Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucosedependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [ 14 C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14 C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial ␤-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Diabetes 50:315-321, 2001A ccording to the lipotoxicity hypothesis, chronic exposure to elevated lipid levels impairs pancreatic ␤-cell function in type 2 diabetic patients (1,2). We (3) and others (4,5) have previously shown that prolonged (>1 day) culture of normal islets in the presence of supraphysiologic concentrations of palmitate decreases insulin content and impairs insulin gene expression only in the presence of elevated glucose levels. This occurs, at least in part, via decreased insulin gene promoter activity in HIT-T15 cells (3) and decreased binding of the transcription factor pancreas-duodenum homeobox-1 (PDX-1) to the insulin gene in islets (4). In Zucker diabetic fatty (ZDF) rats, it has been postulated that ␤-cell dysfunction is due to increased triacylglycerol (TAG) content in islets (6-8), which leads to increased production of nitric oxide (9) and ceramide synthesis (10). However, the ZDF rat is an extremely obese genetic model of type 2 diabetes bearing a mutation in the leptin receptor gene. It remains...

Chronic exposure to elevated levels of fatty acids impairs pancreatic beta cell function, a phenomenon thought to contribute to the progressive deterioration of insulin secretion in type 2 diabetes. We have previously demonstrated that prolonged exposure of isolated islets to elevated levels of palmitate inhibits preproinsulin mRNA levels in the presence of high glucose concentrations. However, whether this occurs via transcriptional or post-transcriptional mechanisms has not been determined. In addition, the nature of the lipid metabolites involved in palmitate inhibition of insulin gene expression is unknown. In this study, we show that palmitate decreases glucose-stimulated preproinsulin mRNA levels in isolated rat islets, an effect that is not mediated by changes in preproinsulin mRNA stability, but is associated with inhibition of glucose-stimulated insulin promoter activity. Prolonged culture of isolated islets with palmitate is associated with increased levels of intracellular ceramide. Palmitate-induced ceramide generation is prevented by inhibitors of de novo ceramide synthesis. Further, exogenous ceramide inhibits insulin mRNA levels, whereas blockade of de novo ceramide synthesis prevents palmitate inhibition of insulin gene expression. We conclude that prolonged exposure to elevated levels of palmitate affects glucose-stimulated insulin gene expression via transcriptional mechanisms and ceramide synthesis.

Chronic elevations in plasma levels of fatty acids (FAs) adversely affect pancreatic ␤-cell function in type 2 diabetes. In vitro, we have previously shown that deleterious effects of prolonged exposure of isolated islets to FAs were dependent on the presence of elevated glucose concentration. This led us to hypothesize that both hyperlipidemia and hyperglycemia must be present simultaneously for FAs to affect ␤-cell function. To test this hypothesis in vivo, we administered a high-fat diet for 6 weeks to Goto-Kakizaki (GK) rats. High-fat feeding had no effect on insulin secretion, insulin content, or insulin mRNA levels in islets from normoglycemic Wistar rats. In contrast, high-fat feeding markedly impaired glucose-induced insulin secretion in islets from GK rats. High-fat feeding did not affect triglyceride (TG) content or the rate of glucose oxidation in islets. It was, however, accompanied by a twofold increase in uncoupling protein (UCP)-2 levels in GK rat islets. Insulin treatment completely normalized glucoseinduced insulin secretion and prevented the increase in UCP-2 expression in islets from high-fat-fed GK rats. We conclude that hyperlipidemia induced by high-fat feeding affects insulin secretion in islets from hyperglycemic GK rats only, by a mechanism which may involve, at least in part, modulation of UCP-2 expression.

The mechanisms by which prolonged exposure to elevated levels of fatty acids (FA) adversely affects pancreatic beta-cell function remain unclear. Studies in the Zucker diabetic fatty rat have suggested that excessive accumulation of triglycerides (TG) in islets plays a key role in the deleterious effects of FA. However, a direct relationship between TG accumulation and defective beta-cell function has not been established. The aim of the present study was therefore to determine whether increasing TG synthesis in isolated rat islets of Langerhans impairs insulin secretion. To this end, we infected isolated rat islets with an adenovirus encoding for the enzyme catalyzing the last step of triglyceride synthesis, acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGAT). DGAT overexpression did not modify glucose oxidation nor palmitate oxidation, but increased palmitate incorporation into triglycerides by approximately 2-fold. Islets overexpressing DGAT and cultured in elevated glucose levels for 72 h had markedly impaired insulin secretion in response to glucose, but responded normally to the nonglucose secretagogues glyburide and potassium chloride. The deleterious effects of DGAT overexpression were not additive to those of prolonged exposure to palmitate. We conclude that a selective increase in TG content impairs glucose-induced insulin secretion, a mechanism likely to mediate, at least in part, the deleterious effects of FA on pancreatic beta-cell function.