京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22725号 / 医博第4643号 / 新制||医||1045(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 長船 健二, 教授 伊藤 貴浩, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19604号 / 医博第4111号 / 新制||医||1014(附属図書館) / 32640 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 松田 道行, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

No description available.

No description available.

Glucagon-like peptide-1 (GLP-1) synthetic analog therapies are prescribed for type 2 diabetes due to their effects on insulin and glucagon secretion, and glycemic control. Recent studies also suggest that they may have cardiovascular benefits; however, the mechanism responsible for this is unknown. To examine this question, we evaluated the effects of GLP-1 and the GLP-1 synthetic analog, liraglutide on cell signaling and function in human aortic endothelial cells (HAECs). The results indicate that both agents inhibit TNFα and LPS induced cellular adhesion molecule expression and monocyte adhesion. They also show that incubation with 30pM GLP-1 and 100nM liraglutide stimulates an immediate increase in intracellular calcium, which activates calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). This in turn led to a 2.5 fold increase in the phosphorylation of both AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent protein kinase 1 (CaMK1) within 5 minutes. In addition both GLP-1 and liraglutide caused a 2-fold increase in the phosphorylation of the downstream AMPK/CaMK1 targets: endothelial nitric oxide synthase (eNOS) and cAMP response element-binding protein (CREB). Inhibition of CaMKKβ with STO-609 (0.5ug/mL) blocked the phosphorylation of both AMPK and CaMK1, confirming its pivotal role. Incubation of the HAECs for three hours with lipopolysaccharide (LPS, 2ug/mL) and TNFα (10ng/mL) increased the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by 5 and 2 fold, respectively. Comparable increases in THP-1 monocyte adhesion to the HAECs, a putative initiating event in atherogenesis, also occurred. Pre-incubation for one hour with either GLP-1 or liraglutide inhibited these events. Likewise, pre-incubation with the CaMKK inhibitor STO-609, or use of lentivirus shRNA to knock down AMPK, blocked the inhibitory effects of both GLP-1 and liraglutide on monocyte adhesion. These results suggest that the recently observed cardiovascular benefits of GLP-1 and liraglutide could be mediated by their effects on CaMKKβ, AMPK and CaMK1 activation, which lead to decreased adhesion molecule expression and monocyte adhesion in endothelial cells. The finding that these effects occur at concentrations of GLP-1 (30pM) and liraglutide (100nM) observed in vivo also suggests they are physiologically relevant.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

nÃo hà / Diabetes mellitus (DM) à uma sÃndrome associada a secreÃÃo de insulina deficiente, resistÃncia à aÃÃo da insulina ou uma associaÃÃo de ambas. Caracteriza-se por hiperglicemia crÃnica que a longo prazo poderà causar retinopatia, nefropatia, neuropatia perifÃrica e autonÃmica. Este estudo do tipo intervencionista, prospectivo e aberto teve como objetivo avaliar a eficÃcia terapÃutica da sitagliptina no controle metabÃlico do diabetes mellitus do tipo 2 recentemente diagnosticado (menos de seis meses) e nas vias de conduÃÃo nervosa somato-sensitiva e visual. Antes de iniciar o tratamento, foram avaliados os Ãndices antropomÃtricos e a pressÃo arterial. TambÃm foram realizadas as dosagens em jejum de triglicerÃdeos, colesterol total, colesterol de alta densidade (HDL), gama glutamil transferase, aspartato amino transferase, alanina amino transferase, proteÃna C reativa, microalbuminÃria, Ãxido nÃtrico, glicose, hemoglobina glicada (A1C), peptÃdeo C, insulina e glucagon. ApÃs um estÃmulo alimentar de 566 Kcal foram feitas coletas de sangue seriadas durante 3 horas nos tempos de 5, 15, 30, 45, 60, 90, 120, 150 e 180 minutos para a dosagem de glicose, insulina e glucagon com a construÃÃo da Ãrea sob a curva (AUC) destes analitos. Um subgrupo de 20 pacientes realizou ainda, anÃlises seriadas das incretinas (GLP-1 ativo e GIP total) apÃs estÃmulo alimentar e, tambÃm, exames eletroneurofisiolÃgicos (potenciais evocados somato-sensitivos â PESS, e visuais - PEV). ApÃs estas avaliaÃÃes, os pacientes receberam sitagliptina 100 mg (1 comprimido/dia) por um perÃodo de 3 meses, apÃs o qual, eles retornaram para avaliaÃÃo clÃnica e para realizar todas as dosagens bioquÃmicas e os exames de PESS e PEV que haviam realizado no inÃcio do estudo. Dos 41 pacientes avaliados, 28 eram do sexo feminino e 13 do sexo masculino, idade mÃdia de 53,2  9,43 anos, em mÃdia obesos e 50% deles eram hipertensos. ApÃs o tratamento, observou-se a melhora dos Ãndices antropomÃtricos, de todos os parÃmetros laboratoriais com Ãnfase na glicose em jejum (prÃ: 174,43  67,18 e pÃs: 129,07  29,19 mg/dL), A1C (prÃ: 8,76  2,68 e pÃs: 6,64  1,11%) e Ãxido nÃtrico (prÃ: 85,21  44,02 e pÃs: 39,91  20,99 ÂM), todos com P<0,0001 e ainda, aumento da concentraÃÃo da AUC de GLP-1 ativo (P=0,003) e diminuiÃÃo da AUC de GIP total (P=0,001), bem como diminuiÃÃo das latÃncias N9D (P<0,0001), N13E (P=0,036), N20D (P=0,028) e dos tempos de conduÃÃo central N13-N9E (P=0,045), N20-N13D (P=0,044), alÃm de correlaÃÃes com significÃncia estatÃstica entre parÃmetros bioquÃmicos e eletroneurofisiolÃgicos. Este estudo concluiu que houve melhora clÃnica, metabÃlica e nas vias de conduÃÃo do impulso nervoso somato-sensitivo e visual dos pacientes tratados com sitagliptina. / Diabetes mellitus (DM) is a syndrome associated with deficient insulin secretion, resistance to insulin action or the combination of both. It is characterized by chronic hyperglycemia that in a long term can cause retinopathy, nephropathy, peripheral and autonomic neuropathy. The present interventional, prospective and open study was designed to evaluate therapeutic efficacy of sitagliptin in metabolic control of diabetes mellitus type 2 recently diagnosed (less than 6 months) and in pathways of visual and somatosensory nerve conduction. Blood pressure and anthropometric data were colected before treatment. Measures of fasting triglycerides, total cholesterol, high density lipoprotein cholesterol gamma glutamyl transpeptidase, alanine aminotransferase, aspartate aminotransferase, microalbumin, nitric oxide, glucose, glycated hemoglobin (A1C), C-peptide, insulin and glucagon were also collected. Also during the pre-study, after a feeding stimulus of 566 Kcal, biochemical evaluations were conducted of the metabolism in fasting and during 3 hours, in standard intervals of 5, 15, 30, 45, 60, 90, 120, 150 and 180 minutes for glucose, insulin and glucagon for design of the area under curve (AUC) of these analytes. In a subgroup of 20 patients were performed dosages in pre-fixed times of incretins (active Glucagon Like Peptide -1, GLP-1, and total Glucose-dependent Insulinotropic polypeptide, GIP) after feeding stimulus and electroneurophysiological tests (somatosensory evoked potentials - SEP, and visual - VEP). After these evaluations, the patients received sitagliptin 100 mg (1 tablet a day) for 3 months and then returned for clinical evaluation and to repeat all the biochemical dosages, SEP and VEP exams. From 41 patients evaluated, 28 were female and 13 male, the average age was 53.2  9.43 years, in average obese and 50% were hypertensive. After treatment, improvement was observed in all anthropometric and laboratory parameters, specially fasting glucose (pre: 174,43  67,18 mg/dL and post: 129,07  29,19 mg/dL), A1c (pre: 8,76  2,68 and post: 6,64  1,11%), and nitric oxide (pre: 85,21  44,02 and post: 39,91  20,99 ÂM), all with P<0.0001, and also increase in the concentration of the AUC of active GLP-1 (P=0.003), decrease of the AUC of total GIP (P=0.001), and decrease of the N9D (P<0.0001), N13E (P=0.036 ), N20D (P=0.028) latencies and of the central conduction time N13-N9E (P=0.045), N20-N13D (P=0.044). There were also statistically significant correlations between biochemical and electrophysiological parameters. This study concluded that there was clinical, metabolic and somatosensory and visual nerve impulse conduction pathways improvement in patients treated with sitagliptin.

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・環境学) / 甲第18366号 / 人博第679号 / 新制||人||163(附属図書館) / 25||人博||679(吉田南総合図書館) / 31224 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦, 教授 津田 謹輔 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM