Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation.
AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.
Liangpunsakul
Suthat
S
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine and Roudebush Veterans Administration Medical Center, Indianapolis, IN 46202-5124, USA. sliangpu@iupui.edu
Wou
Sung-Eun
SE
Zeng
Yan
Y
Ross
Ruth A
RA
Jayaram
Hiremagalur N
HN
Crabb
David W
DW
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured; D000099221:Protein Kinase C zeta
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D000431:Ethanol; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D011494:Protein Kinases; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases; D000099221:Protein Kinase C zeta; C488755:Stk11 protein, rat