Anti-apoptotic Action of Exendin-4 in INS-1 Beta Cells: Comparative Protein Pattern Analysis of Isolated Mitochondria

Abstract

Glucagon like peptide-1 (Glp-1) exhibits beneficial effects on beta cell mass by both enhancing proliferation and inhibiting apoptosis. The precise mechanism of the anti-apoptotic effect of Glp-1 and Glp-1 mimetics like exendin-4 has remained elusive. Here, we studied cytokine-induced apoptosis in the pancreatic beta cell line INS-1 and performed a comparative mitochondrial protein pattern analysis using two-dimensional difference gel electrophoresis (2D-DIGE). Cytokine incubation of INS-1 cells increased caspase-3 activity about 3-fold, which was reduced by 60% in the presence of exendin-4. Production of reactive oxygen species in response to cytokines was completely prevented after preincubation with exendin-4. Highly purified mitochondria were obtained and mitochondrial proteins were labeled with Cy-dyes and separated on overlapping zoom 2D gels spanning a pH-range of 4–9. Protein spots with significant changes after cytokine and exendin-4 treatment were identified by MALDI mass spectrometry. Comparing all treatment conditions, comparative mitochondrial proteome analysis allowed to identify 33 different proteins, which were significantly altered between comparison groups. Changes in protein patterns revealed involvement of cytokine-induced electron transport chain damage. Thus, cytochrome bc1 complex subunit I and ATP synthase subunit beta were downregulated by 30–40%. This was abrogated by the presence of exendin-4. In conclusion, this study provides further insights into the role of mitochondria in cytokine-induced apoptosis. We show here that exendin-4 significantly counter-regulates the reduced abundance of electron transport chain proteins, leading to a reduction of oxidative stress and most likely contributing to the anti-apoptotic action of this drug.

apoptosis - INS-1 beta cells - exendin-4 - mitochondria - proteome analysis

References

• 1 Saldeen J. Cytokines induce both necrosis and apoptosis via a common Bcl-2-inhibitable pathway in rat insulin-producing cells.  Endocrinology. 2000;  141 2003-2010
• 2 Eizirik DL, Mandrup-Poulsen T. A choice of death–the signal-transduction of immune-mediated beta-cell apoptosis.  Diabetologia. 2001;  44 2115-2133
• 3 Rakatzi I, Mueller H, Ritzeler O, Tennagels N, Eckel J. Adiponectin counteracts cytokine- and fatty acid-induced apoptosis in the pancreatic beta-cell line INS-1.  Diabetologia. 2004;  47 249-258
• 4 Kagan VE, Borisenko GG, Tyurina YY, Tyurin VA, Jiang J, Potapovich AI, Kini V, Amoscato AA, Fujii Y. Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine.  Free Radic Biol Med. 2004;  37 1963-1985
• 5 Fehmann HC, Goke R, Goke B. Cell and molecular biology of the incretin hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide.  Endocr Rev. 1995;  16 390-410
• 6 Fehmann HC, Habener JF. Insulinotropic hormone glucagon-like peptide-I(7–37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.  Endocrinology. 1992;  130 159-166
• 7 Nauck MA, Heimesaat MM, Behle K, Holst JJ, Nauck MS, Ritzel R, Hufner M, Schmiegel WH. Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers.  J Clin Endocrinol Metab. 2002;  87 1239-1246
• 8 Nauck MA, Niedereichholz U, Ettler R, Holst JJ, Orskov C, Ritzel R, Schmiegel WH. Glucagon-like peptide 1 inhibition of gastric emptying outweighs its insulinotropic effects in healthy humans.  Am J Physiol. 1997;  273 E981-E988
• 9 Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans.  J Clin Invest. 1998;  101 515-520
• 10 Gutzwiller JP, Goke B, Drewe J, Hildebrand P, Ketterer S, Handschin D, Winterhalder R, Conen D, Beglinger C. Glucagon-like peptide-1: a potent regulator of food intake in humans.  Gut. 1999;  44 81-86
• 11 Hui H, Nourparvar A, Zhao X, Perfetti R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5′-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway.  Endocrinology. 2003;  144 1444-1455
• 12 Farilla L, Hui H, Bertolotto C, Kang E, Bulotta A, Mario U Di, Perfetti R. Glucagon-like peptide-1 promotes islet cell growth and inhibits apoptosis in Zucker diabetic rats.  Endocrinology. 2002;  143 4397-4408
• 13 Mellado-Gil JM, Aguilar-Diosdado M. High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes.  J Endocrinol. 2004;  183 155-162
• 14 Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis.  J Biol Chem. 2003;  278 471-478
• 15 Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms B, Holst JJ. Both subcutaneously and intravenously administered glucagon-like peptide I are rapidly degraded from the NH2-terminus in type II diabetic patients and in healthy subjects.  Diabetes. 1995;  44 1126-1131
• 16 Goke R, Fehmann HC, Linn T, Schmidt H, Krause M, Eng J, Goke B. Exendin-4 is a high potency agonist and truncated exendin-(9–39)-amide an antagonist at the glucagon-like peptide 1-(7–36)-amide receptor of insulin-secreting beta-cells.  J Biol Chem. 1993;  268 19650-19655
• 17 Chen J, Yu L, Wang L, Fang X, Li L, Li W. Stability of synthetic exendin-4 in human plasma in vitro.  Protein Pept Lett. 2007;  14 19-25
• 18 Wang Q, Li L, Xu E, Wong V, Rhodes C, Brubaker PL. Glucagon-like peptide-1 regulates proliferation and apoptosis via activation of protein kinase B in pancreatic INS-1 beta cells.  Diabetologia. 2004;  47 478-487
• 19 Li L, El Kholy W, Rhodes CJ, Brubaker PL. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis: role of protein kinase B.  Diabetologia. 2005;  48 1339-1349
• 20 Jhala US, Canettieri G, Screaton RA, Kulkarni RN, Krajewski S, Reed J, Walker J, Lin X, White M, Montminy M. cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2.  Genes Dev. 2003;  17 1575-1580
• 21 Kwon G, Pappan KL, Marshall CA, Schaffer JE, MacDaniel ML. cAMP Dose-dependently prevents palmitate-induced apoptosis by both protein kinase A- and cAMP-guanine nucleotide exchange factor-dependent pathways in beta-cells.  J Biol Chem. 2004;  279 8938-8945
• 22 Asfari M, Janjic D, Meda P, Li GD, Halban PA, Wollheim CB. Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell-lines.  Endocrinology. 1992;  130 167-178
• 23 Lehr S, Kotzka J, Avci H, Knebel B, Muller S, Hanisch FG, Jacob S, Haak C, Susanto F, Muller-Wieland D. Effect of sterol regulatory element binding protein-1a on the mitochondrial protein pattern in human liver cells detected by 2D-DIGE.  Biochemistry. 2005;  44 5117-5128
• 24 Rabilloud T, Strub JM, Luche S, Dorsselaer A Van, Lunardi J. A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.  Proteomics. 2001;  1 699-704
• 25 Lenzen S. Oxidative stress: the vulnerable beta-cell.  Biochem Soc Trans. 2008;  36 343-347
• 26 Ferdaoussi M, Abdelli S, Yang JY, Cornu M, Niederhauser G, Favre D, Widmann C, Regazzi R, Thorens B, Waeber G, Abderrahmani A. Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.  Diabetes. 2008;  57 1205-1215
• 27 D’Hertog W, Overbergh L, Lage K, Ferreira GB, Maris M, Gysemans C, Flamez D, Cardozo AK, Van den BG, Schoofs L, Arckens L, Moreau Y, Hansen DA, Eizirik DL, Waelkens E, Mathieu C. Proteomic analysis of cytokine-induced dysfunction and death in insulin producing INS-1E cells: New insights into the pathways involved.  Mol Cell Proteomics. 2007;  6 2180-2199
• 28 Tarze A, Deniaud A, Le Bras M, Maillier E, Molle D, Larochette N, Zamzami N, Jan G, Kroemer G, Brenner C. GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization.  Oncogene. 2007;  26 2606-2620
• 29 Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria.  Biochem J. 1980;  191 421-427
• 30 Iverson SL, Orrenius S. The cardiolipin-cytochrome c interaction and the mitochondrial regulation of apoptosis.  Arch Biochem Biophys. 2004;  423 37-46
• 31 Lenaz G. The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology.  IUBMB Life. 2001;  52 159-164
• 32 Chen J, Couto FM, Minn AH, Shalev A. Exenatide inhibits beta-cell apoptosis by decreasing thioredoxin-interacting protein.  Biochem Biophys Res Commun. 2006;  346 1067-1074
• 33 Zhang Y, Marcillat O, Giulivi C, Ernster L, Davies KJ. The oxidative inactivation of mitochondrial electron transport chain components and ATPase.  J Biol Chem. 1990;  265 16330-16336
• 34 Welsh N, Eizirik DL, Bendtzen K, Sandler S. Interleukin-1 beta-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may lead to inhibition of the Krebs cycle enzyme aconitase.  Endocrinology. 1991;  129 3167-3173
• 35 Saleh MC, Fatehi-Hassanabad Z, Wang R, Nino-Fong R, Wadowska DW, Wright GM, Harper ME, Chan CB. Mutated ATP synthase induces oxidative stress and impaired insulin secretion in beta-cells of female BHE/cdb rats.  Diabetes Metab Res Rev. 2008;  24 392-403
• 36 Cnop M, Welsh N, Jonas JC, Jorns A, Lenzen S, Eizirik DL. Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities.  Diabetes. 2005;  54 ((Suppl. 2)) S97-S107
• 37 Yusta B, Baggio LL, Estall JL, Koehler JA, Holland DP, Li H, Pipeleers D, Ling Z, Drucker DJ. GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress.  Cell Metab. 2006;  4 391-406

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