京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12816号 / 論医博第2078号 / 新制||医||1004(附属図書館) / 31303 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 長田 重一, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DGAM

Diabetes mellitus is a major public health concern. Although the accessible novel drugs, techniques, and surgical intervention has improved the survival rate of individuals with diabetes, the prevalence of diabetes is still rising. Type 2 diabetes (T2D) is a result of chronic insulin resistance (IR) and loss of β-cell mass and function. Therefore, the search for naturally occurring, low-cost, and safe compounds that could enhance insulin sensitivity and protect functional β-cell mass can be an effective strategy to prevent this disease. Kaempferol, a flavonol present in various medicinal herbs and edible plants, has been shown to elicit various pharmacological activities in preclinical studies. However, studies investigating the effect of kaempferol on diabetes are limited. In this dissertation, I explored the anti-diabetic potential of dietary intake of kaempferol in diet-induced obese mice and insulin-deficient diabetic mice. First, kaempferol was supplemented in the diet to determine whether it can prevent IR and hyperglycemia in high fat (HF) diet-induced obese mice or STZ-induced obese diabetic mice. To evaluate its efficacy for treating diabetes, kaempferol was administrated once daily via oral gavage to diet-induced obese and insulin-resistant mice or lean STZ-induced diabetic mice. The results demonstrated that long-term oral administration of kaempferol prevents HFD-induced metabolic disorders in middle-aged obese mice. Oral administration of kaempferol improved glucose intolerance and insulin sensitivity, and this effect was associated with increased Glut4 and AMPKa expression in muscle and adipose tissues. Consistent with our findings from the in iii vitro study in C2C12 muscle cell line, these findings suggest that kaempferol may reduce IR at the molecular level by improving glucose metabolism in peripheral tissues. In the second study, dietary kaempferol supplementation prevented hyperglycemia and glucose intolerance by protecting β-cell against the induced damage in obese STZ-induced diabetic mice. In the third study, the administration of kaempferol by oral gavage significantly ameliorated hyperglycemia and glucose intolerance and reduced the incidence of diabetes from 100 % to 77.8% in lean STZinduced diabetic mice. This kaempferol effect was associated with reduced hepatic glucose production, the primary contributor to hyperglycemia, and increased glucose oxidation in the muscle of diabetic mice. Kaempferol treatment restored hexokinase activity in the liver and skeletal muscle and reduced pyruvate carboxylase (PC) activity and glycogenolysis in the liver. Unlike its effect on T2D mice, kaempferol effect in lean STZ-induced diabetic mice was not associated with changes in plasma insulin levels. In the last study, we found that administration of kaempferol by oral gavage significantly improved blood glucose control by suppressing hepatic glucose production and improving glucose intolerance in obese insulin-resistant mice. Similar to its effect in old obese mice, kaempferol enhanced whole-body insulin sensitivity. Kaempferol increased Akt and hexokinase activity and decreased PC activity in the liver. However, kaempferol did not exert any changes in glucose metabolism or insulin sensitivity when administered to healthy lean mice. Overall, findings from these studies provide new insight into the role of kaempferol in the regulation of glucose homeostasis and suggest that kaempferol may be a naturally occurring anti-diabetic compound by improving insulin sensitivity, improving glucose regulation and metabolism, and preserving functional β-cell mass. / Ph. D.

Early pancreatectomy experiments performed in ducks and pigeons at the end of the 19th century revealed that avians, unlike mammals, do not display signs of diabetes. Relative to mammals, birds are considered hyperglycemic, displaying fasting blood glucose concentrations twice that of a normal human. While circulating levels of insulin are similar in avians and mammals, and structure and function of the insulin receptor are also conserved among vertebrate species, birds do not experience deleterious effects of chronic hyperglycemia as observed in mammals. Understanding avian glucose homeostasis, particularly in chickens, has both agricultural and biomedical implications. Improvement of feed efficiency and accelerated growth in poultry may come from a greater understanding of the physiological processes associated with glucose utilization in muscle and fat. The chicken has also recently been recognized as an attractive model for human diabetes, where there is a great need for preventative and therapeutic strategies. The link between type 2 diabetes and obesity, coupled with the inherent hyperglycemic nature of chickens, make chickens artificially selected for juvenile low (LWS) and high (HWS) body weight a favorable model for investigating glucose regulation and pancreas physiology. Oral glucose tolerance and insulin sensitivity tests revealed differences in threshold sensitivity to insulin and glucose clearance rate between the lines. Results from real-time PCR showed greater pancreatic mRNA expression of four glucose regulatory genes (preproinsulin, PPI; preproglucagon, PPG; glucose transporter 2, GLUT2; and pancreatic duodenal homeobox 1, Pdx1) in LWS, than HWS chickens. Histological analysis of pancreas revealed that HWS chickens have larger pancreatic islets, less pancreatic islet mass, and more pancreatic inflammation than LWS chickens, all of which presumably contribute to impaired glucose metabolism. In summary, results suggest that at selection age, there are differences in pancreas physiology that may explain the differences in glucose regulation between LWS and HWS. These data pave the way for future studies aimed at understanding the developmental regulation of endocrine pancreas function in chickens, as well as how aging affects homeostatic control of blood glucose in chickens. / Ph. D.

<p>Enterovirus (EV) infections have been associated with the pathogenesis of Type 1 Diabetes (T1D). However, the pathway(s) by which EV may induce or accelerate diabetes is not well understood. The purpose of this thesis was to obtain new information on the mechanism by which EV infections, with different strains of EV, could cause damage to the insulin-producing β-cells in isolated human islets and in a rat insulin-producing cell line (RINm5F). </p><p>Infection with EV strains isolated from T1D patients revealed replication/cell destruction in human islets and EV-like particles in the cytoplasm of the β-cell and infection with the isolates affected the release of insulin in response to glucose stimulation as early as three days post infection, before any decrease in cell viability was observed. A decrease in the induction/secretion of the chemokine RANTES in human islets during EV infection was also detected. When islets were cultured with nicotinamide (NA) the secretion of RANTES was increased irrespectively if the islets were infected or not. In addition, the degree of virus-induced cytolysis of human islets was reduced by NA, suggesting an antiviral effect of NA. Infection with EV strains revealed permissiveness to islet-derived cells. </p><p>All EV strains used for infection were able to replicate in the RIN cell clusters (RCC) but not in the RIN cells that were cultured as a monolayer. This might be due to the differences in expression of the Coxsackie-adenovirus receptor (CAR), which only could be detected on the RCC. Infection of RCC with a CBV-4 strain did not affect cell viability and did not induce nitric oxide (NO) production alone or with the addition of IFN-γ. This was in contrast to the results obtained with synthetic dsRNA, poly(IC), which induced NO, suggesting that synthetic dsRNA does not mimic enteroviral intermediate dsRNA.</p><p>During analyses performed with the samples from a family where the mother and one son where diagnosed with T1D on the same day, the results showed that the whole family had a proven EV infection at the time diagnosis.</p><p>To conclude, the ability of EV strains to replicate in RIN cells is dependent on the growth pattern of the cells and this may be due to the upregulation and/or changed expression pattern of CAR in these cells. In the RIN cells, contrary to artificial dsRNA, viral dsRNA does not induce NO. The isolated EV virus strains used were able to infect and affect human pancreatic islets in vitro. The chemokine RANTES is reduced during an EV infection of human pancreatic islets and NA causes upregulation of RANTES in infected and uninfected islets. </p>

Enterovirus (EV) infections have been associated with the pathogenesis of Type 1 Diabetes (T1D). However, the pathway(s) by which EV may induce or accelerate diabetes is not well understood. The purpose of this thesis was to obtain new information on the mechanism by which EV infections, with different strains of EV, could cause damage to the insulin-producing β-cells in isolated human islets and in a rat insulin-producing cell line (RINm5F). Infection with EV strains isolated from T1D patients revealed replication/cell destruction in human islets and EV-like particles in the cytoplasm of the β-cell and infection with the isolates affected the release of insulin in response to glucose stimulation as early as three days post infection, before any decrease in cell viability was observed. A decrease in the induction/secretion of the chemokine RANTES in human islets during EV infection was also detected. When islets were cultured with nicotinamide (NA) the secretion of RANTES was increased irrespectively if the islets were infected or not. In addition, the degree of virus-induced cytolysis of human islets was reduced by NA, suggesting an antiviral effect of NA. Infection with EV strains revealed permissiveness to islet-derived cells. All EV strains used for infection were able to replicate in the RIN cell clusters (RCC) but not in the RIN cells that were cultured as a monolayer. This might be due to the differences in expression of the Coxsackie-adenovirus receptor (CAR), which only could be detected on the RCC. Infection of RCC with a CBV-4 strain did not affect cell viability and did not induce nitric oxide (NO) production alone or with the addition of IFN-γ. This was in contrast to the results obtained with synthetic dsRNA, poly(IC), which induced NO, suggesting that synthetic dsRNA does not mimic enteroviral intermediate dsRNA. During analyses performed with the samples from a family where the mother and one son where diagnosed with T1D on the same day, the results showed that the whole family had a proven EV infection at the time diagnosis. To conclude, the ability of EV strains to replicate in RIN cells is dependent on the growth pattern of the cells and this may be due to the upregulation and/or changed expression pattern of CAR in these cells. In the RIN cells, contrary to artificial dsRNA, viral dsRNA does not induce NO. The isolated EV virus strains used were able to infect and affect human pancreatic islets in vitro. The chemokine RANTES is reduced during an EV infection of human pancreatic islets and NA causes upregulation of RANTES in infected and uninfected islets.

Aging is a universal process that is accompanied by the loss of proliferative potential of cells. However, factors governing this age-dependent decline in proliferation remain largely undefined. The pancreatic β-cells of the islets of Langerhans serve as a unique model to explore the effect of cellular age on proliferation and function within the same organ. During early juvenile stage, the zebrafish islet is rapidly expanding and newly differentiated β-cells are added to the pool of older β-cells that were formed during embryogenesis. In this thesis, using accurate reporters for cell-cycle stages and intra-cellular calcium sensors, it was shown that younger β-cells are more proliferative but less functional compared to older β-cells. Furthermore, as the animal ages, the overall rate of β-cell proliferation declines. Transcriptomic analysis of β-cells from young adult and older adult islets revealed that older cells display an inflammatory signature. Transgenic reporter line for inflammatory NF-kB activity showed that β-cells of younger islets display varying levels of NF-kB activity, which becomes homogenous in older β-cells. Furthermore, the cells with higher NF-kB-activity proliferate less compared to their neighbors with lower activity. Specifically, younger NF-kBhigh cells upregulate socs2, a negative regulator of proliferation that is also enriched in older β-cells. Interestingly, activated macrophages were observed infiltrating the islet during late juvenile stages, thus pointing to an important role of the microenvironment in activation of inflammatory signature in the islet. Overall, this study shows that cells of different ages co-exist within the same micro-organ. This age-related cellular heterogeneity governs the rate of proliferation of the tissue. The loss of cellular heterogeneity with age reduces the proliferative pool of the tissue. Finally, the expression of inflammatory NF-kB activity acts as a marker of this loss of proliferative heterogeneity.

Le pancréas est un organe hétérogène composé d’une partie exocrine, responsable de la synthèse d’enzymes pour la digestion, et d’une partie endocrine, essentielle pour l’homéostasie glucidique. Notamment la sécrétion d’insuline par les cellules β contrôle la glycémie. Les dysfonctionnements des cellules β sont une des causes du diabète, première épidémie non infectieuse au monde. Il est actuellement possible d’en traiter les symptômes mais pas de le guérir. De nombreux laboratoires recherchent un protocole idéal de production de cellules β afin de pouvoir greffer ces cellules aux patients. L’identification des facteurs qui gouvernent chaque étape du développement des cellules β devrait permettre de progresser dans ce sens. Le but de ma thèse a été d’étudier le rôle des Espèces Réactives de l’Oxygène (ROS) au cours du développement pancréatique. Cette question a été soulevée lorsque nous avons analysé l’expression des gènes codant pour les enzymes détoxifiantes des ROS: leur expression était extrêmement réduite dans les pancréas embryonnaires comparés aux pancréas adultes, suggérant que les précurseurs sont particulièrement sensibles aux variations des ROS. Nous avons ensuite montré que la réduction des ROS in vivo, obtenue par un traitement avec un antioxydant (NAC), diminue le développement des cellules β. Une analyse in vitro a permis de détailler les mécanismes de l’action des ROS. En effet, le peroxyde d’hydrogène favorise la différenciation des cellules β en augmentant l’expression du facteur pro-endocrine Ngn3 dans les progéniteurs. Ce processus implique l’activation la voie ERK1/2 par les ROS. Au contraire, la diminution des ROS induite par des méthodes génétiques ou pharmacologiques altère la différenciation des cellules β. Nos résultats indiquent également que la mitochondrie est impliquée dans ce processus. Nous avons donc montré que la présence des ROS est essentielle pour le bon développement du pancréas. Ces recherches devraient donc permettre de progresser vers une thérapie cellulaire du diabète. / The pancreas is an heterogenous gland composed by exocrine tissue, responsible for digestive enzyme secretions, and endocrine tissue, essential for glucose homeostasis. In particular β cells secrete insulin which controls glycemia. Moreover, β cell failure is one of the primary causes of diabetes and this pathology is nowadays considered as the first non infectious worldwide outbreak. There is unfortunately no cure for this disease. Many laboratories are currently improving β cell generation protocols in order to inject those cells into patients. This is the reason why it appears mandatory to be able to identify factors that govern each step of β cell development. The aim of my work was to study the role of the Reactive Oxygen Species (ROS) during pancreatic development. First we found out that the expression of genes coding for antioxidant enzymes was extremely low in embryonic pancreas compared to adult pancreas. This suggested that progenitors could be sensitive to ROS variations. We then showed in vivo using an antioxidant component (NAC) that decreasing ROS level diminishes β cell development. Analysis in vitro allowed us to better describe the role of ROS. Indeed, hydrogen peroxyde favors β cell differentiation by increasing the pro-endocrine marker NGN3 expression in the progenitors. In this process, ROS activate the ERK1/2 signaling pathway. On the contrary, lowering ROS level using both pharmacologic and genetic approaches, decreases β cell differentiation. Our results also point out a role of the mitochondria in this process. Altogether, our data define the effects of ROS on β cell differentiation and open new perspectives to improve protocols of β cell generation.

Le pancréas est un organe hétérogène composé d’une partie exocrine, responsable de la synthèse d’enzymes pour la digestion, et d’une partie endocrine, essentielle pour l’homéostasie glucidique. Notamment la sécrétion d’insuline par les cellules β contrôle la glycémie. Les dysfonctionnements des cellules β sont une des causes du diabète, première épidémie non infectieuse au monde. Il est actuellement possible d’en traiter les symptômes mais pas de le guérir. De nombreux laboratoires recherchent un protocole idéal de production de cellules β afin de pouvoir greffer ces cellules aux patients. L’identification des facteurs qui gouvernent chaque étape du développement des cellules β devrait permettre de progresser dans ce sens. Le but de ma thèse a été d’étudier le rôle des Espèces Réactives de l’Oxygène (ROS) au cours du développement pancréatique. Cette question a été soulevée lorsque nous avons analysé l’expression des gènes codant pour les enzymes détoxifiantes des ROS: leur expression était extrêmement réduite dans les pancréas embryonnaires comparés aux pancréas adultes, suggérant que les précurseurs sont particulièrement sensibles aux variations des ROS. Nous avons ensuite montré que la réduction des ROS in vivo, obtenue par un traitement avec un antioxydant (NAC), diminue le développement des cellules β. Une analyse in vitro a permis de détailler les mécanismes de l’action des ROS. En effet, le peroxyde d’hydrogène favorise la différenciation des cellules β en augmentant l’expression du facteur pro-endocrine Ngn3 dans les progéniteurs. Ce processus implique l’activation la voie ERK1/2 par les ROS. Au contraire, la diminution des ROS induite par des méthodes génétiques ou pharmacologiques altère la différenciation des cellules β. Nos résultats indiquent également que la mitochondrie est impliquée dans ce processus. Nous avons donc montré que la présence des ROS est essentielle pour le bon développement du pancréas. Ces recherches devraient donc permettre de progresser vers une thérapie cellulaire du diabète. / The pancreas is an heterogenous gland composed by exocrine tissue, responsible for digestive enzyme secretions, and endocrine tissue, essential for glucose homeostasis. In particular β cells secrete insulin which controls glycemia. Moreover, β cell failure is one of the primary causes of diabetes and this pathology is nowadays considered as the first non infectious worldwide outbreak. There is unfortunately no cure for this disease. Many laboratories are currently improving β cell generation protocols in order to inject those cells into patients. This is the reason why it appears mandatory to be able to identify factors that govern each step of β cell development. The aim of my work was to study the role of the Reactive Oxygen Species (ROS) during pancreatic development. First we found out that the expression of genes coding for antioxidant enzymes was extremely low in embryonic pancreas compared to adult pancreas. This suggested that progenitors could be sensitive to ROS variations. We then showed in vivo using an antioxidant component (NAC) that decreasing ROS level diminishes β cell development. Analysis in vitro allowed us to better describe the role of ROS. Indeed, hydrogen peroxyde favors β cell differentiation by increasing the pro-endocrine marker NGN3 expression in the progenitors. In this process, ROS activate the ERK1/2 signaling pathway. On the contrary, lowering ROS level using both pharmacologic and genetic approaches, decreases β cell differentiation. Our results also point out a role of the mitochondria in this process. Altogether, our data define the effects of ROS on β cell differentiation and open new perspectives to improve protocols of β cell generation.

Type 2 diabetes mellitus represents a metabolic disease reaching epidemic dimensions in the 21st century. Fatty acid-induced apoptosis of pancreatic β-cells significantly contributes to its pathogenesis. Saturated fatty acids (FAs) are strongly cytotoxic for β-cells, whereas unsaturated FAs are well tolerable by β-cells, they are even able to inhibit proapoptotic effects of saturated FAs when co-incubated. According to recent studies, FAs-induced apoptosis in pancreatic β-cells is partly regulated by autophagy, a catabolic process involved in the degradation and recyclation of cell components in lysosomes. The aim of this diploma thesis was to contribute to the clarification of the role of autophagy in FAs-induced apoptosis regulation. We induced apoptosis in human pancreatic β- cell line NES2Y by 1 mM stearic acid (SA) and inhibited it with 0.2 mM oleic acid (OA) co- incubated with SA. We revealed, that the saturated SA used in apoptosis-inducing concentration simultaneously inhibits the autophagic flux in pancreatic NES2Y cell line. When SA is co- incubated with unsaturated OA in concentration sufficient for inhibition of proapoptotic effect of SA, OA is also able to inhibit the block of autophagy induced by the effect of SA. Application of unsaturated OA alone in this concentration did not...

Le pancréas est un organe hétérogène composé d’une partie exocrine, responsable de la synthèse d’enzymes pour la digestion, et d’une partie endocrine, essentielle pour l’homéostasie glucidique. Notamment la sécrétion d’insuline par les cellules β contrôle la glycémie. Les dysfonctionnements des cellules β sont une des causes du diabète, première épidémie non infectieuse au monde. Il est actuellement possible d’en traiter les symptômes mais pas de le guérir. De nombreux laboratoires recherchent un protocole idéal de production de cellules β afin de pouvoir greffer ces cellules aux patients. L’identification des facteurs qui gouvernent chaque étape du développement des cellules β devrait permettre de progresser dans ce sens. Le but de ma thèse a été d’étudier le rôle des Espèces Réactives de l’Oxygène (ROS) au cours du développement pancréatique. Cette question a été soulevée lorsque nous avons analysé l’expression des gènes codant pour les enzymes détoxifiantes des ROS: leur expression était extrêmement réduite dans les pancréas embryonnaires comparés aux pancréas adultes, suggérant que les précurseurs sont particulièrement sensibles aux variations des ROS. Nous avons ensuite montré que la réduction des ROS in vivo, obtenue par un traitement avec un antioxydant (NAC), diminue le développement des cellules β. Une analyse in vitro a permis de détailler les mécanismes de l’action des ROS. En effet, le peroxyde d’hydrogène favorise la différenciation des cellules β en augmentant l’expression du facteur pro-endocrine Ngn3 dans les progéniteurs. Ce processus implique l’activation la voie ERK1/2 par les ROS. Au contraire, la diminution des ROS induite par des méthodes génétiques ou pharmacologiques altère la différenciation des cellules β. Nos résultats indiquent également que la mitochondrie est impliquée dans ce processus. Nous avons donc montré que la présence des ROS est essentielle pour le bon développement du pancréas. Ces recherches devraient donc permettre de progresser vers une thérapie cellulaire du diabète. / The pancreas is an heterogenous gland composed by exocrine tissue, responsible for digestive enzyme secretions, and endocrine tissue, essential for glucose homeostasis. In particular β cells secrete insulin which controls glycemia. Moreover, β cell failure is one of the primary causes of diabetes and this pathology is nowadays considered as the first non infectious worldwide outbreak. There is unfortunately no cure for this disease. Many laboratories are currently improving β cell generation protocols in order to inject those cells into patients. This is the reason why it appears mandatory to be able to identify factors that govern each step of β cell development. The aim of my work was to study the role of the Reactive Oxygen Species (ROS) during pancreatic development. First we found out that the expression of genes coding for antioxidant enzymes was extremely low in embryonic pancreas compared to adult pancreas. This suggested that progenitors could be sensitive to ROS variations. We then showed in vivo using an antioxidant component (NAC) that decreasing ROS level diminishes β cell development. Analysis in vitro allowed us to better describe the role of ROS. Indeed, hydrogen peroxyde favors β cell differentiation by increasing the pro-endocrine marker NGN3 expression in the progenitors. In this process, ROS activate the ERK1/2 signaling pathway. On the contrary, lowering ROS level using both pharmacologic and genetic approaches, decreases β cell differentiation. Our results also point out a role of the mitochondria in this process. Altogether, our data define the effects of ROS on β cell differentiation and open new perspectives to improve protocols of β cell generation.