Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway

Abstract

Aims/hypothesis The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. Methods The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. Results Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitateinduced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitateinduced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. Conclusions/interpretation Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7-and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

The p66Shc redox adaptor protein is induced by saturated fatty acids and mediates lipotoxicity-induced apoptosis in pancreatic beta cells

“…Rat insulin-secreting INS-1E cells (passage 15-30; a kind gift from C. B. Wollheim, University of Geneva, Switzerland) were grown and treated with or without 0.5 mmol/l palmitic acid (Sigma-Aldrich, St Louis, MO, USA), as previously reported [6,28]. All chemical inhibitors were provided by Calbiochem (Darmstadt, Germany).…”

“…RNA isolation and cDNA synthesis were conducted as previously described [6,27]. Primers were designed using Primer Express version 3.0 (Life Technologies, Carlsbad, CA, USA) and purchased from Life Technologies.…”

“…Cells lysates were obtained and analysed by immunoblotting, as previously described [6]. A list of the antibodies used is shown in ESM Table 3.…”

“…6b). Interestingly, glucagon-like peptide 1 (GLP-1) receptor agonists, which reportedly inhibit palmitate-induced apoptosis by preventing JNK phosphorylation [6], also inhibited p66 Shc phosphorylation on Ser 36 . Pretreatment of Ad/mock cells with 10 nmol/l exendin-4 for 16 h resulted in abrogation of palmitate-induced Ser 36 phosphorylation of p66 Shc (p<0.05; Fig.…”

“…FA-induced beta cell apoptosis involves a variety of signalling mechanisms, including endoplasmic reticulum (ER) stress induction [4], mitochondrial dysfunction [5], activation of specific intracellular kinases such as the members of the mitogen-activated protein kinase (MAPK) family c-Jun N-terminal kinase (JNK) and p38 MAPK [6,7] and protein kinase C (PKC)δ [8], and peroxisome-generated reactive oxygen species (ROS) [7,9]. The tumour suppressor protein p53 is also implicated in FA-induced beta cell apoptosis [7,10], since both palmitate and oleate were shown to stimulate apoptosis of NIT-1 beta cells through p53 [7], and p53 inhibition was found to be involved in growth factor-dependent promotion of beta cell survival via Akt/protein kinase B [10].…”