Up-regulation of Mitochondrial Uncoupling Protein-2 by the AMP-activated Protein Kinase in Endothelial Cells Attenuates Oxidative Stress in Diabetes

Abstract

Objective. Recent evidence suggests that the AMP-activated protein kinase (AMPK) is an important therapeutic target for diabetes. The present study was conducted to determine how AMPK activation suppressed tyrosine nitration of prostacyclin synthase (PGIS) in diabetes.

Research Design and Methods. Confluent human umbilical vein endothelial cells or mice were treated with 5-amino-4-imidazole carboxamide riboside (AICAR) for the detection of AMPK phosphorylation and the expression of mitochondrial uncoupling protein (UCP)-2.

Results. Exposure of HUVECs to high glucose (30 mM, HG) increased superoxide anions (O₂^.−) and PGIS nitration. In addition, overexpression of constitutively active AMPK (Ad-CA-AMPK) or the addition of AICAR, reduced both O₂^.− and PGIS nitration caused by HG, whereas adenoviral overexpression of dominant negative AMPK mutants (Ad-DN-AMPK) enhanced the latter effects of HG. Exposure of HUVECs to either AICAR or metformin caused AMPK-dependent up-regulation of both UCP-2 mRNA and UCP-2 protein. Furthermore, over-expression of UCP-2 significantly ablated both O₂^.− and PGIS nitration triggered by HG. Further, overexpression of Ad-CA-AMPK increased, whereas overexpression of Ad-DN-AMPK inhibited AICAR-induced phosphorylation of p38 kinase at Thr180/tyr 182. Inhibition of p38 kinase with SB239063, which had no effect on AICAR-induced AMPK-Thr172 phosphorylation, dose-dependently suppressed AICAR-induced up-regulation of UCP-2, suggesting that AMPK lies upstream of p38 kinase. Finally, AICAR markedly increased UCP-2 expression and reduced both O₂^.− and PGIS nitration in diabetic wild type mice but not in their AMPK α2 deficient counterparts in vivo.

Conclusion. We conclude that AMPK activation increases UCP-2 resulting in the inhibition of both O₂^.− and PGIS nitration in diabetes.