Early Activation of mTOR in Response to Mechanical Overload Occurs Independent of PI3K/Akt Signaling

Abstract

It had been established that mammalian target of rapamycin complex 1 (TORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth, however, the regulatory mechanisms which determine mTOR activity in skeletal muscle are poorly defined. PURPOSE: The aim of this study was to determine the upstream signaling mechanisms that contribute to mTOR activation during mechanical overload-induced skeletal muscle hypertrophy in vivo. METHODS: C57BL/6J mice (male, 12-14 wk of age) were used in this study. The bilateral synergist ablation model was used to induce hypertrophy of the plantaris muscle by functional overload. Muscle samples were collected at days 1, 2, 3, 5, 7 and 10 after synergist ablation surgery. Insulin (0.75 Unit/kg), wortmannin (3 mg/kg) and rapamycin (1.5 mg/kg) were administered by intraperitoneal injection. RESULTS: Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle (Ex. 1.7-fold increase in total muscle mass, 14.2-fold increase in rates of protein synthesis and 2.1-fold increase in total RNA contents following 7 days of mechanical overload). Phosphorylation states of both Akt and mTOR signaling pathways were determined. Activity of mTOR signaling was monitored by analysis of Thr389 phosphorylation of S6 kinase 1 (S6K1). We found a significant increase in Thr389 phosphorylation of S6K1 by day 1 following mechanical overload. In contrast we saw no change in Akt phosphorylation, no change in Akt kinase activity and we found no change in the phosphorylation of known downstream targets of Akt including glycogen synthase kinase 3, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 at day 1 of mechanical overload. These results were surprising and indicated that activation of phosphoinositide 3-kinase (PI3K)/Akt signaling was not required for mTOR activation in vivo. To determine whether PI3K-dependent signaling was necessary for the early activation of mTOR, we treated mice with the PI3K-specific inhibitor wortmannin, which was sufficient to inhibit insulin-dependent signal inputs, but did not prevent overload-induced mTOR activation in vivo. CONCLUSIONS: These observations demonstrate that mTOR activation at the early phase of mechanical overload occurs independent of PI3K/Akt regulation in skeletal muscle. This suggests that other signal inputs including mechanical strain and/or nutrient status are important for this initial signaling event during overload-induced hypertrophic growth. Supported by NIH Grant ROI AR45617 to KAE and AHA postdoctoral fellowship (0825668D) to MM.