Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability

The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic beta-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and beta-cell survival. We have previously demonstrated loss of beta-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-1 beta (IL-1 beta) combined with or without cycloheximide or actinomycin D. IL-1 beta 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-1 beta 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-1 beta on SERCA2b protein stability. Similarly, IL-1 beta-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 beta-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) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1 beta, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ 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 beta-cell calcium homeostasis and reduced beta-cell survival in diabetes.