Abstract
Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.ObjectivesThe aim of this study is to determine if AMP-activated protein kinase (AMPK), a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC).MethodsBovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.ResultsIn BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC) at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-NG-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor) blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol) pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.ConclusionOur results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.
Highlights
AMP-activated protein kinase (AMPK), a heterotrimeric complex comprised of a catalytic subunit a and two regulatory subunits, b and c, is an evolutionarily conserved serine/threonine protein kinase that is ubiquitously expressed
Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberineinduced AMPK activation in endothelial cells
To investigate whether ONOO2 is critical to Berberine activated AMPK and its downstream target, acetyl CoA carboxylase (ACC) in endothelial cells (EC), confluent Bovine aortic endothelial cells (BAEC) were treated with different concentrations of Berberine (0, 1, 5, 10, 20, 50, 100, 200 mM) for 2 h
Summary
AMP-activated protein kinase (AMPK), a heterotrimeric complex comprised of a catalytic subunit a and two regulatory subunits, b and c, is an evolutionarily conserved serine/threonine protein kinase that is ubiquitously expressed. It was initially characterized as a ‘‘fuel gauge’’, modulating cellular energy flux in eukaryotic cells in response to changes of AMP:ATP ratios [1,2,3]. AMPK can be activated by conditions that increase intracellular AMP such as exercise [4], hypoxia [5], hypoglycemia [6], as well as certain cytokines and drugs such as leptin [7], adiponectin [8], metformin [9], statins [10] and rosiglitazone [11]. In addition to its roles in energy homeostasis, more recent studies have identified some broader protective roles for AMPK in atherosclerosis [16] and inflammation [17] as well as some beneficial angiogenic effects [18,19]
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