Androgen-mediated improvement of body composition and muscle function involves a novel early transcriptional program including IGF1, mechano growth factor, and induction of β-catenin

in Journal of Molecular Endocrinology

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5α-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of β-catenin, were repressed, indicating modulation of the β-catenin pathway. DHT increased total levels of β-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear β-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of β-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

Abstract

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5α-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of β-catenin, were repressed, indicating modulation of the β-catenin pathway. DHT increased total levels of β-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear β-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of β-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

Introduction

Androgens are important endocrine regulators of male sexual development and maintenance of muscle, bone, adipose tissue, and body composition (Vermeulen 1998, Vermeulen et al. 1999). Testosterone, the major circulating androgen, can act directly or be converted to the more potent androgen 5α-dihydrotestosterone (DHT) by 5α-reductase, or to estrogens by aromatase (Russell & Wilson 1994, Simpson et al. 1994). Both testosterone and DHT activate the androgen receptor (AR), a nuclear receptor that functions as a transcription factor in hormone-sensitive cells (Chang et al. 1995, Heinlein & Chang 2002). After reaching peak levels in early adulthood, androgen levels decline with age in both sexes (Tenover 1994, Lamberts et al. 1997). This loss of endogenous androgens parallels several symptoms of aging, including decreased muscle mass and function (Snyder et al. 1999), increased visceral fat (Katznelson et al. 1998) and bone loss (Katznelson et al. 1996). Treatment with testosterone improves muscle mass and strength, bone density, and reduces visceral fat in a variety of subjects (Bardin 1996, Katznelson et al. 1996, Bhasin et al. 1997, 2000, Swerdloff & Wang 2003). Thus, restoring androgens to youthful levels could potentially be used to manage sarcopenia, osteoporosis, visceral obesity, and frailty.

The mechanisms by which androgens promote anabolism in adult animals are largely unknown. AR is detectable in bone and muscle cells, but levels are low compared with reproductive tissues such as the prostate and levator ani muscle (Antonio et al. 1999, Monks et al. 2004). Nevertheless, castration in rats reduces contractile force in muscles of the hindlimb (Brown et al. 2001) and produces ultrastructural signs of degeneration and reduced protein synthesis (Ustunel et al. 2003). Treatment with anti-androgens limits gains in muscle strength during weight training (Ruzic et al. 2003). Conversely, repletion with testosterone increases lean mass and muscle strength, improves nitrogen balance, and induces myofiber hypertrophy (Sinha-Hikim et al. 2002). Thus, testosterone regulates both the mass and function of skeletal muscle in adults.

At the cellular level, the effects of testosterone on body composition might involve recruitment or activation of resident myogenic precursor cells to existing myofibers (Chen et al. 2005b). Satellite cells cultured from porcine muscle express AR, and AR agonists delay their differentiation (Doumit et al. 1996). Furthermore, in muscle biopsies from men that exhibited gains in myofiber volume and strength following 20 weeks of testosterone treatment, satellite cell number was increased (Sinha-Hikim et al. 2003). Other studies report changes in the local expression of insulin-like growth factor 1 (IGF1) in muscle samples from patients receiving anabolic androgens (Sheffield-Moore 2000, Ferrando et al. 2002). As IGF1 and its related splice variants stimulate satellite cell proliferation and promote muscle hypertrophy (Musaro et al. 2001, Hill & Goldspink 2003, Goldspink & Yang 2004), these data suggest that androgens regulate muscle mass by this mechanism. Some in vitro data support the concept that androgens act directly on satellite or other precursor cells. Murine C2C12 cells, which resemble myogenic precursors, do not express AR, but when AR expression is produced by transfection, AR-selective ligands increase myogenin expression and accelerate myoblast differentiation and fusion (Lee 2002, Vlahopoulos et al. 2005). Mouse C3H10T1/2 fibroblast cells express endogenous AR, and DHT inhibits their differentiation into adipocytes and promotes the expression of MyoD and myosin heavy chain type II (MHC2; Compston 2001, Singh et al. 2003). In addition to satellite cells, AR is expressed in mature myofibers (Saartok et al. 1984, Sar et al. 1990) in several types of motoneurons (Lumbroso et al. 1996, Piccioni et al. 2001), and in intramuscular fibroblasts (Monks et al. 2004), and thus could influence growth and function through activation in these cells. Finally, androgens regulate systemic levels of IGF1, GH, and thyroid hormone, and may oppose the actions of glucocorticoids, any of which could contribute to the effects of androgen on muscle (Link et al. 1986, Hickson et al. 1990, Banu et al. 2002, Ferrando et al. 2002).

It has been proposed that testosterone could promote the differentiation of mesenchymal multipotent cells into the myogenic lineage while inhibiting adipogenic differentiation by modulating nuclear translocation of β-catenin (Bhasin et al. 2006). β-Catenin, through its action in cell adhesion and Wnt signal transduction, plays a critical role in embryonic development and regulation of adult stem cell populations in various tissues (Clevers 2006). In vitro, β-catenin is both necessary and sufficient for myogenesis and inhibits adipogenesis (Ross et al. 2000, Petropoulos & Skerjanc 2002). Moreover, β-catenin is essential for adult skeletal muscle growth and regeneration in vivo (Polesskaya et al. 2003, Reya & Clevers 2005, Armstrong et al. 2006), and myonuclear β-catenin is up-regulated during overload-induced muscle hypertrophy in adults (Armstrong & Esser 2005). Muscle regrowth following atrophy is associated with downregulation of glycogen synthase kinase-3β (GSK3β), a negative regulator of β-catenin (van der Velden et al. 2007, Schakman et al. 2008). Finally, β-catenin promotes self-renewal of satellite stem cells (Perez-Ruiz et al. 2008) Together, these findings suggest that Wnt/β-catenin signaling plays an active role in the maintenance of body composition in adults. However, the role for β-catenin in androgen-mediated muscle growth has not been studied.

An important step towards understanding androgenic signaling in regulating body composition will be to define the processes governed by AR and the cell type(s) in which AR exerts its anabolic effect. Since many studies use testosterone, the relative contributions of the AR and estrogen receptors are ambiguous, and some report that estrogen is an essential component of androgenic anabolism (Bilezikian et al. 1998, Vandenput et al. 2002). Furthermore, the genes targeted by AR in muscle, fat, and bone are unknown. To address these issues, we validated the castrated rat model and characterized the effects of DHT in the soleus muscle and identified genes that respond to DHT within the first week of treatment and are potential downstream effectors of anabolic action.

Materials and methods

Animal studies and analysis of body composition

All animal studies described in this report were approved by the Institutional Animal Care and Use Committee. Sprague–Dawley rats (Taconic, Hudson, NY, USA) were purchased following orchidectomy (ORX) or sham orchidectomy (SHAM) at 10 weeks of age and maintained for 11 weeks post-surgery with ad libitum access to food and water. Animals aged 21 weeks were then analyzed by dual photon emission X-ray absorptiometry (DEXA) to quantify lean, fat, and bone mass using a Hologic 4500A instrument at baseline and at indicated times during treatment by s.c. injection with 3 mg/kg per day DHT, 10 mg/kg per day testosterone, or vehicle (0.4 ml propylene glycol; n=9 per group). In a second time-course experiment, 11-week-post-ORX rats were treated with DHT or vehicle as above for 4, 7, 14, and 21 days (n=10 per group) with a vehicle group matched with each time point, and then the soleus muscles were collected for RNA, DNA, protein extraction, or formalin fixed for histological examination. Finally, a shorter experiment was conducted with older ORX rats aged 6 months. They were treated with vehicle or DHT for 1, 4, and 7 days (n=4 per group) to focus on earlier gene expression changes and confirm previous quantitative real-time PCR (qRT-PCR) results.

Contractile measurements

Animals were sedated with 3:1 ketamine:xylazine, shaved at the left hind limb, and placed on a 39 °C heating pad. The sciatic nerve was placed over a platinum bipolar electrode connected to an Astro-Med S48 stimulator. Then the soleus was surgically isolated at its insertion point, and the tendon was severed and sutured to a T10 force transducer housed in a micromanipulator and connected to a P220 amplifier using 2.0 silk. The Achilles tendon was severed to minimize the influence of contraction by the gastrocnemius. The sciatic nerve and soleus were bathed in 37 °C mineral oil and Rat Ringer's respectively. A force–tension curve was obtained by stimulating the sciatic nerve at supramaximal voltage (1.2 V) for 0.5 ms while stretching the muscle across 1 mm increments. Once the stretch distance that produced maximal twitch strength was identified, the sciatic nerve was stimulated with 1.2 V, 100 Hz, 400 ms, and square pulse waves to obtain peak tetanic tension. The data were collected and analyzed using Astro-Med software (Warwick, RI, USA). The soleus was dissected and weighed after recording. Peak tetonic tension was then normalized per unit mass as previously described (Brown et al. 2001, Brown & Taylor 2005).

Histomorphometry

Soleus muscles were fixed in formalin and embedded in paraffin, and multiple serial cross-sections were produced. Hemotoxylin- and eosin-stained muscle sections were examined and photographed using a Nikon Eclipse E1000M microscope/digital camera system. The number of nuclei was counted in an area covering 10–20 fibers three times per muscle section (n=10 sections/group). The number of fibers in that area was counted, and the nuclei per fiber and μm2 of area were calculated using Bioquant software (San Diego, CA, USA). Differences in fiber size or nuclear number were tested by statistical t-test. Nuclei labeled by hemotoxylin or by anti-β-catenin antibodies by immunohistochemistry were counted per muscle cross-sectional area of soleus muscles. Sections were visualized and photographed using a Nikon Eclipse E1000M microscope/digital camera system.

Immunohistochemistry

Immunostaining for β-catenin was performed on formalin-fixed, paraffin-embedded tissue sections of rat soleus muscles (n=10 rats/sections per group). Rat duodenum sections were used as a positive control. Sections were dewaxed and rehydrated. Mouse monoclonal anti-β-catenin antibodies (Sigma, clone 6F9, 1:500) were detected using biotinylated anti-mouse IgG secondary antibody and avidin-conjugated HRP system (Vectastain ABC, Invitrogen), and stained using nickel diaminobenzidene (DAB). For counting the proportion of β-catenin-stained nuclei, sections were immunostained by DAB (without nickel and therefore brown) and counterstained with hemotoxylin (blue). The sample sections were counterstained with eosin and visualized using a Nikon Eclipse E1000M microscope/digital camera system. The ratio of DAB stained to hemotoxylin-stained nuclei was reported as percent β-catenin-positive nuclei.

C2C12 myoblast cell culture

Mouse C2C12 myoblasts (American Type Culture Collection, ATCC, Rockford, MD, USA) were maintained in DMEM with 1 g/l d-glucose, sodium pyruvate (110 mg/l), fetal bovine serum 10%, l-glutamine (2 mM), and penicillin–streptomycin 1% (all Gibco, Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. Cells were passed at 60–70% confluence and tested in OptiMem (Invitrogen) media at 100% confluence.

Immunocytochemistry

Confluent C2C12 myoblasts were treated with recombinant mouse IGF1 (Sigma) at 10 and 30 ng/ml, or the C-terminal peptide 1–24 of mechano growth factor (ctMGF, Phoenix Pharmaceuticals, Burlingame, CA, USA) at 30 and 60 ng/ml for 25 min, fixed with 4% paraformaldehyde in PBS, permeablized with 0.5% Triton X-100 in PBS, and blocked with 5% normal goat serum. The fixed cells were immunostained for myonuclear β-catenin using primary mouse monoclonal anti-β-catenin antibodies (clone 6F9, Sigma) 1:50, goat anti-mouse Alexa Fluor 488 secondary antibodies (Invitrogen) 1:100, counterstained with the fluorochrome 4′,6-di-amidino-phenyl-indole (DAPI) nuclear stain, and photographed using a Nikon Eclipse E1000M microscope camera system. Photos were processed for false color for DAPI nuclear staining and merged using Adobe Photoshop 7.0.

Western immunoblot

Whole soleus muscle lysates were prepared in lysis buffer (2% SDS, 62.5 mM Tris, pH 6.8, 10% glycerol, 1% 2-mercaptoethanol, 1× complete protease inhibitors (Roche), and phosphatase inhibitor cocktails (Sigma)) and assayed for total protein using the BCA method (Pierce Biotechnology, Rockford, IL, USA). About 40 μg of protein were subjected to SDS-PAGE using 12.5% polyacrylamide gels (Pierce Biotechnology), transferred to nitrocellulose, and detected using mouse monoclonal IgG1 anti-β-catenin (clone 6F9, Sigma), polyclonal rabbit anti-phospho-β-catenin Ser33/37 Thr41 IgG1 1:400 (#9561 Cell Signaling, Danvers, MA, USA), rabbit monoclonal anti-GSK3β IgG1 1:400 (#9315 Cell Signaling), rabbit polyclonal anti-phospho-GSK3β (Ser9) IgG1 1:400 (#9336 Cell Signaling), mouse monoclonal anti-axin (H98) IgG1 1:400, rabbit polyclonal anti-axin2/conductin IgG1 1:200, polyclonal mouse anti-β-tubulin (D10) IgG1 1:200, mouse anti-actin 1:400, and mouse anti-lamin IgG1 1:200 primary antibodies (all Santa Cruz Biotechnology, Palo Alto, CA, USA), followed by HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology) 1:17 000 and processed for enhanced chemiluminescence (Pierce Biotechnology). Bands were visualized using Biomax MR Kodak film. Nuclear protein from C2C12 myoblasts was prepared using the nuclear protein isolation kit (NE-PER, Pierce Biotechnology) according to the manufacturer's instructions. Control and ctMGF or IGF1-treated C2C12 cells were homogenized in 750 μl cytoplasmic extraction reagent and centrifuged at 16 000 g for 5 min. The supernatant, consisting of cytosolic protein, was collected and the remaining pellet was treated with 150 μl nuclear extraction reagent. The samples were then centrifuged at 16 000 g for 10 min, and the supernatant was collected for nuclear proteins. Protein concentrations were determined using BCA method with a NanoDrop ND-1000 spectrophotometer. Band optical density measurements were generated by a Bio-Rad GS-800 calibrated densitometer (Bio-Rad Labs, Hercules, CA, USA), and the data were analyzed by Alphaease software.

TOPFLASH β-catenin plasmid reporter transfection and assay

To measure β-catenin nuclear translocation, the TCF/LEF β-catenin TOPFLASH firefly and control Renilla luciferase reporter plasmids (Milipore, Bedford, MA, USA) were cotransfected into suspended C2C12 myoblasts using 1 μg DNA with Lipofectamine 2000 (Invitrogen), and plated into 96-well plates at 10 000 cells per well. After 24 h, the growth media were replaced with serum-free media, and the confluent myoblasts were treated with water vehicle, IGF1, or ctMGF as above. After 16 h, both firefly and Renilla luciferase activity were measured using a commercially available luciferase assay system kit (Dual-Glo, Promega) and quantified using an EnVision luminescence detector (Perkin Elmer, Shelton, CT, USA). All values were first normalized to control Renilla luciferase activity, and IGF1 and ctMGF values normalized to vehicle-treated wells.

Quantitative RT-PCR

Total RNA was prepared from soleus muscle using TRIzol (Gibco) following the manufacturer's protocol. Quantitative RT-PCR (qRT-PCR) was performed using the Perkin Elmer Taqman 7700 (Perkin-Elmer) with gene-specific primers and fluorescence-labeled probes (5′-reporter dye, 6-carboxyfluorescein (FAM); 3′-quencher dye, 6-carboxy-N,N,N′,N′-tetramethylrhodamine (TAMRA)), which were designed using Primer Express (version 1.5) software and synthesized by Applied Biosystems (Foster City, CA, USA). The primer and probe sequences are listed in Table 1. Mgf and Igf1ea were detected by SYBR Green (Stratagene, Cedar Creek, TX, USA) as described elsewhere (Hill & Goldspink 2003). Three aliquots of RNA from each sample underwent three independent reverse transcription reactions, resulting in nine measurements. From these measurements, a mean and s.d. of measurement were derived, and both vehicle- and DHT-treated samples were normalized to glucuronidase expression. DHT values were compared with time-matched vehicle values and analyzed by t-test. Values with P values <0.05 were considered significantly different. Data were confirmed in multiple independent experiments.

Table 1

Gene primers and probe sets

Accession numberPrimer/probe sequence
Gene
Axin2NM_024355Forward 5′-GGAGCACCGGTCCAACAC-3′
Reverse 5′-TGGTTTGTGGGTCCTCTTCATA-3′
Probe 5′-CCCTGGCCCTCTTACCCTCTGGC-3′
Ctnnb1 (β-catenin)NM_053357Forward 5′-GCCACAGCTCCCCTGACA-3′
Reverse 5′-ATATGTTGCCACGCCTTCATT-3′
Probe 5′-AGTTGCTCCACTCCAG-3′
Ccl2 (Mcp1)NM_031530Forward 5′-CCAATGAGTCGGCTGGAGAA-3′
Reverse 5′-GAGCTTGGTGACAAATACTACAGCTT-3′
Probe 5′-AATCACCAGCAGCAGGTGTCCCAAA-3′
Ccl7 (Mcp3)NM_001007612Forward 5′-GCCGCGCTTCTGTGTGT-3′
Reverse 5′-TGGATGAATTGGTCCCATCTG-3′
Probe 5′-CTGCTCACAGCTGCTGCTTTCACCG-3′
Egr1NM_012551Forward 5′-CCATGA ACGCCCGTATGC-3′
Reverse 5′-CATGCAGATTCGACACTGGAA-3′
Probe 5′-TCGCCGCTTTTCTCGCTCGGAT-3′
GusbNM_017015Forward 5′-GGAGTCGGGCCCAACCT-3′
Reverse 5′-GCTCTGCTTCTTGGGTGATGT-3′
Probe 5′-VIC-ATGCCGGTCCCTTCCAGCTTCAA-3′
Igf1EaNM_001082478Forward 5′-GCTTGCTCACCTTTACCAGC-3′
Reverse 5′-AATGTACTTCCTTCTGGGTCT-3′
Detected by SYBR Green method
MgfNM_001082478Forward 5′-GCTTGCTCACCTTTACCAGC-3′
Reverse 5′-AAATGTACTTCCTTTCCTTCTC-3′
Detected by SYBR Green method
MnfNM_010911Forward 5′-TACCGCTTCGTCCAGAATGTG-3′
Reverse 5′-GCCTTCGCGGCGAACT-3′
Probe 5′-CCTCTGACCTTCAGCTGGCCGC-3′
Myh4NM_019325Forward 5′-GGAGCGGGCCGACATC-3′
Reverse 5′-TTCGCTTATGACTTTGGTGTGAA-3′
Probe 5′-CCGAGTCCCAGGTCAACAAGCTGC-3′
MyoDNM_176079Forward 5′-GCCCGGTCTGCACTCATG-3′
Reverse 5′-GAGTGTCATTTAAGCTTCATTTTTGG-3′
Probe 5′-ATGGTGCCCCTGGGTCCTTCATG-3′
MstnNM_019151Forward 5′-TTGGATGAGAATGGGCATGAT-3′
Reverse 5′-AAAAAGGGATTCAGCCCATCTT-3′
Probe 5′-TTGCTGTAACCTTCCCAGGACCAGGA-3′
Nr4a3 (Nor-1)NM_031628Forward 5′-TGAAGGAAGTTGTGCGTACAGATAG-3′
Reverse 5′-TCATCATACAGATCGGAGGAGATG-3′
Probe 5′-GTCTGCCTTCCAAACCAAAGAGCC-3′
PvalbNM_022499Forward 5′-GACACCACTCTTCTGGAAAATGC-3′
Reverse 5′-GCCTGGGTCCTCCCTACAG-3′
Probe 5′-AAACAATAAAGGCTGTACCCATCGGACACC-3′

Microarrays

Total RNA was collected from soleus (Trizol, Gibco) from rats treated with vehicle or DHT (3 mg/kg per day, n=6 per group) from two separate experiments and treated with DNAse I (as directed by the manufacturer, Genehunter Nashville, TN, USA), and purified using Qiagen RNeasy (Qiagen) columns. Complete details of the microarray protocol are available (van't Veer et al. 2002). Briefly, RNA samples were labeled in an in vitro transcription reaction with the fluorescent dyes Cy3 and Cy5. RNA from vehicle-treated samples labeled with Cy3 was then mixed with RNA from DHT samples labeled with Cy5 and competitively hybridized to 25 000 feature rat oligonucleotide arrays (Agilent Technologies, Palo Alto, CA, USA). Samples were also labeled in reverse and hybridized to a second microarray. Extensive quality control and normalization measures assure the overall validity of the experiment and have been previously described (van't Veer et al. 2002). The two fluorescent measurements (Cy5 (red) and Cy3 (green)) provide two intensities for comparison. Cy5 and Cy3 ratio intensities were converted to fold change. P values for differences between hybridization signals were calculated using an error ratio model (Weng et al. 2006). Genes were selected by applying a filter using absolute fold change >1.5 and P<0.05 for both ratio experiments, with genes regulated the same direction both times, which yielded 70 genes.

Results

Effects of castration on body composition

To compare testosterone, which can be converted to estrogen, with DHT, which cannot be converted to estrogen, we examined both hormones' effects on body composition and muscle strength in castrated male rats (orchidectomized, ORX). Age- and weight-matched male rats aged 10 weeks underwent ORX or sham operation and were left untreated for 11 weeks. DEXA revealed that ORX significantly increased fat mass, and decreased lean body mass (LBM) and bone mineral content (BMC), a measure of the extent of mineralized skeleton (Fig. 1A). These data confirm previous observations showing that androgen depletion in rats (Vanderschueren et al. 2000) and in humans (Wang et al. 2000) negatively affects body composition, and establishes these animals as suitable models for investigating androgenic modulation of body composition.

Figure 1

Download Figure

Figure 1

Effect of androgen depletion on lean body mass, fat mass, and bone mineral content (BMC). Graphed values are mean body composition values of the 21-week-old rats, 11 weeks after SHAM surgery or orchidectomy (ORX) as measured by DEXA (±s.e.m). (A) Mean pretreatment values for SHAM (n=9) versus all ORX animals (n=27). *Indicates P<0.05, t-test for SHAM versus all ORX rats. All ORX animals were randomized to three groups prior to treatment. Androgen induced changes in body composition. (B) Mean group DEXA measurements of change in lean mass, fat mass, and bone mineral content graphed as a function of time (error bar represents s.e.m). Data are different from baseline measurements taken immediately before treatment. ▵, ORX+testosterone (10 mg/kg per day); □, ORX+12 DHT (3 mg/kg per day); ▪, ORX+ VEH; •, SHAM+ VEH. *Indicates different from ORX 13 (P<0.05, one-way ANOVA, Fisher's PLSD only shown at final time point). N=9 per group. (C) Soleus weight and strength after androgen treatment. All values are mean±s.e.m., and asterisks indicate different from ORX (P<0.05, one-way ANOVA, Fisher's PLSD). Peak tetanic tension (Po) of the right soleus, soleus wet weight, and Po divided by soleus weight. Note Y-axis scale does not begin at the value 0 for visual clarity in (A) for BMC and (C).

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Effects of androgen treatment on body composition

We then determined whether treatment with androgens influences body composition in ORX rats. ORX rats were randomized into groups (n=9) based on LBM and were then treated daily for 8 weeks with 3 mg/kg per day DHT or 10 mg/kg per day testosterone. The doses were selected based on pilot pharmacokinetic studies in ORX rats showing them as the minimal doses required to maintain prostate weights to that measured in SHAM rats while producing the maximal increase in periosteal bone formation rate. The average Cmax and AUC for this dose were 1.8 ng/ml and 32 000 pg*h/ml respectively, which is near the physiologic range for testosterone in rats (Oliva et al. 2006) and is similar to that used previously (Hanada et al. 2003, Gao et al. 2005). DEXA scans were performed at 4, 6, and 8 weeks to determine the effects on body composition. After 6 weeks of treatment, sham-operated rats, testosterone, and DHT showed similar increases in LBM (Fig. 1B). By 8 weeks, both testosterone and DHT fully restored LBM accretion rate to that observed in intact animals. Likewise, both testosterone and DHT were equivalent in their ability to inhibit increases in fat mass, beginning 4 weeks after treatment (Fig. 1B). In contrast, only testosterone treatment increased BMC compared with control animals, and only after the full 8 weeks of treatment (Fig. 1B). Seminal vesicle and prostate weights were measured and show that both testosterone and DHT were fully effective in restoring these organs (data not shown). These data confirm that the doses of testosterone and DHT given were roughly equivalent, in that they equally supported the growth and maintenance of androgen-dependent organs.

Effects of androgens on contractile force of the soleus

To characterize the effects of androgens on muscle function, the contractile properties of the soleus muscle were measured at the end of the 8-week treatment. The soleus was chosen because it is a relatively homogenous type I fiber muscle in the hind limb widely used in regeneration studies. ORX rats exhibited a significant loss of muscle strength compared with sham rats as measured by peak tetanic tension (Fig. 1C). Treatment of ORX rats with either testosterone or DHT restored peak tetanic strength to sham levels. Soleus mass measurements from ORX rats were significantly lower than in sham rats, indicating that androgen deficiency for a total of 19 weeks produces significant atrophy. Androgen treatment did not produce statistically meaningful increases in soleus mass after 8 weeks of treatment (8.3% increase, P=0.34, testosterone; 8.8% increase, P=0.33, DHT; Fig. 1C). Both testosterone and DHT increased muscle quality as a function of strength to mass ratio (Fig. 1C). There were no significant differences in soleus muscle relaxation time half-life after withdrawal of electrical stimulus or the time to peak tetanic tension after initiation of electrical stimulation. Thus, both testosterone and DHT restore contractile strength to the soleus of ORX rats.

Histological examination of androgen-treated soleus

Several reports suggest that androgens modulate the number of myogenic precursors and affect fiber number and/or diameter (Bhasin et al. 1997, 2003, Sinha-Hikim et al. 2002). To examine the sequence of events that precede androgenic myoanabolism, ORX rats were treated daily with vehicle or 3 mg/kg per day DHT for 4, 7, 14, or 21 days. At each time point, soleus was collected and cross-sections were prepared. As expected from the timing of the experiment (before changes in lean mass are evident), no consistent effect was observed during this time frame on fiber area. When the total number of nuclei per area was examined, DHT transiently increased nuclear density at day 7 (Fig. 2). These visual data were confirmed by measuring DNA content, which was also transiently increased by DHT (data not shown). These data suggest that DHT increases myonuclear number in the soleus without affecting fiber diameter during the initial phases of anabolism.

Figure 2

Download Figure

Figure 2

Histology of soleus muscle 7 days after DHT treatment. ORX rats were treated for 4, 7, 14, or 21 days with vehicle or DHT (3 mg/kg per day) n=10 per group, and the soleus was collected and processed for histological examination. Representative cross-sectional fields of day 7 samples are shown above. Nuclear number per square micron (mean±s.e.m.) at each time point (* different from vehicle P<0.05, t-test) is shown below.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Gene expression changes in the soleus with androgen treatment

We next examined the possibility that the early phases of androgenic anabolism involve in the regulation of myogenic regulatory factors (MRFs) such as MyoD, myogenin, the satellite cell marker monocyte nuclear factor (MNF), the negative regulator of myogenesis myostatin (McPherron et al. 1997) or IGF1, or its splice variant MGF, which are critically involved in soleus muscle regeneration (DeVol et al. 1990, Adams et al. 1999, Semsarian et al. 1999, Hameed et al. 2003). The RNA abundance for each was measured in soleus total RNA during the first 3 weeks of androgen treatment by qRT-PCR. There were no significant changes in the RNA expression of the classic MRF genes (Fig. 3). In contrast, we observed increased expression of both IGF1Ea (classic circulating) and its splice variant MGF (Fig. 4). Thus, DHT does not regulate, within the first 21 days of treatment, the expression of these myogenic factors associated with muscle except for IGF1 and MGF (Cornelison & Wold 1997, Liu et al. 2003, Polesskaya et al. 2003, Seale et al. 2003).

Figure 3

Download Figure

Figure 3

Expression of classic myogenic regulators during DHT treatment time-course experiments. RNA was collected from the soleus of animals treated for the indicated time points with vehicle or DHT (3 mg/kg per day), and RNA levels of MyoD, myostatin, Mnf, and myogenin were determined by quantitative qRT-PCR. All values were first normalized within samples to glucuronidase, a housekeeping gene not affected by treatment or time in this experiment (not shown), and then to vehicle. For each time point, the DHT value normalized to the vehicle value, which was set to 1. Error bars represent s.e.m.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Figure 4

Download Figure

Figure 4

Expression of Igf1ea (classic circulating) and Igf1ec (mechano growth factor, Mgf). Aged ORX rats (n=4 per group) were treated with vehicle or DHT (3 mg/kg per day) for 1, 4, and 7 days in one experiment (left panel) and soleus RNA collected individually, and the rats were again treated with vehicle or DHT for 4, 7, and 21 days in the second experiment (right panel) and soleus RNA collected and pooled in three replicates of 3–4 (from n=10 rats per group) and processed for total RNA. RNA for Igf1ea and IGF1 splice variant Mgf was assayed by qRT-PCR for gene expression relative to vehicle. All values were first normalized within samples to glucuronidase gene expression. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. Error bars represent s.e.m. *P<0.005, t-test.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Microarray screen for androgen-responsive genes in the soleus

To characterize the immediate early response to DHT, a nonbiased microarray approach was employed. ORX rats aged 21 weeks were treated with a single injection of 3 mg/kg per day DHT or with vehicle (n=6 each). After 24 h, the soleus was collected and total RNA was prepared as above. A second experiment was then performed using the same design for confirmation. Genes were selected for further analysis if their RNA was altered by DHT treatment (absolute 1.5-fold change and P<0.05) in both experiments. Selected results were confirmed by qRT-PCR in independent experiments (see below). The genes, accession numbers, mean fold change results, P values, and functional annotations are listed in Tables 2 and 3.

Table 2

5α-Dihydrotestosterone (3 mg/kg) up-regulated genes, soleus, aged castrated rats after 24 h

Sequence descriptionAccession numberClass/pathway(s)Fold changeP value
Sequence name
Myh4Myosin heavy chain type-2b, fast twitchL24897ATPase/muscle contraction, fast twitch9.49<0.001
PvalbParvalbuminNM_022499Calcium-binding protein/fast twitch, neurogenesis4.63<0.001
Actn3Actinin, α-3 skeletal muscle specificNM_133244Muscle structure2.16<0.001
CtgfConnective tissue growth factorNM_022266Receptor ligand/ECM remodeling/inflammation2.140.016
Ggps1Geranylgeranyl diphosphate synthase 1NM_001007626Isoprenyl synthase/protein lipidation2.08<0.001
Ccl7Chemokine (C–C motif) ligand 7 (Mcp3)NM_001007612Receptor ligand/ECM remodeling, inflammation2.01<0.001
Mcpt4Mast cell protease 4NM_019321Protease/ECM remodeling, inflammation1.980.017
Ccl2Chemokine (C–C motif) ligand 2 (Mcp1)AF058786Receptor ligand/ECM remodeling, inflammation1.95<0.001
Slc6a18Solute carrier family 6, member 18NM_017163Neurotransmitter transporter/hypotonic stress response1.87<0.001
Pard3Par-3 partitioning defective 3NM_031235PKC-binding protein/axonogenesis1.860.005
Cyp2cCytochrome P-450 2cJ02657Male-specific CYP450 oxidase/testosterone metabolism1.840.031
Grm2Metabotropic glutamate receptor 2M92075Amino acid receptor/pain signaling1.84<0.001
Agtr1Angiotensin II receptor, type-1NM_031009Surface receptor/angiogenesis1.780.019
Slco1b2Solute carrier organic anion transporter member 1b2AF147740Surface receptor/steroid hormone and anion transport1.780.043
Ptger2Prostaglandin E receptor EP2 subtypeU48858Receptor ligand/chondrogenesis, inflammation1.75<0.001
Slc16a13Solute carrier family 16 member 13NM_001005530Surface receptor/pyruvate transport1.72<0.001
Serpine1Serine proteinase inhibitor, clade E, member 1NM_012620Serine protease inhibitor/inflammation1.700.003
Kim-1Kidney injury molecule-1AF035963Cell adhesion molecule/kidney regeneration1.690.018
Cntn3Contactin 3, fibronectin type-IIINM_019329Cell adhesion molecule/ECM remodeling/inflammation1.670.016
Cnr2Cannabinoid receptor 2NM_020543Surface receptor/ECM remodeling/inflammation1.660.001
Hspa1aHeat shock protein 70L16764Chaperone and nuclear receptor cofactor/signaling1.61<0.001
GzmbGranzyme B, serine proteaseM34097Serine protease/inflammation, circadian rhythm1.580.007
Mybpc2Myosin-binding protein-c, fast twitchXM_214945Myosin-binding protein/muscle structure, fast twitch1.580.011
Col17a1Procollagen, type XVII, α-1XM_219976Extracellular matrix molecule/structural1.580.002
Tpm1Tropomyosin 1 (α)NM_019131Calcium-binding ATPase/muscle contraction1.57<0.001
Tnfaip6TNF-α-induced proteinXM_001065494Unknown class/ECM remodeling/inflammation1.57<0.001
Pck1Phosphoenol pyruvate carboxykinase-1NM_198780Kinase/gluconeogenesis, glucose homeostasis1.560.048
Mbnl2Muscleblind-like 2XM_214253Unknown class/muscle develpoment1.560.020
Syn1Synapsin INM_019133Synapse cytoskeleton anchor/neurotransmitter release1.550.025
Spinlw1Eppin precursor, serine protease inhibitorXM_001071681Serine protease inhibitor/ECM remodeling/inflammation1.520.011
Arid1bAT-rich interactive domain 1bNM_172157Nuclear transcription factor/ischemic stress1.520.001
Igsf7Immunoglobulin superfamily, member 7CD300dXM_213514Surface receptor/ECM remodeling, inflammation1.51<0.001
Slc2a3Solute carrier family 2 member 3(GLUT3)NM_017102Glucose transporter/glucose homeostasis1.510.005
LyzLysozymeNM_012771Polysaccharide hydrolase/anti-inflammatory1.510.003
Table 3

5α-Dihydrotestosterone (3 mg/kg) down-regulated genes, soleus, aged castrated rats after 24 h

Sequence descriptionAccession numberClass/pathway(s)Fold changeP value
Sequence name
PrkceProtein kinase C epsilonNM_017171Protein kinase/inflammation, insulin secretion−1.500.003
Axin2Axin 2 (conductin), axilNM_024355Signal transduction/Wnt inhibitor, β-catenin degradation−1.50<0.001
GnaoGuanine nucleotide-binding protein, α activating activity polypeptide oNM_017327G-protein/muscuranic cholinergic signal−1.510.001
Itga6Integrin α-6AJ312934Cell adhesion receptor/cell contact and recognition−1.510.025
Pfkfb36-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3NM_057135Metabolic enzyme/glycolysis−1.52<0.001
Gstt2Glutathione S-transferase theta 2D10026GST/xenobiotic metabolism−1.52<0.001
HntNeurotriminNM_017354Cell adhesion molecule/axonogenesis−1.520.025
Sertad2Serta domain containing 2NM_001024903E2F transcription factor/unknown function−1.530.002
Grm4Glutamate receptor, metabotropic 4NM_022666Surface receptor/GABAergic inhibitor−1.56<0.001
Irx5Iroquois homeobox protein 5NM_001030044Transcription factor/mesoderm development−1.56<0.001
Ches1Checkpoint suppressor 1XM_234377Transcription factor/mesoderm development−1.570.049
Kng1Kininogen 1NM_012696Protease inhibitor/inflammation/senescence−1.590.024
Col10a1Collagen α-1 type XAJ131848Extracelluar matrix protein/structural−1.610.030
Phactr1Phosphatase and actin regulator 1NM_214457Phosphatase/actin cytoskeletal organization−1.610.003
Aox1Aldehyde oxidase 1NM_019363Oxidase/retinoic acid synthesis (ALS pathophysiology)−1.610.005
Arhgef5Rho guanine nucleotide exchange factor 5140898G-protein/oxidation, neurogenesis−1.620.003
LpdLiposidin, acyl-CoA synthaseAF208125Fatty acid synthase/fatty acid metabolism−1.620.031
Fbp1Fructose-1,6-biphosphatase 1NM_012558Phosphatase enzyme/gluconeogenesis−1.630.039
KalrnKalirin-12A RhoGEF kinase84009Kinase/cell adhesion, axonogenesis−1.64<0.001
JunJun oncogeneX17215Transcription factor/oncogene−1.640.005
Tnfsf13bTumor necrosis factor superfamily, member 13bAI059288Unknown class/ECM remodeling, inflammation−1.650.008
Cyp26b1Cytochrome P-450, family 26, subfam b, polypeptide 1NM_181087Oxidase/retinoic acid inactivation−1.70<0.001
Nr4a2Nurr1U01146Nuclear orphan receptor/neurogenesis−1.710.013
Egr2Early growth response-2, zinc finger protein krox-20AB032419Nuclear transcription factor/synaptic transmission−1.730.001
Nppcc-type natriuretic peptideD90219Receptor ligand/vasoactive neuropeptide−1.740.008
Mmp14Matrix metalloproteinase 14NM_031056Protease/ECM remodeling, inflammation−1.76<0.001
ArntlAryl hydrocarbon receptor nuclear translocator-likeAF015953Unknown class/circadian rhythm/protein catabolism−1.86<0.001
Cldn4Claudin 4304407Cell adhesion molecule/tight junctions−1.86<0.001
Sema6aSemaphorin 6aXM_341612Transmembrane protein/neurogenesis/axon guidance−1.910.001
Tacstd1Tumor-associated calcium signal transducer 1AJ001044Transmembrane protein/cell adhesion, calcium signaling−1.950.001
Trp63Transformation-related protein 63NM_019221p53-like nuclear transcription factor/differentiation−1.950.016
Nr4a3Nuclear receptor subfam 4, grp A, member 3 (NOR-1)NM_017352Nuclear orphan receptor/proliferation mesoderm−1.98<0.001
Egr1Early growth response 1NM_012551Nuclear transcription factor/proliferation/differentiation−2.23<0.001
Fabp1Fatty acid-binding protein 1M35991Fatty acid-binding protein/fatty acid metabolism−2.420.005
Slc15a1Solute carrier family 15 member 1D50664Surface receptor/peptide transporter−2.450.002
Cldn3Claudin3NM_031700Cell adhesion molecule/tight junctions−2.580.036

Identification of early androgen-responsive genes in muscle

Seventy genes met the above criteria. These genes were categorized by their putative functions as those affecting tissue remodeling, inflammation/immune modulation, glucose metabolism, neurogenesis, and transcriptional and signaling cascades. The regulated genes include fast twitch MHC subtype-4/fiber type-2b (Myh4) and parvalbumin (Pvalb); both components of type-2 fast twitch muscle that is selectively lost in hypogonadal men and replaced during androgen treatment (Table 2; Anderson et al. 1988, Sinha-Hikim et al. 2002, Racay et al. 2006). Multiple genes implicated in tissue remodeling (Holmbeck et al. 1999, Ichimura et al. 2004, Koh et al. 2005) were identified and tabulated separately (Table 4), including plasminogen activator inhibitor-1 (Serpine1), matrix metalloproteinase-14 (Mmp14), connective tissue growth factor (Ctgf), kidney injury molecule-1 (Kim1), mast cell protease-4 (Mcpt4), and heat shock protein 70 (Hspa1a). Several of these genes are proposed to play important roles in muscle homeostasis, for example the IGF1-binding protein, CTGF, is involved in muscle, bone, and liver cell regeneration, and inhibits the transforming growth factor (TGF)/bone morphogenetic protein (BMP) pathway that limits muscle growth (Ohnishi et al. 1998, Pummila et al. 2007, Hayata et al. 2008, Smerdel-Ramoya et al. 2008). In terms of inflammation, chemokine C–C ligand-7, Ccl7 and chemokine C–C ligand-2, Ccl2 were up-regulated by microarray and confirmed by qRT-PCR (Fig. 5). These RNAs code for cytokine molecules involved in local inflammation signaling and monocyte recruitment via chemotaxis (Van Damme et al. 1993, Wuyts et al. 1994).

Table 4

Microarray identified early genes involved in muscle or tissue regeneration

Common nameUp/down regulationRT-QPCR confirmedCitation
Sequence name
Myh4Myosin heavy chain type-2bUpYesSinha-Hikim et al. (2002)
PvalbParvalbuminUpYesAnderson et al. (1988) and Racay et al. (2006)
CtgfConnective tissue growth factorUpNDOhnishi et al. (1998) and Hayata et al. (2008)
Ccl2Monocyte chemoattractant protein-1UpYesShireman et al. (2007)
Ccl7Monocyte chemoattractant protein-3UpYesSchenk et al. (2007)
Serpine1Plasminogen activator inhibitor-1UpNDKoh et al. (2005)
Kim-1Kidney injury molecule-1UpNDIchimura et al. (2004)
Hspa1aHeat shock protein 70UpNDMiyabara et al. (2006)
Mcpt4Mast cell protease- 4UpNDZweifel et al. (2005)
Mmp14Matrix metalloprotease-14DownNDHolmbeck et al. (1999)
Figure 5

Download Figure

Figure 5

Confirmation of microarray expression of genes involved in muscle regeneration. Aged ORX rats (n=4 per group) were treated for the times indicated with vehicle or DHT (3 mg/kg per day). The soleus was collected from individual rats (n=4 per group) and processed for total RNA. RNA for myosin heavy chain type-2b (Myh4), parvalbumin (Pvalb), monocyte chemotactic protein-1 and -3 (Ccl2 and Ccl7) were assayed by qRT-PCR for gene expression relative to vehicle. All values were first normalized within samples to glucuronidase gene expression. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005 t-test. Error bars represent s.e.m.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Another subset of genes we identified suggests a role for specific transcription and signaling cascades. This set of genes includes BMP receptor type-II (Bmpr2), which was up-regulated in only one of the 24-h microarray screens; however, it was up-regulated by DHT in other experiments and, due to its important function, it was manually selected for confirmation. BMP receptors transduce signals from the BMP and TGF-β superfamily, which includes several inhibitors of myogenesis such as BMP2, TGF-β2, and myostatin (Shi & Massague 2003). Other genes with less clear function in muscle were regulated, including the nuclear orphan receptor (Nr4a3, Nor-1) and the transcription factor early growth response-1 (Egr1, EGR1), both of which were repressed at 24 h (Table 3). Axin2 (Axin2), a constituent of the Wnt-regulatory complex involved in the degradation of β-catenin, was down-regulated (Table 3). Though typically regulated largely at the post-translational level and not detected by the microarray screen, this observation prompted us to measure β-catenin RNA, which showed modest upregulation at 1, 4, and 7 days by qRT-PCR (Fig. 6). Therefore, during the first 24 h, DHT regulates the expression of a specific set of genes that might be downstream mediators of AR in muscle tissue.

Figure 6

Download Figure

Figure 6

DHT upregulates expression of Wnt pathway regulator β-catenin through transcription and repression of phosphorylation. Rats were treated with vehicle or DHT (3 mg/kg per day) for the times indicated. The soleus was collected individually (n=4 per group, top panels) or pooled (n=6 per group, bottom panels), and processed for total RNA and protein. (A) Gene expression levels of β-catenin were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.05, t-test. Error bars represent s.e.m. (B) Immunoblot of total β-catenin and phospho-β-catenin (top panel). Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values. *P<0.05, t-test.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Based on the microarray results, the expression of Myh4, Parvl, Ccl2, and Ccl7 were confirmed by qRT-PCR (Fig. 5). Other potentially myogenic transcription factors were confirmed by qRT-PCR to be regulated by DHT including Nr4a3, Egr1, and Bmpr2. These genes were studied in separate time-course experiments ranging from 1 to 21 days after DHT stimulation in soleus muscle (Fig. 7). The Bmpr2 gene is induced twofold after 1–4 days and falls to basal levels through 21 days (Fig. 7). Nr4a3 exhibits a biphasic expression pattern, with levels suppressed after 1–4 days and induced twofold afterwards (Fig. 7). Egr1 expression was repressed throughout both experiments (Fig. 7). Thus, the microarray experiment detected real and reproducible changes in gene expression, as all seven genes were in fact androgen responsive.

Figure 7

Download Figure

Figure 7

Expression of regulated genes in the soleus of ORX rats treated with vehicle or DHT (3 mg/kg per day) in two time-course experiments for the times indicated. The soleus was collected individually for the short term (n=4 per group, left panels) and in three pooled replicates of 2 (from n=6 rats per group, right panels) and processed for total RNA. Gene expression levels of nuclear orphan receptor-1 (Nr4a3), bmp type II receptor (Bmpr2), and early growth response-1 (Egr1) were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005, t-test. Error bars represent s.e.m.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Androgenic repression of Axin and Axin2 in the soleus

The microarray observation of decreased Axin2 expression was confirmed by qRT-PCR (Fig. 8A). Axin2 acts as a negative feedback regulator of the Wnt-signaling pathway in the colon (Lustig et al. 2002). Axin1 and Axin2 have 45% homology and are functionally redundant in mice (Chia & Costantini 2005). Thus, we examined protein levels of AXIN and AXIN2 in soleus extracts and found that DHT repressed expression (Fig. 8A and B). As both axin types inhibit β-catenin, these findings further suggested to us that β-catenin is functionally induced.

Figure 8

Download Figure

Figure 8

DHT downregulates expression of Wnt pathway negative regulators, Axin2 and Axin. (A) Rats were treated with vehicle or DHT (3 mg/kg per day) for the times indicated. The soleus was collected individually (n=4 per group top panels) and processed for total RNA and protein. Gene expression levels of Axin2 were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005, t-test. Error bars represent s.e.m. (B) Immunoblot of total AXIN and AXIN2. Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values. (C) Immunoblot for total GSK3β and serine-9-phophorylated GSK3β. DHT upregulates expression of serine-9-phosphorylated GSK3β. Rats were treated with vehicle or DHT (3 mg/kg per day) for 7 days. The soleus was collected individually (n=4) and processed for protein extraction. Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Finally, it has been reported that growing muscle features inactivation via serine-9 phosphorylation of the β-catenin inhibitor GSK3β (Chin et al. 2005). If true with DHT treatment, such an observation would also suggest activation of β-catenin signaling. Thus, we measured serine-9 phosphorylation of GSK3β after DHT treatment and found increased serine-9 phosphorylated (and thus inactivated) GSK3β after 7 days (Fig. 8C).

Androgenic induction of β-catenin in the soleus

The above data suggested that β-catenin protein, which is regulated post-translationally by axin expression, IGF1 and GSK3β phosphorylation, is induced by DHT in muscle. Western blot data confirmed the accumulation of β-catenin protein, with levels ∼20-fold greater than those in controls during the first 21 days of response to DHT (Fig. 6B). We also observed decreased levels of serine 33/37/41 phosphorylated β-catenin, which triggers β-catenin degradation and is consistent with downregulation of axin-dependent GSK3β activity and upregulation of β-catenin levels. To determine the subcellular localization of β-catenin, sections of soleus muscle from rats treated with DHT or with vehicle for 7 days were examined by immunohistochemistry. Antibodies against β-catenin labeled nuclei with a significantly higher proportion of β-catenin-positive nuclei evident in the DHT-treated specimens (Fig. 9). Though we did not definitively identify the cell types expressing β-catenin, we were careful to exclude nuclei that were not intimately associated with myofibers such as cells in or around fatty deposits or blood vessels. The specificity of the staining was confirmed by omitting the primary antibody, which resulted in loss of all signals and parallel staining of intestinal sections, which showed typical β-catenin patterns (data not shown). These data demonstrate that β-catenin protein is rapidly induced by DHT in the soleus and accumulates in nuclei during the early stages of anabolism.

Figure 9

Download Figure

Figure 9

Expression of β-catenin in muscle nuclei with DHT treatment. ORX rats were treated for 4, 7, 14, or 21 days with vehicle or DHT (3 mg/kg per day), and then soleus was collected and processed for histological examination. (A) Representative cross-sectional fields of day 7 samples are shown above. Muscle nuclei labeled with β-catenin-specific antibody as visualized by immunohistochemistry using nickel DAB. (B) The number of β-catenin-positive nuclei was counted per mm2 of cross-sectional area. N=10 rats/sections per group. *P<0.05, t-test. (C) The total number of nuclei was counted (without nickel) following hemotoxylin counterstaining for each section and the proportion calculated and graphed. *P<0.05, t-test.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

IGF1 and ctMGF induce β-catenin nuclear translocation in C2C12 myoblasts

Our findings of androgenic induction of Igf1 and Mgf prompted us to explore the potential direct link of these IGF1 variants to β-catenin in cultured C2C12 myoblasts since IGF1 induces β-catenin nuclear translocation in other cells (Satyamoorthy et al. 2001, Chen et al. 2005a). Both IGF1 and ctMGF rapidly increased total nuclear β-catenin protein expression in C2C12 myoblasts (Fig. 10A and C) and induced β-catenin-mediated TCF/LEF signaling using the TOPFLASH β-catenin reporter assay (Fig. 10B). Finally, nuclear localization of β-catenin in C2C12 cells after IGF1 and ctMGF treatment was confirmed by immunocytochemistry (Fig. 10C). Thus, in myoblast-like cells, IGF1 and MGF are sufficient to upregulate β-catenin protein and induce nuclear localization and transcriptional activity.

Figure 10

Download Figure

Figure 10

IGF1 and its splice variant mechano growth factor C-terminal peptide (ctMGF) promote nuclear translocation of myogenic Wnt regulator β-catenin in C2C12 myoblasts. Cells were treated with vehicle, IGF1 (10 and 30 ng/ml), and ctMGF (30 and 60 ng/ml). (A) Immunoblot of total nuclear β-catenin after 25 min of treatment. Lamin is load control. (B) The β-catenin TOPFLASH reporter plasmid containing TCF/LEF promoter linked to the firefly luciferase gene was cotransfected into C2C12 myoblasts with a constitutively active Renilla luciferase plasmid, and the cells were plated in 96-well plates and stimulated with IGF1 and ctMGF at the above concentrations for 16 h. β-Catenin-responsive firefly luciferase reporter activity was first normalized to Renilla luciferase activity, and the IGF1 and ctMGF values were normalized to the vehicle value, which was set to 1. *P<0.01, one-way ANOVA, Fisher's PLSD. (C) Near confluent C2C12 myoblasts in chamber slides were washed and placed in serum-free media with vehicle, IGF1 (30 ng/ml), or ctMGF (60 ng/ml) for 25 min just prior to fixation. The cells were prepared for immunocytochemistry, stained with anti-β-catenin antibodies, and counterstained with nuclear DAPI stain.

Citation: Journal of Molecular Endocrinology 44, 1; 10.1677/JME-09-0048

Discussion

Androgens are important regulators of body composition during postnatal development and aging in both genders. In clinical studies, testosterone maintains, restores and/or increases LBM and BMC, and decreases fat mass. However, testosterone can be converted to estradiol (E2); thus, clinical studies involving testosterone remain equivocal for understanding the molecular basis of androgenic myoanabolism. We explored this problem by establishing a suitable animal model for clarifying the role of AR on body composition and identifying genomic and molecular responses in muscle tissue downstream of androgen stimulation.

ORX-induced androgen deficiency results in decreased LBM, greater fat mass, and decreased BMC (Fig. 1A). Examination of the soleus confirmed that previous data showing muscle weight and strength are decreased by androgen loss (Boissonneault 2001, Brown et al. 2001). Both testosterone and DHT restored muscle contractile strength and induced LBM accumulation (Fig. 1B and C). Moreover, both testosterone and DHT suppressed the accumulation of fat mass similar to that of sham-operated rats. However, only testosterone increased whole-body BMC above that of vehicle-treated ORX rats, suggesting an important role for aromatization to E2 for maintenance of bone mass. This observation is in agreement with other studies suggesting that DHT is less effective than testosterone in preventing cancellous bone loss (Vanderschueren et al. 1992) and that aromatization to estrogens is important for skeletal homeostasis (Vanderschueren et al. 1997). Note that whole-body BMC measurements are not likely to detect small but important changes in the extent of mineralization in the periosteum, as it constitutes a small fraction of total bone and is stimulated by androgens in rats (Hanada et al. 2003).

The role of AR in muscle growth at the cellular and molecular level was then examined in the soleus, a common model for regeneration studies. Interestingly, soleus contractile strength was restored in the absence of large gains in soleus mass after 8 weeks of treatment (Fig. 1C), suggesting a positive effect on muscle efficiency. Seventeen-week studies in OVX rats show that DHT induces gains in soleus weight (data not shown); thus, the experiments presented here were probably not long enough to detect significant gains in muscle weight. To characterize the sequence of events preceding, and therefore likely involved in initiating, muscle anabolism, we focused on the first 21 days following DHT treatment. We find that DHT might promote the proliferation or recruitment of cells early during the regeneration process, as an increase in nuclear density was detected only in 7 days post-treatment (Fig. 2). We then examined whether key myogenic factors are regulated, but over this time we found no differences in the expression of MyoD or myogenin, the satellite cell marker MNF (Garry et al. 2000), or myostatin (Fig. 3). This observation is in agreement with recent findings in ARKO mice where classic MRF genes were not regulated, and the overall gene profile suggested that androgens delay differentiation allowing clonal expansion of myoblasts (MacLean et al. 2008). These data do not exclude the possibility that these genes are involved in androgenic anabolism, as they could be regulated in a small proportion of cells, by a post-transcriptional mechanism, or at later time points. It will be important to determine whether and when the classical MRF transcriptional cascade and satellite cell activation occur.

The microarray analysis revealed that a relatively small number of genes respond to DHT in the soleus after 24 h. Using our statistical criteria, 70 genes responded to DHT (Tables 2 and 3). In a parallel experiment in which prostate was studied, approximately ten times as many genes were DHT-responsive at 24 h, even though more stringent statistical criteria were applied (Nantermet et al. 2004). Thus, the soleus is relatively unresponsive, possibly reflecting the lower levels of AR in muscle compared with prostate (Krieg 1976, Michel & Baulieu 1980).

Examination of the function of these genes provides insight into the initial response of the soleus to DHT. MHC type-2b and parvalbumin were induced, both of them are type II muscle-specific proteins, lending validity to the model since androgen depletion results in the loss of type II muscle and repletion increases (Sinha-Hikim et al. 2002). When taken together, these gene expression data suggest an initiation of events similar to muscle regeneration, which initially involves cytokine-mediated inflammation, extracellular matrix, vascular remodeling, and, at later stages, recruitment of myogenic progenitor cells and satellite cells followed by differentiation into new muscle myofibers (Seale & Rudnicki 2000, Goetsch et al. 2003, Charge & Rudnicki 2004). In terms of cytokine-mediated inflammation, two cytokine-encoding RNAs, Ccl2 and Ccl7, were induced. Their proteins are potent chemotactic agents and thus could recruit cells to the regenerating muscle, as has been proposed for CCL2 (Warren et al. 2004) and CCL7 after myocardial infarction (Schenk et al. 2007). Interestingly, CCL2 knockout mice exhibit impaired regeneration and abnormal fat accumulation after muscle injury (Shireman et al. 2007, Contreras-Shannon et al. 2007). Moreover, side population and CD45+ cells induced by Wnt/β-catenin signaling are important for regeneration of the soleus in response to toxin-induced muscle damage (Asakura et al. 2002, Polesskaya et al. 2003) and represent specific subsets of immune cells distinct from satellite cells. We also demonstrate the androgenic induction of Mgf, an IGF1 splice variant, up-regulated with muscle damage and exercise (Hameed et al. 2003, Rigamonti et al. 2009). There is also evidence for matrix remodeling, as Mmp14 rapidly responded to DHT. Since muscle matrix undergoes significant changes during embryogenesis (Visse & Nagase 2003) and regeneration (Charge & Rudnicki 2004), this gene could function in that process. It is interesting to note the induction of genes that are also induced during exercise, which includes IGF1, MGF, β-catenin, HSPA1A, SERPINE1, CCL2, and CCL7 (Tables 2 and 4), since the rats were single-housed and not allowed vigorous exercise.

The gene induction for a receptor for BMP-signaling molecules, Bmpr2, is interesting, given the role of this family in repressing myogenesis (e.g. myostatin (Thomas et al. 2000) and TGF-β (Massague et al. 1986)). However, the type II BMP receptor requires a type I receptor for classical Smad-mediated signal transduction (Foletta et al. 2003); thus, this observation does not necessarily indicate enhanced myoinhibitory BMP/TGF-β signaling. In fact, the Drosophila homolog of Bmpr2, wishful thinking, is required for proper formation of the neuromuscular junction (Marques et al. 2002), potentially suggesting a neuromodulatory function of this gene.

The microarray study also identified genes whose function in muscle tissue has not been studied. These include the transcription factors EGR1, which functions in inflammation, proliferation, differentiation, and apoptosis, and NR4A3, an orphan nuclear receptor that acts as a transcriptional activator in a ligand-independent manner (Thiel & Cibelli 2002, Wansa et al. 2003). qRT-PCR reveals that both Egr1 and Nr4a3 RNAs are initially repressed, and while Egr1 exhibited sustained repression, Nr4a3 was induced during the time nuclear content was elevated (see Figs 2 and 7). Two Nr4a3 gene-disrupted mouse strains have been reported, one of which reports embryonic lethality due to accumulation of mesodermal cells in the primitive streak (DeYoung et al. 2003). NR4A3 also regulates in oxidative metabolism in muscle (Pearen et al. 2008), suggesting that androgens could modulate metabolism through control of NR4A3 expression.

The most notable finding from the microarray was inhibition of the Wnt-signaling molecule Axin2, an inhibitor of β-catenin. This phenomenon has been reported in the testosterone-treated ORX mouse prostate (Wang et al. 2008). Axin mRNA and protein were repressed through 7 days (Fig. 8) corresponding to increased GSK3β serine-9 phosphorylation, and decreased phosphorylation and nuclear accumulation of β-catenin protein (Figs 9 and 10). β-Catenin is both essential and sufficient for P19 cell myogenesis and inhibits adipogenesis in vitro (Ross et al. 2000, Petropoulos & Skerjanc 2002). Furthermore, β-catenin signaling is required for regeneration of the soleus (Polesskaya et al. 2003). Upregulation of both total muscle and myonuclear β-catenin occurs during exercise, load-induced muscle hypertrophy, and myocardial recovery after heart failure in vivo (Sakamoto et al. 2004, Armstrong & Esser 2005, Braz et al. 2009), and the Wnt/β-catenin pathway promotes insulin/IGF1-mediated reserve cell activation and myotube hypertrophy in vitro (Rochat et al. 2004). The anti-atrophy effects of IGF1 in glucocorticoid-treated rats are via the AKT/GSK3β/β-catenin pathway (Schakman et al. 2008). Moreover, β-catenin expression is necessary for physiological growth of muscle in adult animals (Armstrong et al. 2006). Our data apparently oppose the current view that Axin2 expression is a direct indicator of β-catenin signaling. However, the observation of DHT-mediated IGF induction, AXIN protein downregulation, and serine-9 phosphorylation of GSK3β (Fig. 8C) indicates that multiple mechanisms are in effect, which could result in a special case where Axin2 downregulation may not signify reduced β-catenin signaling. Interestingly, β-catenin and AR physically and functionally interact in prostate and neuronal cells (Pawlowski et al. 2002, Yang et al. 2002), suggesting crosstalk between androgen and Wnt signaling. To explore how androgens affect β-catenin signaling, we stimulated mouse myoblasts with IGF1 or ctMGF, and observed that both promote rapid upregulation and nuclear translocation of β-catenin (Fig. 10). Thus, we suggest that by inducing IGF1 and its muscle-specific splice variants, AR promotes the growth of muscle by activating the β-catenin pathway. Downregulation of β-catenin attenuates myocardial remodeling by promoting precursor cell differentiation, while upregulation induces precursor cell proliferation during muscle regeneration (Otto et al. 2008, Perez-Ruiz et al. 2008, Zelarayan et al. 2008). Thus, these data are consistent with the current understanding of β-catenin in muscle remodeling. Though the current data do not prove this hypothesis, the role of β-catenin in promoting myogenesis and muscle regeneration and repressing adipogenesis provides a unifying concept for androgen-mediated changes in body composition.

Declaration of interest

There are no conflicts of interest that would compromise the impartiality of this research.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Acknowledgements

We thank Dr Mary Beth Brown, University of Missouri, for advice regarding muscle force measurements and Dr Steve Alves, Merck, for advice regarding histochemistry. We also thank Jill Williams, Merck, for invaluable help with generation of figures and the late Dr Shun-ichi Harada for his advice and support.

References

  • AdamsGRHaddadFBaldwinKM1999Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. Journal of Applied Physiology8717051712.

  • AndersonJEBresslerBHOvalleWK1988Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. Journal of Muscle Research and Cell Motility9499515.

  • AntonioJWilsonJDGeorgeFW1999Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles. Journal of Applied Physiology8720162019.

  • ArmstrongDDEsserKA2005Wnt/β-catenin signaling activates growth-control genes during overload induced skeletal muscle hypertrophy. American Journal of Physiology. Cell Physiology289C853C859.

  • ArmstrongDDWongVLEsserKA2006Expression of β-catenin is necessary for physiological growth of adult skeletal muscle. American Journal of Physiology. Cell Physiology291C185C188.

  • AsakuraASealePGirgis-GabardoARudnickiMA2002Myogenic specification of side population cells in skeletal muscle. Journal of Cell Biology159123134.

  • BanuSKGovindarajuluPAruldhasMM2002Testosterone and estradiol differentially regulate TSH-induced thyrocyte proliferation in immature and adult rats. Steroids67573579.

  • BardinCW1996The anabolic action of testosterone. New England Journal of Medicine3355253.

  • BhasinSStorerTWBermanNYarasheskiKEClevengerBPhillipsJLeeWPBunnellTJCasaburiR1997Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. Journal of Clinical Endocrinology and Metabolism82407413.

  • BhasinSStorerTWJavanbakhtMBermanNYarasheskiKEPhillipsJDikeMSinha-HikimIShenRHaysRD2000Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testosterone levels. Journal of the American Medical Association283763770.

  • BhasinSTaylorWESinghRArtazaJSinha-HikimIJasujaRChoiHGonzalez-CadavidNF2003The mechanisms of androgen effects on body composition: mesenchymal pluripotent cell as the target of androgen action. Journals of Gerontology. Series A Biological Sciences and Medical Sciences58M1103M1110.

  • BhasinSCalofOMStorerTWLeeMLMazerNAJasujaRMontoriVMGaoWDaltonJT2006Drug insight: testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging. Nature Clinical Practice. Endocrinology and Metabolism2146159.

  • BilezikianJPMorishimaABellJGrumbachMM1998Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. New England Journal of Medicine339599603.

  • BoissonneaultG2001Evidence of apoptosis in the castration-induced atrophy of the rat levator ani muscle. Endocrine Research27317328.

  • BrazJCGillRMCorblyAKJonesBDJinNVlahosCJWuQShenW2009Selective activation of PI3Kα/Akt/GSK-3β signalling and cardiac compensatory hypertrophy during recovery from heart failure. European Journal of Heart Failure11739748.

  • BrownMTaylorJ2005Prehabilitation and rehabilitation for attenuating hindlimb unweighting effects on skeletal muscle and gait in adult and old rats. Archives of Physical Medicine and Rehabilitation8622612269.

  • BrownMFisherJSHasserEM2001Gonadectomy and reduced physical activity: effects on skeletal muscle. Archives of Physical Medicine and Rehabilitation829397.

  • ChangCSaltzmanAYehSYoungWKellerELeeHJWangCMizokamiA1995Androgen receptor: an overview. Critical Reviews in Eukaryotic Gene Expression597125.

  • ChargeSBRudnickiMA2004Cellular and molecular regulation of muscle regeneration. Physiological Reviews84209238.

  • ChenJWuASunHDrakasRGarofaloCCascioSSurmaczEBasergaR2005aFunctional significance of type 1 insulin-like growth factor-mediated nuclear translocation of the insulin receptor substrate-1 and β-catenin. Journal of Biological Chemistry2802991229920.

  • ChenYZajacJDMacLeanHE2005bAndrogen regulation of satellite cell function. Journal of Endocrinology1862131.

  • ChiaIVCostantiniF2005Mouse axin and axin2/conductin proteins are functionally equivalent in vivo. Molecular and Cellular Biology2543714376.

  • ChinPCMajdzadehND'MelloSR2005Inhibition of GSK3β is a common event in neuroprotection by different survival factors. Brain Research. Molecular Brain Research137193201.

  • CleversH2006Wnt/β-catenin signaling in development and disease. Cell127469480.

  • CompstonJE2001Sex steroids and bone. Physiological Reviews81419447.

  • Contreras-ShannonVOchoaOReyes-ReynaSMSunDMichalekJEKuzielWAMcManusLMShiremanPK2007Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2−/− mice following ischemic injury. American Journal of Physiology. Cell Physiology292C953C967.

  • CornelisonDDWoldBJ1997Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Developmental Biology191270283.

  • Van DammeJProostPLenaertsJPConingsROpdenakkerGBilliauA1993Monocyte chemotactic proteins related to human MCP-1. Advances in Experimental Medicine and Biology351111118.

  • DeVolDLRotweinPSadowJLNovakofskiJBechtelPJ1990Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. American Journal of Physiology259E89E95.

  • DeYoungRABakerJCCadoDWinotoA2003The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis. Journal of Biological Chemistry2784710447109.

  • DoumitMECookDRMerkelRA1996Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology13713851394.

  • FerrandoAASheffield-MooreMYeckelCWGilkisonCJiangJAchacosaALiebermanSATiptonKWolfeRRUrbanRJ2002Testosterone administration to older men improves muscle function: molecular and physiological mechanisms. American Journal of Physiology. Endocrinology and Metabolism282E601E607.

  • FolettaVCLimMASoosairajahJKellyAPStanleyEGShannonMHeWDasSMassagueJBernardO2003Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. Journal of Cell Biology16210891098.

  • GaoWReiserPJCossCCPhelpsMAKearbeyJDMillerDDDaltonJT2005Selective androgen receptor modulator treatment improves muscle strength and body composition and prevents bone loss in orchidectomized rats. Endocrinology14648874897.

  • GarryDJMeesonAEltermanJZhaoYYangPBassel-DubyRWilliamsRS2000Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF. PNAS9754165421.

  • GoetschSCHawkeTJGallardoTDRichardsonJAGarryDJ2003Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration. Physiological Genomics14261271.

  • GoldspinkGYangSY2004The splicing of the IGF-1 gene to yield different muscle growth factors. Advances in Genetics522349.

  • HameedMOrrellRWCobboldMGoldspinkGHarridgeSD2003Expression of IGF-1 splice variants in young and old human skeletal muscle after high resistance exercise. Journal of Physiology547247254.

  • HanadaKFuruyaKYamamotoNNejishimaHIchikawaKNakamuraTMiyakawaMAmanoSSumitaYOguroN2003Bone anabolic effects of S-40503, a novel nonsteroidal selective androgen receptor modulator (SARM), in rat models of osteoporosis. Biological & Pharmaceutical Bulletin2615631569.

  • HayataNFujioYYamamotoYIwakuraTObanaMTakaiMMohriTNonenSMaedaMAzumaJ2008Connective tissue growth factor induces cardiac hypertrophy through Akt signaling. Biochemical and Biophysical Research Communications370274278.

  • HeinleinCAChangC2002Androgen receptor (AR) coregulators: an overview. Endocrine Reviews23175200.

  • HicksonRCCzerwinskiSMFaldutoMTYoungAP1990Glucocorticoid antagonism by exercise and androgenic-anabolic steroids. Medicine and Science in Sports and Exercise22331340.

  • HillMGoldspinkG2003Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. Journal of Physiology549409418.

  • HolmbeckKBiancoPCaterinaJYamadaSKromerMKuznetsovSAMankaniMRobeyPGPooleARPidouxI1999MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell998192.

  • IchimuraTHungCCYangSAStevensJLBonventreJV2004Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. American Journal of Physiology. Renal Physiology286F552F563.

  • KatznelsonLFinkelsteinJSSchoenfeldDARosenthalDIAndersonEJKlibanskiA1996Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. Journal of Clinical Endocrinology and Metabolism8143584365.

  • KatznelsonLRosenthalDIRosolMSAndersonEJHaydenDLSchoenfeldDAKlibanskiA1998Using quantitative CT to assess adipose distribution in adult men with acquired hypogonadism. American Journal of Roentgenology170423427.

  • KohTJBryerSCPucciAMSissonTH2005Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration. American Journal of Physiology. Cell Physiology289C217C223.

  • KriegM1976Characterization of the androgen receptor in the skeletal muscle of the rat. Steroids28261274.

  • LambertsSWvan den BeldAWvan der LelyAJ1997The endocrinology of aging. Science278419424.

  • LeeDK2002Androgen receptor enhances myogenin expression and accelerates differentiation. Biochemical and Biophysical Research Communications294408413.

  • LinkKBlizzardRMEvansWSKaiserDLParkerMWRogolAD1986The effect of androgens on the pulsatile release and the twenty-four-hour mean concentration of growth hormone in peripubertal males. Journal of Clinical Endocrinology and Metabolism62159164.

  • LiuWThomasSGAsaSLGonzalez-CadavidNBhasinSEzzatS2003Myostatin is a skeletal muscle target of growth hormone anabolic action. Journal of Clinical Endocrinology and Metabolism8854905496.

  • LumbrosoSSandillonFGeorgetVLobaccaroJMBrinkmannAOPrivatASultanC1996Immunohistochemical localization and immunoblotting of androgen receptor in spinal neurons of male and female rats. European Journal of Endocrinology134626632.

  • LustigBJerchowBSachsMWeilerSPietschTKarstenUvan de WeteringMCleversHSchlagPMBirchmeierW2002Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Molecular and Cellular Biology2211841193.

  • MacLeanHEChiuWSNotiniAJAxellAMDaveyRAMcManusJFMaCPlantDRLynchGSZajacJD2008Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB Journal2226762689.

  • MarquesGBaoHHaerryTEShimellMJDuchekPZhangBO'ConnorMB2002The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function. Neuron33529543.

  • MassagueJCheifetzSEndoTNadal-GinardB1986Type β transforming growth factor is an inhibitor of myogenic differentiation. PNAS8382068210.

  • McPherronACLawlerAMLeeSJ1997Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature3878390.

  • MichelGBaulieuEE1980Androgen receptor in rat skeletal muscle: characterization and physiological variations. Endocrinology10720882098.

  • MiyabaraEHMartinJLGriffinTMMoriscotASMestrilR2006Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning. American Journal of Physiology Cell Physiology290C1128C1138.

  • MonksDAO'BryantELJordanCL2004Androgen receptor immunoreactivity in skeletal muscle: enrichment at the neuromuscular junction. Journal of Comparative Neurology4735972.

  • MusaroAMcCullaghKPaulAHoughtonLDobrowolnyGMolinaroMBartonERSweeneyHLRosenthalN2001Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nature Genetics27195200.

  • NantermetPVXuJYuYHodorPHolderDAdamskiSGentileMAKimmelDBHaradaSGerholdD2004Identification of genetic pathways activated by the androgen receptor during the induction of proliferation in the ventral prostate gland. Journal of Biological Chemistry27913101322.

  • OhnishiHOkaTKusachiSNakanishiTTakedaKNakahamaMDoiMMurakamiTNinomiyaYTakigawaM1998Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats. Journal of Molecular and Cellular Cardiology3024112422.

  • OlivaSUMessiasAGSilvaDAPereiraOCGerardinDCKempinasWG2006Impairment of adult male reproductive function in rats exposed to ethanol since puberty. Reproductive Toxicology22599605.

  • OttoASchmidtCLukeGAllenSValasekPMuntoniFLawrence-WattDPatelK2008Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. Journal of Cell Science12129392950.

  • PawlowskiJEErtelJRAllenMPXuMButlerCWilsonEMWiermanME2002Liganded androgen receptor interaction with β-catenin: nuclear co-localization and modulation of transcriptional activity in neuronal cells. Journal of Biological Chemistry2772070220710.

  • PearenMAMyersSARaichurSRyallJGLynchGSMuscatGE2008The orphan nuclear receptor, NOR-1, a target of β-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle. Endocrinology14928532865.

  • Perez-RuizAOnoYGnocchiVFZammitPS2008β-Catenin promotes self-renewal of skeletal-muscle satellite cells. Journal of Cell Science12113731382.

  • PetropoulosHSkerjancIS2002β-Catenin is essential and sufficient for skeletal myogenesis in P19 cells. Journal of Biological Chemistry2771539315399.

  • PiccioniFSimeoniSAndriolaIArmaturaEBassaniniSPozziPPolettiA2001Polyglutamine tract expansion of the androgen receptor in a motoneuronal model of spinal and bulbar muscular atrophy. Brain Research Bulletin56215220.

  • PolesskayaASealePRudnickiMA2003Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell113841852.

  • PummilaMFliniauxIJaatinenRJamesMJLaurikkalaJSchneiderPThesleffIMikkolaML2007Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression. Development134117125.

  • RacayPGregoryPSchwallerB2006Parvalbumin deficiency in fast-twitch muscles leads to increased ‘slow-twitch type’ mitochondria, but does not affect the expression of fiber specific proteins. FEBS Journal27396108.

  • ReyaTCleversH2005Wnt signalling in stem cells and cancer. Nature434843850.

  • RigamontiAELocatelliLCellaSGBonomoSMGiuntaMMolinariFSartorioAMullerEE2009Muscle expressions of MGF, IGF-IEa, and myostatin in intact and hypophysectomized rats: effects of rhGH and testosterone alone or combined. Hormone and Metabolic Research412329.

  • RochatAFernandezAVandrommeMMolesJPBouschetTCarnacGLambNJ2004Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy. Molecular Biology of the Cell1545444555.

  • RossSEHematiNLongoKABennettCNLucasPCEricksonRLMacDougaldOA2000Inhibition of adipogenesis by Wnt signaling. Science289950953.

  • RussellDWWilsonJD1994Steroid 5 alpha-reductase: two genes/two enzymes. Annual Review of Biochemistry632561.

  • RuzicLMatkovicBRLekoG2003Antiandrogens in hormonal contraception limit muscle strength gain in strength training: comparison study. Croatian Medical Journal446568.

  • SaartokTDahlbergEGustafssonJA1984Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology11421002106.

  • SakamotoKArnoldsDEEkbergIThorellAGoodyearLJ2004Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle. Biochemical and Biophysical Research Communications319419425.

  • SarMLubahnDBFrenchFSWilsonEM1990Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology12731803186.

  • SatyamoorthyKLiGVaidyaBPatelDHerlynM2001Insulin-like growth factor-1 induces survival and growth of biologically early melanoma cells through both the mitogen-activated protein kinase and β-catenin pathways. Cancer Research6173187324.

  • SchakmanOKalistaSBertrandLLausePVerniersJKetelslegersJMThissenJP2008Role of Akt/GSK-3β/β-catenin transduction pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-treated rats. Endocrinology14939003908.

  • SchenkSMalNFinanAZhangMKiedrowskiMPopovicZMcCarthyPMPennMS2007Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor. Stem Cells25245251.

  • SealePRudnickiMA2000A new look at the origin, function, and “stem-cell” status of muscle satellite cells. Developmental Biology218115124.

  • SealePPolesskayaARudnickiMA2003Adult stem cell specification by Wnt signaling in muscle regeneration. Cell Cycle2418419.

  • SemsarianCWuMJJuYKMarciniecTYeohTAllenDGHarveyRPGrahamRM1999Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature400576581.

  • Sheffield-MooreM2000Androgens and the control of skeletal muscle protein synthesis. Annals of Medicine32181186.

  • ShiYMassagueJ2003Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell113685700.

  • ShiremanPKContreras-ShannonVOchoaOKariaBPMichalekJEMcManusLM2007MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration. Journal of Leukocyte Biology81775785.

  • SimpsonERMahendrooMSMeansGDKilgoreMWHinshelwoodMMGraham-LorenceSAmarnehBItoYFisherCRMichaelMD1994Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews15342355.

  • SinghRArtazaJNTaylorWEGonzalez-CadavidNFBhasinS2003Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology14450815088.

  • Sinha-HikimIArtazaJWoodhouseLGonzalez-CadavidNSinghABLeeMIStorerTWCasaburiRShenRBhasinS2002Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. American Journal of Physiology. Endocrinology and Metabolism283E154E164.

  • Sinha-HikimIRothSMLeeMIBhasinS2003Testosterone-induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. American Journal of Physiology. Endocrinology and Metabolism285E197E205.

  • Smerdel-RamoyaAZanottiSDeregowskiVCanalisE2008Connective tissue growth factor enhances osteoblastogenesis in vitro. Journal of Biological Chemistry2832269022699.

  • SnyderPJPeacheyHHannoushPBerlinJALohLLenrowDAHolmesJHDlewatiASantannaJRosenCJ1999Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. Journal of Clinical Endocrinology and Metabolism8426472653.

  • SwerdloffRSWangC2003Three-year follow-up of androgen treatment in hypogonadal men: preliminary report with testosterone gel. Aging Male6207211.

  • TenoverJS1994Androgen administration to aging men. Endocrinology and Metabolism Clinics of North America23877892.

  • ThielGCibelliG2002Regulation of life and death by the zinc finger transcription factor Egr-1. Journal of Cellular Physiology193287292.

  • ThomasMLangleyBBerryCSharmaMKirkSBassJKambadurR2000Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. Journal of Biological Chemistry2754023540243.

  • UstunelIAkkoyunluGDemirR2003The effect of testosterone on gastrocnemius muscle fibres in growing and adult male and female rats: a histochemical, morphometric and ultrastructural study. Anatomia Histologia Embryologia327079.

  • VandenputLBoonenSVan HerckESwinnenJVBouillonRVanderschuerenD2002Evidence from the aged orchidectomized male rat model that 17β-estradiol is a more effective bone-sparing and anabolic agent than 5α-dihydrotestosterone. Journal of Bone and Mineral Research1720802086.

  • VanderschuerenDVan HerckESuikerAMVisserWJSchotLPBouillonR1992Bone and mineral metabolism in aged male rats: short and long term effects of androgen deficiency. Endocrinology13029062916.

  • VanderschuerenDvan HerckENijsJEderveenAGDe CosterRBouillonR1997Aromatase inhibition impairs skeletal modeling and decreases bone mineral density in growing male rats. Endocrinology13823012307.

  • VanderschuerenDVandenputLBoonenSVan HerckESwinnenJVBouillonR2000An aged rat model of partial androgen deficiency: prevention of both loss of bone and lean body mass by low-dose androgen replacement. Endocrinology14116421647.

  • van't VeerLJDaiHvan de VijverMJHeYDHartAAMaoMPeterseHLvan der KooyKMartonMJWitteveenAT2002Gene expression profiling predicts clinical outcome of breast cancer. Nature415530536.

  • van der VeldenJLangenRKeldersMWillemsJWoutersEJanssen-HeiningerYScholsA2007Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3β. American Journal of Physiology. Cell Physiology29216361644.

  • VermeulenA1998Plasma androgens in women. Journal of Reproductive Medicine43725733.

  • VermeulenAGoemaereSKaufmanJM1999Testosterone, body composition and aging. Journal of Endocrinological Investigation22110116.

  • VisseRNagaseH2003Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circulation Research92827839.

  • VlahopoulosSZimmerWEJensterGBelaguliNSBalkSPBrinkmannAOLanzRBZoumpourlisVCSchwartzRJ2005Recruitment of the androgen receptor via serum response factor facilitates expression of a myogenic gene. Journal of Biological Chemistry28077867792.

  • WangCSwedloffRSIranmaneshADobsASnyderPJCunninghamGMatsumotoAMWeberTBermanN2000Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Testosterone Gel Study Group. Journal of Clinical Endocrinology and Metabolism8528392853.

  • WangBEWangXDErnstJAPolakisPGaoWQ2008Regulation of epithelial branching morphogenesis and cancer cell growth of the prostate by Wnt signaling. PLoS ONE3e2186.

  • WansaKDHarrisJMYanGOrdentlichPMuscatGE2003The AF-1 domain of the orphan nuclear receptor NOR-1 mediates trans-activation, coactivator recruitment, and activation by the purine anti-metabolite 6-mercaptopurine. Journal of Biological Chemistry2782477624790.

  • WarrenGLO'FarrellLSummanMHuldermanTMishraDLusterMIKuzielWASimeonovaPP2004Role of CC chemokines in skeletal muscle functional restoration after injury. American Journal of Physiology. Cell Physiology286C1031C1036.

  • WengLDaiHZhanYHeYStepaniantsSBBassettDE2006Rosetta error model for gene expression analysis. Bioinformatics2211111121.

  • WuytsAProostPPutWLenaertsJPPaemenLvan DammeJ1994Leukocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes. Journal of Immunological Methods174237247.

  • YangFLiXSharmaMSasakiCYLongoDLLimBSunZ2002Linking β-catenin to androgen-signaling pathway. Journal of Biological Chemistry2771133611344.

  • ZelarayanLCNoackCSekkaliBKmecovaJGehrkeCRengerAZafiriouMPvan der NagelRDietzRde WindtLJ2008β-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation. PNAS1051976219767.

  • ZweifelMBreuKMatozanKRennerEWelleMSchaffnerTClavienPA2005Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy. Immunology and Cell Biology83587595.

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

An official journal of

Society for Endocrinology

Sections

Figures

  • View in gallery

    Effect of androgen depletion on lean body mass, fat mass, and bone mineral content (BMC). Graphed values are mean body composition values of the 21-week-old rats, 11 weeks after SHAM surgery or orchidectomy (ORX) as measured by DEXA (±s.e.m). (A) Mean pretreatment values for SHAM (n=9) versus all ORX animals (n=27). *Indicates P<0.05, t-test for SHAM versus all ORX rats. All ORX animals were randomized to three groups prior to treatment. Androgen induced changes in body composition. (B) Mean group DEXA measurements of change in lean mass, fat mass, and bone mineral content graphed as a function of time (error bar represents s.e.m). Data are different from baseline measurements taken immediately before treatment. ▵, ORX+testosterone (10 mg/kg per day); □, ORX+12 DHT (3 mg/kg per day); ▪, ORX+ VEH; •, SHAM+ VEH. *Indicates different from ORX 13 (P<0.05, one-way ANOVA, Fisher's PLSD only shown at final time point). N=9 per group. (C) Soleus weight and strength after androgen treatment. All values are mean±s.e.m., and asterisks indicate different from ORX (P<0.05, one-way ANOVA, Fisher's PLSD). Peak tetanic tension (Po) of the right soleus, soleus wet weight, and Po divided by soleus weight. Note Y-axis scale does not begin at the value 0 for visual clarity in (A) for BMC and (C).

  • View in gallery

    Histology of soleus muscle 7 days after DHT treatment. ORX rats were treated for 4, 7, 14, or 21 days with vehicle or DHT (3 mg/kg per day) n=10 per group, and the soleus was collected and processed for histological examination. Representative cross-sectional fields of day 7 samples are shown above. Nuclear number per square micron (mean±s.e.m.) at each time point (* different from vehicle P<0.05, t-test) is shown below.

  • View in gallery

    Expression of classic myogenic regulators during DHT treatment time-course experiments. RNA was collected from the soleus of animals treated for the indicated time points with vehicle or DHT (3 mg/kg per day), and RNA levels of MyoD, myostatin, Mnf, and myogenin were determined by quantitative qRT-PCR. All values were first normalized within samples to glucuronidase, a housekeeping gene not affected by treatment or time in this experiment (not shown), and then to vehicle. For each time point, the DHT value normalized to the vehicle value, which was set to 1. Error bars represent s.e.m.

  • View in gallery

    Expression of Igf1ea (classic circulating) and Igf1ec (mechano growth factor, Mgf). Aged ORX rats (n=4 per group) were treated with vehicle or DHT (3 mg/kg per day) for 1, 4, and 7 days in one experiment (left panel) and soleus RNA collected individually, and the rats were again treated with vehicle or DHT for 4, 7, and 21 days in the second experiment (right panel) and soleus RNA collected and pooled in three replicates of 3–4 (from n=10 rats per group) and processed for total RNA. RNA for Igf1ea and IGF1 splice variant Mgf was assayed by qRT-PCR for gene expression relative to vehicle. All values were first normalized within samples to glucuronidase gene expression. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. Error bars represent s.e.m. *P<0.005, t-test.

  • View in gallery

    Confirmation of microarray expression of genes involved in muscle regeneration. Aged ORX rats (n=4 per group) were treated for the times indicated with vehicle or DHT (3 mg/kg per day). The soleus was collected from individual rats (n=4 per group) and processed for total RNA. RNA for myosin heavy chain type-2b (Myh4), parvalbumin (Pvalb), monocyte chemotactic protein-1 and -3 (Ccl2 and Ccl7) were assayed by qRT-PCR for gene expression relative to vehicle. All values were first normalized within samples to glucuronidase gene expression. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005 t-test. Error bars represent s.e.m.

  • View in gallery

    DHT upregulates expression of Wnt pathway regulator β-catenin through transcription and repression of phosphorylation. Rats were treated with vehicle or DHT (3 mg/kg per day) for the times indicated. The soleus was collected individually (n=4 per group, top panels) or pooled (n=6 per group, bottom panels), and processed for total RNA and protein. (A) Gene expression levels of β-catenin were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.05, t-test. Error bars represent s.e.m. (B) Immunoblot of total β-catenin and phospho-β-catenin (top panel). Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values. *P<0.05, t-test.

  • View in gallery

    Expression of regulated genes in the soleus of ORX rats treated with vehicle or DHT (3 mg/kg per day) in two time-course experiments for the times indicated. The soleus was collected individually for the short term (n=4 per group, left panels) and in three pooled replicates of 2 (from n=6 rats per group, right panels) and processed for total RNA. Gene expression levels of nuclear orphan receptor-1 (Nr4a3), bmp type II receptor (Bmpr2), and early growth response-1 (Egr1) were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005, t-test. Error bars represent s.e.m.

  • View in gallery

    DHT downregulates expression of Wnt pathway negative regulators, Axin2 and Axin. (A) Rats were treated with vehicle or DHT (3 mg/kg per day) for the times indicated. The soleus was collected individually (n=4 per group top panels) and processed for total RNA and protein. Gene expression levels of Axin2 were determined by qRT-PCR. All values were normalized within samples to glucuronidase. For each time point, the DHT value was normalized to the vehicle value, which was set to 1. *P<0.005, t-test. Error bars represent s.e.m. (B) Immunoblot of total AXIN and AXIN2. Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values. (C) Immunoblot for total GSK3β and serine-9-phophorylated GSK3β. DHT upregulates expression of serine-9-phosphorylated GSK3β. Rats were treated with vehicle or DHT (3 mg/kg per day) for 7 days. The soleus was collected individually (n=4) and processed for protein extraction. Bands represent individual rats at day 7 of treatment. The optical density (OD) values of DHT bands were normalized to vehicle values.

  • View in gallery

    Expression of β-catenin in muscle nuclei with DHT treatment. ORX rats were treated for 4, 7, 14, or 21 days with vehicle or DHT (3 mg/kg per day), and then soleus was collected and processed for histological examination. (A) Representative cross-sectional fields of day 7 samples are shown above. Muscle nuclei labeled with β-catenin-specific antibody as visualized by immunohistochemistry using nickel DAB. (B) The number of β-catenin-positive nuclei was counted per mm2 of cross-sectional area. N=10 rats/sections per group. *P<0.05, t-test. (C) The total number of nuclei was counted (without nickel) following hemotoxylin counterstaining for each section and the proportion calculated and graphed. *P<0.05, t-test.

  • View in gallery

    IGF1 and its splice variant mechano growth factor C-terminal peptide (ctMGF) promote nuclear translocation of myogenic Wnt regulator β-catenin in C2C12 myoblasts. Cells were treated with vehicle, IGF1 (10 and 30 ng/ml), and ctMGF (30 and 60 ng/ml). (A) Immunoblot of total nuclear β-catenin after 25 min of treatment. Lamin is load control. (B) The β-catenin TOPFLASH reporter plasmid containing TCF/LEF promoter linked to the firefly luciferase gene was cotransfected into C2C12 myoblasts with a constitutively active Renilla luciferase plasmid, and the cells were plated in 96-well plates and stimulated with IGF1 and ctMGF at the above concentrations for 16 h. β-Catenin-responsive firefly luciferase reporter activity was first normalized to Renilla luciferase activity, and the IGF1 and ctMGF values were normalized to the vehicle value, which was set to 1. *P<0.01, one-way ANOVA, Fisher's PLSD. (C) Near confluent C2C12 myoblasts in chamber slides were washed and placed in serum-free media with vehicle, IGF1 (30 ng/ml), or ctMGF (60 ng/ml) for 25 min just prior to fixation. The cells were prepared for immunocytochemistry, stained with anti-β-catenin antibodies, and counterstained with nuclear DAPI stain.

References

AdamsGRHaddadFBaldwinKM1999Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. Journal of Applied Physiology8717051712.

AndersonJEBresslerBHOvalleWK1988Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. Journal of Muscle Research and Cell Motility9499515.

AntonioJWilsonJDGeorgeFW1999Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles. Journal of Applied Physiology8720162019.

ArmstrongDDEsserKA2005Wnt/β-catenin signaling activates growth-control genes during overload induced skeletal muscle hypertrophy. American Journal of Physiology. Cell Physiology289C853C859.

ArmstrongDDWongVLEsserKA2006Expression of β-catenin is necessary for physiological growth of adult skeletal muscle. American Journal of Physiology. Cell Physiology291C185C188.

AsakuraASealePGirgis-GabardoARudnickiMA2002Myogenic specification of side population cells in skeletal muscle. Journal of Cell Biology159123134.

BanuSKGovindarajuluPAruldhasMM2002Testosterone and estradiol differentially regulate TSH-induced thyrocyte proliferation in immature and adult rats. Steroids67573579.

BardinCW1996The anabolic action of testosterone. New England Journal of Medicine3355253.

BhasinSStorerTWBermanNYarasheskiKEClevengerBPhillipsJLeeWPBunnellTJCasaburiR1997Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. Journal of Clinical Endocrinology and Metabolism82407413.

BhasinSStorerTWJavanbakhtMBermanNYarasheskiKEPhillipsJDikeMSinha-HikimIShenRHaysRD2000Testosterone replacement and resistance exercise in HIV-infected men with weight loss and low testosterone levels. Journal of the American Medical Association283763770.

BhasinSTaylorWESinghRArtazaJSinha-HikimIJasujaRChoiHGonzalez-CadavidNF2003The mechanisms of androgen effects on body composition: mesenchymal pluripotent cell as the target of androgen action. Journals of Gerontology. Series A Biological Sciences and Medical Sciences58M1103M1110.

BhasinSCalofOMStorerTWLeeMLMazerNAJasujaRMontoriVMGaoWDaltonJT2006Drug insight: testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging. Nature Clinical Practice. Endocrinology and Metabolism2146159.

BilezikianJPMorishimaABellJGrumbachMM1998Increased bone mass as a result of estrogen therapy in a man with aromatase deficiency. New England Journal of Medicine339599603.

BoissonneaultG2001Evidence of apoptosis in the castration-induced atrophy of the rat levator ani muscle. Endocrine Research27317328.

BrazJCGillRMCorblyAKJonesBDJinNVlahosCJWuQShenW2009Selective activation of PI3Kα/Akt/GSK-3β signalling and cardiac compensatory hypertrophy during recovery from heart failure. European Journal of Heart Failure11739748.

BrownMTaylorJ2005Prehabilitation and rehabilitation for attenuating hindlimb unweighting effects on skeletal muscle and gait in adult and old rats. Archives of Physical Medicine and Rehabilitation8622612269.

BrownMFisherJSHasserEM2001Gonadectomy and reduced physical activity: effects on skeletal muscle. Archives of Physical Medicine and Rehabilitation829397.

ChangCSaltzmanAYehSYoungWKellerELeeHJWangCMizokamiA1995Androgen receptor: an overview. Critical Reviews in Eukaryotic Gene Expression597125.

ChargeSBRudnickiMA2004Cellular and molecular regulation of muscle regeneration. Physiological Reviews84209238.

ChenJWuASunHDrakasRGarofaloCCascioSSurmaczEBasergaR2005aFunctional significance of type 1 insulin-like growth factor-mediated nuclear translocation of the insulin receptor substrate-1 and β-catenin. Journal of Biological Chemistry2802991229920.

ChenYZajacJDMacLeanHE2005bAndrogen regulation of satellite cell function. Journal of Endocrinology1862131.

ChiaIVCostantiniF2005Mouse axin and axin2/conductin proteins are functionally equivalent in vivo. Molecular and Cellular Biology2543714376.

ChinPCMajdzadehND'MelloSR2005Inhibition of GSK3β is a common event in neuroprotection by different survival factors. Brain Research. Molecular Brain Research137193201.

CleversH2006Wnt/β-catenin signaling in development and disease. Cell127469480.

CompstonJE2001Sex steroids and bone. Physiological Reviews81419447.

Contreras-ShannonVOchoaOReyes-ReynaSMSunDMichalekJEKuzielWAMcManusLMShiremanPK2007Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2−/− mice following ischemic injury. American Journal of Physiology. Cell Physiology292C953C967.

CornelisonDDWoldBJ1997Single-cell analysis of regulatory gene expression in quiescent and activated mouse skeletal muscle satellite cells. Developmental Biology191270283.

Van DammeJProostPLenaertsJPConingsROpdenakkerGBilliauA1993Monocyte chemotactic proteins related to human MCP-1. Advances in Experimental Medicine and Biology351111118.

DeVolDLRotweinPSadowJLNovakofskiJBechtelPJ1990Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. American Journal of Physiology259E89E95.

DeYoungRABakerJCCadoDWinotoA2003The orphan steroid receptor Nur77 family member Nor-1 is essential for early mouse embryogenesis. Journal of Biological Chemistry2784710447109.

DoumitMECookDRMerkelRA1996Testosterone up-regulates androgen receptors and decreases differentiation of porcine myogenic satellite cells in vitro. Endocrinology13713851394.

FerrandoAASheffield-MooreMYeckelCWGilkisonCJiangJAchacosaALiebermanSATiptonKWolfeRRUrbanRJ2002Testosterone administration to older men improves muscle function: molecular and physiological mechanisms. American Journal of Physiology. Endocrinology and Metabolism282E601E607.

FolettaVCLimMASoosairajahJKellyAPStanleyEGShannonMHeWDasSMassagueJBernardO2003Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. Journal of Cell Biology16210891098.

GaoWReiserPJCossCCPhelpsMAKearbeyJDMillerDDDaltonJT2005Selective androgen receptor modulator treatment improves muscle strength and body composition and prevents bone loss in orchidectomized rats. Endocrinology14648874897.

GarryDJMeesonAEltermanJZhaoYYangPBassel-DubyRWilliamsRS2000Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF. PNAS9754165421.

GoetschSCHawkeTJGallardoTDRichardsonJAGarryDJ2003Transcriptional profiling and regulation of the extracellular matrix during muscle regeneration. Physiological Genomics14261271.

GoldspinkGYangSY2004The splicing of the IGF-1 gene to yield different muscle growth factors. Advances in Genetics522349.

HameedMOrrellRWCobboldMGoldspinkGHarridgeSD2003Expression of IGF-1 splice variants in young and old human skeletal muscle after high resistance exercise. Journal of Physiology547247254.

HanadaKFuruyaKYamamotoNNejishimaHIchikawaKNakamuraTMiyakawaMAmanoSSumitaYOguroN2003Bone anabolic effects of S-40503, a novel nonsteroidal selective androgen receptor modulator (SARM), in rat models of osteoporosis. Biological & Pharmaceutical Bulletin2615631569.

HayataNFujioYYamamotoYIwakuraTObanaMTakaiMMohriTNonenSMaedaMAzumaJ2008Connective tissue growth factor induces cardiac hypertrophy through Akt signaling. Biochemical and Biophysical Research Communications370274278.

HeinleinCAChangC2002Androgen receptor (AR) coregulators: an overview. Endocrine Reviews23175200.

HicksonRCCzerwinskiSMFaldutoMTYoungAP1990Glucocorticoid antagonism by exercise and androgenic-anabolic steroids. Medicine and Science in Sports and Exercise22331340.

HillMGoldspinkG2003Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. Journal of Physiology549409418.

HolmbeckKBiancoPCaterinaJYamadaSKromerMKuznetsovSAMankaniMRobeyPGPooleARPidouxI1999MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell998192.

IchimuraTHungCCYangSAStevensJLBonventreJV2004Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury. American Journal of Physiology. Renal Physiology286F552F563.

KatznelsonLFinkelsteinJSSchoenfeldDARosenthalDIAndersonEJKlibanskiA1996Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. Journal of Clinical Endocrinology and Metabolism8143584365.

KatznelsonLRosenthalDIRosolMSAndersonEJHaydenDLSchoenfeldDAKlibanskiA1998Using quantitative CT to assess adipose distribution in adult men with acquired hypogonadism. American Journal of Roentgenology170423427.

KohTJBryerSCPucciAMSissonTH2005Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration. American Journal of Physiology. Cell Physiology289C217C223.

KriegM1976Characterization of the androgen receptor in the skeletal muscle of the rat. Steroids28261274.

LambertsSWvan den BeldAWvan der LelyAJ1997The endocrinology of aging. Science278419424.

LeeDK2002Androgen receptor enhances myogenin expression and accelerates differentiation. Biochemical and Biophysical Research Communications294408413.

LinkKBlizzardRMEvansWSKaiserDLParkerMWRogolAD1986The effect of androgens on the pulsatile release and the twenty-four-hour mean concentration of growth hormone in peripubertal males. Journal of Clinical Endocrinology and Metabolism62159164.

LiuWThomasSGAsaSLGonzalez-CadavidNBhasinSEzzatS2003Myostatin is a skeletal muscle target of growth hormone anabolic action. Journal of Clinical Endocrinology and Metabolism8854905496.

LumbrosoSSandillonFGeorgetVLobaccaroJMBrinkmannAOPrivatASultanC1996Immunohistochemical localization and immunoblotting of androgen receptor in spinal neurons of male and female rats. European Journal of Endocrinology134626632.

LustigBJerchowBSachsMWeilerSPietschTKarstenUvan de WeteringMCleversHSchlagPMBirchmeierW2002Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Molecular and Cellular Biology2211841193.

MacLeanHEChiuWSNotiniAJAxellAMDaveyRAMcManusJFMaCPlantDRLynchGSZajacJD2008Impaired skeletal muscle development and function in male, but not female, genomic androgen receptor knockout mice. FASEB Journal2226762689.

MarquesGBaoHHaerryTEShimellMJDuchekPZhangBO'ConnorMB2002The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function. Neuron33529543.

MassagueJCheifetzSEndoTNadal-GinardB1986Type β transforming growth factor is an inhibitor of myogenic differentiation. PNAS8382068210.

McPherronACLawlerAMLeeSJ1997Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature3878390.

MichelGBaulieuEE1980Androgen receptor in rat skeletal muscle: characterization and physiological variations. Endocrinology10720882098.

MiyabaraEHMartinJLGriffinTMMoriscotASMestrilR2006Overexpression of inducible 70-kDa heat shock protein in mouse attenuates skeletal muscle damage induced by cryolesioning. American Journal of Physiology Cell Physiology290C1128C1138.

MonksDAO'BryantELJordanCL2004Androgen receptor immunoreactivity in skeletal muscle: enrichment at the neuromuscular junction. Journal of Comparative Neurology4735972.

MusaroAMcCullaghKPaulAHoughtonLDobrowolnyGMolinaroMBartonERSweeneyHLRosenthalN2001Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nature Genetics27195200.

NantermetPVXuJYuYHodorPHolderDAdamskiSGentileMAKimmelDBHaradaSGerholdD2004Identification of genetic pathways activated by the androgen receptor during the induction of proliferation in the ventral prostate gland. Journal of Biological Chemistry27913101322.

OhnishiHOkaTKusachiSNakanishiTTakedaKNakahamaMDoiMMurakamiTNinomiyaYTakigawaM1998Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats. Journal of Molecular and Cellular Cardiology3024112422.

OlivaSUMessiasAGSilvaDAPereiraOCGerardinDCKempinasWG2006Impairment of adult male reproductive function in rats exposed to ethanol since puberty. Reproductive Toxicology22599605.

OttoASchmidtCLukeGAllenSValasekPMuntoniFLawrence-WattDPatelK2008Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. Journal of Cell Science12129392950.

PawlowskiJEErtelJRAllenMPXuMButlerCWilsonEMWiermanME2002Liganded androgen receptor interaction with β-catenin: nuclear co-localization and modulation of transcriptional activity in neuronal cells. Journal of Biological Chemistry2772070220710.

PearenMAMyersSARaichurSRyallJGLynchGSMuscatGE2008The orphan nuclear receptor, NOR-1, a target of β-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle. Endocrinology14928532865.

Perez-RuizAOnoYGnocchiVFZammitPS2008β-Catenin promotes self-renewal of skeletal-muscle satellite cells. Journal of Cell Science12113731382.

PetropoulosHSkerjancIS2002β-Catenin is essential and sufficient for skeletal myogenesis in P19 cells. Journal of Biological Chemistry2771539315399.

PiccioniFSimeoniSAndriolaIArmaturaEBassaniniSPozziPPolettiA2001Polyglutamine tract expansion of the androgen receptor in a motoneuronal model of spinal and bulbar muscular atrophy. Brain Research Bulletin56215220.

PolesskayaASealePRudnickiMA2003Wnt signaling induces the myogenic specification of resident CD45+ adult stem cells during muscle regeneration. Cell113841852.

PummilaMFliniauxIJaatinenRJamesMJLaurikkalaJSchneiderPThesleffIMikkolaML2007Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression. Development134117125.

RacayPGregoryPSchwallerB2006Parvalbumin deficiency in fast-twitch muscles leads to increased ‘slow-twitch type’ mitochondria, but does not affect the expression of fiber specific proteins. FEBS Journal27396108.

ReyaTCleversH2005Wnt signalling in stem cells and cancer. Nature434843850.

RigamontiAELocatelliLCellaSGBonomoSMGiuntaMMolinariFSartorioAMullerEE2009Muscle expressions of MGF, IGF-IEa, and myostatin in intact and hypophysectomized rats: effects of rhGH and testosterone alone or combined. Hormone and Metabolic Research412329.

RochatAFernandezAVandrommeMMolesJPBouschetTCarnacGLambNJ2004Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy. Molecular Biology of the Cell1545444555.

RossSEHematiNLongoKABennettCNLucasPCEricksonRLMacDougaldOA2000Inhibition of adipogenesis by Wnt signaling. Science289950953.

RussellDWWilsonJD1994Steroid 5 alpha-reductase: two genes/two enzymes. Annual Review of Biochemistry632561.

RuzicLMatkovicBRLekoG2003Antiandrogens in hormonal contraception limit muscle strength gain in strength training: comparison study. Croatian Medical Journal446568.

SaartokTDahlbergEGustafssonJA1984Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology11421002106.

SakamotoKArnoldsDEEkbergIThorellAGoodyearLJ2004Exercise regulates Akt and glycogen synthase kinase-3 activities in human skeletal muscle. Biochemical and Biophysical Research Communications319419425.

SarMLubahnDBFrenchFSWilsonEM1990Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology12731803186.

SatyamoorthyKLiGVaidyaBPatelDHerlynM2001Insulin-like growth factor-1 induces survival and growth of biologically early melanoma cells through both the mitogen-activated protein kinase and β-catenin pathways. Cancer Research6173187324.

SchakmanOKalistaSBertrandLLausePVerniersJKetelslegersJMThissenJP2008Role of Akt/GSK-3β/β-catenin transduction pathway in the muscle anti-atrophy action of insulin-like growth factor-I in glucocorticoid-treated rats. Endocrinology14939003908.

SchenkSMalNFinanAZhangMKiedrowskiMPopovicZMcCarthyPMPennMS2007Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor. Stem Cells25245251.

SealePRudnickiMA2000A new look at the origin, function, and “stem-cell” status of muscle satellite cells. Developmental Biology218115124.

SealePPolesskayaARudnickiMA2003Adult stem cell specification by Wnt signaling in muscle regeneration. Cell Cycle2418419.

SemsarianCWuMJJuYKMarciniecTYeohTAllenDGHarveyRPGrahamRM1999Skeletal muscle hypertrophy is mediated by a Ca2+-dependent calcineurin signalling pathway. Nature400576581.

Sheffield-MooreM2000Androgens and the control of skeletal muscle protein synthesis. Annals of Medicine32181186.

ShiYMassagueJ2003Mechanisms of TGF-β signaling from cell membrane to the nucleus. Cell113685700.

ShiremanPKContreras-ShannonVOchoaOKariaBPMichalekJEMcManusLM2007MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration. Journal of Leukocyte Biology81775785.

SimpsonERMahendrooMSMeansGDKilgoreMWHinshelwoodMMGraham-LorenceSAmarnehBItoYFisherCRMichaelMD1994Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews15342355.

SinghRArtazaJNTaylorWEGonzalez-CadavidNFBhasinS2003Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology14450815088.

Sinha-HikimIArtazaJWoodhouseLGonzalez-CadavidNSinghABLeeMIStorerTWCasaburiRShenRBhasinS2002Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. American Journal of Physiology. Endocrinology and Metabolism283E154E164.

Sinha-HikimIRothSMLeeMIBhasinS2003Testosterone-induced muscle hypertrophy is associated with an increase in satellite cell number in healthy, young men. American Journal of Physiology. Endocrinology and Metabolism285E197E205.

Smerdel-RamoyaAZanottiSDeregowskiVCanalisE2008Connective tissue growth factor enhances osteoblastogenesis in vitro. Journal of Biological Chemistry2832269022699.

SnyderPJPeacheyHHannoushPBerlinJALohLLenrowDAHolmesJHDlewatiASantannaJRosenCJ1999Effect of testosterone treatment on body composition and muscle strength in men over 65 years of age. Journal of Clinical Endocrinology and Metabolism8426472653.

SwerdloffRSWangC2003Three-year follow-up of androgen treatment in hypogonadal men: preliminary report with testosterone gel. Aging Male6207211.

TenoverJS1994Androgen administration to aging men. Endocrinology and Metabolism Clinics of North America23877892.

ThielGCibelliG2002Regulation of life and death by the zinc finger transcription factor Egr-1. Journal of Cellular Physiology193287292.

ThomasMLangleyBBerryCSharmaMKirkSBassJKambadurR2000Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. Journal of Biological Chemistry2754023540243.

UstunelIAkkoyunluGDemirR2003The effect of testosterone on gastrocnemius muscle fibres in growing and adult male and female rats: a histochemical, morphometric and ultrastructural study. Anatomia Histologia Embryologia327079.

VandenputLBoonenSVan HerckESwinnenJVBouillonRVanderschuerenD2002Evidence from the aged orchidectomized male rat model that 17β-estradiol is a more effective bone-sparing and anabolic agent than 5α-dihydrotestosterone. Journal of Bone and Mineral Research1720802086.

VanderschuerenDVan HerckESuikerAMVisserWJSchotLPBouillonR1992Bone and mineral metabolism in aged male rats: short and long term effects of androgen deficiency. Endocrinology13029062916.

VanderschuerenDvan HerckENijsJEderveenAGDe CosterRBouillonR1997Aromatase inhibition impairs skeletal modeling and decreases bone mineral density in growing male rats. Endocrinology13823012307.

VanderschuerenDVandenputLBoonenSVan HerckESwinnenJVBouillonR2000An aged rat model of partial androgen deficiency: prevention of both loss of bone and lean body mass by low-dose androgen replacement. Endocrinology14116421647.

van't VeerLJDaiHvan de VijverMJHeYDHartAAMaoMPeterseHLvan der KooyKMartonMJWitteveenAT2002Gene expression profiling predicts clinical outcome of breast cancer. Nature415530536.

van der VeldenJLangenRKeldersMWillemsJWoutersEJanssen-HeiningerYScholsA2007Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3β. American Journal of Physiology. Cell Physiology29216361644.

VermeulenA1998Plasma androgens in women. Journal of Reproductive Medicine43725733.

VermeulenAGoemaereSKaufmanJM1999Testosterone, body composition and aging. Journal of Endocrinological Investigation22110116.

VisseRNagaseH2003Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circulation Research92827839.

VlahopoulosSZimmerWEJensterGBelaguliNSBalkSPBrinkmannAOLanzRBZoumpourlisVCSchwartzRJ2005Recruitment of the androgen receptor via serum response factor facilitates expression of a myogenic gene. Journal of Biological Chemistry28077867792.

WangCSwedloffRSIranmaneshADobsASnyderPJCunninghamGMatsumotoAMWeberTBermanN2000Transdermal testosterone gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. Testosterone Gel Study Group. Journal of Clinical Endocrinology and Metabolism8528392853.

WangBEWangXDErnstJAPolakisPGaoWQ2008Regulation of epithelial branching morphogenesis and cancer cell growth of the prostate by Wnt signaling. PLoS ONE3e2186.

WansaKDHarrisJMYanGOrdentlichPMuscatGE2003The AF-1 domain of the orphan nuclear receptor NOR-1 mediates trans-activation, coactivator recruitment, and activation by the purine anti-metabolite 6-mercaptopurine. Journal of Biological Chemistry2782477624790.

WarrenGLO'FarrellLSummanMHuldermanTMishraDLusterMIKuzielWASimeonovaPP2004Role of CC chemokines in skeletal muscle functional restoration after injury. American Journal of Physiology. Cell Physiology286C1031C1036.

WengLDaiHZhanYHeYStepaniantsSBBassettDE2006Rosetta error model for gene expression analysis. Bioinformatics2211111121.

WuytsAProostPPutWLenaertsJPPaemenLvan DammeJ1994Leukocyte recruitment by monocyte chemotactic proteins (MCPs) secreted by human phagocytes. Journal of Immunological Methods174237247.

YangFLiXSharmaMSasakiCYLongoDLLimBSunZ2002Linking β-catenin to androgen-signaling pathway. Journal of Biological Chemistry2771133611344.

ZelarayanLCNoackCSekkaliBKmecovaJGehrkeCRengerAZafiriouMPvan der NagelRDietzRde WindtLJ2008β-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation. PNAS1051976219767.

ZweifelMBreuKMatozanKRennerEWelleMSchaffnerTClavienPA2005Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy. Immunology and Cell Biology83587595.

Index Card

Cited By

PubMed

Google Scholar

Related Articles

Altmetrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 611 611 137
PDF Downloads 196 196 62