Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Abstract

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...

Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload

“…Although it is tempting to speculate that the trafficking defect described here may contribute to this impairment, specific alterations in post-Golgi processing have also been described [11,12]. Evaluating the relative contribution of these alternative mechanisms would therefore require a better understanding of how palmitate actually reduces protein trafficking, since this would facilitate investigation of whether rescuing the trafficking defect could improve proinsulin conversion.…”

Ubiquitin C-terminal hydrolase L1 is required for pancreatic beta cell survival and function in lipotoxic conditions

“…Pancreatic islet isolation and cell culture Mouse islets were isolated and cultured using a previously described protocol [27]. Islets were lysed immediately after isolation for Western blot analysis or subjected to other treatments in RPMI medium as indicated or dispersed into single cells for intracellular Ca 2+ measurement.…”

“…Type 2 diabetes, where obesity is a major risk factor [24,25], is characterised by beta cell death in the lipotoxic milieu, as well as pathology associated with protein misfolding and accumulation [26], reminiscent of neurodegenerative diseases. Moreover, endoplasmic reticulum (ER) and oxidative stress have been suggested as common mediators of both beta cell [27][28][29] and neuronal [30,31] death. Obesity and hyperlipidaemia cause ER [27,32] and oxidative stress, and may promote both diabetes and neurodegenerative diseases [30,31].…”

“…Moreover, endoplasmic reticulum (ER) and oxidative stress have been suggested as common mediators of both beta cell [27][28][29] and neuronal [30,31] death. Obesity and hyperlipidaemia cause ER [27,32] and oxidative stress, and may promote both diabetes and neurodegenerative diseases [30,31]. As part of our efforts to determine the mechanisms of lipotoxicity, UCHL1 was identified in a proteomic screen as the most upregulated protein in MIN6 beta cells treated with the fatty acid, palmitate [27].…”

“…Obesity and hyperlipidaemia cause ER [27,32] and oxidative stress, and may promote both diabetes and neurodegenerative diseases [30,31]. As part of our efforts to determine the mechanisms of lipotoxicity, UCHL1 was identified in a proteomic screen as the most upregulated protein in MIN6 beta cells treated with the fatty acid, palmitate [27]. Here, we test the hypothesis that UCHL1 is essential for beta cell function and survival under lipotoxic conditions using a combination of in vivo and in vitro studies and a mouse model lacking Uchl1.…”