Nitric Oxide Stress and Activation of AMP-activated Protein Kinase Impair β-cell Sarcoendoplasmic Reticulum Calcium ATPase 2b Activity and Protein Stability

Cell Death Dis. 2015 Jun 18;6(6):e1790. doi: 10.1038/cddis.2015.154.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • AMP-Activated Protein Kinases / antagonists & inhibitors
  • AMP-Activated Protein Kinases / biosynthesis
  • AMP-Activated Protein Kinases / metabolism*
  • Aminoimidazole Carboxamide / analogs & derivatives
  • Aminoimidazole Carboxamide / pharmacology
  • Animals
  • Apoptosis / physiology
  • Calcium / metabolism
  • Cycloheximide / pharmacology
  • Dactinomycin / pharmacology
  • Diabetes Mellitus / pathology*
  • Endoplasmic Reticulum / metabolism
  • Enzyme Activation
  • Fura-2 / analogs & derivatives
  • Fura-2 / pharmacology
  • Humans
  • Insulin-Secreting Cells / cytology
  • Insulinoma / pathology
  • Interleukin-1beta / pharmacology
  • Male
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type II / biosynthesis
  • Nitric Oxide Synthase Type II / metabolism*
  • Oxidative Stress / physiology
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Rats
  • Rats, Wistar
  • Ribonucleotides / pharmacology
  • S-Nitroso-N-Acetylpenicillamine / pharmacology
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / genetics
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism*
  • Signal Transduction / genetics
  • omega-N-Methylarginine / pharmacology

Substances

  • Atp2a2 protein, rat
  • IL1B protein, rat
  • Interleukin-1beta
  • RNA, Messenger
  • Ribonucleotides
  • fura-2-am
  • Dactinomycin
  • omega-N-Methylarginine
  • Nitric Oxide
  • Aminoimidazole Carboxamide
  • S-Nitroso-N-Acetylpenicillamine
  • Cycloheximide
  • Nitric Oxide Synthase Type II
  • AMP-Activated Protein Kinases
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • AICA ribonucleotide
  • Calcium
  • Fura-2