Endoplasmic Reticulum Stress–Induced Apoptosis Is Partly Mediated by Reduced Insulin Signaling Through Phosphatidylinositol 3-Kinase/Akt and Increased Glycogen Synthase Kinase-3 in Mouse Insulinoma Cells

An imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum (ER) results in stress that has been increasingly implicated in pancreatic islet -cell apoptosis and diabetes. Because insulin/IGF/Akt signaling has been implicated in -cell survival, we sought to determine whether this pathway is involved in ER stress–induced apoptosis. Mouse insulinoma cells treated with pharmacological agents commonly used to induce ER stress exhibited apoptosis within 48 h. ER stress–induced apoptosis was inhibited by cotreatment of the cells with IGF-1. Stable cell lines were created by small-interfering RNA (siRNA) with graded reduction of insulin receptor expression, and these cells had enhanced susceptibility to ER stress– induced apoptosis and reduced levels of phospho–glycogen synthase kinase 3 (GSK3 ). In control cells, ER stress–induced apoptosis was associated with a reduction in phospho-Akt and phospho-GSK3 . To further assess the role of GSK3 in ER stress–induced apoptosis, stable cell lines were created by siRNA with up to 80% reduction in GSK3 expression. These cells were found to resist ER stress–induced apoptosis. These results illustrate that ER stress–induced apoptosis is mediated at least in part by signaling through the phosphatidylinositol 3-kinase/Akt/GSK3 pathway and that GSK3 represents a novel target for agents to promote -cell survival. Diabetes 54:968–975, 2005 Molecular mechanisms involved in various forms of pancreatic islet -cell failure are being discovered, and most recently the endoplasmic reticulum (ER) has been shown to mediate signals that may contribute to this process (1,2). All cells regulate the capacity of the ER to fold and process proteins and thereby control the balance between protein demand and folding capacity. An imbalance in this process triggers an aberrant process referred to as ER stress, which if unabated can lead to apoptosis. Pancreatic -cells have highly developed ER, and they also abundantly express ER stress transducer proteins including Ire1 , PERK (pancreatic ER kinase or PKR-like ER kinase), and BiP (3). Recent studies have shown that these cells may be particularly vulnerable to ER stress. A targeted disruption of Chop, a C/EBP homologous protein strongly implicated in ER stress–induced apoptosis, resulted in resistance to nitric oxide–induced apoptosis in -cells as well as amelioration of -cell failure caused by a mutated insulin gene (Akita mouse) (4,5). PERK is activated by ER stress, and it in turn phosphorylates eukaryotic initiation factor 2 (eIF2 ), which leads to attenuation in protein synthesis. Loss of PERK (3,6) or a mutant eIF2 incapable of undergoing phosphorylation by PERK (eIF2 ) in mice (7) leads to diabetes due to destruction of pancreatic -cells. Mutations in the human EIF2AK3 (PERK) gene are the cause of a rare recessive disorder, the Wolcott-Rallison syndrome, which is characterized by early-onset diabetes (8). These studies highlight that ER stress is a likely contributor to the -cell dysfunction in diabetes. Recent evidence has indicated the importance of ER stress and reduced insulin signaling in the fat-feeding model of diabetes (9). In these experiments, it was shown that fat feeding was associated with markers of ER stress, C-Jun NH2-terminal kinase (JNK) activation, and insulin resistance in the liver (9). Genetic mouse models deficient in insulin or IGF-1 receptors, or in insulin receptor substrate-1 or -2, exhibit various impairments in -cell mass and/or function (10–19). Insulin/IGF signaling through phosphatidylinositol 3 (PI3)-kinase and Akt are well-established activators of survival in numerous cell types, and overexpression of Akt specifically in pancreatic islet -cells resulted in marked expansion of cell number and From the Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, St. Louis, Missouri. Address correspondence and reprint requests to M. Alan Permutt, MD, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail: [email protected]. Received for publication 28 June 2004 and accepted in revised form 1 January 2005. eIF2 , eukaryotic initiation factor 2 ; ER, endoplasmic reticulum; GSK3 , glycogen synthase kinase 3 ; JNK, c-Jun NH2-terminal kinase; IKRD, insulin receptor knock down; PARP, poly-ADP-ribose polymerase; PERK, PKR-like ER kinase/pancreatic eIF2 kinase; PI3, phosphatidylinositol 3; Q-VD-OPh, N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)methyl ketone; siRNA, small-interfering RNA. S.S. is currently affiliated with the Division of Digestive Diseases, Emory University School of Medicine, Atlanta, Georgia. S.S., M.O., and Z.L. contributed equally to this study. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.