Loss of glucose-stimulated insulin secretion (GSIS) from the pancreatic beta-cells is one of the earliest detectable defects in the pathogenesis of type 2 diabetes. However, despite its relevance, the mechanisms that govern GSIS are still not completely understood. ARNT/HIF-1β is a member of the bHLH-PAS family of transcription factors, with a prominent role in the transcriptional regulation of enzymes required for the metabolism of xenobiotics as well as regulation of genes that are critical for cellular responses to hypoxia. Recent research has uncovered a previously unknown function for ARNT/HIF-1β in the pancreatic beta-cells, where the gene was found to be 90% down-regulated in human type 2 diabetic islets and loss of ARNT/HIF-1β protein leads to defective GSIS in pancreatic beta-cells of mice. The main focus of this thesis was to understand the mechanisms by which ARNT/HIF-1β maintains normal GSIS from pancreatic beta-cells and understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice. ARNT/HIF-1β was found to positively regulate GSIS in both INS-1 derived 832/13 cell line and mice islets. In the 832/13 cells, loss of ARNT/HIF-1β leads to a reduction in glycolysis without affecting the glucose oxidation and the ATP/ADP ratio suggesting that the regulation of GSIS takes place in a manner that is independent of the KATP channels. In order to further assess the mechanism of lowered GSIS in the absence of ARNT/HIF-1β in the 832/13 cells, a metabolite profiling was performed which revealed a significant reduction in the metabolite levels of glycolysis and the TCA cycle intermediates and glucose-induced fatty acid production, suggesting the involvement of ARNT/HIF-1β in regulating glucose-stimulated anaplerosis, which is believed to play a key role in the regulation of GSIS from the pancreatic beta-cells. The changes in metabolite levels in the absence of ARNT/HIF-1β were associated with corresponding changes in the gene expression pattern of key enzymes regulating glycolysis, the TCA cycle and fatty acid synthesis in beta-cells. In an attempt to understand how loss of ARNT/HIF-1β leads to beta-cell dysfunction and type 2 diabetes in mice, a pancreatic beta-cell specific ARNT/HIF-1β knock out mouse (β-ARNT KO) was generated using the Cre-loxP technology. Functional characterization of islets from both male and female β-ARNT KO mice revealed a significant impairment in GSIS, which was attributed due to a small, but significant reduction in rise in intracellular calcium upon glucose stimulation. Further analysis revealed reduced secretory response to glucose in the presence of KCl and diazoxide indicating a defect in the amplifying pathway of GSIS in β-ARNT KO islets. Expression of pyruvate carboxylase (PC) was significantly reduced in β-ARNT KO islets suggesting possible impairments in anaplerosis and consistent with this, defect in GSIS in β-ARNT KO islets could be almost completely rescued by treatment with membrane permeable TCA intermediates. Surprisingly, both male and female β-ARNT KO mice have normal glucose homeostasis. In an attempt to assess how β-ARNT KO mice maintained normal blood glucose levels, indirect calorimetry was used to understand changes in whole-body energy expenditure. This investigation revealed that β-ARNT KO mice exhibited a small but significant increase in respiratory exchange ratio (RER), suggesting a preference in utilizing carbohydrates as a fuel source, possibly leading to improved glucose uptake from the blood stream. Response to exogenous insulin was completely normal in β-ARNT KO mice suggesting intact functioning of the skeletal muscles. To conclude, based on our in vitro data, we believe that ARNT/HIF-1β plays an indispensable role in maintaining normal beta-cell secretory function, however, results from β-ARNT KO mice indicates that these mice are protected from the adverse effects of hyperglycemia. Although loss of ARNT/HIF-1β alone is not sufficient for the genesis of type 2 diabetes, it creates a perfect storm in the pancreatic beta-cells that may eventually lead to an imbalance in the whole body glucose homeostasis. Our study provides significant information to the scientific community that engages in assessing the pharmacological potential of gene targets for the treatment of type 2 diabetes.

The present project investigated the effect of epidural blockade with local anesthetic on the catabolic stress response during and immediately after abdominal surgery in fasting patients and during infusion of glucose at 2 mg·kg-1·min-1. The kinetics of glucose and protein metabolism were assessed by the stable isotope tracers [6,6-2H2]glucose and L-[1-13C]-leucine. / Epidural blockade was associated with a lower plasma glucose concentration and glucose production when compared to control subjects in the fasted state. Whole body protein breakdown, amino acid oxidation and protein synthesis were suppressed during surgery, and epidural blockade had no modifying effect on perioperative protein metabolism. The suppression of endogenous glucose production by exogenous glucose was more pronounced in the presence of epidural blockade. Perioperative protein metabolism, however, was not influenced by epidural blockade during glucose infusion. / Although epidural blockade suppressed glucose metabolism both in the fasted state and during glucose administration, it failed to exert a modifying effect on perioperative protein metabolism.

Glucose is a principle precursor for milk lactose and de novo synthesis of milk fat; therefore exercising during lactation could create competition for glucose between exercising muscle and lactating mammary gland. This study investigated the combined effects of maternal dietary glucose (20%, 40%, 60%), exercise (chronically exercised, sedentary) and litter size (8, 12 pups) on rat mammary gland composition, milk composition, milk yield and pup growth. Chronic exercise increased milk fat concentrations and an interaction between chronic exercise and 20% dietary glucose decreased milk lactose concentrations compared to 40% or 60% glucose diets. Restricting maternal dietary glucose also decreased milk fat concentrations and exercise decreased mammary fat. In addition, pups of dams fed the 40% glucose diet were heavier on lactation day 15 than pups of dams fed the 60% diet. These results suggest that competition for glucose occurs and that a 40% glucose maternal diet may be more appropriate for pup growth.

Diss. (sammanfattning) Stockholm : Karol. inst. / Titel på diss.-titelbl.: Islet glucose metabolism and insulin secretion in two animal models of glucose intolerance. Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

This thesis describes several aspects of the role for extracellular adenosine in Drosophila. Reverse genetic, molecular and microscopic methods together with the most forefront Drosophila research techniques have been applied to elucidate the role of adenosine signaling in the regulation of development, physiology and metabolism of Drosophila larvae. The thesis helps to establish the model for extracellular adenosine as a stress-signal for the release of energy stores. It also describes the elucidation of Drosophila extracellular adenosine production pathway by functional characterization of extracellular ATP/adenosine converting enzymes. Further, this thesis describes the attempt to study adenosine signaling by creation of tissue specific knock-down mutation of Drosophila adenosine receptor. Finally, it provides results of the utilization of in vivo fluorescent reporters for studying of the localization, function and interaction of enzymes implicated in adenosine signaling pathway.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21959号 / 医博第4501号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 小杉 眞司, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Glycemic control in patients with type 2 diabetes (T2D) can be achieved through potentiation of the signalling by glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Both incretin hormones have been traditionally characterized to be secreted by distinct enteroendocrine cells within the gut in response to nutrients. Signalling through the incretin receptors stimulates islet hormone release by potentiating glucose-stimulated insulin secretion from the β-cell and decreasing glucagon secretion from the α-cell. However, the bioactivity of GLP-1 and GIP is controlled by post-translational, N-terminal cleavage by the widely expressed serine protease dipeptidyl peptidase 4 (DPP4). As such, DPP4 inhibitors (DPP4i) have been successfully used to treat millions of patients with T2D. DPP4i target the catalytic active site of DPP4 and prevent the cleavage of the incretin hormones, thus prolonging their action. Recently, studies in genetically modified mice have demonstrated that GLP-1 is not solely an intestinally-derived peptide hormone and proposed that islet-derived GLP-1 is required for proper glucose homeostasis. Therefore, with the current study, we sought to assess whether β-cell-derived DPP4 is an important target for the regulation of glycemia. Treatment of Glp1r/Gipr^(β-cell-/-) mice with the DPP4 inhibitor sitagliptin demonstrated that β-cell incretin receptor signaling is required to mediate the beneficial effects of this class of drugs on glucose homeostasis. Additionally, Dpp4^(-/-) mice exhibited a significant reduction in hepatic glucose production during hyperinsulinemic-euglycemic clamps. Dpp4 mRNA, DPP4 protein and activity are present in isolated mouse islets, further supporting the islet as an important potential site of DPP4i action. In this study, we show that both DPP4i-treated wildtype islets and islets isolated from Dpp4^(β-cell-/-) mice exhibit increased glucose-stimulated insulin secretion (GSIS) during perifusion after a high-fat diet feeding. Genetic elimination of Dpp4 from islet β-cells also improved oral glucose tolerance and insulin sensitivity in female mice, but had no effects on circulating DPP4 or incretin levels. Finally, eliminating Dpp4 from β-cells or the whole pancreas did not improve whole-body glucose tolerance, response to DPP4i, insulin tolerance, or body weight in male mice fed chow or a high-fat diet. Therefore, we provide evidence for islet-derived DPP4 to have a role in local hormone responses to glucose; however, its role in systemic glucose metabolism is shown to be sex-dependent.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第18197号 / 人健博第14号 / 新制||人健||2(附属図書館) / 31055 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12936号 / 論農博第2816号 / 新制||農||1033(附属図書館) / 学位論文||H27||N4895(農学部図書室) / 32146 / (主査)教授 伏木 亨, 教授 保川 清, 教授 安達 修二 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM