Castration alters protein balance after high-frequency muscle contraction

Abstract

Resistance exercise increases muscle mass by shifting protein balance in favor of protein accretion. Androgens independently alter protein balance, but it is unknown whether androgens alter this measure after resistance exercise. To answer this, male mice were subjected to sham or castration surgery 7-8 wk before undergoing a bout of unilateral, high-frequency, electrically induced muscle contractions in the fasted or refed state. Puromycin was injected 30 min before euthanasia to measure protein synthesis. The tibialis anterior was analyzed 4 h postcontraction. In fasted mice, neither basal nor stimulated rates of protein synthesis were affected by castration despite lower phosphorylation of mechanistic target of rapamycin in complex 1 (mTORC1) substrates [p70S6K1 (Thr389) and 4E-BP1 (Ser65)]. Markers of autophagy (LC3 II/I ratio and p62 protein content) were elevated by castration, and these measures remained elevated above sham values after contractions. Furthermore, in fasted mice, the protein content of Regulated in Development and DNA Damage 1 (REDD1) was correlated with LC3 II/I in noncontracted muscle, whereas phosphorylation of uncoordinated like kinase 1 (ULK1) (Ser757) was correlated with LC3 II/I in the contracted muscle. When mice were refed before contractions, protein synthesis and mTORC1 signaling were not affected by castration in either the noncontracted or contracted muscle. Conversely, markers of autophagy remained elevated in the muscles of refed, castrated mice even after contractions. These data suggest the castration-mediated elevation in baseline autophagy reduces the absolute positive shift in protein balance after muscle contractions in the refed or fasted states.

New & noteworthy: In the absence of androgens, markers of autophagy were elevated, and these could not be normalized by muscle contractions. In the fasted state, REDD1 was identified as a potential contributor to autophagy in noncontracted muscle, whereas phosphorylation of ULK1 may contribute to this process in the contracted muscle. In the refed state, markers of autophagy remain elevated in both noncontracted and contracted muscles, but the relationship with REDD1 and ULK1 (Ser757) no longer existed.

Keywords: autophagy; protein degradation; protein synthesis; resistance exercise.

Fig. 1.

Rates of muscle protein synthesis and mTORC1 signaling in the fasted metabolic state. A: muscle protein synthesis was determined by the SUnSET method. Phosphorylated to total protein ratio of p70S6K1 (Thr389) (B) and 4E-BP1 (Ser65) (C) was determined by Western blot analysis. D: representative Western blots. Significance set at P < 0.05. ME, main effect; P, phosphorylated protein; T, total protein. N = 7 or 8/group from 3 independent experiments.

Fig. 2.

Markers of protein degradation in the fasted metabolic state. LC3 II/I ratio (A), p62 protein content (B), and the content of ubiquitylated proteins (C) were determined by Western blot analysis. Relative mRNA abundance of MAFbx (D) and MuRF-1 (E) were determined by qRT-PCR using GAPDH as an internal control. F: representative Western blots. Significance set at P < 0.05. ME, main effect. N = 6–8/group from 3 independent experiments. *Significance between noncontracted and contracted muscle within a treatment group. #Significance between sham-castrated and castrated groups for specified condition.

Fig. 3.

Analysis of autophagy regulatory factors in the fasted metabolic state. Phosphorylated to total protein ratio of ULK1 (Ser317) (A), ULK1 (Ser757) (B), Akt (Thr308) (C), FoxO3a (Ser253) (D), and AMPK (Thr172) (E) were determined by Western blot analysis. Total protein content of BNIP3 (F) and REDD1 (G) protein content were determined by Western blot analysis. H: representative Western blots. Significance set at P < 0.05. ME, main effect; P, phosphorylated protein; T, total protein. N = 8/group from 3 independent experiments.

Fig. 4.

Protein synthesis and mTORC1 signaling in the refed metabolic state. A: muscle protein synthesis was determined by the SUnSET method. Phosphorylated to total protein ratio of p70S6K1 (Thr389) (B) and 4E-BP1 (Ser65) (C) were determined by Western blot analysis. D: representative Western blots. Significance set at P < 0.05. ME, main effect; P, phosphorylated protein; T, total protein. N = 7–10/group from 4 independent experiments.

Fig. 5.

Markers of protein degradation in the refed metabolic state. LC3 II/I ratio (A), p62 protein content (B), and content of ubiquitylated proteins (C) were determined by Western blot analysis. Phosphorylated to total protein ratio of ULK1 (Ser757) (D) and the protein content of REDD1 (E) were determined by Western blot analysis. F: representative Western blots. ME, main effect; P, phosphorylated protein; T, total protein. N = 8–10/group from 4 independent experiments.