Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

AIMS/HYPOTHESIS: The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. METHODS: Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute 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 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. CONCLUSIONS/INTERPRETATION: Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.

The lack of functional nicotinamide nucleotide transhydrogenase only moderately contributes to the impairment of glucose tolerance and glucose-stimulated insulin secretion in C57BL/6J vs C57BL/6N mice.

AIMS/HYPOTHESIS: Nicotinamide nucleotide transhydrogenase (NNT) is involved in mitochondrial NADPH production and its spontaneous inactivating mutation (NntTr [Tr, truncated]) is usually considered to be the main cause of the lower glucose tolerance of C57BL/6J vs C57BL/6N mice. However, the impact of this mutation on glucose tolerance remains disputed. Here, we singled out the impact of NntTr from that of other genetic variants between C57BL/6J and C57BL/6N mice on mitochondrial glutathione redox state (EGSH), glucose-stimulated insulin secretion (GSIS) and glucose tolerance. METHODS: Male and female N5BL/6J mice that express wild-type Nnt (NntWT) or NntTr (N5-WT and N5-Tr mice) on the C57BL/6J genetic background were obtained by crossing N5BL/6J NntWT/Tr heterozygous mice. C57BL/6J and C57BL/6N mice were from Janvier Labs. The Nnt genotype was confirmed by PCR and the genetic background by whole genome sequencing of one mouse of each type. Glucose tolerance was assessed by IPGTT, ITT and fasting/refeeding tests. Stimulus-secretion coupling events and GSIS were measured in isolated pancreatic islets. Cytosolic and mitochondrial EGSH were measured using the fluorescent redox probe GRX1-roGFP2 (glutaredoxin 1 fused to redox-sensitive enhanced GFP). RESULTS: The Nnt genotype and genetic background of each type of mouse were confirmed. As reported previously in C57BL/6N vs C57BL/6J islets, the glucose regulation of mitochondrial (but not cytosolic) EGSH and of NAD(P)H autofluorescence was markedly improved in N5-WT vs N5-Tr islets, confirming the role of NNT in mitochondrial redox regulation. However, ex vivo GSIS was only 1.2-1.4-times higher in N5-WT vs N5-Tr islets, while it was 2.4-times larger in C57BL/6N vs N5-WT islets, questioning the role of NNT in GSIS. In vivo, the ITT results did not differ between N5-WT and N5-Tr or C57BL/6N mice. However, the glucose excursion during an IPGTT was only 15-20% lower in female N5-WT mice than in N5-Tr and C57BL/6J mice and remained 3.5-times larger than in female C57BL/6N mice. Similar observations were made during a fasting/refeeding test. A slightly larger (~30%) impact of NNT on glucose tolerance was found in males. CONCLUSIONS/INTERPRETATION: Although our results confirm the importance of NNT in the regulation of mitochondrial redox state by glucose, they markedly downsize the role of NNT in the alteration of GSIS and glucose tolerance in C57BL/6J vs C57BL/6N mice. Therefore, documenting an NntWT genotype in C57BL/6 mice does not provide proof that their glucose tolerance is as good as in C57BL/6N mice.

Disruption of Beta-Cell Mitochondrial Networks by the Orphan Nuclear Receptor Nor1/Nr4a3.

Nor1, the third member of the Nr4a subfamily of nuclear receptor, is garnering increased interest in view of its role in the regulation of glucose homeostasis. Our previous study highlighted a proapoptotic role of Nor1 in pancreatic beta cells and showed that Nor1 expression was increased in islets isolated from type 2 diabetic individuals, suggesting that Nor1 could mediate the deterioration of islet function in type 2 diabetes. However, the mechanism remains incompletely understood. We herein investigated the subcellular localization of Nor1 in INS832/13 cells and dispersed human beta cells. We also examined the consequences of Nor1 overexpression on mitochondrial function and morphology. Our results show that, surprisingly, Nor1 is mostly cytoplasmic in beta cells and undergoes mitochondrial translocation upon activation by proinflammatory cytokines. Mitochondrial localization of Nor1 reduced glucose oxidation, lowered ATP production rates, and inhibited glucose-stimulated insulin secretion. Western blot and microscopy images revealed that Nor1 could provoke mitochondrial fragmentation via mitophagy. Our study unveils a new mode of action for Nor1, which affects beta-cell viability and function by disrupting mitochondrial networks.

The orphan nuclear receptor Nor1/Nr4a3 is a negative regulator of ß-cell mass.

The Nr4a subfamily of nuclear receptor comprises three members in mammalian cells: Nur77/Nr4a1, Nurr1/Nr4a2, and Nor1/Nr4a3. Nr4a proteins play key roles in the regulation of glucose homeostasis in peripheral metabolic tissues. However, their biological functions in ß-cells remain relatively uncharacterized. Here we sought to investigate the potential role of Nor1 in the regulation of ß-cell mass and, in particular, ß-cell survival/apoptosis. We used histological analysis to examine the consequences of genetic deletion of either Nur77 and Nor1 on ß-cell mass, investigated the expression patterns of Nr4as in human islets and INS cells and performed gain- and loss-of-function experiments to further characterize the role of Nor1 in ß-cell apoptosis. Surprisingly, Nor1 knockout mice displayed increased ß-cell mass, whereas mice with genetic deletion of Nur77 did not exhibit any significant differences compared with their WT littermates. The increase in ß-cell mass in Nor1 knockout mice was accompanied by improved glucose tolerance. A gene expression study performed in both human islets and INS cells revealed that Nor1 expression is significantly increased by pro-inflammatory cytokines and, to a lesser extent, by elevated concentrations of glucose. Nor1 overexpression in both INS and human islet cells caused apoptosis, whereas siRNA-mediated Nor1 knockdown prevented cytokine-induced ß-cell death. Finally, Nor1 expression was up-regulated in islets of individuals with type 2 diabetes. Altogether, our results uncover that Nor1 negatively regulates ß-cell mass. Nor1 represents a promising molecular target in diabetes treatment to prevent ß-cell destruction.

A critical role for the neural zinc factor ST18 in pancreatic ß-cell apoptosis.

The tumor suppressor gene ST18 was originally characterized as the third member of the neural zinc finger transcription factor family. However, little is known about its biological functions. Herein, we demonstrate that, in the pancreas, ST18 expression is restricted to endocrine cells. The detection of ST18 expression in pancreatic ß-cells prompted us to investigate its regulation and its role in ß-cell mass and function. We show that ST18 expression and activity are increased by cytotoxic concentrations of fatty acids and cytokines in INS832/13 cells. Furthermore, ST18 is also increased in islets of diet-induced obese animals. Overexpression and RNA interference knockdown studies demonstrate that ST18 induces ß-cell apoptosis and curtails ß-cell replication. Finally, our data suggest that ST18 impairs insulin secretion. Taken together, our findings indicate that ST18 could represent a novel transcriptional mediator of lipotoxicity and cytokine-induced ß-cell death. We suggest that genetic or pharmacologic manipulations of ST18 could help maintain a functional ß-cell mass.

Protective antioxidant and antiapoptotic effects of ZnCl2 in rat pancreatic islets cultured in low and high glucose concentrations.

AIM/HYPOTHESIS: Rat pancreatic islet cell apoptosis is minimal after prolonged culture in 10 mmol/l glucose (G10), largely increased in 5 mmol/l glucose (G5) and moderately increased in 30 mmol/l glucose (G30). This glucose-dependent asymmetric V-shaped profile is preceded by parallel changes in the mRNA levels of oxidative stress-response genes like Metallothionein 1a (Mt1a). In this study, we tested the effect of ZnCl(2), a potent inducer of Mt1a, on apoptosis, mitochondrial oxidative stress and alterations of glucose-induced insulin secretion (GSIS) induced by prolonged exposure to low and high vs. intermediate glucose concentrations. METHODS: Male Wistar rat islets were cultured in RPMI medium. Islet gene mRNA levels were measured by RTq-PCR. Apoptosis was quantified by measuring islet cytosolic histone-associated DNA fragments and the percentage of TUNEL-positive ß-cells. Mitochondrial thiol oxidation was measured in rat islet cell clusters expressing "redox sensitive GFP" targeted to the mitochondria (mt-roGFP1). Insulin secretion was measured by RIA. RESULTS: As observed for Mt1a mRNA levels, ß-cell apoptosis and loss of GSIS, culture in either G5 or G30 vs. G10 significantly increased mt-roGFP1 oxidation. While TPEN decreased Mt1a/2a mRNA induction by G5, addition of 50-100 µM ZnCl(2) to the culture medium strongly increased Mt1a/2a mRNA and protein levels, reduced early mt-roGFP oxidation and significantly decreased late ß-cell apoptosis after prolonged culture in G5 or G30 vs. G10. It did not, however, prevent the loss of GSIS under these culture conditions. CONCLUSION: ZnCl(2) reduces mitochondrial oxidative stress and improves rat ß-cell survival during culture in the presence of low and high vs. intermediate glucose concentrations without improving their acute GSIS.