Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Kubota Hiroyuki Last modified date:2019.12.24

Professor / Research Center for Transomics Medicine / Medical Institute of Bioregulation


Papers
1. Ohigashi I, Tanaka Y, Kondo K, Fujimori S, Kondo H, Palin AC, Hoffmann V, Kozai M, Matsushita Y, Uda S, Motosugi R, Hamazaki J, Kubota H, Murata S, Tanaka K, Katagiri T, Kosako H, Takahama Y., Trans-omics Impact of Thymoproteasome in Cortical Thymic Epithelial Cells., Cell Reports, 10.1016/j.celrep.2019.10.079., 2019.11.
2. Fujii M, Murakami Y, Karasawa Y, Sumitomo Y, Fujita S, Koyama M, Uda S, Kubota H, Inoue H, Konishi K, Oba S, Ishii S, Kuroda S., Logical design of oral glucose ingestion pattern minimizing blood glucose in humans., NPJ Syst Biol Appl., 10.1038/s41540-019-0108-1., 2019.09.
3. Kentaro Kawata, Katsuyuki Yugi, Atsushi Hatano, Toshiya Kokaji, Yoko Tomizawa, Masashi Fujii, Shinsuke Uda, Hiroyuki Kubota, Masaki Matsumoto, Keiichi Nakayama, Shinya Kuroda, Reconstruction of global regulatory network from signaling to cellular functions using phosphoproteomic data, Genes to Cells, 10.1111/gtc.12655, 24, 1, 82-93, 2019.01, Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase–substrate relationships (KSRs). We used phosphoproteomic data from insulin-stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over-represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k-Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data..
4. Kentaro Kawata, Atsushi Hatano, Katsuyuki Yugi, Hiroyuki Kubota, Takanori Sano, Masashi Fujii, Yoko Tomizawa, Toshiya Kokaji, Kaori Y. Tanaka, Shinsuke Uda, Yutaka Suzuki, Masaki Matsumoto, Keiichi Nakayama, Kaori Saitoh, Keiko Kato, Ayano Ueno, Maki Ohishi, Akiyoshi Hirayama, Tomoyoshi Soga, Shinya Kuroda, Trans-omic Analysis Reveals Selective Responses to Induced and Basal Insulin across Signaling, Transcriptional, and Metabolic Networks, Food Science and Human Wellness, 10.1016/j.isci.2018.07.022, 7, 212-229, 2018.09, The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions..
5. Hiroyuki Kubota, Shinsuke Uda, Fumiko Matsuzaki, Yukiyo Yamauchi, Shinya Kuroda, In Vivo Decoding Mechanisms of the Temporal Patterns of Blood Insulin by the Insulin-AKT Pathway in the Liver, Cell Systems, 10.1016/j.cels.2018.05.013, 7, 1, 118-128.e3, 2018.07, Cells respond to various extracellular stimuli through a limited number of signaling pathways. One strategy to process such stimuli is to code the information into the temporal patterns of molecules. Although we showed that insulin selectively regulated molecules depending on its temporal patterns using Fao cells, the in vivo mechanism remains unknown. Here, we show how the insulin-AKT pathway processes the information encoded into the temporal patterns of blood insulin. We performed hyperinsulinemic-euglycemic clamp experiments and found that, in the liver, all temporal patterns of insulin are encoded into the insulin receptor, and downstream molecules selectively decode them through AKT. S6K selectively decodes the additional secretion information. G6Pase interprets the basal secretion information through FoxO1, while GSK3β decodes all secretion pattern information. Mathematical modeling revealed the mechanism via differences in network structures and from sensitivity and time constants. Given that almost all hormones exhibit distinct temporal patterns, temporal coding may be a general principle of system homeostasis by hormones. Kubota et al. show that the insulin-AKT pathway in the liver processes the information encoded into the temporal patterns of blood insulin and selectively regulates downstream molecules. Given that almost all hormones exhibit distinct temporal patterns, our study demonstrates the possibility of temporal coding as a general principle of systemic homeostasis by hormones..
6. Shinsuke Uda, Hiroyuki Kubota, Sparse Gaussian graphical model with missing values, 21st Conference of Open Innovations Association, FRUCT 2017 Proceedings of the 21st Conference of Open Innovations Association, FRUCT 2017, 10.23919/FRUCT.2017.8250201, Part F134240, 336-343, 2018.01, Recent advances in measurement technology have enabled us to measure various omic layers, such as genome, transcriptome, proteome, and metabolome layers. The demand for data analysis to determine the network structure of the interaction between molecular species is increasing. The Gaussian graphical model is one method of estimating the network structure. However, biological omics data sets tend to include missing values, which is conventionally handled by preprocessing. We propose a novel method by which to estimate the network structure together with missing values by combining a sparse graphical model and matrix factorization. The proposed method was validated by artificial data sets and was applied to a signal transduction data set as a test run..
7. Kaoru Ohashi, Masashi Fujii, Shinsuke Uda, Hiroyuki Kubota, Hisako Komada, Kazuhiko Sakaguchi, Wataru Ogawa, Shinya Kuroda, Increase in hepatic and decrease in peripheral insulin clearance characterize abnormal temporal patterns of serum insulin in diabetic subjects, npj Systems Biology and Applications, 10.1038/s41540-018-0051-6, 4, 2018, Insulin plays a central role in glucose homeostasis, and impairment of insulin action causes glucose intolerance and leads to type 2 diabetes mellitus (T2DM). A decrease in the transient peak and sustained increase of circulating insulin following an infusion of glucose accompany T2DM pathogenesis. However, the mechanism underlying this abnormal temporal pattern of circulating insulin concentration remains unknown. Here we show that changes in opposite direction of hepatic and peripheral insulin clearance characterize this abnormal temporal pattern of circulating insulin concentration observed in T2DM. We developed a mathematical model using a hyperglycemic and hyperinsulinemic-euglycemic clamp in 111 subjects, including healthy normoglycemic and diabetic subjects. The hepatic and peripheral insulin clearance significantly increase and decrease, respectively, from healthy to borderline type and T2DM. The increased hepatic insulin clearance reduces the amplitude of circulating insulin concentration, whereas the decreased peripheral insulin clearance changes the temporal patterns of circulating insulin concentration from transient to sustained. These results provide further insight into the pathogenesis of T2DM, and thus may contribute to develop better treatment of this condition..
8. Takaho Tsuchiya, Masashi Fujii, Naoki Matsuda, Katsuyuki Kunida, Shinsuke Uda, Hiroyuki Kubota, Katsumi Konishi, Shinya Kuroda, System identification of signaling dependent gene expression with different time-scale data, PLoS Computational Biology, 10.1371/journal.pcbi.1005913, 13, 12, 2017.12, Cells decode information of signaling activation at a scale of tens of minutes by downstream gene expression with a scale of hours to days, leading to cell fate decisions such as cell differentiation. However, no system identification method with such different time scales exists. Here we used compressed sensing technology and developed a system identification method using data of different time scales by recovering signals of missing time points. We measured phosphorylation of ERK and CREB, immediate early gene expression products, and mRNAs of decoder genes for neurite elongation in PC12 cell differentiation and performed system identification, revealing the input–output relationships between signaling and gene expression with sensitivity such as graded or switch-like response and with time delay and gain, representing signal transfer efficiency. We predicted and validated the identified system using pharmacological perturbation. Thus, we provide a versatile method for system identification using data with different time scales..
9. Takanori Sano, Kentaro Kawata, Satoshi Ohno, Katsuyuki Yugi, Hiroaki Kakuda, Hiroyuki Kubota, Shinsuke Uda, Masashi Fujii, Katsuyuki Kunida, Daisuke Hoshino, Atsushi Hatano, Yuki Ito, Miharu Sato, Yutaka Suzuki, Shinya Kuroda, Selective control of up-regulated and down-regulated genes by temporal patterns and doses of insulin, Science Signaling, 10.1126/scisignal.aaf3739, 9, 455, 2016.11, Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the downregulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii) transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness..
10. 久保田 浩行, 柚木 克之, 幡野 敦, 黒田 真也, Trans-Omics: How To Reconstruct Biochemical Networks Across Multiple 'Omic' Layers., 2016.05.
11. Katsuyuki Yugi, Hiroyuki Kubota, Atsushi Hatano, Shinya Kuroda, Trans-Omics
How To Reconstruct Biochemical Networks Across Multiple 'Omic' Layers, Trends in Biotechnology, 10.1016/j.tibtech.2015.12.013, 34, 4, 276-290, 2016.04, We propose 'trans-omic' analysis for reconstructing global biochemical networks across multiple omic layers by use of both multi-omic measurements and computational data integration. We introduce technologies for connecting multi-omic data based on prior knowledge of biochemical interactions and characterize a biochemical trans-omic network by concepts of a static and dynamic nature. We introduce case studies of metabolism-centric trans-omic studies to show how to reconstruct a biochemical trans-omic network by connecting multi-omic data and how to analyze it in terms of the static and dynamic nature. We propose a trans-ome-wide association study (trans-OWAS) connecting phenotypes with trans-omic networks that reflect both genetic and environmental factors, which can characterize several complex lifestyle diseases as breakdowns in the trans-omic system..
12. Shinji Fukuda, Hisayo Nishida-Fukuda, Daisuke Nanba, Koh Ichi Nakashiro, Hironao Nakayama, Hiroyuki Kubota, Shigeki Higashiyama, Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system, Scientific Reports, 10.1038/srep20209, 6, 2016.02, Epithelial cell plasticity is controlled by extracellular cues, but the underlying mechanisms remain to be fully understood. Epidermal growth factor (EGF) and amphiregulin (AREG) are high- and low-affinity ligands for EGF receptor (EGFR), respectively. EGFR signaling is known to promote epithelial-mesenchymal transition (EMT) by the activation of ERK and the induction of an EMT transcription factor, ZEB1. Here, we demonstrate that ligand-switching between EGF and AREG at equivalent molarity reversibly interconverts epithelial and mesenchymal-like states of EGFR signal-dependent mammary epithelial cells. The EGF- and AREG-cultured cells also differ in their epithelial characteristics, including the expression of cell surface markers, the mode of migration and the ability for acinus-formation. The ligand-switching between EGF and AREG temporally alters strength of the shared EGFR-ERK signaling. This alteration inverts relative expression levels of ZEB1 and its antagonizing microRNAs, miR-205 and miR-200c, those are critical determinants of the epithelial phenotype. Further, AREG-induced EGFR accumulation on the plasma membrane compensates for the weak association between AREG and EGFR. The EGFR dynamics enables AREG to support proliferation as efficiently as EGF at equivalent molarity and to maintain epithelial characteristics. Our findings reveal a role of EGFR ligands-generated signal strength in the regulation of mammary epithelial cell plasticity..
13. 久保田 浩行, 福田 信治, 福田 尚代, 難波 大輔, 中山 寛尚, 東山 繁樹, Reversible interconversion and maintenance of mammary epithelial cell characteristics by the ligand-regulated EGFR system., 20209 , 2016.01.
14. 久保田 浩行, 大橋  郁, 駒田久子, 宇田 新介, 岩木 俊直, 福沢 紘揮, 小森 靖則, 藤井 雅史, 豊島 有, 坂口 一彦, 小川 渉, 黒田 真也, Glucose Homeostatic Law: Insulin Clearance Predicts the Progression of Glucose Intolerance in Humans., e0143880, 2015.12.
15. Kaoru Ohashi, Hisako Komada, Shinsuke Uda, Hiroyuki Kubota, Toshinao Iwaki, Hiroki Fukuzawa, Yasunori Komori, Masashi Fujii, Yu Toyoshima, Kazuhiko Sakaguchi, Wataru Ogawa, Shinya Kuroda, Glucose homeostatic law
Insulin clearance predicts the progression of glucose intolerance in humans, PLoS One, 10.1371/journal.pone.0143880, 10, 12, 2015.12, Homeostatic control of blood glucose is regulated by a complex feedback loop between glucose and insulin, of which failure leads to diabetes mellitus. However, physiological and pathological nature of the feedback loop is not fully understood. We made a mathematical model of the feedback loop between glucose and insulin using time course of blood glucose and insulin during consecutive hyperglycemic and hyperinsulinemic-euglycemic clamps in 113 subjects with variety of glucose tolerance including normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). We analyzed the correlation of the parameters in the model with the progression of glucose intolerance and the conserved relationship between parameters. The model parameters of insulin sensitivity and insulin secretion significantly declined from NGT to IGT, and from IGT to T2DM, respectively, consistent with previous clinical observations. Importantly, insulin clearance, an insulin degradation rate, significantly declined from NGT, IGT to T2DM along the progression of glucose intolerance in the mathematical model. Insulin clearance was positively correlated with a product of insulin sensitivity and secretion assessed by the clamp analysis or determined with the mathematical model. Insulin clearance was correlated negatively with postprandial glucose at 2h after oral glucose tolerance test. We also inferred a square-law between the rate constant of insulin clearance and a product of rate constants of insulin sensitivity and secretion in the model, which is also conserved among NGT, IGT and T2DM subjects. Insulin clearance shows a conserved relationship with the capacity of glucose disposal among the NGT, IGT and T2DM subjects. The decrease of insulin clearance predicts the progression of glucose intolerance..
16. Yoshihiro Katsura, Hiroyuki Kubota, Katsuyuki Kunida, Akira Kanno, Shinya Kuroda, Takeaki Ozawa, An optogenetic system for interrogating the temporal dynamics of Akt, Scientific Reports, 10.1038/srep14589, 5, 2015.10, The dynamic activity of the serine/threonine kinase Akt is crucial for the regulation of diverse cellular functions, but the precise spatiotemporal control of its activity remains a critical issue. Herein, we present a photo-activatable Akt (PA-Akt) system based on a light-inducible protein interaction module of Arabidopsis thaliana cryptochrome2 (CRY2) and CIB1. Akt fused to CRY2phr, which is a minimal light sensitive domain of CRY2 (CRY2-Akt), is reversibly activated by light illumination in several minutes within a physiological dynamic range and specifically regulates downstream molecules and inducible biological functions. We have generated a computational model of CRY2-Akt activation that allows us to use PA-Akt to control the activity quantitatively. The system provides evidence that the temporal patterns of Akt activity are crucial for generating one of the downstream functions of the Akt-FoxO pathway; the expression of a key gene involved in muscle atrophy (Atrogin-1). The use of an optical module with computational modeling represents a general framework for interrogating the temporal dynamics of biomolecules by predictive manipulation of optogenetic modules..
17. Hiroyuki Kubota, Shinya Kuroda, Temporal coding of insulin signaling, Protein Modifications in Pathogenic Dysregulation of Signaling, 10.1007/978-4-431-55561-2_7, 95-109, 2015.09, During recent years, it has become clearer that temporal patterns of stimuli and molecules are important in the regulation of cellular functions. For example, many hormones show distinct temporal patterns in vivo, which are important for homeostasis. One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions depending on their temporal patterns. Therefore, one of the major advances in understanding the "pathogenic dysregulation of signaling" is to reveal the temporal coding mechanisms of signaling pathways related to pathogenesis. A systems biological approach combining experiments and computational analysis is necessary to address this issue. In this chapter, we will introduce the concept that the insulin- dependent AKT pathway uses temporal patterns multiplexing for selective regulation of signaling molecules and metabolites, which depend on their network structures and kinetics, using rat hepatoma Fao cells. These results represent a huge step forward in our understanding of insulin actions and type II diabetes mellitus ..
18. 久保田 浩行, 桂 嘉宏, 小澤 岳昌, 国田 勝行, 黒田 真也, An optogenetic system for interrogating the temporal dynamics of Akt, 5, 14589, 2015.05.
19. 久保田 浩行, 柚木 克之, 黒田 真也, 豊島 有, 野口 怜, 川田 健太郎, 小森 靖典, 宇田 新介, 国田 勝行, 富沢 瑶子, 船戸 洋佑, 三木 裕明, 松本 雅記, 中山 敬一, 柏倉 風純, 遠藤 慶子, 池田 和貴, 曽我 朋義, Reconstruction of insulin signal flow from phosphoproteome and metabolome data, 2014.08, Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis have yet to be explored. We developed a reconstruction method for the signal flow based on time course phosphoproteome and metabolome data using multiple databases and applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flowed through a network; it flowed through signaling pathways that involved 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network revealed that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We provide a versatile method of reconstruction of signal flow through the network using phosphoproteome andmetabolome data..
20. Katsuyuki Yugi, Hiroyuki Kubota, Yu Toyoshima, Rei Noguchi, Kentaro Kawata, Yasunori Komori, Shinsuke Uda, Katsuyuki Kunida, Yoko Tomizawa, Yosuke Funato, Hiroaki Miki, Masaki Matsumoto, Keiichi Nakayama, Kasumi Kashikura, Keiko Endo, Kazutaka Ikeda, Tomoyoshi Soga, Shinya Kuroda, Reconstruction of insulin signal flow from phosphoproteome and metabolome data, Cell Reports, 10.1016/j.celrep.2014.07.021, 8, 4, 1171-1183, 2014.08, Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data..
21. Yuki Akimoto, Katsuyuki Yugi, Shinsuke Uda, Takamasa Kudo, Yasunori Komori, Hiroyuki Kubota, Shinya Kuroda, The Extraction of Simple Relationships in Growth Factor-Specific Multiple-Input and Multiple-Output Systems in Cell-Fate Decisions by Backward Elimination PLS Regression, PloS one, 10.1371/journal.pone.0072780, 8, 9, 2013.09, Cells use common signaling molecules for the selective control of downstream gene expression and cell-fate decisions. The relationship between signaling molecules and downstream gene expression and cellular phenotypes is a multiple-input and multiple-output (MIMO) system and is difficult to understand due to its complexity. For example, it has been reported that, in PC12 cells, different types of growth factors activate MAP kinases (MAPKs) including ERK, JNK, and p38, and CREB, for selective protein expression of immediate early genes (IEGs) such as c-FOS, c-JUN, EGR1, JUNB, and FOSB, leading to cell differentiation, proliferation and cell death; however, how multiple-inputs such as MAPKs and CREB regulate multiple-outputs such as expression of the IEGs and cellular phenotypes remains unclear. To address this issue, we employed a statistical method called partial least squares (PLS) regression, which involves a reduction of the dimensionality of the inputs and outputs into latent variables and a linear regression between these latent variables. We measured 1,200 data points for MAPKs and CREB as the inputs and 1,900 data points for IEGs and cellular phenotypes as the outputs, and we constructed the PLS model from these data. The PLS model highlighted the complexity of the MIMO system and growth factor-specific input-output relationships of cell-fate decisions in PC12 cells. Furthermore, to reduce the complexity, we applied a backward elimination method to the PLS regression, in which 60 input variables were reduced to 5 variables, including the phosphorylation of ERK at 10 min, CREB at 5 min and 60 min, AKT at 5 min and JNK at 30 min. The simple PLS model with only 5 input variables demonstrated a predictive ability comparable to that of the full PLS model. The 5 input variables effectively extracted the growth factor-specific simple relationships within the MIMO system in cell-fate decisions in PC12 cells..
22. Shinsuke Uda, Takeshi H. Saito, Takamasa Kudo, Toshiya Kokaji, Takaho Tsuchiya, Hiroyuki Kubota, Yasunori Komori, Yu Ichi Ozaki, Shinya Kuroda, Robustness and compensation of information transmission of signaling pathways, Science, 10.1126/science.1234511, 341, 6145, 558-561, 2013.01, Robust transmission of information despite the presence of variation is a fundamental problem in cellular functions. However, the capability and characteristics of information transmission in signaling pathways remain poorly understood. We describe robustness and compensation of information transmission of signaling pathways at the cell population level. We calculated the mutual information transmitted through signaling pathways for the growth factor-mediated gene expression. Growth factors appeared to carry only information sufficient for a binary decision. Information transmission was generally more robust than average signal intensity despite pharmacological perturbations, and compensation of information transmission occurred. Information transmission to the biological output of neurite extension appeared robust. Cells may use information entropy as information so that messages can be robustly transmitted despite variation in molecular activities among individual cells..
23. Rei Noguchi, Hiroyuki Kubota, Katsuyuki Yugi, Yu Toyoshima, Yasunori Komori, Tomoyoshi Soga, Shinya Kuroda, The selective control of glycolysis, gluconeogenesis and glycogenesis by temporal insulin patterns, Molecular Systems Biology, 10.1038/msb.2013.19, 9, 1, 2013.01, Insulin governs systemic glucose metabolism, including glycolysis, gluconeogenesis and glycogenesis, through temporal change and absolute concentration. However, how insulin-signalling pathway selectively regulates glycolysis, gluconeogenesis and glycogenesis remains to be elucidated. To address this issue, we experimentally measured metabolites in glucose metabolism in response to insulin. Step stimulation of insulin induced transient response of glycolysis and glycogenesis, and sustained response of gluconeogenesis and extracellular glucose concentration (GLCex). Based on the experimental results, we constructed a simple computational model that characterises response of insulin-signalling-dependent glucose metabolism. The model revealed that the network motifs of glycolysis and glycogenesis pathways constitute a feedforward (FF) with substrate depletion and incoherent feedforward loop (iFFL), respectively, enabling glycolysis and glycogenesis responsive to temporal changes of insulin rather than its absolute concentration. In contrast, the network motifs of gluconeogenesis pathway constituted a FF inhibition, enabling gluconeogenesis responsive to absolute concentration of insulin regardless of its temporal patterns. GLCex was regulated by gluconeogenesis and glycolysis. These results demonstrate the selective control mechanism of glucose metabolism by temporal patterns of insulin..
24. Hiroyuki Kubota, Rei Noguchi, Yu Toyoshima, Yu ichi Ozaki, Shinsuke Uda, Kanako Watanabe, Wataru Ogawa, Shinya Kuroda, Temporal Coding of Insulin Action through Multiplexing of the AKT Pathway, Molecular Cell, 10.1016/j.molcel.2012.04.018, 46, 6, 820-832, 2012.06, One of the unique characteristics of cellular signaling pathways is that a common signaling pathway can selectively regulate multiple cellular functions of a hormone; however, this selective downstream control through a common signaling pathway is poorly understood. Here we show that the insulin-dependent AKT pathway uses temporal patterns multiplexing for selective regulation of downstream molecules. Pulse and sustained insulin stimulations were simultaneously encoded into transient and sustained AKT phosphorylation, respectively. The downstream molecules, including ribosomal protein S6 kinase (S6K), glucose-6-phosphatase (G6Pase), and glycogen synthase kinase-3β (GSK3β) selectively decoded transient, sustained, and both transient and sustained AKT phosphorylation, respectively. Selective downstream decoding is mediated by the molecules' network structures and kinetics. Our results demonstrate that the AKT pathway can multiplex distinct patterns of blood insulin, such as pulse-like additional and sustained-like basal secretions, and the downstream molecules selectively decode secretion patterns of insulin..
25. Yu Ichi Ozaki, Shinsuke Uda, Takeshi H. Saito, Jaehoon Chung, Hiroyuki Kubota, Shinya Kuroda, A quantitative image cytometry technique for time series or population analyses of signaling networks, PloS one, 10.1371/journal.pone.0009955, 5, 4, 2010.09, Background: Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. Methodology/Principal Findings: We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. Conclusions/Significance: The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling..
26. Kazuhiro A. Fujita, Yu Toyoshima, Shinsuke Uda, Yu Ichi Ozaki, Hiroyuki Kubota, Shinya Kuroda, Decoupling of receptor and downstream signals in the Akt pathway by its low-pass filter characteristics, Science Signaling, 10.1126/scisignal.2000810, 3, 132, 2010.07, In cellular signal transduction, the information in an external stimulus is encoded in temporal patterns in the activities of signaling molecules; for example, pulses of a stimulus may produce an increasing response or may produce pulsatile responses in the signaling molecules. Here, we show how the Akt pathway, which is involved in cell growth, specifically transmits temporal information contained in upstream signals to downstream effectors. We modeled the epidermal growth factor (EGF)-dependent Akt pathway in PC12 cells on the basis of experimental results. We obtained counterintuitive results indicating that the sizes of the peak amplitudes of receptor and downstream effector phosphorylation were decoupled; weak, sustained EGF receptor (EGFR) phosphorylation, rather than strong, transient phosphorylation, strongly induced phosphorylation of the ribosomal protein S6, a molecule downstream of Akt. Using frequency response analysis, we found that a three-component Akt pathway exhibited the property of a low-pass filter and that this property could explain decoupling of the peak amplitudes of receptor phosphorylation and that of downstream effectors. Furthermore, we found that lapatinib, an EGFR inhibitor used as an anticancer drug, converted strong, transient Akt phosphorylation into weak, sustained Akt phosphorylation, and, because of the low-pass filter characteristics of the Akt pathway, this led to stronger S6 phosphorylation than occurred in the absence of the inhibitor. Thus, an EGFR inhibitor can potentially act as a downstream activator of some effectors..