In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.

It has been widely proposed that signaling by mammalian target of rapamycin (mTOR) is both necessary and sufficient for the induction of skeletal muscle hypertrophy. Evidence for this hypothesis is largely based on studies that used stimuli that activate mTOR via a phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)-dependent mechanism. However, the stimulation of signaling by PI3K/PKB also can activate several mTOR-independent growth-promoting events; thus, it is not clear whether signaling by mTOR is permissive, or sufficient, for the induction of hypertrophy. Furthermore, the presumed role of mTOR in hypertrophy is derived from studies that used rapamycin to inhibit mTOR; yet, there is very little direct evidence that mTOR is the rapamycin-sensitive element that confers the hypertrophic response. In this study, we determined that, in skeletal muscle, overexpression of Rheb stimulates a PI3K/PKBindependent activation of mTOR signaling, and this is sufficient for the induction of a rapamycin-sensitive hypertrophic response. Transgenic mice with muscle specific expression of various mTOR mutants also were used to demonstrate that mTOR is the rapamycin-sensitive element that conferred the hypertrophic response and that the kinase activity of mTOR is necessary for this event. Combined, these results provide direct genetic evidence that a PI3K/PKB-independent activation of mTOR signaling is sufficient to induce hypertrophy. In summary, overexpression of Rheb activates mTOR signaling via a PI3K/PKB-independent mechanism and is sufficient to induce skeletal muscle hypertrophy. The hypertrophic effects of Rheb are driven through a rapamycin-sensitive (RS) mechanism, mTOR is the RS element that confers the hypertrophy, and the kinase activity of mTOR is necessary for this event.

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SUnSET (SUrface SEnsing of Translation) involves the immunological detection of puromycin in nascent peptides as a means for quantifying rates of protein synthesis (PS). To validate SUnSET against an established technique, plantaris muscles were subjected to synergist ablation (SA) or sham surgery. After 7d, muscles were incubated ex vivo with a flooding dose of 3H‐Phenylalanine (3H‐P) or with puromycin. According to the established 3H‐P method, SA induced a 3.4 fold increase in PS while a western blot (WB) version of SUnSET revealed a 3.6 fold increase, indicating that both techniques produce quantitatively similar results. Furthermore, SUnSET revealed that SA induces a 2.9 fold increase in PS when measurements were performed in vivo. SUnSET was also used to demonstrate that food deprivation induces a ~70% decrease in PS in vivo when measured with WB analysis and similar results were obtained when an immunohistochemical (IHC) version of SUnSET was applied to cross‐sections from control and food deprived samples. Finally, the IHC version of SUnSET was applied to muscles electroporated with constitutively active Akt (CA‐AKT) and it was determined that PS rates were 80% higher in CA‐AKT transfected fibers when compared to non‐transfected fibers in the same section. In conclusion, SUnSET is a simple and inexpensive alternative for measuring PS and allows for measurements to be performed at the single fiber level.

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