Lyndon Olvera
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As expected, the expression of AR targets, such as PSA and KLK2, decreased significantly. AR was knocked down successfully by siRNA in both testosterone conditions (Figure 2A). Total protein level of the unphosphorylated substrates was not affected by bicalutamide.
The results suggest that testosterone and DHT may have differential effects on the cross-talk between mTOR and AR. However, the increase at 5 nM testosterone appeared unrelated to a change of AR level. Rapamycin completely blocked the phosphorylation of p70S6K and S6, and decreased only marginally the phosphorylation of 4EBP-1 (Figure 4A). The next step was to address whether mTOR regulates AR in a reciprocal manner and if testosterone modulates the signal from mTOR to AR. PSA was used as a target gene of AR to assess the successful inhibition of AR activity by AR knockdown.
Myostatin production is also induced by food deprivation in a glucocorticoid-dependent manner (Allen et al., 2010). Human myostatin promoter is reported to have a putative GRE and is responsive to dexamethasone and RU-486, an antagonist of GR (Ma et al., 2001). KlF15 plays a critical role in muscle catabolism through the transcriptional upregulation of atrogen-1, MuRF-1, and branched-chain aminotransferase 2 (BCAT2). Exogenous administration of glucocorticoids induces muscle atrophy and the blockage of GR; adrenalectomy or treatment with the GR antagonist RU486 diminishes muscle atrophy in sepsis, cachexia, starvation, and severe insulinopenia (Menconi et al., 2007; Schakman et al., 2008).
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.
Since mTOR is sensitive to nutrient levels, its activity would be diminished as a result of nutrient deprivation. Chen et al. (18) demonstrated that increased AR protein may amplify the output from residual ligand and alter the response to antagonist. Wang et al. (7) found that rapamycin inhibition of mTORC1 increases AR transcriptional activity via an Akt-dependent pathway downstream of mTORC2. The role of testosterone in glucose deprivation-induced apoptosis was therefore studied. Another experiment with the same protocol was carried out, with the exception that the trypan blue method was used to asses the percentage of dead cells (Figure 6B). To test this hypothesis, an adjuvant bicalutamide protocol was designed in which cells were subjected first to glucose deprivation for three days, followed by bicalutamide treatment for another day.
In this study, treatment with rapamycin does not affect myofibrillar synthesis, while it decreases the phosphorylation of p70S6K1 and S6, implying that mTOR is not involved in myostatin-mediated myofibrillar synthesis (Welle et al., 2009). The injection of a myostatin antibody enhances phosphorylation of p70S6K1 and S6 in muscle, but does not change phosphorylation of Akt and 4EBP1 in the concomitant increase of myofibrillar synthesis (Welle et al., 2009). Myostatin, a transforming growth factor-β (TGF-β) family member, plays a critical role in inhibiting the growth of muscle mass and muscle cell differentiation (McPherron et al., 1997). In line with mTOR function as a positive regulator of muscle hypertrophy, mTOR signaling is negatively regulated by muscle atrophy-inducing signals or blocks muscle atrophy signals. The loss of skeletal muscle, muscle atrophy, stems from an increase in the rate of protein degradation or the decrease of protein synthesis under various conditions, such as disuse, diseases, and aging. Muscle-specific expression of IGF-I in transgenic mice results in at least a 2-fold increase in muscle hypertrophy (Coleman et al., 1995; Musaro et al., 2001), suggesting that the IGF-I/Akt/mTORC1 pathway is indispensable to muscle hypertrophy. Instead, Akt /mTOR signaling by IGF-I/IGFR/IRS-1 has been shown to be indispensable in prompting muscle hypertrophy (Glass, 2003).
E, estrogen; OVX, ovariectomized; T, testosterone; WGA, wheat germ agglutinin; WKY, Wistar Kyoto. (B, C, D, and E) mRNA expression of β-MHC, ANP, MMP-9 and TIMP-1 by real-time RT-PCR. BW, body weight; E, estrogen; HW, heart weight; OVX, ovariectomized; TL, tibial length; T, testosterone; UW, uterine weight; WKY, Wistar Kyoto. MTOR, 4EBP1 and eIF4E expression was augmented by OVX in comparison to sham group (Fig. 3D, E, G and H).
There was no difference in SBP and DBP levels between the medium-dose group and the low-dose group at the end of day 21 after intervention. The levels of IVST, LVPWT, LVM, E/A, and E/eâ in the OVX + Eâ+âT group were greater than those in the OVXâ+âE group (Fig. 2A and Table 2). There were statistically significant differences in blood pressure at various time points after testosterone intervention, and the effect of testosterone on blood pressure remained stable (Fig. 1A and B). The testosterone level in OVXâ+âEâ+âT group was higher than that in other groups (Table 1). Before drug intervention, the baseline level of blood pressure levels was same in each group.
The results suggest that testosterone and DHT may have differential effects on the cross-talk between mTOR and AR. However, the increase at 5 nM testosterone appeared unrelated to a change of AR level. Rapamycin completely blocked the phosphorylation of p70S6K and S6, and decreased only marginally the phosphorylation of 4EBP-1 (Figure 4A). The next step was to address whether mTOR regulates AR in a reciprocal manner and if testosterone modulates the signal from mTOR to AR. PSA was used as a target gene of AR to assess the successful inhibition of AR activity by AR knockdown.
Myostatin production is also induced by food deprivation in a glucocorticoid-dependent manner (Allen et al., 2010). Human myostatin promoter is reported to have a putative GRE and is responsive to dexamethasone and RU-486, an antagonist of GR (Ma et al., 2001). KlF15 plays a critical role in muscle catabolism through the transcriptional upregulation of atrogen-1, MuRF-1, and branched-chain aminotransferase 2 (BCAT2). Exogenous administration of glucocorticoids induces muscle atrophy and the blockage of GR; adrenalectomy or treatment with the GR antagonist RU486 diminishes muscle atrophy in sepsis, cachexia, starvation, and severe insulinopenia (Menconi et al., 2007; Schakman et al., 2008).
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.
Since mTOR is sensitive to nutrient levels, its activity would be diminished as a result of nutrient deprivation. Chen et al. (18) demonstrated that increased AR protein may amplify the output from residual ligand and alter the response to antagonist. Wang et al. (7) found that rapamycin inhibition of mTORC1 increases AR transcriptional activity via an Akt-dependent pathway downstream of mTORC2. The role of testosterone in glucose deprivation-induced apoptosis was therefore studied. Another experiment with the same protocol was carried out, with the exception that the trypan blue method was used to asses the percentage of dead cells (Figure 6B). To test this hypothesis, an adjuvant bicalutamide protocol was designed in which cells were subjected first to glucose deprivation for three days, followed by bicalutamide treatment for another day.
In this study, treatment with rapamycin does not affect myofibrillar synthesis, while it decreases the phosphorylation of p70S6K1 and S6, implying that mTOR is not involved in myostatin-mediated myofibrillar synthesis (Welle et al., 2009). The injection of a myostatin antibody enhances phosphorylation of p70S6K1 and S6 in muscle, but does not change phosphorylation of Akt and 4EBP1 in the concomitant increase of myofibrillar synthesis (Welle et al., 2009). Myostatin, a transforming growth factor-β (TGF-β) family member, plays a critical role in inhibiting the growth of muscle mass and muscle cell differentiation (McPherron et al., 1997). In line with mTOR function as a positive regulator of muscle hypertrophy, mTOR signaling is negatively regulated by muscle atrophy-inducing signals or blocks muscle atrophy signals. The loss of skeletal muscle, muscle atrophy, stems from an increase in the rate of protein degradation or the decrease of protein synthesis under various conditions, such as disuse, diseases, and aging. Muscle-specific expression of IGF-I in transgenic mice results in at least a 2-fold increase in muscle hypertrophy (Coleman et al., 1995; Musaro et al., 2001), suggesting that the IGF-I/Akt/mTORC1 pathway is indispensable to muscle hypertrophy. Instead, Akt /mTOR signaling by IGF-I/IGFR/IRS-1 has been shown to be indispensable in prompting muscle hypertrophy (Glass, 2003).
E, estrogen; OVX, ovariectomized; T, testosterone; WGA, wheat germ agglutinin; WKY, Wistar Kyoto. (B, C, D, and E) mRNA expression of β-MHC, ANP, MMP-9 and TIMP-1 by real-time RT-PCR. BW, body weight; E, estrogen; HW, heart weight; OVX, ovariectomized; TL, tibial length; T, testosterone; UW, uterine weight; WKY, Wistar Kyoto. MTOR, 4EBP1 and eIF4E expression was augmented by OVX in comparison to sham group (Fig. 3D, E, G and H).
There was no difference in SBP and DBP levels between the medium-dose group and the low-dose group at the end of day 21 after intervention. The levels of IVST, LVPWT, LVM, E/A, and E/eâ in the OVX + Eâ+âT group were greater than those in the OVXâ+âE group (Fig. 2A and Table 2). There were statistically significant differences in blood pressure at various time points after testosterone intervention, and the effect of testosterone on blood pressure remained stable (Fig. 1A and B). The testosterone level in OVXâ+âEâ+âT group was higher than that in other groups (Table 1). Before drug intervention, the baseline level of blood pressure levels was same in each group.
