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;...

Over the past decade, important roles for the 84–88 kDa Group VIA Ca2+-independent phospholipase A2 (iPLA2β) in various organs have been described. We demonstrated that iPLA2β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA2β, and certain stimuli promote perinuclear localization of iPLA2β. To gain a better understanding of its mobilization, iPLA2β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA2β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA2β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA2β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA2β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA2β in β-cells and we find here that these stimuli promote differential localization of iPLA2β in subcellular organelles. Further, mass spectrometric analyses identified iPLA2β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA2β and redistribution of iPLA2β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA2β facilitates its participation in multiple biological processes.

Evidence that group VIA cytosolic calcium-independent phospholipase A 2 (iPLA 2 ␤) participates in ␤-cell signal transduction includes the observations that inhibition of iPLA 2 ␤ with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA 2 ␤ amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA 2 2 ) is a diverse group of enzymes that catalyze hydrolysis of the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1,2). Among the PLA 2 s is an 84-kDa cytosolic PLA 2 that does not require Ca 2ϩ for catalysis and is designated group VIA cytosolic calcium-independent phospholipase A 2 (iPLA 2 ␤) (3-5).A role for iPLA 2 ␤ in signal transduction in insulinsecreting ␤-cells is suggested by the observations that inhibition of iPLA 2 ␤ activity with the bromoenol lactone (BEL) suicide substrate of iPLA 2 ␤ suppresses insulin secretion, and overexpression of iPLA 2 ␤ amplifies glucoseand forskolin-stimulated insulin secretion from pancreatic islets and INS-1 insulinoma cells (6,7). Stimulation of iPLA 2 ␤-overexpressing INS-1 cells with cAMP-elevating agents also induces translocation of iPLA 2 ␤ to the perinuclear region (7). This is of interest because glucose promotes both ␤-cell insulin secretion and proliferation, and glucose-induced INS-1 cell mitogenesis is cAMP dependent (8). To characterize iPLA 2 ␤ subcellular movements further, we developed INS-1 cell lines that overexpress iPLA 2 ␤ as a fusion protein with enhanced green fluorescent protein (EGFP) so that green fluorescence associated with EGFP reflects the location of iPLA 2 ␤. Simultaneous monitoring of florescence associated with various tracking molecules allowed us to identify specific subcellular organelles with which iPLA 2 ␤ associates when cells are stimulated. RESEARCH DESIGN AND METHODSPreparation of a construct for expressing iPLA 2 ␤ as a fusion protein with EGFP and selection of stably transfected clones. Full-length iPLA 2 ␤ cDNA was amplified by PCR using the following primer set: sense, 5Ј AGCTTCGAATTCATGCAGTTCTTTGGACGC-3, and antisense, 5Ј-TTC GATATCGGGAGATAGCAGCAGCTGG-3Ј. The amplified full-length iPLA 2 ␤ from the pMSCV-neo-iPLA 2 ␤ constructs were then subcloned into the pEGFP-N2 (Clontech, Palo Alto, CA) after the immediate early promoter of cytomegalovirus major and before EGFP coding sequences in the same code-reading frame with EGFP. The EGFP-N2 control vector and the construct encoding iPLA 2 ␤-EGFP (FPN2) fusion protein were transfected into INS-1 cells with a Gene PORTER transfection system according to the manufacture's instructions (Gene Therapy Systems, San Diego, CA). Stably transfected clones were selected using G418 (0.4 mg/ml This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier.BEL, bromoenol lactone; EGFP, enha...

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2 ␤) does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate. Several potential functions have been proposed for iPLA 2 ␤. Our studies indicate that iPLA 2 ␤ is expressed in ␤-cells and participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. If iPLA 2 ␤ plays a signaling role in glucosestimulated insulin secretion, then conditions that impair iPLA 2 ␤ functions might contribute to the diminished capacity of ␤-cells to secrete insulin in response to glucose, which is a prominent characteristic of type 2 diabetes. Our recent studies suggest that iPLA 2 ␤ might also participate in ␤-cell proliferation and apoptosis and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the iPLA 2 ␤ protein level reveals that ␤-cells express multiple isoforms of the enzyme, and our studies involve the hypothesis that different isoforms have different functions. Diabetes 53 (Suppl. 1)