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Background and aims: The rapid reversibility of recent type 2 diabetes (T2D) by very low-calorie diet in some patients correlates with a marked improvement of glucose-stimulated insulin secretion (GSIS), emphasizing the role of β-cell dysfunction in the early stages of the disease. In search of human-specific mechanisms of β-cell dysfunction, we extensively characterized the glucotoxic alterations of insulin secretion and upstream coupling events in human pancreatic islets cultured for 1 to 3 weeks at ~5, 8, 10 or 20 mmol/l glucose and acutely stimulated by a stepwise increase in glucose concentration. Materials and methods: Islets from 46 non-diabetic (ND) and 6 type 2 diabetic (T2D) donors were obtained from 5 isolation centers over the last 10 years. The islets were precultured 3-7 days in RPMI containing 5 mmol/l glucose and 10% FBS. They were then cultured for 1-3 weeks in the same medium containing 5.5, 8.5, 10.5 or 20.5 mmol/l glucose before measurements of insulin secretion during culture, islet insulin/DNA content ratio, β-cell apoptosis, cytosolic and mitochondrial thiol redox state, and assessment of dynamic insulin secretion and upstream coupling events during stepwise stimulation with glucose (NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)). Results: Culture of ND-islets for 1 to 3 weeks at ~8, 10 or 20 vs 5 mmol/l glucose did not increase β-cell apoptosis or oxidative stress but concentration-dependently decreased insulin content and increased the β-cell sensitivity to subsequent stimulation with glucose. The islet glucose responsiveness (max amplitude of GSIS per islet) was larger after culture at 8 or 10 vs 5 mmol/l glucose but was markedly reduced after culture at 20 vs 5 mmol/l glucose. In the latter islets, [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were bell-shaped, with a maximal stimulation at 5 to 10 mmol/l glucose followed by a rapid sustained inhibition above that concentration. This glucotoxic alteration was a robust characteristic of human islets. It resulted from long-term stimulation of glucose metabolism and was fully reversible after culture at 5 mmol/l glucose. Finally, acute activation/inhibition of glucokinase during perifusion of islets cultured at 20 mmol/l glucose indicated that the acute reduction of [Ca2+]c and insulin secretion above 10 mmol/l glucose was due to overstimulation rather than inhibition of glucose metabolism. Similar results were obtained in islets from T2D-donors. Conclusion: Long-term exposure of human islets to mildly elevated glucose concentrations markedly increases their glucose sensitivity and reveals a bell-shaped glucose response curve for changes in [Ca2+]c and insulin secretion. This human-specific glucotoxic alteration may contribute to β-cell dysfunction in T2D independently from a detectable increase in β-cell apoptosis and oxidative stress.

Background and aims: The mechanisms responsible for β cell compensation in obesity and for β cell failure in type 2 diabetes (T2D) are poorly defined. Metallothioneins play a role in both Zn2+ homeostasis and the regulation of cellular redox state. The mRNA levels of several metallothionein genes are upregulated in islets from subjects with T2D, but their role in β cells is not clear. Here we examined: 1) the temporal changes of islet Mt1 and Mt2 gene expression in models of β cell compensation and failure, and 2) the role of Mt1 and Mt2 in β cell function and glucose homeostasis. Materials and methods: Mt1 and Mt2 expression was assessed in islets from control lean (chow diet) and diet-induced obese (DIO) mice (8 weeks high fat diet), and pre-diabetic (6-week-old) and diabetic (16-week-old) db/db mice and age-matched db/+ (control) mice. Mt1-Mt2 double knockout (KO) mice, Mt1 overexpressing transgenic mice (Tg-Mt1) and corresponding control mice were studied. Mt1 and Mt2 were inhibited in MIN6 cells by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet metallothionein levels by ELISA, glucose tolerance by i.p. glucose tolerance tests (ipGTT) and fasting-1h refeeding tests, insulin secretion by RIA, cytosolic free Ca2+ with Fura-2 LR, NAD(P)H by autofluorescence and cytosolic redox state using roGFP1 ratiometric thiol redox probe. Results: Increased plasma insulin levels (β cell compensation) correlated with marked downregulation of Mt1 and Mt2 mRNA levels in islets of DIO mice (Mt1: ~4-fold, p

International audience; 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.

OBJECTIVE: Sarco-endoplasmic reticulum Ca(2+)-ATPase 2b (SERCA2b) and SERCA3 pump Ca(2+) in the endoplasmic reticulum (ER) of pancreatic β-cells. We studied their role in the control of the free ER Ca(2+) concentration ([Ca(2+)](ER)) and the role of SERCA3 in the control of insulin secretion and ER stress. RESEARCH DESIGN AND METHODS: β-Cell [Ca(2+)](ER) of SERCA3(+/+) and SERCA3(-/-) mice was monitored with an adenovirus encoding the low Ca(2+)-affinity sensor D4 addressed to the ER (D4ER) under the control of the insulin promoter. Free cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [Ca(2+)](ER) were simultaneously recorded. Insulin secretion and mRNA levels of ER stress genes were studied. RESULTS: Glucose elicited synchronized [Ca(2+)](ER) and [Ca(2+)](c) oscillations. [Ca(2+)](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 [Ca(2+)](ER) rise in SERCA3(+/+) and SERCA3(-/-) β-cells. In a Ca(2+)-free medium, glucose moderately raised [Ca(2+)](ER) from a highly buffered cytosolic Ca(2+) pool. Increasing [Ca(2+)](c) with high [K] elicited a [Ca(2+)](ER) rise that was larger but more transient in SERCA3(+/+) than SERCA3(-/-) β-cells because of the activation of a Ca(2+) release from the ER in SERCA3(+/+) β-cells. Glucose-induced insulin release was larger in SERCA3(-/-) than SERCA3(+/+) islets. SERCA3 ablation did not induce ER stress. CONCLUSIONS: [Ca(2+)](c) and [Ca(2+)](ER) oscillate in phase in response to glucose. Upon [Ca(2+)](c) increase, Ca(2+) is taken up by SERCA2b and SERCA3. Strong Ca(2+) influx triggers a Ca(2+) release from the ER that depends on SERCA3. SERCA3 deficiency neither impairs Ca(2+) uptake by the ER upon cell metabolism acceleration and insulin release nor induces ER stress.

Effects of deglycating enzyme Fructosamine-3-kinase gene knockout on pancreatic beta cell glucotoxicity

Background and Aim: We previously tested the effects of a 18h culture in 2, 5, 10 and 30 mmol/l glucose (G2, G5, G10 and G30) on the transcriptome of cultured rat islets and identified 18 clusters with distinct glucose-dependent mRNA profiles. Of these, genes that were up-regulated between G10 and G30 are particularly interesting, as they may contribute to beta-cell glucotoxicity or protection against it. Besides genes involved in the unfolded protein response, this cluster contains most glycolytic enzymes and other genes typically induced by hypoxia, such as Adrenomedullin (Adm). Hypothesis: High glucose, which increases O2 consumption in β-cells, may induce hypoxia and thereby cause glucotoxicity. In this study, we tested whether overnight culture in high glucose activates the hypoxia-inducible transcription factor HIF in cultured rat beta-cells. Material and Methods: Male Wistar rat islets were precultured for one week in serum-free RPMI medium containing 10 mmol/l glucose (G10) and 5 g/l BSA, during which islets with signs of central necrosis were systematically discarded. INS1-E cells were cultured as monolayers (passage 72 to 80) in the presence of 10% FBS. Rat islets and INS1-E cells (70% confluence) were then cultured 18h in the presence of increasing glucose concentrations (G2, G5, G10 and G30) with various test substances and at different O2 concentrations (1%, 20% or 60% corresponding to a pO2 of 7.6, 152 and 456 mmHg). Results: Compared with G2, culture in G30 significantly increased 4-fold the protein levels of HIF1α and HIF2α, and 2-fold their dimerization partner HIF1β in INS1-E cell nuclear extracts while significantly decreasing HIF1β protein levels by ~50% in cytosolic extracts. High glucose also significantly up-regulated the mRNA levels of several HIF target genes (Adm, Glyceraldehyde phosphate dehydrogenase, Aldolase A, Lactate dehydrogenase A). These glucose effects, which were confirmed in whole rat islets, were mimicked by 6h exposure to a low pO2 (1% O2) or by 18h treatment with 10-30 µmol/l CoCl2, a known activator of HIF. In contrast, they were almost completely inhibited by 60% O2 or by various agents that inhibit Ca2+ influx and insulin secretion (250 µmol/l diazoxide, 1 µmol/l nimodipine and 1 µmol/l clonidine) and thereby likely reduce without suppressing the glucose stimulation of β-cell O2 consumption. In comparison with HIF target genes, Thioredoxin interacting protein (Txnip), one of the most glucose-responsive genes in cultured rat islets, was regulated in a completely different manner. Thus, Txnip mRNA levels were not increased by CoCl2 and 1% O2, and their increase by G30 was not reduced by 60% O2 and was markedly enhanced by diazoxide, nimodipine and clonidine. Conclusion: High glucose induces HIF1α and HIF2α protein stabilization, HIF1/2α-HIF1β dimer translocation to the nucleus, and increased expression of glycolytic enzymes and other HIF target genes in cultured rat beta-cells. These effects likely result from the glucose stimulation of ATP utilization and O2 consumption which may induce beta-cell hypoxia. These effects could contribute to in vitro β-cell glucotoxicity not only in whole islets but also in insulin-secreting cells cultured as monolayers.

The contribution of Ca(2+) release from intracellular stores to the rise in the free cytosolic Ca(2+) concentration ([Ca(2+)](c)) triggered by Ca(2+) influx was investigated in mouse pancreatic beta-cells. Depolarization of beta-cells by 45 mm K(+) (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca(2+)](c) responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca(2+)](c) peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca(2+)](c) and voltage-dependent Ca(2+) current established that the TCP did not result from a larger Ca(2+) current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca(2+) mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of beta-cells but required a rise in [Ca(2+)](c) pointing to a Ca(2+)-induced Ca(2+) release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified beta-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP(3)R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K(+), but plays little role in response to glucose in non-obese mouse pancreatic beta-cells.

BACKGROUND AND AIMS: The low-affinity Ca2+-ATPase SERCA3 is highly expressed in p-cells. It mediates uptake of cytosolic Ca2+ by the endoplasmic reticulum during each period of Ca2+ influx from the extracellular medium, whereas the high-affinity SERCA2b controls basal [Ca'+t For unknown reasons, p-cells within mouse islets display variable patterns of [Ca2 +], oscillations during glucose (G) stimulation. These oscillations can be either regular and fast, or mixed with rapid oscillations superimposed on slow ones. We studied the role of the membrane potential (MP) and the possible intervention of SERCA3 in the generation of both types of [Ca2 +], oscillations. [...]

BACKGROUND AND AIMS: In pancreatic ß-cells, Ca2+ influx through voltagedependent Ca2+ channels plays a prominent role in the depolarizationinduced rise in free cytosolic Ca2+ concentration ([Ca2+]c). The contribution of Ca2+ release from the endoplasmic reticulum (ER) to this [Ca2+]c elevation is largely debated and was investigated here [...]

Chronic hyperglycemia exerts deleterious effects on glucose-induced insulin secretion by pancreatic beta cells. In 90% pancreatectomized rats, chronic hyperglycemia induces beta cell hypertrophy and loss of differentiation associated with increased expression of the early response gene c-Myc. Hyperglycemia also stimulates c-Myc expression in vivo in islets from glucose-infused rats and in vitro in cultured rat islets. This effect requires the elevation of cytosolic Ca2+ concentration produced by glucose. Our results suggest that overstimulation of c-Myc expression by chronic hyperglycemia may be the cause of beta cell hypertrophy and loss of differentiation and function observed in animal models of type 2 diabetes.