Molecular brakes regulating mTORC1 activation in skeletal muscle following synergist ablation

The goal of the current work was to profile positive (mTORC1 activation, autocrine/paracrine growth factors) and negative [AMPK, unfolded protein response (UPR)] pathways that might regulate overload-induced mTORC1 activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supra-physiological model of hypertrophy. To achieve this, mTORC1-IRS1/2 signaling, BiP/CHOP/IRE1a, and AMPK activation were determined in rat plantaris muscle following synergist ablation (SA). SA resulted in significant increases in muscle mass of ~4% per day throughout the 21 days of the experiment. The expression of the insulin-like growth factors were high throughout the 21d of overload. However, IGF signaling was limited since IRS1 and 2 were undetectable in the overloaded muscle from day 3 to day 9. The decreases in IRS1/2 protein were paralleled by increases in GRB10(Ser501/503) and S6K1(Thr389) phosphorylation, two mTORC1 targets that can destabilize IRS proteins. PKB(Ser473) phosphorylation was higher from 3-6 days and this was associated with increased TSC2(Thr939) phosphorylation. The phosphorylation of TSC2(Thr1345) (an AMPK site) was also elevated whereas phosphorylation at the other PKB site, Thr(1462), was unchanged at 6d. In agreement with the phosphorylation of Thr(1345), synergist ablation led to activation of a1-AMPK during the initial growth phase, lasting the first 9 days before returning to baseline by day 12. The UPR markers CHOP and BiP were elevated over the first 12 days following ablation, whereas IRE1a levels decreased. These data suggest that during supra-physiological muscle loading, at least three potential molecular brakes engage to down-regulate mTORC1.