Immunosuppressants that target the serine/threonine phosphatase calcineurin are commonly administered following organ transplantation. Their chronic use is associated with reduced insulin secretion and new onset diabetes in a subset of patients, suggestive of pancreatic β cell dysfunction. Calcineurin plays a critical role in the activation of CREB, a key transcription factor required for β cell function and survival. CREB activity in the islet is activated by glucose and cAMP, in large part due to activation of Crtc2, a critical coactivator for CREB. Previous studies have demonstrated that Crtc2 activation is dependent on dephosphorylation regulated by calcineurin. In this study, we sought to evaluate the impact of calcineurin-inhibiting immunosuppressants on Crtc2-CREB activation in the primary β cell. In addition, we further characterized the role and regulation of Crtc2 in the β cell. We demonstrate that Crtc2 is required for glucose dependent up-regulation of CREB target genes. The phosphatase calcineurin and kinase regulation by LKB1 contribute to the phosphorylation status of Crtc2 in the β cell. CsA and FK506 block glucose-dependent dephosphorylation and nuclear translocation of Crtc2. Overexpression of a constitutively active mutant of Crtc2 that cannot be phosphorylated at Ser171 and Ser275 enables CREB activity under conditions of calcineurin inhibition. Furthermore, β cells lacking Crtc2 display impaired glucose-stimulated insulin secretion and cell survival. Together, these results demonstrate that phosphorylation of Crtc2 plays a critical role in regulating CREB activity and contributes to β cell dysfunction and death caused by chronic immunosuppression.

Type 1 diabetes (T1D) is an autoimmune disorder, targeting the β-cells of the pancreas. Processes occurring in the gut and pancreas are inferred to be involved. The pre-diabetic expression signature in these tissues is largely uncharacterized. HYPOTHESIS: Spontaneous models of T1D, the LEW.1AR1/Ztm-iddm rat (LEW-DP) and BioBreeding diabetes-prone rat (BBdp) exhibit a distinct transcriptional signature prior to T1D onset. Transcriptional profiling was used to elucidate the expression signatures of the LEW-DP gut and BBdp pancreas. The LEW-DP gut displayed decreased expression of markers of anti-inflammatory M2 macrophages. The LEW-DP rats showed an upregulation of markers of pro-inflammatory signaling when fed a diabetes-promoting cereal diet compared with LEW-DP rats fed a protective hydrolyzed casein diet. Prospective pancreatectomy was used to analyze T1D development in the BBdp rat. Significant upregulation of β-cell markers Reg3α, Reg3β, and Trim26 was observed in pre-diabetic rats. Thus, it was shown that environment modifies the transcriptional program and the transcriptional profile is programmed early to affect T1D development.

It is estimated by the World Health Organization (WHO) that 422 million people had diabetes worldwide in 2014, including 30.3 million people in the US. The cost of treating the disease is has tripled from 2003-2013 due to the increased number of patients. One of the genes strongly associated with type 2 diabetes (T2D) is the transcription factor 7 like 2 (TCF7L2). A single nucleotide polymorphism (SNP) of the TCF7L2 results in increased expression of long chain acyl-CoA synthetase 5 (ACSL5) while deletion of this part of the TCF7L2 gene reduces ACSL5 mRNA level. The regulation of ACSL5 gene expression by the high risk TCF7L2 allele highlights the importance of investigating the role of ACSL5 in T2D. ACSL5 is one of a family of enzymes that activates FA to its CoA ester and is required for FA metabolism within cells. Mice lacking this protein have reduced fat mass and are more insulin sensitive. Chronic exposure of clonal pancreatic ß-cells to excess nutrients has been shown to result in increased intrinsic lipid droplets, reduced insulin content, a left-shift in glucose dose-dependent insulin secretion curve characterized by basal insulin hypersecretion (IH) and blunted glucose stimulated insulin secretion (GSIS). We tested the hypothesis that the use of a putative ACSL5 inhibitor (Adipo C) can reduce accumulated lipid droplets, rescue insulin content and reverse the left-shift in glucose dose-dependent insulin secretion curve. INS-1 (823/13) cells were cultured in either 4 mM or 11 mM glucose media representing physiological and excess nutrients environment. Adipo C (10-25 µM) was added to cells to both acutely (2 hrs) and chronically (72 hrs) inhibit ACSL5 activity. Thin layer chromatography with C11 Bodipy fatty acid (BFA) was used to detect acute fatty acid incorporation into neutral lipids. Nile red was used to visualize intrinsic lipid droplets inside cells. Intracellular Ca2+ activity was detected using fura 2. Insulin assay was measured by HTRF. Acute fatty acid incorporation and lipid accumulation were reduced in cells exposed to Adipo C. An Adipo C concentration dependent right shift of glucose dose-dependent insulin release and increased insulin content were observed. 11 mM glucose cells cultured in 25 µM Adipo C showed decreased intracellular Ca2+ activity at 3 mM glucose and increased Ca2+ activity at 12 mM glucose, which are characteristic of cells cultured in 4 mM glucose having reduced lipid stores. These results all indicate possible protective effects on -cells exposed to excess nutrients. Islets of T2D patients who have a physiologically elevated blood glucose level are exposed to a similar excess nutrient environment. Therefore, the results illustrated here warrant further research on Adipo C compound to explore its therapeutic potential on T2D.

Le diabète de type II (T2D) est une pathologie caractérisée par une hyperglycémie chronique due au dysfonctionnement ainsi qu’à l’apoptose des cellules β pancréatiques, associée à la résistance à l’action de l’insuline. Dans le cas d’un T2D accompagné d’une obésité, l’hyperglycémie chronique potentialise les effets délétères des acides gras saturés sur la cellule β. Ce phénomène est défini comme étant la gluco-lipotoxicité (GL). Aujourd’hui, peu de cibles thérapeutiques existent afin de contrecarrer les effets de la GL et de traiter/prévenir définitivement le diabète, ceci étant dû en partie au manque de connaissances sur la régulation de la cellule β dans des conditions pathologiques. Dans ce but, le consortium Européen IMIDIA (http://www.imidia.org) a réalisé une analyse multiparamétrique permettant l’identification de gènes exprimés dans les îlots de Langerhans qui sont associés à la tolérance au glucose ainsi que la capacité de l’îlot à sécréter l’insuline chez des souris obèses. Parmi ces gènes, je me suis intéressée au rôle de l’élongase 2 (ELOVL2), enzyme impliquée dans la synthèse d’acides gras ω3-poly-insaturés (PUFAs) et en particulier l’acide docosahexaénoïque (DHA). J’ai pu mettre en évidence que la GL diminue l’expression d’Elovl2 et la quantité de DHA dans les cellules β. J’ai pu montrer que le DHA et la surexpression d’Elovl2 restaurent la sécrétion d’insuline induite par le glucose inhibée par la GL, suggérant qu’une augmentation de la quantité endogène de DHA, via la surexpression d’Elovl2 serait capable de contrecarrer le défaut de sécrétion d’insuline associé à la GL. J’ai pu ensuite montrer qu’une sous-expression d’Elovl2 accroît encore plus l’apoptose des cellules β induite par la GL. Ceci étant contrecarré par une surexpression d’Elovl2 et l’addition de DHA. L’axe Elovl2/DHA diminue drastiquement l’accumulation de céramides, responsable de l’apoptose des cellules β induite par la GL. Néanmoins, cet axe ne semble pas affecter la synthèse de novo. En revanche, l’étomoxir (inhibiteur de l’oxydation des acides gas) inhibe totalement l’effet protecteur de l’axe Elovl2/DHA. Ceci suggère donc que l’axe Elovl2/DHA altère le devenir des acides gras dans la cellule en favorisant la dégradation des acides gras par la β-oxydation afin de protéger la cellule β de l’apoptose. En conclusion, mes résultats ont permis de mettre en évidence le rôle de l’axe Elovl2/DHA dans le disfonctionnement et l’apoptose induits par la GL. L’existence de cet axe pourrait conduire à développer de nouvelles thérapies qui cibleraient la synthèse de DHA afin de protéger la cellule β contre les effets délétères de la GL. Enfin, bien que je me sois focalisée uniquement sur la validation d’Elovl2, la base de données créée au cours de cette étude pour identifier de nouveaux gènes impliqués dans le T2D représente une nouvelle ressource importante pour mieux comprendre la défaillance de la cellule β durant un stress métabolique / Type 2 diabetes (T2D) is a disease characterised by a dysfunction of pancreatic β cell survival and function associated with insulin resistance. In the case of T2D associated with obesity, chronic hyperglycaemia potentiates the deleterious effect of saturated free fatty acids on β cell. This phenomenon is defined as gluco-lipotoxicity (GL). Up to now, limited therapeutic options exist to fight against GL and treat diabetes and none to cure or prevent this disease, in part due to the limited knowledge of β-cell biology in health and disease. To face to the lack of knowledge regarding β cell function in diabetes, the European consortium IMIDIA (http://www.imidia.org) had conducted a multi-parameter analysis that led to the identification of a sub-network of islet-expressed genes associated with glucose tolerance and insulin secretory capacity during development of obesity in mice. Among these genes, I decided to further investigate the role of the very long chain fatty acid elongase 2 (ELOVL2). ELOVL2 is an enzyme involved in the synthesis of ω3-poly-unsaturated fatty acids (PUFAs), especially Docosahexaenoic acid (DHA). I have found that GL decreases Elovl2 expression and DHA levels in β-cells. I showed that DHA and Elovl2 over-expression rescues glucose-induced insulin secretion and cytosolic Ca2+ influx impaired by GL, suggesting that increased endogenous DHA levels resulting from Elovl2 up-regulation counteracts the insulin secretion defect associated with GL. In a second part, I found that down-regulation of Elovl2 drastically potentiates apoptosis induced by GL. In contrast DHA and over-expressed Elovl2 counteract β cell apoptosis induced by GL. Interestingly, I found that ELOVL2/DHA axis inhibits accumulation of ceramide, which normally mediate β cell apoptosis under GL. It appears that ELOVL2/DHA axis did not inhibit enzyme function involved in de novo ceramide synthesis. In contrast, the fat oxidation inhibitor, etomoxir, which markedly enhanced GL-induced cell death, completely inhibits the beneficial effect of ELOVL2/DHA axis. These results suggest that ELOVL2/DHA alter fatty acid partitioning, in favour of mitochondrial fatty acid β-oxydation in order to protect β-cells from apoptosis. Collectively, my results show for the first time a role of the ELOVL2/DHA axis in β-cell dysfunction and apoptosis induced by GL. The existence of this axis could lead to develop new therapies that target DHA synthesis to protect β-cells against the deleterious effect of GL. Finally, although I focus experimental validation on Elovl2, the comprehensive data set and integrative network model used to identify this candidate gene represents an important novel resource to dissect the molecular aetiology of β-cell failure following metabolic stress.

OBJECTIVE: The close relationship between obesity and type 2 diabetes (T2D) highlights the fact that most diabetes patients are overweight or obese. We propose that elevated glucose and free fatty acid levels in those patients cause beta-cell dysfunction. Chronic exposure to excess nutrients (glucose and free fatty acid) leads to glucolipotoxicity, characterized by basal insulin hypersecretion, a left-shift in the glucose dose-dependent insulin secretion curve, and blunted glucose-stimulated insulin secretion. One of the suggested reasons for this defect is elevated intracellular lipid. In this study, our objective was to investigate whether reducing beta-cell lipid levels can reverse basal insulin hypersecretion. METHODS: INS-1 (823/13) cells were cultured in 4 or 11 mM glucose media. Elevated glucose and KCl doses were added to cells in the insulin secretion experiments. In the KCl-induced insulin secretion experiment, cells were treated with a combination of 12 mM glucose and 250 μM diazoxide, then assigned to different test concentrations with elevated KCl doses. Insulin release and content were measured by the insulin ultra-sensitive homogenous time-resolved fluorescence (HTRF) kit (Cisbio). Following that, we monitored intracellular Ca2+ activity of KCl-induced insulin secretion on a fluorescence spectrophotometer F-2000 (Hitachi). Additionally, we acutely added Adipo C (20 µM) or fatty acid-free BSA to cells to reduce the lipids levels in the ß-cells. We also stained with Nile Red (Sigma) to examine the intrinsic lipid droplets in those cells. RESULTS: ß-cells cultured in excess nutrients (11 mM glucose) exhibited a left shift in the glucose dose-dependent response curve. The hypersecretion at low glucose could be blocked by the KATP channel activator, diazoxide, indicating that Ca2+ influx drives the increase in secretion at glucose concentrations normally considered basal. Here we extend this left shift to include KCl-induced insulin secretion, supporting a role for Ca2+ in the observed hypersensitivity. KCl-induced Ca2+ influx was also left-shifted. Interestingly, we found acute exposure to Adipo C or fatty acid-free BSA reversed the basal hypersecretion in cells cultured in excess nutrients. CONCLUSION: The work presented in this study provided supporting evidence that ß-cells cultured in excess nutrients were hypersensitive to glucose while extending these studies to KCl-induced insulin release. The excess nutrient-induced left shift in both glucose and KCl-stimulated insulin secretion was mediated by increased Ca2+. Thus, we postulate that excess nutrient exposure increases ß-cell plasma membrane lipids that alter Ca2+ handling to allow increased Ca2+ influx at inappropriate low glucose concentrations. Our results demonstrated that cells acutely exposed to the putative long-chain acyl-CoA synthetase inhibitor Adipo C or fatty acid-free BSA reversed basal insulin hypersecretion and supports a role for lipids mediating the adverse effect. T2D patients with obesity have a similar physiologically elevated fasting blood glucose and lipid. Thus, our findings suggest lowering lipid levels in ß-cells may have therapeutic potential in treating hyperinsulinemia leading to T2D.

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

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24519号 / 医博第4961号 / 新制||医||1065(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 川口 義弥, 教授 辻川 明孝, 教授 大鶴 繁 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24200号 / 医博第4894号 / 新制||医||1061(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 中本 裕士, 教授 江木 盛時 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p> Fetal exposure to cigarette smoke is associated with an increased risk of adult-onset metabolic abnormalities. In Canada, nicotine replacement therapy (NRT) is recommended as a safe smoking cessation aid for pregnant women. However, our laboratory has demonstrated that fetal and neonatal nicotine exposure results in glucose intolerance in adult rats. The goal of this thesis was to determine the mechanism(s) underlying the observed dysglycemia following fetal and neonatal nicotine exposure, with a specific focus on the effects of nicotine on pancreatic development and postnatal beta cell function.</p> <p> Nulliparous female Wistar rats received daily subcutaneous injections of either saline or nicotine bitartrate (1 mg/kg/d) for 2 weeks prior to mating until weaning (postnatal day 21 - PND21 ). Pancreatic tissue was collected from male offspring at birth (PND1), 3, 7, 15 and 26 weeks of age. For the critical windows study, dams received nicotine or saline during different stages of pancreatic development, including: A) pre-mating only, B) pre-mating + pregnancy only, C) pre-mating, pregnancy and lactation, or D) pre-mating + lactation only. For the intervention study, nicotine-exposed dams received either normal chow or diet containing antioxidants (1000 IU/kg vitamin E, 0.25% w/w coenzyme Q10 and 0.05% w/w α-lipoic acid) during mating, pregnancy and lactation.</p> <p> Results from this thesis demonstrate that exposure to nicotine during both fetal and neonatal development (but neither stage alone) causes a permenant loss of beta cell mass beginning at birth, and adult-onset dysglycemia in rodents. Furthermore, nicotine exposure induces pancreatic oxidative stress and mitochondrial-mediated beta cell apoptosis in neonates, followed by a progressive decline in mitochondrial structure and function. Maternal treatment with a dietary antioxidant cocktail during pregnancy and lactation protected the developing beta cells from nicotine-induced apoptosis and mitochondrial swelling. These data indicate that the safety of NRT use during pregnancy should be re-evaluated.</p> / Thesis / Doctor of Philosophy (PhD)