However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. The outcome is predictable because the low testosterone-acclimated cells are able to up-regulate AR protein and activity, and are therefore better equipped for survival in a stress situation. The data of the scrambled siRNA control cells presented in Figure 2B show that the phosphorylation of p70S6K and S6 was increased by testosterone stimulation (lane 1 vs. lane 3). These results indicated that the mTOR pathway plays a key role in testosterone-induced OVX SHR myocardial hypertrophy. Finally, the relationship between the total elevated levels of these proteins (mTOR, S6K1 and 4E-BP1) induced by testosterone and their phosphorylated form remains unclear and requires further investigation. First, this study effectively identified the mTOR signaling pathway as a potential target of testosterone-induced OVX SHR cardiac hypertrophy, but it did not explore mTOR upstream regulatory molecules. MTOR is localized on the lysosome in the basal state and the mTOR-LAMP2 complex is translocated to the cell periphery under resistance exercise, which provide close proximity to the capillaries. However, the kinase for TSC phosphorylation was unclear in this study since mechanical stimulation was previously shown to activate mTOR in PI3K/Akt-independent manner (Hornberger et al., 2004; O'Neil et al., 2009). The Hornberger group found that mTOR and TSC2 were highly enriched in the lysosome of the muscle in the resting state (Jacobs et al., 2013). It has been established that mTORC1 translocates to the lysosome through regulation of Ragulator-Rag in amino acid signaling (Sancak et al., 2008, 2010). (D, E, F, G, and H) T increased the expression level of mTOR/S6K1/4EBP1/eIF4E signaling pathway protein in OVX SHR myocardial tissue. Thus, in this study, we focused on ovariectomized (OVX) SHR to validate that the mTOR/ribosomal protein S6 kinase (S6K1)/4E-binding protein 1 (4EBP1) pathway was involved in the development of testosterone-induced OVXSHR myocardial hypertrophy. Other studies have also suggested that the expression of mTOR protein in myocardial tissue is increased during exercise-mediated physiological hypertrophy (Zhang et al. 2010). Several miRNAs are identified as myomiRNAs, which are enriched in skeletal muscle and known to modulate the cellular processes involved in muscle growth, development, and maintenance, including hypertrophy and atrophy. A recent report also suggests that glucocorticoids increase phosphorylation of CEBP by decreasing PDE3/4 and activating PKA through inhibiting Akt activity, resulting in increased myostatin expression (Xie et al., 2017). Effect of glucose deprivation on induction of cell death in high testosterone- or low testosterone-acclimated cells. The low testosterone-acclimated cells were much more resistant to apoptotic death than the high testosterone-acclimated cells (Figure 7). As noted above, the induction of AR protein by inhibition of mTOR activity is only operative in a low testosterone condition. Thus, in the face of an energy crisis when mTOR activity is greatly compromised, AR function is needed to keep cells in a survival mode. The data suggest that the growth inhibitory effect of bicalutamide may be reversible, and cells are able to recover from growth arrest over time. First, the effects of glucose deprivation, bicalutamide, or the combination treatment on cell growth were evaluated in a low testosterone condition. LNCaP cells were treated with 10 nM rapamycin in the presence of 0.03, 1 or 5 nM DHT. AR inhibition generally suppresses cell growth and slows down metabolism, thereby reducing energy demand, which may in turn lessen the sensitivity to glucose deprivation. In these experiments, glucose deprivation was achieved by incubating cells in a glucose-free medium. In order to interpret the above finding, evidence of apoptosis in the surviving cells was sought using the ELISA cell death assay after three days of treatment. Effect of concomitant glucose deprivation and bicalutamide treatment on cell growth and cell death. Glucose deprivation inhibited cell growth by ~40–50% for the five-day duration (Figure 5A).
