Mechanisms of Beta-Cell Apoptosis in Type 2 Diabetes-Prone Situations and Potential Protection by GLP-1-Based Therapies.

Type 2 diabetes (T2D) is characterized by chronic hyperglycemia secondary to the decline of functional beta-cells and is usually accompanied by a reduced sensitivity to insulin. Whereas altered beta-cell function plays a key role in T2D onset, a decreased beta-cell mass was also reported to contribute to the pathophysiology of this metabolic disease. The decreased beta-cell mass in T2D is, at least in part, attributed to beta-cell apoptosis that is triggered by diabetogenic situations such as amyloid deposits, lipotoxicity and glucotoxicity. In this review, we discussed the molecular mechanisms involved in pancreatic beta-cell apoptosis under such diabetes-prone situations. Finally, we considered the molecular signaling pathways recruited by glucagon-like peptide-1-based therapies to potentially protect beta-cells from death under diabetogenic situations.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

AIMS/HYPOTHESIS: Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. METHODS: NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. RESULTS: Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001). CONCLUSIONS/INTERPRETATION: ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.

ß-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice.

AIMS/HYPOTHESIS: Beta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of ß-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors. METHODS: ß-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca(2+)] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting. RESULTS: Arrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway. CONCLUSIONS/INTERPRETATION: ARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Frequency-dependent mitochondrial Ca(2+) accumulation regulates ATP synthesis in pancreatic ß cells.

Pancreatic ß cells respond to increases in glucose concentration with enhanced metabolism, the closure of ATP-sensitive K(+) channels and electrical spiking. The latter results in oscillatory Ca(2+) influx through voltage-gated Ca(2+) channels and the activation of insulin release. The relationship between changes in cytosolic and mitochondrial free calcium concentration ([Ca(2+)]cyt and [Ca(2+)]mit, respectively) during these cycles is poorly understood. Importantly, the activation of Ca(2+)-sensitive intramitochondrial dehydrogenases, occurring alongside the stimulation of ATP consumption required for Ca(2+) pumping and other processes, may exert complex effects on cytosolic ATP/ADP ratios and hence insulin secretion. To explore the relationship between these parameters in single primary ß cells, we have deployed cytosolic (Fura red, Indo1) or green fluorescent protein-based recombinant-targeted (Pericam, 2mt8RP for mitochondria; D4ER for the ER) probes for Ca(2+) and cytosolic ATP/ADP (Perceval) alongside patch-clamp electrophysiology. We demonstrate that: (1) blockade of mitochondrial Ca(2+) uptake by shRNA-mediated silencing of the uniporter MCU attenuates glucose- and essentially blocks tolbutamide-stimulated, insulin secretion; (2) during electrical stimulation, mitochondria decode cytosolic Ca(2+) oscillation frequency as stable increases in [Ca(2+)]mit and cytosolic ATP/ADP; (3) mitochondrial Ca(2+) uptake rates remained constant between individual spikes, arguing against activity-dependent regulation ("plasticity") and (4) the relationship between [Ca(2+)]cyt and [Ca(2+)]mit is essentially unaffected by changes in endoplasmic reticulum Ca(2+) ([Ca(2+)]ER). Our findings thus highlight new aspects of Ca(2+) signalling in ß cells of relevance to the actions of both glucose and sulphonylureas.

GLP-1 and GIP receptors signal through distinct ß-arrestin 2-dependent pathways to regulate pancreatic ß cell function.

Glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIPR) receptors are G-protein-coupled receptors involved in glucose homeostasis. Diabetogenic conditions decrease ß-arrestin 2 (ARRB2) levels in human islets. In mouse ß cells, ARRB2 dampens insulin secretion by partially uncoupling cyclic AMP (cAMP)/protein kinase A (PKA) signaling at physiological doses of GLP-1, whereas at pharmacological doses, the activation of extracellular signal-related kinase (ERK)/cAMP-responsive element-binding protein (CREB) requires ARRB2. In contrast, GIP-potentiated insulin secretion needs ARRB2 in mouse and human islets. The GIPR-ARRB2 axis is not involved in cAMP/PKA or ERK signaling but does mediate GIP-induced F-actin depolymerization. Finally, the dual GLP-1/GIP agonist tirzepatide does not require ARRB2 for the potentiation of insulin secretion. Thus, ARRB2 plays distinct roles in regulating GLP-1R and GIPR signaling, and we highlight (1) its role in the physiological context and the possible functional consequences of its decreased expression in pathological situations such as diabetes and (2) the importance of assessing the signaling pathways engaged by the agonists (biased/dual) for therapeutic purposes.

The nuclear receptor REV-ERBα is implicated in the alteration of ß-cell autophagy and survival under diabetogenic conditions.

Pancreatic ß-cell failure in type 2 diabetes mellitus (T2DM) is associated with impaired regulation of autophagy which controls ß-cell development, function, and survival through clearance of misfolded proteins and damaged organelles. However, the mechanisms responsible for defective autophagy in T2DM ß-cells remain unknown. Since recent studies identified circadian clock transcriptional repressor REV-ERBα as a novel regulator of autophagy in cancer, in this study we set out to test whether REV-ERBα-mediated inhibition of autophagy contributes to the ß-cell failure in T2DM. Our study provides evidence that common diabetogenic stressors (e.g., glucotoxicity and cytokine-mediated inflammation) augment ß-cell REV-ERBα expression and impair ß-cell autophagy and survival. Notably, pharmacological activation of REV-ERBα was shown to phenocopy effects of diabetogenic stressors on the ß-cell through inhibition of autophagic flux, survival, and insulin secretion. In contrast, negative modulation of REV-ERBα was shown to provide partial protection from inflammation and glucotoxicity-induced ß-cell failure. Finally, using bioinformatic approaches, we provide further supporting evidence for augmented REV-ERBα activity in T2DM human islets associated with impaired transcriptional regulation of autophagy and protein degradation pathways. In conclusion, our study reveals a previously unexplored causative relationship between REV-ERBα expression, inhibition of autophagy, and ß-cell failure in T2DM.

Metabolic Stress Impairs Pericyte Response to Optogenetic Stimulation in Pancreatic Islets.

Pancreatic islets are highly vascularized micro-organs ensuring whole body glucose homeostasis. Islet vascular cells play an integral part in sustaining adequate insulin release by beta cells. In particular, recent studies have demonstrated that islet pericytes regulate local blood flow velocity and are required for maintenance of beta cell maturity and function. In addition, increased metabolic demand accompanying obesity alters islet pericyte morphology. Here, we sought to explore the effects of metabolic stress on islet pericyte functional response to stimulation in a mouse model of type 2 diabetes, directly in the pancreas in vivo . We found that high fat diet induced islet pericyte hypertrophy without alterations in basal local blood flow. However, optogenetic stimulation of pericyte activity revealed impaired islet vascular responses, despite increased expression of genes encoding proteins directly or indirectly involved in cell contraction. These findings suggest that metabolic stress impinges upon islet pericyte function, which may contribute to beta cell failure during T2D.

Glucose controls cytosolic Ca2+ and insulin secretion in mouse islets lacking adenosine triphosphate-sensitive K+ channels owing to a knockout of the pore-forming subunit Kir6.2.

Glucose-induced insulin secretion is classically attributed to the cooperation of an ATP-sensitive potassium (K ATP) channel-dependent Ca2+ influx with a subsequent increase of the cytosolic free Ca2+ concentration ([Ca2+]c) (triggering pathway) and a K ATP channel-independent augmentation of secretion without further increase of [Ca2+]c (amplifying pathway). Here, we characterized the effects of glucose in beta-cells lacking K ATP channels because of a knockout (KO) of the pore-forming subunit Kir6.2. Islets from 1-yr and 2-wk-old Kir6.2KO mice were used freshly after isolation and after 18 h culture to measure glucose effects on [Ca2+]c and insulin secretion. Kir6.2KO islets were insensitive to diazoxide and tolbutamide. In fresh adult Kir6.2KO islets, basal [Ca2+]c and insulin secretion were marginally elevated, and high glucose increased [Ca2+]c only transiently, so that the secretory response was minimal (10% of controls) despite a functioning amplifying pathway (evidenced in 30 mm KCl). Culture in 10 mm glucose increased basal secretion and considerably improved glucose-induced insulin secretion (200% of controls), unexpectedly because of an increase in [Ca2+]c with modulation of [Ca2+]c oscillations. Similar results were obtained in 2-wk-old Kir6.2KO islets. Under selected conditions, high glucose evoked biphasic increases in [Ca2+]c and insulin secretion, by inducing K ATP channel-independent depolarization and Ca2+ influx via voltage-dependent Ca2+ channels. In conclusion, Kir6.2KO beta-cells down-regulate insulin secretion by maintaining low [Ca2+]c, but culture reveals a glucose-responsive phenotype mainly by increasing [Ca2+]c. The results support models implicating a K ATP channel-independent amplifying pathway in glucose-induced insulin secretion, and show that K ATP channels are not the only possible transducers of metabolic effects on the triggering Ca2+ signal.

Imaging a target of Ca2+ signalling: dense core granule exocytosis viewed by total internal reflection fluorescence microscopy.

Ca2+ ions are the most ubiquitous second messenger found in all cells, and play a significant role in controlling regulated secretion from neurons, endocrine, neuroendocrine and exocrine cells. Here, we describe microscopic techniques to image regulated secretion, a target of Ca2+ signalling. The first of these, total internal reflection fluorescence (TIRF), is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation (<150 nm). It is thus particularly useful for studies of regulated hormone secretion. A brief summary of this approach is provided, as well as a description of the physical basis for the technique and the tools to implement TIRF using a standard fluorescence microscope. We also detail the different fluorescent probes which can be used to detect secretion and how to analyze the data obtained. A comparison between TIRF and other imaging modalities including confocal and multiphoton microscopy is also included.

Methods to Study Roles of ß-Arrestins in the Regulation of Pancreatic ß-Cell Function.

Novel findings reveal important functional roles for ß-arrestin 1 and ß-arrestin 2 in the regulation of insulin secretion, ß-cell survival, and ß-cell mass plasticity not only by glucose but also by G-protein-coupled receptors, such as the glucagon-like peptide-1 (GLP-1) and the pituitary adenylate cyclase-activating polypeptide (PACAP) receptors or GPR40, or tyrosine kinase receptors, such as the insulin receptor. Here, we describe experimental protocols to knock down ß-arrestins by small interference RNA, to follow subcellular localization of ß-arrestins in the cytosol and nucleus of the insulinoma INS-1E rat pancreatic ß-cell line, and to analyze ß-arrestin protein expression by Western blot using INS-1E cells and isolated mouse or human pancreatic islets. We also provide details on how to genotype ß-arrestin 2 knockout (Arrb2-/-) mice and to evaluate ß-arrestin-mediated roles in ß-cell mass plasticity and ß-cell signaling using immunocytochemistry on pancreatic sections or on primary dispersed ß-cells from wild-type mice and Arrb2-/- mice.

Glucose-dependent regulation of gamma-aminobutyric acid (GABA A) receptor expression in mouse pancreatic islet alpha-cells.

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, gamma-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABA(A) receptor (GABA(A)R) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABA(A)R alpha4, beta3, and gamma2 subunit mRNAs were detected in mouse islets and clonal alphaTC1-9 cells, and immunocytochemistry confirmed the presence of GABA(A)Rs at the plasma membrane of primary alpha-cells. Glucose dose-dependently increased GABA(A)R expression in both islets and alphaTC1-9 cells such that mRNA levels at 16 mmol/l glucose were approximately 3.0-fold (alpha4), 2.0-fold (beta3), or 1.5-fold (gamma2) 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 Ca(2+) channel blocker nimodipine. We conclude that 1) release of GABA from neighboring beta-cells contributes substantially to the acute inhibition of glucagon secretion from mouse islets by glucose and 2) that changes in GABA(A)R expression, mediated by changes in intracellular free Ca(2+) concentration, may modulate this response in the long term.

Proteasomal degradation of the histone acetyl transferase p300 contributes to beta-cell injury in a diabetes environment.

In type 2 diabetes, amyloid oligomers, chronic hyperglycemia, lipotoxicity, and pro-inflammatory cytokines are detrimental to beta-cells, causing apoptosis and impaired insulin secretion. The histone acetyl transferase p300, involved in remodeling of chromatin structure by epigenetic mechanisms, is a key ubiquitous activator of the transcriptional machinery. In this study, we report that loss of p300 acetyl transferase activity and expression leads to beta-cell apoptosis, and most importantly, that stress situations known to be associated with diabetes alter p300 levels and functional integrity. We found that proteasomal degradation is the mechanism subserving p300 loss in beta-cells exposed to hyperglycemia or pro-inflammatory cytokines. We also report that melatonin, a hormone produced in the pineal gland and known to play key roles in beta-cell health, preserves p300 levels altered by these toxic conditions. Collectively, these data imply an important role for p300 in the pathophysiology of diabetes.

Sustained exposure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic beta-cells.

The mechanism(s) by which chronic hyperglycemia impairs glucose-stimulated insulin secretion is poorly defined. Here, we compare the "nanomechanics" of single exocytotic events in primary rat pancreatic beta-cells cultured for 48 h at optimal (10 mmol/l) or elevated (30 mmol/l) glucose concentrations. Cargo release was imaged by total internal reflection fluorescence microscopy of lumen-targeted probes (neuropeptide Y [NPY]-pH-insensitive yellow fluorescent protein [NPY-Venus] or NPY-monomeric red fluorescent protein), while the fate of the vesicle membrane was reported simultaneously with phosphatase-on-the-granule-of-insulinoma-enhanced green fluorescent protein. Under all conditions studied, exocytosis proceeded via a "cavity recapture" mechanism in which the vesicle and plasma membranes fused transiently. While essentially complete release of NPY-Venus was observed in 24 +/- 1% of glucose-stimulated exocytotic events in cells maintained at 10 mmol/l glucose, this value was reduced reversibly to 5 +/- 2% of events by culture at 30 mmol/l glucose, in line with decreases in Glut2 and glucokinase gene expression, and attenuated glucose-stimulated increases in NADPH and intracellular [Ca2+]. Since vesicle release in response to cell depolarization with KCl was not affected by culture at 30 mmol/l glucose, we conclude that hyperglycemia causes the abnormal termination of individual insulin release events principally by inhibiting glucose signaling.

Ca2+ microdomains and the control of insulin secretion.

Nutrient-induced increases in intracellular free Ca(2+) concentrations are the key trigger for insulin release from pancreatic islet beta-cells. These Ca(2+) changes are tightly regulated temporally, occurring as Ca(2+) influx-dependent baseline oscillations. We explore here the concept that locally high [Ca(2+)] concentrations (i.e. Ca(2+) microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca(2+) indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca(2+) microdomains in beta-cells. We summarise here some of the evidence indicating that Ca(2+) microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to "ATP-sensitive K(+)-channel independent" effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca(2+) from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca(2+) uptake in beta-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca(2+) are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process.

Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic alpha-cells.

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 beta-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) alpha-cells within the intact mouse islet, 2) dissociated alpha-cells, and 3) clonal alphaTC1-9 cells. Free cytosolic concentrations of ATP ([ATP](c)) or Ca(2+) ([Ca(2+)](c)) were imaged using alpha-cell-targeted firefly luciferase or a green fluorescent protein-based Ca(2+) probe ("pericam"), respectively. Consistent with a direct effect of glucose on alpha-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 alpha-cells within the intact islet and by approximately 4% in alphaTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca(2+)](c) oscillations in both dissociated alpha-cells and alphaTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl(2) slightly increased the frequency of [Ca(2+)](c) oscillations but failed to affect glucagon release from either islets or alphaTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn(2+) ions, independently suppress glucagon secretion in the mouse.

Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release.

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-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 beta-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 ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.

Control mechanisms of the oscillations of insulin secretion in vitro and in vivo.

The mechanisms driving the pulsatility of insulin secretion in vivo and in vitro are still unclear. Because glucose metabolism and changes in cytosolic free Ca(2+) ([Ca(2+)](c)) in beta-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 beta-cells, pulsatile insulin secretion could theoretically result from [Ca(2+)](c) or metabolism oscillations. We could not detect metabolic oscillations independent from [Ca(2+)](c) changes in beta-cells, and imposed metabolic oscillations were poorly effective in inducing oscillations of secretion when [Ca(2+)](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 [Ca(2+)](c) and insulin release strongly suggest that [Ca(2+)](c) oscillations are the direct drivers of insulin secretion oscillations. Metabolism may play a dual role, inducing [Ca(2+)](c) oscillations (via changes in ATP-sensitive K(+) channel activity and membrane potential) and amplifying the secretory response by increasing the efficiency of Ca(2+) on exocytosis. The mechanisms underlying the oscillations of insulin secretion by the isolated pancreas and those observed in vivo remain elusive. It is not known how the functioning of distinct islets is synchronized, and the possible role of intrapancreatic ganglia in this synchronization requires confirmation. That pulsatile insulin secretion is beneficial in vivo, by preventing insulin resistance, is suggested by the greater hypoglycemic effect of exogenous insulin when it is infused in a pulsatile rather than continuous manner. The observation that type 2 diabetic patients have impaired pulsatile insulin secretion has prompted the suggestion that such dysregulation contributes to the disease and justifies the efforts toward understanding of the mechanism underlying the pulsatility of insulin secretion both in vitro and in vivo.

Do oscillations of insulin secretion occur in the absence of cytoplasmic Ca2+ oscillations in beta-cells?

That oscillations of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in beta-cells induce oscillations of insulin secretion is not disputed, but whether metabolism-driven oscillations of secretion can occur in the absence of [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i) and insulin secretion in response to repetitive depolarizations with 30 mmol/l K(+) in the presence of 0.1 mmol/l diazoxide and 12 mmol/l glucose. Continuous depolarization with high K(+) steadily elevated [Ca(2+)](i) in all types of islets, with no significant oscillation, and caused a biphasic insulin response. In islets from young (4-month-old) NMRI mice and 7-month-old lean mice, the insulin profile did not show significant peaks when [Ca(2+)](i) was stable. In contrast, two or more peaks were detected over 20 min in the response of most ob/ob islets. Similar insulin peaks appeared in the insulin response of 10-month-old lean and NMRI mice. However, the size of the insulin peaks detected in the presence of stable [Ca(2+)](i) was small, so that no more than 10-13% of total insulin secretion occurred in a pulsatile manner. In conclusion, insulin secretion does not oscillate when [Ca(2+)](i) is stably elevated in beta-cells from young normal mice. Some oscillations are observed in aged mice and are seen more often in ob/ob islets. These fluctuations of the insulin secretion rate at stably elevated [Ca(2+)](i), however, are small compared with the large oscillations induced by [Ca(2+)](i) oscillations in beta-cells.

Signals and pools underlying biphasic insulin secretion.

Rapid and sustained stimulation of beta-cells with glucose induces biphasic insulin secretion. The two phases appear to reflect a characteristic of stimulus-secretion coupling in each beta-cell rather than heterogeneity in the time-course of the response between beta-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 Ca(2+) concentration ([Ca(2+)](i)) induced by glucose is important to shape the two phases of secretion. The first phase requires a rapid and marked elevation of [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i). The species characteristics of biphasic insulin secretion and its perturbations in pathological situations are discussed.

Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1.

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 a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.