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Updated: Jun 6, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Redox regulation of the AMP-activated protein kinase
Yingying Han1, Qilong Wang, Ping Song
1Department of Biochemistry and Department of Medicine, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, United States of America.
Unlabelled:
Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.
Objectives:
The 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).
Methods:
Bovine aortic endothelial cells (BAEC) were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.
Results:
In 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-N(G)-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.
Conclusion:
Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.
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