SIRT1 induction in the skeletal muscle of male mice partially preserves limb muscle mass but not contractile force in response to androgen deprivation.

In males, the factors that decrease limb muscle mass and strength in response to androgen deprivation are largely unknown. Sirtuin1 (SIRT1) protein levels are lower in the limb muscle of male mice subjected to androgen deprivation. The present study aimed to assess whether SIRT1 induction preserved limb muscle mass and force production in response to androgen deprivation. Physically mature male mice containing an inducible muscle-specific SIRT1 transgene were subjected to a sham or castration surgery and compared to sham and castrated male mice where the SIRT1 transgene was not induced. SIRT1 induction partially preserved whole-body lean mass, tibialis anterior (TA) mass and triceps surae muscle mass in response to castration. Further analysis of the TA muscle showed that muscle-specific SIRT1 induction partially preserved limb muscle soluble protein content and fibre cross-sectional area. Unilateral AAV9-mediated SIRT1 induction in the TA muscle showed that SIRT1 partially preserved mass by acting directly in the muscle. Despite those positive outcomes to limb muscle morphology, muscle-specific SIRT1 induction did not preserve the force generating capacity of the TA or triceps surae muscles. Interestingly, SIRT1 induction in females did not alter limb muscle mass or limb muscle strength even though females have naturally low androgen levels. SIRT1 also did not alter the androgen-mediated increase in limb muscle mass or strength in females. In all, these data suggest that decreases in SIRT1 protein in the limb muscle of males may partially contribute to the loss of limb muscle mass in response to androgen deprivation. KEY POINTS: SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation partially preserved limb muscle mass and fibre cross-sectional area. SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation did not prevent preserve limb muscle force generating capacity. SIRT1 induction in skeletal muscle of females did not alter baseline limb muscle mass, nor did it affect the androgen-mediated increase in limb muscle mass.

Binge alcohol disrupts skeletal muscle core molecular clock independent of glucocorticoids.

Circadian rhythms are central to optimal physiological function, as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle [Zeitgeber time (ZT12)], and gastrocnemius was collected every 4 h from control and EtOH-treated mice for the next 48 h following isoflurane anesthetization. In addition, metyrapone was administered before alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100 mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1, Per1/2, and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1, Dbp, Tef, and Bhlhe40 (P < 0.05), in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1, in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1, Cry1 and Cry2, and Myod1 mRNA expression. Core clock gene expression (Bmal, Per1/2, and Cry1/2) was not changed following 4, 8, or 12 h of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle.NEW & NOTEWORTHY Alcohol is a myotoxin that impairs skeletal muscle metabolism and function following either chronic consumption or acute binge drinking; however, mechanisms underlying alcohol-related myotoxicity have not been fully elucidated. Herein, we demonstrate that alcohol acutely interrupts oscillation of skeletal muscle core clock genes, and this is neither a direct effect of ethanol on the skeletal muscle, nor an effect of elevated serum corticosterone, a major clock regulator.

Androgen depletion alters the diurnal patterns to signals that regulate autophagy in the limb skeletal muscle.

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy protein breakdown pathway in the limb muscle that persist throughout the diurnal cycle. However, the regulatory signals underpinning the increase in autophagy markers remain ill-defined. The purpose of this study was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. Male mice were subjected to a castration surgery or a sham surgery as a control. Seven weeks post-surgery, a subset of mice from each group was sacrificed every 4 hr over a 24 hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. Consistent with an overall increase in autophagy, the phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice. Factors that induce the progression of autophagy were also increased in the TA following androgen depletion including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL-2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). Moreover, we observed an increase in the protein expression pattern of p53 and the mRNA of the p53 target genes Cyclin-Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These data characterize novel changes to autophagy regulatory signals in the limb skeletal muscle following androgen deprivation.

The Effects of Multiple Sets of Squats and Jump Squats on Mechanical Variables.

Rossetti, ML, Munford, SN, Snyder, BW, Davis, SE, and Moir, GL. The effects of multiple sets of squats and jump squats on mechanical variables. J Strength Cond Res 34(4): 1017-1023, 2020-The mechanical responses to 2 nonballistic squat and 2 ballistic jump squat protocols performed over multiple sets were investigated. One protocol from each of the 2 nonballistic and ballistic conditions incorporated a pause between the eccentric and concentric phases of the movements in order to determine the influence of the coupling time on the mechanical variables and postactivation potentiation (PAP). Eleven men (age: 21.9 ± 1.8 years; height: 1.79 ± 0.05 m; mass: 87.0 ± 7.4 kg) attended 4 sessions where they performed multiple sets of squats and jump squats with a load equivalent to 30% 1-repetition maximum under one of the following conditions: (a) 3 × 4 repetitions of nonballistic squats (30N-B); (b) 3 × 4 repetitions of nonballistic squats with a 3-second pause between the eccentric and concentric phases of each repetition (30PN-B); (c) 3 × 4 repetitions of ballistic jump squats (30B); (d) 3 × 4 repetitions of ballistic jump squats with a 3-second pause between the eccentric and concentric phases of each repetition (30PB). Force plates were used to calculate variables including average vertical velocity, average vertical force (GRF), and average power output (PO). Vertical velocities during the ballistic conditions were significantly greater than those attained during the nonballistic conditions (mean differences: 0.21-0.25 m·s, p < 0.001, effect sizes [ES]: 1.70-1.89) as were GRFs (mean differences: 478-526 N, p < 0.001, ES: 1.61-1.63), and PO (mean differences: 711-869 W, p < 0.001, ES: 1.66-1.73). Moreover, the increase in PO across the 3 sets in 30B was significantly greater than the changes observed during 30N-B, 30PN-B, and 30PB (p ≤ 0.015). The pause reduced the mechanical variables during both the nonballistic and ballistic conditions, although the differences were not statistically significant (p > 0.05). Ballistic jump squats may be an effective exercise for developing PO given the high velocities and forces generated in these exercises. Furthermore, the completion of multiple sets of jump squats may induce PAP to enhance PO. The coupling times between the eccentric and concentric phases of the jump squats should be short in order to maximize the GRF and PO across the sets.

Arrdc2 and Arrdc3 elicit divergent changes in gene expression in skeletal muscle following anabolic and catabolic stimuli.

Skeletal muscle is a highly plastic organ regulating various processes in the body. As such, loss of skeletal muscle underlies the increased morbidity and mortality risk that is associated with numerous conditions. However, no therapies are available to combat the loss of muscle mass during atrophic conditions, which is due in part to the incomplete understanding of the molecular networks altered by anabolic and catabolic stimuli. Thus, the current objective was to identify novel gene networks modulated by such stimuli. For this, total RNA from the tibialis anterior muscle of mice that were fasted overnight or fasted overnight and refed the next morning was subjected to microarray analysis. The refeeding stimulus altered the expression of genes associated with signal transduction. Specifically, expression of alpha arrestin domain containing 2 (Arrdc2) and alpha arrestin domain containing 3 (Arrdc3) was significantly lowered 70-85% by refeeding. Subsequent analysis showed that expression of these genes was also lowered 50-75% by mechanical overload, with the combination of nutrients and mechanical overload acting synergistically to lower Arrdc2 and Arrdc3 expression. On the converse, stimuli that suppress growth such as testosterone depletion or acute aerobic exercise increased Arrdc2 and Arrdc3 expression in skeletal muscle. While Arrdc2 and Arrdc3 exhibited divergent changes in expression following anabolic or catabolic stimuli, no other member of the Arrdc family of genes exhibited the consistent change in expression across the analyzed conditions. Thus, Arrdc2 and Arrdc3 are a novel set of genes that may be implicated in the regulation of skeletal muscle mass.

Disruptions to the limb muscle core molecular clock coincide with changes in mitochondrial quality control following androgen depletion.

Androgen depletion in humans leads to significant atrophy of the limb muscles. However, the pathways by which androgens regulate limb muscle mass are unclear. Our laboratory previously showed that mitochondrial degradation was related to the induction of autophagy and the degree of muscle atrophy following androgen depletion, implying that decreased mitochondrial quality contributes to muscle atrophy. To increase our understanding of androgen-sensitive pathways regulating decreased mitochondrial quality, total RNA from the tibialis anterior of sham and castrated mice was subjected to microarray analysis. Using this unbiased approach, we identified significant changes in the expression of genes that compose the core molecular clock. To assess the extent to which androgen depletion altered the limb muscle clock, the tibialis anterior muscles from sham and castrated mice were harvested every 4 h throughout a diurnal cycle. The circadian expression patterns of various core clock genes and known clock-controlled genes were disrupted by castration, with most genes exhibiting an overall reduction in phase amplitude. Given that the core clock regulates mitochondrial quality, disruption of the clock coincided with changes in the expression of genes involved with mitochondrial quality control, suggesting a novel mechanism by which androgens may regulate mitochondrial quality. These events coincided with an overall increase in mitochondrial degradation in the muscle of castrated mice and an increase in markers of global autophagy-mediated protein breakdown. In all, these data are consistent with a novel conceptual model linking androgen depletion-induced limb muscle atrophy to reduced mitochondrial quality control via disruption of the molecular clock.

Hormonal regulation of core clock gene expression in skeletal muscle following acute aerobic exercise.

Exercise increases skeletal muscle health in part by altering the types of genes that are transcribed. Previous work suggested that glucocorticoids signal through the protein Regulated in Development and DNA Damage 1 (REDD1) to regulate gene expression following acute aerobic exercise. The present study shows that expression of the core clock gene, Period1, is among those modulated by the glucocorticoid-REDD1 signaling pathway in skeletal muscle. We also provide evidence that Aldosterone and Epinephrine contribute to the regulation of Period1 expression via REDD1. These data show that adrenal stress hormones signal through REDD1 to regulate skeletal muscle gene expression, specifically those of the core clock, following acute aerobic exercise.

Androgens induce growth of the limb skeletal muscles in a rapamycin-insensitive manner.

Signaling through the mechanistic target of rapamycin complex 1 (mTORC1) has been well defined as an androgen-sensitive transducer mediating skeletal muscle growth in vitro; however, this has yet to be tested in vivo. As such, male mice were subjected to either sham or castration surgery and allowed to recover for 7 wk to induce atrophy of skeletal muscle. Then, castrated mice were implanted with either a control pellet or a pellet that administered rapamycin (~2.5 mg·kg-1·day-1). Seven days postimplant, a subset of castrated mice with control pellets and all castrated mice with rapamycin pellets were given once weekly injections of nandrolone decanoate (ND) to induce muscle growth over a six-week period. Effective blockade of mTORC1 by rapamycin was noted in the skeletal muscle by the inability of insulin to induce phosphorylation of ribosomal S6 kinase 1 70 kDa (Thr389) and uncoordinated-like kinase 1 (Ser757). While castration reduced tibialis anterior (TA) mass, muscle fiber cross-sectional area, and total protein content, ND administration restored these measures to sham levels in a rapamycin-insensitive manner. Similar findings were also observed in the plantaris and soleus, suggesting this rapamycin-insensitive effect was not specific to the TA or fiber type. Androgen-mediated growth was not due to changes in translational capacity. Despite these findings in the limb skeletal muscle, rapamycin completely prevented the ND-mediated growth of the heart. In all, these data indicate that mTORC1 has a limited role in the androgen-mediated growth of the limb skeletal muscle; however, mTORC1 was necessary for androgen-mediated growth of heart muscle.

REDD1 induction regulates the skeletal muscle gene expression signature following acute aerobic exercise.

The metabolic stress placed on skeletal muscle by aerobic exercise promotes acute and long-term health benefits in part through changes in gene expression. However, the transducers that mediate altered gene expression signatures have not been completely elucidated. Regulated in development and DNA damage 1 (REDD1) is a stress-induced protein whose expression is transiently increased in skeletal muscle following acute aerobic exercise. However, the role of this induction remains unclear. Because REDD1 altered gene expression in other model systems, we sought to determine whether REDD1 induction following acute exercise altered the gene expression signature in muscle. To do this, wild-type and REDD1-null mice were randomized to remain sedentary or undergo a bout of acute treadmill exercise. Exercised mice recovered for 1, 3, or 6 h before euthanization. Acute exercise induced a transient increase in REDD1 protein expression within the plantaris only at 1 h postexercise, and the induction occurred in both cytosolic and nuclear fractions. At this time point, global changes in gene expression were surveyed using microarray. REDD1 induction was required for the exercise-induced change in expression of 24 genes. Validation by RT-PCR confirmed that the exercise-mediated changes in genes related to exercise capacity, muscle protein metabolism, neuromuscular junction remodeling, and Metformin action were negated in REDD1-null mice. Finally, the exercise-mediated induction of REDD1 was partially dependent upon glucocorticoid receptor activation. In all, these data show that REDD1 induction regulates the exercise-mediated change in a distinct set of genes within skeletal muscle.

Effects of chronic alcohol intoxication on aerobic exercise-induced adaptations in female mice.

Chronic alcohol intoxication decreases muscle strength/function and causes mitochondrial dysfunction. Aerobic exercise training improves mitochondrial oxidative capacity and increases muscle mass and strength. Presently, the impact of chronic alcohol on aerobic exercise-induced adaptations was investigated. Female C57BL/6Hsd mice were randomly assigned to one of four groups: control sedentary (CON SED; n = 26), alcohol sedentary (ETOH SED; n = 27), control exercise (CON EX; n = 28), and alcohol exercise (ETOH EX; n = 25). Exercise mice had running wheel access for 2 h a day, 7 days a week. All mice were fed either control or an alcohol-containing liquid diet. Grip strength testing and EchoMRI were performed before and after the interventions. After 6 wk, hindlimb muscles were collected for molecular analyses. A subset of mice performed a treadmill run to fatigue (RTF), then abstained from alcohol for 2 wk and repeated the RTF. Alcohol decreased lean mass and forelimb grip strength compared with control-fed mice. Alcohol blunted the exercise-induced increase in muscle mass (plantaris and soleus), type IIa fiber percentage in the plantaris, and run time to fatigue. Mitochondrial markers (Citrate synthase activity and Complex I-IV, COXIV and Cytochrome C protein expression) were increased with exercise regardless of ETOH in the gastrocnemius but not tibialis anterior muscle. Two weeks of alcohol abstinence improved RTF time in ETOH EX but not in ETOH SED. These data suggest that alcohol impairs some exercise-induced adaptations in skeletal muscle, but not all were negatively affected, indicating that exercise may be a beneficial behavior even while consuming alcohol.NEW & NOTEWORTHY Alcohol consumption during an aerobic exercise training period prevented training-induced increases in run to fatigue time and grip strength. Cessation of alcohol allowed for recovery of endurance performance within 2 wk. The worsened exercise performance after alcohol was unrelated to impairments in markers of mitochondrial health. Therefore, some adaptations to exercise training are impaired with alcohol use (endurance performance, muscle growth, and strength), while others remain mostly unaffected (mitochondrial health).

Impact of prior alcohol use on the subsequent development of cancer cachexia in male and female mice.

BACKGROUND: Alcohol is a carcinogen and its intake prior to developing cancer and throughout its duration exacerbates cancer cachexia in rodent models. However, the effects on cancer cachexia of stopping alcohol prior to tumor establishment are unknown. METHODS: Male and female mice consumed either a nonalcohol control liquid diet (CON) or a 20% ethanol (kcal/day) liquid diet (EtOH) for 6 weeks. All mice then consumed a control diet and mice in the cancer groups were inoculated with C26 colon cancer cells. Gastrocnemius muscles were collected and analyzed after ~2 weeks. RESULTS: Skeletal muscle weight and male epididymal and female perigonadal fat mass were reduced more by the combination of cancer and prior EtOH than either exposure alone in both males and females. In males, protein synthesis was reduced by 30% following alcohol exposure, while no reductions were observed in female mice. AMPK Thr172 phosphorylation was increased in both male and female EtOH-Cancer groups, while Akt Thr308 phosphorylation was reduced only among males in EtOH-Cancer mice. Substrates in the mTORC1 pathway were reduced by cancer in both males and females, but prior alcohol intake only reduced phosphorylation of 4E-BP1 Ser65 and rpS6 Ser240/244 to a greater extent in male, but not female, mice. Autophagic and proteasomal signaling were largely unaffected by prior alcohol intake in cancer mice, despite a greater increase in Murf1 mRNA in both sexes. CONCLUSIONS: Prior alcohol consumption accelerates or worsens the onset of certain aspects of cancer cachexia in a sex-dependent manner, with males being more sensitive to these exposures, even with abstinence from alcohol prior to tumor initiation.

Long-term administration of resveratrol and MitoQ stimulates cavernosum antioxidant gene expression in a mouse castration model of erectile dysfunction.

AIMS: Erectile dysfunction is a common complication within many pathological conditions associated with low testosterone. Testosterone deficiency increases oxidative stress in the penile tissue that contributes to endothelial dysfunction and subsequent erectile dysfunction. Current therapies do not ameliorate oxidative stress so targeting oxidative stress may improve erectile dysfunction. Resveratrol and MitoQ are two prospective drugs that have antioxidant-like properties and may be useful to improve erectile dysfunction induced by androgen deprivation. MATERIALS AND METHODS: We castrated 12-week-old male C57BL/6 mice and performed an eight-week intervention with oral delivery of resveratrol or MitoQ at low and high doses. We assessed vascular reactivity of the corpus cavernosum and internal pudendal arteries (IPA) through dose-dependent responses to vasodilatory, vasocontractile, and neurogenic stimuli in a myograph system. We performed qRT-PCR to measure expression changes of 18 antioxidant genes in the corpus cavernosum. KEY FINDINGS: Castration significantly impaired erectile function via impaired endothelial-dependent and-independent relaxation, and increased constriction of the corpus cavernosum, and induced severe endothelial dysfunction of the IPA. Castration decreased expression of 8 of the antioxidant genes investigated. Resveratrol and MitoQ were ineffective in reversing the effects of androgen deprivation on vascular reactivity, however high-dose resveratrol treatment upregulated several key antioxidant genes, including Cat, Sod1, Gstm1, and Prdx3. SIGNIFICANCE: Our findings suggest that oral resveratrol and MitoQ treatment may provide protection to the corpus cavernosum under androgen deprived conditions by stimulating endogenous antioxidant systems. However, they may need to be paired with vasoactive drugs to reverse erectile dysfunction under androgen deprived conditions.

Spermidine is not an independent factor regulating limb muscle mass in mice following androgen deprivation.

Maintaining a critical amount of skeletal muscle mass is linked to reduced morbidity and mortality. In males, testicular androgens regulate muscle mass with a loss of androgens being critical as it is associated with muscle atrophy. Atrophy of the limb muscles is particularly important, but the pathways by which androgens regulate limb muscle mass remain equivocal. We used microarray analysis to identify changes to genes involved with polyamine metabolism in the tibialis anterior (TA) muscle of castrated mice. Of the polyamines, the concentration of spermidine (SPD) was significantly reduced in the TA of castrated mice. To assess whether SPD was an independent factor by which androgens regulate limb muscle mass, we treated castrated mice with SPD for 8 weeks and compared them with sham operated mice. Though this treatment paradigm effectively restored SPD concentrations in the TA muscles of castrated mice, mass of the limb muscles (i.e., TA, gastrocnemius, plantaris, and soleus) were not increased to the levels observed in sham animals. Consistent with those findings, muscle force production was also not increased by SPD treatment. Overall, these data demonstrate for the first time that SPD is not an independent factor by which androgens regulate limb skeletal muscle mass. Novelty: Polyamines regulate growth in various cells/tissues. Spermidine concentrations are reduced in the limb skeletal muscle following androgen depletion. Restoring spermidine concentrations in the limb skeletal muscle does not increase limb muscle mass or force production.

A clinically relevant decrease in contractile force differentially regulates control of glucocorticoid receptor translocation in mouse skeletal muscle.

Muscle atrophy decreases physical function and overall health. Increased glucocorticoid production and/or use of prescription glucocorticoids can significantly induce muscle atrophy by activating the glucocorticoid receptor, thereby transcribing genes that shift protein balance in favor of net protein degradation. Although mechanical overload can blunt glucocorticoid-induced atrophy in young muscle, those affected by glucocorticoids generally have impaired force generation. It is unknown whether contractile force alters the ability of resistance exercise to mitigate glucocorticoid receptor translocation and induce a desirable shift in protein balance when glucocorticoids are elevated. In the present study, mice were subjected to a single bout of unilateral, electrically induced muscle contractions by stimulating the sciatic nerve at 100 Hz or 50 Hz frequencies to elicit high or moderate force contractions of the tibialis anterior, respectively. Dexamethasone was used to activate the glucocorticoid receptor. Dexamethasone increased glucocorticoid signaling, including nuclear translocation of the receptor, but this was mitigated only by high force contractions. The ability of high force contractions to mitigate glucocorticoid receptor translocation coincided with a contraction-mediated increase in muscle protein synthesis, which did not occur in the dexamethasone-treated mice subjected to moderate force contractions. Though moderate force contractions failed to increase protein synthesis following dexamethasone treatment, both high and moderate force contractions blunted the glucocorticoid-mediated increase in LC3 II:I marker of autophagy. Thus, these data show that force generation is important for the ability of resistance exercise to mitigate glucocorticoid receptor translocation and promote a desirable shift in protein balance when glucocorticoids are elevated.NEW & NOTEWORTHY Glucocorticoids induce significant skeletal muscle atrophy by activating the glucocorticoid receptor. Our work shows that muscle contractile force dictates glucocorticoid receptor nuclear translocation. We also show that blunting nuclear translocation by high force contractions coincides with the ability of muscle to mount an anabolic response characterized by increased muscle protein synthesis. This work further defines the therapeutic parameters of skeletal muscle contractions to blunt glucocorticoid-induced atrophy.

Systemic delivery of a mitochondria targeted antioxidant partially preserves limb muscle mass and grip strength in response to androgen deprivation.

Muscle mass is important for health. Decreased testicular androgen production (hypogonadism) contributes to the loss of muscle mass, with loss of limb muscle being particularly debilitating. Androgen replacement is the only pharmacological treatment, which may not be feasible for everyone. Prior work showed that markers of reactive oxygen species and markers of mitochondrial degradation pathways were higher in the limb muscle following castration. Therefore, we tested whether an antioxidant preserved limb muscle mass in male mice subjected to a castration surgery. Subsets of castrated mice were treated with resveratrol (a general antioxidant) or MitoQ (a mitochondria targeted antioxidant). Relative to the non-castrated control mice, lean mass, limb muscle mass, and grip strength were partially preserved only in castrated mice treated with MitoQ. Independent of treatment, markers of mitochondrial degradation pathways remained elevated in all castrated mice. Therefore, a mitochondrial targeted antioxidant may partially preserve limb muscle mass in response to hypogonadism.

Increased mitochondrial turnover in the skeletal muscle of fasted, castrated mice is related to the magnitude of autophagy activation and muscle atrophy.

Androgen-deficiency promotes muscle atrophy in part by increasing autophagy-mediated muscle protein breakdown during the fasted state, but factors contributing to this remain undefined. To identify novel factors, mice were subjected to sham or castration surgery. Seven-weeks post-surgery, mice were fasted overnight, refed for 30 min, and fasted another 4.5 h before sacrifice. BNIP3-mediated turnover of mitochondria was increased within the atrophied tibialis anterior (TA) of castrated mice and related to the magnitude of muscle atrophy and autophagy activation (i.e. decreased p62 protein content), thus linking turnover of potentially dysfunctional mitochondria with autophagy-mediated atrophy. Autophagy induction was likely facilitated by AMPK activation as a stress survival mechanism since phosphorylation of AMPK (Thr172), as well as the pro survival kinases Akt (Thr308) and (ERK1/2 Thr202/Tyr204), were increased by castration. Together, these data identify a novel relationship between mitochondrial turnover in the fasted state with autophagy activation and muscle atrophy following androgen depletion.

The role of androgens in the regulation of muscle oxidative capacity following aerobic exercise training.

Reduced production or bioavailability of androgens, termed hypogonadism, occurs in a variety of pathological conditions. While androgens target numerous tissues throughout the body, hypogonadism specifically reduces the ability of skeletal muscle to produce adenosine triphosphate aerobically, i.e., muscle oxidative capacity. This has important implications for overall health as muscle oxidative capacity impacts a number of metabolic processes. Although androgen replacement therapy is effective at restoring muscle oxidative capacity in hypogonadal individuals, this is not a viable therapeutic option for all who are experiencing hypogonadism. While aerobic exercise may be a viable alternative to increase muscle oxidative capacity, it is unknown whether androgen depletion affects this adaptation. To determine this, sham and castrated mice were randomized to remain sedentary or undergo 8 weeks of aerobic treadmill exercise training. All mice were fasted overnight prior to sacrifice. Though exercise increased markers of muscle oxidative capacity independent of castration (cytochrome c oxidase subunit IV and cytochrome c), these measures were lower in castrated mice. This reduction was not due to a difference in peroxisome proliferator activated receptor gamma coactivator 1 alpha protein content, as expression was increased to a similar absolute value in sham and castrated animals following exercise training. However, markers of BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 (BNIP3)-mediated mitophagy were increased by castration independent of exercise. Together, these data show that exercise training can increase markers of muscle oxidative capacity following androgen depletion. However, these values are reduced by androgen depletion likely due in part to elevated BNIP3-mediated mitophagy.

Androgen-mediated regulation of skeletal muscle protein balance.

Androgens significantly alter muscle mass in part by shifting protein balance in favor of net protein accretion. During various atrophic conditions, the clinical impact of decreased production or bioavailability of androgens (termed hypogonadism) is important as a loss of muscle mass is intimately linked with survival outcome. While androgen replacement therapy increases muscle mass in part by restoring protein balance, this is not a comprehensive treatment option due to potential side effects. Therefore, an understanding of the mechanisms by which androgens alter protein balance is needed for the development of androgen-independent therapies. While the data in humans suggest androgens alter protein balance (both synthesis and breakdown) in the fasted metabolic state, a predominant molecular mechanism(s) behind this observation is still lacking. This failure is likely due in part to inconsistent experimental design between studies including failure to control nutrient/feeding status, the method of altering androgens, and the model systems utilized.

Castration alters protein balance after high-frequency muscle contraction.

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.