Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce ␤-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in ␤-cell death. The most significantly altered protein in both human islets and MIN6 ␤-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca 2؉ flux were also required for CPE proteolysis and ␤-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by Ϸ30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued ␤-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced ␤-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and ␤-cell death pathways in diabetes.2D difference gel electrophoresis proteomics ͉ free fatty acids ͉ hyperproinsulinemia ͉ mechanisms of ␤-cell lipotoxicity ͉ type 2 diabetes T here is a strong association between type 2 diabetes and obesity.High levels of circulating lipids, including free fatty acids, are a prominent clinical feature of type 2 diabetes and represent an important risk factor for this disease (1, 2). But exactly how elevated lipids might lead to diabetes remains unresolved. Fatty acids increase basal insulin secretion (3) and the relative levels of circulating proinsulin (4). Chronic exposure to the free fatty acid palmitate has been shown to impair glucose-stimulated insulin release (i.e., lipotoxicity) (5-10). ␤-Cell apoptosis can be initiated by high levels of palmitate (6,7,(11)(12)(13)(14), which may account in part for alterations in insulin secretory function (13). A number of studies have established palmitate targets in the ␤-cell, including lipid metabolism (15, 16), mitochondrial function (17-23), and prosurvival transcription factors such as Pdx1 (24,25). Recently, a role for endoplasmic reticulum (ER) stress in lipotoxicity has been demonstrated in multiple cell types, including ␤-cells (11,26,27). The effects of palmitate on ␤-cell survival are likely mediated by a number of mechanisms.In the present study, we conducted unbiased proteomic screens using human islets and MIN6 ␤-cells to elucidate targets of palmitate. Carboxypeptidase E (CPE) was the most significantly changed protein in both screens. Mice lacking CPE develop hyperproinsulinemia and hyperglycemia (28), but the involvement of this protein in ␤-cell apoptosis has not been reported. Palmitate caused the rapid intracellular redistribution and degradation of CPE via mechanisms that required palmitate metabolism, K ATP channel closure, Ca 2ϩ influx, and protease activity. We further showed that CPE levels control ␤-cell ER stress and apoptosis. Thus, CPE is a cri...

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular Ca2+, and in particular the role of the endoplasmic reticulum (ER). In human β-cells and MIN6 cells, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells. In keeping with this observed ER Ca2+ depletion, palmitate induced rapid phosphorylation of the ER Ca2+ sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and β-cell death. We detected little palmitate-induced insulin secretion, suggesting that these Ca2+ signals are poorly coupled to exocytosis. In summary, we have characterized Ca2+-dependent mechanisms involved in altered β-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER Ca2+ and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes.

Elevated extracellular lipids, such as the free fatty acid palmitate, can induce pancreatic beta cell endoplasmic reticulum (ER) stress and apoptosis, thereby contributing to the initiation and progression of type 2 diabetes. ATP-citrate lyase (ACLY), a key enzyme in cellular lipid production, was identified as a palmitate target in a proteomic screen. We investigated the effects of palmitate on ACLY activity and phosphorylation and its role in beta cell ER stress and apoptosis. We demonstrated that treatment of MIN6 cells, mouse islets and human islets with palmitate reduced ACLY protein levels. These in vitro results were validated by our finding that islets from high fat-fed mice had a significant decrease in ACLY, similar to that previously observed in type 2 diabetic human islets. Palmitate decreased intracellular acetyl-CoA levels to a similar degree as the ACLY inhibitor, SB-204990, suggesting a reduction in ACLY activity. ACLY inhibitors alone were sufficient to induce CCAAT/enhancer-binding protein homologues protein (CHOP)-dependent ER stress and caspase-3-dependent apoptosis. Similarly, even modest shRNA-mediated knockdown of ACLY caused a significant increase in beta cell apoptosis and ER stress. The effects of chemical ACLY inhibition and palmitate were nonadditive and therefore potentially mediated by a common mechanism. Indeed, overexpression of ACLY prevented palmitateinduced beta cell death. These observations provide new evidence that ACLY expression and activity can be suppressed by exogenous lipids and demonstrate a critical role for ACLY in pancreatic beta cell survival. These findings add to the emerging body of evidence linking beta cell metabolism with programmed cell death.

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