Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes mellitus is a chronic disorder characterized by hyperglycemia, insulin resistance, and insufficient insulin secretion from the pancreatic β cells. To maintain adequate levels of insulin secretion, β cells rely on highly coordinated control of luminal ER Ca2+. Stromal Interaction Molecule 1 (STIM1) is an ER Ca2+ sensor that serves to replenish ER Ca2+ stores in response to depletion by gating plasmalemmal Orai1 channels in a process known as store-operated calcium entry (SOCE). We developed a method for the direct measurement of SOCE in pancreatic β cells and found that deletion of STIM1 in INS-1 cells (STIM1KO) is sufficient to block Ca2+ influx in response to store-depletion. To determine the physiological importance of β cell STIM1, we created mice with pancreatic β cell specific deletion of STIM1 (STIM1Δβ) and placed them on a high fat diet (HFD) with 60% of kilocalories derived from fat. After 8 weeks of HFD, female, but not male, STIM1Δβ mice exhibited increased body weight and fat mass as well as significant glucose intolerance and impaired insulin secretion without observable differences in insulin tolerance. Immunohistochemical analysis revealed a reduction of β cell mass and an increase of α cell mass; ELISA of islet lysates revealed a similar significant reduction in insulin content and increased glucagon content. RNA-sequencing performed on STIM1Δβ islets revealed differentially expressed genes for functions related to apoptosis, lipid metabolism, and epithelial cell differentiation, as well as loss of β cell identity. Proteomics analysis of STIM1KO cells phenocopied the metabolic findings, revealing significantly increased glucagon expression. Analysis of islet RNA-sequencing results showed modulation of pathways related to 17-β estradiol (E2) signaling, with notable downregulation of G-protein coupled estrogen receptor 1 (GPER1) expression. Consistently, treatment of female wild-type islets with pharmacological SOCE inhibitors led to reduced expression GPER1, while STIM1KO cells showed lower mobilization of intracellular cAMP levels in response to GPER agonist treatment. Taken together, these findings identify a novel interaction between SOCE and E2 signaling in the female islet and suggest that loss of STIM1 and impairments in SOCE may contribute to diabetes pathophysiology through loss of β cell identity. / 2022-12-28

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20238号 / 医博第4197号 / 新制||医||1019(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 河本 宏, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

A thorough understanding of beta-cell proliferation, function, death and regeneration under normal condition as well as in the progression of diabetes is crucial to the conquest of this disease. The work presented in this thesis aimed to investigate the expression and role of a novel transcription factor, Zinc finger BED domain-containing protein 6 (ZBED6), in beta-cells. ZBED6 was present in mouse βTC-6 cells and human islets as a double nuclear band at 115/120 kDa and as a single cytoplasmic band at 95-100 kDa, which lacked N-terminal nuclear localization signals. Lentiviral shRNA-mediated stable silencing of ZBED6 in βTC-6 cells resulted in altered morphology, decreased proliferation, a partial S/G2 cell cycle arrest, increased expression of beta-cell specific genes, and higher rates of apoptosis. ChIP sequencing of human islets showed that ZBED6 binding was preferentially to genes that control transcription, macromolecule biosynthesis and apoptosis. We proposed that ZBED6 supported proliferation and survival of beta-cells, possibly at the expense of specialized beta-cell function, i.e. insulin production. To further investigate the role of ZBED6 in beta-cells, ChIP sequencing and whole transcriptome analysis were performed using MIN6 cells. More than 4000 putative target genes of ZBED6 were identified, including Pdx1, MafA and Nkx6.1. ZBED6-silencing resulted in differential expression of more than 700 genes, which was paralleled by an increase in the content and release of insulin in response to a high glucose concentration. Altered morphology/growth patterns as indicated by increased cell clustering were observed in ZBED6 silenced cells. We found also that ZBED6 decreased the ratio between N- and E-cadherin. A lower N- to E-cadherin ratio may hamper the formation of three-dimensional beta-cell clusters and cell-to-cell junctions with neural crest stem cells, and instead promote efficient attachment to a laminin support and monolayer growth. Thus, by controlling beta-cell adhesion and cell-to-cell junctions, ZBED6 might play an important role in beta-cell differentiation, proliferation and survival.

<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)

OBJECTIVE: Obesity is a chronic disease with high incidence worldwide, which promotes the risk of incidence of type 2 diabetes (T2D). Obesity-induced adipocyte expansion promotes local chronic inflammation in the adipose tissue which is considered a contributing factor to insulin resistance, hyperinsulinemia, and T2D. Many organs, including adipose tissue, involve in the dysregulation of glucose homeostasis in T2D. The crosstalk between adipose tissue/adipocytes and pancreatic ß-cells has provoked scientists' interest for years. Here in this thesis, we focused on the effect carried out by adipocyte-specific GPS2 depletion on insulin secretion from pancreatic ß-cells. METHODS: Conditioned media collected over 24 h from both primary adipocyte and adipose tissue explant cultures from high fat diet (HFD)-fed WT and adipocyte-specific GPS2 knock-out (GPS2-AKO) mice were used to treat INS-1 clonal pancreatic ß-cells or primary islets from chow-diet WT mice. Conditioned media was diluted 1:8 in culture media of clonal INS-1 cells (cultured in media with 4 mM or 11 mM glucose chronically) and primary islets (cultured in media with 11 mM glucose) and incubated for 18 h before measuring insulin secretion. The isolated islets from chow-diet WT mice were also be treated with the primary adipocytes conditioned media from eWAT (epididymal white adipose tissue) of HFD-fed WT and GPS2-AKO mice. In addition, the effect of exosomes extracted from primary adipocyte conditioned media of HFD-fed WT and GPS2-AKO mice on GSIS was investigated in clonal INS-1 cells. Glucose-stimulated insulin secretion (GSIS) was measured to assess differences in insulin secretion by INS-1 cells and islets from mice in response to signaling from WT or GPS2-AKO adipocytes. RESULTS: Adipocyte conditioned media from both WT and GPS2-AKO mice reduced GSIS from INS-1 cells by the same extent compared to a non-treated control. The same result was obtained using media conditioned by adipose tissue explant culture. Exosomes isolated from adipocyte conditioned media from both WT and GPS2-AKO mice also reduced GSIS from INS-1 cells with no significant difference between WT and GPS2-AKO. Islets isolated from chow-diet WT mice treated with adipocyte conditioned media from eWAT of WT and GPS2-AKO mice also showed no significant difference between WT and GPS2-AKO in GSIS compared to our non-treated control. CONCLUSIONS: Both conditioned media and exosomes from primary adipocytes of HFD-fed mice inhibits GSIS from INS-1 cells and isolated islets, but no difference was observed between WT and GPS2-AKO mice. We conclude that the deletion of GPS2 in adipocytes does not influence GSIS from pancreatic ß-cells under our experimental conditions. Conditioned media-induced inhibition of GSIS is mediated by factors that may contribute to adipocyte-ß-cell crosstalk in-vivo. / 2025-11-02T00:00:00Z

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown. Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis. In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%). The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. The third objective of this work was to obtain a broad picture of the pancreatic beta-cell molecular responses during and after (recovery period) a severe ER stress. For this purpose, we utilized an “in home” spotted microarray, the APOCHIP, containing nearly 600 probes selected for the study of beta-cell apoptosis. Time-dependent gene expression profiles were measured in INS-1E cells exposed to CPA. CPA-induced ER-stress modified expression of 183 genes in at least one of the time points studied. Most of theses genes returned to control levels 3h after CPA removal from the culture medium. We observed full beta-cell recovery and survival, indicating that these cells trigger efficient defenses against ER stress. Beta-cell recovery is associated with a sustained increase in the expression of ER chaperones and a rapid decrease of pro-apoptotic mRNAs following CPA removal. Two groups of genes were particularly affected by CPA, namely those related to the cellular responses to ER stress, which were mostly up-regulated, and those related to differentiated beta-cell functions, which were down-regulated. Among this last group, we observed a 40-90% decrease of the mRNAs for insulin-1 and -2. These findings were confirmed in INS-1E cells exposed to cytokines or thapsigargin (another SERCA blocker), and in primary beta-cells exposed to the same treatments. This decrease in insulin mRNA expression is due to transcript degradation, most probably caused by IRE-1 activation and triggering of its endoribonuclease activity, as recently described in Drosophila cells. In conclusion, our work enabled a better understanding of the pancreatic beta-cell responses to ER stress: 1.)We identified a sensitizing effect of IFN-gamma to ER stress in beta-cells via downregulation of key ER chaperones. 2.)We observed a differential regulation of Chop transcription by different treatments suggesting distinct responses of pancreatic beta-cells to diverse ER stress inducers. 3.)We provided the first global analysis of gene expression modifications in pancreatic beta-cells following ER stress. 4.)We demonstrated a high capacity of beta-cells to cope and recover from a severe ER stress. 5.)We identified a new protective mechanism against ER stress, namely the degradation of insulin mRNA which limits the load posed on the ER by insulin synthesis. This, coupled to a marked increase in ER chaperones and a fast degradation of pro-apoptotic mRNAs, enables beta cells to recover from ER stress after the causes of this stress are removed.

Les pertes de la fonction et de la masse des cellules beta pancréatiques jouent un rôle central dans le diabète de type 2 (DT2). Les beta-arrestines 1 et 2 (ARRB1 et ARRB2), sont impliquées dans la sécrétion et/ou la survie des cellules beta pancréatiques.Dans une première étude, afin de caractériser précisément la fonction d’ARRB1 dans les cellules beta pancréatiques, nous avons eu pour objectif de générer des souris invalidées spécifiquement dans ces cellules en utilisant le système Cre/lox sous le contrôle du promoteur Ins1. Des études avaient été publiées à partir des deux lignées Ins1Cre-/+ et Arrb1f/f. Nous avons généré et travaillé sur les souris Arrb1f/f :Ins1Cre-/+. Le phénotype des souris Arrb1f/f :Ins1Cre-/+ était faible et surtout non reproductible comparé aux souris Arrb1f/f :Ins1Cre-/- utilisées comme témoins. Le faible niveau d’expression d'Arrb1 dans les cellules beta et le manque d'anticorps spécifique pour l'immunocytochimie ont rendu difficile la vérification de l'absence d'expression de ARRB1 dans ces cellules. Après séquençage du gène modifié Arrb1 des souris “floxées“, nous avons pu montrer que l'insertion du premier site loxP avait induit un décalage du cadre de lecture introduisant un codon stop et, par conséquent, la non-expression du gène Arrb1. Étant donné que les souris Arrb1 “floxées“ utilisées comme témoins étaient déjà knockout (KO), le projet utilisant ces souris a dû être arrêté.Notre équipe a rapporté l'implication d'ARRB2 dans la régulation de la masse des cellules bêta pancréatique, mais son rôle dans la signalisation du récepteur du Glucagon-Like Peptide-1 (GLP-1R), une cible thérapeutique majeure du DT2, n'avait pas encore été exploré.Nous avons montré, dans une deuxième étude, une meilleure tolérance orale au glucose ainsi qu’une augmentation de la sécrétion d’insuline chez les souris Arrb2 KO par rapport aux souris témoins sur les îlots en présence des concentrations physiologiques circulantes de GLP-1. Ceci est corrélé à une production d’AMPc et un recrutement de la PKA plus élevés dans les cellules beta Arrb2 KO. A l’inverse, l’activation des kinases ERK1/2 est diminuée indiquant un recrutement majeur des ERK1/2 par ARRB2 au GLP-1R. En parallèle, j’ai montré que les taux de ARRB1 et ARRB2 des îlots pancréatiques sont altérés par des conditions diabétogènes et diabétiques. Mes résultats démontrent clairement un rôle critique de ARRB2 dans la signalisation du GLP-1R. Un défaut d’expression de la protéine pourrait participer au déficit des mécanismes de compensation de la masse fonctionnelle des cellules beta conduisant au DT2. / The loss of function and mass of pancreatic beta-cells play a central role in type 2 diabetes (T2D). Beta-arrestin 1 and 2 (ARRB1 and ARRB2) are involved in insulin secretion and/or beta-cell survival. In a first study, in order to characterize the role of ARRB1 in beta-cells, we aimed to invalidate the Arrb1 gene specifically in these cells using the Cre/lox system under the control of the Ins1 promoter. Studies had been published with both Ins1Cre-/+ and Arrb1f/f lines. We generated Arrb1f/f:Ins1Cre-/+ mice. The phenotype of Arrb1f/f :Ins1Cre-/+ mice was weak with a lack of reproducibility compared to Arrb1f/f :Ins1Cre-/- mice used as controls. The low expression level of Arrb1 in beta-cells and the lack of specific antibody for immunocytochemistry made it difficult to verify the absence of expression of ARRB1 in these cells. After sequencing the modified Arrb1 gene of the “floxed” mice, we observed that the insertion of the first loxP site induced a shift in the reading frame introducing a stop codon and, consequently, the non-expression of the Arrb1 gene. Since the “floxed“ Arrb1 mice used as controls were already knockout (KO), the project using these mice was stopped.Our team has reported the involvement of ARRB2 in the regulation of beta-cell mass, but its role in Glucagon-Like Peptide-1 (GLP-1) receptor signaling, a major therapeutic target for T2D, remained to be explored. In a second study, we showed a better glucose tolerance and an increase in insulin secretion from isolated islets in Arrb2KO compared to control mice in the presence of physiological circulating concentrations of GLP-1. This was correlated with higher cAMP production and PKA activation in Arrb2KO beta-cells. By contrast, the activation of ERK1/2 kinases was decreased indicating a major recruitment of ERK1/2 by ARRB2 to GLP-1R. In parallel, we showed that the expression levels of ARRB1 and ARRB2 in pancreatic islets were altered in diabetogenic and diabetic conditions. My results clearly demonstrate a critical role of ARRB2 in GLP-1R singaling which could impact the function, maintenance and plasticity of beta-cell mass in response to GLP-1. A lack of expression of ARRB2 could participate in the deficit of compensatory mechanisms of the functional beta-cell mass leading to T2D.

Notre travail de thèse avait pour objet l’étude du potentiel anti-diabétique des urolithines A, B, C et D, métabolites de polyphénols formés par le microbiote colique à partir des tanins de l’acide ellagique (présents notamment dans la grenade et les noix).La première partie, bibliographique, constitue un rappel :• de la régulation de l’équilibre glycémique et le rôle de la sécrétion d’insuline dans cette régulation ; •de l ‘épidémiologie et la physiopathologie du diabète type 2 (DT2) ; •des polyphénols et leurs métabolites, ainsi que de leurs effets antidiabétiques potentiels.La seconde partie décrit les effets des urolithines sur différents modèles expérimentaux : •Sur un modèle de cellules β insulino-sécrétrices (lignée INS-1), les urolithines induisent une amplification concentration-dépendante de la sécrétion d’insuline induite par le glucose, mais également par d’autres sécrétagogues comme un analogue du GLP-1 ou une sulfonylurée (médicaments utilisés dans le diabète). Les urolithines préviennent également l’altération sécrétoire induite par un stress oxydant. •L’effet insulino-sécrétoire des urolithines a été confirmé sur îlots de Langerhans isolés. •L’urolithine C étant apparu comme le composé le plus prometteur, nous avons poursuivi la caractérisation de son activité sur un modèle ex vivo mimant la situation physiologique, le pancréas isolé perfusé. Alors que l’effet sécrétoire de l’urolithine C n’apparaît pas en présence de 5mM de glucose, l’urolithine C (20µM) a stimulé la sécrétion d’insuline dans des conditions de stimulation modérée de la sécrétion d’insuline par le glucose (8.3mM). Cet effet est strictement dépendant du glucose, la sécrétion d’insuline retournant immédiatement à son niveau basal lors du passage de 8,3 à 5mM de glucose en présence d’urolithine C. •Des études de pharmacocinétique ont permis de mettre au point une méthodologie de dosage plasmatique de l’urolithine C dans le plasma de rat par chromatographie liquide / ionisation electrospray /spectrographie de masse en tandem. Cette méthodologie a été appliquée à une première étude pharmacocinétique chez le rat après injection de 10mg/kg d’urolithine C par voie intra-péritonéale. Cette étude montre notamment que le profil pharmacocinétique suit un modèle à 3 compartiments et suggère un stockage tissulaire du composé.D’autres résultats (confidentiels) ne peuvent être évoqués dans ce résumé mais confirment l’intérêt potentiel de l’urolithine C dans le traitement du diabète de type 2 en tant que médicament insulinotrope dépendant du glucose. / The objective of our thesis was to study the anti-diabetic potential of metabolites of ellagic acid tanins, present notably in pomegranate and nuts, that are formed by the colon microbiote. The metabolites are urolithins A, B, C and D.The first part of thesis is bibliographic and reviews: •The control of glycemic plasma levels, and in particular the role of insulin secretion in this process; • The pathophysiology of Type 2 Diabetes (T2D); •The various polyphenols and their metabolites, along with their potential anti-diabetic activity.The second part describes the effects of urolithins on various experimental models: •On a model of insulin secreting beta cells (the INS-1cell line), urolithins concentration-dependently amplified insulin secretion induced by glucose, but also by insulinotropic drugs used in the treatment of T2D such as a GLP-1 analogue or a sulfonylurea. In addition, urolithins were able to induce insulin secretion on cells rendered unresponsive to glucose by oxidative stress. • The insulinotropic effect of urolithins was also confirmed on isolated rat islets of Langerhans. •As urolithin C appeared to be the most promising antidiabetic compound, we further characterized its activity on an ex vivo model mimicking the physiological situation, the isolated infused pancreas. While urolithin C (20µM) had no effect in the presence of 5 mM glucose concentration, it amplified the stimulation of insulin secretion in the presence of 8.3mM glucose. The effect of urolithin C was also strictly glucose-dependent, as insulin secretion immediately returned to basal level when glucose concentration was switched from 8.3 to 5mM glucose in the presence of urolithin C. •We also conducted studies aiming at designing a validated methodology for rat plasma urolithin C determination using a liquid chromatography-electrospray ionization-tandem mass spectrometry method. The applicability of this assay was demonstrated in a preclinical pharmacokinetic study carried out in rats receiving intraperitoneal administration of urolithin C (10mg/kg). We found that the urolithin C followed a three-compartment model, suggesting a long-term tissue storage of urolithin C.Some other (confidential) results, not described in this abstract, confirmed urolithin C as a potential glucose-dependent insulinotropic treatment for type 2 diabetes.