Selective androgen receptor modulator, S42 has anabolic and anti-catabolic effects on cultured myotubes

Abstract

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague-Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1-p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.

Keywords: C2C12; Muscle; Myotube; SARM.

Fig. 1

Effects of S42 or DHT on Ar expression on C2C12 myotubes. C2C12 myotubes were incubated with 1–10 µM of S42 or 100 nM of DHT or appropriate vehicle (DMSO) for 24 h. (A) Comparison of mRNA expression levels of Ar relative to those of Gapdh by qPCR. Data are expressed as mean ± SE of triplicate samples. (B)Western blot analysis showing Ar and Gapdh. (C) Statistical comparison of the expression levels of Ar relative to Gapdh by Western blot analysis. Data are expressed as mean ± SE of triplicate samples. In statistical comparisons in (A) and (C), the data of treated groups with DHT or S42 were compared with that of untreated group. **P < 0.01 vs DMSO by one-way ANOVA.

Fig. 2

Effects of S42 or DHT on atrogin1, MuRF1, Igf1 expression on C2C12 myotubes. C2C12 myotubes were incubated with 1–10 µM of S42 or 100 nM of DHT or appropriate vehicle (DMSO) for 24 h. (A), (B), (C) Comparison of mRNA expression levels of atrogin1, MuRF1 and Igf1 relative to those of Gapdh, respectively are shown. Data are expressed as mean ± SE of triplicate samples. In statistical comparisons, the data of treated groups with DHT or S42 were compared with that of untreated group. *P < 0.05 vs DMSO by one-way ANOVA.

Fig. 3

S42 increases phosphorylation of p70S6K (Thr389) in C2C12 cells. Effects of S42 or insulin on phosphorylation of p70S6K (p-p70S6K) on C2C12 myotubes by Western blot analysis (A) and their statistical evaluations (B). C2C12 myotubes were incubated with 1–10 μM of S42 or 2 μM of insulin or appropriate vehicle for 24 h. Effect of S42 or insulin on phosphorylation of p70S6K (p-p70S6K) on C2C12 myotubes in the presence or absence of rapamycin by Western blotting (C) and their statistical evaluations (D). C2C12 myotubes were incubated with 1–10 μM of S42 or 2 μM of insulin or appropriate vehicle in the presence or absence of 1 nM of rapamycin for 24 h. In statistical comparisons, expressions of p-p70S6K protein relative to p70S6K protein were determined and the data of treated groups with S42 or insulin were compared with that of untreated group. Data are expressed as mean ± SE of triplicate samples. ^*P < 0.05, ^**P < 0.01 vs DMSO, ^# P < 0.05, ^##P < 0.01 in indicated comparisons by one-way ANOVA. ns, not significant.

Fig. 4

Effects of S42 on phosphorylation of Akt (Ser473) or Erk on C2C12 myotubes by Western blot analysis (A, B) and their statistical evaluations (C, D). C2C12 myotubes were incubated with 1–10 μM of S42 or 2 μM of insulin or appropriate vehicle for 24 h. In statistical comparisons, expressions of p-Akt relative to Akt (B) or p-Erk relative to Erk (D) were determined. Data of treated groups with S42 or insulin were compared with those of untreated group. Data are expressed as mean ± SE of triplicate samples. ^**P < 0.01 vs DMSO by one-way ANOVA.