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.

Fig. 1

Effect of WY-14,643 on AMP phosphorylation and AMP activity in rat hepatoma H4IIEC3 cells. (A and B) Immunoblot analysis. H4IIEC3 cells were treated with WY-14,643 (100 μM) or metformin (2 mM), as indicated. (C) Bar graph demonstrating quantification of the Western blot (B). There were no changes in AMPK phosphorylation with WY-14,643 after 1 to 20-h of treatment. However, at 24 h, WY-14,643 increased the levels of phospho-AMPK and its downstream target protein, p-ACC by 59% and 25%, respectively. There were no effects of WY-14,643 on PKC-ζ and LKB1. (D) AMPK activity measured using a synthetic peptide substrate. The experiments with metformin (2 mM) served as positive controls. The activity of AMPK correlated with the levels of phospho-AMPK from the Western blot analyses. *Significant difference vs control; p < 0.05, by 1-way ANOVA. The results are representative of blots from four separate experiments.

Fig. 2

Effects of WY-14,643 on AMPKα1 and AMPKα2 gene expression. WY-14,643 increased the abundance of mRNA of AMPKα1 and α2 by 2- to 2.5-fold. The results were expressed as average fold change ± SD. *p < 0.05 vs control. (B) There were no changes in the levels of AMPKα1 and AMPKα2 subunit proteins after treatment with WY-14,643. The results are representative of blots from three separate experiments.

Fig. 3

Effects of WY-14,643 on DHCF oxidation. (A) Cells were treated with WY-14,643 (100 μM) for 5 min, 3 or 24 h. Incubation of H4IIEC3 cells with WY-14,643 for 5 min and 3 h led to 2-fold increase in the formation of ROS. However, no change in ROS production was seen at 24-h treatment. H₂O₂ (0.5 mM) was used as a positive control. Results are from several experiments (n = 4) and expressed as average % of control ± SD. *p < 0.05 vs control. (B) Pretreatment with apocynin (10 μM) did not affect the baseline p-AMPK and the ability of WY-14,643 to stimulate AMPK phosphorylation after 24-h treatment. H4IIEC3 cells were treated with or without 10 μM apocynin for 2 h and then stimulated with WY-14,643 as indicated. The results are representative of blots from three separate experiments.