Elsevier

Metabolism

Volume 60, Issue 8, August 2011, Pages 1081-1089
Metabolism

Glucagon-like peptide–1 and candesartan additively improve glucolipotoxicity in pancreatic β-cells

https://doi.org/10.1016/j.metabol.2010.11.004Get rights and content

Abstract

Glucagon-like peptide–1 (GLP-1) and angiotensin II type 1 receptor blocker reduce β-cell apoptosis in diabetes, but the underlying mechanisms are not fully understood. We examined the combination effects of GLP-1 and candesartan, an angiotensin II type 1 receptor blocker, on glucolipotoxicity-induced β-cell apoptosis; and we explored the possible mechanisms of the antiapoptotic effects. The effects of GLP-1 and/or candesartan on glucolipotoxicity-induced apoptosis and the phosphorylation of insulin receptor substrate–2 (IRS-2), protein kinase B (PKB), and forkhead box O1 (FoxO1) were evaluated by using MIN6 cells and isolated mouse pancreatic islets. Although palmitate significantly enhanced the high-glucose–induced apoptosis in both islets and MIN6 cells, GLP-1 and candesartan significantly inhibited apoptosis; and combination treatment additively prevented apoptosis. Whereas palmitate significantly decreased the phosphorylation of IRS-2, PKB, and FoxO1 in MIN6 cells, these changes were significantly inhibited by treatment with GLP-1 and/or candesartan. In addition, wortmannin, an inhibitor of phosphoinositide 3-kinase, markedly inhibited GLP-1– and/or candesartan-mediated PKB and FoxO1 phosphorylation. The present results suggest that GLP-1 and candesartan additively prevent glucolipotoxicity-induced apoptosis in pancreatic β-cells through the IRS-2/phosphoinositide 3-kinase/PKB/FoxO1 signaling pathway.

Introduction

Glucolipotoxicity plays an important role in the development and progression of type 2 diabetes mellitus. Chronic hyperglycemia causes pancreatic β-cell dysfunction characterized by reduced insulin biosynthesis [1] and increased levels of apoptosis (glucotoxicity) [2], [3], [4]. In addition, long-term exposure of β-cells to high concentrations of free fatty acid triggers β-cell apoptosis (lipotoxicity) [5], [6], [7]. The combination of glucotoxicity and lipotoxicity (glucolipotoxicity) has been postulated to contribute to the worsening of β-cell function over time, creating a vicious cycle by which metabolic abnormalities impair insulin secretion, thereby further aggravating metabolic perturbation [8], [9]. Therefore, the stabilization of metabolic changes induced by glucolipotoxicity in β-cells represents a potential new avenue for the treatment of patients with type 2 diabetes mellitus [10].

The rennin-angiotensin system (RAS) in isolated pancreatic islets includes angiotensinogen, angiotensin-converting enzymes, and angiotensin II type 1 receptor (AT1R) [11]. Activation of AT1R stimulates superoxide formation, inflammatory cascades, and cell apoptosis [12], whereas blockade of AT1R improves islet structure and function by decreasing oxidative stress–mediated apoptosis [13]. Angiotensin II type 1 receptor blockers (ARBs) decrease insulin resistance in obese diabetic animal models [14]. In addition, RAS activation induces superoxide-producing NAD(P)H oxidase in a rat model of acute pancreatitis [15], suggesting that up-regulation of islet RAS enhances oxidative stress and damages β-cell function. We recently reported that telmisartan, an ARB, attenuates fatty-acid–induced oxidative stress and NAD(P)H oxidase activity in pancreatic β-cells [16]. Therefore, blockade of RAS may preserve β-cell function and be a useful therapy for type 2 diabetes mellitus.

Glucagon-like peptide–1 (GLP-1), which is secreted from intestinal L cells in response to nutrient ingestion, is a potential therapeutic substance in the treatment of diabetes [17], [18]. Glucagon-like peptide–1 receptor (GLP-1R), a G-protein–coupled receptor, was first cloned from rat pancreatic islets [19] and later from human pancreatic islets [20]. Combined with GLP-1R, GLP-1 stimulates insulin secretion in a glucose-dependent manner [21], decreases β-cell apoptosis, and increases islet cell mass [22]. Exendin-4, a GLP-1 receptor agonist, promotes β-cell growth and survival; these effects are mainly mediated by insulin receptor substrate-2 (IRS-2) induction via increased intracellular cyclic adenosine monophosphate levels [23]. Previous studies have reported that IRS2 plays a crucial role in β-cell growth and survival [24], [25]. Glucagon-like peptide–1 reportedly promotes β-cell growth and survival by increasing protein kinase B (PKB; also called Akt) levels in β-cells both in vivo in db/db mice and in vitro in INS-1 cells [26], [27], [28]. Protein kinase B plays a major role in phosphoinositide 3-kinase (PI3K)–mediated survival effects [29]. Activated PKB can directly phosphorylate and, thereby, inactivate several components of the apoptotic machinery, including members of the transcription factor forkhead family [30]. Indeed, PKB is being increasingly implicated as a key player in the regulation of β-cell growth and survival [31]. Phosphorylation of forkhead box O1 (FoxO1) by PKB causes redistribution of FoxO1 from the nucleus to the cytoplasm, and the resulting decrease in nuclear FoxO1 has been proposed as a possible mechanism for the inhibition of FoxO1-mediated transcription [32]. Forkhead box O1 inhibition plays a role in the proliferative and antiapoptotic actions of GLP-1 in β-cells [33].

Although GLP-1 prevents apoptosis in pancreatic β-cells [22] and ARB improves islet structure and function [13], the additive effects of GLP-1 and ARB on glucolipotoxicity in pancreatic β-cells are unclear. In the present study, we examined the additive effects and possible mechanisms of GLP-1 and candesartan, an ARB, on glucolipotoxicity in pancreatic β-cells by using an apoptosis assay, immunoblotting, and immunoprecipitation. We found that GLP-1 and/or candesartan significantly prevented both high-glucose– and palmitate-induced apoptosis in MIN6 cells and that stronger effects were induced by combination treatment. Moreover, GLP-1 and/or candesartan significantly prevented high glucose levels via a palmitate-induced decrease in phosphorylation of IRS-2, PKB, and FoxO1 in MIN6 cells, suggesting that GLP-1 and candesartan play important roles in the prevention of β-cell apoptosis via the IRS-2/PI3K/PKB/FoxO1 signaling pathway.

Section snippets

Materials

Dulbecco modified Eagle medium (DMEM), palmitate (sodium salt), and protease inhibitor cocktail were purchased from Sigma (St Louis, MO); and candesartan was kindly provided by Takeda Chemical Industries (Osaka, Japan). Human GLP-1 fragment 7-36 amide was obtained from CS Bio (Tokyo, Japan). Penicillin G, streptomycin, amphotericin B, fetal bovine serum, and RPMI medium 1640 were obtained from Gibco (Auckland, New Zealand). Trizol was purchased from Life Technologies (Oslo, Norway). Wortmannin,

GLP-1 and candesartan protected islets and MIN6 cells from high-glucose–/palmitate-induced apoptosis

As compared with control group (low glucose, 5.6 mmol/L), high glucose (25 mmol/L) significantly increased apoptosis levels in both islets and MIN6 cells (Fig. 1). The addition of palmitate enhanced the high-glucose–induced apoptosis in mouse islets by 99% and in MIN6 cells by 68% (P < .01) (Fig. 1). In contrast, the addition of GLP-1 or candesartan significantly inhibited apoptosis in mouse islets (by 26% and 23%, respectively, vs high glucose plus palmitate) and in MIN6 cells (by 48% and 38%,

Discussion

We examined the additive effects of GLP-1 and candesartan on glucolipotoxicity in pancreatic β-cells, and we explored the possible mechanisms. Our data showed that GLP-1 and candesartan not only significantly prevented glucolipotoxicity-induced apoptosis, but also prevented the glucolipotoxicity-induced decrease in the phosphorylation of IRS-2, PKB, and FoxO1 in MIN6 cells. These results suggest that GLP-1 and candesartan play important roles in the prevention of β-cell apoptosis via a

Acknowledgment

This work was supported by Japan Science and Technology Agency.

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    Author contributions: Masamitsu Nakazato, Masanari Mizuta, and Hiroaki Ueno conceived the experimental plan and discussed analyses and interpretation. Hong-Wei Wang, Yukie Saitoh, and Kenji Noma performed the experiments. Hong-Wei Wang performed statistical analysis and wrote the manuscript.

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