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 (mTOR complex 1) activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supraphysiological model of hypertrophy. To achieve this, mTORC1-IRS-1/2 signaling, BiP/CHOP/IRE1α, 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 (IGF) were high throughout the 21st day of overload. However, IGF signaling was limited, since IRS-1 and -2 were undetectable in the overloaded muscle from day 3 to day 9. The decreases in IRS-1/2 protein were paralleled by increases in GRB10 Ser^501/503 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, Thr¹⁴⁶², was unchanged at 6 days. In agreement with the phosphorylation of Thr¹³⁴⁵, SA led to activation of AMPKα1 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 IRE1α levels decreased. These data suggest that during supraphysiological muscle loading at least three potential molecular brakes engage to downregulate mTORC1.