Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine

Abstract

Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.

Aging accentuates alcohol-induced decrease in protein synthesis in gastrocnemius

“…Whereas the increased raptor phosphorylation is typically attributed to activation of the cellular energy sensor AMPK (33), this pathway does not appear activated because the phosphorylation of AMPK and its upstream kinase LKB1 and a downstream substrate REDD1 did not differ between adult control-and alcohol-fed rats. We also detected a reduced binding of RagA with raptor in response to alcohol, a situation that would be expected to impair protein synthesis (76) and that has only been previously reported in myocytes incubated short-term with alcohol (38). Relatively less is known regarding alcohol-induced changes in muscle proteolysis.…”

“…As a result of these changes, we detected an increased formation of the Deptor-raptor complex, with a reduction in the 4E-BP1-raptor complex. We speculate these changes in mTORC1 are causally related to the accentuated decrease in muscle protein synthesis in the alcohol-fed aged rats (38,43,44). These changes in protein-protein inter- action within mTORC1 may be LKB1-AMPK-REDD1-dependent, as this signaling pathway was only activated in muscle from alcohol-fed aged rats.…”

Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs

“…This process is partly regulated by the eEF2 pathway (8). Phosphorylation of eEF2 has been reported to be increased during leucine deprivation in myoblast and myotubes (72) and reduced with leucine administration in cell culture (39,61) in rats (77) and in humans (22,34). In the current study, eEF2 phosphorylation was not affected by CONϩLEU feeding compared with the CON and BOL groups.…”

“…In vitro studies using C 2 C 12 cells (24) and myocytes (39) and a recent study in rats (77) suggest that leucine decreases AMPK activation, at least in part, by decreasing the AMP-to-ATP ratio. In this study, pulsatile leucine infusion during continuous feeding had no effect on AMPK␣ phosphorylation, consistent with our previous studies which showed no effect of …”

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs

“…It has been reported that leucine deprivation in myoblast and myotubes increased (78) and leucine administration in cell culture (44,66), rats (87,88), and humans (29,41) decreased the phosphorylation of eEF2, a key component of the elongation process. In the current study, phosphorylation of eEF2 did not differ between the CON ϩ LEU and the CON ϩ ALA groups.…”