Chronic hyperglycemia has been shown to induce either a lack of response or an increased sensitivity to glucose in pancreatic beta-cells. We reinvestigated this controversial issue in a single experimental model by culturing rat islets for 1 wk in 10 or 30 mmol/l glucose (G10, Controls; or G30, High-glucose islets) before testing the effect of stepwise glucose stimulation from G0.5 to G20 on key beta-cell stimulus-secretion coupling events. Compared with Controls, the glucose sensitivity of High-glucose islets was markedly increased, leading to maximal stimulation of oxidative metabolism and both triggering and amplifying pathways of insulin secretion in G6 rather than G20, hence to loss of glucose effect above G6. This enhanced glucose sensitivity occurred despite an approximately twofold increase in islet uncoupling protein 2 mRNA expression. Besides this increased glucose sensitivity, the maximal glucose stimulation of insulin secretion in High-glucose islets was reduced by approximately 50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in (45)Ca(2+) influx induced by glucose and diazoxide were qualitatively similar but quantitatively smaller than in Control islets and, paradoxically, did not lead to detectable changes in the intracellular Ca(2+) concentration measured by microspectrofluorimetry (fura PE 3). In conclusion, after 1 wk of culture in G30, the loss of glucose stimulation of insulin secretion in the physiological range of glucose concentrations (G5-G10) results from the combination of an increased sensitivity to glucose of both triggering and amplifying pathways of insulin secretion and an approximately 50% reduction in the maximal glucose stimulation of insulin secretion.

Aims/hypothesis: The study was designed to identify the key metabolic signals of glucose-stimulated proinsulin gene transcription and translation, focusing on the mechanism of succinate stimulation of insulin production. Methods: Wistar rat islets were incubated in 3.3 mmol/l glucose with and without esters of different mitochondrial metabolites or with 16.7 mmol/l glucose. Proinsulin biosynthesis was analysed by tritiated leucine incorporation into newly synthesised proinsulin. Preproinsulin gene transcription was evaluated following transduction with adenoviral vectors expressing the luciferase reporter gene under the control of the rat I preproinsulin promoter. Steady-state preproinsulin mRNA was determined using relative quantitative PCR. The mitochondrial membrane potential was measured by microspectrofluorimetry using rhodamine-123. Results: Succinic acid monomethyl ester, but not other mitochondrial metabolites, stimulated preproinsulin gene transcription and translation. Similarly to glucose, succinate increased specific preproinsulin gene transcription and biosynthesis. The inhibitor of succinate dehydrogenase (SDH), 3-nitropropionate, abolished glucose-and succinate-stimulated mitochondrial membrane hyperpolarisation and proinsulin biosynthesis, indicating that stimulation of proinsulin translation depends on SDH activity. Partial inhibition of SDH activity by exposure to fumaric acid monomethyl ester abolished the stimulation of preproinsulin gene transcription, but only partially inhibited the stimulation of proinsulin biosynthesis by glucose and succinate, suggesting that SDH activity is particularly important for the transcriptional response to glucose. Conclusions/interpretation: Succinate is a key metabolic mediator of glucose-stimulated preproinsulin gene transcription and translation. Moreover, succinate stimulation of insulin production depends on its metabolism via SDH. The differential effect of fumarate on preproinsulin gene transcription and translation suggests that these processes have different sensitivities to metabolic signals.

N-acetyl-L-cysteine and manganese(III)tetrakis (4-benzoic acid)porphyrin do not prevent ␤-cell dysfunction in rat islets cultured in high glucose for 1 wk. Am J Physiol Endocrinol Metab 291: E137-E146, 2006. First published February 7, 2006 doi:10.1152/ajpendo.00145.2005.-We previously showed that the stimulation of heme oxygenase-1 expression by high glucose and hydrogen peroxide (H2O2) in cultured rat islets is prevented by antioxidants and suggested that this effect of high glucose results from an oxidative stress. However, the role of oxidative stress in highglucose-induced ␤-cell dysfunction is unclear. We therefore compared the preventative effects of N-acetyl-L-cysteine (NAC), a free radical scavenger, and manganese(III)tetrakis (4-benzoic acid)porphyrin (MnTBAP), a superoxide dismutase/catalase mimetic agent, on the alteration of stimulus-secretion coupling induced in rat islets by overnight exposure to hydrogen peroxide (H 2O2-treated islets) or 1-wk culture in 30 vs. 10 mmol/l glucose (High-glucose vs. Control islets). The features of ␤-cell dysfunction differed between the two groups: reduced glucose-induced insulin secretion without changes in glucose sensitivity in H 2O2-treated islets; increased sensitivity to glucose with parallel reductions in insulin content and maximal rate of glucose-induced insulin secretion in High-glucose islets. The latter alterations were accompanied by a decrease in preproinsulin without changes in pancreatic and duodenal homeobox gene 1 mRNA levels. The functional alterations induced by H 2O2 were significantly prevented by addition of NAC or MnTBAP in the culture medium. In contrast, neither NAC nor MnTBAP affected the functional alterations induced by high glucose. These results suggest that ␤-cell dysfunction induced by 1-wk culture in high glucose does not result from an increase in oxidative stress. cytosolic calcium concentration; heme oxygenase-1; insulin secretion; mitochondrial membrane potential; oxidative stress; pancreatic ␤-cell; superoxide dismutase mimetic THE CAUSAL ROLE OF CHRONIC HYPERGLYCEMIA in the onset and progression of diabetic complications is well established (7,58). At the cellular level, the deleterious effects of hyperglycemia may result from several mechanisms, including increased glucose flux through the polyol and hexosamine pathways, protein glycation, activation of receptors for advanced glycation end products, protein kinase C and mitogen-activated protein kinase activation, and excessive mitochondrial production of reactive oxygen species (ROS) (28,57,61). It has been proposed that an increase in oxidative stress, defined as a serious imbalance between enhanced ROS production and reduced antioxidant capacity, may be a common denominator for these various mechanisms of diabetic complications (1,4,5,39,50,63).In insulin-secreting pancreatic ␤-cells, chronic hyperglycemia or in vitro exposure to supraphysiological glucose concentrations progressively induces reversible and irreversible phenotypic alterations, including loss of glucose-i...