Δ-4-Androstene-3,17-Dione Binds Androgen Receptor, Promotes Myogenesisin Vitro, and Increases Serum Testosterone Levels, Fat-Free Mass, and Muscle Strength in Hypogonadal Men

Abstract

Previous studies of Delta 4-androstene-3,17-dione (4-androstenedione) administration in men have not demonstrated sustained increments in testosterone levels, fat-free mass (FFM), and muscle strength, and failure to demonstrate androstenedione's androgenic/anabolic effects has stifled efforts to regulate its sales. To determine whether 4-androstenedione has androgenic/anabolic properties, we evaluated its association with androgen receptor (AR) and its effects on myogenesis in vitro. Additionally, we studied the effects of a high dose of 4-androstenedione on testosterone levels, FFM, and muscle strength in hypogonadal men. We determined the dissociation constant (K(d)) for 4-androstenedione using fluorescence anisotropy measurement of competitive displacement of fluorescent androgen from AR ligand-binding domain. AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, pluripotent C3H10T1/2 cells, in which androgens stimulate myogenesis through an AR pathway. We determined effects of a high dose of androstenedione (500 mg thrice daily) given for 12 wk on FFM, muscle strength, and hormone levels in nine healthy, hypogonadal men. 4-Androstenedione competitively displaced fluorescent androgen from AR ligand-binding domain with a lower affinity than dihydrotestosterone (K(d), 648 +/- 21 and 10 +/- 0.4 nm, respectively). In C3H10T1/2 cells, 4-androstenedione caused nuclear translocation of AR and stimulated myogenesis, as indicated by a dose-dependent increase in myosin heavy chain II+ myotube area and up-regulation of MyoD protein. Stimulatory effects of 4-androstenedione on myosin heavy chain II+ myotubes and myogenic determination factor expression were attenuated by bicalutamide, an AR antagonist. Administration of 1500 mg 4-androstenedione daily to hypogonadal men significantly increased serum androstenedione, total and free testosterone, estradiol, and estrone levels and suppressed SHBG and high-density lipoprotein cholesterol levels. 4-androstenedione administration was associated with significant gains in FFM (+1.7 +/- 0.5 kg; P = 0.012) and muscle strength in bench press (+4.3 +/- 3.1 kg; P = 0.006) and leg press exercises (+18.8 +/- 17.3 kg; P = 0.045). 4-androstenedione is an androgen that binds AR, induces AR nuclear translocation, and promotes myogenesis in vitro, with substantially lower potency than dihydrotestosterone. 4-androstenedione administration in high doses to hypogonadal men increases testosterone levels, FFM, and muscle strength, although at the dose tested, the anabolic effects in hypogonadal men are likely because of its conversion to testosterone.

Myostatin promotes a fibrotic phenotypic switch in multipotent C3H 10T1/2 cells without affecting their differentiation into myofibroblasts

“…Starting with this experiment, cells were always treated with AZCT, even if this drug did not affect the number of myofibroblasts that originated from C3H10T1/2 cells. This was done in order to facilitate comparisons with studies, including our previous results (Singh et al 2003, Jasuja et al 2005 where azacytidine was routinely employed to stimulate multipotent stem cell differentiation (Schmittwolf et al 2005). Incubation with recombinant myostatin protein clearly stimulated the intensity of immunocytochemical staining for the four selected fibrotic markers PAI-1, TGF-b1, collagen I, and collagen III (Fig.…”

“…Cells grown in eight-well chamber slides were fixed in 2% p-formaldehyde, quenched with H 2 O 2 , blocked with normal goat or horse serum and incubated with specific antibodies (Artaza et al 2002, Singh et al 2003, Jasuja et al 2005. These consisted of a a-smooth muscle actin immunohistology kit (Sigma-Aldrich), and antibodies against pSmad2/3 (1:500); Smad3 (1:500), Smad4 (1:500) and Smad7 (1:500) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); PAI-1 (1:200; Abcam Inc.); TGF-b1 (1:200) (Promega Corporation); collagen I (1:100) and collagen III (1:100; Chemicon International Inc., Temecula, CA, USA).…”

“…After images were calibrated for background lighting, wherein integrated OD (IODZarea!average intensity) was calculated using at least ten pictures per treatment group per well done in duplicate. These experiments were repeated at least thrice (Singh et al 2003, Jasuja et al 2005.…”

“…It has been shown to modulate multipotent cell differentiation, specifically in the C3H10T1/2 mouse embryonic cell line of fibroblast origin, where myostatin stimulates adipogenic commitment while inhibiting myogenic lineage, whereas androgens exert the opposite effect (Singh et al 2003, Jasuja et al 2005, Feldman et al 2006. C3H10T1/2 is a well-known and widely used multipotent mesenchymal cell line that undergoes differentiation into several cell lineages (Taylor & Jones 1979, Atkinson et al 1997, Fischer et al 2002, Wilson & Rotwein 2006.…”

Hormone-mediated maternal effects in birds:mechanismsmatter but what do we know of them?

“…Even when acting through the androgen receptor pathway, the three androgens have different affinities for the androgen receptor (5a-DHTOT[A 4 ). The androgen receptor's affinity for A 4 is low (0.1% of 5a-DHT; Holterhus et al 2002;Chen et al 2004;Jasuja et al 2005) and A 4 has a relatively low biological activity. For example, the relative agonistic activity of A 4 in cell-line based in vivo assays is 5.7% of 5a-DHT, while that of T is 14.6% of 5a-DHT (Sonneveld et al 2005(Sonneveld et al , 2006.…”