Upregulation of mitochondrial uncoupling protein-2 by the AMP-activated protein kinase in endothelial cells attenuates oxidative stress in diabetes.
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 in diabetes.


METHODS
Confluent human umbilical vein endothelial cells (HUVECs) 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 mmol/l) increased superoxide anions (O(2).(-)) and prostacyclin synthase nitration. In addition, overexpression of constitutively active AMPK (Ad-CA-AMPK) or the addition of AICAR reduced both O(2).(-) and prostacyclin synthase nitration caused by high glucose, whereas adenoviral overexpression of dominant-negative AMPK mutants (Ad-DN-AMPK) enhanced the latter effects of high glucose. Exposure of HUVECs to either AICAR or metformin caused AMPK-dependent upregulation of both UCP-2 mRNA and UCP-2 protein. Furthermore, overexpression of UCP-2 significantly ablated both O(2).(-) and prostacyclin synthase nitration triggered by high glucose. Furthermore, overexpression of Ad-CA-AMPK increased, whereas overexpression of Ad-DN-AMPK inhibited AICAR-induced phosphorylation of p38 kinase at Thr180/Tyr182. Inhibition of p38 kinase with SB239063, which had no effect on AICAR-induced AMPK-Thr172 phosphorylation, dose dependently suppressed AICAR-induced upregulation of UCP-2, suggesting that AMPK lies upstream of p38 kinase. Finally, AICAR markedly increased UCP-2 expression and reduced both O(2).(-) and prostacyclin synthase nitration in diabetic wild-type mice but not in their AMPKalpha2-deficient counterparts in vivo.


CONCLUSIONS
We conclude that AMPK activation increases UCP-2, resulting in the inhibition of both O(2).(-) and prostacyclin synthase nitration in diabetes.
Xie
Zhonglin
Z
Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Zhang
Junhua
J
Wu
Jiliang
J
Viollet
Benoit
B
Zou
Ming-Hui
MH
D055372:AMP-Activated Protein Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D054412:Chemokine CCL8 / Q000235:genetics*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012265:Ribonucleotides / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism; D014471:Umbilical Veins; D015854:Up-Regulation / Q000235:genetics*
C515453:CCL8 protein, human; D054412:Chemokine CCL8; D065607:Cytochrome P-450 Enzyme Inhibitors; D017382:Reactive Oxygen Species; D012265:Ribonucleotides; D013481:Superoxides; D000620:Aminoimidazole Carboxamide; D003577:Cytochrome P-450 Enzyme System; D055372:AMP-Activated Protein Kinases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase; C031143:AICA ribonucleotide; D005947:Glucose