Introduction Parmi les mecanismes impliques dans la regulation des genes controlant la fonction et la survie des cellules beta, le remodelage de la chromatine par des mecanismes epigenetiques, tels que l'acetylation des histones, est... more
Introduction Parmi les mecanismes impliques dans la regulation des genes controlant la fonction et la survie des cellules beta, le remodelage de la chromatine par des mecanismes epigenetiques, tels que l'acetylation des histones, est un processus fondamental. L'histone acetyl-transferase p300 est un activateur cle de la machinerie transcriptionnelle. L'objectif de cette etude est d'evaluer les modes de regulation de p300 et son impact « epigenetique » vis-a-vis d'un programme de differenciation/survie beta-cellulaire dans des conditions physiologiques et dans des situations physiopathologiques plus particulierement liees a la proteotoxicite induite par la proteine amyloide de l'ilot. Materiels et Methodes Les experiences sont realisees avec des cellules beta en culture (lignee INS-1E), des ilots pancreatiques isoles et des sections pancreatiques de souris [souris controles vs souris transgeniques exprimant la forme humaine toxique du polypeptide amyloide de l'ilot (h-TG)]. Les partenaires proteiques de p300 sont identifies par immunoprecipitation. Les niveaux proteiques de p300 sont evalues par western-blot et immunofluorescence. L'apoptose est mise en evidence par l'activation/le clivage de la caspase-3. Resultats L'invalidation de p300 par siRNA entraine l'apoptose des cellules beta. Parmi les mecanismes impliques dans le role anti-apoptotique de p300, nous montrons qu'une stimulation aigue par le glucose et/ou le GLP-1 favorise l'interaction de p300 avec la forme phosphorylee/active de CREB (cAMP-Responsive Element Binding protein), un facteur de transcription crucial pour la survie des cellules beta. De maniere interessante, les niveaux nucleaires de p300 evalues par fractionnement subcellulaire et immunofluorescence, sont diminues dans les cellules beta de souris h-TG, un modele murin de proteotoxicite recapitulant les deficits beta-cellulaires du diabete de type 2 humain. Conclusions Cette etude revele pour la premiere fois l'implication de la fonction histone acetyl-transferase p300 dans la survie beta-cellulaire, ainsi que son alteration dans les cellules beta soumises a un environnement diabetogene.
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Introduction Recemment, nous avons rapporte l'implication de la β-arrestine2 (ARRB2), une proteine d'echafaudages moleculaires, dans la regulation de la masse des cellules β pancreatiques et identifie un defaut de la signalisation... more
Introduction Recemment, nous avons rapporte l'implication de la β-arrestine2 (ARRB2), une proteine d'echafaudages moleculaires, dans la regulation de la masse des cellules β pancreatiques et identifie un defaut de la signalisation insulinique. L'insuline exerce des effets autocrines via son propre recepteur dont le couplage a la voie PI3K/Akt joue un role majeur dans le maintien de la masse des cellules β, notamment par la phosphorylation du facteur de transcription FoxO1, conduisant a son inhibition par exclusion nucleaire. Notre etude vise a determiner le(s) mecanisme(s) moleculaire(s) impliquant ARRB2 dans la signalisation insulinique dans la cellule β. Materiels et Methodes Les experiences sont realisees dans la lignee INS-1E et les cellules β de souris (controles ou Arrb2-/-). Les niveaux d'expression, de phosphorylation ou la distribution subcellulaire des proteines sont mesures par western blot et/ou immunocytochimie. Resultats Dans les cellules INS-1E, l'insuline active Akt par un mecanisme dependant non seulement de la PI3K (inhibition pharmacologique, wortmanine) comme attendu, mais egalement de Src (inhibition pharmacologique, PP2) et de ARRB2 (SiRNA). Ce processus d'activation est associe a une interaction rapide et durable de ARRB2 avec le recepteur de l'insuline, et les kinases Src et Akt. Dans les cellules β de souris controles, l'inhibition de Src reduit le niveau de phosphorylation et d'exclusion nucleaire de FoxO1 en reponse a l'insuline. Dans les cellules β de souris Arrb2-/-, la reduction de l'activation d'Akt par l'insuline est egalement correlee a la sequestration nucleaire de FoxO1. En revanche, la reexpression de ARRB2 par infection adenovirale, retablit l'exclusion nucleaire de Foxo1 par l'insuline. De facon interessante, l'expression de ARRB2 est reduite dans les cellules β dans des conditions d'insulinoresistance in vitro (palmitate) et in vivo (regime gras, souris diabetiques db/db). Conclusions Notre etude revele un nouveau mecanisme moleculaire dans la signalisation insulinique impliquant ARRB2 dans l'echafaudage d'un complexe comprenant le recepteur, Src et Akt pour une pleine activation d'Akt dans les cellules β. Un defaut d'expression de ARRB2 dans les etats d'insulinoresistance pourrait participer au deficit de la signalisation insulinique dans la cellule β lors du diabete de type 2.
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Introduction Les kinases ERK1/2 sont connues pour etre impliquees dans la survie des cellules β et pourraient reguler la secretion d'insuline. Neanmoins le role specifique de chaque kinase n'a jamais ete etudie dans les cellules... more
Introduction Les kinases ERK1/2 sont connues pour etre impliquees dans la survie des cellules β et pourraient reguler la secretion d'insuline. Neanmoins le role specifique de chaque kinase n'a jamais ete etudie dans les cellules β. Le but de cette etude est de determiner le role de ERK1 en utilisant des souris ERK1-/-. Materiels et methodes La [Ca 2 + ] c et la secretion d'insuline ont ete mesurees sur des ilots perifuses. La masse des ilots a ete quantifiee sur coupes histologiques. Les niveaux d'expression et l'activation des kinases ont ete mesures par western blot et immunocytochimie. Resultats In vivo , les souris ERK1–/– presentent des glycemies comparables aux souris ERK1 +/+. In vitro , les changements de la [Ca 2 + ] c et de la secretion d'insuline en reponse au glucose sont similaires dans les ilots ERK1 +/+ et ERK1–/–. En revanche l'analyse morphometrique des coupes de pancreas de souris ERK1–/– âgees de 5 mois, a montre un nombre comparable mais un appauvrissement en petits ilots et un enrichissement en gros ilots en comparaison des souris ERK1 +/+. Chez les animaux plus âges (10 mois) ce decalage de taille ne s'observe plus. Neanmoins, les souris ERK1–/– semblent protegees de l'inflammation pancreatique liee a l'âge. L'absence de ERK1 n'a pas ete com-pensee par ERK2 (messager, proteine et activation). Par contre les activations de MSK1 ( p p p p Conclusion La kinase ERK1 semble jouer un role dans la regulation de la masse des cellules s mais pas dans la regulation de la secretion d'insuline. Declaration d’interet Les auteurs declarent ne pas avoir d'interet direct ou indirect (financier ou en nature) avec un organisme prive, industriel ou commercial en relation avec le sujet presente.
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The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS)... more
The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as...
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Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 −/− )... more
Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout ( ZnT8 −/− ) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8 −/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8 +/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8 −/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging...
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Research Interests: Endocrinology, Fluorescence Microscopy, Biology, Calcium, Immunohistochemistry, and 13 moreConfocal Microscopy, Medicine, Internal Medicine, Glucose, Insulin, Mice, Female, Animals, Clinical Sciences, Public health systems and services research, Beta Cell, Paediatrics and reproductive medicine, and In Vitro Techniques
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ABSTRACT Introduction La résistance à l’insuline au cours de l’obésité induit une augmentation compensatoire de la masse des cellules β pancréatiques. Nous avons récemment montré que la β-arrestine-2 (β-arr2), protéine d’échafaudage, est... more
ABSTRACT Introduction La résistance à l’insuline au cours de l’obésité induit une augmentation compensatoire de la masse des cellules β pancréatiques. Nous avons récemment montré que la β-arrestine-2 (β-arr2), protéine d’échafaudage, est requise dans la signalisation insulinique connue pour réguler la masse fonctionnelle des cellules β. Notre but est d’évaluer le rôle de la β-arr2 dans l’expansion de la masse des cellules β induite par un régime riche en graisses (HFD), en utilisant des souris invalidées pour la β-arr2 (β-arr2-/-). Matériels et méthodes Les expériences ont été réalisées sur des souris mâles β-arr2+/+ et β-arr2-/-. Les animaux ont été nourris par un régime standard (5 % kcal % graisse) ou par un HFD (45 % kcal % graisse) pendant 19 semaines. La prolifération (anti-Ki67) et la masse des cellules β (anti-insuline) ont été évaluées par immunofluorescence. Résultats Sous régime standard, les souris β-arr2-/- présentent une architecture normale des îlots mais une masse réduite des cellules β (40 %). Sous HFD, les souris β-arr2-/- montrent une surcharge pondérale comparable aux β-arr2+/+ mais une hyperinsulinémie compensatoire déficiente. De plus, alors que les souris β-arr2+/+ présentent un doublement de la masse des cellules β associée à une augmentation de la prolifération, les souris β-arr2-/- sont incapables de présenter cette adaptation compensatrice. De manière intéressante, l’expression de la β-arr2 dans les cellules β est réduite dans des conditions d’insulino-résistance induites par le palmitate (in vitro) et l’obésité (in vivo). Conclusion La β-arr2 est impliquée dans la plasticité de la masse des cellules β, non seulement en condition normale, mais également dans l’expansion compensatoire en réponse au régime gras. Un défaut d’expression de cette protéine dans les états d’insulino-résistance pourrait participer au déficit de l’expansion compensatoire de la masse des cellules β survenant au cours du diabète de type 2.
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ABSTRACT Introduction Le glucose stimule la sécrétion d’insuline (SI) en élevant la [Ca2+] dans le cytosol des cellules β (voie déclenchante) et en augmentant l’efficacité du Ca2+ sur l’exocytose (voie amplificatrice). Le concept... more
ABSTRACT Introduction Le glucose stimule la sécrétion d’insuline (SI) en élevant la [Ca2+] dans le cytosol des cellules β (voie déclenchante) et en augmentant l’efficacité du Ca2+ sur l’exocytose (voie amplificatrice). Le concept d’amplification repose sur l’observation qu’à [Ca2+]c élevée et stable, le glucose augmente la SI sans augmenter davantage la [Ca2+]c. Toutefois, ces mesures globales de [Ca2+]c n’excluent pas la possibilité qu’une élévation de la [Ca2+] sous la membrane plasmique seulement ([Ca2+]SM) explique l’effet amplificateur du glucose. C’est l’hypothèse que nous avons testée. Matériels et Méthodes La [Ca2+]SM a été mesurée par « Total Internal Reflection fluorescence Microscopy » (TIRF) après expression adenovirale du rapporteur D3cpv (cytosolique) dans des amas de cellules β de souris ou du LynD3cpv (membranaire) dans les cellules β d’îlots intacts (stratégie Rip-Cre-Lox). Résultats L’expression du D3cpv n’a pas altéré les changements de [Ca2+] et de SI dans les amas de cellules β. L’effet amplificateur a été étudié en présence de diazoxide (pour ouvrir les canaux KATP) et 30 mM KCl (pour stimuler un influx de Ca2+). Dans ces conditions, une augmentation de glucose a abaissé la [Ca2+]SM mais doublé la SI. Une inhibition de la capture du Ca2+ par le réticulum endoplasmique a atténué la baisse de [Ca2+]SM produite par 15 mM glucose mais n’a pas démasqué d’augmentation, même localisée, de la [Ca2+]SM. En revanche, des augmentations significatives de la [Ca2+]SM ont été détectées lors de changements répétitifs de la concentration de KCl entre 30 et 32-35 mM, attestant de la sensibilité adéquate de notre système de détection. Des résultats similaires ont été obtenus dans les cellules β d’îlots intacts. Conclusion L’amplification de la SI par le glucose n’est pas due à une augmentation sous-membranaire de la [Ca2+] dans la cellule β et peut donc être attribuée à une augmentation de l’action du [Ca2+] sur l’exocytose.
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Normal insulin secretion requires the coordinated functioning of β-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of β-cells with extracellular signals and neighboring... more
Normal insulin secretion requires the coordinated functioning of β-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of β-cells with extracellular signals and neighboring cells. In particular, adjacent β-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36−/−) featured β-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36−/− mice did not display the regular oscillations of intracellular calcium concentrations ([Ca2+]i) seen in controls due to the loss of cell-to-cell synchronization of [Ca2+]i changes. The same islets did no...
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The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by β-cell secretory products is unresolved.... more
The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by β-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) α-cells within the intact mouse islet, 2) dissociated α-cells, and 3) clonal αTC1-9 cells. Free cytosolic concentrations of ATP ([ATP]c) or Ca2+ ([Ca2+]c) were imaged using α-cell–targeted firefly luciferase or a green fluorescent protein–based Ca2+ probe (“pericam”), respectively. Consistent with a direct effect of glucose on α-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP]c by 7–9% in α-cells within the intact islet and by ∼4% in αTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca2+]c oscillations in both dissociated α-cells and αTC1-9 cells. Although the effects of glucose were mimicked by exogenous insul...
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Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets... more
Glucose-induced insulin secretion depends on an acceleration of glucose metabolism, requires a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i), and is modulated by activation of protein kinases in beta-cells. Normal mouse islets were used to determine whether oscillations of these three signals are able and necessary to trigger oscillations of insulin secretion. The approach was to minimize or abolish spontaneous oscillations and to compare the impact of forced oscillations of each signal on insulin secretion. In a control medium, repetitive increases in the glucose concentration triggered oscillations in metabolism [NAD(P)H fluorescence], [Ca2+]i (fura-PE3 method), and insulin secretion. In the presence of diazoxide, metabolic oscillations persisted, but [Ca2+]i and insulin oscillations were abolished. When the islets were depolarized with high K+ with or without diazoxide, [Ca2+]i was elevated, and insulin secretion was stimulated. Forced metabolic oscillations transient...
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Rapid and sustained stimulation of β-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each β-cell rather than heterogeneity in the time-course of... more
Rapid and sustained stimulation of β-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each β-cell rather than heterogeneity in the time-course of the response between β-cells or islets. There is no evidence indicating that biphasic secretion can be attributed to an intrinsically biphasic metabolic signal. In contrast, the biphasic rise in cytoplasmic Ca2+ concentration ([Ca2+]i) induced by glucose is important to shape the two phases of secretion. The first phase requires a rapid and marked elevation of [Ca2+]i and corresponds to the release of insulin granules from a limited pool. The magnitude of the second phase is determined by the elevation of [Ca2+]i, but its development requires production of another signal. This signal corresponds to the amplifying action of glucose and may serve to replenish the pool of granules that are releasable at the prevailing [Ca2+]i. The species characteristics...
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That oscillations of the cytoplasmic free Ca2+ concentration ([Ca2+]i) in β-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca2+]i... more
That oscillations of the cytoplasmic free Ca2+ concentration ([Ca2+]i) in β-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca2+]i oscillations is still debated. Because this possibility is based partly on the results of experiments using islets from aged, hyperglycemic, hyperinsulinemic ob/ob mice, we compared [Ca2+]i and insulin secretion patterns of single islets from 4- and 10-month-old, normal NMRI mice to those of islets from 7- and 10-month- old ob/ob mice (Swedish colony) and their lean littermates. The responses were subjected to cluster analysis to identify significant peaks. Control experiments without islets and with a constant insulin concentration were run to detect false peaks. Both ob/ob and NMRI islets displayed large synchronous oscillations of [Ca2+]i and insulin secretion in response to repetitive depolarizations with 30 mmol/l K+ in the presence of 0.1 mmol/l dia...
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The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca2+ ([Ca2+]c) in β-cells play a key role in the control of insulin secretion,... more
The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca2+ ([Ca2+]c) in β-cells play a key role in the control of insulin secretion, and because oscillations of these two factors have been observed in single isolated islets and β-cells, pulsatile insulin secretion could theoretically result from [Ca2+]c or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca2+]c changes in β-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca2+]c was kept stable, which suggests that metabolic oscillations are not the direct regulator of the oscillations of secretion. By contrast, tight temporal and quantitative correlations between the changes in [Ca2+]c and insulin release strongly suggest that [Ca2+]c oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role,...
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The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, γ-aminobutyric acid (GABA) inhibited glucagon release... more
The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, γ-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABAA receptor (GABAAR) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABAAR α4, β3, and γ2 subunit mRNAs were detected in mouse islets and clonal αTC1-9 cells, and immunocytochemistry confirmed the presence of GABAARs at the plasma membrane of primary α-cells. Glucose dose-dependently increased GABAAR expression in both islets and αTC1-9 cells such that mRNA levels at 16 mmol/l glucose were ∼3.0-fold (α4), 2.0-fold (β3), or 1.5-fold (γ2) higher than at basal glucose concentrations (2.5 or 1.0 mmol/l, respectively). These effects were mimicked by depolarizing concentrations of K+ and reversed by the L-type Ca2+ channel blocker nim...
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Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the... more
Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events (‘transient’ versus ‘full fusion’) are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 β-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with rou...
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OBJECTIVE Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of... more
OBJECTIVE Sarco-endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and SERCA3 pump Ca2+ in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca2+ concentration ([Ca2+]ER) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS β-Cell [Ca2+]ER of SERCA3+/+ and SERCA3−/− mice was monitored with an adenovirus encoding the low Ca2+-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca2+ concentration ([Ca2+]c) and [Ca2+]ER were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS Glucose elicited synchronized [Ca2+]ER and [Ca2+]c oscillations. [Ca2+]ER oscillations were smaller in SERCA3−/− than in SERCA3+/+ β-cells. Stimulating cell metabolism with various [glucose] in the presence of diazoxide induced a similar dose-dependent [Ca2+]ER rise in SERCA3+/+ and SERCA3−/− β-cells. In a Ca2+-free ...