Abstract
Accumulating evidence suggests that AMP-activated protein kinase (AMPK) activation exerts anti-apoptotic effects in multiple types of cells. However, the underlying mechanisms remain poorly defined. The aim of the present study was to determine how AMPK suppresses apoptosis in endothelial cells exposed to hypoxia and glucose deprivation (OGD). AMPK activity, NF-kappaB activation, and endothelial cell apoptosis were assayed in cultured endothelial cells and mouse common carotid artery with or without OGD treatment. OGD markedly activated AMPK as early as 30 min, and AMPK activity reached maximal at 2 h of OGD. Endothelial apoptosis was not detected until 2 h of OGD but became markedly elevated at 6 h of OGD treatment. Furthermore, AMPK inhibition by Compound C or overexpression of dominant negative AMPK (AMPK-DN) exacerbated, whereas AMPK activation by pharmacologic (aminoimidazole carboxamide ribonucleotide (AICAR)) or genetic means (overexpression of constitutively active AMPK) suppressed endothelial cell apoptosis caused by OGD. Concomitantly, AMPK activation increased the expression of both Bcl-2 and Survivin, two potent anti-apoptotic proteins. Furthermore, AMPK activation significantly enhanced IkappaBalpha kinase activation, NF-kappaB nuclear translocation, and DNA binding activity of NF-kappaB. Consistently, selective inhibition of NF-kappaB, which abolished OGD-enhanced expression of Bcl-2 and Survivin, accentuated endothelial apoptosis caused by OGD. Finally, we found that genetic deletion of the AMPKalpha1, but not AMPKalpha2, suppressed OGD-enhanced NF-kappaB activation, the expression of Bcl-2 and Survivin, and endothelial apoptosis. Overall, our results suggest that AMPKalpha1, but not AMPKalpha2 activation, promotes cell survival by increasing NF-kappaB-mediated expression of anti-apoptotic proteins (Bcl-2 and Survivin) and intracellular ATP contents.
Highlights
Time Course of AMPK Activation in Endothelial Cells Subjected to Oxygen and Glucose Deprivation—As defined by its name, AMPK is known to be activated by increased ratio of AMP to ATP
AICAR together, these results suggest that AMPK activation supalleviated oxygen and glucose deprivation (OGD)-induced cell death, it alone did not pressed caspase-3 activation caused by OGD in endothelial affect endothelial viability in endothelial cells without OGD. cells
In this study we have demonstrated that hypoxia combined with glucose deprivation in endothelial cells induces AMPK activation, and this appears to afford protection to these cells by preventing apoptosis via both an up-regulation of the NF-B-dependent expression of anti-apoptotic gene products and maintaining intracellular ATP in endothelial cells
Summary
NF-B Reporter Assay kits were obtained from SABioscience Corp. (Frederick, MD). AMPK by their impact on cellular metabolism and bioenerget- ground were housed in temperature-controlled cages under a ics and these include hypoxia [27, 28] and glucose deprivation 12-h light-dark cycle and given free access to water and normal [29, 30]. Others, such as oxidative stress [28, 31], have been chow. Mice aged 8 –10 weeks were used for the ligation of the demonstrated to activate AMPK without strict dependence on common carotid artery. AMPK activation results in increased apoptosis and injury in common carotid artery without ligation served as control.
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