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.