Fig. 4.

EtOH-induced PLD activity is mediated by AMPK and PI3 kinase pathways. C2C12 myocytes were incubated overnight in the presence or absence of 100 mM EtOH. The cells were then pre-incubated for 1 h in SFM with either the AMPK inhibitor compound C (CC, 20 µM) or the PI3 kinase inhibitor wortmannin (wort, 100 nM). Thereafter, EtOH was added to the media for an additional 50 min in the presence or absence of inhibitors. Cells were collected and phospholipase D activity was determined (A–B) as described in “Materials and Methods.” (C) Equal amounts of protein from cell lysates were analyzed via Western blotting using an antibody against the phosphorylated form of AMPKα. Results were normalized with total protein and expressed as percentage of control levels. Each bar represents the mean ± SE of 3–4 independent experiments consisting of 3–5 replicate samples per experiment. Groups with different letters are significantly different from one another (P< 0.05). Groups with the same letters are not significantly different.