Jean-Christophe Jonas

Hierarchy of the beta-cell signals controlling insulin secretion

The main function of pancreatic β cells is to synthesize and secrete insulin at appropriate rates to limit blood glucose fluctuations within a narrow range. Any alteration in β -cell functioning has a profound impact on glucose homeostasis: excessive secretion of insulin causes hypoglycaemia, and insufficient secretion leads to diabetes. It is therefore not surprising that insulin secretion is subject to very tight control. This control is primarily ensured by glucose itself but also involves an array of metabolic, neural, hormonal and sometimes pharmacological factors (Fig. 1). To integrate all these stimulatory and inhibitory influences, β cells rely on an astonishingly complex stimulus-secretion coupling. This review discusses how the hierarchy between two intracellular pathways, producing triggering and amplifying signals [1], optimizes adequate insulin secretion to changes in blood glucose concentration and enables the β cell to grade the numerous extracellular messages that it receives.

The molecular mechanisms of pancreatic β-cell glucotoxicity: Recent findings and future research directions

It is well established that regular physiological stimulation by glucose plays a crucial role in the maintenance of the β-cell differentiated phenotype. In contrast, prolonged or repeated exposure to elevated glucose concentrations both in vitro and in vivo exerts deleterious or toxic effects on the β-cell phenotype, a concept termed as glucotoxicity. Evidence indicates that the latter may greatly contribute to the pathogenesis of type 2 diabetes. Through the activation of several mechanisms and signaling pathways, high glucose levels exert deleterious effects on β-cell function and survival and thereby, lead to the worsening of the disease over time. While the role of high glucose-induced β-cell overstimulation, oxidative stress, excessive Unfolded Protein Response (UPR) activation, and loss of differentiation in the alteration of the β-cell phenotype is well ascertained, at least in vitro and in animal models of type 2 diabetes, the role of other mechanisms such as inflammation, O-GlcNacylation, PKC activation, and amyloidogenesis requires further confirmation. On the other hand, protein glycation is an emerging mechanism that may play an important role in the glucotoxic deterioration of the β-cell phenotype. Finally, our recent evidence suggests that hypoxia may also be a new mechanism of β-cell glucotoxicity. Deciphering these molecular mechanisms of β-cell glucotoxicity is a mandatory first step toward the development of therapeutic strategies to protect β-cells and improve the functional β-cell mass in type 2 diabetes.

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets.

Background. Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. Methods. Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. Results. After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2–3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. Conclusions. The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.

Influence of cell number on the characteristics and synchrony of Ca2+ oscillations in clusters of mouse pancreatic islet cells.

1 The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single cells and cell clusters of different sizes prepared from mouse pancreatic islets. 2 During stimulation with 15 mM glucose, 20 % of isolated cells were inert, whereas 80 % showed [Ca2+]i oscillations of variable amplitude, duration and frequency. Spectral analysis identified a major frequency of 0.14 min−1 and a less prominent one of 0.27 min−1. 3 In contrast, practically all clusters (2–50 cells) responded to glucose, and no inert cells were identified within the clusters. As compared to single cells, mean [Ca2+]i was more elevated, [Ca2+]i oscillations were more regular and their major frequency was slightly higher (but reached a plateau at ≈0.25 min−1). In some cells and clusters, faster oscillations occurred on top of the slow ones, between them or randomly. 4 Image analysis revealed that the regular [Ca2+]i oscillations were well synchronized between all cells of the clusters. Even when the Ca2+ response was irregular, slow and fast [Ca2+]i oscillations induced by glucose were also synchronous in all cells. 5 In contrast, [Ca2+]i oscillations resulting from mobilization of intracellular Ca2+ by acetylcholine were restricted to certain cells only and were not synchronized. 6 Heptanol and 18α‐glycyrrhetinic acid, two agents widely used to block gap junctions, altered glucose‐induced Ca2+ oscillations, but control experiments showed that they also exerted effects other than a selective uncoupling of the cells. 7 The results support theoretical models predicting an increased regularity of glucose‐dependent oscillatory events in clusters as compared to isolated islet cells, but contradict the proposal that the frequency of the oscillations increases with the number of coupled cells. Islet cell clusters function better as electrical than biochemical syncytia. This may explain the co‐ordination of [Ca2+]i oscillations driven by depolarization‐dependent Ca2+ influx during glucose stimulation.

Imidazoline antagonists of alpha 2-adrenoceptors increase insulin release in vitro by inhibiting ATP-sensitive K+ channels in pancreatic beta-cells.

1 Islets from normal mice were used to study the mechanisms by which imidazoline antagonists of α2‐adrenoceptors increase insulin release in vitro. 2 Alinidine, antazoline, phentolamine and tolazoline inhibited 86Rb efflux from islets perifused with a medium containing 3 mm glucose, i.e. under conditions where many adenosine 5′‐triphosphate (ATP)‐sensitive K+ channels are open in the β‐cell membrane. They also reduced the acceleration of 86Rb efflux caused by diazoxide, an opener of ATP‐sensitive K+ channels. 3 ATP‐sensitive and voltage‐sensitive K+ currents were measured in single β‐cells by the whole‐cell mode of the patch‐clamp technique. Antazoline more markedly inhibited the ATP‐sensitive than the voltage‐sensitive current, an effect previously observed with phentolamine. Alinidine and tolazoline partially decreased the ATP‐sensitive K+ current. 4 The four imidazolines reversed the inhibition of insulin release caused by diazoxide (through opening of ATP‐sensitive K+ channels) or by clonidine (through activation of α2‐adrenoceptors) in a concentration‐dependent manner. Only the former effect correlated with the ability of each drug to increase control insulin release stimulated by 15 mm glucose alone. 5 It is concluded that the ability of imidazoline antagonists of α2‐adrenoceptors to increase insulin release in vitro can be ascribed to their blockade of ATP‐sensitive K+ channels in β‐cells rather than to their interaction with the adrenoceptor.

MicroRNAs contribute to compensatory β cell expansion during pregnancy and obesity

Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional β cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory β cell mass expansion is largely unknown. We found in rodents that β cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in β cells using specific anti-miR molecules mimicked gene expression changes occurring during β cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory β cell mass expansion occurring under different insulin resistance states.

Temporal and quantitative correlations between insulin secretion and stably elevated or oscillatory cytoplasmic Ca2+ in mouse pancreatic beta-cells

An increase in cytoplasmic Ca2+ in β-cells is a key step in glucose-induced insulin secretion. However, whether changes in cytoplasmic free Ca2+ ([Ca2+]i) directly regulate secretion remains disputed. This question was addressed by investigating the temporal and quantitative relationships between [Ca2+]i and insulin secretion. Both events were measured simultaneously in single mouse islets loaded with fura-PE3 and perifused with a medium containing diazoxide (to prevent any effect of glucose on the membrane potential) and either 4.8 or 30 mmol/l K+. Continuous depolarization with 30 mmol/l K+ in the presence of 15 mmol/l glucose induced a sustained rise in [Ca2+]i and insulin release. No oscillations of secretion were detected even after mathematical analysis of the data (pulse, spectral and sample distribution analysis). In contrast, alternating between 30 and 4.8 mmol/l K+ (1 min/2 min or 2.5 min/5 min) triggered synchronous [Ca2+]i and insulin oscillations of regular amplitude in each islet. A good correlation was found between [Ca2+]i and insulin secretion, and it was independent of the presence or absence of oscillations. This quantitative correlation between [Ca2+]i and insulin secretion was confirmed by experiments in which extracellular Ca2+ was increased or decreased (0.1–2.5 mmol/l) stepwise in the presence of 30 mmol/l K+. This resulted in parallel stepwise increases or decreases in [Ca2+]i and insulin secretion. However, while the successive [Ca2+]i levels were unaffected by glucose, each plateau of secretion was much higher in 20 than in 3 mmol/l glucose. In conclusion, in our preparation of normal mouse islets, insulin secretion oscillates only when [Ca2+]i oscillates in β-cells. This close temporal relationship between insulin secretion and [Ca2+]i changes attests of the regulatory role of Ca2+. There also exists a quantitative relationship that is markedly influenced by the concentration of glucose.

Glucose regulation of islet stress responses and β-cell failure in type 2 diabetes

Pancreatic β‐cells exposed to high glucose concentrations display altered gene expression, function, survival and growth that may contribute to the slow deterioration of the functional β‐cell mass in type 2 diabetes. These glucotoxic alterations may result from various types of stress imposed by the hyperglycaemic environment, including oxidative stress, endoplasmic reticulum stress, cytokine‐induced apoptosis and hypoxia. The glucose regulation of oxidative stress‐response and integrated stress‐response genes in cultured rat islets follows an asymmetric V‐shaped profile parallel to that of β‐cell apoptosis, with a large increase at low glucose and a moderate increase at high vs. intermediate glucose concentrations. These observations suggest that both types of stress could play a role in the alteration of the functional β‐cell mass under states of prolonged hypoglycaemia and hyperglycaemia. In addition, β‐cell demise under glucotoxic conditions may also result from β‐cell hypoxia and, in vivo, from their exposure to inflammatory cytokines released locally by non‐endocrine islet cells. A better understanding of the relative contribution of each type of stress to β‐cell glucotoxicity and of their pathophysiological cause in vivo may lead to new therapeutic strategies to prevent the slow deterioration of the functional β‐cell mass in glucose intolerant and type 2 diabetic patients.

Glucose-Induced O2 Consumption Activates Hypoxia Inducible Factors 1 and 2 in Rat Insulin-Secreting Pancreatic Beta-Cells

Background Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner. Methodology/Principal Findings Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO2 values or treated with the HIF activator CoCl2. HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl2 or a four-fold reduction in pO2. Islets also showed signs of HIF activation in diabetic Leprdb/db but not non-diabetic Leprdb/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO2 or by inhibitors of Ca2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1α and Hif2α, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. Conclusions/Significance Glucose-induced O2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.

Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins : confounding effects of pH with HyPer but not roGFP1

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.