Evaluation of early biomarkers of muscle anabolic response to testosterone

Abstract

BACKGROUND: Early biomarkers of skeletal muscle anabolism will facilitate the development of therapies for sarcopenia and frailty. METHODS AND RESULTS: We examined plasma type III collagen N-terminal propeptide (P3NP), skeletal muscle protein fractional synthesis rate, and gene and protein expression profiles to identify testosterone-induced changes in muscle anabolism. Two placebo-controlled studies enrolled community-dwelling men (study 1, 60-75 years; study 2, 18-40 years) with low to normal testosterone levels. Men were randomized to lower dose (study 1, 100 mg; study 2, 200 mg) or higher dose (study 1, 300 mg; study 2, 600 mg) single intramuscular testosterone or saline injection. After 1 week, testosterone acutely increased plasma P3NP levels in a dose-dependent manner and altered the expression of several skeletal muscle transcripts and proteins. Though not statistically significant, mixed muscle protein fractional synthesis rate tended to increase (1.08-fold with 100 mg testosterone, 1.12-fold with 300 mg testosterone). Testosterone exposure also increased skeletal muscle expression of the collagen type III gene that encodes P3NP. CONCLUSION: P3NP is a potentially useful early biomarker for muscle anabolic therapy. Skeletal muscle protein and RNA profiling are useful tools for the discovery of novel muscle anabolic biomarkers.

Figures

Fig. 1

Testosterone administration significantly increased both serum total and free testosterone levels in both young and older men after 1-week treatment. Serum total (a) and free testosterone (b) increased significantly with 300 and 100 mg testosterone IM compared with placebo in study 1 involving healthy 60- to 75-year-old men. Serum total (c) and free testosterone (d) increased significantly with 600 and 200 mg testosterone IM compared with placebo in study 2 involving healthy 18- to 40-year-old men. Y-axis is in log scale. Values of p, sample size (n), mean and 90% confidence intervals are shown

Fig. 2

Plasma P3NP changes from baseline after 1-week testosterone treatment. a Plasma P3NP concentration is significantly increased from baseline with 300 mg testosterone (T) relative to placebo (study 1). b Plasma P3NP concentration is significantly increased from baseline with 600 mg T relative to placebo (study 2). c Scatter plot of change in P3NP from baseline vs. change in total testosterone from baseline (study 1, filled circle; study 2, empty triangle). A line fitted to the points is shown (r = 0.518, p < 0.001). d Change in skeletal muscle FSR after 1-week treatment with 300 mg testosterone, 100 mg testosterone, and placebo. There were no statistically significant changes in FSR relative to placebo. Y-axis is in log scale. X-axis in log scale (c). Values of p, sample size (n), mean, and 90% confidence intervals are shown

Fig. 3

Skeletal muscle protein expression changes after 600-mg testosterone treatment. A plot of estimates of difference of relative intensity for the placebo, 200-mg, and 600-mg testosterone treatment groups at baseline (day 0) and after 1-week treatment (day 7) are shown. Two tryptic peptides of sequence LAPNIPLEMELPGVK and VQLLHTQNTSLINTK that correspond to the PDZ and LIM domain protein 3 and skeletal myosin heavy chain–light meromyosin region are shown. Y-axis is the area under the curve (AUC) on a natural log scale for the mass spectrometry feature. Values of p determined by a linear mixed model are shown

Fig. 4

The MTR index gene signature demonstrates a dose-dependent increase with testosterone comparing change from baseline and 1-week treatment. a In study 1, the MTR was assessed twice at baseline and twice after 1-week treatment; muscle biopsy performed at 3 and 12 h after beginning the stable isotope infusion for measuring fractional synthetic rate. For comparison between groups, p < 3.9 × 10−5 (3 h) and p < 2.7 × 10−4 (12 h). b In study 2, MTR was assessed once at baseline and after 1-week treatment. For comparison between groups, p < 6.2 × 10−4. c Collagen 3 (COL3A1) RNA expression is increased with the testosterone 600-mg dose compared with testosterone 200-mg dose and placebo. The box has horizontal lines at the lower, median, and upper quartile values. Whiskers extend from each end of the box to the adjacent values in the data, the most extreme values within 1.5 times the interquartile range from the ends of the box. Outliers displayed are data with values beyond the ends of the whiskers