Molecular Regulators of Muscle Mass and Mitochondrial Remodeling Are Not Influenced by Testosterone Administration in Young Women

Abstract

Testosterone (T) administration has previously been shown to improve muscle size and oxidative capacity. However, the molecular mechanisms underlying these adaptations in human skeletal muscle remain to be determined. Here, we examined the effect of moderate-dose T administration on molecular regulators of muscle protein turnover and mitochondrial remodeling in muscle samples collected from young women. Forty-eight healthy, physically active, young women (28 ± 4 years) were assigned in a random double-blind fashion to receive either T (10 mg/day) or placebo for 10-weeks. Muscle biopsies collected before and after the intervention period were divided into sub-cellular fractions and total protein levels of molecular regulators of muscle protein turnover and mitochondrial remodeling were analyzed using Western blotting. T administration had no effect on androgen receptor or 5α-reductase levels, nor on proteins involved in the mTORC1-signaling pathway (mTOR, S6K1, eEF2 and RPS6). Neither did it affect the abundance of proteins associated with proteasomal protein degradation (MAFbx, MuRF-1 and UBR5) and autophagy-lysosomal degradation (AMPK, ULK1 and p62). T administration also had no effect on proteins in the mitochondria enriched fraction regulating mitophagy (Beclin, BNIP3, LC3B-I, LC3B-II and LC3B-II/I ratio) and morphology (Mitofilin), and it did not alter the expression of mitochondrial fission- (FIS1 and DRP1) or fusion factors (OPA1 and MFN2). In summary, these data indicate that improvements in muscle size and oxidative capacity in young women in response to moderate-dose T administration cannot be explained by alterations in total expression of molecular factors known to regulate muscle protein turnover or mitochondrial remodeling.

Trial registration: ClinicalTrials.gov NCT03210558.

Keywords: androgen receptor; fission; fusion; mTORC1-signaling; ubiquitin-proteasome system.

Figure 1

Protein levels of androgen receptor (A), 5α-reductase (B), mTOR (C), S6K1 (D), eEF2 (E) and RPS6 (F) before (black dots) and after the intervention (red dots). Representative blots for each individual protein target are shown above each graph. Loading control is represented by a band at ~ 95 kDa from the corresponding total protein stain (Memcode™). The values presented are means ± SD and individual data points from 18 and 16 individuals (DF 32) in the placebo group and T group, respectively. The ANOVA revealed a significant main effect of time with respect to changes in protein levels for 5α-reductase and a significant main effect of time and group for RPS6.

Figure 2

Protein levels of MAFbx (A), MuRF-1 (B), UBR5 (C), AMPK (D), ULK1 (E) and p62 (F) before (black dots) and after the intervention (red dots). Representative blots for each individual protein target are shown above each graph. Loading control is represented by a band at ~ 95 kDa from the corresponding total protein stain (Memcode™). The values presented are means ± SD and individual data points from 18 and 16 individuals (DF 32) in the placebo group and T group, respectively.

Figure 3

Protein levels of Beclin (A), BNIP3 (B), LC3B-I (C), LC3B-II (D), LC3B-II/I ratio (E) and Mitofilin (F) in the mitochondrial fraction before (black dots) and after the intervention (red dots), as well as the assessment of mitochondrial fraction purity (G). Representative blots for each individual protein target and Porin are shown above each graph. The values presented are means ± SD and individual data points from 22 and 20 individuals (DF 40) in the placebo group and T group, respectively. The ANOVA revealed a significant main effect of time with respect to changes in protein levels for BNIP3 and LC3B-II. For illustrative purposes, two subjects from the placebo group displaying extreme values were removed from (A) (pre-post; 6.7 to 18.5 and 12.3 to 23.6, respectively) and one subject from the placebo group was removed from (D) (pre-post; 6.1 to 19.4), but these values were included in the statistical analysis.

Figure 4

Protein levels of OPA1 (A), MFN2 (B), FIS1 (C), and DRP1 (D) in the mitochondrial fraction before (black dots) and after the intervention (red dots). Representative blots for each individual protein target and Porin are shown above each graph. The values presented are means ± SD and individual data points from 22 and 20 individuals (DF 40) in the placebo group and T group, respectively. For illustrative purposes, one subject from the placebo group displaying extreme values was removed from (A) (pre-post; 6.1 to 19.4), but these values were included in the statistical analysis.