Testosterone supplementation reverses sarcopenia in aging through regulation of myostatin, c-Jun NH2-terminal kinase, Notch, and Akt signaling pathways

Abstract

Aging in rodents and humans is characterized by loss of muscle mass (sarcopenia). Testosterone supplementation increases muscle mass in healthy older men. Here, using a mouse model, we investigated the molecular mechanisms by which testosterone prevents sarcopenia and promotes muscle growth in aging. Aged mice of 22 months of age received a single sc injection of GnRH antagonist every 2 wk to suppress endogenous testosterone production and were implanted subdermally under anesthesia with 0.5 or 1.0 cm testosterone-filled implants for 2 months (n = 15/group). Young and old mice (n = 15/group), of 2 and 22 months of age, respectively, received empty implants and were used as controls. Compared with young animals, a significant (P < 0.05) increase in muscle cell apoptosis coupled with a decrease in gastrocnemius muscles weight (by 16.7%) and muscle fiber cross-sectional area, of both fast and slow fiber types, was noted in old mice. Importantly, such age-related changes were fully reversed by higher dose (1 cm) of testosterone treatment. Testosterone treatment effectively suppressed age-specific increases in oxidative stress, processed myostatin levels, activation of c-Jun NH(2)-terminal kinase, and cyclin-dependent kinase inhibitor p21 in aged muscles. Furthermore, it restored age-related decreases in glucose-6-phosphate dehydrogenase levels, phospho-Akt, and Notch signaling. These alterations were associated with satellite cell proliferation and differentiation. Collectively these results suggest involvement of multiple signal transduction pathways in sarcopenia. Testosterone reverses sarcopenia through stimulation of cellular metabolism and survival pathway together with inhibition of death pathway.

Figure 1

Testosterone supplementation results in suppression of 4-HNE levels and apoptosis in aged muscles. Panel A, Western blots of muscle lysates from young, old, and old mice treated with testosterone (T) show suppression of age-related increase in 4-HNE levels by both doses of testosterone. The gels are representative of two animals at each group from one of three separate experiments. GAPDH in the immunoblot is shown as a loading control. B, Quantification of band intensities. Values are mean ± sem. Means with unlike letters are significantly (P < 0.05) different. Means with superscript letter A are different from means with letter B. However, means with letters AB are not different from means with either letter A or B. Panel C, In situ detection of muscle cell apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL). Compared with young, in which no apoptosis is detected, a distinct increase in the incidence of muscle cell apoptosis is evident in aged muscles. Scale bar, 15 μm. Panel D, Quantitation of muscle cell apoptosis in young, old, and old mice treated with testosterone. Apoptotic rate was expressed as the percentage of TUNEL-positive nuclei per total nuclei (apoptotic plus nonapoptotic nuclei) counted in a unit reference area. Testosterone treatment at both doses significantly prevents age-related increase in muscle cell apoptosis. Values are mean ± sem. Means with unlike letters are significantly (P < 0.001) different.

Figure 2

Panel A, Western blots of muscle lysates show the presence of unprocessed (45–55 kDa) and the processed (32 kDa) myostatin in young and old mice, respectively. Testosterone (T) effectively suppresses the processed myostatin levels in the aged muscles. The gels are representative of two animals at each group from one of three separate experiments. GAPDH in the immunoblot is shown as a loading control. Panel B, Quantification of band intensities shows significant suppression of the processed myostatin levels after treatment with 1 cm testosterone. Values are mean ± sem. Means with letters BC are not different from means with either superscript letter B or C but are significantly (P < 0.05) different from means with letter A. Panel C, EIA assay reveals a significant increase in phospho-JNK levels in old animals when compared with young animals, which can be significantly prevented by testosterone treatment at both doses. Values are mean ± sem. Means with unlike letters are significantly (P < 0.001) different. Panel D, Phopho-p38 MAPK levels were significantly increased in aged muscles compared with that of young animals. However, testosterone treatment had no effect on aging-associated activation of p38 MAPK. Values are mean ± sem. Means with unlike letters are significantly (P < 0.05) different.

Figure 3

Testosterone-induced muscle fiber hypertrophy in old mice is associated with stimulation of Notch signaling and suppression of age-specific induction of CDK inhibitor p21. Panel A, Western blots of muscle lysates show increased levels of Notch 1 in muscle lysates from old animals after testosterone (T) treatment when compared with both untreated young and old mice, in which a modest expression of Notch is detected. Activation of Notch signaling after testosterone treatment is associated with increased expression of PCNA in muscle lysates. Compared with young animals, in which little or no expression is detected, a distinct age-related increase in p21 expression is detected in aged mice. Testosterone treatment at both doses effectively up-regulates both Notch 1 and PCNA but attenuates age-specific increase in p21 levels. Panels B–D, Quantification of band intensities shows robust up-regulation of Notch 1 and PCNA and complete restoration of p21 to its young levels in aged mice after testosterone treatment. Values are mean ± sem. Means with unlike letters are significantly (P < 0.05) different.

Figure 4

A, Double-immunofluorescence staining for PAX3/7 (green) and PCNA (red) from young (upper panels), old (middle panels), and old+testosterone-treated gastrocnemius muscles (lower panels) shows colocalization of PAX3/7 and PCNA (yellow). Scale bar, 50 μm. Panels B and C, Quantitation of the numbers of PCNA (B)- and myogenin-positive (Panel C) shows a significant increase in the number of these cells in aged muscles after testosterone treatment. Values are mean ± sem. Means with unlike letters are significantly (P < 0.05) different.

Figure 5

Panel A, Western blot analysis shows decreased phospho-Akt and G6PDH levels in aged gastrocnemius muscles compared with that of young muscles. Supplementation with 1 cm testosterone (T) prevents such age-related decrease in phospho-Akt and G6PDH levels. The gels are representative of two animals at each group from one of three separate experiments. GAPDH in the immunoblot is shown as a loading control. Panels B and C, Quantification of band intensities shows complete restoration of phospho-Akt and G6PDH, respectively, to their young levels after treatment with 1 cm testosterone. Values are mean ± sem. Means with unlike letters are significantly (P < 0.05) different.

Figure 6

Key signaling pathways involved in testosterone-mediated mitigation of sarcopenia in aging. Testosterone, though suppression myostatin, inhibits JNK but stimulates Akt signaling. Suppression of JNK promotes muscle growth by not only inhibiting muscle cell apoptosis but also stimulating cellular proliferation by attenuating myostatin-induced up-regulation of p21. Akt can promote muscle growth through direct activation of Notch signaling (67) as well as through modulation of multiple signaling molecules involved in both apoptotic and survival pathways in muscle remodeling (24). Akt can also restrain caspase-2-mediated death pathway and promote muscle growth by stimulation of cellular metabolism (47). Thus, testosterone through stimulation of survival pathway together with the inhibition of death pathway possibly restores the microenvironment and promotes muscle growth in aging.