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;...

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca2؉ -independent phospholipase A 2 (iPLA 2 ␤)-mediated ceramide generation, but the mechanism by which iPLA 2 ␤ and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA 2 ␤ overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA 2 ␤ inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA 2 ␤ participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA 2 ␤ accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (⌬⌿) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in ⌬⌿ and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA 2 ␤ or NSMase, suggesting that iPLA 2 ␤-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA 2 ␤ or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA 2 ␤-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.Diabetes mellitus is the most prevalent human metabolic disease resulting from the loss and/or dysfunction of ␤-cells in pancreatic islets. Type 1 diabetes mellitus (T1DM) 2 is caused by autoimmune ␤-cell destruction (1) and apoptosis plays a prominent role in the loss of ␤-cells during development of T1DM (1, 2). Type 2 diabetes mellitus (T2DM) results from a progressive decline in ␤-cell function and chronic insulin resistance (3, 4) that is also associated with decreases in ␤-cell mass due to increased ␤-cell apoptosis (5, 6). Autopsy studies indicate that the ␤-cell mass in obese T2DM subjects is smaller than that in obese non-diabetic subjects (7,8) and that the loss in ␤-cell function in non-obese T2DM is associated with decreases in ␤-cell mass (5, 6). ␤-Cell mass is regulated by a balance between ␤-cell replication/neogenesis and ␤-cell death resulting from apoptosis (9, 10). Findings in rodent models of T2DM (10, 11) and in human T2DM (5, 6) indicate that the decrease in ␤-cell mass in T2DM is not attributable to reduced ␤-cell proliferation or neogenesis but to increased ␤-cell apoptosis. Emerging evidence also suggests that cytokine-mediated ␤-cell apoptosis is a contributor to ␤-cell death...

Obesity predisposes to human type 2 diabetes, the most common cause of diabetic retinopathy. To determine if high-fat diet–induced diabetes in mice can model retinal disease, we weaned mice to chow or a high-fat diet and tested the hypothesis that diet-induced metabolic disease promotes retinopathy. Compared with controls, mice fed a diet providing 42% of energy as fat developed obesity-related glucose intolerance by 6 months. There was no evidence of microvascular disease until 12 months, when trypsin digests and dye leakage assays showed high fat–fed mice had greater atrophic capillaries, pericyte ghosts, and permeability than controls. However, electroretinographic dysfunction began at 6 months in high fat–fed mice, manifested by increased latencies and reduced amplitudes of oscillatory potentials compared with controls. These electroretinographic abnormalities were correlated with glucose intolerance. Unexpectedly, retinas from high fat–fed mice manifested striking induction of stress kinase and neural inflammasome activation at 3 months, before the development of systemic glucose intolerance, electroretinographic defects, or microvascular disease. These results suggest that retinal disease in the diabetic milieu may progress through inflammatory and neuroretinal stages long before the development of vascular lesions representing the classic hallmark of diabetic retinopathy, establishing a model for assessing novel interventions to treat eye disease.

Studies involving pharmacologic inhibition or transient reduction of Group

Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca2؉ -independent phospholipase A 2 (iPLA 2 ␤)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA 2 ␤ and ER stress, we compared ␤-cell lines generated from wild type (WT) and Akita mice. The Akita mouse is a spontaneous model of ER stress that develops hyperglycemia/ diabetes due to ER stress-induced ␤-cell apoptosis. Consistent with a predisposition to developing ER stress, basal phosphorylated PERK and activated caspase-3 are higher in the Akita cells than WT cells. Interestingly, basal iPLA 2 ␤, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (⌬⌿) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates ⌬⌿ loss and apoptosis of Akita cells and is associated with increases in iPLA 2 ␤, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA 2 ␤ small interfering RNA, however, suppresses NSMase message, ⌬⌿ loss, and apoptosis. The iPLA 2 ␤ gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA 2 ␤ in the Akita cells and suppresses increases in mSREBP-1 and iPLA 2 ␤ due to thapsigargin. These findings suggest that ER stress leads to generation of mSREBP-1, which can bind to the sterol-regulatory element in the iPLA 2 ␤ gene to promote its transcription. Consistent with this, SREBP-1, iPLA 2 ␤, and NSMase messages in Akita mouse islets are higher than in WT islets.␤-Cell loss due to apoptosis contributes to the progression and development of Type 1 or Type 2 diabetes mellitus (T1DM 2 or T2DM, respectively). This is supported by autopsy studies that reveal reduced ␤-cell mass in obese T2DM subjects in comparison with obese non-diabetic subjects (1, 2) and reveal that the loss in ␤-cell function in non-obese T2DM is associated with decreases in ␤-cell mass (3, 4). Other evidence suggests that cytokines cause ␤-cell apoptosis during the development of autoimmune T1DM (5-8). It is therefore important to understand the mechanisms underlying ␤-cell apoptosis if this process is to be prevented or delayed.␤-Cell mass is regulated by a balance between ␤-cell replication/neogenesis and ␤-cell death resulting from apoptosis (9, 10). Findings in rodent models of T2DM (10, 11) and in human T2DM (3, 4) indicate that the decrease in ␤-cell mass in T2DM is not attributable to reduced ␤-cell proliferation or neogenesis but to increased ␤-cell apoptosis (12). In addition to the intrinsic and extrinsic apoptotic pathways, apoptosis due to prolonged endoplasmic reticulum (ER) stress (12, 13) has been reported in various diseases, including Alzheimer and Parkinson diseases (14).Evidence from the Akita (15, 16) and NOD.k iHEL (17) mouse model...

Many cells express a group VIA 84 kDa phospholipase A 2 (iPLA 2 β) that is sensitive to inhibition by a bromoenol lactone (BEL) suicide substrate. Inhibition of iPLA 2 β in pancreatic islets and insulinoma cells suppresses, and overexpression of iPLA 2 β in INS-1 insulinoma cells amplifies, glucose-stimulated insulin secretion, suggesting that iPLA 2 β participates in secretion. Western blotting analyses reveal that glucose-responsive 832/13 INS-1 cells express essentially no 84 kDa iPLA 2 β-immunoreactive protein but predominantly express a previously unrecognized immunoreactive iPLA 2 β protein in the 70 kDa region that is not generated by a mechanism of alternate splicing of the iPLA 2 β transcript. To determine if the 70 kDa-immunoreactive protein is a short isoform of iPLA 2 β, protein from the 70 kDa region was digested with trypsin and analyzed by mass spectrometry. Such analyses reveal several peptides with masses and amino acid sequences that exactly match iPLA 2 β tryptic peptides. Peptide sequences identified in the 70 kDa tryptic digest include iPLA 2 β residues 7-53, suggesting that the N-terminus is preserved. We also report here that the 832/13 INS-1 cells express iPLA 2 β catalytic activity and that BEL inhibits secretagogue-stimulated insulin secretion from these cells but not the incorporation of arachidonic acid into membrane PC pools of these cells. These observations suggest that the catalytic iPLA 2 β † This research was supported in part by grants from National Institutes of Health (R37-DK34388, P41-RR00954, P01-HL57278, P60-DK20579, P30-DK56341) and by an Award (S.R.) from the American Diabetes Association. 1 Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone suicide inhibitor of iPLA 2 β; BME, β-mercaptoethanol; BSA, bovine serum albumin; bp, base pairs; cPLA 2 , Group IV cytosolic phospholipase A 2 ; dpm, disintegrations per minute; ECL, enhanced chemiluminescence; FBS, fetal bovine serum; GPC, glycerophosphocholine; IF, immunofluorescence; iPLA 2 β, β-isoform of Group VIA calcium-independent phospholipase A 2 ; kDa, kilodaltons; LC/ESI, liquid chromatography/electrospray ionization; MALDI/TOF, matrix-assisted laser desorption ionization/time-of-flight; MS, mass spectrometry; OE, iPLA 2 β overexpressing cells; O/N, overnight; PAGE, polyacrylamide gel electrophoresis; Q-TOF, quadrupole-time-of-flight; PBS, phosphate-buffered saline; PC, phosphatidylcholine; piPLA 2 , polyclonal iPLA 2 ; PIC, protease inhibitor cocktail; PLA 2 , phospholipase A 2 ; SDS, sodium dodecyl sulfate; TBS-T, Tris-buffered saline-tween; TLC, thin-layer chromatography; RT, room temperature; RT-PCR, reverse transcriptionpolymerase chain reactions; V, empty vector transfected cells. activity expressed in 832/13 INS-1 cells is attributable to a short isoform of iPLA 2 β and that this isoform participates in insulin secretory but not in membrane phospholipid remodeling pathways. Further, the finding that pancreatic islets also express predominantly a 70 kDa iPLA 2 β-immunoreactive protein suggests that...

Death of β-cells due to apoptosis is an important contributor to β-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)β) in apoptosis of insulinoma cells due to ER stress. To examine whether islet β-cells are similarly susceptible to ER stress and undergo iPLA(2)β-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)β protein is expressed predominantly in the β-cells of human islets and that thapsigargin-induced ER stress promotes β-cell apoptosis, as reflected by increases in activated caspase-3 in the β-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)β message, protein, and activity, iPLA(2)β-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that β-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)β protein in human islet β-cells and that induction of iPLA(2)β during ER stress contributes to human islet β-cell apoptosis. We hypothesize that modulation of iPLA(2)β activity might reduce β-cell apoptosis and this would be beneficial in delaying or preventing β-cell dysfunction associated with diabetes.