Effect of long-term testosterone therapy on molecular regulators of skeletal muscle mass and fibre-type distribution in aging men with subnormal testosterone.

BACKGROUND: Long-term testosterone replacement therapy (TRT) increases muscle mass in elderly men with subnormal testosterone levels. However, the molecular mechanisms underlying this effect of TRT on protein balance in human skeletal muscle in vivo remain to be established. METHODS: Here, we examined skeletal muscle biopsies obtained before and 24-h after the last dose of treatment with either testosterone gel (n = 12) or placebo (n = 13) for 6 months in aging men with subnormal bioavailable testosterone levels. The placebo-controlled, testosterone-induced changes (ß-coefficients) in mRNA levels, protein expression and phosphorylation were examined by quantitative real-time PCR and western blotting. RESULTS: Long-term TRT increased muscle mass by ß = 1.6 kg (p = 0.01) but had no significant effect on mRNA levels of genes involved in myostatin/activin/SMAD or IGF1/FOXO3 signalling, muscle-specific E3-ubiquitin ligases, upstream transcription factors (MEF2C, PPARGC1A-4) or myogenic factors. However, TRT caused a sustained decrease in protein expression of SMAD2 (ß = -36%, p = 0.004) and SMAD3 (ß = -32%, p = 0.001), which was accompanied by reduced protein expression of the muscle-specific E3-ubiquitin ligases, MuRF1 (ß = -26%, p = 0.004) and Atrogin-1/MAFbx (ß = -20%, p = 0.04), but with no changes in FOXO3 signalling. Importantly, TRT did not affect muscle fibre type distribution between slow-oxidative (type 1), fast-oxidative (type 2a) and fast-glycolytic (type 2×) muscle fibres. CONCLUSIONS: Our results indicate that long-term TRT of elderly men with subnormal testosterone levels increases muscle mass, at least in part, by decreasing protein breakdown through the ubiquitin proteasome pathway mediated by a sustained suppression of SMAD-signalling and muscle-specific E3-ubiquitin ligases.

Marker-dependent associations among oxidative stress, growth and survival during early life in a wild mammal.

Oxidative stress (OS) is hypothesized to be a key physiological mechanism mediating life-history trade-offs, but evidence from wild populations experiencing natural environmental variation is limited. We tested the hypotheses that increased early life growth rate increases OS, and that increased OS reduces first-winter survival, in wild Soay sheep (Ovis aries) lambs. We measured growth rate and first-winter survival for four consecutive cohorts, and measured two markers of oxidative damage (malondialdehyde (MDA), protein carbonyls (PC)) and two markers of antioxidant (AOX) protection (total AOX capacity (TAC), superoxide dismutase (SOD)) from blood samples. Faster lamb growth was weakly associated with increased MDA, but not associated with variation in the other three markers. Lambs with higher SOD activity were more likely to survive their first winter, as were male but not female lambs with lower PC concentrations. Survival did not vary with MDA or total TAC. Key predictions relating OS to growth and survival were therefore supported in some OS markers, but not others. This suggests that different markers capture different aspects of the complex relationships between individual oxidative state, physiology and fitness, and that overarching hypotheses relating OS to life-history variation cannot be supported or refuted by studying individual markers.

Plasma markers of oxidative stress are uncorrelated in a wild mammal.

Oxidative stress, which results from an imbalance between the production of potentially damaging reactive oxygen species versus antioxidant defenses and repair mechanisms, has been proposed as an important mediator of life-history trade-offs. A plethora of biomarkers associated with oxidative stress exist, but few ecological studies have examined the relationships among different markers in organisms experiencing natural conditions or tested whether those relationships are stable across different environments and demographic groups. It is therefore not clear to what extent studies of different markers can be compared, or whether studies that focus on a single marker can draw general conclusions regarding oxidative stress. We measured widely used markers of oxidative damage (protein carbonyls and malondialdehyde) and antioxidant defense (superoxide dismutase and total antioxidant capacity) from 706 plasma samples collected over a 4-year period in a wild population of Soay sheep on St Kilda. We quantified the correlation structure among these four markers across the entire sample set and also within separate years, age groups (lambs and adults), and sexes. We found some moderately strong correlations between some pairs of markers when data from all 4 years were pooled. However, these correlations were caused by considerable among-year variation in mean marker values; correlation coefficients were small and not significantly different from zero after accounting for among-year variation. Furthermore, within each year, age, and sex subgroup, the pairwise correlation coefficients among the four markers were weak, nonsignificant, and distributed around zero. In addition, principal component analysis confirmed that the four markers represented four independent axes of variation. Our results suggest that plasma markers of oxidative stress may vary dramatically among years, presumably due to environmental conditions, and that this variation can induce population-level correlations among markers even in the absence of any correlations within contemporaneous subgroups. The absence of any consistent correlations within years or demographic subgroups implies that care must be taken when generalizing from observed relationships with oxidative stress markers, as each marker may reflect different and potentially uncoupled biochemical processes.

Differential effects of strength training and testosterone treatment on soluble CD36 in aging men: Possible relation to changes in body composition.

PURPOSE: We measured soluble CD36 (sCD36) and body composition to determine the effects of testosterone treatment (TT) and/or strength training (ST) on cardiovascular risk in men with low normal testosterone levels. METHODS: Double-blinded, placebo-controlled study in 54 men aged 60-78 years with bioavailable testosterone < 7.3 nmol/L and waist > 94 cm randomized to TT (gel, 50-100 mg/day, n = 20), placebo (n = 18) or ST (n = 16) for 6 months. Moreover, the ST group was randomized to TT (ST + TT, n = 7) or placebo (ST + placebo, n = 9) after 3 months. OUTCOMES: sCD36, total and regional fat mass were established by Dual X-ray absorptiometry and magnetic resonance imaging. Data are presented as median (quartiles). Kruskal-Wallis and Mann-Whitney tests were performed on delta values at 0, 3 and 6 months. RESULTS: ST + placebo decreased sCD36 levels by 21% [from 0.80 (0.68-1.22) to 0.63 (0.51-0.73) rel. units] vs. TT and vs. placebo (p < 0.05). ST + placebo did not change bioavailable testosterone and lean body mass. Fat mass measures significantly improved during ST + placebo, ST + TT, and TT vs. placebo. During ST + placebo, delta sCD36 was associated with delta total fat mass (r = 0.81) and delta central fat mass (r = 0.84). CONCLUSIONS: Compared to testosterone treatment, six months of strength training reduced sCD36 levels suggesting decreased cardiovascular risk, possibly due to a reduction in central fat mass.

Oxidative stress and life histories: unresolved issues and current needs.

Life-history theory concerns the trade-offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life-history trade-offs, but the details remain obscure. As life-history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life-history trade-offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life-history information is available, cannot generally be performed without compromising the aims of the studies that generated the life-history data. There is a need therefore for novel non-invasive measurements of multi-tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life-history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life-history trade-offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life-history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade-offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other.

Effect of testosterone on markers of mitochondrial oxidative phosphorylation and lipid metabolism in muscle of aging men with subnormal bioavailable testosterone.

OBJECTIVE: Recent studies have indicated that serum testosterone in aging men is associated with insulin sensitivity and expression of genes involved in oxidative phosphorylation (OxPhos), and that testosterone treatment increases lipid oxidation. Herein, we investigated the effect of testosterone therapy on regulators of mitochondrial biogenesis and markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels. METHODS: Skeletal muscle biopsies were obtained before and after treatment with either testosterone gel (n=12) or placebo (n=13) for 6 months. Insulin sensitivity and substrate oxidation were assessed by euglycemic-hyperinsulinemic clamp and indirect calorimetry. Muscle mRNA levels and protein abundance and phosphorylation of enzymes involved in mitochondrial biogenesis, OxPhos, and lipid metabolism were examined by quantitative real-time PCR and western blotting. RESULTS: Despite an increase in lipid oxidation (P<0.05), testosterone therapy had no effect on insulin sensitivity or mRNA levels of genes involved in mitochondrial biogenesis (PPARGC1A, PRKAA2, and PRKAG3), OxPhos (NDUFS1, ETFA, SDHA, UQCRC1, and COX5B), or lipid metabolism (ACADVL, CD36, CPT1B, HADH, and PDK4). Consistently, protein abundance of OxPhos subunits encoded by both nuclear (SDHA and UQCRC1) and mitochondrial DNA (ND6) and protein abundance and phosphorylation of AMP-activated protein kinase and p38 MAPK were unaffected by testosterone therapy. CONCLUSION: The beneficial effect of testosterone treatment on lipid oxidation is not explained by increased abundance or phosphorylation-dependent activity of enzymes known to regulate mitochondrial biogenesis or markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels.

Mechanical muscle function and lean body mass during supervised strength training and testosterone therapy in aging men with low-normal testosterone levels.

OBJECTIVES: To examine the effect of strength training and testosterone therapy on mechanical muscle function and lean body mass (LBM) in aging men with low-normal testosterone levels in a randomized, double-blind, placebo-controlled 24-week study. DESIGN: Randomized, double-blind, placebo-controlled. SETTING: Odense, Denmark. PARTICIPANTS: Men aged 60 to 78, with bioavailable testosterone levels of less than 7.3 nmol/L and a waist circumference greater than 94 cm were randomized to testosterone (50-100 mg/d, n = 22) placebo (n = 23) or strength training (n = 23) for 24 weeks. The strength training group was randomized to addition of testosterone or placebo after 12 weeks. Subjects performed supervised strength training (2-3 sets with 6- to 10-repetition maximum loads, 3 times per week). MEASUREMENTS: Testosterone levels, maximal voluntary contraction and rate of force development, and LBM were obtained at 0 and at Weeks 12 and 24 of the intervention. RESULTS: No changes in any variables were recorded with placebo. In the strength training group, maximal voluntary contraction increased 8% after 12 weeks (P = .005). During the following 12 weeks of strength training rate of force development increased by 10% (P = .04) and maximal voluntary contraction further increased (P < .001). Mechanical muscle function was unchanged in men receiving only testosterone for 24 weeks. LBM increased only in men receiving testosterone (P = .004). CONCLUSION: Strength training in aging men with low-normal testosterone levels may improve mechanical muscle function, but this effect occurs without a significant increase in LBM. Clinically, only the combination of testosterone therapy and strength training resulted in an increase in mechanical muscle function and LBM.