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LIVER AND BILIARY TRACT

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation

¹Division of Gastroenterology and Hepatology, Department of Medicine, ²Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Roudebush Veterans Administration Medical Center, Indianapolis, Indiana

Submitted 23 May 2008 ; accepted in final form 29 September 2008

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 (H₂O₂, 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. H₂O₂ markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-, LKB1, and AMPK caused by exposure to H₂O₂. This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H₂O₂-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- and LKB1 phosphorylation and the activation of PP2A.

protein phosphatase