Role of PLD and PA in alcohol (EtOH)‐mediated mTOR signaling in C2C12 myocytes

Previously, we reported that EtOH decreases muscle protein synthesis, with this being mediated by reduced mTORC1 and increased mTORC2 activities. In contrast to EtOH, phospholipase D (PLD) and phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less defined. Here, we examine the role that PLD and PA play in EtOH‐mediated mTOR signaling in C2C12 myocytes. EtOH (100 mM) increased levels of PA and PLD activity in myocytes. This was associated with increased mTORC2 activity, as indicated by elevated Akt (S473) phosphorylation. Suppression of PLD function with an inhibitor blocked the EtOH‐induced increase in Akt phosphorylation, but it decreased mTORC1 activity, as evidenced by decreased phosphorylation of S6K1 (T389). Mechanistically, PLD inhibition affected the composition of mTORC2 and mTORC1. It also affected the interaction of these complexes with the negative regulatory cytosolic protein 14–3‐3. Addition of PA (30 μM) to myocytes decreased mTORC2 activity, but it increased mTORC1 activity. PA also reduced phosphorylation of AMPK and the AMPK target TSC2, thereby allowing for increased Rheb, RagA and RagC interactions with mTOR. Collectively, our results show that PA stimulates mTORC1 function, whereas it suppresses activation of mTORC2. In the presence of EtOH, however, PLD activity is an essential mediator of mTORC2 stimulation. (Grant AA11290)