Effects of WY-14,643 on the phosphorylation and activation of AMP-dependent protein kinase

Abstract

Background: AMP-dependent protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) alpha facilitate fatty acid oxidation. We have shown that treatment of hepatoma cells with ethanol or feeding ethanol-containing diets to mice inhibited both PPARalpha and AMPK activity. Importantly, WY-14,643 reversed the development of fatty liver in alcohol-fed mice. Whether WY-14,643, a PPARalpha agonist, has any effects on AMPK is not known. The aim of this study was to investigate the effect of WY-14,643 on AMPK activity. Methods: The effect of WY-14,643 on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3). The effect of WY-14,643 on upstream kinases of AMPK, PKC-zeta/LKB1, intracellular AMP:ATP ratio, oxidative stress, and AMPK gene expression were studied. Results: Treatment of the H4IIEC3 cells with WY-14,643 for 24h led to 60% increase in the phosphorylation of AMPK. The effect of WY-14,643 on AMPK phosphorylation is PKC-zeta/LKB1 independent. WY-14,643 did not alter the levels of intracellular AMP:ATP ratio and it did not increase the levels of reactive oxygen species at 24-h of treatment. WY-14,643-induced AMPK alpha subunit expression by 2- to 2.5-fold, but there was no change in AMPKalpha subunit protein at 24h. The effect of WY-14,643 on AMPK phosphorylation did not altered by the presence of an NADPH oxidase inhibitor. Conclusions: WY-14,643 induced AMPKalpha subunit phosphorylation and the activity of the enzyme. This was associated with induction of AMPKalpha1 and alpha2 mRNA, but the mechanism for this activation is uncertain.