Apoptosis of Insulin-Secreting Cells Induced by Endoplasmic Reticulum Stress Is Amplified by Overexpression of Group VIA Calcium-Independent Phospholipase A₂ (iPLA₂β) and Suppressed by Inhibition of iPLA₂β

Abstract

The death of insulin-secreting β-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive β-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to β-cell loss in diabetes. Agents that deplete ER calcium levels induce β-cell apoptosis by a process that is independent of increases in [Ca 2+ ] i . Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A 2 (iPLA 2 β). Overexpression of iPLA 2 β amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA 2 β expression in various cell lines, and apoptosis is associated with stimulation of iPLA 2 β activity, perinuclear accumulation of iPLA 2 β protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA 2 β to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA 2 β. These findings indicate that iPLA 2 β participates in ER stress-induced apoptosis, a pathway that promotes β-cell death in diabetes.Diabetes mellitus (DM) 1 is the most prevalent human endocrine disease, and it results from loss and/or dysfunction of insulin-secreting β-cells in pancreatic islets. Type I DM is caused † This research was supported in part by grants from the National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, and P30-DK56341) and by an Award (to S.R.) from the American Diabetes Association.© 2004 American Chemical Society * To whom correspondence should be addressed: Department of Medicine, Washington University School of Medicine, Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-8194. Fax: (314) 362-8188. sramanad@im.wustl.edu. 1 Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone suicide inhibitor of iPLA 2 β; BME, β-mercaptoethanol; BSA, bovine serum albumin; CAD, collisionally activated dissociation; CM, ceramide; CNL, constant neutral loss; C3-I, caspase-3 inhibitor; cPLA 2 , group IV cytosolic phospholipase A2; ECL, enhanced chemiluminescence; EGFP, enhanced green fluorescence protein; ER, endoplasmic reticulum; ESI, electrospray ionization; FBS, fetal bovine serum; IF, immunocytofluorescence; iPLA 2 β, β-isoform of group VIA calcium-independent phospholipase A 2 ; IS, internal standard; MS, mass spectrometry; OE, iPLA 2 β-overexpressing cells; O/N, overnight; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIC, protease inhibitor cocktail; PLA 2 , phospholipase A 2 ; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; SERCA, sarcoen-doplasmic reticulum Ca 2+ -ATPase; TBS-T, Tris-buffered saline-tween; TIC, total ion current; TLC, thin-layer chromatography;...

Activation Mechanism for CRAC Current and Store-operated Ca2+ Entry

“…Another prominent feature of some variants of iPLA 2 ␤ is multiple ankyrin repeats in the N terminus, which may be important for their cellular localization and assembly into specific signaling domains. In vascular smooth muscle cells we found that iPLA 2 ␤ remains functional at the inner leaflet of excised plasma membrane patches (2), which was consistent with its plasma membrane localization reported in some cell types (25,26). However, in numerous studies iPLA 2 ␤ was found in cytosol (27)(28)(29), the inner membrane of mitochondria (30), and nucleus (25).…”

“…In vascular smooth muscle cells we found that iPLA 2 ␤ remains functional at the inner leaflet of excised plasma membrane patches (2), which was consistent with its plasma membrane localization reported in some cell types (25,26). However, in numerous studies iPLA 2 ␤ was found in cytosol (27)(28)(29), the inner membrane of mitochondria (30), and nucleus (25). There is also some evidence for its possible translocation to perinuclear and plasma membranes (25,31,32).…”

“…However, in numerous studies iPLA 2 ␤ was found in cytosol (27)(28)(29), the inner membrane of mitochondria (30), and nucleus (25). There is also some evidence for its possible translocation to perinuclear and plasma membranes (25,31,32). The existence of multiple splice variants of iPLA 2 ␤ isoform (33) as well as their post-translational modifications (34) may be some of the reasons for inconsistence in iPLA 2 ␤ localization.…”

Stromal Cell–Derived Factor-1 (SDF-1)/CXCL12 Attenuates Diabetes in Mice and Promotes Pancreatic β-Cell Survival by Activation of the Prosurvival Kinase Akt

“…Glucose deprivation induces apoptosis in ␤-cells (30,31). Thapsigargin inhibits endoplasmic reticulum Ca 2ϩ ATPase, produces endoplasmic reticulum stress, and induces apoptosis in INS-1 cells (32,33). Glucose deprivation induced caspase-3 activity by 80%, and this effect was completely reversed by the concurrent incubation with SDF-1 (Fig.…”

Male Mice That Do Not Express Group VIA Phospholipase A2 Produce Spermatozoa with Impaired Motility and Have Greatly Reduced Fertility

“…Physiological roles for PLA 2 s can be studied with genetic gainor loss-of-function manipulations. Overexpressing iPLA 2 ␤ in insulinoma cells, for example, provides evidence for its participation in exocytosis, cell proliferation, and apoptosis (12)(13)(14), and cPLA 2 ␣ gene disruption by homologous recombination has produced cPLA 2 ␣-null mice that reveal a role for cPLA 2 ␣ in parturition, allergic responses, and post-ischemic brain injury (15,16).…”