title: Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.
abstract: The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.
authors.affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: X
authors.initials: X
authors.lastname: Tong
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: T
authors.initials: T
authors.lastname: Kono
authors.affiliation: 1] Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA [2] Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA [3] Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA [4] The Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA [5] The Roudebush VA Medical Center, Indianapolis, IN 46202, USA.
authors.forename: C
authors.initials: C
authors.lastname: Evans-Molina
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003920:Diabetes Mellitus / Q000473:pathology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D016257:Fura-2 / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology; D007340:Insulinoma / Q000473:pathology; D053583:Interleukin-1beta / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012265:Ribonucleotides / Q000494:pharmacology; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: C506948:Atp2a2 protein, rat; C000594089:IL1B protein, rat; D053583:Interleukin-1beta; D012333:RNA, Messenger; D012265:Ribonucleotides; C049925:fura-2-am; D003609:Dactinomycin; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D000620:Aminoimidazole Carboxamide; D026423:S-Nitroso-N-Acetylpenicillamine; D003513:Cycloheximide; D052247:Nitric Oxide Synthase Type II; D055372:AMP-Activated Protein Kinases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C031143:AICA ribonucleotide; D002118:Calcium; D016257:Fura-2