| 1. |
Rongzhi Li, Tomomi Sano, Akiko Mizokami, Takao Fukuda, Takanori Shinjo, Misaki Iwashita, Akiko Yamashita, Terukazu Sanui, Yusuke Nakatsu, Yusuke Sotomaru, Tomoichiro Asano, Takashi Kanematsu, Fusanori Nishimura, miR-582-5p targets Skp1 and regulates NF-κB signaling-mediated inflammation., Archives of biochemistry and biophysics, 10.1016/j.abb.2022.109501, 109501-109501, 2022.12, A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3'-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.. |
| 2. |
Jing Gao, Akiko Mizokami, Hiroshi Takeuchi, Aonan Li, Fei Huang, Haruki Nagano, Takashi Kanematsu, Eijiro Jimi, Masato Hirata, Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes., Journal of cell science, 10.1242/jcs.258584, 135, 1, 2022.01, Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.. |
| 3. |
Kayo Mori, Akiko Mizokami, Tomomi Sano, Satoru Mukai, Fumitaka Hiura, Yasunori Ayukawa, Kiyoshi Koyano, Takashi Kanematsu, Eijiro Jim, RANKL elevation activates NIK/NF-κB pathway, inducing obesity in ovariectomized mice, Journal of Endocrinology, https://doi.org/10.1530/JOE-21-0424, 2022.04, Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized wild-type obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.. |
| 4. |
Jing Gao, Akiko Mizokami, Hiroshi Takeuchi, Aonan Li, Fei Huang, Haruki Nagano, Takashi Kanematsu, Eijiro Jimi, Masato Hirata, Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes, JOURNAL OF CELL SCIENCE, 10.1242/jcs.258584, 135, 1, 2022.01. |
| 5. |
Tomoyo Kawakubo-Yasukochi, Ena Yano, Soi Kimura, Takuya Nishinakagawa, Akiko Mizokami, Yoshikazu Hayashi, Yuji Hatakeyama, Kenji Ohe, Atsushi Yasukochi, Seiji Nakamura, Eijiro Jimi, Masato Hirata, Hepatic glycogenolysis is determined by maternal high-calorie diet via methylation of Pygl and this is modified by osteocalcin administration in mice, Molecular Metabolism, doi: 10.1016/j.molmet.2021.101360., 2021.10. |
| 6. |
Mizokami A., Mukai S., Gao J., Kawakubo-Yasukochi T., Otani T., Takeuchi H., Jimi E., Hirata M., GLP-1 signaling is required for improvement of glucose tolerance by osteocalcin, Journal of Endocrinology, 10.1530/JOE-19-0288, 2020.01. |
| 7. |
Kobayakawa M., Matsubara M., Mizokami A., Hiura F., Takakura N., Kokabu S., Matsuda M., Yasuda H., Nakamura I., Takei Y., Honda H., Hosokawa R., Jimi E., Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas, Cell Biochemistry & Function, 10.1002/cbf.3476, 2020.01. |
| 8. |
Yosuke Yamawaki, Satomi Shirawachi, Akiko Mizokami, Kanako Nozaki, Hikaru Ito, Satoshi Asano, Kana Oue, Hidenori Aizawa, Shigeto Yamawaki, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein regulates lipopolysaccharide-induced hypothalamic inflammation-mediated anorexia in mice, Neurochemistry International, 10.1016/j.neuint.2019.104563, 131, 2019.12, Peripheral lipopolysaccharide (LPS) injection induces systemic inflammation through the activation of the inhibitor of nuclear factor kappa B (NF-κB) kinase (IKK)/NF-κB signaling pathway, which promotes brain dysfunction resulting in conditions including anorexia. LPS-mediated reduction of food intake is associated with activation of NF-κB signaling and phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the hypothalamus. We recently reported phospholipase C-related catalytically inactive protein (PRIP) as a new negative regulator of phosphatidylinositol 3-kinase/AKT signaling. AKT regulates the IKK/NF-κB signaling pathway; therefore, this study aimed to investigate the role of PRIP/AKT signaling in LPS-mediated neuroinflammation-induced anorexia. PRIP gene (Prip1 and Prip2) knockout (Prip-KO) mice intraperitoneally (ip) administered with LPS exhibited increased anorexia responses compared with wild-type (WT) controls. Although few differences were observed between WT and Prip-KO mice in LPS-elicited plasma pro-inflammatory cytokine elevation, hypothalamic pro-inflammatory cytokines were significantly upregulated in Prip-KO rather than WT mice. Hypothalamic AKT and IKK phosphorylation and IκB degradation were significantly increased in Prip-KO rather than WT mice, indicating further promotion of AKT-mediated NF-κB signaling. Consistently, hypothalamic STAT3 was further phosphorylated in Prip-KO rather than WT mice. Furthermore, suppressor of cytokine signaling 3 (Socs3), a negative feedback regulator for STAT3 signaling, and cyclooxogenase-2 (Cox2), a candidate molecule in LPS-induced anorexigenic responses, were upregulated in the hypothalamus in Prip-KO rather than WT mice. Pro-inflammatory cytokines were upregulated in hypothalamic microglia isolated from Prip-KO rather than WT mice. Together, these findings indicate that PRIP negatively regulates LPS-induced anorexia caused by pro-inflammatory cytokine expression in the hypothalamus, which is mediated by AKT-activated NF-κB signaling. Importantly, hypothalamic microglia participate in this PRIP-mediated process. Elucidation of PRIP-mediated neuroinflammatory responses may provide novel insights into the pathophysiology of many brain dysfunctions.. |
| 9. |
Takahito Otani, Miho Matsuda, Akiko Mizokami, Norio Kitagawa, Hiroshi Takeuchi, Eijiro Jimi, Tetsuichiro Inai, Masato Hirata, Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300, Cell Death and Disease, 10.1038/s41419-018-1257-7, 9, 12, 2018.12, The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP–PKA–ERK–CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca
2+
influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.. |
| 10. |
Kana Oue, Yosuke Yamawaki, Satoshi Asano, Akiko Mizokami, Masato Hirata, Masahiro Irifune, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein-knockout mice exhibit uncoupling protein 1 upregulation in adipose tissues following chronic cold exposure, Journal of Oral Biosciences, 10.1016/j.job.2017.04.001, 59, 2, 108-112, 2017.05, Objectives We have previously demonstrated that phospholipase C-related catalytically inactive protein (PRIP) is involved in fat metabolism and energy consumption. However, whether PRIP participates in body energy metabolism in vivo remains to be determined. Therefore, we examined whether PRIP deficiency affects whole-body energy homeostasis, which is modulated by non-shivering thermogenesis in brown adipose tissue, using a cold exposure animal model. Methods Fasting plasma triacylglycerol levels were measured to evaluate fat metabolism in wild-type and Prip-KO mice. In addition, a glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed. To determine changes in energy consumption, mice were exposed to a cold environment for 7 days, and expression of uncoupling protein 1 (UCP1) in brown adipose tissue was analyzed via western blotting. Results Fasting plasma levels of triacylglycerols were significantly higher in Prip-KO mice than in wild-type mice. However, Prip-KO mice showed a healthy phenotype based on GTT and ITT. UCP1 expression was significantly upregulated in the brown and white adipose tissues of Prip-KO mice exposed to cold conditions. Conclusion Prip-KO mice exhibit greater ability to consume lipids as an energy source, indicating that PRIP modulates of systemic energy expenditure. Our findings provide increased understanding of PRIP-mediated non-shivering thermogenic mechanisms and offers important insights for the treatment and control of obesity.. |
| 11. |
Yoshikazu Hayashi, Tomoyo Kawakubo-Yasukochi, Akiko Mizokami, Mai Hazekawa, Tomiko Yakura, Munekazu Naito, Hiroshi Takeuchi, Seiji Nakamura, Masato Hirata, Uncarboxylated osteocalcin induces antitumor immunity against mouse melanoma cell growth, Journal of Cancer, 10.7150/jca.18648, 8, 13, 2478-2486, 2017.01, Because of the poor response to chemotherapy and radiation therapy, new treatment approaches by immune-based therapy involving activated T cells are required for melanoma. We previously reported that the uncarboxylated form of osteocalcin (GluOC), derived from osteoblasts, potentially suppresses human prostate cancer cell proliferation by direct suppression of cell growth. However, the mechanisms in vivo have not been elucidated. In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma transplants in C57Bl/6N wild-type mice. Our data demonstrated that GluOC suppressed cell growth by downregulating phosphorylation levels of receptor tyrosine kinases and inducing apoptosis in vitro. Additionally, stimulation of primary mouse splenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC increased T cell proliferation and their interferon-γ production. Taken together, we demonstrate that GluOC exerts multiple antitumor effects not only in vitro, but also in vivo through cellular immunostimulatory effects against B16 mouse melanoma cells.. |
| 12. |
Kondo A., Kawakubo-Yasukochi T., Mizokami A., Chishaki S., Takeuchi H., Hirata M., Uncarboxylated osteocalcin increases serum nitric oxide levels and ameliorates hypercholesterolemia in mice fed an atherogenic diet, 2016.12. |
| 13. |
Akiko Mizokami, Da Guang Wang, Mitsuru Tanaka, Jing Gao, Hiroshi Takeuchi, Toshiro Matsui, Masato Hirata, An extract from pork bones containing osteocalcin improves glucose metabolism in mice by oral administration, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2016.1214530, 80, 11, 2176-2183, 2016.11, Osteocalcin (OC) is a bone-derived hormone that regulates energy metabolism. OC exists in two forms, carboxylated (GlaOC) and uncaboxylated (GluOC), but only the latter appears to have an endocrine function. In this study, we prepared an extract containing both Gla- and GluOC from boiled pork bone using 0.2 M carbonate buffer at pH 9.5, and tested whether the extract had beneficial effects on improving metabolic parameters in obese mice. The extract equivalent of 1.2 μg of GluOC/mouse was orally administrated to C57BL/6 female mice fed a high-fat, high-sucrose diet. Daily oral administration of the extract for four weeks decreased blood glucose levels and promoted glucose tolerance as well as insulin sensitivity. Our study shows for the first time that boiled pork bones are a source material for osteocalcin in the large-scale production of supplements designed to improve glucose metabolism.. |
| 14. |
Jing Gao, Makiko Hirata, Akiko Mizokami, Jin Zhao, Ichiro Takahashi, Hiroshi Takeuchi, Masato Hirata, Differential role of SNAP-25 phosphorylation by protein kinases A and C in the regulation of SNARE complex formation and exocytosis in PC12 cells, Cellular Signalling, 10.1016/j.cellsig.2015.12.014, 28, 5, 425-437, 2016.05, The final step of regulated exocytosis, membrane fusion, is mediated by formation of the SNARE complex by syntaxin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and VAMP (vesicle-associated membrane protein). Phosphorylation of SNARE and accessory proteins contributes to regulation of exocytosis. We previously identified residues of SNAP-25 phosphorylated by protein kinase A (PKA) and PKC. However, the physiological role of SNAP-25 phosphorylation in exocytosis, in particular with regard to SNARE complex formation, has remained elusive. SNARE complex formation by purified recombinant SNAP-25, syntaxin-1, and VAMP-in vitro was inhibited or promoted as a result of the phosphorylation at Thr138 by PKA or at Ser187 by PKC, respectively. SNARE complex formation in intact PC12 cells was similarly inhibited by forskolin (activator of PKA) and promoted by phorbol 12-myristate 13-acetate (PMA, activator of PKC). Noradrenaline secretion from PC12 cells induced by a high K+ concentration was enhanced by forskolin or PMA. Stable depletion of SNAP-25 inhibited high-K+-induced noradrenaline secretion. Forced expression of WT SNAP-25 restored the secretory response of the SNAP-25-depleted cells to high-K+, and this response was enhanced by forskolin or PMA. Expression of the nonphosphorylatable T138A or S187A mutants of SNAP-25 similarly rescued the secretory response to high-K+, but the augmentation of this response by forskolin was more pronounced in the cells expressing SNAP-25 (T138A) than in those expressing SNAP-25 (WT), whereas that by PMA was less pronounced in those expressing SNAP-25 (S187A). Our results thus suggest that SNAP-25 phosphorylation by PKA or PKC contributes differentially to the control of exocytosis in PC12 cells by regulating SNARE complex formation.. |
| 15. |
Yu Yasutake, Akiko Mizokami, Tomoyo Kawakubo-Yasukochi, Sakura Chishaki, Ichiro Takahashi, Hiroshi Takeuchi, Masato Hirata, Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet, American Journal of Physiology - Endocrinology and Metabolism, 10.1152/ajpendo.00334.2015, 310, 8, E662-E675, 2016.04, Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic β-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic β-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.. |
| 16. |
Tomoyo Kawakubo-Yasukochi, Akihiko Kondo, Akiko Mizokami, Yoshikazu Hayashi, Sakura Chishaki, Seiji Nakamura, Hiroshi Takeuchi, Masato Hirata, Maternal oral administration of osteocalcin protects offspring from metabolic impairment in adulthood, Obesity, 10.1002/oby.21447, 24, 4, 895-907, 2016.04, Objective Maternal diet during pregnancy has been found to influence the health of offspring. However, strategies for modulation of maternal energy metabolism without an adverse effect on the fetus have remained limited. It was recently shown that oral administration of uncarboxylated osteocalcin (GluOC) improves metabolic status in adult female mice. Whether maternal GluOC administration during gestation might improve the metabolic status of offspring was investigated. Methods Female C57BL/6 mice were fed a normal diet (ND) or high-fat, high-sucrose diet (HFS) and were given saline or GluOC by oral administration during pregnancy. The resulting offspring were in turn assigned to ND- or HFS-fed groups immediately after weaning, and their body weight, glucose metabolism, serum lipid parameters, and level of adipose tissue inflammation were subsequently assessed. Results Maternal HFS feeding during gestation had adverse effects on glucose and lipid parameters, body weight, and adipose tissue inflammation in female offspring fed the same diet, and these effects were attenuated by maternal oral GluOC administration. Conclusions Maternal oral administration of GluOC protects HFS-fed female offspring from metabolic disorders induced by maternal obesity.. |
| 17. |
Yoshikazu Hayashi, Tomoyo Kawakubo-Yasukochi, Akiko Mizokami, Hiroshi Takeuchi, Seiji Nakamura, Masato Hirata, Differential roles of carboxylated and uncarboxylated osteocalcin in prostate cancer growth, Journal of Cancer, 10.7150/jca.15523, 7, 12, 1605-1609, 2016.01, Serum levels of osteocalcin (OC), a bone matrix non-collagenous protein secreted by osteoblasts, are correlated with pathological bone remodeling such as the bone metastasis of cancer, as well as physiological bone turnover. The pathological roles in prostate cancer growth of the two existing types of serum OC, γ-carboxylated (GlaOC) and lower- (or un-) carboxylated (GluOC), have not yet been discriminatively examined. In the present study, we demonstrate that normal prostate epithelial cell growth was promoted by both types of OC, while growth of cancer cells in the prostate was accelerated by GlaOC but suppressed by GluOC. We suggest that OC regulates prostate cancer growth depending on the γ-carboxylation, in part by triggering reduced phosphorylation of receptor tyrosine kinases.. |
| 18. |
Shintaro Tsuka, Fumiko Aonuma, Sen Higashi, Tomoko Ohsumi, Koki Nagano, Akiko Mizokami, Tomoyo Kawakubo-Yasukochi, Chihiro Masaki, Ryuji Hosokawa, Masato Hirata, Hiroshi Takeuchi, Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2015.02.123, 459, 3, 437-442, 2015.04, A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca2+ levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.. |
| 19. |
Takahito Otani, Akiko Mizokami, Yoshikazu Hayashi, Jing Gao, Yoshihide Mori, Seiji Nakamura, Hiroshi Takeuchi, Masato Hirata, Signaling pathway for adiponectin expression in adipocytes by osteocalcin, Cellular Signalling, 10.1016/j.cellsig.2014.12.018, 27, 3, 532-544, 2015.03, In addition to providing skeletal support, the bone is an endocrine organ that produces osteocalcin, whose uncarboxylated form (GluOC) increases insulin secretion either directly or indirectly by promoting incretin secretion. We have now investigated the signaling pathway by which GluOC increases expression of adiponectin in adipocytes. Activation of its putative receptor GPRC6A by GluOC induced the intracellular accumulation of cAMP and consequent activation of protein kinase A (PKA) in differentiated 3T3-L1 adipocytes. It also induced phosphorylation of CREB (cAMP response element binding protein), but this effect appeared to be mediated indirectly by extracellular signal-regulated kinase (ERK) rather than directly by PKA, given that it was attenuated by the ERK signaling inhibitor U0126. Activated PKA also induced activation of the tyrosine kinase Src, the small GTPase Rap1, an upstream of ERK and CREB phosphorylation. Activated CREB up-regulated the expression of peroxisome proliferator-activated receptor γ (PPARγ), which in turn led to induction of adiponectin expression. Finally, intermittent oral administration of GluOC in mice reduced the size of gonadal white adipocytes as well as increased the expression of PPARγ and adiponectin in these cells. Our results have thus revealed the signaling pathway by which GluOC induces adiponectin expression in adipocytes.. |
| 20. |
Akiko Mizokami, Yu Yasutake, Sen Higashi, Tomoyo Kawakubo-Yasukochi, Sakura Chishaki, Ichiro Takahashi, Hiroshi Takeuchi, Masato Hirata, Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion, Bone, 10.1016/j.bone.2014.09.006, 69, 68-79, 2014.09, Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion and pancreatic β-cell proliferation. We previously showed that the effect of GluOC on insulin secretion is mediated largely by glucagon-like peptide-1 (GLP-1) secreted from the intestine in response to GluOC exposure. We have now examined the effect of oral administration of GluOC on glucose utilization as well as the fate of such administered GluOC in mice. Long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level and improved glucose tolerance in mice without affecting insulin sensitivity. It also increased the fasting serum insulin concentration as well as the β-cell area in the pancreas. A small proportion of orally administered GluOC reached the small intestine and remained there for at least 24. h. GluOC also entered the general circulation, and the serum GLP-1 concentration was increased in association with the presence of GluOC in the intestine and systemic circulation. The putative GluOC receptor, GPRC6A was detected in intestinal cells, and was colocalized with GLP-1 in some of these cells. Our results suggest that orally administered GluOC improved glucose handling likely by acting from both the intestinal lumen and the general circulation, with this effect being mediated in part by stimulation of GLP-1 secretion. Oral administration of GluOC warrants further study as a safe and convenient option for the treatment or prevention of metabolic disorders.. |
| 21. |
Toshiya Okumura, Kae Harada, Kana Oue, Jun Zhang, Satoshi Asano, Masaki Hayashiuchi, Akiko Mizokami, Hiroto Tanaka, Masahiro Irifune, Nobuyuki Kamata, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein (PRIP) regulates lipolysis in adipose tissue by modulating the phosphorylation of hormone-sensitive lipase, PLoS One, 10.1371/journal.pone.0100559, 9, 6, 2014.06, Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP -KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis. Copyright:. |
| 22. |
Hisanori Umebayashi, Akiko Mizokami, Miho Matsuda, Kae Harada, Hiroshi Takeuchi, Isei Tanida, Masato Hirata, Takashi Kanematsu, Phospholipase C-related catalytically inactive protein, a novel microtubule-associated protein 1 light chain 3-binding protein, negatively regulates autophagosome formation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2013.01.119, 432, 2, 268-274, 2013.03, Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.. |
| 23. |
Akiko Mizokami, Yu Yasutake, Jing Gao, Miho Matsuda, Ichiro Takahashi, Hiroshi Takeuchi, Masato Hirata, Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice, PLoS One, 10.1371/journal.pone.0057375, 8, 2, 2013.02, The uncarboxylated form (ucOC), but not the γ-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide-1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1.. |
| 24. |
Koshiro Tsutsumi, Miho Matsuda, Miho Kotani, Akiko Mizokami, Ayako Murakami, Ichiro Takahashi, Yoshihiro Terada, Takashi Kanematsu, Kiyoko Fukami, Tadaomi Takenawa, Eijiro Jimi, Masato Hirata, Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation, Journal of Biological Chemistry, 10.1074/jbc.M111.235903, 286, 35, 31032-31042, 2011.09, PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.. |
| 25. |
Akiko Mizokami, Hiroto Tanaka, Hitoshi Ishibashi, Hisanori Umebayashi, Kiyoko Fukami, Tadaomi Takenawa, Keiichi Nakayama, Takeshi Yokoyama, Junichi Nabekura, Takashi Kanematsu, Masato Hirata, GABAA receptor subunit alteration-dependent diazepam insensitivity in the cerebellum of phospholipase C-related inactive protein knockout mice, Journal of Neurochemistry, 10.1111/j.1471-4159.2010.06754.x, 114, 1, 302-310, 2010.07, The GABAA receptor, a pentamer composed predominantly of α, β, and γ subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABAA receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The α subunit affects diazepam sensitivity; α1, 2, 3, and 5 subunits assemble with any form of β and the γ2 subunits to produce diazepam-sensitive receptors, whereas α4 or α6/β/γ2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the α6 subunit. The expression of α1/β/γ2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the α6 subunit were increased, the α1/β/γ2 receptors might be replaced with α6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of α6/δ receptors was decreased in cerebellar granule neurons, while that of α6/γ2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (α1/γ2) and increase in diazepam-insensitive (α6/γ2) GABAA receptors in the cerebellar granule cells.. |
| 26. |
Takashi Kanematsu, Akiko Mizokami, Keiko Watanabe, Masato Hirata, Regulation of GABAA-receptor surface expression with special reference to the involvement of GABARAP (GABAA receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein), Journal of Pharmacological Sciences, 10.1254/jphs.CP0070063, 104, 4, 285-292, 2007.09, GABAA receptors are heteropentameric ligand-gated chloride channels composed of a variety of subunits, including α1 - 6, β1 - 3, γ1 - 3, δ, ε, θ, and π, and play a key role in controlling inhibitory neuronal activity. Modification of the efficacy of the synaptic strength is produced by changes in both the number of neuronal surface receptors and pentameric molecular assembly, leading to differences of sensitivity to neurotransmitters and neuromimetic drugs. Therefore, it is important to understand the molecular mechanisms regulating the so-called "life cycle of GABAA receptors" including sequential pentameric assembly at the site synthesized, intracellular transport through the Golgi apparatus and the cytoplasm, insertion into the cell membrane, functional modulation at the cell surface, and finally internalization, followed by either recycling back to the surface membrane or lysosomal degradation. This review is focused on events related to the surface expression of the receptor containing the γ2 subunit and clathrin /AP2 complex-mediated phospho-regulated endocytosis of the receptor, with special reference to the function of novel GABAA receptor modulators, GABARAP (GABAA receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein).. |
| 27. |
Takashi Kanematsu, Makoto Fujii, Akiko Mizokami, Josef T. Kittler, Junichi Nabekura, Stephen J. Moss, Masato Hirata, Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex, Journal of Neurochemistry, 10.1111/j.1471-4159.2006.04399.x, 101, 4, 898-905, 2007.05, A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABAA receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABAA receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABAA receptors through association with receptor β subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABAA receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABAA receptor α/β/γ subunits and PRIP. PRIP was internalized together with GABAA receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to β subunits. The clathrin heavy chain, μ2 and β2 subunits of AP2 and PRIP-1, were complexed with GABAA receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-β2/3 GABAA receptor antibody or by pull-down assay using β subunits of GABAA receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABAA receptor that requires clathrin and AP2 protein complex.. |
| 28. |
Akiko Mizokami, Takashi Kanematsu, Hitoshi Ishibashi, Taku Yamaguchi, Isei Tanida, Kei Takenaka, Keiichi Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Eiki Kominami, Stephen J. Moss, Tsuneyuki Yamamoto, Junichi Nabekura, Masato Hirata, Phosholipase C-related inactive protein is involved in trafficking of γ2 subunit-containing GABAA receptors to the cell surface, Journal of Neuroscience, 10.1523/JNEUROSCI.3155-06.2007, 27, 7, 1692-1701, 2007.02, The subunit composition of GABAA receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABAA receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABAA receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABAA receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABAA receptors and GABAA receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABAA receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABAA receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABAA receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.. |
| 29. |
Takashi Kanematsu, Akiko Mizokami, Miho Terunuma, Hiroshi Takeuchi, Masato Hirata, Identification of a Novel Signaling Molecule and Elucidation of Its Cellular Functions —Development of an Interface between Neuroscience and Oral Health Science—, journal of oral biosciences, 10.2330/joralbiosci.49.244, 49, 4, 244-258, 2007.01, The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and-2. Regarding its ability to bind Ins (1,4,5)-P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABAA receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABAA receptor signaling. PRIP-1 and-2 double knock-out (DKO) mice were analyzed for GABAA receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors; sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABAA receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABAA receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP.. |
| 30. |
Akiko Mizokami, Takashi Kanematsu, Masato Hirata, Roles of PRIP in GABAA Receptor Signaling, journal of oral biosciences, 10.2330/joralbiosci.49.105, 49, 2, 105-112, 2007.01, GABAA receptors are a family of ligand-gated ion channels that are pentamer composed pre-dominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter (GABA, γ-aminobutyric acid) and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABAA receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABAA receptors. We have first isolated PRIP (phospholipase C-related, but catalytically inactive protein) as a novel inositol 1,4,5-trisphosphate binding protein, and subsequently found GABARAP (GABAA receptor associated protein) as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c (catalytic subunit of protein phosphatase 1). These findings have prompted us to explore the possible involvement of PRIP in modulation of GABAA receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABAA receptor signaling on the basis of the characteristics of these interacting molecules.. |
