Maintenance of muscle mass in adult male mice is independent of testosterone

Abstract

Testosterone is considered a potent anabolic agent in skeletal muscle with a well-established role in adolescent growth and development in males. However, the role of testosterone in the regulation of skeletal muscle mass and function throughout the lifespan has yet to be fully established. While some studies suggest that testosterone is important for the maintenance of skeletal muscle mass, an understanding of the role this hormone plays in young, adult, and old males with normal and low serum testosterone levels is lacking. We investigated the role testosterone plays in the maintenance of muscle mass by examining the effect of orchiectomy-induced testosterone depletion in C57Bl6 male mice at ages ranging from early postnatal through old age (1.5-, 5-, 12-, and 24-month old mice). Following 28 days of testosterone depletion, we assessed mass and fiber cross-sectional-area (CSA) of the tibialis anterior, gastrocnemius, and quadriceps muscles. In addition, we measured global rates of protein synthesis and degradation using the SuNSET method, western blots, and enzyme activity assays. Twenty-eight days of testosterone depletion resulted in reduced muscle mass in the two youngest cohorts, but had no effect in the two oldest cohorts. Mean CSA decreased only in the youngest cohort and only in the tibialis anterior muscle. Testosterone depletion resulted in a general increase in proteasome activity at all ages. No change in protein synthesis was detected at the terminal time point. These data suggest that within physiological serum concentrations, testosterone may not be critical for the maintenance of muscle mass in mature male mice; however, in young mice testosterone is crucial for normal growth.

Fig 1. 28 days of testosterone depletion selectively decrease muscle mass in young mice and have no effect in older mice.

(A) body mass following one month of castration or sham castration surgery. (B) Serum total testosterone measures using an ELISA kit following one month of castration or sham castration surgery. Wet mass of the right perirenal fat pad (C), tibialis anterior (D), gastrocnemius (E), and quadriceps (F) following one month of castration or sham castration surgery. * indicates p < 0.05 between treatment groups of the same age.

Fig 2. 28 days of testosterone depletion decreases fiber CSA only in the TA muscle and only at 1.5-month-old mice.

(A) Tibialis anterior CSA following one month of castration or sham castration surgery. (B) Representative images from tibialis anterior histological section stained for laminin. (C) Gastrocnemius CSA following one month of castration or sham castration surgery. (D) Representative images from gastrocnemius histological sections stained for laminin. * indicates p < 0.05 between treatment groups of the same age.

Fig 3. One month of testosterone depletion has no effect on fiber CSA distribution in older mice.

(A) Distribution of the tibialis anterior fiber cross section area per animal and group. (B) Distribution of the gastrocnemius fiber cross section area per animal and group. * indicates p < 0.05 between treatment groups of the same age.

Fig 4. Protein degradation via the ubiquitin proteasome activity decreases with age and castration tends to increase the activity of this system.

Proteasome activity of the 26S β-5 subunit in the tibialis anterior (A), quadriceps (C), and gastrocnemius (E). Proteasome activity of the 20S β-5 subunit in the tibialis anterior (B), and quadriceps (D), and gastrocnemius (F). LC3BII/LC3BI ratio (G) and protein level of P62 (H). Representative images for LC3B (I) and P62 (J) blots. * indicates p < 0.05 between treatment groups of the same age.

Fig 5. Basal levels of protein synthesis protein synthesis are not affected by testosterone depletion across life span and in different muscles.

Quantification of protein synthesis rate using SUnSET in the tibialis anterior (A), quadriceps (D) and gastrocnemius (G) and quantification of 4E-BP1^The37/46 phosphorylation in the tibialis anterior (B) quadriceps (E) and gastrocnemius (H) muscles. Representative SUnSET blots are shown to the right (C, F, I). * indicates p < 0.05 between treatment groups of the same age.