Myostatin, a member of the transforming growth factor-␤ superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene's expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Testosterone supplementation increases skeletal muscle mass and decreases fat mass; however, the underlying mechanisms are unknown. We hypothesized that testosterone regulates body composition by promoting the commitment of mesenchymal pluripotent cells into myogenic lineage and inhibiting their differentiation into adipogenic lineage. Mouse C3H 10T1/2 pluripotent cells were treated with testosterone (0-300 nM) or dihydrotestosterone (DHT, 0-30 nM) for 0-14 d, and myogenic conversion was evaluated by immunocytochemical staining for early (MyoD) and late (myosin heavy chain II; MHC) myogenic markers and by measurements of MyoD and MHC mRNA and protein. Adipogenic differentiation was assessed by adipocyte counting and by measurements of peroxisomal proliferator-activated receptor gamma 2 (PPAR gamma 2) mRNA and PPAR gamma 2 protein and CCAAT/enhancer binding protein alpha. The number of MyoD+ myogenic cells and MHC+ myotubes and MyoD and MHC mRNA and protein levels increased dose dependently in response to testosterone and DHT treatment. Both testosterone and DHT decreased the number of adipocytes and down-regulated the expression of PPAR gamma 2 mRNA and PPAR gamma 2 protein and CCAAT/enhancer binding protein alpha. Androgen receptor mRNA and protein levels were low at baseline but increased after testosterone or DHT treatment. The effects of testosterone and DHT on myogenesis and adipogenesis were blocked by bicalutamide. Therefore, testosterone and DHT regulate lineage determination in mesenchymal pluripotent cells by promoting their commitment to the myogenic lineage and inhibiting their differentiation into the adipogenic lineage through an androgen receptor-mediated pathway. The observation that differentiation of pluripotent cells is androgen dependent provides a unifying explanation for the reciprocal effects of androgens on muscle and fat mass in men.

. Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression. Am J Physiol Endocrinol Metab 285: E363-E371, 2003. First published April 29, 2003 10.1152/ajpendo.00487.2002The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (Ϫ4.0, Ϫ13.4, Ϫ17.2%, respectively, P Ͻ 0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P Ͻ 0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P Ͻ 0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 g ⅐ kg Ϫ1 ⅐ day Ϫ1 ) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to ϳ2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasonetreated rats were significantly lower (Ϫ43% after 5-day treatment, Ϫ14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.regulation;

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli(Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.

Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/beta-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-alpha and -delta and peroxisome proliferator-activated receptor-gamma2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of beta-catenin and AR. Immunoprecipitation studies demonstrated association of beta-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-alpha in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of beta-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.

. Testosteroneinduced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. Am J Physiol Endocrinol Metab 283: E154-E164, 2002. First published March 12, 2002 10.1152/ajpendo.00502.2001.-Administration of replacement doses of testosterone to healthy hypogonadal men and supraphysiological doses to eugonadal men increases muscle size. To determine whether testosteroneinduced increase in muscle size is due to muscle fiber hypertrophy, 61 healthy men, 18-35 yr of age, received monthly injections of a long-acting gonadotropin-releasing hormone (GnRH) agonist to suppress endogenous testosterone secretion and weekly injections of 25, 50, 125, 300, or 600 mg testosterone enanthate (TE) for 20 wk. Thigh muscle volume was measured by magnetic resonance imaging (MRI) scan, and muscle biopsies were obtained from vastus lateralis muscle in 39 men before and after 20 wk of combined treatment with GnRH agonist and testosterone. Administration of GnRH agonist plus TE resulted in mean nadir testosterone concentrations of 234, 289, 695, 1,344, and 2,435 ng/dl at the 25-, 50-, 125-, 300-, and 600-mg doses, respectively. Graded doses of testosterone administration were associated with testosterone dose and concentration-dependent increase in muscle volume measured by MRI (changes in vastus lateralis volume, Ϫ4, ϩ7, ϩ15, ϩ32, and ϩ48 ml at 25-, 50-, 125-, 300-, and 600-mg doses, respectively). Changes in cross-sectional areas of both type I and II fibers were dependent on testosterone dose and significantly correlated with total (r ϭ 0.35, and 0.44, P Ͻ 0.0001 for type I and II fibers, respectively) and free (r ϭ 0.34 and 0.35, P Ͻ 0.005) testosterone concentrations during treatment. The men receiving 300 and 600 mg of TE weekly experienced significant increases from baseline in areas of type I (baseline vs. 20 wk, 3,176 Ϯ 186 vs. 4,201 Ϯ 252 m 2 , P Ͻ 0.05 at 300-mg dose, and 3,347 Ϯ 253 vs. 4,984 Ϯ 374 m 2 , P ϭ 0.006 at 600-mg dose) muscle fibers; the men in the 600-mg group also had significant increments in cross-sectional area of type II (4,060 Ϯ 401 vs. 5,526 Ϯ 544 m 2 , P ϭ 0.03) fibers. The relative proportions of type I and type II fibers did not change significantly after treatment in any group. The myonuclear number per fiber increased significantly in men receiving the 300-and 600-mg doses of TE and was significantly correlated with testosterone concentration and muscle fiber cross-sectional area. In conclusion, the increases in muscle volume in healthy eugonadal men treated with graded doses of testosterone are associated with concentration-dependent increases in cross-sectional areas of both type I and type II muscle fibers and myonuclear number. We conclude that the testosterone induced increase in muscle volume is due to muscle fiber hypertrophy.androgen; muscle hypertrophy; satellite cells; mechanism of action TESTOSTERONE ADMINISTRATION in replacement doses to hypogonadal men (9,12,26,43,46) and in supraphysiological doses to healthy eugonadal men (8,16,18,49) is associated wit...

Cadavid. Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin. Am J Physiol Endocrinol Metab 285: E876-E888, 2003. First published June 24, 2003 10.1152 10. /ajpendo.00107.2003 in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind-and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified. sarcopenia; wasting; skeletal muscle growth A NUMBER OF GENETIC FACTORS, growth factors, hormones, and nutritional factors are important in the regulation of skeletal muscle mass; however, their precise role in the integrated, in vivo regulation of skeletal muscle homeostasis and muscle wasting associated with chronic illness and aging remains poorly understood. Considerable interest has focused recently on the role of myostatin, or growth differentiation factor (GDF) 8 (16, 35), a novel regulator of muscle mass that is produced predominantly in the skeletal muscle. A targeted deletion of the entire COOH terminus of myostatin (23) or a selected natural mutation leading to a truncated and inactive myostatin protein (38) causes a considerable increase in muscle mass in mice. Naturally occurring mutations in certain breeds of cattle that elicit an out-of-frame truncated protein or an inactive full-length protein ...

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