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...

Free fatty acids (FFA) have been reported to reduce pancreatic ␤-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic ␤-cell line, INS-1, over a 18-h period as assessed by Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the phosphorylation activation of protein kinase B (PKB) showed this was reduced in the presence of FFA that correlated with the incidence of apoptosis. At stimulatory 15 mM glucose and/or in the added presence of insulin-like growth factor 1, FFA-induced ␤-cell apoptosis was lessened compared with that at a basal 5 mM glucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a "kinase-dead" PKB variant, essentially prevented FFA-induced ␤-cell apoptosis under all glucose/insulin-like growth factor 1 conditions. Further analysis of pro-apoptotic downstream targets of PKB, implicated a role for PKB-mediated phosphorylation inhibition of glycogen synthase kinase-3␣/␤ and the forkhead transcription factor, FoxO1, in protection of FFA-induced ␤-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppresser protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting ␤-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced ␤-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited ␤-cell apoptosis by ϳ50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic ␤-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3␣/␤, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced ␤-cell apoptosis.

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70 S6K , in ␤-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70 S6K phosphorylation activation is undefined.