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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

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.

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 [...]