implications of leptin for obesity-associated diabetes, we crossed transgenic skinny mice overexpressing leptin (Tg/+), which we have developed recently, and lethal yellow KK(y) mice (A(y)/+), a genetic model for obesity-diabetes syndrome, and examined the metabolic phenotypes of F1 animals. At 6 weeks of age, plasma leptin concentrations in Tg/+ mice with the A(y) allele (Tg/+:A(y)/+) were significantly higher than those in A(y)/+ mice. Although no significant differences in body weight were noted among Tg/+:A(y)/+ mice, A(y)/+ mice, and their wild-type lean littermates (+/+), glucose and insulin tolerance tests revealed increased glucose tolerance and insulin sensitivity in Tg/+:A(y)/+ compared with A(y)/+ mice. However, at 12 weeks of age, when plasma leptin concentrations in A(y)/+ mice were comparable to those in Tg/+:A(y)/+ mice, Tg/+:A(y)/+ mice developed obesity-diabetes syndrome similar to that of A(y)[+ mice. Body weights of 12-week-old Tg/+:A(y)/+ and A(y)/+ mice were reduced to those of +/+ mice by a 3-week food restriction; when plasma leptin concentrations remained high in Tg/+:A(y)/+ mice but were markedly reduced in A(y)/+ and +/+ mice, glucose tolerance and insulin sensitivity in Tg/+:A(y)/+ mice were markedly improved as compared with A(y)/+ and +/+ mice. The present study demonstrates that hyperleptinemia can delay the onset of impaired glucose metabolism and accelerate the recovery from diabetes during caloric restriction in Tg/+:A(y)/+ mice, thereby suggesting the potential usefulness of leptin in combination with a long- term caloric restriction for the treatment of obesity-associated diabetes..
440. Masafumi Nakayama, Hirofumi Yasue, Michihiro Yoshimura, Yukio Shimasaki, Kiyotaka Kugiyama, Hisao Ogawa, Takeshi Motoyama, Yoshihiko Saito, Yoshihiro Ogawa, Yoshihiro Miyamoto, Kazuwa Nakao, T-786 → C mutation in the 5'-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm, Circulation, 10.1161/01.CIR.99.22.2864, 99, 22, 2864-2870, 1999.06, Background - Coronary spasm plays an important role in the pathogenesis of ischemic heart diseases in general. However, the precise mechanism(s) responsible for coronary spasm remains to be elucidated, and we examined the molecular genetics of coronary spasm. Methods and Results - We Searched for the possible mutations in the endothelial nitric oxide synthase (eNOS) gene in patients with coronary spasm. In this study, we demonstrate the existence of 3 linked mutations in the 5'-flanking region of the eNOS gene (T-786 → C, A-922 → G, and T-1468 → A). The incidence of the mutations was significantly greater in patients with coronary spasm than in the control group (P < 0.0001). Multiple logistic regression analysis with forward stepwise selection using the environmental risk factors and the eNOS gene variant revealed that the most predictive independent risk factor for coronary spasm was the mutant allele (P < 0.0001). As assessed by luciferase reporter gene assays, the T-786 → C mutation resulted in a significant reduction in eNOS gene promoter activity (P < 0.05), whereas neither the A- 922 → G nor the T-1468 → A mutation had any affect. Conclusions - Taken together, these findings strongly suggest that the T-786 → C mutation in the eNOS gene reduces the endothelial NO synthesis and predisposes the patients with the mutation to coronary spasm..
441. Koichiro Kuwahara, Yoshihiko Saito, Osamu Nakagawa, Ichiro Kishimoto, Masaki Harada, Emiko Ogawa, Yoshihiro Miyamoto, Ichiro Hamanaka, Noboru Kajiyama, Nobuki Takahashi, Takehiko Izumi, Rika Kawakami, Naohisa Tamura, Yoshihiro Ogawa, Kazuwa Nakao, The effects of the selective ROCK inhibitor, Y27632, on ET-1-induced hypertrophic response in neonatal rat cardiac myocytes - Possible involvement of Rho/ROCK pathway in cardiac muscle cell hypertrophy, FEBS Letters, 10.1016/S0014-5793(99)00680-8, 452, 3, 314-318, 1999.06, A small GTPase, Rho, participates in agonist-induced cytoskeletal organization and gene expression in many cell types including cardiac myocytes. However, little is known about the functions of Rho's downstream targets in cardiac myocytes. We examined the role of ROCK, a downstream target of Rho, in ET-1-induced hypertrophic response. Y27632, a selective ROCK inhibitor, inhibited ET-1-induced increases in natriuretic peptide production, cell size, protein synthesis, and myofibrillar organization. In addition, a dominant-negative mutant of p160ROCK suppressed ET-1-induced transcription of the BNP gene. These findings suggest that the Rho/ROCK pathway is an important component of ET-1-induced hypertrophic signals in cardiac myocytes. Copyright (C) 1999 Federation of European Biochemical Societies..
442. Kiyoshi Mori, Yoshihiro Ogawa, Ken Ebihara, Naohisa Tamura, Kei Tashiro, Takashi Kuwahara, Masashi Mukoyama, Akira Sugawara, Shoichi Ozaki, Issei Tanaka, Kazuwa Nakao, Isolation and characterization of CA XIV, a novel membrane-bound carbonic anhydrase from mouse kidney, Journal of Biological Chemistry, 10.1074/jbc.274.22.15701, 274, 22, 15701-15705, 1999.05, Carbonic anhydrase (CA) is involved in various physiological processes such as acid-base balance and transport of carbon dioxide and ions. In this study, we have succeeded in the isolation of a novel CA from the mouse kidney by use of the signal sequence trap method. It is a 337-amino acid polypeptide with a calculated molecular mass of 37.5 kDa, consisting of a putative amino- terminal signal sequence, a CA domain, a transmembrane domain, and a short hydrophilic carboxyl terminus, which we designated CA XIV. The CA domain of CA XIV is highly homologous with those of known CAs, especially extracellular CAs including CA XII, IX, VI, and IV. The expression study of an epitope- tagged protein has suggested that CA XIV is located on the plasma membrane. When expressed in COS-7 cells, CA XIV exhibits CA activity that is predominantly associated with the membrane fraction. By Northern blot analysis, the gene expression of CA XIV is most abundant in the kidney and heart, followed by the skeletal muscle, brain, lung, and liver. In situ hybridization has revealed that, in the kidney, the gene is expressed intensely in the proximal convoluted tubule, which is the major segment for bicarbonate reabsorption and also in the outer border of the inner stripe of the outer medulla. In conclusion, we have cloned a functional cDNA encoding a novel membrane-bound CA. This study will bring new insights into our understanding of carbon dioxide metabolism and acid-base balance..
443. Yoshihiro Ogawa, Hiroaki Masuzaki, Kazuwa Nakao, 5. Clinical implication of leptin, the obese gene product, Internal Medicine, 10.2169/internalmedicine.38.210, 38, 2, 210-212, 1999.02.
444. Mitsuyo Shintani, Haruo Nishimura, Takashi Akamizu, Shin Yonemitsu, Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Gen Inoue, Yasunao Yoshimasa, Kazuwa Nakao, Thyrotropin decreases leptin production in rat adipocytes, Metabolism: Clinical and Experimental, 10.1016/S0026-0495(99)90247-0, 48, 12, 1570-1574, 1999.01, Leptin, which is secreted from adipocytes, has a role in the regulation of appetite and energy expenditure. The thyrotropin receptor (TSH-R) was recently found in adipocytes. We examined the effects of TSH on leptin production and lipolysis in rat epididymal adipocytes. TSH decreased the concentration of leptin in the medium time (~24 hours), and dose (~10-7 mol/L)-dependently (half-maximal inhibition [IC50] ≃ 10-9 mol/L). TSH also decreased the ob mRNA level approximately 55% in adipocytes. We confirmed the presence of TSH-R mRNA in the adipocytes by reverse transcription-polymerase chain reaction (RT-PCR). TSH stimulated glycerol release dose-dependently (IC50 ≃ 10-8 mol/L) in adipocytes. This TSH- induced glycerol release was further enhanced by adenosine deaminase (ADA). In summary, TSH reduced leptin production and stimulated lipolysis in rat epididymal adipocytes. Although the pathophysiological relevance of the regulation of leptin production and lipolysis by TSH is unknown, we speculate that TSH may affect the regulation of appetite and energy expenditure in pathophysiological states..
445. M. Suda, Y. Komatsu, K. Tanaka, A. Yasoda, Y. Sakuma, N. Tamura, Y. Ogawa, K. Nakao, C-type natriuretic peptide/guanylate cyclase B system in rat osteogenic ROB-C26 cells and its down-regulation by dexamethazone, Calcified Tissue International, 10.1007/s002239900735, 65, 6, 472-478, 1999, There is recent evidence that natriuretic peptides are important regulators of bone and cartilage, although they were originally identified as the cardiac hormones causing natriuresis and hypotension. Three members of natriuretic peptide family are known: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biologically active receptors for these peptides are particulate guanylate cyclases; the two known types are GC-A and GC-B. ANP and BNP have high affinities for GC-A, and CNP is the preferred ligand for GCB. In this paper we report the results of our study of the expression and possible role(s) of natriuretic peptides in the ROB-C26 cell, which is an osteogenic cell line with multiple potentials for differentiating into myoblast, osteoblast, and adipocyte. ROB-C26 cells produced cGMP in response to natriuretic peptides at both their basal state and after enhanced differentiation into osteoblast which was induced by bone morphogenetic protein [(BMP)-2]. CNP was far more potent than ANP in cGMP production. In contrast, enhanced differentiation into adipocyte by dexamethasone resulted in the marked decrease in their responsiveness to natriuretic peptides. Although the messages for GC-A and GC-B were demonstrated by Northern blot analysis at both the basal stage and after BMP treatment, they were downregulated after dexamethasone treatment. The presence of CNP was shown by RT-PCR and immunohistochemistry in ROB-C26 cells. C3H10T1/2, which is another and more primitive mesenchymal cell line, also produced cGMP in response to CNP, and less potently to ANP. Culturing ROB-C26 cells with CNP or 8-bromo cGMP decreased [3H]thymidine uptake and slightly increased the message for alkaline phosphatase, which is a marker for osteoblast differentiation. These results suggest that the CNP/GC-B system is preferentially expressed in the cells of osteogenic lineage and their expression is down-regulated with differentiation into adipocyte lineage. The CNP/GC-B system is likely to be an autocrine/paracrine regulator of osteoblast growth and differentiation..
446. K. Yahata, I. Tanaka, M. Kotani, M. Mukoyama, Y. Ogawa, M. Goto, M. Nakagawa, A. Sugawara, K. Tanaka, A. Shimatsu, K. Nakao, Identification of a novel R642C mutation in Na/Cl cotransporter with Gitelman's syndrome, American Journal of Kidney Diseases, 10.1016/S0272-6386(99)70041-7, 34, 5, 845-853, 1999, Gitelman's syndrome, a variant of Bartter's syndrome, is an inherited disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria, and these abnormalities have recently been linked to the thiazide-sensitive Na/Cl cotransporter (TSC) gene. We evaluated three unrelated patients affected with this syndrome whose diagnosis was made based on clinical and biochemical features. The data of clearance studies in these patients were compatible with Gitelman's syndrome. We then investigated possible mutations of the TSC gene. In one patient whose parents are consanguineous, we identified a novel missense mutation in the TSC gene, which causes alteration of arginine to cysteine at codon 642 (R642C mutation) located in the cytoplasmic tail of the product. This mutation results in the loss of an Mspl site in exon 15 of the TSC gene. Mspl digestion analysis of genomic DNA fragments from the family was consistent with the autosomal recessive inheritance of the disorder, and presence of this mutation correlated with the clinical manifestations. Such mutation was not detected in 47 normal healthy subjects, in the second patient, we found another missense mutation in one allele of the TSC gene, which results in alteration of arginine to glutamine at codon 955. In the third patient, no mutation causing amino acid substitution was found in the TSC gene. These results indicate that the R642C mutation in TSC is critically important for impairment of this cotransporter function and also suggest the necessity of further investigations in the genetic background of Gitelman's syndrome..
447. Keiichi Imagawa, Yoshito Numata, Goro Katsuura, Isako Sakaguchi, Atsushi Morita, Shino Kikuoka, Yayoi Matumoto, Tetsuo Tsuji, Mikio Tamaki, Kazuyuki Sasakura, Hiroshi Teraoka, Kiminori Hosoda, Yoshihiro Ogawa, Kazuwa Nakao, Structure-function studies of human leptin, Journal of Biological Chemistry, 10.1074/jbc.273.52.35245, 273, 52, 35245-35249, 1998.12, To elucidate the structural requirement of human leptin for its functions, the wild-type, mutant-type, C-terminal deletion, and N-terminal deletion were expressed in Escherichia coli and purified in soluble forms. These leptin analogs were intracerebroventrically injected into C57BL/6J ob/ob mice, and their in vivo biological activities were evaluated. The mutant-type leptin lacking a C-terminal disulfide bond reduced food intake at doses of more than 15 pmol/mouse, which was as effective as the wild-type leptin. C-terminal deletion without the loop structure, also significantly, but to a lesser extent, reduced food intake at doses of more than 90 pmol/mouse. However, N-terminal deletions showed no effect on food intake. We also evaluated the effects of the leptin analogs on radiolabeled leptin binding to its receptor in the choroid plexus using autoradiography. An excess of unlabeled mutant-type leptin as well as wild-type leptin led to complete inhibition of binding. C-terminal deletions led to weak inhibitory activity, whereas N-terminal deletions caused no inhibitory activity. These results clearly demonstrate that the N-terminal region of leptin is essential for both its biological and receptor binding activities. The amino acid sequence of the C-terminal loop structure is also important for enhancing these actions, whereas the C-terminal disulfide bond is not needed..
448. Shigeo Yura, Norimasa Sagawa, Yoshihiro Ogawa, Hiroaki Masuzaki, Hiroko Mise, Tsunekazu Matsumoto, Ken Ebihara, Shingo Fujii, Kazuwa Nakao, Augmentation of leptin synthesis and secretion through activation of protein kinases A and C in cultured human trophoblastic cells, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.83.10.3609, 83, 10, 3609-3614, 1998.11, Leptin is a fat cell-derived hormone that regulates food intake and energy expenditure. We previously demonstrated that leptin is produced by nonadipose cells, i.e. by placental trophoblasts. We also reported that a human trophoblastic cell line, BeWo cells, expresses leptin gene and secretes leptin into culture media. To elucidate the regulatory mechanisms of leptin production by human trophoblasts, we investigated synthesis and secretion of leptin in BeWo cells and in explant cultures of human placental tissue. Leptin production and gene expression in BeWo cells were increased by treatment with forskolin. The forskolin-induced increase in leptin production was completely suppressed by H89, an inhibitor of protein kinase A. Leptin production and gene expression in BeWo cells were increased by treatment with phorbol myristate acetate (PMA). The PMA-induced increase in leptin production was completely suppressed by H7 and staurosporine, both of which are inhibitors of protein kinase C. Leptin secretion from first trimester chorionic tissue was approximately 50-fold greater than that from term placental tissue. Leptin production and gene expression in explant cultures of placental tissue at both stages of pregnancy were augmented markedly by treatment with forskolin or PMA. The present study demonstrated augmentation of leptin production by protein kinase A and protein kinase C in cultured human trophoblasts, thereby leading to a better understanding of the regulatory mechanisms of leptin production in human trophoblasts in vivo..
449. Mayumi Inoue, Hiroshi Itoh, Makiko Ueda, Takahiko Naruko, Akiko Kojima, Ryushi Komatsu, Kentaro Doi, Yoshihiro Ogawa, Naohisa Tamura, Kazuhiko Takaya, Toshio Igaki, Jun Yamashita, Tae Hwa Chun, Ken Masatsugu, Anton E. Becker, Kazuwa Nakao, Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions
Possible pathophysiological significance of VEGF in progression of atherosclerosis, Circulation, 10.1161/01.CIR.98.20.2108, 98, 20, 2108-2116, 1998.11, Background - Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. Methods and Results - Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription-polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. Conclusions - These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases..
450. Michihiro Yoshimura, Hirofumi Yasue, Masafumi Nakayama, Yukio Shimasaki, Hitoshi Sumida, Seigo Sugiyama, Kiyotaka Kugiyama, Hisao Ogawa, Yoshihiro Ogawa, Yoshihiko Saito, Yoshihiro Miyamoto, Kazuwa Nakao, A missense Glu298Asp variant in the endothelial nitric oxide synthase gene is associated with coronary spasm in the Japanese, Human Genetics, 10.1007/s004390050785, 103, 1, 65-69, 1998.09, Coronary spasm plays an important role in the pathogenesis of not only variant angina but also ischemic heart disease in general. However, the precise mechanism(s) by which coronary spasm occurs remains to be elucidated. Coronary spasm may arise from interactions between environmental and genetic factors. Endothelial-derived nitric oxide (NO) has been implicated in the control of vascular tone. We have recently shown that both basal and acetylcholine (ACh)-induced NO activities are impaired in the coronary arteries of patients with coronary spasm. The purpose of this study has been to elucidate the possible variants that occur in the coding region of the endothelial nitric oxide synthase (eNOS) gene and that may be associated with coronary spasm. After initial screening in the entire 26 coding regions of the eNOS gene, we found a missense Glu298Asp variant in exon 7 in patients with coronary spasm. We subsequently performed a larger scale study involving 113 patients with coronary spasm and 100 control subjects, who were all diagnosed by intracoronary injection of ACh. The analysis revealed a significant difference in the distribution of the variant between the coronary spasm group (21.2%) and control group (9.0%; P = 0.014 for dominant effect). Thus, we have found the missense Glu298Asp variant in the eNOS gene by the analysis of its entire 26 coding regions. The variant is significantly associated with coronary spasm..
451. Yukio Shimasaki, Hirofumi Yasue, Michihiro Yoshimura, Masafumi Nakayama, Kiyotaka Kugiyama, Hisao Ogawa, Eisaku Harada, Takenobu Masuda, Wasaku Koyama, Yoshihiko Saito, Yoshihiro Miyamoto, Yoshihiro Ogawa, Kazuwa Nakao, Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with myocardial infarction, Journal of the American College of Cardiology, 10.1016/S0735-1097(98)00167-3, 31, 7, 1506-1510, 1998.06, Objectives. We examined the possible association between the missense Glu298Asp variant of the endothelial nitric oxide synthase (eNOS) gene and myocardial infarction (MI). Background. Endothelium-derived nitric oxide (NO) plays a key role in the regulation of vascular tone. Recently, we reported that a missense Glu298Asp variant in exon 7 of the eNOS gene is a possible genetic factor involved in the pathogenesis of coronary spasm. Endothelium- derived NO also has vasoprotective effects by suppressing platelet aggregation, leukocyte adhesion and smooth muscle cell proliferation. Methods. We screened 285 patients with an MI and 607 control subjects in Kumamoto Prefecture, Japan. Genotypes were determined by polymerase chain reaction-restriction fragment-length polymorphism analysis. Results. The frequency of the missense Glu298Asp variant was significantly higher in the MI group than in the control group (21.1% vs. 13.3%, p = 0.003, odds ratio 1.73 for the dominant effect of the eNOS T allele). Multiple logistic regression analysis showed that the missense Glu298Asp variant was an independent risk factor for MI, as was diabetes mellitus, hypertension, cigarette smoking, hypercholesterolemia and body mass index. Conclusions. There was a significant association of the missense Glu298Asp variant of the eNOS gene with MI. This marker-disease association may be due to the impaired effects of NO on the cardiovascular system: dysregulation of vascular tone, platelet aggregation and leukocyte adhesion and smooth muscle cell proliferation, all of which promote coronary atherosclerosis and thrombosis..
452. J. Yamashita, T. Yoshimasa, H. Arai, J. Hiraoka, K. Takaya, Y. Miyamoto, Y. Ogawa, H. Itoh, K. Nakao, Identification of cis-elements of the human endothelin-A receptor gene and inhibition of the gene expression by the decoy strategy, Journal of Biological Chemistry, 10.1074/jbc.273.26.15993, 273, 26, 15993-15999, 1998.06, Previously, we succeeded in molecular cloning of the cDNA and the gene for human endothelin-A receptor (ET-AR). In the present study, we define cis- elements in the 5'-flanking region of the ET-AR gene. Deletion analyses were performed in A7r5 cells, rat vascular smooth muscle cell line, and Chinese hamster ovary cells using ET-AR promoter-luciferase gene constructs including 5 kilobases of the 5'-flanking region. These analyses demonstrated the existence of one negative regulatory element (-2.0 kilobases to -857 bases) and two positive regulatory elements (-137 to -53 and -53 to +251). Gel mobility shift assay revealed a nuclear protein binding to the region (-104 to -78) (R1). DNase I footprinting analysis showed a footprint spanning from -91 to -83 whose sequence is CCCCACCTT (ETA-P1). When a plasmid including R1 fragments (R1 decoy) was co-transfected into A7r5 cells with ET-AR (-137 to +251)-luciferase gene construct, it significantly reduced the luciferase activity in a dose-dependent manner. Moreover, R1 decoy down-regulated the endogenous ET-AR mRNA in A7r5 cells by a maximum of 75%. Thus, we identified cis-elements that regulate basal transcriptional activity of the ET-AR gene and proved the feasibility to suppress the expression of the ET-AR gene by the DNA decoy strategy using the positive regulatory element we identified..
453. Noriko Satoh, Yoshihiro Ogawa, Goro Katsuura, Yoshito Numata, Hiroaki Masuzaki, Yasunao Yoshimasa, Kazuwa Nakao, Satiety effect and sympathetic activation of leptin are mediated by hypothalamic melanocortin system, Neuroscience Letters, 10.1016/S0304-3940(98)00401-7, 249, 2-3, 107-110, 1998.06, Leptin is an adipocyte-derived blood-borne satiety factor that decreases food intake and increases energy expenditure, thereby leading to a substantial decrease in body weight. To explore the possible roles of the hypothalamic melanocortin system in leptin action, we examined the effects of intracerebroventricular (ICV) injection of leptin with or without SHU9119, a potent antagonist of α-melanocyte stimulating hormone, on food intake, body weight, and mitochondrial uncoupling protein-1 (UCP-1) mRNA expression in the brown adipose tissue (BAT) in rats. A single ICV injection of leptin decreased cumulative food intake and body weight gain, and increased UCP-1 mRNA expression during 3 h at the onset of the dark phase. Inhibition of food intake and body weight change with leptin was reversed by co-injection of SHU9119 in a dose-dependent manner. Co-injection of SHU9119 also inhibited completely the leptin-induced increase in UCP-1 mRNA expression in the BAT. Treatment with SHU9119 alone did not affect food intake, body weight, and UCP-1 mRNA expression in rats. The present study provides evidence that the hypothalamic melanocortin system plays a central role in both satiety effect and sympathetic activation of leptin..
454. Akihiro Yasoda, Yoshihiro Ogawa, Michio Suda, Naohisa Tamura, Kiyoshi Mori, Yoko Sakuma, Hideki Chusho, Kohei Shiota, Kiyoshi Tanaka, Kazuwa Nakao, Natriuretic peptide regulation of endochondral ossification
Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway, Journal of Biological Chemistry, 10.1074/jbc.273.19.11695, 273, 19, 11695-11700, 1998.05, The natriuretic peptide family consists of three structurally related endogenous ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biological actions of natriuretic peptides are thought to be mediated through the activation of two guanylyl cyclase (GC)-coupled receptor subtypes (GC-A and GC-B). In this study, we examined the effects of ANP and CNP, which are endogenous ligands for GC-A and GC-B, respectively, on bone growth using an organ culture of fetal mouse tibias, an in vitro model of endochondral ossification. CNP increased the cGMP production much more potently than ANP, thereby resulting in an increase in the total longitudinal bone length. Histological examination revealed an increase in the height of the proliferative and hypertrophic chondrocyte zones in fetal mouse tibias treated with CNP. The natriuretic peptide stimulation of bone growth, which was mimicked by 8- bromo-cGMP, was inhibited by HS-142-1, a non-peptide GC-coupled natriuretic peptide receptor antagonist. The spontaneous increase in the total longitudinal bone growth and cGMP production was also inhibited significantly by HS-142-1. CNP mRNA was expressed abundantly in fetal mouse tibias, where no significant amounts of ANP and BNP mRNAs were detected. A considerable amount of GC-B mRNA was present in fetal mouse tibias. This study suggests the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification..
455. Michio Suda, Yoshihiro Ogawa, Kiyoshi Tanaka, Naohisa Tamura, Akihiro Yasoda, Toshiya Takigawa, Masahiro Uehira, Hirofumi Nishimoto, Hiroshi Itoh, Yoshihiko Saito, Kohei Shiota, Kazuwa Nakao, Skeletal overgrowth in transgenic mice that overexpress brain natriuretic peptide, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.95.5.2337, 95, 5, 2337-2342, 1998.03, Longitudinal bone growth is determined by the process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones and involves many systemic hormones and local regulators. Natriuretic peptides organize a family of three structurally related peptides: atrial natriuretic peptide, brain natriuretic peptide (BNP), and C-type natriuretic peptide. Atrial natriuretic peptide and BNP are cardiac hormones that are produced predominantly by the atrium and ventricle, respectively. C-type natriuretic peptide occurs in a wide variety of tissues, where it acts as a local regulator. These peptides can influence body fluid homeostasis and blood pressure control through the activation of two guanylyl cyclase (GC)-coupled natriuretic peptide receptor subtypes - GC-A and GC-B. We report here marked skeletal overgrowth in transgenic mice that overexpress BNP. Transgenic mice with elevated plasma BNP concentrations exhibited deformed bony skeletons characterized by kyphosis, elongated limbs and paws, and crooked tails, Bone abnormalities resulted from a high turnover of endochondral ossification accompanied by overgrowth of the growth plate. Studies using an in vitro organ culture of embryonic mouse tibias revealed that BNP increases the height of cartilaginous primordium directly, thereby stimulating the total longitudinal bone growth. The present study demonstrates that natriuretic peptides can affect the process of endochondral ossification..
456. Toshio Igaki, Hiroshi Itoh, Shin Ichi Suga, Norio Hama, Yoshihiro Ogawa, Yasato Komatsu, Jun Yamashita, Kentaro Doi, Tae Hwa Chun, Kazuwa Nakao, Effects of intravenously administered C-type natriuretic peptide in humans
Comparison with atrial natriuretic peptide, Hypertension Research, 10.1291/hypres.21.7, 21, 1, 7-13, 1998.01, We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells and suggested that CNP might be a local regulator of vascular tone and growth. To evaluate the biological actions of CNP as compared with human a-trial natriuretic peptide (hANP), we intravenously administered synthetic CNP (0.43 nmol/kg) and α-hANP (0.43 and 0.043 nmol/kg) to healthy humans. The experiments were done on different days in the same five healthy volunteers (31±1 yr old). CNP injection caused a transient but significant decrease in both systolic and diastolic blood pressure (-4.3±1.3, -4.1±1.0 mmHg) with a significant increase in heart rate (+7.6±2.6 bpm), and exerted significant diuretic and natriuretic activities (+130±80%, +160±100% over the basal level). These effects of CNP (0.43 nmol/kg) were comparable to, or less than, those of α-hANP (0.043 nmol/kg). CNP injection also significantly suppressed aldosterone secretion (22% reduction as compared with the basal level). Our results demonstrate that intravenously-administered CNP acts as a natriuretic peptide with less potency than ANP..
457. J. Hiraoka, Y. Ogawa, H. Masuzaki, K. Ikeda, S. Yura, M. Sawamura, N. Katoh, K. Hosoda, A. Kitamura, Y. Nara, N. Sagawa, Y. Yamori, K. Nakao, Elevated plasma leptin concentrations in rat pregnancy, japanese heart journal, 10.1536/ihj.39.574, 39, 4, 1998.01.
458. Koichiro Kuwahara, Yoshihiko Saito, Yoshihiro Ogawa, Naohisa Tamura, Masahiro Ishikawa, Masaki Harada, Emiko Ogawa, Yoshihiro Miyamoto, Ichiro Hamanaka, Shigeki Kamitani, Noboru Kajiyama, Nobuki Takahashi, Osamu Nakagawa, Izuru Masuda, Kazuwa Nakao, Endothelin-1 and cardiotrophin-1 induce brain natriuretic peptide gene expression by distinct transcriptional mechanisms, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199800001-00099, 31, SUPPL. 1, 1998.01, Cardiotrophin-1 (CT-1) a novel IL-6-related cytokine, induces distinct hypertrophic responses to endothelin-1 (ET-1) on cultured neonatal rat cardiac myocytes. We found that ET-1 and CT-1 show a distinct pattern of gene induction of natriuretic peptides. Elucidation of the transcriptional mechanisms of brain natriuretic peptide (BNP) gene induction by ET-1 or CT-1 will provide better information for our understanding of the molecular mechanisms of cardiac hypertrophy. In this study, reporter constructs containing the human BNP 5′ flanking sequence were transfected into neonatal rat cardiac myocytes and the cells were stimulated with ET-1 or CT-1. A total of 1813 bp of the human BNP 5′ flanking sequence conferred an ET-1 inducibility on the reporter gene. However, it did not confer CT-1 inducibility. These results show that distinct mechanisms are involved in BNP gene induction by ET-1 or CT-1, and (in this study) that the CT-1 responsive element is not located in the region examined..
459. K. Takaya, Y. Ogawa, H. Masuzaki, N. Matsuoka, K. Hosoda, K. Nakao, Leptin Receptor, Nippon rinsho. Japanese journal of clinical medicine, 56, 7, 1925-1930, 1998.01, Leptin is a fat cell-derived satiety factor that regulates food intake and energy expenditure. Its effects are mediated by interactions with the leptin receptor (Ob-R) that is alternatively spliced to encode at least five isoforms(a-e), which are distributed in a wide range of tissues including the hypothalamus. Ob-R is a member of cytokine receptors and involves the JAK-STAT signal transduction system. We found Ob-R mutations in Zucker fatty rats and obese Koletsky rats and demonstrated that Ob-R dysfunction brings around hyperphagia and obesity. However we and others have not found any Ob-R mutation in human obese subjects..
460. Yoshihiro Ogawa, K. Nakao, Obesity and diseases. 6. Clinical significance of obesity gene product (leptin), Nihon Naika Gakkai zasshi. The Journal of the Japanese Society of Internal Medicine, 10.2169/naika.87.1811, 87, 9, 1811-1816, 1998.01.
461. S. Yura, N. Sagawa, H. Mise, T. Mori, H. Masuzaki, Y. Ogawa, K. Nakao, A positive umbilical venous-arterial difference of leptin level and its rapid decline after birth, American Journal of Obstetrics and Gynecology, 10.1016/S0002-9378(98)70525-3, 178, 5, 926-930, 1998, OBJECTIVE: We investigated the site of leptin production in the fetoplacental circulation. STUDY DESIGN: We simultaneously determined plasma leptin levels in cord vessels and maternal peripheral veins of 38 healthy pregnant women. We also compared plasma leptin levels in 20 neonates at birth and on the fifth day after birth. RESULTS: Leptin levels in cord vessels were significantly (p < 0.001) lower than those in maternal veins (mean 29.5 mg/ml). Leptin levels in umbilical arteries (mean 9.8 ng/ml) were significantly (p < 0.01) lower than those in umbilical veins (mean 12.9 ng/ml). Leptin levels in neonatal veins on the fifth day (mean 3.0 ng/ml) were markedly (p < 0.001) lower than those in umbilical arteries of the same neonates (mean 10.9 ng/ml). CONCLUSIONS: The higher leptin levels in umbilical veins that in umbilical arteries and the marked decrease during the neonatal period suggest that the placenta is one of the major sources of leptin in the fetal circulation..
462. Hiroko Mise, Norimasa Sagawa, Tsunekazu Matsumoto, Shigeo Yura, Hidetaka Nanno, Hiroaki Itoh, Takahide Mori, Hiroaki Masuzaki, Kiminori Hosoda, Yoshihiro Ogawa, Kazuwa Nakao, Augmented placental production of leptin in preeclampsia
Possible involvement of placental hypoxia, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.83.9.3225, 83, 9, 3225-3229, 1998, Preeclampsia (PE) is a hypertensive disorder, which develops in late pregnancy and is usually associated with placental hypoxia and dysfunction. We have recently demonstrated that leptin is a novel placenta-derived hormone in humans and suggested its significance in human pregnancy (see Ref. 19). To explore the changes in the leptin production in placenta in PE, we measured the plasma leptin level and placental leptin messenger RNA expression in pregnant women with PE. Plasma leptin levels in preeclamptic women were elevated significantly, compared with gestational age- and body mass index- matched normal pregnant women (P < 0.0001). Plasma leptin levels in the severe PE group were significantly higher than those in the mild PE group (P < 0.0001). Plasma leptin levels in preeclamptic women were reduced, soon after the placental delivery, to those expected for their body mass indices. Northern blot analysis revealed that leptin messenger RNA levels are increased in the placentas from preeclamptic women, compared with normal pregnant women. Leptin secretion was increased significantly in a human trophoblastic cell line (BeWo cells) cultured under hypoxic conditions (5% O2), compared with those cultured under standard conditions (20% O2; P < 0.01). The present study demonstrated that placental production of leptin is augmented in severe PE, probably because of placental hypoxia, thereby suggesting the possible significance of leptin as a marker of placental hypoxia in severe PE..
463. Keiichi Imagawa, Yayoi Matsumoto, Yoshito Numata, Atsushi Morita, Shino Kikuoka, Mikio Tamaki, Chie Higashikubo, Tetsuo Tsuji, Kazuyuki Sasakura, Hiroshi Teraoka, Hiroaki Masuzaki, Kiminori Hosoda, Yoshihiro Ogawa, Kazuwa Nakao, Development of a sensitive ELISA for human leptin, using monoclonal antibodies, Clinical Chemistry, 44, 10, 2165-2171, 1998, A new, sensitive ELISA for human leptin in plasma and cerebrospinal fluid (CSF) was developed, using monoclonal antibodies. The lower limit of detection of this ELISA was 0.78 pg/assay. Both intra- and interassay imprecision values were <7%. The dilution curves of plasma and CSF showed good linearity, and the recovery was 83.2-95.6%. There was good correlation between plasma leptin concentrations by the ELISA and a commercially available RIA (r = 0.99). Our ELISA is advantageous because it does not require radioisotopes, it produces results in hours rather than days, and more importantly, it improves on the detection limit and plasma interference of the RIA kit. The new ELISA enables measurement of low concentrations of leptin, as are seen in CSF and in plasma of patients with anorexia nervosa..
464. Ken Masatsugu, Hiroshi Itoh, Tae Hwa Chun, Yoshihiro Ogawa, Naoshita Tamura, Jun Yamashita, Kentaro Doi, Mayumi Inoue, Yasutomo Fukunaga, Naoki Sawada, Takatoshi Saito, Risa Korenaga, Joji Ando, Kazuwa Nakao, Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199800001-00014, 31, SUPPL. 1, S42-S45, 1998, Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1α and ECE-1β. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1α mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1α mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress..
465. Ken Ebihara, Yoshihiro Ogawa, Naohi Isse, Kiyoshi Mori, Naohisa Tamura, Hiroaki Masuzaki, Ken ichi Kohno, Shigeo Yura, Kiminori Hosoda, Norimasa Sagawa, Kazuwa Nakao, Identification of the human leptin 5'-flanking sequences involved in the trophoblast-specific transcription, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1997.7869, 241, 3, 658-663, 1997.12, Leptin is an adipocyte-derived blood-borne satiety factor that is involved in the regulation of energy homeostasis. We have recently demonstrated nonadipose tissue production of leptin; leptin is synthesized in and secreted from placental trophoblasts. To understand the transcriptional regulation of the human leptin gene in placental trophoblasts, we examined the promoter activity of various lengths of the human leptin 5'-flanking sequences in BeWo cells, a human trophoblastic cell line. The 2080-bp human leptin gene promoter region (-2080 to +108) showed a high-level transcription activity in BeWo cells. When DNA sequences between -1885 and -1830 were deleted, the promoter activity was reduced dramatically in BeWo cells. No significant changes in the promoter activity were noted when tested in primary cultures of rat mature adipocytes. Electrophoretic mobility shift assays revealed the presence of nuclear protein(s) binding to the sequences in BeWo cells but not in isolated rat mature adipocytes. The present study provides new insight into the trophoblast-specific transcription of the human leptin gene..
466. Kiyoshi Mori, Yoshihiro Ogawa, Ken Ebihara, Tomohiro Aoki, Naohisa Tamura, Akira Sugawara, Takashi Kuwahara, Shoichi Ozaki, Masashi Mukoyama, Kei Tashiro, Issei Tanaka, Kazuwa Nakao, Kidney-specific expression of a novel mouse organic cation transporter-like protein, FEBS Letters, 10.1016/S0014-5793(97)01325-2, 417, 3, 371-374, 1997.11, Using the signal sequence trap method, we have cloned a novel 12-membrane-spanning transporter-like protein, termed renal-specific transporter (RST), from the mouse kidney. RST is a 553-amino-acid protein highly homologous to recently cloned organic cation transporters, e.g. it is 30% identical to rat organic cation transporter 1 at the amino acid level. Northern blot analysis has revealed that the RST gene is expressed abundantly and specifically in the kidney. In situ hybridization analysis has shown that RST gene expression is restricted to the renal proximal tubule, where various organic cations such as endogenous catecholamines and choline or clinically used cationic drugs are known to he actively excreted..
467. Norimasa Sagawa, Takahide Mori, Hiroaki Masuzaki, Yoshihiro Ogawa, Kazuwa Nakao, Leptin production by hydatidiform mole, Lancet, 10.1016/S0140-6736(05)63940-2, 350, 9090, 1518-1519, 1997.11.
468. M. Shigemoto, S. Nishi, Y. Ogawa, N. Isse, N. Matsuoka, T. Tanaka, N. Azuma, H. Masuzaki, H. Nishimura, Y. Yoshimasa, K. Hosoda, K. Nakao, Molecular screening of both the promoter and the protein coding regions in the human ob gene in Japanese obese subjects with non-insulin-dependent diabetes mellitus, European journal of endocrinology, 10.1530/eje.0.1370511, 137, 5, 511-513, 1997.11, Objective: Although the molecular mechanism of obesity has been poorly understood, recent studies indicate that leptin plays a critical role in regulating both food intake and body weight. Because obesity decreases the sensitivity to insulin, the human ob gene is presumed to be one of the candidate genes for non-insulin-dependent diabetes mellitus (NIDDM) associated with obesity. Although the protein coding region in the ob gene has been screened for mutations, the promoter region and the noncoding first exon have not yet been studied. We investigated the involvement of the human ob gene, especially mutations at the promoter region and the non-coding first exon, in the development of NIDDM associated with obesity. Subjects: The study group comprised 60 Japanese obese subjects with NIDDM (body mass index (BMI) 43.6≤BMI≤26.4, 29.0±0.41 (mean±S.E.M.)) and 24 obese individuals with impaired glucose tolerance (IGT) (30≤BMI≤26.4, 27.1±0.22). Methods: Mutations at both the promoter region and all three exons in the human ob gene were screened by the single-stranded conformational polymorphism analysis. When aberrantly migrated bands were recognized, the PCR-amplified DNA fragment was directly sequenced. Results: In the protein coding region a silent mutation in the second exon was detected. The noncoding first exon and the about 100 bp 5'-flanking region of the gene which contains a proximal CCAAT/enhancer-binding protein site were screened, but no mutations were found. Conclusion: These results suggest that no mutations in either the promoter region at the about 100 bp 5'-flanking region of the gene, or in any of the three exons, are involved in the development of NIDDM or IGT associated with obesity..
469. Masaki Harada, Hiroshi Itoh, Osamu Nakagawa, Yoshihiro Ogawa, Yoshihiro Miyamoto, Koichiro Kuwahara, Emiko Ogawa, Toshio Igaki, Jun Yamashita, Izuru Masuda, Takaaki Yoshimasa, Issei Tanaka, Yoshihiko Saito, Kazuwa Nakao, Significance of ventricular myocytes and nonmyocytes interaction during cardiocyte hypertrophy
Evidence for endothelin-1 as a paracrine hypertrophic factor from cardiac nonmyocytes, Circulation, 10.1161/01.CIR.96.10.3737, 96, 10, 3737-3744, 1997.11, Background: In cardiac hypertrophy, both excessive enlargement of cardiac myocytes and progressive interstitial fibrosis are well known to occur simultaneously. In the present study, to investigate the interaction between ventricular myocytes (MCs) and cardiac nonmyocytes (NMCs), mostly fibroblasts, during cardiocytes hypertrophy, we examined the change in cell size and gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cultured MCs as markers for hypertrophy in the neonatal rat ventricular cardiac cell culture system. Methods and Results: The size of cultured MCs significantly increased in the MC-NMC coculture. Concomitantly, secretions of ANP and BNP into culture media were significantly increased in the MC-NMC coculture compared with in the MC culture (with the possible contamination of NMC <1% of MC). Moreover, in the MC culture, enlargement of MC and an increase in ANP and BNP secretions were induced by treatment with conditioned media of the NMC culture. A considerable amount of endothelin (ET)-1 production was detected in the NMC- conditioned media. BQ-123, an ET-A receptor antagonist, and bosentan, a nonselective ET receptor antagonist, significantly blocked the hypertrophic response of MCs induced by treatment with NMC-conditioned media. Angiotensin II (Ang II) (10-10 to 10-6 mol/L) and transforming growth factor-β1 (TGF-β1) (10-13 to 10-9 mol/L), both of which are known to be cardiac hypertrophic factors, did not induce hypertrophy in MC culture, but both Ang II and TGF-β1 increased the size of MCs and augmented ANP and BNP productions in the MC-NMC coculture. This hypertrophic activity of Ang II and TGF-β1 was associated with the potentiation of ET-1 production in the MC- NMC coculture, and the effect of Ang II or TGF-β1 on the secretions of ANP and BNP in the coculture was significantly suppressed by pretreatment with BQ-123. Conclusions: These results demonstrate that NMCs regulate MC hypertrophy at least partially via ET-1 secretion and that the interaction between MCs and NMCs plays a critical role during the process of Ang II-or TGF-β1-induced cardiocyte hypertrophy..
470. Kentaro Doi, Hiroshi Itoh, Tadashi Ikeda, Kiminori Hosoda, Yoshihiro Ogawa, Toshio Igaki, Jun Yamashita, Tae Hwa Chun, Mayumi Inoue, Ken Masatsugu, Katsuhiko Matsuda, Katsuyuki Ohmori, Kazuwa Nakao, Adenovirus-mediated gene transfer of C-type natriuretic peptide causes G1 growth inhibition of cultured vascular smooth muscle cells, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1997.7576, 239, 3, 889-894, 1997.10, We have proposed the 'vascular natriuretic peptide system', in which C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, can control vascular tone and growth as an endothelium-derived relaxing peptide. We aimed at overexpression of CNP gene in vascular smooth muscle cells (SMCs) by adenovirus-mediated gene transfer to examine the growth characteristics of SMCs via the augmentation of cGMP production. Rat aortic SMCs infected with Ad.CNP, a replication deficient adenovirus driving rat CNP cDNA, produced 162 ± 55 fmol/mL of CNP, which was 4000 times higher than that produced by endothelial cells. cGMP production was also augmented in Ad.CNP-infected SMCs (2200 ± 270 fmol/104 cells). Accordingly, significant growth inhibition was observed in SMCs infected with Ad.CNP. The flow cytometry analysis revealed that the population of the S and G2 + M phases was reduced by 60% of the control in Ad.CNP-infected SMCs. The gene expression of ANP-B receptor, which is expressed abundantly in SMCs with the synthetic phenotype, was suppressed in Ad.CNP infected SMCs, while the gene expression of ANP-A receptor, which is expressed predominantly in SMCs with the contractile phenotype, became detectable in Ad.CNP-infected SMCs. In addition, the gene expression of smooth muscle myosin heavy chain-2 (SM-2), which is the molecular marker of highly-differentiated SMCs, was also induced in Ad.CNP-treated SMCs. These results suggest that cGMP cascade activation induces re-differentiation of SMCs. The present study demonstrated that overexpression of CNP induced growth inhibition of SMCs at the G1 phase with possible alteration of the phenotype..
471. N. Matsuoka, Y. Ogawa, K. Hosoda, J. Matsuda, H. Masuzaki, T. Miyawaki, N. Azuma, K. Natsui, H. Nishimura, Y. Yoshimasa, S. Nishi, D. B. Thompson, K. Nakao, Human leptin receptor gene in obese Japanese subjects
Evidence against either obesity-causing mutations or association of sequence variants with obesity, Diabetologia, 10.1007/s001250050808, 40, 10, 1204-1210, 1997.10, Leptin is an adipocyte-derived bloodborne satiety factor that acts on its cognate leptin receptor (Ob-R) in the hypothalamus, thereby regulating food intake and energy expenditure. To explore whether mutations in the Ob-R gene cause obesity in humans, we have searched for mutations in the gene for Ob-Rb, a biologically active receptor isoform, in obese Japanese subjects. We have also examined associations between such mutants and obesity in the Japanese. Genomic DNAs were used as templates in polymerase chain reaction (PCR) with primers selected to amplify exons 2 to 20 of the human Ob-Rb gene. Direct sequence analysis of the PCR products revealed 7 nucleotide sequence variants (Lys109Arg, Gln223Arg, Ser343Ser, Ser492Thr, Lys656Asn, Ala976Asp, and Pro1019Pro) in the Ob-Rb coding region from 17 obese Japanese subjects with a family history of obesity (BMI 39.3±8.4 kg/m2). No missense and nonsense mutations were found such as those in Zucker fatty (fa/fa) rats and Koletsky (fa(k)/fa(k)) rats. Nucleotide substitutions occurred at relatively high frequencies at codons 109, 223, 976, and 1019 (79, 91, 100, and 85%, respectively). Allele frequency of each variant determined by PCR-RFLP and PCR-single strand conformation polymorphism analyses showed no significant differences between 47 obese (BMI 35.1±6.5 kg/m2) and 68 non-obese (BMI 21.6±2.2 kg/m2) subjects. The present study represents the first report of sequence variants of the Ob-Rb gene in the Japanese and provides evidence against either obesity-causing mutations or association of sequence variants with obesity in obese Japanese subjects..
472. H. Nishimura, K. Hosoda, Y. Ogawa, K. Nakao, ob gene (leptin gene), Nippon rinsho. Japanese journal of clinical medicine, 55 Suppl, 482-489, 1997.10.
473. Hiroaki Masuzaki, Yoshihiro Ogawa, Norimasa Sagawa, Kiminori Hosoda, Tsunekazu Matsumoto, Hiroko Mise, Haruo Nishimura, Yasunao Yoshimasa, Issei Tanaka, Takahide Mori, Kazuwa Nakao, Nonadipose tissue production of leptin
Leptin as a novel placenta- derived hormone in humans, Nature medicine, 10.1038/nm0997-1029, 3, 9, 1029-1033, 1997.09, Leptin is a circulating hormone that is expressed abundantly and specifically in the adipose tissue1-5. It is involved in the regulation of energy homeostasis, as well as the neuroendocrine and reproductive systems6-11. Here, we demonstrate production of leptin by nonadipose tissue, namely, placental trophoblasts and amnion cells from uteri of pregnant women. We show that pregnant women secrete a considerable amount of leptin from the placenta into the maternal circulation as compared with nonpregnant obese women. Leptin production was also detected in a cultured human choriocarcinoma cell line, BeWo cells, and was augmented during the course of forskolin-induced differentiation of cytotrophoblasts into syncytiotrophoblasts. Plasma leptin levels were markedly elevated in patients with hydatidiform mole or choriocarcinoma and were reduced after surgical treatment or chemotherapy. Leptin is also produced by primary cultured human amnion cells and is secreted into the amniotic fluid. The present study provides evidence for leptin as a novel placenta-derived hormone in humans and suggests the physiologic and pathophysiologic significance of leptin in normal pregnancy and gestational trophoblastic neoplasms..
474. Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Takashi Miyawaki, Ikuko Hanaoka, Junko Hiraoka, Akiko Yasuno, Haruo Nishimura, Yasunao Yoshimasa, Shigeo Nishi, Kazuwa Nakao, Glucocorticoid regulation of leptin synthesis and secretion in humans
Elevated plasma leptin levels in Cushing's syndrome, Journal of Clinical Endocrinology and Metabolism, 10.1210/jc.82.8.2542, 82, 8, 2542-2547, 1997.08, Leptin, the obese (ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food ingestion and body weight. To investigate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing's syndrome with adrenal or pituitary adenoma and in patients with iatrogenic Cushing's syndrome. Plasma leptin levels in patients with Cushing's syndrome wore significantly elevated compared to those in nonobese healthy subjects and obese subjects without any metabolic or endocrine diseases at a given percentage of body fat by analysis of covariance. In patients with adrenal or pituitary adenoma, after the tumor resection, plasma leptin levels were reduced, with a concurrent decrease in plasma cortisol levels. With no significant changes in body weight, plasma leptin levels were also elevated significantly in lean healthy volunteers 24 h after the admistration of 1 mg dexamethasone. Dexamethasone potently induced ob gene expression and leptin secretion in the organ culture of human adipose tissue. The data demonstrate that glucocorticoids act, at least in part, directly on the adipose tissue and increase leptin synthesis and secretion in humans..
475. Noriko Satoh, Yoshihiro Ogawa, Goro Katsuura, Minoru Hayase, Tetsuo Tsuji, Keiichi Imagawa, Yasunao Yoshimasa, Shigeo Nishi, Kiminori Hosoda, Kazuwa Nakao, The arcuate nucleus as a primary site of satiety effect of leptin in rats, Neuroscience Letters, 10.1016/S0304-3940(97)00163-8, 224, 3, 149-152, 1997.04, The obese (ob) gene encodes a fat cell-derived circulating satiety factor (leptin) that is involved in the regulation of energy homeostasis. In the present study, we examined effects of i.c.v. injection of recombinant human leptin on food intake and body weight gain in rats. We also studied effects of direct microinjections of leptin into the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH). A single i.c.v. injection of recombinant human leptin (0.25-2.0 μg/rat) reduced significantly and dose-dependently food intake and body weight gain in rats. Microinjections (0.125-0.5 μg/site) into the bilateral Arc, VMH, and LH caused dose-related decreases in food intake and body weight gain as compared with vehicle-treated groups with a rank order of potency; Arc > VMH = LH. The present study provides the first direct evidence that the Arc is a primary site of satiety effect of leptin..
476. Masato Kotani, Issei Tanaka, Yoshihiro Ogawa, Takeshi Usui, Naohisa Tamura, Kiyoshi Mori, Shuh Narumiya, Teruya Yoshimi, Kazuwa Nakao, Structural organization of the human prostaglandin EP3 receptor subtype gene (PTGER3), Genomics, 10.1006/geno.1996.4585, 40, 3, 425-434, 1997.03, Prostaglandin EP3 receptor subtype is a seven-membrane-spanning protein with multiple C-terminal tails generated by alternative mRNA splicing. We report here the structural organization of the human EP3 gene (PTGER3). The human EP3 gene spanned more than 80 kb and was composed of 10 exons separated by nine introns. Exon 1 and the 5' 180-bp portion of exon 2 (exon 2a) encoded the seven transmembrane domains and 10 amino acid residues of the cytoplasmic tail, which are common to all EP3 isoforms. The 3' 3461-bp portion of exon 2 (exon 2b) or combinations of exons 3-10 encoded the EP3 isoform-specific C termini and formed their 3'-untranslated regions by multiple fashions of alternative mRNA splicing. Exons 2b, 4, 6, and 10 contained polyadenylation sites. The EP3 gene formed nine distinct mRNAs encoding eight EP3 isoforms, two of which were novel ones tentatively designated EP(3-V) and EP(3-VI). The transcription initiation sites of the human EP3 gene were mapped 227 to ~231 bp upstream of the ATG start codon. The 360-bp 5'flanking region contained a TATA box-like sequence, a GC box, and several cis-acting regulatory elements. The present study provides insight into the molecular mechanisms underlying the prostanoid receptor family..
477. Kosei Yoshimine, Masahisa Horiuchi, Syusaku Suzuki, Keiko Kobayashi, Jalil Md Abdul, Mina Masuda, Mineko Tomomura, Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, Mituhiro Osame, Takeyori Saheki, Altered expression of atrial natriuretic peptide and contractile protein genes in hypertrophied ventricle of JVS mice with systemic carnitine deficiency, Journal of Molecular and Cellular Cardiology, 10.1006/jmcc.1996.0300, 29, 2, 571-578, 1997.02, To characterize cardiac hypertrophy in juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency, we investigated how the hypertrophy develops and whether it is associated with altered expression of any specific genes, especially atrial natriuretic peptide (ANP) and contractile protein genes, in the hypertrophied ventricle. Cardiac hypertrophy in JVS mice became apparent at 10 days after birth and progressed during development. The hypertrophy was observed in the ventricles but not in the atria. ANP mRNA was more intensively expressed in JVS ventricles than in control even at 5 days. Carnitine administration ameliorated the cardiac hypertrophy and suppressed the augmentation of ANP mRNA in the ventricles. Isoform change of expression of α-actin genes from cardiac to skeletal was seen in the ventricles of JVS mice at 2 weeks. There was no difference in the ratio of β-myosin heavy chain mRNA to α-myosin heavy chain mRNA between control and JVS mice at 5 days, but at 2 weeks the ratio was significantly lower in JVS mice than in control. These results suggest that the molecular characteristics of cardiac hypertrophy caused by carnitine deficiency are different from those of cardiac hypertrophy caused by aortic constriction..
478. Junko Hiraoka, Kiminori Hosoda, Yoshihiro Ogawa, Katsumi Ikeda, Yasuo Nara, Hiroaki Masuzaki, Kazuhiko Takaya, Kiyoshi Nakagawa, Tomoshi Mashimo, Makoto Sawamura, Richard J. Koletsky, Yukio Yamori, Kazuwa Nakao, Augmentation of obese (ob) gene expression and leptin secretion in obese spontaneously hypertensive rats (Obese SHR or Koletsky rats), Biochemical and Biophysical Research Communications, 10.1006/bbrc.1997.6145, 231, 3, 582-585, 1997.02, To explore the pathophysiologic roles of the obese (ob) gene product, leptin, in the development of obesity and hypertension, we examined ob gene expression and leptin secretion in obese spontaneously hypertensive rats (obese SHR or Koletsky rats) at the stage of established obesity and hypertension. Expression of the ob gene was augmented in the epididymal, mesenteric, subcutaneous, and retroperitoneal white adipose tissue (WAT) from 20-week-old male obese SHR compared to their lean littermates (lean SHR). Using a radioimmunoassay for rat leptin, we also measured plasma leptin levels in 20-week-old lean and obese SHR. Plasma leptin levels in obese SHR (292.5 ± 37.1 ng/ml) were more than 100-fold higher than those in lean SHR (2.8 ± 1.0 ng/ml). The present study demonstrates that ob gene expression and leptin secretion are markedly augmented in obese SHR..
479. J. Hiraoka, K. Hosoda, Yoshihiro Ogawa, K. Ikeda, Y. Nara, H. Masuzaki, M. Sawamura, K. Takaya, Y. Yamori, K. Nakao, Augmented plasma leptin levels in obese spontaneously hypertensive koletsky (fa(k)/fa(k)) rats (Obese SHR), Japanese Heart Journal, 10.1536/ihj.38.591, 38, 4, 1997.01.
480. Kiyoshi Mori, Yoshihiro Ogawa, Naohisa Tamura, Ken Ebihara, Tomohiro Aoki, Seiji Muro, Shoichi Ozaki, Issei Tanaka, Kei Tashiro, Kazuwa Nakao, Molecular cloning of a novel mouse aspartic protease-like protein that is expressed abundantly in the kidney, FEBS Letters, 10.1016/S0014-5793(96)01473-1, 401, 2-3, 218-222, 1997.01, By use of the signal sequence trap method, we isolated a cDNA encoding a novel aspartic protease-like protein from the mouse kidney, and termed it 'kidney-derived aspartic protease-like protein (KAP)'. The protein, a 419-amino-acid polypeptide with a 16-amino-acid signal sequence, had 47% identity with mouse cathepsin D, and its overall structure was closely related to known aspartic proteases. Northern blot analysis revealed that KAP mRNA is expressed at the highest level in the kidney, at a moderate level in the lung, and at low levels in the spleen and adipose tissue. In situ hybridization analysis demonstrated that the mRNA is expressed abundantly in the proximal straight tubule and slightly, but significantly, in the proximal convoluted tubule in the kidney. This intra-renal distribution differs distinctly from those of previously reported proteases such as cathepsins B, D, and H..
481. Jun Yamashita, Hiroshi Itoh, Yoshihiro Ogawa, Naohisa Tamura, Kazuhiko Takaya, Toshio Igaki, Kentaro Doi, Tae Hwa Chun, Mayumi Inoue, Ken Masatsugu, Kazuwa Nakao, Opposite regulation of Gax homeobox expression by angiotensin II and C- type natriuretic peptide, Hypertension, 10.1161/01.hyp.29.1.381, 29, 1 II, 381-387, 1997.01, Growth arrest specific homeobox (Gax) gene was isolated from rat aorta cDNA library and its expression was largely confined to the cardiovascular tissues. Gax gene was rapidly downregulated by platelet-derived growth factor in vascular smooth muscle cells (VSMCs) and overexpressed Gax was reported to reduce the neointimal thickening after balloon injury in vivo. We have demonstrated that angiotensin II (Ang II) stimulates vascular growth. In contrast, we also reported that C-type natriuretic peptide (CNP) is secreted from vascular endothelial cells to act as a novel endothelium derived relaxing peptide and inhibits vascular growth via cGMP cascade. In the present study, we examined the effects of Ang II and CNP on Gax gene expression in VSMCs. In quiescent rat aortic VSMCs, Gax mRNA (2.3 kb) level became negligible 6 hours after the addition of Ang II (10-6 mol/L). The inhibitory action of Ang II on Gax mRNA expression (ED50: 10-11 mol/L) was almost completely blocked by an AT1R antagonist, CV11974. In contrast, CNP 10-6 mol/L augmented Gax mRNA expression to exhibit 1.8-fold increase of the control 12 hours after the stimulation. This effect of CNP was mimicked by the addition of 8-bromoadenosine 3':5'-cyclic monophosphate. The addition of C-ANF[4-23], an atrial natriuretic peptide-C receptor-specific agonist and devoid of stimulating cGMP production, exhibited no effect on Gax mRNA expression. Simultaneous administration of Ang II and CNP revealed that CNP (10-6 mol/L) significantly attenuated the inhibitory action of Ang II (10-10 mol/L) on Gax mRNA expression. These results suggest that Gax is a common transcription factor involved in the signaling pathway of vascular growth for Ang II and CNP and regulates the cell cycle and/or phenotype of VSMCs for vascular remodeling in hypertension and atherosclerosis..
482. Noriko Satoh, Yoshihiro Ogawa, Goro Katsuura, Tetsuo Tsuji, Hiroaki Masuzaki, Junko Hiraoka, Taku Okazaki, Mikio Tamaki, Minoru Hayase, Yasunao Yoshimasa, Shigeo Nishi, Kiminori Hosoda, Kazuwa Nakao, Pathophysiological significance of the obese gene product, leptin, in ventromedial hypothalamus (VMH)lesioned rats
Evidence for loss of its satiety effect in VMH-lesioned rats, Endocrinology, 10.1210/endo.138.3.4989, 138, 3, 947-954, 1997.01, To explore the pathophysiological significance of the obese (ob) gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats, we examined the synthesis and secretion of leptin and its satiety effect in VMH- lesioned rats compared with those in sham-operated rats. Northern blot analysis revealed that ob gene expression is markedly augmented in the mesenteric and sc white adipose tissue, but remained unchanged in the epididymal white adipose tissue during the development of obesity in VMH- lesioned rats. Plasma leptin levels were relatively constant in sham-operated rats, but were elevated during the development of obesity in VMH-lesioned rats. In sham-operated rats, a single iv (1.0 mg/rat) or intracerebroventricular (2.0 μg/rat) injection of recombinant human leptin reduced food intake and body weight gain in sham-operated rats. By contrast, no significant effect on food intake or body weight gain was observed in VMH- lesioned rats. The present study provides evidence that VMH-lesioned rats overproduce leptin and increase its release but cannot respond to it and suggests that the loss of its satiety effect contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats..
483. Tae Hwa Chun, Hiroshi Itoh, Yoshihiro Ogawa, Naohisa Tamura, Kazuhiko Takaya, Toshio Igaki, Jun Yamashita, Kentaro Doi, Mayumi Inoue, Ken Masatsugu, Risa Korenaga, Joji Ando, Kazuwa Nakao, Shear stress augments expression of C-type natriuretic peptide and adrenomedullin, Hypertension, 10.1161/01.HYP.29.6.1296, 29, 6, 1296-1302, 1997.01, Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls; these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium- derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm2, 2.5-fold increase of static; 15 dyne/cm2, 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radio-immunoassay for CNP, Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, 1.2-fold increase of static; 24 hours, threefold increase) and shear stress intensity-dependent manner (15 dyne/cm2, threefold increase compared with that at 5 dyne/cm2). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress-dependent vasodilator and antiproliferative effects..
484. Hiroshi Itoh, Shin Ichi Suga, Yoshihiro Ogawa, Yasato Komatsu, Naohisa Tamura, Toshio Igaki, Jun Yamashita, Tadashi Ikeda, Kentaro Doi, Tae Hwa Chun, Mayumi Inoue, Katsuhiko Matsuda, Takaaki Yoshimasa, Makiko Ueda, Toshihiko Ban, Kazuwa Nakao, Significance of vascular natriuretic peptide system in vascular remodeling in humans and its application to gene therapy, Annals of the New York Academy of Sciences, 10.1111/j.1749-6632.1997.tb52037.x, 811, 533-541, 1997.01.
485. Toshio Igaki, Hiroshi Itoh, Shin Ichi Suga, Yasato Komatsu, Yoshihiro Ogawa, Kentaro Doi, Takaaki Yoshimasa, Kazuwa Nakao, Insulin suppresses endothelial secretion of C-type natriuretic peptide, a novel endothelium-derived relaxing peptide, Diabetes, 45, 3 SUPPL., S62-S64, 1996.12, We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells (ECs) and acts as an endothelium-derived relaxing peptide. We further demonstrated the detection of the gene transcripts of CNP and atrial natriuretic peptide (ANP) B receptor, a specific receptor for CNP, in human blood vessels. We thus propose the existence of a vascular natriuretic peptide system (NPS). CNP secretion was also demonstrated to be stimulated by various growth factors and cytokines. To clarify the significance of vascular NPS in proliferative vascular complications associated with diabetes, hypertension, or atherosclerosis, in the present study we examined the effect of insulin on CNP secretion from cultured ECs. Insulin at a concentration in the physiological range (10-10-10-7 mol/l) potently suppressed CNP secretion, whereas insulin at the same concentration did not suppress endothelin (ET) secretion from EC. IGF-I had no significant effect on CNP secretion. Insulin, therefore, can be a potent inhibitor of CNP secretion through the activation of insulin receptor. Since CNP has been shown to be a potent inhibitor of vascular smooth muscle cell proliferation, the present study suggests the possibility that attenuated activity of vascular NPS is associated with hyperinsulinemia, which might result in proliferative vascular lesions..
486. Taku Okazaki, Yoshihiro Ogawa, Naohisa Tamura, Kiyoshi Mori, Naohi Isse, Tomohiro Aoki, Julie M. Rochelle, Makoto M. Taketo, Michael F. Seldin, Kazuwa Nakao, Genomic organization, expression, and chromosomal mapping of the mouse adrenomedullin gene, Genomics, 10.1006/geno.1996.0576, 37, 3, 395-399, 1996.11, We have isolated and characterized the mouse adrenomedullin (AM) gene (Adm) and determined its chromosomal location. The gene spans approximately 2.1 kb and is organized into four exons separated by three introns. The transcription start site was determined to be the adenine nucleotide at - 618. The mouse AM 5'-flanking region contains a TATA box-like sequence and several cis-acting regulatory elements. Analysis of the nucleotide and deduced amino acid sequences revealed that mouse preproAM is a 184-amino- acid polypeptide, from which AM and proAM N-terminal 20 peptide are cleaved. Using restriction fragment length variants on a DNA panel of interspecific backcross mice, we mapped Adm to a distal region of mouse chromosome 7..
487. Kazuhiko Takaya, Yoshihiro Ogawa, Naohi Isse, Taku Okazaki, Noriko Satoh, Hiroaki Masuzaki, Kiyoshi Mori, Naohisa Tamura, Kiminori Hosoda, Kazuwa Nakao, Molecular cloning of rat leptin receptor isoform complementary DNAs-identification of a missense mutation in Zucker fatty (fa/fa) rats, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1996.1133, 225, 1, 75-83, 1996.08, We cloned the full-length rat leptin receptor (Ob-R) isoform complementary DNAs (cDNAs) and examined the gene expression in rats. We also identified a mutation in Ob-R in Zucker fatty (fa/fa) rats. Three alternatively spliced isoforms (Ob-Ra, Ob-Rb, and Ob-Re) have been identified, which are closely related to the gp130 signal-transduction component of class I cytokine receptors. Rat Ob-Ra and Ob-Rb were single transmembrane proteins, which differ in the C-terminal amino acid sequences. On the other hand, Ob-Re had no transmembrane domain and was a soluble form of the receptor. Reverse transcription-polymerase chain reaction analysis revealed that Ob-R isoform messenger RNAs (mRNAs) are expressed in a wide variety of rat tissues in tissue-specific manners. A missense mutation (an A to C conversion at nucleotide position 806) was found in the extracellular domain of all the isoforms in Zucker fatty (fa/fa) rats, which resulted in an amino acid change from Gin to Pro at + 269 (the Gln269Pro mutation). These Ob-R isoform mRNAs were present in the brain from Zucker fatty (fa/fa)) rats at comparable amounts to those in their lean littermates. The present study provides new insight into the molecular mechanisms for Ob-R..
488. Minoru Hayase, Yoshihiro Ogawa, Goro Katsuura, Haruyuki Shintaku, Kiminori Hosoda, Kazuwa Nakao, Regulation of obese gene expression in KK mice and congenic lethal yellow obese KKA(y) mice, American Journal of Physiology - Endocrinology and Metabolism, 10.1152/ajpendo.1996.271.2.e333, 271, 2 34-2, E333-E339, 1996.08, To elucidate the regulation of obese (ob) gene expression in obesity and diabetes, we examined ob gene expression in KK mice and congenic lethal yellow obese KKA(y) mice. Northern blot analysis revealed that the ob mRNA levels are roughly equivalent in each of the epididymal, mesenteric, and subcutaneous white adipose tissue (WAT) from KK and KKA(y) mice at 4 wk of age, when the obese phenotype of KKA(y) mice was not apparent. Expression of the ob gene was augmented in the mesenteric and subcutaneous WAT but was unchanged in the epididymal WAT in KKA(y) mice at 12 wk of age, when KKA(y) mice developed marked obesity with hyperglycemia, hyperlipidemia, and hyperinsulinemia. The ob gene expression was also examined during fasting in 12-wk-old KK and KKA(y) mice. After 24 or 72 h of fasting in both mouse strains, ob gene expression was downregulated in the epididymal and mesenteric WAT but was unchanged in the subcutaneous WAT. The present study demonstrates that adipose tissue expression of the ob gene is regulated depending on the nutritional status in KK and KKA(y) mice..
489. Naohisa Tamura, Yoshihiro Ogawa, Akihiro Yasoda, Hiroshi Itoh, Yoshihiko Saito, Kazuwa Nakao, Two cardiac natriuretic peptide genes (Atrial natriuretic peptide and brain natriuretic peptide) are organized in tandem in the mouse and human genomes, Journal of Molecular and Cellular Cardiology, 10.1006/jmcc.1996.0170, 28, 8, 1811-1815, 1996.08, Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones, are produced mainly by the atrium and ventricle, respectively, and are involved in body fluid homeostasis and blood pressure control. The ANP and BNP gene expressions are markedly augmented in ventricles of patients with a wide variety of cardiovascular diseases. It has been demonstrated that the ANP and BNP genes are tightly linked on mouse chromosome 4 and on the distal short arm of human chromosome 1. However, the precise physical map of the ANP and BNP genes has never been elucidated. In the present study, we characterized the genomic DNA fragment containing the ANP and BNP genes in mice and humans. Three genomic DNA clones harboring the entire mouse BNP gene were isolated from a 129/ Sv mouse genomic DNA library. Nucleotide sequence analysis revealed that a phage clone (λmBNP3) contains at its 3'-end the 5'-flanking region and the first 209-bp sequence of the first exon of the mouse ANP gene. In mice, the BNP gene was located about 12kb upstream of the ANP gene. By polymerase chain reaction, we isolated an approximately 11-kb human genomic DNA fragment containing the third exon of the BNP gene and the first and second exons of the ANP gene. In humans, the BNP gene was located upstream of the ANP gene, approximately 8 kb apart. The present study provides the direct evidence that the ANP and BNP genes are organized in tandem in the mouse and human genomes..
490. Michio Suda, Kiyoshi Tanaka, Mitsuo Fukushima, Koshi Natsui, Akihiro Yasoda, Yasato Komatsu, Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, C-type natriuretic peptide as an autocrine/paracrine regulator of osteoblast
Evidence for possible presence of bone natriuretic peptide system, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1996.0836, 223, 1, 1-6, 1996.06, C-type natriuretic peptide (CNP) is a local regulator in the brain and vascular wall. We present data to demonstrate the production and action of CNP in the osteoblast. CNP increased cGMP production, far more potently than atrial natriuretic peptide (ANP) in an osteoblastic cell line, MC3T3-E1. Since ANP and CNP are the ligands for two particulate guanylate cyclases, guanylate cyclase-A (GC-A) and guanylate cyclase-B (GC-B), respectively, these results reveal the expression of GC-B in MC3T3-E1. In addition, CNP mRNA and CNP-like immunoreactivity were detected in cell extracts from MC3T3-E1 and its culture medium, respectively. Both CNP and 8-bromo cGMP dose-dependently decreased [3H]thymidine uptake, without affecting alkaline phosphatase activity. These results indicate that CNP is a novel autocrine/paracrine regulator of osteoblast and suggest the presence of "bone natriuretic peptide system.".
491. Kiminori Hosoda, Hiroaki Masuzaki, Yoshihiro Ogawa, Takashi Miyawaki, Junko Hiraoka, Ikuko Hanaoka, Akiko Yasuno, Toshiyuki Nomura, Yukio Fujisawa, Yasunao Yoshimasa, Shigeo Nishi, Yukio Yamori, Kazuwa Nakao, Development of radioimmunoassay for human leptin, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1996.0579, 221, 2, 234-239, 1996.04, Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma human leptin-LI concentration in nonobese subjects (17.6 < body mass index (BMI) < 23.0) was 5.6 ± 1.3 (mean ± SE) ng/ml, while that in obese subjects (29.0 < BMI) was 43.0 ± 43.0 ± 9.4 (mean ± SE) ng/ml. In gel permeation chromatographic analyses, leptin-LI in culture media and plasma consisted of single component emerging at the elution position of recombinant human leptin. These findings indicate that leptin is secreted from the adipose tissue into the circulating blood as a large molecular form corresponding to recombinant leptin. The RIA developed in the present study will be a powerful tool to investigate the physiological and pathophysiological significance of leptin in human..
492. Naohisa Tamura, Hiroshi Itoh, Yoshihiro Ogawa, Osamu Nakagawa, Masaki Harada, Tae Hwa Chun, Shin Ichi Suga, Takaaki Yoshimasa, Kazuwa Nakao, cDNA cloning and gene expression of human type Iα cGMP-dependent protein kinase, Hypertension, 10.1161/01.hyp.27.3.552, 27, 3 II, 552-557, 1996.03, The type I cGMP-dependent protein kinase (cGK) is one of the major pathways for the cGMP cascade and has been demonstrated to inhibit platelet aggregation, relax smooth muscle cells, and control cardiocyte contractility. There are two subtypes of the type I cGK, cGKIα and cGKIβ. The former is more sensitive to cGMP than the latter. In humans, cGKIβ cDNA was isolated, but the full structure and tissue-specific gene expression of cGKIα have not been determined. The significance of cGK in human cardiovascular diseases has not been investigated at the molecular level. In the present study, we isolated the full-length human cGKIα cDNA (-36 to +2177: the translation start site: +1) encoding the 671-amino acid protein. Nucleotides +267 to +2177 of the isolated cDNA were identical to the corresponding nucleotides of human cGKIβ cDNA. Southern blot analysis suggested that human cGKIα and cGKIβ are generated by alternative splicing of a single gene assigned to chromosome 10. By Northern blot analysis, we detected abundant human cGKIα mRNA (7.0 kb) in the aorta, heart, kidneys, and adrenals. In contrast, human cGKIβ mRNA (7.0 kb) was detected abundantly only in the uterus. In cultured vascular smooth muscle cells, the type I cGK mRNA concentration was reduced to 10% of the basal level by 4 x 10-10 mol/L platelet-derived growth factor. Angiotensin II (10-8 mol/L), transforming growth factor-β (4 x 10-11 mol/L), and tumor necrosis factor-α (6 x 10-6 mol/L) also exhibited an inhibitory effect on type I cGK gene expression. These findings suggest a pathophysiological implication of the type I cGK in cardiovascular diseases, including hypertension and atherosclerosis..
493. Yoshihiro Miyamoto, Takaaki Yoshimasa, Hiroshi Arai, Kazuhiko Takaya, Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts, Biochemical Journal, 10.1042/bj3130795, 313, 3, 795-801, 1996.02, In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular-mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts result from alternative RNA splicing (designated ET-ARΔ4 and ET-ARΔ3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-ARΔ4 and ET-ARΔ3,4, as well as ET-AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression..
494. Hiroaki Masuzaki, Kiminori Hosoda, Yoshihiro Ogawa, Michika Shigemoto, Noriko Satoh, Kiyoshi Mori, Naohisa Tamura, Shigeo Nishi, Yasunao Yoshimasa, Yukio Yamori, Kazuwa Nakao, Augmented expression of obese (ob) gene during the process of obesity in genetically obese-hyperglycemic Wistar fatty (falfa) rats, FEBS Letters, 10.1016/0014-5793(95)01472-1, 378, 3, 267-271, 1996.01, Expression of the obese (ob) gene is up-regulated in the adipose tissue in several obese rodent models. To study the regulation of the ob gene expression during the development of obesity, we examined the ob gene expression in genetically obese-hyperglycemic Wistar fatty (falfa) rats at several stages of obesity. The ob mRNA levels in the adipose tissue from Wistar fatty rats was unequivocally augmented and continued to rise in the process of obesity. Furthermore, the ob gene expression in this obese model was much more rapidly enhanced in the mesenteric fat than in the subcutaneous fat. Moreover, the ob gene expression was more greatly augmented in the mesenteric fat than the lipoprotein lipase gene expression. These results suggest the presence of obesity-linked and region-specific regulation of the ob gene expression..
495. T. Igaki, H. Itoh, S. Suga, N. Hama, Y. Ogawa, Y. Komatsu, M. Mukoyama, A. Sugawara, T. Yoshimasa, I. Tanaka, K. Nakao, C-type natriuretic peptide in chronic renal failure and its action in humans, Kidney International, Supplement, 55, S-144-S-147, 1996.01, We have previously reported that C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is produced in vascular endothelial cells and acts as an endothelium-derived relaxing peptide. To clarify the clinical significance of CNP in disorders, we examined the plasma level of CNP in patients with various cardiovascular diseases, including chronic renal failure (CRF) patients who were under hemodialysis therapy. We also investigated biological effects of intravenously-administered CNP (0.43 nmol/kg) by bolus injection from the peripheral vein in healthy volunteers and measured systemic hemodynamic variables, plasma levels of CNP, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), cGMP, aldosterone and also urine volume, urinary excretions of sodium, potassium, chloride and cGMP. The plasma CNP levels in healthy humans (N = 13) was 1.4 ± 0.6 fmol/ml. In CRF patients, the plasma CNP significantly increased up to 3.0 ± 1.1 fmol/ml. The administration of CNP elicited significant increase of plasma cGMP level (from 4.77 ± 1.25 to 8.33 ± 1.59 pmol/ml 15 min after the administration) and of urinary cGMP excretion (from 30.7 ± 4.3 to 74.9 ± 13.4 nmol/30 min). Intravenously-administered CNP exerted significant diuretic (%increase: +117 ± 85.0), natriuretic, kalliuretic and chloriuretic actions with the increase of endogenous creatinine clearance. CNP also elicited significant hypotensive actions ((Δ)BPs/(Δ)BPd: -4.3 ± 1.3/-4.1 ± 1.0 mm Hg) with the concomitant increase of heart rate (+7.6 ± 2.6 bpm). Plasma aldosterone concentration significantly decreased from 45.4 ± 2.3 to 35.4 ± 4.9 pg/ml 30 minutes after the administration. Taken together, these results suggest a role for CNP in human renal function..
496. K. Kikuta, H. Yasue, M. Yoshimura, E. Morita, H. Sumida, H. Kato, K. Kugiyama, H. Ogawa, K. Okumura, Y. Ogawa, K. Nakao, Increased plasma levels of B-type natriuretic peptide in patients with unstable angina, American heart journal, 10.1016/S0002-8703(96)90396-8, 132, 1 I, 101-107, 1996.01, This study was designed to examine the plasma levels of B-type or brain natriuretic peptide (BNP), as well as A-type or atrial natriuretic peptide (ANP) in patients with unstable angina as compared with those in patients with stable exertional angina end control subjects. We measured the plasma levels of BNP and ANP in 33 patients with unstable angina, 20 patients with stable exertional angina, and 20 control subjects. The plasma levels of BNP were significantly increased in patients with unstable angina compared with those in patients with stable exertional angina and control subjects, respectively (39.5 ± 29.4 pg/ml vs 15.1 ± 8.0 pg/ml; p < 0.01 and 39.5 ± 29.4 pg/ml vs 19.3 ± 6.4 pg/ml; p < 0.01, respectively). On the other hand, there was no significant difference in the plasma levels of ANP among the three groups. Furthermore, in patients with unstable angina, the plasma levels of BNP decreased significantly after the medical treatment (from 39.5 ± 29.4 pg/ml to 15.8 ± 11.0 pg/ml; p < 0.01), whereas the plasma levels of ANP did not change. We conclude that the plasma levels of BNP are increased in the majority of patients with unstable angina and that the increased levels decrease toward normal after treatment..
497. Kazuhiko Takaya, Yoshihiro Ogawa, Junko Hiraoka, Kiminori Hosoda, Yukio Yamori, Kazuwa Nakao, Richard J. Koletsky, Nonsense mutation of leptin receptor in the obese spontaneously hypertensive koletsky rat, Nature genetics, 10.1038/ng1096-130, 14, 2, 130-131, 1996.01.
498. Yasato Komatsu, Hiroshi Itoh, Shin Ichi Suga, Yoshihiro Ogawa, Norio Hama, Ichiro Kishimoto, Osamu Nakagawa, Toshio Igaki, Kentaro Doi, Takaaki Yoshimasa, Kazuwa Nakao, Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells
Role of the vascular natriuretic peptide system in vascular growth inhibition, Circulation research, 10.1161/01.RES.78.4.606, 78, 4, 606-614, 1996.01, Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-β (TGF-β) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-β abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-β. Furthermore, the conditioned medium from ECs stimulated by TGF-β was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs..
499. Yasato Komatsu, Hiroshi Itoh, Shin Ichi Suga, Toshio Igaki, Yoshihiro Ogawa, Ichiro Kishimoto, Osamu Nakagawa, Takaaki Yoshimasa, Kazuwa Nakao, Regulation of secretion and clearance of C-type natriuretic peptide in the interaction of vascular endothelial cells and smooth muscle cells, Journal of hypertension, 10.1097/00004872-199605000-00007, 14, 5, 585-592, 1996.01, Objective. To clarify the significance of C-type natriuretic peptide in the interaction between endothelial cells and vascular smooth muscle cells by investigating the endothelial production of C-type natriuretic peptide and the clearance mechanism of C-type natriuretic peptide using the endothelial cells-smooth muscle cells co-culture system. Results. Secretion of C-type natriuretic peptide in the direct co-culture of endothelial cells with smooth muscle cells elicited as much as a 60-fold increase compared with endothelial cells alone. The accumulation of intracellular cyclic GMP in the co-culture was consequently increased and the elevation of cyclic GMP level in the co-culture was abolished by the anti-C-type natriuretic peptide monoclonal antibody. The elevated cyclic GMP production in the co-culture was abolished by the anti-transforming growth factor-β neutralizing antibody. Candoxatrilat (10-6-10-4 mol/l), a neutral endopeptidase inhibitor, dose-dependently increased the concentrations of C-type natriuretic peptide in the culture medium with endothelial cells alone, but not in the endothelial cells-smooth muscle cells co-culture. The transcript of neutral endopeptidase messenger RNA was detected in endothelial cells but not in smooth muscle cells by reverse transcriptase polymerase chain reaction. Treatment with C-atrial natriuretic factor (10-9-10-6 mol/l), the specific ligand for the clearance receptor of the natriuretic peptides, resulted in dose-dependent augmentation of C-type natriuretic peptide concentration and concomitant intracellular cyclic GMP production in the endothelial cells-smooth muscle cells co-culture but not in endothelial cells alone. Conclusion. The present study demonstrated that direct interaction between endothelial cells and smooth muscle cells augments C-type natriuretic peptide secretion from endothelial cells through transforming growth factor-β activation, and revealed that the enzymatic degradation is responsible for the steady state level of C-type natriuretic peptide in endothelial cells alone and that the receptor-mediated clearance mainly determines the augmented level of C-type natriuretic peptide in the interaction between endothelial cells and smooth muscle cells. The results taken together raise the possibility that endothelial C-type natriuretic peptide might play a role in regulation of vascular tone and remodelling..
500. Hiroaki Masuzaki, Hisato Jingami, Naoki Matsuoka, Osamu Nakagawa, Yoshihiro Ogawa, Megumi Mizuno, Yasunao Yoshimasa, Tokuo Yamamoto, Kazuwa Nakao, Regulation of very-low-density lipoprotein receptor in hypertrophic rat heart, Circulation Research, 10.1161/01.RES.78.1.8, 78, 1, 8-14, 1996.01, To elucidate the regulation of very-low-density lipoprotein (VLDL) receptor, we have studied its gene expression in the heart of spontaneously hypertensive rat-stroke prone (SHR-SP, an animal model for hypertension- induced cardiac hypertrophy) compared with Wistar-Kyoto rats. RNase protection assay showed that ventricular VLDL receptor mRNA falls to 41% of normal levels at 4 weeks, when hypertensions is not yet fully developed, and drops further to 14% at 13 weeks, when cardiac hypertrophy is established. Lipoprotein lipase mRNA decreases in parallel with VLDL receptor mRNA. In cultured neonatal rat ventricular cardiomyocytes. VLDL receptor mRNA decreases in parallel with the process of cardiocyte hypertrophy during the 24 hours after treatment with 10-8 mol/L endothelin-1, falling to 40% of the initial value. These results demonstrate that there is downregulation of VLDL receptor gene expression in cardiac hypertrophy both in vivo and in vitro and suggest that the regulation of the VLDL receptor is possibly linked with the switch in energy substrate from lipid to glucose known to occur in cardiac hypertrophy..
501. Kazuwa Nakao, Yoshihiko Saito, Hiroshi Itoh, Yoshihiro Ogawa, Osamu Nakagawa, Masaki Harada, Izuru Masuda, Takaaki Yoshimasa, Roles of natriuretic peptides in heart failure, Journal of Cardiac Failure, 10.1016/S1071-9164(96)80068-6, 2, SUPPL. 1, S129-S133, 1996.01.
502. Kazuwa Nakao, Hiroshi Itoh, Yoshihiko Saito, Masashi Mukoyama, Yoshihiro Ogawa, The natriuretic peptide family, Current opinion in nephrology and hypertension, 10.1097/00041552-199601000-00003, 5, 1, 4-11, 1996.01, The natriuretic peptide system is a complicated system comprising at least three endogenous peptides (atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide) and three receptors [the atrial natriuretic peptide-A receptor (guanylyl cyclase A), the atrial natriuretic peptide-B receptor (guanylyl cyclase B) and the clearance receptor]. The accumulated evidence indicates that this system is implicated in the control of blood pressure, body fluid homeostasis and vascular remodelling as cardiac hormone and local regulator..
503. Masaaki Hasegawa, Norimasa Sagawa, Hidetaka Nanno, Hiroaki Itoh, Kumiko Inamori, Yoshiyuki Ihara, Fuminori Kobayashi, Takahide Mori, Juri Yano, Gotaro Shirakami, Shin Ichi Suga, Yoshihiro Ogawa, Takaaki Yoshimasa, Kazuwa Nakao, Endothelin-1-like immunoreactivity and endothelin receptors in the human placenta from normotensive and hypertensive pregnancies, Journal of Perinatal Medicine, 10.1515/jpme.1996.24.5.451, 24, 5, 451-460, 1996, The levels of endothelin-1-like immunoreactivity (ET-1-LI) and characteristics of endothelin receptors in the chorionic villous tissue of human placenta were determined. The ET-1-LI level in chorionic villous tissue obtained from normal term placenta was 2,450 ± 940 pg/g wet weight (mean ± SD, n = 4). Further analysis using gel permeation chromatography and reverse-phase high performance liquid chromatography showed that the main ET-1-LI constituent of ET-1-LI in this tissue was ET-1. Scatchard analysis of [125I]ET-1 binding to the membrane fraction of chorionic villous tissue obtained from term placenta showed high affinity receptor sites with an apparent dissociation constant (Kd) of 23.6 ± 11.1 pM and a Bmax value of 388 ± 238 fmol/mg protein (n = 5). The same binding study with [125I]ET-3 showed a Kd of 13.9 ± 3.8 pM and a Bmax value of 176 ± 78 fmol/mg protein (n = 5). These results suggest that both ET-A and ET-B receptors (ET-AR and ET-BR) are expressed in chorionic villous tissue. This finding was further confirmed by Northern blot analysis showing the expression of both ET-AR and ET-BR mRNAs in this tissue. ET-1-LI in the umbilical venous plasma of the newborns from women with pregnancy-induced hypertension (PIH) (38.3 ± 10.4 pg/mL, n = 5) was significantly (P < 0.05) higher than that in the normal newborns from normotensive pregnant women (26.3 ± 5.2 pg/mL, n = 12). However, in placental chorionic villous tissue obtained from PIH women, both ET-1-LI level and ET binding profile were not different from those in chorionic villous tissue from normotensive pregnant women. These results suggest that the abundant ET-ET receptor system is present in the placental chorionic villous tissue and that this system is not the major factor of the pathogenesis of placental dysfunction occurring in PIH because these systems are similar in normotensive and hypertensive pregnancies..
504. K. Mori, I. Tanaka, M. Kotani, F. Miyaoka, T. Sando, S. Muro, Y. Sasaki, O. Nakagawa, Y. Ogawa, T. Usui, S. Ozaki, A. Ichikawa, S. Narumiya, K. Nakao, Gene expression of the human prostaglandin E receptor EP4 subtype
Differential regulation in monocytoid and lymphoid lineage cells by phorbol ester, Journal of Molecular Medicine, 10.1007/BF00207510, 74, 6, 333-336, 1996, We isolated a cDNA clone encoding the human prostaglandin (PG) E receptor EP4 subtype and examined the gene expression in human blood cells. Northern blot analysis revealed that the EP4 gene is expressed at a high level in peripheral blood mononuclear cells, and at lower levels in cultured human blood cell lines, THP-1 and U937 (monocytoid cell lines), MOLT-4 and Jurkat (T-cell lines), and Raji (B-cell line). To examine regulation of the EP4 gene expression in the immune system, we studied the effects of phorbol 12-myristate 13-acetate (PMA) on these cell lines. Gene expression was upregulated in THP-1, U937, and Raji cells by PMA, and was downregulated in MOLT-4 and Jurkat cells. In THP-1 cells the effects of PMA were further analyzed, and the upregulation of the EP4 gene was shown to be followed by an increase in PGE2 binding sites and in PGE2-induced cAMP accumulation. In the striking contrast, other PGE receptor subtypes (EP1, EP2 and EP3) and other prostanoid receptors (IP and DP) were shown not to be upregulated by PMA. Therefore, this is the first demonstration of a highly specific upregulation of the EP4 subtype in THP-1 cells treated with PMA, suggesting the importance of the EP4 subtype in the immune system. In the present study we also clarified that EP4 gene expression is regulated differently among human monocytoid and lymphoid lineage cells, thus leading to the better understanding of the regulatory mechanisms for the human EP4 gene expression in the immune system..
505. Junko Hiraoka, Hiroshi Arai, Takaaki Yoshimasa, Kazuhiko Takaya, Yoshihiro Miyamoto, Jun Yamashita, Shin‐ichi ‐i Suga, Yoshihiro Ogawa, Gotaro Shirakami, Hiroshi Itoh, Yukio Yamori, Kazuwa Nakao, AUGMENTED EXPRESSION OF THE ENDOTHELIN‐A RECEPTOR GENE IN CULTURED MESANGIAL CELLS FROM STROKE‐PRONE SPONTANEOUSLY HYPERTENSIVE RATS, Clinical and Experimental Pharmacology and Physiology, 10.1111/j.1440-1681.1995.tb02876.x, 22, S191-S192, 1995.11, 1. To elucidate the role of endothelin (ET) receptor in hypertension, we studied the expression of the ET‐A receptor (ET‐ AR) gene and the ET‐B receptor (ET‐BR) gene in cultured mesangial cells isolated from stroke‐prone spontaneously hypertensive rats (SHRSP) and Wistar‐Kyoto (WKY) rats. 2. The mesangial cells from both SHRSP and WKY expressed ET‐AR predominantly. The level of the ET‐AR mRNA in mesangial cells from SHRSP was 5‐fold higher than that in the cells from WKY. The ET‐BR mRNA in the mesangial cells from both strains was hardly detectable by northern blot analysis. 3. These results demonstrate that the expression of the ET‐AR gene was markedly augmented in mesangial cells from SHRSP..
506. Hiroaki Masuzaki, Yoshihiro Ogawa, Kiminori Hosoda, Teruo Kawada, Toru Fushiki, Kazuwa Nakao, Augmented expression of the obese gene in the adipose tissue from rats fed high-fat diet, Biochemical and Biophysical Research Communications, 10.1006/bbrc.1995.2631, 216, 1, 355-358, 1995.11, Expression of the obese (ob) gene is augmented in the adipose tissue in several rodent models of genetic obesity. In the present study, we examined the ob gene expression in a rodent model of acquired obesity obtained by pure overfeeding of normal rats. Male Sprague-Dawley rats at 8 weeks of age were fed standard diet or high-fat diet. Rats fed high-fat diet developed moderate degree of obesity, hyperglycemia, and hyperlipidemia as compared with rats fed standard diet. Northern blot analysis revealed that the ob gene is expressed abundantly in the adipose tissue obtained from the epididymal, mesenteric, subcutaneous, retroperitoneal, and interscapular fat pads in rats fed standard diet. Expression of the ob gene was augmented in all the adipose tissue examined in rats fed high-fat diet. The present study demonstrates that the ob gene expression is augmented in the adipose tissue in diet-induced obesity, thereby suggesting the pathophysiologic roles of the ob gene in acquired obesity..
507. H. Jingami, H. Masuzaki, N. Matsuoka, O. Nakagawa, Y. Ogawa, M. Mizuno, T. Yamamoto, K. Nakao, DECREASED EXPRESSION OF THE VERY LOW DENSITY LIPOPROTEIN RECEPTOR mRNA IN THE CARDIAC VENTRICLE OF SPONTANEOUSLY HYPERTENSIVE RATS, Clinical and Experimental Pharmacology and Physiology, 10.1111/j.1440-1681.1995.tb02902.x, 22, S246-S248, 1995.11, 1. To elucidate the functional implication of very low density lipoprotein (VLDL) receptor, we studied the gene expression of VLDL receptor in rats. The VLDL receptor mRNA was highly expressed in the cardiac ventricle and skeletal muscle. Intermediate amounts of VLDL receptor mRNA were detected in adipose tissue, adrenal gland, brain and lung. Thus the tissue distribution of VLDL receptor mRNA in rats was similar to that reported previously in rabbits. 2. We studied the gene expression of the VLDL receptor in the heart of stroke‐prone spontaneously hypertensive rats (SHRSP), an animal model for hypertension‐induced cardiac hypertrophy. RNase protection assay showed that the level of ventricular VLDL receptor mRNA was already decreased to one half when hypertension was not fully developed, and further diminished to one fifth when cardiac hypertrophy was established. 3. It is reported that energy utilization in SHRSP hypertrophied myocardium is impaired. Our results suggest that inactive fatty acid metabolism in the ventricle of SHRSP is related to the lowered expression of the VLDL receptor which is postulated as a gate for triglyceride‐rich lipoprotein particle..
508. Y. Ogawa, H. Itoh, O. Nakagawa, G. Shirakami, N. Tamura, T. Yoshimasa, K. Nagata, N. Yoshida, K. Nakao, Characterization of the 5′-flanking region and chromosomal assignment of the human brain natriuretic peptide gene, Journal of Molecular Medicine, 10.1007/BF00202264, 73, 9, 457-463, 1995.09, Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle, and synthesis and secretion of BNP are greatly augmented in patients with congestive heart failure and in animal models of ventricular hypertrophy. In order to elucidate the molecular mechanisms underlying the human BNP gene expression in the heart, the human BNP gene was isolated from a size-selected genomic minilibrary. The 1.9-kb human BNP 5′-flanking region (-1813 to +110) contained an array of putative cis-acting regulatory elements. Various lengths of the cloned 5′-flanking sequences were linked upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene, and their promoter activities were assayed. The 1.9-kb promoter region showed a high-level CAT activity in cultured neonatal rat ventricular cardiocytes. When the CT-rich sequences (-1288 to -1095) were deleted, the high-level activity was reduced to approximately 30%. The 399-bp BNP 5′ flanking region (-289 to +110) showed approximately 10% activity of the 1.9-kb region. Furthermore, using human-rodent somatic hybrid cell lines, the BNP gene was assigned to human chromosome 1, on which the atrial natriuretic peptide gene is localized. The present study leads to a better understanding of the molecular mechanisms for the human BNP gene expression in the heart..
509. Yoshihiro Ogawa, Hiroaki Masuzaki, Naohi Isse, Taku Okazaki, Kiyoshi Mori, Michika Shigemoto, Noriko Satoh, Naohisa Tamura, Kiminori Hosoda, Yasunao Yoshimasa, Hisato Jingami, Teruo Kawada, Kazuwa Nakao, Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats, Journal of Clinical Investigation, 10.1172/JCI118204, 96, 3, 1647-1652, 1995.09, The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue ≥ interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromal- vascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene..
510. Osamu Nakagawa, Yoshihiro Ogawa, Hiroshi Itoh, Shin Ichi Suga, Yasato Komatsu, Ichiro Kishimoto, Kazuyoshi Nishino, Takaaki Yoshimasa, Kazuwa Nakao, Rapid transcriptional activation and early mRNA turnover of brain natriuretic peptide in cardiocyte hypertrophy
Evidence for brain natriuretic peptide as an "emergency" cardiac hormone against ventricular overload, Journal of Clinical Investigation, 10.1172/JCI118162, 96, 3, 1280-1287, 1995.09, We previously demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone mainly produced in the ventricle, while the major production site of atrial natriuretic peptide (ANP) is the atrium. To assess the pathophysiological role of BNP in ventricular overload, we have examined the gene expression of BNP, in comparison with that of ANP, in a model of cardiac hypertrophy using cultured neonatal rat ventricular cardiocytes. During cardiocyte hypertrophy evoked by endothelin-1, phenylephrine, or PMA, the steady state level of BNP mRNA increased as rapidly as the "immediate-early" induction of the c-fos gene expression, and reached a maximal level within 1 h. Actinomycin D, a transcriptional inhibitor, completely diminished the response, while the translational blockade with cycloheximide did not inhibit it. In contrast, ANP mRNA began to increase 3 h after the stimulation, and accumulated during cardiocyte hypertrophy. The BNP secretion from ventricular cardiocytes was also stimulated more rapidly than the ANP secretion. Furthermore, the turnover of BNP mRNA was significantly faster than that of ANP mRNA, being consistent with the existence of AUUUA motif in the 3′-untranslated region of BNP mRNA. These results demonstrate that the gene expression of BNP is distinctly regulated from that of ANP at transcriptional and posttranscriptional levels, and indicate that the characteristics of the BNP gene expression are suitable for its possible role as an "emergency" cardiac hormone against ventricular overload..
511. Norio Hama, Hiroshi Itoh, Gotaro Shirakami, Osamu Nakagawa, Shin Ichi Suga, Yoshihiro Ogawa, Izuru Masuda, Kuniaki Nakanishi, Takaaki Yoshimasa, Yukiya Hashimoto, Masayuki Yamaguchi, Ryouhei Hori, Hirofumi Yasue, Kazuwa Nakao, Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction, Circulation, 10.1161/01.CIR.92.6.1558, 92, 6, 1558-1564, 1995.09, Background: We have demonstrated that brain natriuretic peptide (BNP) is a cardiac hormone predominantly synthesized in and secreted from the ventricle. We have also reported that, compared with atrial natriuretic peptide (ANP), the plasma concentration of BNP is increased to a greater degree in patients with congestive heart failure and more rapidly in patients with acute myocardial infarction (AMI). Methods and Results: To investigate ventricular gent expression of BNP in AMI, we analyzed plasma and ventricular BNP concentrations along with ventricular BNP mRNA in rats with AMI produced by coronary artery ligation. The BNP concentration in the left ventricle increased about 2-fold as early as 12 hours postinfarction and 5-fold 1 day postinfarction compared with sham-operated rats, whereas left ventricular ANP concentration remained unchanged within 1 day. The tissue concentration of BNP increased in the noninfarcted region as well as in the infarcted region. The surviving myocytes in and around the necrotic tissues in the infarcted region were intensely stained with the anti-BNP antiserum, indicating augmented production in the remaining myocytes in the infarcts. The BNP concentration in the right ventricle also increased about 10-fold 12 hours postinfarction, whereas the ANP concentration remained unchanged within 12 hours. Northern blot analysis revealed that BNP mRNA expression was augmented 3-fold in the left ventricle as early as 4 hours postinfarction. In contrast, ANP mRNA expression was unchanged. Reflecting the rapid induction of ventricular BNP production, the plasma BNP concentration rose to about 100 pg/mL 12 hours postinfarction (sham-operated rats, <70 pg/mL). Conclusions: These results demonstrate the rapid induction of ventricular BNP gene expression in rats with AMI compared with ANP and suggest that BNP gene expression in the ventricle is regulated distinctively from ANP gene expression against acute ventricular overload. They also suggest that the BNP gene can be one of the acutely responsive cardiac genes for the ventricular overload and suggest a possible pathophysiological role of BNP distinct from ANP in AMI..
512. Yoshihiro Ogawa, Issei Tanaka, Miho Inoue, Yuka Yoshitake, Naohi Isse, Osamu Nakagawa, Takeshi Usui, Hiroshi Itoh, Takaaki Yoshimasa, Shuh Narumiya, Kazuwa Nakao, Structural organization and chromosomal assignment of the human prostacyclin receptor gene, Genomics, 10.1006/geno.1995.1016, 27, 1, 142-148, 1995.05, Prostacyclin receptor is a member of the prostanoid receptor family in the G protein-coupled receptor superfamily with seven transmembrane domains. We report here the isolation and structural organization of the human prostacyclin receptor gene. Southern blot analysis demonstrated a single copy of the human prostacyclin receptor gene in the human genome. The human prostacyclin receptor gene spanned approximately 7.0 kb and was composed of three exons separated by two introns. The first intron occurred in the 5′-untranslated region, 13 bp upstream to the ATG start codon. The second intron was located at the end of the sixth transmembrane domain, thereby separating it from the downstream coding region and the 3′-untranslated region. By primer extension analysis, the transcription initiation sites were mapped 870-872 bp upstream to the ATG start codon. The 1.2-kb human prostacyclin receptor 5′-flanking region lacked conventional TATA and CCAAT boxes, but it contained several cis -acting regulatory elements including an inverted CCAAT box (Y box) and two copies of SP-1 binding sites. Using human-rodent somatic hybrid cell DNA, the human prostacyclin receptor gene was assigned to human chromosome 19. The present study helps establish the genetic basis for prostacyclin receptor research and provides further insight into the molecular mechanisms underlying the prostanoid receptor family..
513. Masahisa Goto, Hiroshi Itoh, Issei Tanaka, Shin‐ichi ‐i Suga, Yoshihiro Ogawa, Ichiro Kishimoto, Masayo Nakagawa, Akira Sugawara, Takaaki Yoshimasa, Masashi Mukoyama, Kazuwa Nakao, ALTERED GENE EXPRESSION OF NATRIURETIC PEPTIDE RECEPTOR SUBTYPES IN THE KIDNEY OF STROKE‐PRONE SPONTANEOUSLY HYPERTENSIVE RATS, Clinical and Experimental Pharmacology and Physiology, 10.1111/j.1440-1681.1995.tb02871.x, 22, S177-S179, 1995.01, 1. To elucidate the physiological and pathophysiological role of the natriuretic peptide system in the progression of hypertensive renal disease, we examined the gene expression of natriuretic peptide receptor subtypes, guanylate cyclase‐A (GC‐A), guanylate cyclase‐B (GC‐B) and clearance receptor (C receptor), in the kidney of stroke‐prone spontaneously hypertensive rats (SHRSP) at 8 and 20 weeks of age, and compared them with their gene expression in age‐matched Wistar‐Kyoto (WKY) rats. 2. Northern blot analyses revealed that messages for three natriuretic peptide receptor subtypes were expressed in the kidney, and their expressions were higher in the glomeruli than in the whole kidney in each strain. 3. In 20 week old rats with established hypertension, the glomerular concentration of GC‐A mRNA was significantly higher in SHRSP than in WKY. The concentrations of GC‐B and C receptor mRNA in the glomeruli tended to increase and decrease, respectively, but they were not statistically significant in SHRSP. 4. In 8 week old rats, the glomerular concentrations of GC‐A, GC‐B and C receptor mRNA were not significantly different between SHRSP and WKY. 5. This study demonstrates that in the progression of hypertension, the expression of GC‐A, which mediates biological actions of natriuretic peptides, is enhanced in the kidney of SHRSP compared to that of WKY. Together with the augmented secretion of the ligands previously revealed, altered expression of natriuretic peptide receptor subtypes in SHRSP may have a deterrent role in the development of hypertension and its renal complications..
514. Hiroaki Masuzaki, Yoshihiro Ogawa, Michika Shigemoto, Noriko Satoh, Kiyoshi Mori, Naohisa Tamura, Kiminori Hosoda, Yasunao Yoshimasa, Hisato Jingami, Kazuwa Nakao, Adipose Tissue-specific Expression of the Obese (ob) Gene in Rats and Its Marked Augmentation in Genetically Obese-hyperglycemic Wistar Fatty Rats, Proceedings of the Japan Academy Series B: Physical and Biological Sciences, 10.2183/pjab.71.148, 71, 5, 148-152, 1995.01, The obese (ob) gene has recently been isolated by positional genetics, the mutation of which causes marked obesity and diabetes in mice. In the present study, we elucidated the tissue distribution of the ob gene expression in rats and examined the gene expression in genetically obese-hyperglycemic Wistar fatty rats. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kilobase in size in the adipose tissue, while no significant amount of ob mRNA was detected in other tissues in Sprague-Dawley and Wistar rats. The ob gene expression occurred in the adipose tissue with region-specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue ⑥ interscapular brown adipose tissue in both rat strains. Furthermore, expression of the ob gene was augmented in all the adipose tissue examined in Wistar fatty rats at the stage of established obesity..
515. M. Goto, H. Itoh, S. I. Suga, Y. Ogawa, I. Kishimoto, M. Nakagawa, M. Mukoyama, A. Sugawara, T. Yoshimasa, I. Tanaka, K. Nakao, Gene expression of natriuretic peptide receptor subtypes in the kidney of SHR-stroke prone, japanese heart journal, 10.1536/ihj.36.532, 36, 4, 1995.01.
516. Hiroaki Masuzaki, Yoshihiro Ogawa, Naohi Isse, Noriko Satoh, Taku Okazaki, Michika Shigemoto, Kiyoshi Mori, Naohisa Tamura, Kiminori Hosoda, Yasunao Yoshimasa, Hisato Jingami, Teruo Kawada, Kazuwa Nakao, Human obese gene expression
Adipocyte-specific expression and regional differences in the adipose tissue, Diabetes, 10.2337/diab.44.7.855, 44, 7, 855-858, 1995.01, The obese (ob) gene, the mutation of which results in severe hereditary obesity and diabetes in mice, has recently been isolated through positional cloning. In this study, we isolated a full-length human ob complementary DNA (cDNA) clone and examined the tissue distribution of ob gene expression in humans. The nucleotide sequences of the human ob cDNA coding region were 83% identical to those of the mouse and rat ob cDNA coding regions. Analysis of the deduced amino acid sequences revealed that the human ob protein is a 166- amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively. Northern blot analysis using the cloned human ob cDNA fragment as a probe identified a single messenger RNA (mRNA) species 4.5 kb in size found abundantly in the adipose tissues obtained from the subcutaneous, omental, retroperitoneal, perilymphatic, and mesenteric fat pads. However, no significant amount of ob mRNA was present in the brain, heart, lung, liver, stomach, pancreas, spleen, small intestine, kidney, prostate, testis, colon, or skeletal muscle. The ob mRNA level in the adipose tissue varied from region to region even in the same individual. Furthermore, in the human adipose tissue, ob gene expression occurred in mature adipocytes rather than in stromal-vascular cells. This study is the first report of the elucidation of ob gene expression in human tissues, thereby leading to better understanding of the physiological and clinical implications of the ob gene..
517. Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, MOLECULAR BIOLOGY AND BIOCHEMISTRY OF NATRIURETIC PEPTIDE FAMILY, Clinical and Experimental Pharmacology and Physiology, 10.1111/j.1440-1681.1995.tb01918.x, 22, 1, 49-54, 1995.01, The natriuretic peptide family consists of three endogenous ligands; atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C‐type natriuretic peptide (CNP), and is involved in the regulation of cardiovascular homeostasis. Both ANP and BNP act mainly as cardiac hormones and are produced predominantly by the atrium and ventricle, respectively. Expression of the BNP and ANP genes is greatly augmented in patients with congestive heart failure and animal models of ventricular hypertrophy or cardiomyopathy. In the heart, the BNP gene expression is regulated differently from the ANP gene expression both at transcriptional and post‐transcriptional levels. Transgenic technology has provided the direct evidence that BNP as well as ANP is involved in the chronic blood pressure control. Contrasting with ANP and BNP, CNP does not act as a cardiac hormone but as a neuropeptide or an endothelium‐derived autocrine/paracrine regulator. Endothelial production of CNP is remarkably augmented by various cytokines and growth factors such as transforming growth factor‐β and tumour necrosis factor‐α, suggesting the pathophysiological significance of CNP in the process of various vascular disorders. Chromosomal mapping of natriuretic peptides has revealed that the CNP gene is localized on mouse chromosome 1, while ANP and BNP are tightly linked on mouse chromosome 4, suggesting that CNP, a local regulator, is functionally and evolutionarily distinct from ANP and BNP, both of which are cardiac hormones. Understanding the molecular biology and biochemistry of the natriuretic peptide family will lead to the better understanding of its physiological and pathophysiological implication, and the clinical application in cardiorenal regulation..
518. M. Kotani, I. Tanaka, Y. Ogawa, T. Usui, K. Mori, A. Ichikawa, S. Narumiya, T. Yoshimi, K. Nakao, Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing
Multiple second messenger systems and tissue-specific distributions, Molecular Pharmacology, 48, 5, 869-879, 1995.01, Five distinct cDNA clones encoding four different isoforms of human prostaglandin (PG) E receptor EP3 subtype were isolated from a human kidney cDNA library. Two cDNA clones differed only in their 3'-untranslated regions. The four isoforms, tentatively named EP(3-I), EP(3-II), EP(3-III), and EP(3- IV), which were generated by alternative mRNA splicing, had identical amino acid sequences except for their different carboxyl-terminal tails. Transfection experiments revealed that all the four isoforms show high binding affinities to PGE2, PGE1, and M and B28767, an EP3-specific agonist, whereas their downstream signaling pathways are divergent. M and B28767 increased cAMP concentrations in cells expressing EP(3-II) and EP(3- IV), whereas it inhibited forskolin-induced cAMP accumulations in cells expressing all EP3 isoforms. M and B28767 also stimulated phosphoinositide turnover in cells expressing EP(3-I) and EP(3-II). Northern blot analysis revealed that the EP3 gene is expressed in a wide variety of human tissues. The human EP3 mRNA was present most abundantly in the kidney, pancreas, and uterus. A substantial expression was also detected in the heart, liver, skeletal muscle, small intestine, colon, prostate, ovary, and testis. Furthermore, reverse transcription-polymerase chain reaction analysis demonstrated tissue-specific expressions of the five different EP3 mRNA species. The present study suggests the presence of the multiple systems of PGE2/EP3 isoforms and leads to the better understanding of its physiological and pathophysiological implications in humans..
519. Masayo Nakagawa, Issei Tanaka, Shin‐ichi ‐i Suga, Yoshihiro Ogawa, Naohisa Tamura, Masahisa Goto, Akira Sugawara, Takaaki Yoshimasa, Hiroshi Itoh, Masashi Mukoyama, Kazuwa Nakao, PREPARATION OF A MONOCLONAL ANTIBODY AGAINST MOUSE BRAIN NATRIURETIC PEPTIDE (BNP) AND TISSUE DISTRIBUTION OF BNP IN MICE, Clinical and Experimental Pharmacology and Physiology, 10.1111/j.1440-1681.1995.tb02874.x, 22, S186-S187, 1995.01, 1. In order to explore the significance of brain natriuretic peptide (BNP), a cardiac hormone secreted from the ventricle, in mice, we prepared a monoclonal antibody against mouse BNP (mBNP) and established a specific radioimmunoassay (RIA) for mBNP. 2. A monoclonal antibody, KY‐mBNP‐I, was prepared by the fusion of mouse myeloma cells X63‐Ag8.653 with spleen cells of the BALB/c mouse immunized with synthetic mBNP[108–121] conjugated to bovine thyroglobulin. KY‐mBNP‐I belonged to an IgG2a subclass and showed a high affinity for mBNP(Ka = 1.8 × 1011 mol/L−l). 3. The RIA established that using KY‐mBNP‐I was highly sensitive and specific for mBNP, with an IC50 value of 3 fmol/ tube and cross‐reactivities of less than 0.003% with related natriuretic peptides. mBNP‐like immunoreactivity (mBNP‐LI) was detected in the mouse atrium (0.35 ± 0.02 nmol/g), ventricle (20.5 ± 0.5 pmol/g) and kidney (0.50 ± 0.05 pmol/g), but not in other tissues including brain. 4. Gel filtration analysis revealed that the major component of tissue mBNP‐LI was co‐eluted with synthetic mBNP[77–1211, a 45‐amino acid mature peptide. 5. The monoclonal antibody and RIA for mBNP established here will provide useful tools to investigate the functional significance of BNP in mice, coupled with the genetic engineering approach..
520. N. Isse, Y. Ogawa, N. Tamura, H. Masuzaki, K. Mori, T. Okazaki, N. Satoh, M. Shigemoto, Y. Yoshimasa, S. Nishi, K. Hosoda, J. Inazawa, K. Nakao, Structural organization and chromosomal assignment of the human obese gene, Journal of Biological Chemistry, 10.1074/jbc.270.46.27728, 270, 46, 27728-27733, 1995.01, The obese (ob) gene has been identified through a positional cloning approach; the mutation of this gene causes marked hereditary obesity and diabetes mellitus in mice. We report here the isolation and characterization of the human ob gene. Southern blot analysis demonstrated a single copy of the ob gene in the human genome. The human ob gene spanned 20 kilobases (kb) and contained three exons separated by two introns. The first intron, 10.6 kb in size, occurred in the 5'-untranslated region, 29 base pair (bp) upstream of the ATG start codon. The second intron of 2.3 kb in size was located at glutamine +49. By rapid amplification of 5'-cDNA ends, the transcription initiation sites were mapped 54~57 bp upstream of the ATG start codon. The 172-bp 5'-flanking region of the human ob gene contained a TATA box-like sequence and several cis-acting regulatory elements (three copies of GC boxes, an AP-2-binding site, and a CCAAT/enhancer-binding protein-binding site). By the fluorescence in situ hybridization technique, the ob gene was assigned to human chromosome 7q31.3. This study should establish the genetic basis for ob gene research in humans, thereby leading to the better understanding of the molecular mechanisms underlying the ob gene..
521. Jun Yamashita, Takaaki Yoshimasa, Hiroshi Arai, Junko Hiraoka, Kazuhiko Takaya, Yoshihiro Miyamoto, Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, Cis elements for transcriptional regulation of the human endothelin-A receptor gene, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199526003-00008, 26, S26-S28, 1995, To elucidate the transcriptional regulation of the endothelin-A receptor (ET-AR) gene, we defined the cis elements in the 5'-flanking region by functional analysis. Chimeric gene constructs were prepared using serial deletion mutants of a 5-kilobase (kb) fragment of the human ET-AR 5'-flanking region and the luciferase gene and were transfected into a rat vascular smooth-muscle cell line, A7r5. Deletion of the region between -2.0 kb and - 857 bases caused a twofold increase in luciferase activity, and deletion from - 137 bases to -53 bases reduced the activity by 50%. These results indicate the existence of a negative regulatory element and a positive regulatory element, respectively. The gel shift assay revealed the existence of nuclear proteins in A7r5 cells that bind to the region containing the positive regulatory element. Thus, we identified cis regulatory elements and nuclear factors that regulate the expression of the ET-AR gene..
522. Masaaki Hasegawa, Norimasa Sagawa, Hiroaki Itoh, Kumiko Inamori, Takahide Mori, Juri Yano, Yoshihiro Ogawa, Takaaki Yoshimasa, Kazuwa Nakao, Endothelin receptors in the human amnion, chorion laeve, decidua vera and placenta, Reproduction, Fertility and Development, 10.1071/RD9951585, 7, 6, 1585-1589, 1995, The levels of endothelin-1 (ET-1) and the characteristics of endothelin (ET) receptors were investigated in the amnion, chorion laeve, decidua vera and placenta by using a specific radioimmunoassay for ET-1 and a saturation binding assay for ET. ET receptor gene expression in these tissues was also examined by Northern blot analysis. The levels of ET-1-like immunoreactivity (ET-1-LI) in the human amnion, chorion laeve, decidua vera and placenta obtained at elective Caesarean section before labour onset at term (mean +/- s.e.m.) were 1260 +/- 380 (n = 6), 3740 +/- 980 (n = 4), 4550 +/- 780 (n = 4) and 2450 +/- 470 (n = 4) pg g-1 wet weight, respectively. The levels of ET-1-LI in the tissues obtained after spontaneous vaginal deliveries at term did not differ from these. Gel-permeation chromatography and reverse-phase high-performance liquid chromatography revealed that the ET-1-LI in these tissues was mainly composed of ET-1. Scatchard analysis of the saturation binding assay for [125I]-labelled ET-1 and [125I]-labelled ET-3 indicated that high concentrations of both ET-A and ET-B subtypes of receptor were present in membrane fractions from the chorion laeve, decidua vera and placenta. However, in membrane fractions from the amnion, no ET receptors could be detected. These results were confirmed by Northern blot analysis using human ET-A and ET-B receptor cDNA probes. Taken together, these results suggest that the amnion is not the site of action of amniotic ET, and that ET may be involved in the regulation of functions of the chorion laeve or decidua vera..
523. Yoshihiro Miyamoto, Takaaki Yoshimasa, Hiroshi Arai, Kazuhiko Takaya, Yoshihiro Ogawa, Hiroshi Itoh, Kazuwa Nakao, Multiple transcripts of human endothelin-A receptor gene detected by reverse transcription and the polymerase chain reaction, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199506263-00009, 26, S29-S31, 1995, To elucidate the regulatory mechanism of gene expression of the human endothelin-A receptor (hET-AR), we characterized the hET-AR transcripts using reverse transcription (RT) and polymerase chain reaction (PCR) analysis in a variety of human tissues. The RT-PCR using a set of primers in exons 2 and 5 showed two lower-molecular-weight transcripts in addition to the expected fragment. PCR cloning of these two novel transcripts revealed that these transcripts contain a 199-base pair (bp) and a 327-bp deletion compared with the previously described hET-AR cDNA, respectively. Comparison of their sequences with that of the hET-AR gene showed that the lacking sequences exactly correspond to exon 4 and exons 3 and 4, respectively, suggesting that these lower-molecular-weight ET-AR transcripts may result from alternative RNA splicing. Therefore, we isolated the cDNAs of novel transcripts of hET-AR that might be generated by alternative RNA splicing. These results suggest that the alternative RNA splicing might contribute to the regulation of ET-AR gene expression..
524. Yoshihiro Ogawa, Hiroshi Itoh, Yuka Yoshitake, Miho Inoue, Takaaki Yoshimasa, Tadao Serikawa, Kazuwa Nakao, Molecular cloning and chromosomal assignment of the mouse C-Type natriuretic peptide (CNP) gene (NPPC)
Comparison with the human CNP gene (NPPC), Genomics, 10.1006/geno.1994.1633, 24, 2, 383-387, 1994.11, The mouse C-type natriuretic peptide (CNP) genomic fragment was isolated from a mouse genomic DNA library. The mouse CNP gene is composed of at least two exons and one intron. The 5′-flanking region contains an array of cis -acting regulatory elements and a dinucleotide CA repeat (microsatellite). Analysis of the deduced amino acid sequences revealed that mouse preproCNP is a 126-amino-acid peptide and that its C-terminal 22-residue peptide preceded by Lys-Lys is identical to porcine, rat, and human CNPs. On the basis of the polymerase chain reaction-analyzed microsatellite length polymorphisms among recombinant inbred strains of mice, the CNP gene (Nppc) was assigned to mouse chromosome 1. Furthermore, the human CNP 5′-flanking region was extended for sequencing, and comparison of the mouse and human CNP genomic sequences revealed regions of conservation and diversity. Using somatic hybrid cell methodology, the CNP gene (NPPC) was assigned to human chromosome 2. The present study has added another locus to the conserved syntenic group in mice and humans..
525. Ichiro Kishimoto, Takaaki Yoshimasa, Shin Ichi Suga, Yoshihiro Ogawa, Yasato Komatsu, Osamu Nakagawa, Hiroshi Itoh, Kazuwa Nakao, Natriuretic peptide clearance receptor is transcriptionally down-regulated by β2-adrenergic stimulation in vascular smooth muscle cells, Journal of Biological Chemistry, 269, 45, 28300-28308, 1994.11, Three natriuretic peptide receptors (the ANP-A, ANP-B, and clearance (C) receptors) have been reported. The regulation of these receptors by catecholamines was examined in cultured rat vascular smooth muscle cells. Treatment with norepinephrine decreased the maximum 125I-ANP binding. The competitive binding assay with des[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4-23)-NH2 (C-ANF-4-23), a specific ligand for the C receptor, revealed that the decrease in the 125I-ANP binding by norepinephrine was caused by the down-regulation of the C receptor. Isoproterenol also down-regulated the C receptor in a time- and dose-dependent manner. The catecholamine-induced down-regulation of the C receptor was antagonized by a β2-selective adrenergic antagonist, ICI 118,551 but not by an α1-, α2-, or β1-adrenergic antagonist. Forskolin, NaF and, 8-bromo-cyclic AMP also decreased the C receptor density. The isoproterenol-induced decrease in the C receptor level was further confirmed by affinity cross-linking and Western blot analysis. Northern blot analysis revealed that isoproterenol and 8-bromo-cyclic AMP decreased the steady- state level of C receptor mRNA. By contrast, neither the ANP-A receptor nor the ANP-B receptor mRNA level was affected by 8-bromo-cyclic AMP. The nuclear run-on assay showed that the transcriptional rate of the C receptor gene was decreased by isoproterenol, whereas those of the ANP-A and ANP-B receptor genes were unchanged. Isoproterenol attenuated the clearance of exogenously added ANP and augmented the ANP-stimulated intracellular cyclic GMP production to the same extent as the selective occupancy of the C receptor with C-ANF-(4-23), suggesting that the isoproterenol-induced enhancement of responsiveness to ANP could result not from the sensitization of the ANP-A or ANP-B receptor but from the down-regulation of the C receptor, which leads to the attenuated clearance of ANP. These findings suggest that the β2- adrenergic receptor stimulation down-regulates the C receptor through the decrease in the transcriptional rate of the C receptor gene and that the activation of the sympathetic nervous system augments the biological responsiveness to natriuretic peptides by attenuating their metabolic clearance in vascular walls..
526. Yoshihiro Ogawa, Hiroshi Itoh, Naohisa Tamura, Shin Ichi Suga, Takaaki Yoshimasa, Masahiro Uehira, Saburo Matsuda, Shozo Shiono, Hirofumi Nishimoto, Kazuwa Nakao, Molecular cloning of the complementary DNA and gene that encode mouse brain natriuretic peptide and generation of transgenic mice that overexpress the brain natriuretic peptide gene, Journal of Clinical Investigation, 10.1172/JCI117182, 93, 5, 1911-1921, 1994.05, Brain natriuretic peptide (BNP) is a cardiac hormone that occurs predominantly in the ventricle. To study the roles of BNP in chronic cardiovascular regulation, we isolated mouse BNP cDNA and genomic clones, and generated transgenic mice with elevated plasma BNP concentration. The mouse BNP gene was organized into three exons and two introns. Two BNP mRNA species were identified, which were generated by the alternative mRNA splicing. The ventricle was a major site of BNP production in mice. Mouse preproBNP was a 121- (or 120-) residue peptide, and its COOH-terminal 45-residue peptide was the major storage form in the heart. Transgenic mice carrying the human serum amyloid P component/mouse BNP fusion gene were generated so that the hormone expression is targeted to the liver. In the liver of these mice, considerable levels of BNP mRNA and peptide were detected, reaching up to 10-fold greater than in the ventricle. These animals showed 10- to 100-fold increase in plasma BNP concentration accompanied by elevated plasma cyclic GMP concentration, and had significantly lower blood pressure than their nontransgenic littermates. The present study demonstrates that these mice provide a useful model system with which to assess the roles of BNP in cardiovascular regulation and suggests the potential usefulness of BNP as a long-term therapeutic agent..
527. Yoshihiro Ogawa, H. Itoh, K. Nakao, Molecular biology of brain natriuretic peptide, Japanese Heart Journal, 10.1536/ihj.35.496, 35, 4, 496-497, 1994.01.
528. N. Tamura, H. Itoh, A. Takahashi, K. Iwai, T. Yoshimasa, I. Masuda, H. Arai, S. Suga, Y. Ogawa, I. Kishimoto, Y. Komatsu, O. Nakagawa, N. Hama, K. Takaya, Y. Miyamoto, T. Igaki, M. Harada, J. Yamashita, K. Nakao, Plasma atrial and brain natriuretic peptide concentrations in a case of malignant hypertension, Therapeutic Research, 15, 7, 197-204, 1994.01, We examined the alternation of the plasma atrial and brain natriuretic peptide (ANP and BNP) concentrations during clinical course of a 57-year-old male with malignant hypertension. The patient exhibited marked elevation of diastolic blood pressure up to 152 mmHg with progressive organ damages snch as concentric cardiac hypertrophy, hypertensive retinopathy, and renal insufficiency. In the present case, extreme hypertension and markedly activated renin-angiotensin system (the plasma renin activity, 35.3 ng/ml/h; the plasma aldosterone concentration, 321.4 pg/ml) caused cardiac overload and hypertrophy (the left ventricular mass index, 161 g/m2) which was considered to lead to marked increase of the plasma BNP concentration (1195.5 pg/ml), much higher than the concomitantly elevated plasma ANP concentration (197.1 pg/ml). During the clinical course, the blood pressure decreased trom 268/152 mmHg to 148/82 mmHg, and the left ventricular mass index decreased from 161 g/m2 to 136 g/m2, in response to the anti-hypertensive therapy with Ca-entry blockers and angiotensin converting enzyme inhibitors. Following the decrease of blood pressure and regression of cardiac hypertrophy, the elevated plasma BNP concentration significantly decreased to 47.7 pg/ml, lower than the concomitantly decreased plasma ANP concentration (68.8 pg/ml). The present report indicates the usefulness of the plasma BNP concentration as a marker to determine the severity of cardiac overload and hypertrophy during the clinical course of hypertensive patients..
529. N. Tamura, Y. Ogawa, H. Itoh, H. Arai, S. I. Suga, O. Nakagawa, Y. Komatsu, I. Kishimoto, K. Takaya, T. Yoshimasa, S. Shiono, K. Nakao, Molecular cloning of hamster brain and atrial natriuretic peptide cDNAs. Cardiomyopathic hamsters are useful models for brain and atrial natriuretic peptides, Journal of Clinical Investigation, 10.1172/jci117420, 94, 3, 1059-1068, 1994, Brain and atrial natriuretic peptides (BNP and ANP) are cardiac hormones with diuretic, natriuretic, and vasodilatory activities. Cardiomyopathic hamsters are widely used animal models of heart failure. Due to the structural divergence of BNP among species, examination on pathophysiological roles of BNP using cardiomyopathic hamsters is so far impossible. We therefore isolated hamster BNP and ANP cDNAs, and investigated synthesis and secretion of these peptides in normal and cardiomyopathic hamsters. The COOH- terminal 32-residue peptide of cloned hamster pre-proBNP with 122 amino acids, preceded by a single arginine residue, supposedly represents hamster BNP showing <50% homology to rat BNP. Alpha-hamster ANP, 28-residue peptide, is identical to α-rat ANP. In hamsters, BNP and ANP occur mainly in the ventricle and the atrium, respectively. The 32-wk-old hypertrophic cardiomyopathic BIO14.6 strain exhibited ventricular hypertrophy. The 32-wk- old dilated cardiomyopathic BIO53.58 strain remained at the stage without apparent heart failure. In BIO14.6 and BIO53.58 strains at this age, ventricular BNP and ANP gene expressions are augmented, and the plasma BNP concentration is elevated to 136 and 108 fmol/ml, respectively, three times greater than the elevated plasma ANP concentration, which well mimics changes of the plasma BNP and ANP concentrations in human heart failure. Cardiomyopathic hamsters, therefore, are useful models to investigate the implication of BNP in human cardiovascular diseases..
530. Shin Ichi Suga, Hiroshi Itoh, Yasato Komatsu, Yoshihiro Ogawa, Norio Hama, Takaaki Yoshimasa, Kazuwa Nakao, Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells--evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells, Endocrinology, 10.1210/endo.133.6.8243333, 133, 6, 3038-3041, 1993.12, We previously demonstrated that C-type natriuretic peptide (CNP), originally isolated from the porcine brain, is produced by endothelial cells and proposed that CNP can exert local control over vascular tone and growth as a local regulator from endothelial cells. Since cytokines play pivotal roles in the control of vascular tone and structure, we have examined effects of various cytokines on CNP secretion from endothelial cells using the specific radioimmunoassay for CNP. While interleukin (IL)-2 had no significant effect on CNP secretion, IL-1 α, IL-1β and tumor necrosis factor (TNF)-α stimulated CNP secretion in a time- and dose-dependent manner. Among them, TNF-α, one of the key mediators for inflammation and vascular remodeling, induced more than two orders of magnitude increase in CNP secretion. In addition, lipopolysaccharide (LPS) potently stimulated CNP secretion. These results indicate that IL-1, TNF-α and LPS, the endotoxin itself, can regulate local vascular tone and growth through the activation of CNP secretion from endothelial cells. Therefore, CNP could be of clinical relevance as an autocrine/paracrine regulator from endothelial cells for systemic and local cytokine-associated disorders, such as endotoxin shock and atherosclerosis..
531. Hiroo Imura, Kazuwa Nakao, Akira Shimatsu, Yoshihiro Ogawa, Takehiro Sando, Ichiro Fujisawa, Hirohiko Yamabe, Lymphocytic Infundibuloneurohypophysitis as a Cause of Central Diabetes Insipidus, New England Journal of Medicine, 10.1056/NEJM199309023291002, 329, 10, 683-689, 1993.09, Central diabetes insipidus may be familial, secondary to hypothalamic or pituitary disorders, or idiopathic. Idiopathic central diabetes insipidus is characterized by selective hypofunction of the hypothalamic-neurohypophysial system, but its cause is unknown. We studied 17 patients with idiopathic diabetes insipidus, in whom the duration of the disorder ranged from 2 months to 20 years. Only four patients had been treated with vasopressin before the study began. All the patients underwent endocrinologic studies and magnetic resonance imaging (MRI) with a 1.5-T superconducting unit, and two patients had biopsies of the neurohypophysis or the pituitary stalk. Nine of the 17 patients had thickening of the pituitary stalk, enlargement of the neurohypophysis, or both and lacked the hyperintense signal of the normal neurohypophysis. In the remaining eight patients, the pituitary stalk and the neurohypophysis were normal, although the hyperintense signal was absent. The abnormalities of thickening and enlargement were seen on MRI only in the patients who had had diabetes insipidus for less than two years, and the abnormalities disappeared during follow-up, suggesting a self-limited process. In addition to vasopressin deficiency, two patients had mild hyperprolactinemia and nine had impaired secretory responses of growth hormone to insulin-induced hypoglycemia. The two biopsies revealed chronic inflammation, with infiltration of lymphocytes (mainly T lymphocytes) and plasma cells. Diabetes insipidus can be caused by lymphocytic infundibuloneurohypophysitis, which can be detected by MRI. The natural course of the disorder is self-limited., Central diabetes insipidus is a chronic disorder characterized by polyuria and polydipsia due to vasopressin deficiency. The disorder may be familial, idiopathic, or secondary. Familial diabetes insipidus is characterized by autosomal dominant inheritance and, at least in some families, mutations of the vasopressin-neurophysin II genes1,2. Secondary diabetes insipidus, the most common form of the disorder, is caused by tumors, infections, trauma, or other processes (such as histiocytosis and vascular lesions) that damage the hypothalamic-neurohypophysial system. Idiopathic diabetes insipidus, which accounts for 10 to 30 percent of cases of central diabetes insipidus,3,4 is characterized by selective hypofunction of….
532. Hiroaki Itoh, Norimasa Sagawa, Masaaki Hasegawa, Atsuhiko Okagaki, Kumiko Inamori, Yoshiyuki Ihara, Takahide Mori, Yoshihiro Ogawa, Shin Ichi Suga, Masashi Mukoyama, Kazuwa Nakao, Hiroo Imura, Brain natriuretic peptide is present in the human amniotic fluid and is secreted from amnion cells, Journal of Clinical Endocrinology and Metabolism, 10.1210/jcem.76.4.8473404, 76, 4, 907-911, 1993.04, The presence and biochemical characteristics of human brain natriuretic peptide (hBNP) in the amniotic fluid at various gestational ages were investigated. The hBNP-like immunoreactivity (hBNP-LI) levels in amniotic fluid, determined by RIA, were 118.7 ± 57.6 pmol/L (mean ± SEM; n = 5) and 107.7 ± 8.7 pmol/L (n = 9) in the first and second trimesters of pregnancy, respectively; it was significantly decreased to 28.4 ± 5.1 pmol/L (n = 9) in the third trimester. However, human atrial natriuretic peptide-like immunoreactivity (hANP-LI) was not detected (< 0.3 pmol/L) in any of these samples. Northern blot analysis demonstrated hBNP mRNA in human amnion tissue. Moreover, cultured amnion cells secreted a significant amount of hBNP-LI (100-200 fmol/106 cells/day), but not hANP-LI, into the culture medium. The synthesis of hBNP in cultured amnion cells was further confirmed using the polymerase chain reaction. High performance gel permeation chromatography of hBNP-LI extracted from human amniotic fluid and the culture medium of amnion cells revealed that the predominant molecular form of hBNP-LI in both samples was the hBNP precursor, with an approximate mol wt of 12 kilodaltons. These findings indicate that hBNP is present in the human amniotic fluid, and that amnion cells synthesize hBNP and secrete it into the amniotic cavity..
533. H. Itoh, S. Suga, Yoshihiro Ogawa, I. Tanaka, K. Nakao, Molecular biology and pharmacology of natriuretic peptide system, Nippon rinsho. Japanese journal of clinical medicine, 51, 6, 1548-1556, 1993.01, The natriuretic peptide system comprises at least three endogenous ligands, namely, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), and three receptors for natriuretic peptides (NPR), that is, NPR-A, NPR-B and clearance receptor (NPR-C). Three natriuretic peptides derived from the distinct genes share a common ring structure with 17 amino acids formed by a disulfide linkage which confers the unique biological property on these peptides. ANP and BNP are elucidated to be the cardiac hormone mainly secreted from the atrium, and from the ventricle, respectively. CNP, first recognized as the neuropeptide, is now identified within the vascular wall, especially in endothelial cells and considered to be the peptidic endothelium-derived relaxing factor (EDRF). While NPR-A shows the high affinity to ANP and BNP, NPR-B is the selective receptor for CNP. These two types of "biologically active" NPR are the membrane-bound guanylate cyclase itself, and mediate the wide range of biological actions of natriuretic peptides through cyclic GMP-dependent cascade. The clearance receptor shows the ligand-binding affinity with the rank order of ANP > CNP > BNP and is considered to be involved in the clearance of the peptides. The natriuretic peptide system as an endocrine and paracrine/autocrine system serves as one of the key modulatory systems for blood pressure, body fluid homeostasis and vascular remodeling..
534. Hiroshi Arai, Kazuwa Nakao, Kiminori Hosoda, Yoshihiro Ogawa, Osamu Nakagawa, Yasato Komatsu, Hiroo Imura, Molecular cloning of human endothelin receptors and their expression in vascular endothelial cells and smooth muscle cells, JAPANESE CIRCULATION JOURNAL, 10.1253/jcj.56.SupplementV_1303, 56, 1993.01.
535. Y. Ogawa, K. Nakao, O. Nakagawa, N. Tamura, H. Imura, The gene expression of brain natriuretic peptide (BNP) in hearts of spontaneously hypertensive rats-stroke prone (SHR-SP), japanese heart journal, 10.1536/ihj.34.486, 34, 4, 1993.01.
536. H. Arai, K. Nakao, K. Takaya, K. Hosoda, Y. Ogawa, S. Nakanishi, H. Imura, The human endothelin-B receptor gene. Structural organization and chromosomal assignment, Journal of Biological Chemistry, 268, 5, 3463-3470, 1993.01, The gene encoding the human endothelin-B receptor (hET-BR) has been isolated, and its structural organization and chromosomal assignment have been determined. Southern blot analysis demonstrated a single copy of the hET-BR gene in the human genome. The hET-BR gene spans 24 kilobases and consists of seven exons and six introns. The size range for exons is 109- 2855 base pairs, although that for introns is 0.2-14.5 kilobases. Every intron occurs near the border of the putative transmembrane domain in the coding region. The major transcription initiation sites were mapped to the positions 258 and 229 base pairs upstream of the ATG initiation codon by primer extension and nuclease S1 protection experiments. The 5'-flanking region of the hET-BR gene lacks conventional TATA and CCAAT boxes but contains a sequence of potential Sp1 binding sites upstream of the transcription initiation sites. There are some canonical consensus sequences of cis-elements including GATA motif, acute phase reactant regulatory element, and E box. Using human-rodent somatic hybrid cell lines, the hET-BR gene was assigned to human chromosome 13. The present study will lead to a better understanding of the mechanism for the transcriptional regulation of the hET-BR gene and will give a clue as to how to search for possible genetic disorders of hET-BR..
537. K. Nakao, H. Itoh, S. Suga, Yoshihiro Ogawa, H. Imura, The natriuretic peptide family., Current opinion in nephrology and hypertension, 10.1097/00041552-199301000-00007, 2, 1, 45-50, 1993.01, The natriuretic peptide system is a complicated system comprising at least three endogenous peptides, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide and three receptors, the atrial natriuretic peptide-A receptor (guanylyl cyclase A), the atrial natriuretic peptide-B receptor (guanylyl cyclase B), and the clearance receptor. The accumulated evidence indicates that this system is implicated in the control of blood pressure, body fluid homeostasis, and vascular remodeling as both cardiac hormone and local regulator..
538. Y. Ogawa, K. Nakao, H. Itoh, S. Suga, H. Imura, Molecular biology of the natriuretic peptide system, Nippon rinsho. Japanese journal of clinical medicine, 50, 12, 2885-2892, 1992.12, The natriuretic peptide system is composed of at least three distinct endogenous peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), and three receptors: ANP-A receptor/GC-A, ANP-B receptor/GC-B and the clearance receptor. This system influences the control of body fluid and blood pressure as cardiac hormones and local regulators. Recent advances in molecular biology have unravel the molecular mechanism of the natriuretic peptide system and have facilitated our understanding of it. The present review gives the current knowledge of the molecular biology of the natriuretic peptide system..
539. Kazuwa Nakao, Yoshihiro Ogawa, Shin Ichi Suga, Hiroo Imura, Molecular biology and biochemistry of the natriuretic peptide system. I
Natriuretic peptides, Journal of hypertension, 10, 9, 907-912, 1992.09.
540. Yasato Komatsu, Kazuwa Nakao, Itoh Hiroshi Itoh, Shin Ichi Suga, Yoshihiro Ogawa, Hiroo Imura, Vascular natriuretic peptide, The Lancet, 10.1016/0140-6736(92)92167-E, 340, 8819, 1992.09.
541. Shin Ichi Suga, Kazuwa Nakao, Masashi Mukoyama, Hiroshi Arai, Kiminori Hosoda, Yoshihiro Ogawa, Hiroo Imura, Characterization of natriuretic peptide receptors in cultured cells, Hypertension, 10.1161/01.HYP.19.6.762, 19, 6, 762-765, 1992.06, To elucidate physiological and clinical implications of the natriuretic peptide family, the expression of receptors for natriuretic peptides has been examined in cultured cells (a rat pheochromocytoma cell line [PC12], bovine endothelial cells, rat aortic smooth muscle cells, human mesangial cells, and a porcine kidney epithelial cell line [LLC-PK1]) by Northern blot analysis and cyclic GMP production method for the ANP-A and ANP-B receptors and by Northern blot analysis and binding assay for the clearance receptor. The ANP-A receptor was predominantly expressed in PC12 cells, bovine endothelial cells, and LLC-PK, cells but was barely expressed in rat aortic smooth muscle cells and human mesangial cells. By contrast, the ANP-B receptor was the major subtype of the biologically active receptors in rat aortic smooth muscle cells and human mesangial cells. Only a small amount of the ANP-B receptor was detected in PC12 cells, bovine endothelial cells, and LLC-PK, cells. The clearance receptor was abundantly expressed in rat aortic smooth muscle cells and human mesangial cells and was also present in bovine endothelial cells, but it was undetectable in PC12 cells and LLC-PK1 cells. These results demonstrate that the expression of three natriuretic peptide receptors varies from cell to cell, which is relevant to cell- or tissue-specific action of the natriuretic peptide family..
542. Yoshihiro Ogawa, Kazuwa Nakao, Osamu Nakagawa, Yasato Komatsu, Kiminori Hosoda, Shin Ichi Suga, Hiroshi Arai, Kiyoshi Nagata, Nobuo Yoshida, Hiroo Imura, Human C-type natriuretic peptide, Hypertension, 10.1161/01.hyp.19.6.809, 19, 6, 809-813, 1992.06, We isolated the human C-type natriuretic peptide gene and identified the peptide in the brain. The human C-type natriuretic peptide gene appeared to be composed of at least two exons and one intron. In the 5′-flanking region, there is an array of cis elements (an inverted CCA AT box, two GC boxes, and a cyclic AMP response element-like sequence) that is not present in upstream sequences of the atrial and brain natriuretic peptide genes. Analysis of the deduced amino acid sequence revealed that human prepro C-type natriuretic peptide comprises 126 amino acids and that the C-terminal 22-residue peptide (G-L-S-K-G-C-F-G-L-K-L-D-R-I-G-S-M-S-G-L-G-C) preceded by Lys-Lys is identical to the porcine counterpart. However, replacement of two amino acids took place in the C-terminal 53-residue sequence, corresponding to another endogenous form of the peptide. Reverse-phase high-performance liquid chromatography coupled with a radioimmunoassay for C-type natriuretic peptide demonstrated that it occurs in the human brain. C-type natriuretic peptide–like immunoreactivity was detected in discrete regions of the human brain, and its level was 10-fold higher than the atrial and brain natriuretic peptide levels, raising the possibility that C-type natriuretic peptide is the major natriuretic peptide in the human brain..
543. Y. Ogawa, K. Nakao, H. Imura, Molecular biology of the natriuretic peptide family, Nippon rinsho. Japanese journal of clinical medicine, 50 Suppl, 46-53, 1992.04.
544. Kazuwa Nakao, Yoshihiro Ogawa, Shi nichi Suga, Hiroo Imura, Molecular biology and biochemistry of the natriuretic peptide system. II
Natriuretic peptide receptors, Journal of hypertension, 10.1097/00004872-199210000-00002, 10, 10, 1111-1114, 1992.01.
545. K. Hosoda, K. Nakao, N. Tamura, H. Arai, Y. Ogawa, S. I. Suga, S. Nakanishi, H. Imura, Organization, structure, chromosomal assignment, and expression of the gene encoding the human endothelin-A receptor, Journal of Biological Chemistry, 267, 26, 18797-18804, 1992.01, We have isolated and characterized the gene for the human endothelin-A receptor. Southern blot analyses demonstrated a single copy gene for the receptor. The gene spans more than 40 kilobases and contains eight exons and seven introns. Intron 1 exists in the 5'-noncoding region, and introns 2-7 occur in the coding region. The locations of introns 2-7 exist before or after the regions encoding the membrane-spanning domains. The transcription start site, determined by primer extension experiments, is 502 base pairs upstream of the methionine initiation codon. The 5'-flanking region lacks a typical TATA box but contains a potential SP-1-binding site 27 base pairs upstream of the transcription start site. Using human-rodent somatic hybrid cell DNA, the gene was assigned to human chromosome 4. Northern blot analyses revealed a 4.3-kilobase mRNA in a wide variety of human tissues, at the highest level in the aorta and at a substantial level in the cultured human mesangial cells. This is the first report of cloning of a gene for a member of the endothelin receptor family. The present study should give a clue to the discovery of possible disorders of the endothelin-A receptor, as well as facilitate the elucidation of the mechanisms by which the gene expression is regulated..
546. S. I. Suga, K. Nakao, I. Kishimoto, K. Hosoda, M. Mukoyama, H. Arai, G. Shirakami, Y. Ogawa, Y. Komatsu, O. Nakagawa, N. Hama, H. Imura, Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells, Circulation research, 10.1161/01.RES.71.1.34, 71, 1, 34-39, 1992.01, To elucidate the physiological and pathophysiological roles of the natriuretic peptide family in vascular smooth muscle cells, in which the natriuretic peptide family is implicated in growth inhibition as well as vasorelaxation, we have examined the phenotype-related expression of three kinds of natriuretic peptide receptors in rat aortic smooth muscle cells. The expression of natriuretic peptide receptors at the mRNA level was studied by Northern blot hybridization, and the expression at the protein level was determined by the cGMP production method and receptor binding assay. In intact aortic media, atrial natriuretic peptide (ANP)-A receptor mRNA and ANP-B receptor mRNA were detected, and the potency of cGMP production by ANP was at least two orders of magnitude stronger than that by C-type natriuretic peptide. Clearance receptor mRNA was undetectable, and only a small amount of the clearance receptor was detected by the binding assay in intact aortic media. By contrast, in cultured aortic smooth muscle cells at the first, fifth, and 17th passages, the ANP-B receptor mRNA level markedly increased; meanwhile, the expression of the ANP-A receptor mRNA became undetectable. C- type natriuretic peptide was one order of magnitude more potent than ANP in cGMP production in cultured aortic smooth muscle cells. The clearance receptor density and its mRNA level increased tremendously in these cultured cells. These results demonstrate that the marked phenotype-related alteration occurs in the expression of natriuretic peptide receptors in rat aortic smooth muscle cells..
547. Shin Ichi Suga, Kazuwa Nakao, Kiminori Hosoda, Masahi Mukoyama, Yoshihiro Ogawa, Gotaro Shirakami, Hiroshi Arai, Yoshihiko Saito, Yoshikazu Kambayashi, Ken Inouye, Hiroo Imura, Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide, and c-type natriuretic peptide, Endocrinology, 10.1210/endo.130.1.1309330, 130, 1, 229-239, 1992.01, To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclase-A (GC-A), the ANP -B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured cells with the aid of the binding assay, Northern blot technique, and the cGMP production method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP > CNP > BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP greater than or equal to BNP ˃˃ CNP, but that via the ANP-B receptor (GC-B) was CNP ˃ ANP greater than or equal to BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system..
548. S. I. Suga, K. Nakao, H. Itoh, Y. Komatsu, Y. Ogawa, N. Hama, H. Imura, Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-β. Possible existence of 'vascular natriuretic peptide system', Journal of Clinical Investigation, 10.1172/JCI115933, 90, 3, 1145-1149, 1992, C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, is thus far known to be distributed mainly in the central nervous system and is considered to act as a neuropeptide, in contrast to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which act as cardiac hormones. Recently, we and others have demonstrated that the ANP-B receptor, which is selectively activated by CNP, is localized not only in the central nervous system but in peripheral tissues, including blood vessels. This finding has made us speculate regarding the peripheral production of CNP. In the present study, cultured endothelial cells were examined for CNP production by RIA and Northern blot analysis. CNP-like immunoreactivity was detected in the conditioned media of endothelial cells. Northern blot analysis detected CNPmRNA with a size of 1.2 kb. In addition, transforming growth factor (TGF)-β, one of the key growth factors for vascular remodeling, markedly stimulated the expression of CNPmRNA and induced a tremendous increase in CNP secretion. We could also detect CNP transcript in the bovine thoracic aorta using the reverse transcription- polymerase chain reaction method. The present study demonstrates the endothelial production of CNP and suggests that a member of the natriuretic peptide family may act as a local regulator in vascular walls. Since evidence for the pathophysiological importance of the vascular renin-angiotensin system has been accumulating and the natriuretic peptide system is known to be antagonistic to the renin-angiotensin system, the possible existence of 'vascular natriuretic peptide system' may prove to be of physiological and clinical relevance..
549. K. Nakao, H. Arai, K. Hosoda, Y. Ogawa, O. Nakagawa, S. I. Suga, S. Nakanishi, H. Imura, Molecular cloning of two subtypes of human endothelin receptor, Journal of Vascular Medicine and Biology, 3, 4, 303-307, 1991.12, We isolated complementary DNA (cDNA) clones encoding two subtypes of human endothelin receptor (ETR), ET(A)R and ET(B)R. The cDNA for ET(A)R encoded a 427-amino-acid protein. The rank order of the binding affinity to ET(A)R expressed in COS-7 cells was ET-1 ≥ ET-2 >> ET-3. The clones for human ET(B)R, different in the length of their 3'-untranslated regions, encoded the same 442-amino-acid protein. The rank order of the binding affinity of ET isopeptides (ET-1, ET-2, and ET-3) to ET(B)R expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Both ET(A)R and ET(B)R had a transmembrane topology similar to that of other G-protein-coupled receptors. Northern blot analysis of ET(A)R revealed a single band with a size of 4.3 kb in a wide variety of human tissues, especially in the blood vessel. Northern blot analysis of ET(B)R identified three mRNA species, 4.3, 2.7, and 1.7 kb in size. These mRNAs were also expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells..
550. Masashi Mukoyama, Kazuwa Nakao, Kenji Obata, Michihisa Jougasaki, Michihiro Yoshimura, Etsuo Morita, Kiminori Hosoda, Shin ichi Suga, Yoshihiro Ogawa, Hirofumi Yasue, Hiroo Imura, Augmented secretion of brain natriuretic peptide in acute myocardial infarction, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(05)81311-7, 180, 1, 431-436, 1991.10, In order to elucidate biosynthesis and secretion of natriuretic peptides in the early phase of acute myocardial infarction (AMI), we measured the plasma level of brain natriuretic peptide (BNP), a novel cardiac hormone secreted from the ventricle, in patients with AMI and compared with that of atrial natriuretic peptide (ANP). The plasma level of BNP increased rapidly (within hours from the onset of AMI) and markedly (>100 times the normal level) as compared to that of ANP. The plasma ANP level correlated with pulmonary capillary wedge pressure (PCWP), whereas the plasma BNP level did not correlate with PCWP but highly correlated inversely with cardiac index. These results indicate that BNP is secreted from the heart much more acutely and prominently than ANP in the early phase of AMI, in association with left ventricular dysfunction..
551. Yasato Komatsu, Kazuwa Nakao, Shin Ichi Suga, Yoshihiro Ogawa, Masashi Mukoyama, Hiroshi Arai, Gotaro Shirakami, Kiminori Hosoda, Osamu Nakagawa, Norio Hama, Ichiro Kishimoto, Hiroo Imura, C-type natriuretic peptide (CNP) in rats and humans, Endocrinology, 10.1210/endo-129-2-1104, 129, 2, 1104-1106, 1991.08, We have established a specific radioimmunoassay (RIA) for C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, and have elucidated its tissue distribution and molecular form. In rats, high concentrations of CNP-like immunoreactivity (-LI) were detected in the anterior lobe (19.8 t 8.6 pmol/g) and neurointermediate lobe (4.64 ± 0.74 pmol/g) of the pituitary gland. CNP-LI was present throughout the brain with its high concentrations in the hypothalamus and cerebellum. Small amounts of CNP-LI were also detected in the lower part of gastrointestinal tract and the kidney. However, no significant amount of CNP-LI was present in other organs including the heart. Considerable amounts of CNP-LI were detected throughout the human brain. High performance-gel permeation chromatography coupled with the RIA detected two peaks of CNP-LI in the rat brain; CNP and presumably its N-termina1ly elongated form with 53 amino-acid residues, CNP-53. These findings indicate that the tissue distribution and processing pattern of CNP are clearly different from those of atrial natriuretic peptide and brain natriuretic peptide and suggest possible roles of CNP as a neurotransmitter or neuromodulator rather than as a cardiac hormone..
552. Kiminori Hosoda, Kazuwa Nakao, Hiroshi-Arai, Shin ichi Suga, Yoshihiro Ogawa, Masashi Mukoyama, Gotaro Shirakami, Yoshihiko Saito, Shigetada Nakanishi, Hiroo Imura, Cloning and expression of human endothelin-1 receptor cDNA, FEBS Letters, 10.1016/0014-5793(91)80007-P, 287, 1-2, 23-26, 1991.08, We isolated a human endothelin-1 (ET-1) receptor cDNA from a human placenta cDNA library. The cDNA encodes a 427-amino acid protein with seven putative transmembrane domains. The rank order of the binding to the receptor expressed in COS-7 cells was: ET-1 ≥ ET-2 ≫ ET-3. The receptor expressed in Xenopus oocytes showed a potent electrophysiological response to 1 × 10-7 M ET-1 under voltage clamp at -60 mV, while a much weaker response was produced by 1 × 10-7 M ET-3. Northern blot analysis with RNA from human tissues revealed a single band with a size of 4.3 kb in a wide variety of human tissues, especially highly in the blood vessel..
553. Yoshihiro Ogawa, Kazuwa Nakao, Hiroshi Arai, Osamu Nakagawa, Kiminori Hosoda, Shin ichi Suga, Shigetada Nakanishi, Hiroo Imura, Molecular cloning of a non-isopeptide-selective human endothelin receptor, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(91)91806-N, 178, 1, 248-255, 1991.07, We isolated several complementary DNA (cDNA) clones encoding a non-isopeptide-selective human endothelin receptor (ETBR) from a human placenta cDNA library. The clones, different in the length of their 3′-untranslated regions, encoded the same 442-amino acid protein with a transmembrane topology similar to that of other G protein-coupled receptors. The rank order of the binding of ET isopeptides (ET-1, ET-2 and ET-3) to the receptor expressed in COS-7 cells was ET-1 = ET-2 = ET-3. Northern blot analysis identified three mRNA species, 4.3 kb, 2.7 kb and 1.7 kb in size, probably generated by their use of alternative polyadenylation sites. These mRNAs were expressed in a wide variety of human tissues, at the highest level in the brain and at a significant level in cultured endothelial cells..
554. Kiminori Hosoda, Kazuwa Nakao, Masashi Mukoyama, Yoshihiko Saito, Michihisa Jougasaki, Gotaro Shirakami, Shin Ichi Suga, Yoshihiro Ogawa, Hirofumi Yasue, Hiroo Imura, Expression of brain natriuretic peptide gene in human heart
Production in the ventricle, Hypertension, 10.1161/01.hyp.17.6.1152, 17, 6 SUPPL. 2, 1152-1156, 1991.06, To elucidate the expression of the brain natriuretic peptide gene in the human heart, we have measured brain natriuretic peptide mRNA levels in hearts using the Northern blot hybridization method. Brain natriuretic peptide mRNA was present with a size of approximately 0.9 kb in the ventricle as well as in the atrium. The brain natriuretic peptide mRNA level in the ventricle was 52% of that in the atrium, whereas the atrial natriuretic peptide mRNA level in the ventricle was approximately two orders of magnitude lower than that in the atrium. Taking atrial and ventricular weights into account, the total amount of brain natriuretic peptide mRNA in the ventricle represented 77% of that in the whole heart. These results demonstrate that most of brain natriuretic peptide mRNA occurs in the ventricle, in contrast with atrial natriuretic peptide mRNA, which is present mainly in the atrium, indicating that the ventricle is a major production site of brain natriuretic peptide..
555. K. Nakao, M. Mukoyama, K. Hosoda, S. Suga, Yoshihiro Ogawa, Y. Saito, G. Shirakami, H. Arai, M. Jougasaki, H. Imura, Biosynthesis, secretion, and receptor selectivity of human brain natriuretic peptide, Canadian Journal of Physiology and Pharmacology, 10.1139/y91-225, 69, 10, 1500-1506, 1991.01, Biosynthesis, secretion and receptor selectivity of human brain natriuretic peptide (hBNP) were studied. The BNP mRNA level in the ventricle was approximately 40% of that in the atrium and, taking tissue weight into account; the total amount of BNP mRNA in the ventricle was about twofold greater than the total amount in the atrium. The plasma BNP-like immunoreactivity (-LI) level in normal subjects was 0.90 ± 0.07 fmol/mL, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with congestive heart failure, with a progressive rise in proportion to its severity. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root, and the difference in the plasma BNP-LI level between the CS and the aorta (Ao), Δ((CS-Ao))BNP, increased with the severity of congestive heart failure. In addition; the difference in the BNP-LI level between the anterior inverventricular vein (AIV) draining the ventricle and the aorta (Δ((AIV-Ao))BNP) was comparable to Δ((CS-Ao))BNP, indicating that BNP is secreted predominantly from the ventricle. Binding ability to human clearance receptors (C receptors) and cyclic GMP (cGMP) production of hBNP were investigated and compared with those of ANP. hBNP bound to human C receptors very weakly (about 7%), but exerted cGMP production similar to ANP in cultured human mesangial cells and bovine endothelial cells. In conclusion, hBNP is a novel cardiac hormone mainly synthesized in and secreted from the ventricle and plays physiological and pathophysiological roles in the dual cardiac natriuretic peptide system..
556. Yoshihiko Saito, Nakao Kazuwa, Gotaro Shirakami, Masashi Mukoyama, Hiroshi Arai, Kiminori Hosoda, Shin ichi Suga, Yoshihiro Ogawa, Hiroo Imura, Endothelin in patients with chronic renal failure, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199100177-00125, 17, S437-S439, 1991.01, To investigate the pathophysiological significance of endothelin-1 (ET-1) in renal failure, we measured plasma and urine concentrations of ET-1-Like immunoreactivity (LI) by radioimmunoassay (RIA). The plasma ET-1-LI level was significantly increased in hemodialyzed and nonhemodialyzed patients with chronic renal failure (CRF). The increase in the plasma ET-1-LI level in CRF was much larger than that in other diseases in which the plasma ET-1-LI level was reported to be increased. The urine ET-1-LI level was lower, and daily excreted amounts of ET-1-LI were smaller in CRF patients than in normal subjects. These findings indicate that the increased plasma level of ET-1-LI in CRF results from the decreased clearance rate of ET-1-LI in the kidney. Gel permeation chromatographic analysis revealed that ET-1-LI in plasma from CRF consisted of small and large molecular forms of ET-1-LI. The small molecular form is presumably ET-1, and large molecular forms are big ET-1 and another component with the molecular weight of 6,000 as is the case of that in normal plasma (1). However, the ratio of the smaller to large molecular forms of ET-1-LI in CRF was 1:13 and was quite different from that in normal plasma (1:4). These observations clearly indicate that the elevation of plasma ET-1-LI level in CRF was mainly due to the increase in the large molecular form of ET-1-LI..
557. Y. Ogawa, K. Nakao, M. Mukoyama, K. Hosoda, G. Shirakami, H. Arai, Y. Saito, S. I. Suga, M. Jougasaki, H. Imura, Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats
The ventricle is a major site of synthesis and secretion of brain natriuretic peptide, Circulation research, 10.1161/01.RES.69.2.491, 69, 2, 491-500, 1991.01, To study synthesis, storage, and secretion of brain natriuretic peptide (BNP) in the heart, we have measured BNP mRNA and BNP concentrations in the hearts of Wistar-Kyoto rats and also have investigated its secretion from the isolated perfused heart. The atrium expressed the BNP gene at a high level, and a considerable amount of BNP mRNA also was present in the ventricle, which corresponded to approximately 40% of the atrial BNP mRNA concentration. When tissue weight was taken into account, the total content of BNP mRNA in the ventricle was approximately threefold larger than that in the atrium, although the atrial natriuretic peptide (ANP) mRNA content in the ventricle was only 7% of that in the atrium. By contrast, the BNP concentration in the ventricle was 4.07 ± 0.97 pmol/g, which was less than 1% of that in the atrium (451 ± 86 pmol/g). The basal secretory rate of BNP from the isolated perfused whole heart was 49.3 ± 6.1 fmol/min, approximately 60% of which was maintained even after atrial removal, whereas the secretory rate of ANP was reduced to less than 5%. We also studied age-matched spontaneously hypertensive rats-stroke prone. The rank order of the BNP mRNA concentration in the hearts of these rats was left ventricle>right ventricle>right atrium=left atrium, and the total BNP mRNA content and BNP secretory rate in the ventricle were twice as large as in Wistar-Kyoto rats. These results demonstrate that BNP is a novel cardiac hormone in rats and is predominantly synthesized in and secreted from the ventricle. This is in striking contrast to ANP, which occurs mainly in the atrium. The results also suggest possible pathophysiological roles of BNP in certain cardiovascular disorders..
558. Gotaro Shirakami, Kazuwa Nakao, Yoshihiko Saito, Tatsuo Magaribuchi, Michihisa Jougasaki, Masashi Mukoyama, Hiroshi Arai, Kiminori Hosoda, Shin ichi Suga, Yoshihiro Ogawa, Takayuki Yamada, Kenjiro Mori, Hiroo Imura, Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats, Life Sciences, 10.1016/0024-3205(91)90362-F, 48, 10, 969-976, 1991, To investigate the effect of pulmonary alveolar hypoxia on the sunthesis and release of endothelin (ET)-1, ET-1-like immunoreactivity (-LI) levels of the lung and plasma were measured in conscious unrestrained rats under hypoxic conditions. Sixty-min exposure to alveolar hypoxia (10 % O2 or 5 % O2) increased the ET-1-LI level in the lung. The plasma ET-1-LI level in hypoxic rats also increased significantly. The increase of plasma and lung ET-1-LI levels were parallel to the severity of hypoxia. These results demonstrates that acute pulmonary alveolar hypoxia increases lung and plasma ET-1-LI levels in conscious unrestrained rats, suggesting a possible physiological or pathophysiological significance of ET in alveolar hypoxia..
559. M. Mukoyama, K. Nakao, K. Hosoda, S. I. Suga, Y. Saito, Y. Ogawa, G. Shirakami, M. Jougasaki, K. Obata, H. Yasue, Y. Kambayashi, K. Inouye, H. Imura, Brain natriuretic peptide as a novel cardiac hormone in humans
Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide, Journal of Clinical Investigation, 10.1172/JCI115146, 87, 4, 1402-1412, 1991.04, Using a specific radioimmunoassay for human brain natriuretic peptide (hBNP) with a monoclonal antibody, we have investigated its synthesis, secretion, and clearance in comparison with those of atrial natriuretic peptide (ANP) in normal subjects and patients with congestive heart failure (CHF). Mean BNP-like immunoreactivity (-LI) levels in normal atrium and ventricle were 250 and 18 pmol/g, respectively. The plasma BNP-LI level in normal subjects was 0.90 ± 0.07 fmol/ml, which was 16% of the ANP-LI level. In contrast, the plasma BNP-LI level markedly increased in patients with CHF in proportion to its severity, and surpassed the ANP-LI level in severe cases. There was a significant step-up of the plasma BNP-LI level in the coronary sinus (CS) compared with that in the aortic root (Ao) and the difference between these BNP-LI levels, Δ((CS-Ao))BNP, also increased with the severity of CHF. In addition, the step-up of the BNP-LI level in the anterior interventricular vein [Δ((AIV-Ao))BNP] was comparable to Δ((CS-Ao))BNP, indicating that BNP is secreted mainly from the ventricle. Predominant BNP synthesis in the ventricle was also confirmed by Northern blot analysis. Catheterization and pharmacokinetic studies revealed that hBNP is cleared from the circulation more slowly than α-hANP; this was in part attributed to lower (about 7%) binding affinity of hBNP to clearance receptors than that of α-hANP. A predominant molecular form of BNP-LI in the heart and plasma was a 3-kD form corresponding to hBNP. These results indicate that BNP is a novel cardiac hormone secreted predominantly from the ventricle, and that the synthesis, secretion and clearance of BNP differ from those of ANP, suggesting discrete physiological and pathophysiological roles of BNP in a dual natriuretic peptide system..
560. Osamu Nakagawa, Kazuwa Nakao, Yoshihiko Saito, Gotaro Shirakami, Michihisa Jougasaki, Masashi Mukoyama, Kiminori Hosoda, Shin ichi Suga, Yoshihiro Ogawa, Ichiro Kishimoto, Yasato Komatsu, Norio Hama, Hiroo Imura, Isolation and characterization of porcine endothelin-like peptide, Journal of Cardiovascular Pharmacology, 10.1097/00005344-199100177-00005, 17, S13-S16, 1991, Two endogenous molecules possessing an endothelin-like sequence with an apparent molecular weight of 7 kDa were isolated from serum-free culture medium of porcine aortic endothelial cells, using a specific radioimmunoassay. N-terminal sequences of two molecules were identical to each other and were Ser-Leu-Lys-Asp-Leu-Phe-Pro-Ala-Lys-Ala-Ala-Asp-Arg-Arg-Asp-Arg-X-Gln-X-Ala-X-Gln-Lys-Asp, which corresponds to the sequence [94-117] of preproendothelin-1. This finding indicates that these two molecules may be closely related peptides such as the oxidized form or disulfide analogues and also suggests that the endogenous peptide possessing an endothelin-like sequence is generated by proteolytic cleavage at paired basic amino acids Arg92-Arg93Further studies on the structure and function of new endogenous peptides possessing an endothelin-like sequence are ongoing in our laboratory..
561. G. Shirakami, K. Nakao, Y. Saito, T. Magaribuchi, H. Nagata, M. Jougasaki, M. Mukoyama, H. Arai, K. Hosoda, S. Suga, Y. Ogawa, K. Mori, H. Imura, Endothelin in human cerebrospinal fluid, Therapeutic Research, 11, 12, 111-116, 1990.12.
562. Masashi Mukoyama, Kazuwa Nakao, Yoshihiko Saito, Yoshihiro Ogawa, Kiminori Hosoda, Shin Ichi Suga, Gotaro Shirakami, Michihisa Jougasaki, Hiroo Imura, Increased Human Brain Natriuretic Peptide in Congestive Heart Failure, New England Journal of Medicine, 10.1056/NEJM199009133231114, 323, 11, 757-758, 1990.09, To the Editor: Brain natriuretic peptide (BNP), first isolated from the brains of pigs,1 has a striking similarity to atrial natriuretic peptide (ANP) both in structure and in peripheral and central actions.1 2 3 4 BNP is also a cardiac hormone in pigs5 and rats.6,7 Antiserums against porcine or rat BNP, however, failed to detect BNP-like immunoreactivity in human tissues, indicating the structural diversity of BNP among species. Recently, we isolated human BNP from the atrium and determined its 32-amino-acid sequence.8 We have developed a specific radioimmunoassay for human BNP8 with the use of a monoclonal antibody, modeling it on a radioimmunoassay for..
563. Yoshihiko Saito, Kazuwa Nakao, Masashi Mukoyama, Gotaro Shirakami, Hiroshi Itoh, Takayuki Yamada, Hiroshi Arai, Kiminori Hosoda, Shin Ichi Suga, Michihisa Jougasaki, Yoshihiro Ogawa, Shigeyuki Nakajima, Motohiko Ueda, Hiroo Imura, Application of monoclonal antibodies for endothelin to hypertensive research, Hypertension, 10.1161/01.HYP.15.6.734, 15, 6, 734-738, 1990.06, We developed six kinds of monoclonal antibodies against endothelin (ET)-l recognizing different epitopes with high affinities (5x1010 M-1 to 5x1O11 M-1). Using these monoclonal antibodies, we developed radioimmunoassays for ET-1 with different specificities. Crossreactivities with ET-2 ranged from 80% to 100%, and those with ET-3 ranged from 3% to 60%. Patients with essential hypertension (si=20) showed a significant elevation in the plasma ET-l-LI level compared with age-matched control subjects (n = 12) (30.1 ±1.4 pg/ml versus 18.5±0.9 pg/ml, p<0.01). The plasma ET-l-LI level in hypertensive patients in stages II and 111 (World Health Organization classification) was significantly higher than that in those patients in stage I. There was no significant correlation between the plasma ET-l-LI level and systolic blood pressure (r=0.11), diastolic blood pressure (r=-0.13), or age (r=0.24) in all patients studied who had essential hypertension. In the neutralization experiment, monoclonal antibodies attenuated ET-l-induced contraction of rat aortic rings and the pressor action of ET-1 in pithed rats in vivo. The present study demonstrates the elevated plasma ET-l-LI level in patients with essential hypertension. Monoclonal antibodies developed in this study can become powerful tools to investigate the pathophysiological significance of ET in essential hypertension..
564. Kazuwa Nakao, Hiroshi Itoh, Yoshikazu Kambayashi, Kiminori Hosoda, Yoshihiko Saito, Takayuki Yamada, Masashi Mukoyama, Hiroshi Arai, Gotaro Shirakami, Shin Ichi Suga, Michihisa Jougasaki, Yoshihiro Ogawa, Ken Inouye, Hiroo Imura, Rat brain natriuretic peptide
Isolation from rat heart and tissue distribution, Hypertension, 10.1161/01.HYP.15.6.774, 15, 6, 774-778, 1990.06, We have isolated a cardiac natriuretic peptide of 5, 000 d from atrial tissues from 500 rats and determined its amino acid sequence. The 5, 000 d atrial natriuretic factor was elucidated to be a 45 amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-K-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R-L-G-C-D-G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence (51-95) of the rat brain natriuretic peptide precursor deduced from the complementary DNA (cDNA) sequence. The cardiac natriuretic peptide with a molecular weight of 5, 000, or rat brain natriuretic peptide, was identified as the mqjor storage form and as the sole secretory form derived from the brain natriuretic peptide precursor in the rat heart. The rat brain natriuretic peptide level in the atrium was 3.68±0.61 μg/g, which represents about 4% of that of atrial natriuretic factor. Rat brain natriuretic peptide was also detected in the ventricle. The ratio of brain natriuretic peptide to atrial natriuretic peptide in the ventricle was approximately 30% and much higher than that in the atrium. Rat brain natriuretic peptide, however, was not detectable in the brain. We conclude that the 5, 000 d cardiac natriuretic peptide is rat brain natriuretic peptide with 45 amino acids derived from the brain natriuretic peptide precursor and is secreted from the rat heart as a novel cardiac hormone..
565. T. Yamada, K. Nakao, G. Shirakami, Y. Saito, M. Mukoyama, H. Arai, K. Hosoda, S. Suga, M. Jogasaki, Y. Ogawa, Nerve tissue distribution and physiologic role of endogenous opioid peptides and their receptors--POMC-, proenkephalin A, proenkephalin B- derived opioid peptides and kyotorphin, Nippon rinsho. Japanese journal of clinical medicine, 48, 5, 1042-1052, 1990.05.
566. Yoshihiro Ogawa, Kazuwa Nakao, Masashi Mukoyama, Gotaro Shirakami, Hiroshi Itoh, Kiminori Hosoda, Yoshihiko Saito, Hiroshi Arai, Shin Ichi Suga, Michihisa Jougasaki, Takayuki Yamada, Yoshikazu Kambayashi, Ken Inouye, Hiroo Imura, Rat brain natriuretic peptide — tissue distribution and molecular form —, Endocrinology, 10.1210/endo-126-4-2225, 126, 4, 2225-2227, 1990.04, Using an antiserum against the ring structure of rat brain natriuretic peptide (rat BNP), we have established a specific radioimmunoassay (RIA) for rat BNP and elucidated its tissue distribution and molecular form. Rat BNP-like immunoreactivity (-LI) was detected in the highest amount in cardiac extracts (574.0 ± 138.8 pmol/g in the atrium and 4.3 ± 1.1 pmol/g in the ventricle). The secretory rate of rat BNP-LI from the perfused whole heart was.50.0 ± 1.9 fmol/min, about 60% of which was maintained even after atrial removal. We also detected rat BNP-LI throughout the spinal cord (134-175 fmol/g), although no detectable amount was present (less than 100 fmol/g) in other tissues including the brain. High performance-gel permeation chromatography and reverse phase-high performance liquid chromatography coupled with the RIA revealed that rat BNP with 45 amino acids is a major storage form as well as a secretory form of rat BNP-LI in the heart. The major component in the spinal cord was also rat BNP. These findings indicate that the tissue distribution and the processing pattern of rat BNP are different from those of atrial natriuretic peptide and porcine BNP, thereby suggesting the presence of complicated diversity of the natriuretic peptide system..
567. Masashi Mukoyama, Kazuwa Nakao, Yoshihiko Saito, Yoshihiro Ogawa, Kiminori Hosoda, Shin Ichi Suga, Gotaro Shirakami, Michihisa Jougasaki, Hiroo Imura, Human brain natriuretic peptide, a novel cardiac hormone, The Lancet, 10.1016/0140-6736(90)90925-U, 335, 8692, 801-802, 1990.03.
568. Yoshikazu Kambayashi, Kazuwa Nakao, Masashi Mukoyama, Yoshihiko Saito, Yoshihiro Ogawa, Shozo Shiono, Ken Inouye, Nobuo Yoshida, Hiroo Imura, Isolation and sequence determination of human brain natriuretic peptide in human atrium, FEBS Letters, 10.1016/0014-5793(90)80043-I, 259, 2, 341-345, 1990.01, We isolated human brain natriuretic peptide (human BNP) from the human atrium. Sequence analysis has revealed that it is a 32-amino-acid peptide with the sequence S-P-K-M-V-Q-G-S-G-C-F-G-R-K-M-D-R-I-S-S-S-S-G-L-G-C-K-V-L-R-R-H, which is identical to the C-terminal sequence (77-108) of the human BNP precursor deduced from the cDNA sequence. The sequence of human BNP (77-108) is preceded by Pro75-Arg76 in the human BNP precursor, which is the same processing signal as Pro97-Arg98 of the precursor of atrial natriuretic peptide (ANP). The processing of the BNP precursor occurs in the cardiocyte, although that of the ANP precursor in the cardiocyte is unclear at present..
569. Yoshihiko Saito, Kazuwa Nakao, Gotaro Shirakami, Michihisa Jougasaki, Takayuki Yamada, Hiroshi Itoh, Masashi Mukoyama, Hiroshi Arai, Kiminori Hosoda, Shin ichi Suga, Yoshihiro Ogawa, Hiroo Imura, Detection and characterization of endothelin-1-like immunoreactivity in rat plasma, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(89)91151-0, 163, 3, 1512-1516, 1989.09, Using two radioimmunoassays (RIAs) for endothelin-1 (ET-1) with and without a substantial cross-reactivity with ET-3, we have measured the plasma ET-1-like immunoreactivity (-LI) level in rat plasma. ET-1-LI was detected in plasma from male Wistar rats. ET-1-LI in rat plasma consisted of three components with molecular weights of 6K, 4K and 2.5K daltons by gel permeation chromatography. Two of the components were eluted at positions of big ET (4K) and synthetic ET-1 (2.5K). The remaining component was eluted at the preceding fraction (6K). No difference was observed in ET-1-LI of the small molecular form of ET (2.5K) between the two RIAs. Thus, there is little or no ET-3 in rat plasma, which has the sequence found originally in the rat genome. The concentration of the small molecular form of ET, presumably ET-1, in rat plasma was about 4 pg/ml..
570. Yoshikazu Kambayashi, Kazuwa Nakao, Hiroshi Itoh, Kiminori Hosoda, Yoshihiko Saito, Takayuki Yamada, Masashi Mukoyama, Hiroshi Arai, Gotaro Shirakami, Shin ichi Suga, Yoshihiro Ogawa, Michihisa Jougasaki, Naoto Minamino, Kenji Kangawa, Hisayuki Matsuo, Ken Inouye, Hiroo Imura, Isolation and sequence determination of rat cardiac natriuretic peptide, Biochemical and Biophysical Research Communications, 10.1016/0006-291X(89)92126-8, 163, 1, 233-240, 1989.08, We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R-L-G-C-D-G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart..