Spontaneous Development of Endoplasmic Reticulum Stress That Can Lead to Diabetes Mellitus Is Associated with Higher Calcium-independent Phospholipase A2 Expression: A ROLE FOR REGULATION BY SREBP-1

Abstract

Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca2+-independent phospholipase A2 (iPLA2β)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA2β and ER stress, we compared β-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 β-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 iPLA2β, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (ΔΨ) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates ΔΨ loss and apoptosis of Akita cells and is associated with increases in iPLA2β, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA2β small interfering RNA, however, suppresses NSMase message, ΔΨ loss, and apoptosis. The iPLA2β gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA2β in the Akita cells and suppresses increases in mSREBP-1 and iPLA2β 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 iPLA2β gene to promote its transcription. Consistent with this, SREBP-1, iPLA2β, and NSMase messages in Akita mouse islets are higher than in WT islets. Our recent studies indicate that endoplasmic reticulum (ER) stress causes INS-1 cell apoptosis by a Ca2+-independent phospholipase A2 (iPLA2β)-mediated mechanism that promotes ceramide generation via sphingomyelin hydrolysis and subsequent activation of the intrinsic pathway. To elucidate the association between iPLA2β and ER stress, we compared β-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 β-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 iPLA2β, mature SREBP-1 (mSREBP-1), phosphorylated Akt, and neutral sphingomyelinase (NSMase) are higher, relative abundances of sphingomyelins are lower, and mitochondrial membrane potential (ΔΨ) is compromised in Akita cells, in comparison with WT cells. Exposure to thapsigargin accelerates ΔΨ loss and apoptosis of Akita cells and is associated with increases in iPLA2β, mSREBP-1, and NSMase in both WT and Akita cells. Transfection of Akita cells with iPLA2β small interfering RNA, however, suppresses NSMase message, ΔΨ loss, and apoptosis. The iPLA2β gene contains a sterol-regulatory element, and transfection with a dominant negative SREBP-1 reduces basal mSREBP-1 and iPLA2β in the Akita cells and suppresses increases in mSREBP-1 and iPLA2β 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 iPLA2β gene to promote its transcription. Consistent with this, SREBP-1, iPLA2β, and NSMase messages in Akita mouse islets are higher than in WT islets. Introductionβ-Cell loss due to apoptosis contributes to the progression and development of Type 1 or Type 2 diabetes mellitus (T1DM 2The abbreviations used are: T1DMType 1 diabetes mellitusT2DMType 2 diabetes mellitusAKAkitapAktphosphorylated AktPERKpancreatic ER kinasepPERKphosphorylated PERKBELbromoenol lactone suicide inhibitor of iPLA2βDNdominant negativeERendoplasmic reticulumESI/MS/MSelectrospray ionization/tandem mass spectrometryGPCglycerophosphocholineiPLA2ββ-isoform of group VIA Ca2+-independent phospholipase A2NSMaseneutral sphingomyelinasePBSphosphate-buffered salinePLA2phospholipase A2mSREBP-1mature (or processed) SREBP-1TUNELterminal deoxynucleotidyl transferase-mediated (fluorescein) dUTP nick end labelingWTwild typesiRNAsmall interfering RNADAPI4′,6-diamidino-2-phenylindoleqRT-PCRquantitative reverse transcription-PCRSREsterol-regulatory elementDiOC6(3)3,3′-dihexyloxacarbocyanine iodide. or T2DM, respectively). This is supported by autopsy studies that reveal reduced β-cell mass in obese T2DM subjects in comparison with obese non-diabetic subjects (1.Klöppel G. Löhr M. Habich K. Oberholzer M. Heitz P.U. Surv. Synth. Pathol. Res. 1985; 4: 110-125PubMed Google Scholar, 2.Stefan Y. Orci L. Malaisse-Lagae F. Perrelet A. Patel Y. Unger R.H. Diabetes. 1982; 31: 694-700Crossref PubMed Scopus (0) Google Scholar) and reveal that the loss in β-cell function in non-obese T2DM is associated with decreases in β-cell mass (3.Butler A.E. Janson J. Bonner-Weir S. Ritzel R. Rizza R.A. Butler P.C. Diabetes. 2003; 52: 102-110Crossref PubMed Scopus (3225) Google Scholar, 4.Yoon K.H. Ko S.H. Cho J.H. Lee J.M. Ahn Y.B. Song K.H. Yoo S.J. Kang M.I. Cha B.Y. Lee K.W. Son H.Y. Kang S.K. Kim H.S. Lee I.K. Bonner-Weir S. J. Clin. Endocrinol. Metab. 2003; 88: 2300-2308Crossref PubMed Scopus (495) Google Scholar). Other evidence suggests that cytokines cause β-cell apoptosis during the development of autoimmune T1DM (5.Araki E. Oyadomari S. Mori M. Exp. Biol. Med. (Maywood). 2003; 228: 1213-1217Crossref PubMed Scopus (151) Google Scholar, 6.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (420) Google Scholar, 7.Mathis D. Vence L. Benoist C. Nature. 2001; 414: 792-798Crossref PubMed Scopus (755) Google Scholar, 8.Tisch R. McDevitt H. Cell. 1996; 85: 291-297Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). It is therefore important to understand the mechanisms underlying β-cell apoptosis if this process is to be prevented or delayed.β-Cell mass is regulated by a balance between β-cell replication/neogenesis and β-cell death resulting from apoptosis (9.Bernard C. Berthault M.F. Saulnier C. Ktorza A. 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Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1919) Google Scholar).Evidence from the Akita (15.Oyadomari S. Araki E. Mori M. Apoptosis. 2002; 7: 335-345Crossref PubMed Scopus (441) Google Scholar, 16.Oyadomari S. Koizumi A. Takeda K. Gotoh T. Akira S. Araki E. Mori M. J. Clin. Invest. 2002; 109: 525-532Crossref PubMed Scopus (778) Google Scholar) and NOD.k iHEL (17.Socha L. Silva D. Lesage S. Goodnow C. Petrovsky N. Ann. N.Y. Acad. Sci. 2003; 1005: 178-183Crossref PubMed Scopus (22) Google Scholar) mouse models suggests that ER stress can also lead to the development of diabetes mellitus as a consequence of β-cell apoptosis. Further, mutations in genes encoding the ER stress-transducing enzyme pancreatic ER kinase (PERK) (18.Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (992) Google Scholar) and the ER-resident protein involved in degradation of malfolded ER proteins have been clinically linked to diminished β-cell health (19.Delépine M. Nicolino M. Barrett T. Golamaully M. Lathrop G.M. Julier C. Nat. Genet. 2000; 25: 406-409Crossref PubMed Scopus (651) Google Scholar, 20.Takeda K. Inoue H. Tanizawa Y. Matsuzaki Y. Oba J. Watanabe Y. Shinoda K. Oka Y. Hum. Mol. Genet. 2001; 10: 477-484Crossref PubMed Scopus (258) Google Scholar). Other reports suggest that ER stress may also play a prominent role in the autoimmune destruction of β-cells during the development of T1DM (6.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (420) Google Scholar, 21.Fonseca S.G. Fukuma M. Lipson K.L. Nguyen L.X. Allen J.R. Oka Y. Urano F. J. Biol. Chem. 2005; 280: 39609-39615Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 22.Oyadomari S. Takeda K. Takiguchi M. Gotoh T. Matsumoto M. Wada I. Akira S. Araki E. Mori M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10845-10850Crossref PubMed Scopus (511) Google Scholar). Because the secretory function of β-cells endows them with a highly developed ER and the β-cell is one of the cells most sensitive to nitric oxide (23.Kröncke K.D. Brenner H.H. Rodriguez M.L. Etzkorn K. Noack E.A. Kolb H. Kolb-Bachofen V. Biochim. Biophys. Acta. 1993; 1182: 221-229Crossref PubMed Scopus (103) Google Scholar), it is not unexpected that β-cells exhibit a heightened susceptibility to autoimmune-mediated ER stress (24.Corbett J.A. McDaniel M.L. Diabetes. 1992; 41: 897-903Crossref PubMed Scopus (349) Google Scholar, 25.Mandrup-Poulsen T. Diabetologia. 1996; 39: 1005-1029Crossref PubMed Scopus (514) Google Scholar). In support of this, Wolfram syndrome, which is associated with juvenile onset diabetes mellitus, is recognized to be a consequence of chronic ER stress in pancreatic β-cells (21.Fonseca S.G. Fukuma M. Lipson K.L. Nguyen L.X. Allen J.R. Oka Y. Urano F. J. Biol. Chem. 2005; 280: 39609-39615Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 26.Yamada T. Ishihara H. Tamura A. Takahashi R. Yamaguchi S. Takei D. Tokita A. Satake C. Tashiro F. Katagiri H. Aburatani H. Miyazaki J. Oka Y. Hum. Mol. Genet. 2006; 15: 1600-1609Crossref PubMed Scopus (187) Google Scholar).In view of the evidence suggesting that ER stress-induced β-cell apoptosis may be a factor in the development of diabetes mellitus, it was of interest to elucidate the mechanisms involved. Recent work from our laboratory led to the identification of a Ca2+-independent phospholipase A2 (iPLA2β) as a key participant in ER stress-mediated apoptosis of INS-1 insulinoma cells. The iPLA2β, classified as a Group VIA isoform of iPLA2, is a member of a large family of PLA2s (27.Schaloske R.H. Dennis E.A. Biochim. Biophys. Acta. 2006; 1761: 1246-1259Crossref PubMed Scopus (720) Google Scholar) that is cytosolic and does not require Ca2+ for activity (28.Ma Z. Ramanadham S. Wohltmann M. Bohrer A. Hsu F.F. Turk J. J. Biol. Chem. 2001; 276: 13198-13208Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29.Mancuso D.J. Jenkins C.M. Gross R.W. J. Biol. Chem. 2000; 275: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 30.Tanaka H. Takeya R. Sumimoto H. Biochem. Biophys. Res. Commun. 2000; 272: 320-326Crossref PubMed Scopus (78) Google Scholar). It is activated by ATP, is inhibited by the bromoenol lactone suicide substrate (BEL) inhibitor of iPLA2β (31.Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. 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Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (88) Google Scholar).Further work indicated that ER stress activates iPLA2β in INS-1 cells, leading to neutral sphingomyelinase (NSMase) induction and generation of ceramides via hydrolysis of sphingomyelins (41.Lei X. Zhang S. Bohrer A. Bao S. Song H. Ramanadham S. Biochemistry. 2007; 46: 10170-10185Crossref PubMed Scopus (65) Google Scholar). Subsequently, the ceramides trigger mitochondrial apoptotic processes and cell death ensues. Consistent with this proposed mechanism, inhibition of iPLA2β or NSMase suppresses sphingomyelin hydrolysis and ceramide generation, mitochondrial abnormalities, and apoptosis (42.Lei X. Zhang S. Bohrer A. Ramanadham S. J. Biol. Chem. 2008; 283: 34819-34832Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Although a link between iPLA2β and ER stress-induced apoptotic pathway was gleaned from the above observations, it was derived from studies in which a chemical agent was used to induce ER stress and iPLA2β expression levels were genetically manipulated in an insulinoma cell line. Of importance was to determine whether a similar link existed under conditions where ER stress developed in the β-cell in the absence of chemical intervention. Here, we present studies in a β-cell line established from Akita mice (43.Nozaki J. Kubota H. Yoshida H. Naitoh M. Goji J. Yoshinaga T. Mori K. Koizumi A. Nagata K. Genes Cells. 2004; 9: 261-270Crossref PubMed Scopus (101) Google Scholar), which contain a spontaneous mutation of the insulin 2 gene (Ins2) (C96Y) that results in misfolding insulin in the ER leading to development of hyperglycemia/diabetes due to ER stress-induced β-cell apoptosis (44.Kayo T. Koizumi A. J. Clin. Invest. 1998; 101: 2112-2118Crossref PubMed Scopus (79) Google Scholar, 45.Yoshioka M. Kayo T. Ikeda T. Koizumi A. Diabetes. 1997; 46: 887-894Crossref PubMed Scopus (0) Google Scholar). Our findings reveal for the first time that predisposition to ER stress is associated with increased expression of iPLA2β and that its expression is modulated by activation of SREBP-1 (sterol-regulatory element-binding protein-1). We also present the first evidence in Akita mouse islet β-cells that substantiates these findings.DISCUSSIONBoth T1DM and T2DM are associated with losses in β-cells due to apoptosis. It is therefore important to determine the underlying mechanisms that contribute to this process. Recent reports in experimental models and in clinical settings suggest that ER stress is a potential cause of β-cell apoptosis during the development of diabetes mellitus (6.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (420) Google Scholar, 15.Oyadomari S. Araki E. Mori M. Apoptosis. 2002; 7: 335-345Crossref PubMed Scopus (441) Google Scholar, 16.Oyadomari S. Koizumi A. Takeda K. Gotoh T. Akira S. Araki E. Mori M. J. Clin. Invest. 2002; 109: 525-532Crossref PubMed Scopus (778) Google Scholar, 17.Socha L. Silva D. Lesage S. Goodnow C. Petrovsky N. Ann. N.Y. Acad. Sci. 2003; 1005: 178-183Crossref PubMed Scopus (22) Google Scholar, 18.Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (992) Google Scholar, 19.Delépine M. Nicolino M. Barrett T. Golamaully M. Lathrop G.M. Julier C. Nat. Genet. 2000; 25: 406-409Crossref PubMed Scopus (651) Google Scholar, 20.Takeda K. Inoue H. Tanizawa Y. Matsuzaki Y. Oba J. Watanabe Y. Shinoda K. Oka Y. Hum. Mol. Genet. 2001; 10: 477-484Crossref PubMed Scopus (258) Google Scholar, 21.Fonseca S.G. Fukuma M. Lipson K.L. Nguyen L.X. Allen J.R. Oka Y. Urano F. J. Biol. Chem. 2005; 280: 39609-39615Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 22.Oyadomari S. Takeda K. Takiguchi M. Gotoh T. Matsumoto M. Wada I. Akira S. Araki E. Mori M. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10845-10850Crossref PubMed Scopus (511) Google Scholar, 24.Corbett J.A. McDaniel M.L. Diabetes. 1992; 41: 897-903Crossref PubMed Scopus (349) Google Scholar, 25.Mandrup-Poulsen T. Diabetologia. 1996; 39: 1005-1029Crossref PubMed Scopus (514) Google Scholar, 26.Yamada T. Ishihara H. Tamura A. Takahashi R. Yamaguchi S. Takei D. Tokita A. Satake C. Tashiro F. Katagiri H. Aburatani H. Miyazaki J. Oka Y. Hum. Mol. Genet. 2006; 15: 1600-1609Crossref PubMed Scopus (187) Google Scholar). Because β-cells serve a secretory function, they are endowed with a highly developed ER that renders them particularly susceptible to developing ER stress.We found that ER stress causes INS-1 cell apoptosis, in part, by an iPLA2β-dependent mechanism (40.Ramanadham S. Hsu F.F. Zhang S. Jin C. Bohrer A. Song H. Bao S. Ma Z. Turk J. Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (88) Google Scholar). Subsequent studies revealed a previously unrecognized pathway (41.Lei X. Zhang S. Bohrer A. Bao S. Song H. Ramanadham S. Biochemistry. 2007; 46: 10170-10185Crossref PubMed Scopus (65) Google Scholar, 42.Lei X. Zhang S. Bohrer A. Ramanadham S. J. Biol. Chem. 2008; 283: 34819-34832Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) involving accumulation of ceramides via an iPLA2β-mediated induction of NSMase. The ceramides, generated from NSMase-catalyzed hydrolysis of sphingomyelins, promote mitochondrial abnormalities and amplify the apoptosis outcome. The roles for iPLA2β and NSMase in this process are supported by the findings of suppression of sphingomyelin hydrolysis, ceramide generation, mitochondrial activation, and apoptosis following inactivation of iPLA2β or NSMase (41.Lei X. Zhang S. Bohrer A. Bao S. Song H. Ramanadham S. Biochemistry. 2007; 46: 10170-10185Crossref PubMed Scopus (65) Google Scholar, 42.Lei X. Zhang S. Bohrer A. Ramanadham S. J. Biol. Chem. 2008; 283: 34819-34832Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).The above proposed schema of iPLA2β involvement in β-cell apoptosis was derived from studies in INS-1 cells in which ER stress was induced with thapsigargin. The plausibility of the pathway is strengthened by the finding that it is amplified by overexpression of iPLA2β and suppressed by inactivation of iPLA2β. However, identification of a similar role for iPLA2β in a spontaneous ER stress model would significantly strengthen the assertion that iPLA2β participates in ER stress-induced β-cell apoptosis. We therefore chose to address this issue in the Akita mouse model. However, because the islet yield from these mice is limiting, we pursued studies in a β-cell line established from the Akita mice (43.Nozaki J. Kubota H. Yoshida H. Naitoh M. Goji J. Yoshinaga T. Mori K. Koizumi A. Nagata K. Genes Cells. 2004; 9: 261-270Crossref PubMed Scopus (101) Google Scholar). The findings in these cells were compared with a β-cell line derived from isogenic WT littermates.Herein, we find that even under basal conditions, the Akita β-cells are predisposed to developing ER stress, as reflected by the greater induction of pPERK, and to be more prone to undergo apoptosis, as revealed by the greater abundance of TUNEL-positive cells and activated caspase-3, relative to WT β-cells. The greater propensity of the Akita cells to develop ER stress under basal conditions is also revealed by the nearly 2-fold higher basal splicing of XBP1 mRNA in the Akita cells, relative to isogenic WT counterparts (58.Han D. Lerner A.G. Vande Walle L. Upton J.P. Xu W. Hagen A. Backes B.J. Oakes S.A. Papa F.R. Cell. 2009; 138: 562-575Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar).The Akita cells also expressed higher message for NSMase, contained lower abundances of sphingomyelins, and exhibited a higher incidence of loss in mitochondrial membrane potential (ΔΨ). Permeabilization of the outer mitochondrial membrane is considered to be a “point of no return” during apoptosis (59.Chipuk J.E. Bouchier-Hayes L. Green D.R. Cell Death Differ. 2006; 13: 1396-1402Crossref PubMed Scopus (424) Google Scholar), and PTP opening alters outer mitochondrial membrane permeability. Prolonged activation of PTP leads to loss in ΔΨ and release of cytochrome c, a necessary event in the mitochondrial cell death pathway (60.Garrido C. Galluzzi L. Brunet M. Puig P.E. Didelot C. Kroemer G. Cell Death Differ. 2006; 13: 1423-1433Crossref PubMed Scopus (844) Google Scholar, 61.Korsmeyer S.J. Wei M.C. Saito M. Weiler S. Oh K.J. Schlesinger P.H. Cell Death Differ. 2000; 7: 1166-1173Crossref PubMed Scopus (835) Google Scholar), from the mitochondrial intermembrane space into the cytosol. Following its release into the cytosol, the proapoptotic cytochrome c forms the apoptosome complex with apoptosis protease-activating factor-1 to induce caspases and subsequent apoptosis (62.Schafer Z.T. Kornbluth S. Dev. Cell. 2006; 10: 549-561Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). The present findings therefore are consistent with the triggering of mitochondrial abnormalities as a consequence of increased sphingomyelin hydrolysis in Akita β-cells (41.Lei X. Zhang S. Bohrer A. Bao S. Song H. Ramanadham S. Biochemistry. 2007; 46: 10170-10185Crossref PubMed Scopus (65) Google Scholar, 42.Lei X. Zhang S. Bohrer A. Ramanadham S. J. Biol. Chem. 2008; 283: 34819-34832Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Most intriguingly, the Akita cells expressed higher iPLA2β protein than the WT cells. This was reflected by a nearly 3-fold higher basal cytosolic iPLA2β specific activity. These findings strengthen the likelihood that iPLA2β-mediated NSMase activation is a critical factor in ER stress-induced β-cell apoptosis. To further establish this possibility, the effects of accelerating ER stress in the Akita cells were assessed. Exposure to thapsigargin caused elevations in caspase-3 activation and abundance of TUNEL-positive cells that were accompanied by temporal increases in iPLA2β protein and specific activity in both WT and Akita β-cells. However, these outcomes were accelerated in the Akita cells. Thapsigargin also induced NSMase in both WT and Akita cells, and this occurred earlier and the -fold increases in message were greater in the Akita cells. That this was an iPLA2β-dependent effect was evidenced by the suppression in NSMase induction following inactivation of iPLA2β in both WT and Akita cells.To establish that the outcomes measured in the Akita β-cells require iPLA2β, the Akita cells were transfected with iPLA2β siRNA to knock down iPLA2β message and protein levels. The siRNA protocol resulted in dramatic suppression of not only basal but also thapsigargin-induced increases in iPLA2β message and protein. Further, whereas thapsigargin-induced NSMase induction, mitochondrial membrane potential loss, and apoptosis were unaffected by transfection with control siRNA, they were significantly suppressed in Akita cells transfected with the iPLA2β siRNA. In fact, the higher basal apoptosis was also significantly decreased in these cells. These findings indicate that iPLA2β is a critical factor during the development and progression of ER stress-induced abnormalities in the β-cell.Our recent finding of higher iPLA2β protein and activity in the ER and mitochondria of INS-1 cells exposed to thapsigargin was suggestive of increased mobilization of iPLA2β by these organelles with the onset and development of ER stress (41.Lei X. Zhang S. Bohrer A. Bao S. Song H. Ramanadham S. Biochemistry. 2007; 46: 10170-10185Crossref PubMed Scopus (65) Google Scholar, 42.Lei X. Zhang S. Bohrer A. Ramanadham S. J. Biol. Chem. 2008; 283: 34819-34832Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, the surprising finding of higher basal iPLA2β protein expression in Akita cells in the present study raises the possibility that it may in fact be subject to pre- and post-transcriptional regulation under stressful conditions.Other than ATP, only two potential candidates have been identified as regulating iPLA2β: CaMKIIβ and SREBP-1. Whereas CaMKIIβ is reported to activate iPLA2β (63.Wang Z. Ramanadham S. Ma Z.A. Bao S. Mancuso D.J. Gross R.W. Turk J. J. Biol. Chem. 2005; 280: 6840-6849Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), SREBP-1 has been shown to promote transcription of the iPLA2β gene (53.Seashols S.J. del Castillo Olivares A. Gil G. Barbour S.E. Biochim. Biophys. Acta. 2004; 1684: 29-37Crossref PubMed Scopus (23) Google Scholar). The transcription factor ATF6 plays a key role in the unfolded protein response, and ER stress leads to its cleavage by site 1 and site 2 proteases that are also activated by ER stress (64.Werstuck G.H. Lentz S.R. Dayal S. Hossain G.S. Sood S.K. Shi Y.Y. Zhou J. Maeda N. Krisans S.K. Malinow M.R. Austin R.C. J. Clin. Invest. 2001; 107: 1263-1273Crossref PubMed Scopus (590) Google Scholar). These proteases process SREBPs (65.Ye J. Rawson R.B. Komuro R. Chen X. Davé U.P. Prywes R. Brown M.S. Goldstein J.L. Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1332) Google Scholar) into active mature forms that enter the nucleus and transactivate target genes (66.Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar). SREBPs are known to be induced under stressful conditions and are activated in β-cells experiencing ER stress due to glucolipotoxicity or thapsigargin (54.Wang H. Kouri G. Wollheim C.B. J. Cell Sci. 2005; 118: 3905-3915Crossref PubMed Scopus (230) Google Scholar). A fascinating extension to these observations is the recent identification of an SRE in the iPLA2β gene (53.Seashols S.J. del Castillo Olivares A. Gil G. Barbour S.E. Biochim. Biophys. Acta. 2004; 1684: 29-37Crossref PubMed Scopus (23) Google Scholar). That study, performed in Chinese hamster ovary cells that were mutated to constitutively generate mSREBPs, revealed that binding of mSREBPs to SRE leads to iPLA2β transcription. We therefore considered the possibility that SREBP-1 may modulate iPLA2β levels in β-cells that are predisposed to developing ER stress.We find that processing of SREBP-1 to the mSREBP-1 form under basal conditions is higher in the Akita β-cells than in the WT β-cells. This coincided with higher pAkt levels, which is consistent with previous reports that phosphorylation of Akt stimulates the synthesis and nuclear localization of mSREBP-1 (55.Furuta E. Pai S.K. Zhan R. Bandyopadhyay S. Watabe M. Mo Y.Y. Hirota S. Hosobe S. Tsukada T. Miura K. Kamada S. Saito K. Iiizumi M. Liu W. Ericsson J. Watabe K. Cancer Res. 2008; 68: 1003-1011Crossref PubMed Scopus (287) Google Scholar, 56.Menendez J.A. Lupu R. Nat. Rev. Cancer. 2007; 7: 763-777Crossref PubMed Scopus (1963) Google Scholar, 57.Porstmann T. Griffiths B. Chung Y.L. Delpuech O. Griffiths J.R. Downward J. Schulze A. Oncogene. 2005; 24: 6465-6481Crossref PubMed Scopus (334) Google Scholar). Further, exposure to thapsigargin led to temporal increases in mSREBP-1 (and pAkt) in parallel with elevations in iPLA2β protein. These findings provide the first evidence of a correlation between mSREBP-1 and iPLA2β expression with the onset and progression of diminished cellular integrity.To determine if there is a causal r