<section class="abstract" style="box-sizing: border-box; margin: 0px; padding: 1.25rem 1.2rem; border: 0px; font-variant-numeric: inherit; font-stretch: inherit; font-size: medium; line-height: inherit; font-family: Merriweather, serif; vertical-align: baseline; position: relative; background: rgb(239, 242, 247); color: rgb(42, 42, 42);">Glucocorticoids are the most widely used antiinflammatory drugs in the world. However, prolonged use of glucocorticoids results in undesirable side effects such as muscle wasting, osteoporosis, and diabetes. Skeletal muscle wasting, which currently has no approved therapy, is a debilitating condition resulting from either reduced muscle protein synthesis or increased degradation. The imbalance in protein synthesis could occur from increased expression and function of muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle ring finger 1 (MuRF1), or decreased function of the IGF-I and phosphatidylinositol-3 kinase/Akt kinase pathways. We examined the effects of a nonsteroidal tissue selective androgen receptor modulator (SARM) and testosterone on glucocorticoid-induced muscle atrophy and castration-induced muscle atrophy. The SARM and testosterone propionate blocked the dexamethasone-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including Forkhead box O (FoxO). Dexamethasone caused a significant up-regulation in the expression of ubiquitin ligases, but testosterone propionate and SARM administration blocked this effect by phosphorylating FoxO. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of MAFbx and MuRF1 and down-regulation of IGF-I, all of which was attenuated by a SARM. The results suggest that levator ani atrophy caused by hypogonadism may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on up-regulation of MAFbx and MuRF1. Our studies provide the first evidence that glucocorticoid- and hypogonadism-induced muscle atrophy are mediated by distinct but overlapping mechanisms and that SARMs may provide a more effective and selective pharmacological approach to prevent glucocorticoid-induced muscle loss than steroidal androgen therapy.
</section>[FONT="]Topic:
Issue Section:
Glucocorticoids-CRH-ACTH-Adrenal
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Muscle atrophy is a debilitating condition that may result from burns (1), sepsis (2), aging (3), disuse (4), cancer (5), AIDS (6), and long-term glucocorticoid administration for the treatment of rheumatoid arthritis (7) and asthma (8). Muscle atrophy often reduces quality of life and prolongs the recovery of these patients. Skeletal muscle catabolism in all of these disparate conditions is the consequence of decreased rates of protein synthesis and accelerated rates of protein degradation. The ubiquitin ligases muscle atrophy F-box (MAFbx or atrogin-1) and muscle ring finger 1 [MuRF1 (9, 10, 11, 12, 13, 14)] that target muscle specific proteins for degradation by the proteasome are up-regulated and represent two of the most sensitive genes affected by muscle atrophy (14, 15).
Hypertrophy is in part mediated by IGF-I via stimulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. In vivo, load-induced hypertrophy increases the expression of IGF-I and stimulates this pathway (16). In transgenic mice, overexpression of IGF-I and a constitutively active form of Akt are both sufficient to induce hypertrophy in skeletal muscle (17, 18, 19). Likewise, addition of IGF-I to myotubes promotes hypertrophy in vitro (20).
Downstream targets of Akt include glycogen synthase kinase (GSK)-3β, the mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K), and the forkhead family transcription factor Forkhead box O [FoxO (21)]. FoxO, when phosphorylated by Akt, is inactive and excluded from the nucleus (22). In myotubes, treatment with dexamethasone (DEX) causes dephosphorylation of FoxO and leads to its subsequent translocation to the nucleus resulting in increased expression of MAFbx and MuRF1 (22, 23). Cotreatment with IGF-I antagonizes the up-regulation, implicating that the PI3K/Akt pathway induces skeletal muscle hypertrophy through not only increased protein synthesis but also anticatabolic effects (22, 23, 24). Studies confirm that two forms of FoxO are involved in muscle atrophy; activation of FoxO3 induces atrophy in myotubes (22) and transgenic mice overexpressing FoxO1 have reduced skeletal muscle mass compared with wild-type control (25).
The anabolic effects of the steroids testosterone and nandrolone have been demonstrated under conditions related to aging men (3), long-term glucocorticoid treatment (26, 27), HIV (28), and severe burns (29). Testosterone administration is associated with a dose-dependent increase in lean muscle mass and maximal voluntary strength and a decrease in fat mass. The increase in lean muscle mass results from decreased rates of protein catabolism, reestablishing a balance between protein synthesis and degradation (3, 29). Under conditions of DEX or denervation-induced atrophy, testosterone and nandrolone, respectively, prevented the up-regulation of muscle-specific ubiquitin ligases (30, 31). Similar results were reproduced in myotubes (30). These results indicate that anabolic steroids may inhibit muscle atrophy by reducing the rates of protein catabolism by suppressing muscle-specific ubiquitin ligases. However, poor pharmacokinetic profiles and lack of tissue selectivity preclude the frequent use of these steroidal androgens.
The development of selective androgen receptor modulators (SARMs) has advanced tremendously since the first report in 1998 (32). Numerous structural modifications were made to the SARM backbone to optimize in vitro androgen receptor (AR) binding affinities and functional activities and in vivo anabolic and androgenic activities and the pharmacokinetic properties of these nonsteroidal AR ligands (32, 33, 34, 35). SARMs exhibit tissue-selective anabolic activity in castrated rats, demonstrating full AR agonist activity in muscle but only partial activity in the prostate. A lead SARM, S-4, promotes the prostate and levator ani muscle weights to 33.8 and 101% of intact control, respectively (36). In contrast, testosterone propionate (TP) nonselectively increases the size of the prostate and levator ani muscle to 121 and 104% of intact control, respectively (36). In another study, S-4 not only restored the levator ani and soleus muscle mass 12 wk after castration, but skeletal muscle strength was also restored to intact levels. Additionally, a phase IIa study in healthy, elderly men and women given Ostarine demonstrated increased lean muscle mass, decreased fat mass, and significant improvements in their ability to climb stairs (37, 38).
S-23, a modified derivative of S-4, was identified as another SARM with potent anabolic activity. In castrated male rats, S-23 maximally maintained the levator ani muscle weight at 129% of intact control and showed significant improvements in bone mass density, lean mass, and fat mass in intact animals (39). We report herein the results of the first studies to examine the effects of a SARM on signaling involved in protein synthesis and degradation in the levator ani muscle that occurs during DEX-induced and castration-dependent atrophy. The effectiveness of S-23, compared with TP, at inhibiting DEX-induced muscle atrophy and regulating genes and proteins involved in the PI3K/Akt pathway (i.e. MAFbx, MuRF1, FoxO, IGF-I) indicate that SARMs may provide a unique and more selective pharmacological approach to prevent or treat corticosteroid-induced muscle atrophy.
[h=2]Materials and Methods[/h][h=3]Animals and treatment[/h]Male Sprague Dawley rats were purchased from Harlan Bioproducts for Science (Indianapolis, IN). The animals were maintained on a 12-h light, 12-h dark cycle with food and water available ad libitum. All animal studies were conducted under the auspices of an animal protocol approved by the Institutional Laboratory Animal Care and Use Committee at the University of Tennessee. Before and at the end of treatment, body fat, lean body mass (LBM), fluids, and total water were determined by magnetic resonance imaging (MRI; EchoMRI 4-in-1 composition analyzer; Echo Medical Systems, Houston, TX). The animals were 5–6 wk old and weighed 229–238 g. Male Sprague Dawley rats were randomized according to LBM and assigned to one of four groups (n = 5/group). Animals received daily sc injections (200 μl/d) of the compound of interest for 8 d. Each compound was dispersed in vehicle containing 80% Tween 80 (Sigma-Aldrich, St. Louis, MO) and 20% Captex 200 (Abitec, Columbus, OH). Group 1 was the control group and received vehicle alone. Groups 2–4 received 600 μg/kg · d DEX (Sigma-Aldrich). In addition to receiving DEX, groups 3 and 4 were administered 25 mg/kg · d of TP (Sigma-Aldrich) or S-23, respectively. S-23 (39) was synthesized in our laboratories using described methods (40). Chemical purities were confirmed by mass spectrometry and nuclear magnetic resonance and determined to be greater than 99%.
Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate and seminal vesicles were removed, cleared of extraneous tissue, and weighed. The levator ani, gastrocnemius (gastroc), extensor digitorum longus (EDL) and soleus muscles were removed (gastroc, EDL and soleus from the left hind limb), weighed, and a segment preserved in RNAlater (Ambion, Austin, TX) for gene expression analysis and the other portion immediately frozen in dry ice/ethanol (Sigma-Aldrich) for protein expression analysis. All tissue weights were normalized to total body weight (before treatment) and compared. All muscle samples in RNAlater and those that were immediately frozen were stored at 4 or −80 C, respectively, until further analysis.
An additional group of male Sprague Dawley rats was ordered from Harlan Bioproducts for Science for the time-course study. Animals in this study were 5–6 wk old and ranged from 219 to 234 g. Rats were randomized according to weight and assigned to appropriate groups (n = 5/group). Animals were orchidectomized (ORX) via scrotal incision under ketamine/xylazine anesthesia 24 h before drug treatment and received daily sc injections S-23 (200 μl/d), at a dose rate of 1 mg/d. S-23 was dissolved in vehicle containing dimethylsulfoxide (Sigma-Aldrich)/polyethylene glycol 300 (PEG300; Sigma-Aldrich) [10/90 (vol/vol)]. Additional groups of rats (n = 5/group) with or without castration received vehicle only and served as castrate or intact control groups, respectively. Animals were treated for 3, 7, 10, 14, 21, or 28 d. Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, seminal vesicles, levator ani, gastroc, EDL, and soleus muscles were removed (gastroc, EDL and soleus from the left hind limb), cleared of extraneous tissue, weighed, and preserved in RNAlater (Ambion) for gene expression analysis. All organ weights were normalized to total body weight and compared. Percent changes were determined by comparison to intact animals.
Lastly, a separate group was randomized according to weight (n = 5/group) to compare DEX treatment vs. castration on prostate, levator ani, and serum testosterone levels. Animals were 5–6 wk old and weighed 219–231 g. Animals were either castrated as described above (ORX group) or left intact (intact and DEX groups). Animals then received daily sc injections of vehicle (intact and ORX groups) or 600 μg/kg · d DEX for 8 d. Animals were weighed, anesthetized, and serum collected and stored at −20 C until further analysis. The ventral prostate and levator ani muscles were removed, weighed, and normalized to total body weight and compared with the vehicle-treated, intact control group.
[h=3]Serum testosterone analysis[/h]Serum testosterone was quantified using a liquid chromatography and tandem mass spectrometry method, using deuterium-labeled testosterone as an internal standard. The analyses were performed using a quadrupole ion-trap mass spectrometer coupled with an HPLC system and an electrospray ionization source. The samples were prepared by liquid-liquid extraction, using a 3:2 ratio (vol/vol) of ethyl acetate-hexane. An aliquot of sample (10 μl) was injected into a C18 column (100 × 2.1 mm, 3 μm; Alltima HP; Grace Davison Discovery Sciences, Deerfield, IL) at a flow rate of 0.4 ml/min using a gradient mobile phase. Mobile phase A was comprised of 90% water, 10% acetonitrile, and 0.1% formic acid. Mobile phase B consisted of 90% acetonitrile, 10% water, and 0.1% formic acid. The gradient initiated with 30% B at a flow of 0.4 ml/min and the first 3 min was diverted to waste. After 8 min, the gradient changed to 90% B and then back to 30% 6 sec later and remained there until the end of the total run time of 13 min. The lower limit of quantitation for the method was 0.200 ng/ml.
[h=3]Cell culture[/h]Myoblasts from the C2C12 mouse skeletal muscle cell line (American Type Culture Collection, Manassas, VA) were cultured in growth medium consisting of DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all purchased from Hyclone, Logan, UT) in a humidified atmosphere containing 5% CO2 at 37 C. Two independent experiments were performed for both quantitative PCR and Western blotting, and each were performed in triplicate.
[h=3]Quantitative real-time PCR[/h]C2C12 cells were seeded into six-well plates in growth medium at a density of 1 million cells/well and allowed to recover for 8 h. Medium was then replaced with differentiation medium consisting of DMEM supplemented with 2% horse serum (Hyclone). After 24 h, cells were transduced with 250 virus particles/cell of an AR adenovirus (generated by Welgen, Inc., Worcester, MA) and incubated another 24 h. Cells were treated with the compound of interest and ribonucleic acid (RNA) was isolated 24 h later, using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For muscle sample analysis, 50 mg of tissue was removed from RNAlater (Ambion), homogenized using Lysing Matrix D (MP Biomedicals, Solon, OH), and RNA isolated using TRIzol reagent. Total RNA was determined by absorbance at 260 nm. RNA (1 μg) was reverse transcribed to produce cDNA (high capacity cDNA reverse transcription kit with ribonuclease inhibitor; Applied Biosystems, Foster City, CA). Rat and mouse F-box protein 32 (Fbxo32), tripartite motif-containing 63 and IGF-I TaqMan probes and rat AR and GR probes were used (Applied Biosystems). Quantitative PCR was performed (TaqMan fast universal PCR master mix; Applied Biosystems) according to the manufacturer’s instructions. To control for any variation in reverse transcription or PCR efficiency, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The cycle number at which the reaction crossed an arbitrary threshold (CT) was determined for each gene and analyzed using the 2[FONT="]-ΔΔCT method (41). All PCR runs were performed in duplicate.[/FONT]
[h=3]Western blotting[/h]C2C12 cells were seeded into 10-cm dishes in growth medium at a density of 2.5 million cells/dish. Differentiation was induced, and cells were transduced and treated as described above. Cells were washed with Dulbecco’s PBS (Hyclone) and protein was extracted in buffer containing 1 m potassium phosphate, 10 mm sodium molybdate, 50 mm sodium fluoride, 2 mm disodium EDTA, 2 mm EGTA, 0.05% monothioglycerol, 1 mg/ml leupeptin, 1 mg/ml antipain dihydrochloride, 1 mg/ml aprotinin, 1 mg/ml benzamidine hydrochloride, 1 mg/ml chymostatin, 1 mg/ml pepstatin, 4 m sodium chloride, and 100 mm phenylmethanesulfonylfluoride. The samples were subjected to a freeze/thaw cycle and centrifuged. The supernatant was removed and stored at −80 C until analysis. For muscle sample analysis, 50 mg of tissue was homogenized in 750 μl of homogenization buffer using Lysing Matrix D (MP Biomedicals). The samples were subjected to a freeze/thaw cycle, centrifuged, and the supernatant stored at −80 C. Protein concentrations of the supernatant were determined by the Bio-Rad protein assay (Hercules, CA) using BSA (Sigma-Aldrich) as the standard. The protein samples (50 μg per lane) were mixed with loading buffer, heated for 5 min, and resolved by SDS-PAGE on 4–20% Tris-glycine gradient gels (Invitrogen) and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (50 mm Tris, 150 mm NaCl, and 0.1% Tween 20). The primary antibodies were diluted 1:1000 in 5% BSA [phospho-Akt Ser473, phospho-mTOR Ser2488, phospho-GSK-3β Ser 9, phospho-p70S6K Thr389, total Akt, and total FoxO3a; all from Cell Signaling Technology, Danvers, MA; phospho-FoxO3a S253 from Abcam Inc., Cambridge, MA; AR PG21 and antiactin from Upstate, Billerica, MA; glucocorticoid receptor (GR) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA] and the secondary antibodies 1:2000 in 5% nonfat dry milk (horseradish peroxidase conjugated antirabbit IgG and antimouse IgG; Cell Signaling Technology). Immunostaining was visualized by enhanced chemiluminescence (ECL Plus Western blotting detection reagents; Amersham, Piscataway, NJ) and captured on photographic film. The protein band densities were quantified using TotalLab TL100 (Nonlinear Dynamics, Durham, NC). β-Actin was included for loading control.
[h=3]Statistical analyses[/h]All statistical analyses were performed using single-factor ANOVA followed by Dunnett’s multiple comparison test. Differences in which P < 0.05 were considered statistically significant.
[h=2]Results[/h][h=3]Expression of muscle hypertrophy and atrophy markers in C2C12 myotubes[/h]The effects of S-23 and TP on DEX-induced atrophy were examined in differentiated C2C12 myotubes expressing the AR (see Fig. 2A). In the absence of AR in untransfected C2C12 cells, androgens failed to regulate the expression of MAFbx or IGF-I (data not shown). However, in AR-transduced C2C12 cells, overnight incubation with DEX induced a 6.5-fold increase in the expression of MAFbx mRNA (Fig. 1A). Cotreatment with TP and S-23 partially but significantly inhibited the up-regulation, with near maximal effect seen at 1 nm. DEX treatment also caused a 13-fold decrease in expression of IGF-I mRNA, which was significantly inhibited by TP and S-23 observed at all concentrations 1 nm or greater (Fig. 1B).
[FONT="]Fig. 1.
[FONT="]In vitro gene expression. C2C12 cells were differentiated and myotubes treated as indicated for 24 h. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCTmethod. Data are presented as mean ± se (n = 6). *, P < 0.05 compared with the DEX-treated group. A, Fold expression change of muscle atrophy marker MAFbx. B, Fold expression change of muscle hypertrophy marker IFG-1.
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[FONT="]Fig. 2.
[FONT="]In vitro protein expression. C2C12 cells were differentiated and myotubes treated as indicated for 24 h and protein extracted. Fifty micrograms of protein were loaded to each well, resolved by SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk, incubated with primary antibody overnight in 5% BSA (1:1000), and then with secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin, total Akt, and total FoxO3 were included for loading control. Data presented as mean ± se (n = 6). *, P < 0.05 compared with the DEX-treated group. A, Immunoblots of AR in C2C12 cells before and after transduction. B, Immunoblots of proteins in the PI3K/Akt pathway. C, Graphical representation of the change in expression of pAkt compared with total Akt. D, Graphical representation of the change in expression of pFoxO3a compared with total FoxO3.
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Addition of DEX to C2C12 myotubes significantly reduced phosphorylation of many proteins involved in the PI3K/Akt pathway (Fig. 2B). Cotreatment with androgens significantly attenuated the effects of DEX on phosphorylation of Akt, GSK3-β, p70S6K, mTOR, FoxO3a, and FoxO1. Quantification of band densities revealed significant differences observed in the phosphorylation of Akt at 0.1 and 1 nm for S-23 and TP, respectively (Fig. 2C). Additionally, TP and S-23 increased phosphorylation of FoxO3a above that observed for vehicle control (Fig. 2D).
[h=3]DEX-induced muscle atrophy[/h]DEX administration (600 μg/kg · d) significantly reduced the total body weight of intact male rats (Fig. 3A). Cotreatment with 25 mg/kg · d of TP or S-23 partially attenuated the effects of DEX on body weight. However, the weights were still significantly different from vehicle control. DEX treatment also caused a 5% reduction in LBM after 8 d (Fig. 3B). S-23 significantly inhibited the loss of LBM with no significant differences observed vs. vehicle control, whereas TP only partially attenuated this loss. DEX also significantly reduced the size of the prostate and seminal vesicles (Fig. 3, C and D). Coadministration with TP or S-23 increased the weights of these tissues above that observed for vehicle control (Fig. 3, C and D), with TP significantly increasing the seminal vesicles compared with S-23. A variety of muscles were also excised to evaluate their response to glucocorticoid and androgen treatment. The levator ani, a highly androgen-responsive fast-twitch muscle, decreased by 52% on DEX administration. TP and S-23 completely inhibited this reduction, maintaining the weight similar to that observed for intact control (Fig. 3E). The gastrocnemius, a muscle comprised mostly of fast-twitch fibers [94%; (42)], decreased by 37% in response to DEX administration. However, contrary to previously reported data (30), TP and S-23 did not attenuate the effects of DEX on this muscle (Fig. 3F). The EDL, a fast-twitch muscle, also diminished in size by 34% on exposure to DEX, with no inhibition observed upon TP or S-23 administration (Fig. 3G). DEX also caused a reduction in the size of the soleus muscle, a slow-twitch muscle, which was attenuated by S-23 but not TP (Fig. 3H).
[FONT="]Fig. 3.
[FONT="]Effects of treatment on body weight and LBM and androgenic and anabolic tissues. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by magnetic resonance imaging, weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, seminal vesicles, levator ani, gastroc, EDL, and soleus were removed and weighed. All tissues were normalized to total body weight and compared. Data presented as mean ± se (n = 5). #, P < 0.05 compared with the vehicle-treated control group; *, P < 0.05 compared with the DEX-treated group. T, S, P < 0.05 compared with the testosterone- or S-23-treated group, respectively. A, Average body weight. B, Absolute change in LBM. Normalized prostate weights (C), seminal vesicles (D), levator ani muscle (E), gastroc (F), extensor digitorum longus (G), and soleus muscle (H) are shown.
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[h=3]Expression of AR and GR in muscle[/h]The levator ani muscle significantly expresses 6.6-fold more AR than the EDL muscle (Fig. 4, A and B), as determined by immunoblot. Expression of AR in the EDL is also significantly less than that of the soleus muscle. Conversely, the levator ani muscle significantly expresses the least amount of GR. The quantitated immunoblot shows that the soleus significantly expresses more GR than the EDL and levator ani muscles (Fig. 4, C and D). Although the levator ani and soleus muscles may express slightly more AR than the gastroc, these differences are not statistically significant.
[FONT="]Fig. 4.
[FONT="]Expression of AR and GR in anabolic tissues. Male rats were treated with vehicle for 8 d via sc injection. The levator ani, gastroc, extensor digitorum longus, and soleus muscles were removed. A segment of each muscle was frozen in dry ice/ethanol. Protein was extracted, 50 μg loaded to each well, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk, incubated with primary antibody overnight in 5% BSA (1:1000) and then with the secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin included for loading control. Data are presented as mean ± se (n = 3 for AR; n = 5 for GR). L, G, E, S, P < 0.05 compared with the levator ani, gastroc, EDL, and soleus groups, respectively. A, Immunoblot of AR in levator ani, gastroc, EDL, and soleus muscles. B, Graphical representation of the expression of AR present in muscles normalized to β-actin. C, Immunoblot of GR expression in muscle. D, Graphical representation of the expression of GR present in muscles normalized to β-actin.
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[h=3]Effects of S-23 and TP on gene and protein expression in muscle of intact rats[/h]Administration of DEX to intact animals resulted in up-regulation of MAFbx mRNA in the levator ani, gastroc, EDL, and soleus, with the greatest effect observed in the levator ani muscle (60-fold change) (Fig. 5A). Coadministration with androgens completely inhibited this up-regulation in the levator ani. In the gastroc, EDL, and soleus, the up-regulation of MAFbx was slightly attenuated by TP, but S-23 was statistically more effective at inhibiting the up-regulation of MAFbx in the gastroc, EDL, and soleus muscles. A similar trend was observed for MuRF1, with a 25-fold increase in mRNA expression in the levator ani, which was completely blocked by TP and S-23 administration. S-23 was statistically more effective than TP at inhibiting MuRF1 in the soleus (Fig. 5B). DEX administration significantly decreased the levels of IGF-I in the gastroc, EDL, and soleus by 3-, 6.5-, and 5.5-fold, respectively (Fig. 5C). Although IGF-I expression did not decrease in the levator ani, TP and S-23 caused a small but significant increase in expression of IGF-I in this tissue. S-23 was statistically more effective than TP at inhibiting the down-regulation of IGF-I in the gastroc, EDL, and soleus muscles.
[FONT="]Fig. 5.
[FONT="]Effects of treatment on gene expression in the levator ani in intact male rats. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by MRI, weighed, anesthetized, and killed within 24 h after the last dose. The levator ani muscles were removed and weighed. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCT method and presented as mean ± se (n = 5). *, P < 0.05 compared with the DEX-treated group; #, P < 0.05 compared with the vehicle-treated control group; T, S, P < 0.05 compared with the testosterone- or S-23-treated group, respectively. A, Fold expression change of muscle atrophy marker MAFbx. B, Fold expression change of MuRF1. C, Fold expression change of muscle hypertrophy marker IGF-I.
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Proteins upstream of MAFbx and MuRF1 in the PI3K/Akt pathway, phosphorylated Akt, GSK3β, p70S6K, and FoxO3a decreased in the levator ani muscle on DEX exposure (Fig. 6A). As was observed in C2C12 myotubes, cotreatment with TP and S-23 maintained phosphorylated levels of GSK3β, p70S6K, and Akt (Fig. 6, A and B). Down-regulation of phosphorylated FoxO3a was attenuated by TP and S-23 (Fig. 6C).
[FONT="]Fig. 6.
[FONT="]Expression of hypertrophy and atrophy proteins in the levator ani of intact male rats. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by MRI, weighed, anesthetized and killed within 24 h after the last dose. The levator ani muscles were removed and weighed. Protein was extracted, 50 μg loaded to each well, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk, and incubated with primary antibody overnight in 5% BSA (1:1000) and then with the secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin, total Akt, and total FoxO3 were included for loading control. Data presented as mean ± se (n = 5). *, P < 0.05 compared with the DEX-treated group; #, P < 0.05 compared with the vehicle-treated control group. A, Immunoblots of proteins in the PI3K/Akt pathway. p, Phosphorylated. B, Graphical representation of the change in expression of pAkt compared with total Akt (tAkt). C, Graphical representation of the change in expression of pFoxO3a compared with total FoxO3.
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[h=3]Effects of time and S-23 in castrated rats[/h]Castration causes an immediate reduction in size of androgen-dependent tissues. After just 3 d, the prostate and levator ani muscle reduce to 67 and 79% of intact control, respectively (Fig. 7, B and C). Both tissues continue to decrease in size and reach nadir by 14 d, with sizes of the prostate and levator ani leveling off around 15 and 45% of that observed in intact control, respectively. Conversely, when S-23 is administered to ORX animals, the prostate and levator ani are both maintained, and significant increases are observed through d 28 (Fig. 7, B and C). Castration induces a 40-fold increase in MAFbx mRNA expression at 3 d but drops off 14 d after castration (Fig. 7D). Administration of S-23 inhibits this up-regulation at 3, 7, and 10 d, but no change is observed after 14 d (Fig. 7E). Additionally, castration induces a down-regulation in expression of IGF-I (Fig. 7F), which is also attenuated by S-23 (Fig. 7G).
[FONT="]Fig. 7.
[FONT="]Effects of time and S-23 on androgenic and anabolic tissues in castrated male rats and expression of hypertrophy and atrophy markers. Male rats were ORX via scrotal incision under ketamine/xylazine anesthesia 24 h before drug treatment and received daily sc injections of S-23 at a dose rate of 1 mg/d for 3, 7, 10, 14, 21, or 28 d. Additional groups of animals with or without castration received vehicle only and served as castrate or intact control groups, respectively. Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, levator ani, gastroc, EDL, and soleus were removed and weighed. A segment of each muscle was preserved in RNAlater (Ambion). All tissues were normalized to total body weight and compared. Percent changes were determined by comparison with intact animals. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCT method and presented as mean ± se (n = 5). Graphs B, C, D, F: #, P < 0.05 compared with the vehicle-treated, intact control group; graphs E and G: #, P < 0.05 compared with the vehicle-treated, ORX control group on the respective day. A, Body weights of all animal groups before and after treatment. BW, Body weight. B, Normalized prostate weights of ORX control and S-23-treated animals. C, Normalized levator ani weights of ORX control and S-23-treated animals. D, Fold expression change of muscle atrophy marker MAFbx in ORX animals compared with intact control animals. E, Fold expression change of MAFbx in S-23-treated animals compared with ORX at each day. F, Fold expression change of muscle hypertrophy marker IGF-I in ORX animals compared with intact control animals. G, Fold expression change of IGF-I in S-23-treated animals compared with ORX at each day.
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[h=3]Comparison of DEX and ORX on body weight, androgen-dependent tissues, and serum testosterone[/h]Significant reductions in body, prostate and levator ani weights were observed after 8 d of DEX administration or castration (Table 1). Serum testosterone concentrations measured 3.6 ng/ml in vehicle-treated, intact animals. DEX administration induced no significant change in serum testosterone levels. However, castration reduced serum testosterone levels below the limit of quantitation (0.20 ng/ml; Table 1).
[FONT="]TABLE 1.
[FONT="]Effects of DEX treatment and castration on body weight (BW), prostate, levator ani, and serum testosterone
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[FONT="]Animals in the ORX group were castrated on d 0. On d 1–8, animals in the intact and ORX groups received daily sc injections of vehicle, and the DEX group received 600 μg/kg · d DEX. Prostate and levator ani muscle weights were measured at the end of the treatment period and normalized to BW. Serum testosterone concentrations were determined by liquid chromatography and mass spectrometry. Data are presented as mean ± se (n = 5).
a
P < 0.05 compared with the vehicle-treated, intact control group.
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[h=2]Discussion[/h]Corticosteroid-induced protein catabolism predominantly strikes fast-twitch muscle fibers, including the gastrocnemius and levator ani muscles. Atrophy in fast-twitch muscle fibers is highly responsive to androgen administration (43, 44, 45). The Hershberger assay is the method of choice for identifying AR-dependent myoanabolic tissue selectivity (46). Our laboratory and many others have shown that the levator ani muscle of rats rapidly atrophies after castration and quickly hypertrophies on exogenous administration of an anabolic agent (36, 46, 47). Exogenously administered testosterone promotes the growth of anabolic (i.e. muscle) and androgenic (i.e. prostate and seminal vesicles) tissues, whereas SARMs are selectively anabolic. Studies demonstrated the beneficial effects of testosterone in the DEX-induced animal model of muscle atrophy and signaling effects in the PI3K/Akt pathway (30, 48, 49). In this model, most studies reported the effects testosterone on the gastroc muscle (30, 48, 49). The present study provides new information regarding the comparative effects of testosterone and a SARM on muscle atrophy and hypertrophy signaling pathways after corticosteroid administration or castration for not only the gastroc but also the levator ani, extensor digitorum longus, and soleus muscles.
IGF-I mRNA expression decreased in response to DEX treatment, both in vitro and in vivo (Figs. 1B and 5C) as well as castration (Fig. 7F), corroborating the previous reports that the atrophic effects of glucocorticoids are mediated at least partially via suppression of IGF-I signaling (16, 17, 18). This suppression was partially or completely inhibited by TP and S-23. IGF-I activates the PI3K/Akt pathway and downstream targets associated with protein synthesis include Akt, mTOR, GSK3β, and p70S6K. TP and S-23 completely blocked the DEX-dependent dephosphorylation of Akt and downstream proteins, in both skeletal muscle cells and in vivo the levator ani muscle (Figs. 2B and 6A). These results confirm the anabolic effects of TP and S-23 on protein synthesis downstream of IGF-I.
Akt signaling is also involved in inhibiting protein degradation by phosphorylating FoxO and therefore preventing transcription of MAFbx and MuRF1, muscle-specific ubiquitin ligases. Stitt et al. (23) demonstrated that phosphorylation of FoxO1 is required for inhibition of muscle atrophy via the PI3K/Akt pathway. Likewise, Sandri et al. (22) demonstrated that dephosphorylation of FoxO3 is required to activate MAFbx. In the present study, administration of DEX resulted in dephosphorylation of FoxO1 and FoxO3 in skeletal muscle cells and the levator ani muscle (Figs. 2B and 6A). However, TP and S-23 significantly promoted phosphorylation of FoxO, thereby inhibiting muscle atrophy. Gene expression analysis revealed that dephosphorylation of FoxO by DEX significantly increased expression of MAFbx and MuRF1 mRNA but is attenuated by coadministration with TP or S-23 (Figs. 1A and 5, A and B). A coimmunoprecipitation assay was performed to determine whether there is a direct interaction between the AR and FoxO3a to explain the ability of TP to maintain mRNA expression of FoxO3a above that of vehicle control (Fig. 2D). However, the results show that the AR and FoxO3a do not directly interact (data not shown).
Interestingly, the AR ligands did not maintain IGF-I or MAFbx levels as that of control. However, they did bring the proteins downstream of IGF-I back to the control levels. This indicates that the final effectors of anabolism, such as Akt, FoxO3A, and other downstream proteins, are effectively regulated by TP and S-23. Although earlier studies revealed that there was no change in the proliferation and differentiation rate of C2C12 cells stably transfected with AR in response to dihydrotestosterone (50), myotube diameter and size should have increased in the present study due to the extent of regulation observed with the downstream proteins.
Administration of DEX significantly decreased body weight, which was partially inhibited by androgen administration (Fig. 3A). There was a significant loss of LBM observed in DEX-treated animals, which was only partially inhibited by TP (Fig. 3B). However, animals cotreated with S-23 maintained LBM similar to that observed in vehicle control. The levator ani, gastroc, EDL, and soleus muscles all atrophied in response to DEX administration (Fig. 3, E–H). Addition of S-23 completely prevented reduction in weight of the levator ani and soleus muscles. TP protected only against loss of levator ani muscle weight. Minimal effects were observed in the other muscles. Inconsistent with our results, Zhao et al. (30) reported that testosterone (28 mg/kg · d) completely protected against loss of gastroc muscle weight at both 1 and 7 d with significant inhibition of MAFbx expression. In the present study, MAFbx expression in the gastroc was only slightly down-regulated by S-23 at 8 d (Fig. 5A). Protein expression data revealed that the levator ani expresses significantly more AR than the soleus muscle (Fig. 4, A and B) and may explain why this muscle responded to TP and S-23 administration more than the others in the present study. Interestingly, the levator ani muscle expresses the least amount of GR but atrophied the most in response to glucocorticoid administration. The gastroc and soleus muscles express comparable GR and AR; however, the slow-twitch muscle fibers of the soleus muscle responded to SARM treatment, whereas the gastroc did not. Analysis of AR expression in the levator ani, gastroc, EDL, and soleus muscles at the end of the study revealed that there was no significant change in AR expression in DEX-treated animals or castrated animals treated with S-23. However, castration alone significantly increased AR expression in the levator ani, gastroc, and soleus muscles (data not shown). Additionally, previous studies have shown that DEX administration to rats reduces food consumption (51, 52). The animals in the present study were not pair fed to account for differences in food intake, and therefore, this may have an indirect effect on muscle mass and gene expression of animals administered DEX, compared with the other groups.
The levator ani muscle starts atrophying immediately after surgical removal of testosterone and plateaus around 14 d (Fig. 7C). The greatest up-regulation of MAFbx expression in castrated, vehicle-treated animals occurs between d 3 and 10 (Fig. 7D). S-23 showed the most regulation of this gene through 10 d, with no down-regulation observed on d 14–28 (Fig. 7E). This lack of regulation at the later time points is likely related to the muscle no longer atrophying, with minimal up-regulation of MAFbx observed in the ORX animals. Conversely, IGF-I declines in ORX animals throughout the 4 wk but is up-regulated by S-23 with the greatest regulation observed at 28 d (Fig. 7, F and G).
The levator ani muscle atrophies in response to both castration and DEX administration. Stress-induced elevations in plasma glucocorticoid concentrations inhibit secretion of LH and therefore reduces testosterone concentrations in vivo (53). Studies have also shown that glucocorticoids have a direct inhibitory effect on testicular steroidogenesis (54, 55, 56). In humans, significant reductions in circulating testosterone result from DEX administration as well as IGF-I mRNA (57). Yin et al. (49) reported serum testosterone concentrations that were statistically significant between animals administered testosterone and testosterone combined with DEX, compared with both control and DEX-treated animals. In the present study, serum testosterone concentrations reduced by more than 95%, and the levator ani muscle weight decreased by 28%, 5 d after castration (Table 1). Due to variability of the data, there was no reduction in serum testosterone induced by DEX. The highly androgen-responsive levator ani muscle decreased in size in response to both DEX administration and castration possibly due to the reduction in circulating testosterone. This effect may contribute to the myopathy that occurs with glucocorticoid administration.
The mechanisms underlying DEX-induced atrophy appear to be different from castration-induced atrophy. Loss of IGF-I in glucocorticoid-induced muscle atrophy does not appear to be as important in levator ani muscle as in the other muscles (Fig. 5C). In glucocorticoid-induced muscle atrophy, expression of IGF-I in the levator ani did not decline; however, there was a significant reduction observed in the other muscles. Additionally, the levator ani muscle atrophied the most and displayed the greatest up-regulation of MAFbx and MuRF1 compared with the other muscles (Fig. 5, A and B). The inability of the gastroc, EDL, and soleus muscles to maintain IGF-I might account for lack of changes in size because the levator ani was able to maintain both IGF-I levels and size. In castration-induced atrophy, IGF-I mRNA expression declined throughout the 28-d period in the levator ani muscle (Fig. 7F) and was up-regulated after SARM administration (Fig. 7G). However, the levator ani muscle also displayed the greatest up-regulation of MAFbx compared with the other muscles (data not shown). Our studies suggest that levator ani atrophy caused by hypogonadism (i.e. castration) may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on the up-regulation of MAFbx and MuRF1. Although, the gene expression analyses establish this possibility, the direct correlation that the alteration in these pathways result in muscle loss is lacking. This could be established only using knockout or transgenic animal models. Studies are ongoing in our laboratories to elucidate the differences in these two mechanisms of muscle atrophy and their response to TP and SARM treatment.
Previous studies demonstrated that testosterone administration inhibits muscle atrophy by increasing net protein synthesis and reusing intracellular amino acids in skeletal muscle (29, 58, 59). Although we have demonstrated in the present study that AR ligands, testosterone and S-23, induce the anabolic and inhibit the catabolic pathway proteins, studies using radiolabeled amino acids are needed to demonstrate that S-23 has a direct effect on nascent protein synthesis. Importantly, previous publications positively correlate the IGF-I/Akt pathway to protein synthesis in muscle. Recent publications are also bringing clarity on the role of ubiquitination and proteasomal degradation of these anabolic proteins (60, 61). Future experiments with radiolabeled amino acids, cycloheximide, or MG-132 will be helpful to determine the direct effects of AR ligands on the biosynthetic pathways.
The doses of S-23 and TP were chosen to result in equal increases in levator ani weight in the DEX-induced model of muscle atrophy (i.e. a dose that would return the levator ani to the same size as observed in intact controls). Although a high dose of S-23 was used in this study to determine its mechanistic effects on muscle atrophy, S-23 maintains the levator ani muscle around intact levels at a dose of 0.1 mg/d in castrated animals, whereas the prostate was maintained at less than 30% of that observed in intact control (39). Conversely, at the same dose, TP is nonselective and maintains the weights of both the levator ani and prostate at 70% of intact control (36). Thus, the tissue selectivity of S-23, like other SARMs that we have reported, is dose dependent.
In summary, S-23 and TP maintained the weight of the levator ani muscle in the DEX model of muscle atrophy. S-23 and TP blocked the DEX-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including FoxO. DEX caused a significant up-regulation in the expression of MAFbx and MuRF1, but TP and S-23 administration blocked this effect by phosphorylating FoxO. S-23 also attenuated the up-regulation of MAFbx and MuRF1 in the gastroc, EDL, and soleus muscles. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of ubiquitin ligases and down-regulation of IGF-I. S-23 maintained the levator ani muscle at or above intact levels and significantly decreased expression of MAFbx and increased expression of IGF-I on d 3–10, with minimal regulation observed 14 d after castration. Given the observed effects on IGF-I, MAFbx, and Murf1, these studies suggest that SARMs and TP may stimulate protein synthesis and inhibit protein degradation via the IGF-I/PI3K/Akt signaling pathway. SARMs provide the peripheral stimulus necessary to maintain muscle mass with minimal effects on androgenic tissues and represent a viable treatment option for a variety of clinical conditions including glucocorticoid-induced muscle loss and sarcopenia.
</section>[FONT="]Topic:
- testosterone
- dexamethasone
- glucocorticoids
- atrophy
- castration
- down-regulation
- hypogonadism
- insulin-like growth factor i
- ligase
- skeletal muscles
- muscular atrophy
- ubiquitin
- up-regulation (physiology)
- rats
- protein biosynthesis
- levator ani muscle
- proto-oncogene proteins c-akt
- selective androgen receptor modulators
- attenuation
- catabolism
Issue Section:
Glucocorticoids-CRH-ACTH-Adrenal
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Muscle atrophy is a debilitating condition that may result from burns (1), sepsis (2), aging (3), disuse (4), cancer (5), AIDS (6), and long-term glucocorticoid administration for the treatment of rheumatoid arthritis (7) and asthma (8). Muscle atrophy often reduces quality of life and prolongs the recovery of these patients. Skeletal muscle catabolism in all of these disparate conditions is the consequence of decreased rates of protein synthesis and accelerated rates of protein degradation. The ubiquitin ligases muscle atrophy F-box (MAFbx or atrogin-1) and muscle ring finger 1 [MuRF1 (9, 10, 11, 12, 13, 14)] that target muscle specific proteins for degradation by the proteasome are up-regulated and represent two of the most sensitive genes affected by muscle atrophy (14, 15).
Hypertrophy is in part mediated by IGF-I via stimulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. In vivo, load-induced hypertrophy increases the expression of IGF-I and stimulates this pathway (16). In transgenic mice, overexpression of IGF-I and a constitutively active form of Akt are both sufficient to induce hypertrophy in skeletal muscle (17, 18, 19). Likewise, addition of IGF-I to myotubes promotes hypertrophy in vitro (20).
Downstream targets of Akt include glycogen synthase kinase (GSK)-3β, the mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K), and the forkhead family transcription factor Forkhead box O [FoxO (21)]. FoxO, when phosphorylated by Akt, is inactive and excluded from the nucleus (22). In myotubes, treatment with dexamethasone (DEX) causes dephosphorylation of FoxO and leads to its subsequent translocation to the nucleus resulting in increased expression of MAFbx and MuRF1 (22, 23). Cotreatment with IGF-I antagonizes the up-regulation, implicating that the PI3K/Akt pathway induces skeletal muscle hypertrophy through not only increased protein synthesis but also anticatabolic effects (22, 23, 24). Studies confirm that two forms of FoxO are involved in muscle atrophy; activation of FoxO3 induces atrophy in myotubes (22) and transgenic mice overexpressing FoxO1 have reduced skeletal muscle mass compared with wild-type control (25).
The anabolic effects of the steroids testosterone and nandrolone have been demonstrated under conditions related to aging men (3), long-term glucocorticoid treatment (26, 27), HIV (28), and severe burns (29). Testosterone administration is associated with a dose-dependent increase in lean muscle mass and maximal voluntary strength and a decrease in fat mass. The increase in lean muscle mass results from decreased rates of protein catabolism, reestablishing a balance between protein synthesis and degradation (3, 29). Under conditions of DEX or denervation-induced atrophy, testosterone and nandrolone, respectively, prevented the up-regulation of muscle-specific ubiquitin ligases (30, 31). Similar results were reproduced in myotubes (30). These results indicate that anabolic steroids may inhibit muscle atrophy by reducing the rates of protein catabolism by suppressing muscle-specific ubiquitin ligases. However, poor pharmacokinetic profiles and lack of tissue selectivity preclude the frequent use of these steroidal androgens.
The development of selective androgen receptor modulators (SARMs) has advanced tremendously since the first report in 1998 (32). Numerous structural modifications were made to the SARM backbone to optimize in vitro androgen receptor (AR) binding affinities and functional activities and in vivo anabolic and androgenic activities and the pharmacokinetic properties of these nonsteroidal AR ligands (32, 33, 34, 35). SARMs exhibit tissue-selective anabolic activity in castrated rats, demonstrating full AR agonist activity in muscle but only partial activity in the prostate. A lead SARM, S-4, promotes the prostate and levator ani muscle weights to 33.8 and 101% of intact control, respectively (36). In contrast, testosterone propionate (TP) nonselectively increases the size of the prostate and levator ani muscle to 121 and 104% of intact control, respectively (36). In another study, S-4 not only restored the levator ani and soleus muscle mass 12 wk after castration, but skeletal muscle strength was also restored to intact levels. Additionally, a phase IIa study in healthy, elderly men and women given Ostarine demonstrated increased lean muscle mass, decreased fat mass, and significant improvements in their ability to climb stairs (37, 38).
S-23, a modified derivative of S-4, was identified as another SARM with potent anabolic activity. In castrated male rats, S-23 maximally maintained the levator ani muscle weight at 129% of intact control and showed significant improvements in bone mass density, lean mass, and fat mass in intact animals (39). We report herein the results of the first studies to examine the effects of a SARM on signaling involved in protein synthesis and degradation in the levator ani muscle that occurs during DEX-induced and castration-dependent atrophy. The effectiveness of S-23, compared with TP, at inhibiting DEX-induced muscle atrophy and regulating genes and proteins involved in the PI3K/Akt pathway (i.e. MAFbx, MuRF1, FoxO, IGF-I) indicate that SARMs may provide a unique and more selective pharmacological approach to prevent or treat corticosteroid-induced muscle atrophy.
[h=2]Materials and Methods[/h][h=3]Animals and treatment[/h]Male Sprague Dawley rats were purchased from Harlan Bioproducts for Science (Indianapolis, IN). The animals were maintained on a 12-h light, 12-h dark cycle with food and water available ad libitum. All animal studies were conducted under the auspices of an animal protocol approved by the Institutional Laboratory Animal Care and Use Committee at the University of Tennessee. Before and at the end of treatment, body fat, lean body mass (LBM), fluids, and total water were determined by magnetic resonance imaging (MRI; EchoMRI 4-in-1 composition analyzer; Echo Medical Systems, Houston, TX). The animals were 5–6 wk old and weighed 229–238 g. Male Sprague Dawley rats were randomized according to LBM and assigned to one of four groups (n = 5/group). Animals received daily sc injections (200 μl/d) of the compound of interest for 8 d. Each compound was dispersed in vehicle containing 80% Tween 80 (Sigma-Aldrich, St. Louis, MO) and 20% Captex 200 (Abitec, Columbus, OH). Group 1 was the control group and received vehicle alone. Groups 2–4 received 600 μg/kg · d DEX (Sigma-Aldrich). In addition to receiving DEX, groups 3 and 4 were administered 25 mg/kg · d of TP (Sigma-Aldrich) or S-23, respectively. S-23 (39) was synthesized in our laboratories using described methods (40). Chemical purities were confirmed by mass spectrometry and nuclear magnetic resonance and determined to be greater than 99%.
Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate and seminal vesicles were removed, cleared of extraneous tissue, and weighed. The levator ani, gastrocnemius (gastroc), extensor digitorum longus (EDL) and soleus muscles were removed (gastroc, EDL and soleus from the left hind limb), weighed, and a segment preserved in RNAlater (Ambion, Austin, TX) for gene expression analysis and the other portion immediately frozen in dry ice/ethanol (Sigma-Aldrich) for protein expression analysis. All tissue weights were normalized to total body weight (before treatment) and compared. All muscle samples in RNAlater and those that were immediately frozen were stored at 4 or −80 C, respectively, until further analysis.
An additional group of male Sprague Dawley rats was ordered from Harlan Bioproducts for Science for the time-course study. Animals in this study were 5–6 wk old and ranged from 219 to 234 g. Rats were randomized according to weight and assigned to appropriate groups (n = 5/group). Animals were orchidectomized (ORX) via scrotal incision under ketamine/xylazine anesthesia 24 h before drug treatment and received daily sc injections S-23 (200 μl/d), at a dose rate of 1 mg/d. S-23 was dissolved in vehicle containing dimethylsulfoxide (Sigma-Aldrich)/polyethylene glycol 300 (PEG300; Sigma-Aldrich) [10/90 (vol/vol)]. Additional groups of rats (n = 5/group) with or without castration received vehicle only and served as castrate or intact control groups, respectively. Animals were treated for 3, 7, 10, 14, 21, or 28 d. Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, seminal vesicles, levator ani, gastroc, EDL, and soleus muscles were removed (gastroc, EDL and soleus from the left hind limb), cleared of extraneous tissue, weighed, and preserved in RNAlater (Ambion) for gene expression analysis. All organ weights were normalized to total body weight and compared. Percent changes were determined by comparison to intact animals.
Lastly, a separate group was randomized according to weight (n = 5/group) to compare DEX treatment vs. castration on prostate, levator ani, and serum testosterone levels. Animals were 5–6 wk old and weighed 219–231 g. Animals were either castrated as described above (ORX group) or left intact (intact and DEX groups). Animals then received daily sc injections of vehicle (intact and ORX groups) or 600 μg/kg · d DEX for 8 d. Animals were weighed, anesthetized, and serum collected and stored at −20 C until further analysis. The ventral prostate and levator ani muscles were removed, weighed, and normalized to total body weight and compared with the vehicle-treated, intact control group.
[h=3]Serum testosterone analysis[/h]Serum testosterone was quantified using a liquid chromatography and tandem mass spectrometry method, using deuterium-labeled testosterone as an internal standard. The analyses were performed using a quadrupole ion-trap mass spectrometer coupled with an HPLC system and an electrospray ionization source. The samples were prepared by liquid-liquid extraction, using a 3:2 ratio (vol/vol) of ethyl acetate-hexane. An aliquot of sample (10 μl) was injected into a C18 column (100 × 2.1 mm, 3 μm; Alltima HP; Grace Davison Discovery Sciences, Deerfield, IL) at a flow rate of 0.4 ml/min using a gradient mobile phase. Mobile phase A was comprised of 90% water, 10% acetonitrile, and 0.1% formic acid. Mobile phase B consisted of 90% acetonitrile, 10% water, and 0.1% formic acid. The gradient initiated with 30% B at a flow of 0.4 ml/min and the first 3 min was diverted to waste. After 8 min, the gradient changed to 90% B and then back to 30% 6 sec later and remained there until the end of the total run time of 13 min. The lower limit of quantitation for the method was 0.200 ng/ml.
[h=3]Cell culture[/h]Myoblasts from the C2C12 mouse skeletal muscle cell line (American Type Culture Collection, Manassas, VA) were cultured in growth medium consisting of DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all purchased from Hyclone, Logan, UT) in a humidified atmosphere containing 5% CO2 at 37 C. Two independent experiments were performed for both quantitative PCR and Western blotting, and each were performed in triplicate.
[h=3]Quantitative real-time PCR[/h]C2C12 cells were seeded into six-well plates in growth medium at a density of 1 million cells/well and allowed to recover for 8 h. Medium was then replaced with differentiation medium consisting of DMEM supplemented with 2% horse serum (Hyclone). After 24 h, cells were transduced with 250 virus particles/cell of an AR adenovirus (generated by Welgen, Inc., Worcester, MA) and incubated another 24 h. Cells were treated with the compound of interest and ribonucleic acid (RNA) was isolated 24 h later, using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For muscle sample analysis, 50 mg of tissue was removed from RNAlater (Ambion), homogenized using Lysing Matrix D (MP Biomedicals, Solon, OH), and RNA isolated using TRIzol reagent. Total RNA was determined by absorbance at 260 nm. RNA (1 μg) was reverse transcribed to produce cDNA (high capacity cDNA reverse transcription kit with ribonuclease inhibitor; Applied Biosystems, Foster City, CA). Rat and mouse F-box protein 32 (Fbxo32), tripartite motif-containing 63 and IGF-I TaqMan probes and rat AR and GR probes were used (Applied Biosystems). Quantitative PCR was performed (TaqMan fast universal PCR master mix; Applied Biosystems) according to the manufacturer’s instructions. To control for any variation in reverse transcription or PCR efficiency, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The cycle number at which the reaction crossed an arbitrary threshold (CT) was determined for each gene and analyzed using the 2[FONT="]-ΔΔCT method (41). All PCR runs were performed in duplicate.[/FONT]
[h=3]Western blotting[/h]C2C12 cells were seeded into 10-cm dishes in growth medium at a density of 2.5 million cells/dish. Differentiation was induced, and cells were transduced and treated as described above. Cells were washed with Dulbecco’s PBS (Hyclone) and protein was extracted in buffer containing 1 m potassium phosphate, 10 mm sodium molybdate, 50 mm sodium fluoride, 2 mm disodium EDTA, 2 mm EGTA, 0.05% monothioglycerol, 1 mg/ml leupeptin, 1 mg/ml antipain dihydrochloride, 1 mg/ml aprotinin, 1 mg/ml benzamidine hydrochloride, 1 mg/ml chymostatin, 1 mg/ml pepstatin, 4 m sodium chloride, and 100 mm phenylmethanesulfonylfluoride. The samples were subjected to a freeze/thaw cycle and centrifuged. The supernatant was removed and stored at −80 C until analysis. For muscle sample analysis, 50 mg of tissue was homogenized in 750 μl of homogenization buffer using Lysing Matrix D (MP Biomedicals). The samples were subjected to a freeze/thaw cycle, centrifuged, and the supernatant stored at −80 C. Protein concentrations of the supernatant were determined by the Bio-Rad protein assay (Hercules, CA) using BSA (Sigma-Aldrich) as the standard. The protein samples (50 μg per lane) were mixed with loading buffer, heated for 5 min, and resolved by SDS-PAGE on 4–20% Tris-glycine gradient gels (Invitrogen) and then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 (50 mm Tris, 150 mm NaCl, and 0.1% Tween 20). The primary antibodies were diluted 1:1000 in 5% BSA [phospho-Akt Ser473, phospho-mTOR Ser2488, phospho-GSK-3β Ser 9, phospho-p70S6K Thr389, total Akt, and total FoxO3a; all from Cell Signaling Technology, Danvers, MA; phospho-FoxO3a S253 from Abcam Inc., Cambridge, MA; AR PG21 and antiactin from Upstate, Billerica, MA; glucocorticoid receptor (GR) from Santa Cruz Biotechnology, Inc., Santa Cruz, CA] and the secondary antibodies 1:2000 in 5% nonfat dry milk (horseradish peroxidase conjugated antirabbit IgG and antimouse IgG; Cell Signaling Technology). Immunostaining was visualized by enhanced chemiluminescence (ECL Plus Western blotting detection reagents; Amersham, Piscataway, NJ) and captured on photographic film. The protein band densities were quantified using TotalLab TL100 (Nonlinear Dynamics, Durham, NC). β-Actin was included for loading control.
[h=3]Statistical analyses[/h]All statistical analyses were performed using single-factor ANOVA followed by Dunnett’s multiple comparison test. Differences in which P < 0.05 were considered statistically significant.
[h=2]Results[/h][h=3]Expression of muscle hypertrophy and atrophy markers in C2C12 myotubes[/h]The effects of S-23 and TP on DEX-induced atrophy were examined in differentiated C2C12 myotubes expressing the AR (see Fig. 2A). In the absence of AR in untransfected C2C12 cells, androgens failed to regulate the expression of MAFbx or IGF-I (data not shown). However, in AR-transduced C2C12 cells, overnight incubation with DEX induced a 6.5-fold increase in the expression of MAFbx mRNA (Fig. 1A). Cotreatment with TP and S-23 partially but significantly inhibited the up-regulation, with near maximal effect seen at 1 nm. DEX treatment also caused a 13-fold decrease in expression of IGF-I mRNA, which was significantly inhibited by TP and S-23 observed at all concentrations 1 nm or greater (Fig. 1B).
[FONT="]Fig. 1.
[FONT="]In vitro gene expression. C2C12 cells were differentiated and myotubes treated as indicated for 24 h. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCTmethod. Data are presented as mean ± se (n = 6). *, P < 0.05 compared with the DEX-treated group. A, Fold expression change of muscle atrophy marker MAFbx. B, Fold expression change of muscle hypertrophy marker IFG-1.
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[FONT="]Fig. 2.
[FONT="]In vitro protein expression. C2C12 cells were differentiated and myotubes treated as indicated for 24 h and protein extracted. Fifty micrograms of protein were loaded to each well, resolved by SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk, incubated with primary antibody overnight in 5% BSA (1:1000), and then with secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin, total Akt, and total FoxO3 were included for loading control. Data presented as mean ± se (n = 6). *, P < 0.05 compared with the DEX-treated group. A, Immunoblots of AR in C2C12 cells before and after transduction. B, Immunoblots of proteins in the PI3K/Akt pathway. C, Graphical representation of the change in expression of pAkt compared with total Akt. D, Graphical representation of the change in expression of pFoxO3a compared with total FoxO3.
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Addition of DEX to C2C12 myotubes significantly reduced phosphorylation of many proteins involved in the PI3K/Akt pathway (Fig. 2B). Cotreatment with androgens significantly attenuated the effects of DEX on phosphorylation of Akt, GSK3-β, p70S6K, mTOR, FoxO3a, and FoxO1. Quantification of band densities revealed significant differences observed in the phosphorylation of Akt at 0.1 and 1 nm for S-23 and TP, respectively (Fig. 2C). Additionally, TP and S-23 increased phosphorylation of FoxO3a above that observed for vehicle control (Fig. 2D).
[h=3]DEX-induced muscle atrophy[/h]DEX administration (600 μg/kg · d) significantly reduced the total body weight of intact male rats (Fig. 3A). Cotreatment with 25 mg/kg · d of TP or S-23 partially attenuated the effects of DEX on body weight. However, the weights were still significantly different from vehicle control. DEX treatment also caused a 5% reduction in LBM after 8 d (Fig. 3B). S-23 significantly inhibited the loss of LBM with no significant differences observed vs. vehicle control, whereas TP only partially attenuated this loss. DEX also significantly reduced the size of the prostate and seminal vesicles (Fig. 3, C and D). Coadministration with TP or S-23 increased the weights of these tissues above that observed for vehicle control (Fig. 3, C and D), with TP significantly increasing the seminal vesicles compared with S-23. A variety of muscles were also excised to evaluate their response to glucocorticoid and androgen treatment. The levator ani, a highly androgen-responsive fast-twitch muscle, decreased by 52% on DEX administration. TP and S-23 completely inhibited this reduction, maintaining the weight similar to that observed for intact control (Fig. 3E). The gastrocnemius, a muscle comprised mostly of fast-twitch fibers [94%; (42)], decreased by 37% in response to DEX administration. However, contrary to previously reported data (30), TP and S-23 did not attenuate the effects of DEX on this muscle (Fig. 3F). The EDL, a fast-twitch muscle, also diminished in size by 34% on exposure to DEX, with no inhibition observed upon TP or S-23 administration (Fig. 3G). DEX also caused a reduction in the size of the soleus muscle, a slow-twitch muscle, which was attenuated by S-23 but not TP (Fig. 3H).
[FONT="]Fig. 3.
[FONT="]Effects of treatment on body weight and LBM and androgenic and anabolic tissues. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by magnetic resonance imaging, weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, seminal vesicles, levator ani, gastroc, EDL, and soleus were removed and weighed. All tissues were normalized to total body weight and compared. Data presented as mean ± se (n = 5). #, P < 0.05 compared with the vehicle-treated control group; *, P < 0.05 compared with the DEX-treated group. T, S, P < 0.05 compared with the testosterone- or S-23-treated group, respectively. A, Average body weight. B, Absolute change in LBM. Normalized prostate weights (C), seminal vesicles (D), levator ani muscle (E), gastroc (F), extensor digitorum longus (G), and soleus muscle (H) are shown.
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[h=3]Expression of AR and GR in muscle[/h]The levator ani muscle significantly expresses 6.6-fold more AR than the EDL muscle (Fig. 4, A and B), as determined by immunoblot. Expression of AR in the EDL is also significantly less than that of the soleus muscle. Conversely, the levator ani muscle significantly expresses the least amount of GR. The quantitated immunoblot shows that the soleus significantly expresses more GR than the EDL and levator ani muscles (Fig. 4, C and D). Although the levator ani and soleus muscles may express slightly more AR than the gastroc, these differences are not statistically significant.
[FONT="]Fig. 4.
[FONT="]Expression of AR and GR in anabolic tissues. Male rats were treated with vehicle for 8 d via sc injection. The levator ani, gastroc, extensor digitorum longus, and soleus muscles were removed. A segment of each muscle was frozen in dry ice/ethanol. Protein was extracted, 50 μg loaded to each well, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk, incubated with primary antibody overnight in 5% BSA (1:1000) and then with the secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin included for loading control. Data are presented as mean ± se (n = 3 for AR; n = 5 for GR). L, G, E, S, P < 0.05 compared with the levator ani, gastroc, EDL, and soleus groups, respectively. A, Immunoblot of AR in levator ani, gastroc, EDL, and soleus muscles. B, Graphical representation of the expression of AR present in muscles normalized to β-actin. C, Immunoblot of GR expression in muscle. D, Graphical representation of the expression of GR present in muscles normalized to β-actin.
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[h=3]Effects of S-23 and TP on gene and protein expression in muscle of intact rats[/h]Administration of DEX to intact animals resulted in up-regulation of MAFbx mRNA in the levator ani, gastroc, EDL, and soleus, with the greatest effect observed in the levator ani muscle (60-fold change) (Fig. 5A). Coadministration with androgens completely inhibited this up-regulation in the levator ani. In the gastroc, EDL, and soleus, the up-regulation of MAFbx was slightly attenuated by TP, but S-23 was statistically more effective at inhibiting the up-regulation of MAFbx in the gastroc, EDL, and soleus muscles. A similar trend was observed for MuRF1, with a 25-fold increase in mRNA expression in the levator ani, which was completely blocked by TP and S-23 administration. S-23 was statistically more effective than TP at inhibiting MuRF1 in the soleus (Fig. 5B). DEX administration significantly decreased the levels of IGF-I in the gastroc, EDL, and soleus by 3-, 6.5-, and 5.5-fold, respectively (Fig. 5C). Although IGF-I expression did not decrease in the levator ani, TP and S-23 caused a small but significant increase in expression of IGF-I in this tissue. S-23 was statistically more effective than TP at inhibiting the down-regulation of IGF-I in the gastroc, EDL, and soleus muscles.
[FONT="]Fig. 5.
[FONT="]Effects of treatment on gene expression in the levator ani in intact male rats. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by MRI, weighed, anesthetized, and killed within 24 h after the last dose. The levator ani muscles were removed and weighed. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCT method and presented as mean ± se (n = 5). *, P < 0.05 compared with the DEX-treated group; #, P < 0.05 compared with the vehicle-treated control group; T, S, P < 0.05 compared with the testosterone- or S-23-treated group, respectively. A, Fold expression change of muscle atrophy marker MAFbx. B, Fold expression change of MuRF1. C, Fold expression change of muscle hypertrophy marker IGF-I.
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Proteins upstream of MAFbx and MuRF1 in the PI3K/Akt pathway, phosphorylated Akt, GSK3β, p70S6K, and FoxO3a decreased in the levator ani muscle on DEX exposure (Fig. 6A). As was observed in C2C12 myotubes, cotreatment with TP and S-23 maintained phosphorylated levels of GSK3β, p70S6K, and Akt (Fig. 6, A and B). Down-regulation of phosphorylated FoxO3a was attenuated by TP and S-23 (Fig. 6C).
[FONT="]Fig. 6.
[FONT="]Expression of hypertrophy and atrophy proteins in the levator ani of intact male rats. Male rats were treated with vehicle, 600 μg/kg · d DEX, DEX plus 25 mg/kg · d TP, or DEX plus 25 mg/kg · d S-23 for 8 d via sc injection. Rats were scanned by MRI, weighed, anesthetized and killed within 24 h after the last dose. The levator ani muscles were removed and weighed. Protein was extracted, 50 μg loaded to each well, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk, and incubated with primary antibody overnight in 5% BSA (1:1000) and then with the secondary antibody for 1 h in 5% nonfat dry milk (1:2000). Immunostaining was visualized by enhanced chemiluminescence, captured on photographic film, and band densities quantified. β-Actin, total Akt, and total FoxO3 were included for loading control. Data presented as mean ± se (n = 5). *, P < 0.05 compared with the DEX-treated group; #, P < 0.05 compared with the vehicle-treated control group. A, Immunoblots of proteins in the PI3K/Akt pathway. p, Phosphorylated. B, Graphical representation of the change in expression of pAkt compared with total Akt (tAkt). C, Graphical representation of the change in expression of pFoxO3a compared with total FoxO3.
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[h=3]Effects of time and S-23 in castrated rats[/h]Castration causes an immediate reduction in size of androgen-dependent tissues. After just 3 d, the prostate and levator ani muscle reduce to 67 and 79% of intact control, respectively (Fig. 7, B and C). Both tissues continue to decrease in size and reach nadir by 14 d, with sizes of the prostate and levator ani leveling off around 15 and 45% of that observed in intact control, respectively. Conversely, when S-23 is administered to ORX animals, the prostate and levator ani are both maintained, and significant increases are observed through d 28 (Fig. 7, B and C). Castration induces a 40-fold increase in MAFbx mRNA expression at 3 d but drops off 14 d after castration (Fig. 7D). Administration of S-23 inhibits this up-regulation at 3, 7, and 10 d, but no change is observed after 14 d (Fig. 7E). Additionally, castration induces a down-regulation in expression of IGF-I (Fig. 7F), which is also attenuated by S-23 (Fig. 7G).
[FONT="]Fig. 7.
[FONT="]Effects of time and S-23 on androgenic and anabolic tissues in castrated male rats and expression of hypertrophy and atrophy markers. Male rats were ORX via scrotal incision under ketamine/xylazine anesthesia 24 h before drug treatment and received daily sc injections of S-23 at a dose rate of 1 mg/d for 3, 7, 10, 14, 21, or 28 d. Additional groups of animals with or without castration received vehicle only and served as castrate or intact control groups, respectively. Animals were weighed, anesthetized, and killed within 24 h after the last dose. The ventral prostate, levator ani, gastroc, EDL, and soleus were removed and weighed. A segment of each muscle was preserved in RNAlater (Ambion). All tissues were normalized to total body weight and compared. Percent changes were determined by comparison with intact animals. RNA was isolated, reverse transcribed, and quantitative RT-PCR performed. GAPDH was included as internal control. Data were analyzed using the 2-ΔΔCT method and presented as mean ± se (n = 5). Graphs B, C, D, F: #, P < 0.05 compared with the vehicle-treated, intact control group; graphs E and G: #, P < 0.05 compared with the vehicle-treated, ORX control group on the respective day. A, Body weights of all animal groups before and after treatment. BW, Body weight. B, Normalized prostate weights of ORX control and S-23-treated animals. C, Normalized levator ani weights of ORX control and S-23-treated animals. D, Fold expression change of muscle atrophy marker MAFbx in ORX animals compared with intact control animals. E, Fold expression change of MAFbx in S-23-treated animals compared with ORX at each day. F, Fold expression change of muscle hypertrophy marker IGF-I in ORX animals compared with intact control animals. G, Fold expression change of IGF-I in S-23-treated animals compared with ORX at each day.
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[h=3]Comparison of DEX and ORX on body weight, androgen-dependent tissues, and serum testosterone[/h]Significant reductions in body, prostate and levator ani weights were observed after 8 d of DEX administration or castration (Table 1). Serum testosterone concentrations measured 3.6 ng/ml in vehicle-treated, intact animals. DEX administration induced no significant change in serum testosterone levels. However, castration reduced serum testosterone levels below the limit of quantitation (0.20 ng/ml; Table 1).
[FONT="]TABLE 1.
[FONT="]Effects of DEX treatment and castration on body weight (BW), prostate, levator ani, and serum testosterone
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| Group | BW (g) | Prostate (mg/g BW) | Levator ani (mg/g BW) | Serum testosterone (ng/ml) |
|---|---|---|---|---|
| Intact | 251 ± 2.7 | 0.72 ± 0.1 | 0.76 ± 0.04 | 3.6 ± 0.3 |
| DEX | 198 ± 2.2a | 0.56 ± 0.04 | 0.63 ± 0.02a | 2.6 ± 0.7 |
| ORX | 232 ± 4.9a | 0.18 ± 0.05a | 0.55 ± 0.04a | <0.20a |
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[FONT="]Animals in the ORX group were castrated on d 0. On d 1–8, animals in the intact and ORX groups received daily sc injections of vehicle, and the DEX group received 600 μg/kg · d DEX. Prostate and levator ani muscle weights were measured at the end of the treatment period and normalized to BW. Serum testosterone concentrations were determined by liquid chromatography and mass spectrometry. Data are presented as mean ± se (n = 5).
a
P < 0.05 compared with the vehicle-treated, intact control group.
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[h=2]Discussion[/h]Corticosteroid-induced protein catabolism predominantly strikes fast-twitch muscle fibers, including the gastrocnemius and levator ani muscles. Atrophy in fast-twitch muscle fibers is highly responsive to androgen administration (43, 44, 45). The Hershberger assay is the method of choice for identifying AR-dependent myoanabolic tissue selectivity (46). Our laboratory and many others have shown that the levator ani muscle of rats rapidly atrophies after castration and quickly hypertrophies on exogenous administration of an anabolic agent (36, 46, 47). Exogenously administered testosterone promotes the growth of anabolic (i.e. muscle) and androgenic (i.e. prostate and seminal vesicles) tissues, whereas SARMs are selectively anabolic. Studies demonstrated the beneficial effects of testosterone in the DEX-induced animal model of muscle atrophy and signaling effects in the PI3K/Akt pathway (30, 48, 49). In this model, most studies reported the effects testosterone on the gastroc muscle (30, 48, 49). The present study provides new information regarding the comparative effects of testosterone and a SARM on muscle atrophy and hypertrophy signaling pathways after corticosteroid administration or castration for not only the gastroc but also the levator ani, extensor digitorum longus, and soleus muscles.
IGF-I mRNA expression decreased in response to DEX treatment, both in vitro and in vivo (Figs. 1B and 5C) as well as castration (Fig. 7F), corroborating the previous reports that the atrophic effects of glucocorticoids are mediated at least partially via suppression of IGF-I signaling (16, 17, 18). This suppression was partially or completely inhibited by TP and S-23. IGF-I activates the PI3K/Akt pathway and downstream targets associated with protein synthesis include Akt, mTOR, GSK3β, and p70S6K. TP and S-23 completely blocked the DEX-dependent dephosphorylation of Akt and downstream proteins, in both skeletal muscle cells and in vivo the levator ani muscle (Figs. 2B and 6A). These results confirm the anabolic effects of TP and S-23 on protein synthesis downstream of IGF-I.
Akt signaling is also involved in inhibiting protein degradation by phosphorylating FoxO and therefore preventing transcription of MAFbx and MuRF1, muscle-specific ubiquitin ligases. Stitt et al. (23) demonstrated that phosphorylation of FoxO1 is required for inhibition of muscle atrophy via the PI3K/Akt pathway. Likewise, Sandri et al. (22) demonstrated that dephosphorylation of FoxO3 is required to activate MAFbx. In the present study, administration of DEX resulted in dephosphorylation of FoxO1 and FoxO3 in skeletal muscle cells and the levator ani muscle (Figs. 2B and 6A). However, TP and S-23 significantly promoted phosphorylation of FoxO, thereby inhibiting muscle atrophy. Gene expression analysis revealed that dephosphorylation of FoxO by DEX significantly increased expression of MAFbx and MuRF1 mRNA but is attenuated by coadministration with TP or S-23 (Figs. 1A and 5, A and B). A coimmunoprecipitation assay was performed to determine whether there is a direct interaction between the AR and FoxO3a to explain the ability of TP to maintain mRNA expression of FoxO3a above that of vehicle control (Fig. 2D). However, the results show that the AR and FoxO3a do not directly interact (data not shown).
Interestingly, the AR ligands did not maintain IGF-I or MAFbx levels as that of control. However, they did bring the proteins downstream of IGF-I back to the control levels. This indicates that the final effectors of anabolism, such as Akt, FoxO3A, and other downstream proteins, are effectively regulated by TP and S-23. Although earlier studies revealed that there was no change in the proliferation and differentiation rate of C2C12 cells stably transfected with AR in response to dihydrotestosterone (50), myotube diameter and size should have increased in the present study due to the extent of regulation observed with the downstream proteins.
Administration of DEX significantly decreased body weight, which was partially inhibited by androgen administration (Fig. 3A). There was a significant loss of LBM observed in DEX-treated animals, which was only partially inhibited by TP (Fig. 3B). However, animals cotreated with S-23 maintained LBM similar to that observed in vehicle control. The levator ani, gastroc, EDL, and soleus muscles all atrophied in response to DEX administration (Fig. 3, E–H). Addition of S-23 completely prevented reduction in weight of the levator ani and soleus muscles. TP protected only against loss of levator ani muscle weight. Minimal effects were observed in the other muscles. Inconsistent with our results, Zhao et al. (30) reported that testosterone (28 mg/kg · d) completely protected against loss of gastroc muscle weight at both 1 and 7 d with significant inhibition of MAFbx expression. In the present study, MAFbx expression in the gastroc was only slightly down-regulated by S-23 at 8 d (Fig. 5A). Protein expression data revealed that the levator ani expresses significantly more AR than the soleus muscle (Fig. 4, A and B) and may explain why this muscle responded to TP and S-23 administration more than the others in the present study. Interestingly, the levator ani muscle expresses the least amount of GR but atrophied the most in response to glucocorticoid administration. The gastroc and soleus muscles express comparable GR and AR; however, the slow-twitch muscle fibers of the soleus muscle responded to SARM treatment, whereas the gastroc did not. Analysis of AR expression in the levator ani, gastroc, EDL, and soleus muscles at the end of the study revealed that there was no significant change in AR expression in DEX-treated animals or castrated animals treated with S-23. However, castration alone significantly increased AR expression in the levator ani, gastroc, and soleus muscles (data not shown). Additionally, previous studies have shown that DEX administration to rats reduces food consumption (51, 52). The animals in the present study were not pair fed to account for differences in food intake, and therefore, this may have an indirect effect on muscle mass and gene expression of animals administered DEX, compared with the other groups.
The levator ani muscle starts atrophying immediately after surgical removal of testosterone and plateaus around 14 d (Fig. 7C). The greatest up-regulation of MAFbx expression in castrated, vehicle-treated animals occurs between d 3 and 10 (Fig. 7D). S-23 showed the most regulation of this gene through 10 d, with no down-regulation observed on d 14–28 (Fig. 7E). This lack of regulation at the later time points is likely related to the muscle no longer atrophying, with minimal up-regulation of MAFbx observed in the ORX animals. Conversely, IGF-I declines in ORX animals throughout the 4 wk but is up-regulated by S-23 with the greatest regulation observed at 28 d (Fig. 7, F and G).
The levator ani muscle atrophies in response to both castration and DEX administration. Stress-induced elevations in plasma glucocorticoid concentrations inhibit secretion of LH and therefore reduces testosterone concentrations in vivo (53). Studies have also shown that glucocorticoids have a direct inhibitory effect on testicular steroidogenesis (54, 55, 56). In humans, significant reductions in circulating testosterone result from DEX administration as well as IGF-I mRNA (57). Yin et al. (49) reported serum testosterone concentrations that were statistically significant between animals administered testosterone and testosterone combined with DEX, compared with both control and DEX-treated animals. In the present study, serum testosterone concentrations reduced by more than 95%, and the levator ani muscle weight decreased by 28%, 5 d after castration (Table 1). Due to variability of the data, there was no reduction in serum testosterone induced by DEX. The highly androgen-responsive levator ani muscle decreased in size in response to both DEX administration and castration possibly due to the reduction in circulating testosterone. This effect may contribute to the myopathy that occurs with glucocorticoid administration.
The mechanisms underlying DEX-induced atrophy appear to be different from castration-induced atrophy. Loss of IGF-I in glucocorticoid-induced muscle atrophy does not appear to be as important in levator ani muscle as in the other muscles (Fig. 5C). In glucocorticoid-induced muscle atrophy, expression of IGF-I in the levator ani did not decline; however, there was a significant reduction observed in the other muscles. Additionally, the levator ani muscle atrophied the most and displayed the greatest up-regulation of MAFbx and MuRF1 compared with the other muscles (Fig. 5, A and B). The inability of the gastroc, EDL, and soleus muscles to maintain IGF-I might account for lack of changes in size because the levator ani was able to maintain both IGF-I levels and size. In castration-induced atrophy, IGF-I mRNA expression declined throughout the 28-d period in the levator ani muscle (Fig. 7F) and was up-regulated after SARM administration (Fig. 7G). However, the levator ani muscle also displayed the greatest up-regulation of MAFbx compared with the other muscles (data not shown). Our studies suggest that levator ani atrophy caused by hypogonadism (i.e. castration) may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on the up-regulation of MAFbx and MuRF1. Although, the gene expression analyses establish this possibility, the direct correlation that the alteration in these pathways result in muscle loss is lacking. This could be established only using knockout or transgenic animal models. Studies are ongoing in our laboratories to elucidate the differences in these two mechanisms of muscle atrophy and their response to TP and SARM treatment.
Previous studies demonstrated that testosterone administration inhibits muscle atrophy by increasing net protein synthesis and reusing intracellular amino acids in skeletal muscle (29, 58, 59). Although we have demonstrated in the present study that AR ligands, testosterone and S-23, induce the anabolic and inhibit the catabolic pathway proteins, studies using radiolabeled amino acids are needed to demonstrate that S-23 has a direct effect on nascent protein synthesis. Importantly, previous publications positively correlate the IGF-I/Akt pathway to protein synthesis in muscle. Recent publications are also bringing clarity on the role of ubiquitination and proteasomal degradation of these anabolic proteins (60, 61). Future experiments with radiolabeled amino acids, cycloheximide, or MG-132 will be helpful to determine the direct effects of AR ligands on the biosynthetic pathways.
The doses of S-23 and TP were chosen to result in equal increases in levator ani weight in the DEX-induced model of muscle atrophy (i.e. a dose that would return the levator ani to the same size as observed in intact controls). Although a high dose of S-23 was used in this study to determine its mechanistic effects on muscle atrophy, S-23 maintains the levator ani muscle around intact levels at a dose of 0.1 mg/d in castrated animals, whereas the prostate was maintained at less than 30% of that observed in intact control (39). Conversely, at the same dose, TP is nonselective and maintains the weights of both the levator ani and prostate at 70% of intact control (36). Thus, the tissue selectivity of S-23, like other SARMs that we have reported, is dose dependent.
In summary, S-23 and TP maintained the weight of the levator ani muscle in the DEX model of muscle atrophy. S-23 and TP blocked the DEX-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including FoxO. DEX caused a significant up-regulation in the expression of MAFbx and MuRF1, but TP and S-23 administration blocked this effect by phosphorylating FoxO. S-23 also attenuated the up-regulation of MAFbx and MuRF1 in the gastroc, EDL, and soleus muscles. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of ubiquitin ligases and down-regulation of IGF-I. S-23 maintained the levator ani muscle at or above intact levels and significantly decreased expression of MAFbx and increased expression of IGF-I on d 3–10, with minimal regulation observed 14 d after castration. Given the observed effects on IGF-I, MAFbx, and Murf1, these studies suggest that SARMs and TP may stimulate protein synthesis and inhibit protein degradation via the IGF-I/PI3K/Akt signaling pathway. SARMs provide the peripheral stimulus necessary to maintain muscle mass with minimal effects on androgenic tissues and represent a viable treatment option for a variety of clinical conditions including glucocorticoid-induced muscle loss and sarcopenia.
