AMPKalpha2 deletion causes aberrant expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction in vivo: role of 26S proteasomes.
BACKGROUND
AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established.


OBJECTIVE
The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress, and endothelial function.


RESULTS
The markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 to -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of nuclear factor (NF)-kappaB (p50 and p65) were examined in cultured human umbilical vein endothelial cells and mouse aortas isolated from AMPKalpha2 deficient mice. Compared to the wild type, acetylcholine-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKalpha2(-/-) mice. Further, pretreatment of aorta with either superoxide dismutase (SOD) or tempol or apocynin significantly improved acetylcholine-induced endothelium-dependent relaxation in AMPKalpha2(-/-) mice. Analysis of aortic endothelial cells from AMPKalpha2(-/-) mice and human umbilical vein endothelial cells expressing dominant negative AMPK or AMPKalpha2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91(phox), p47(phox), p67(phox), NOX1 and -4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IkappaBalpha degradation, and nuclear translocation of NF-kappaB (p50 and p65), whereas AMPK activation by AICAR or overexpression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKalpha2 in low-density lipoprotein receptor knockout (LDLr(-/-)) strain markedly increased 26S proteasome activity, IkappaB degradation, NF-kappaB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, and endothelial dysfunction, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor.


CONCLUSIONS
We conclude that AMPKalpha2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way, AMPK maintains the nonatherogenic and noninflammatory phenotype of endothelial cells.
Wang
Shuangxi
S
Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
Zhang
Miao
M
Liang
Bin
B
Xu
Jian
J
Xie
Zhonglin
Z
Liu
Chao
C
Viollet
Benoit
B
Yan
Daoguang
D
Zou
Ming-Hui
MH
D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000235:genetics; D000098:Acetophenones / Q000494:pharmacology; D000109:Acetylcholine / Q000494:pharmacology; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000503:physiopathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018836:Inflammation Mediators / Q000097:blood; D007371:Interferon-gamma / Q000097:blood; D007376:Interleukin-2 / Q000097:blood; D007527:Isoenzymes; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D034622:RNA Interference; D011973:Receptors, LDL / Q000172:deficiency / Q000235:genetics; D012265:Ribonucleotides / Q000494:pharmacology; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000494:pharmacology
D000098:Acetophenones; D000975:Antioxidants; D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D018836:Inflammation Mediators; D007376:Interleukin-2; D007527:Isoenzymes; D007976:Leupeptins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D061988:Proteasome Inhibitors; D011973:Receptors, LDL; D012265:Ribonucleotides; D013113:Spin Labels; D014665:Vasodilator Agents; D013481:Superoxides; D000072000:NF-KappaB Inhibitor alpha; D009599:Nitroprusside; D000620:Aminoimidazole Carboxamide; D007371:Interferon-gamma; C056165:acetovanillone; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; C534978:Prkaa2 protein, mouse; C526181:PRKAA2 protein, human; D055372:AMP-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C031143:AICA ribonucleotide; D000109:Acetylcholine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C001803:tempol