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.
The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress and endothelial function.
Methods and Results
AMPK activity, the markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine (Ach)-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2−/− mice. Further, pretreatment of aorta with either superoxide dismutase or Tempol or apocynin significantly improved Ach-induced endothelium-dependent relaxation in AMPKα2−/− mice. Analysis of aortic endothelial cells from AMPKα2−/− mice and human umbilical vein endothelial cells (HUVECs) expressing dominant negative AMPK or AMPK α2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or over-expression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in LDL receptor knockout (LDLr−/−) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, endothelial dysfunction, and atherosclerosis, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor..
We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way AMPK maintains the non-atherogenic and non-inflammatory phenotype of endothelial cells.