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We previously showed that the stimulation of heine oxygenase-1 expression by high glucose and hydrogen peroxide ([H.sub.2][O.sub.2]) 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 high-glucose-induced [beta]-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.sub.2][O.sub.2]-treated islets) or 1-wk culture in 30 vs. 10 mmlo/l glucose (High-glucose vs. Control islets). The features of [beta]-cell dysfunction differed between the two groups: reduced glucose-induced insulin secretion without changes in glucose sensitivity in [H.sub.2][O.sub.2]-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.sub.2][O.sub.2] 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 [beta]-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 [beta]-cell; superoxide dismutase mimetic

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 ~50%, proportionally to the reduction of insulin content. In High-glucose islets, changes in [sup.45][Ca.sup.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.sup.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 ~50% reduction in the maximal glucose stimulation of insulin secretion. pancreatic [beta]-cell; cytosolic calcium concentration; mitochondrial activity; glucose toxicity