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* Department of Medicine, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia; and
Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia
1Correspondence: Austin Hospital, Heidelberg, VIC 3084, Australia. E-mail: hmaclean{at}unimelb.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: hypertrophy • anabolic • knockout • strength • polyamine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Conversely, exogenously administered androgens have anabolic effects on muscle (2)
. Randomized controlled trials demonstrated a dose-dependent response of lean body mass and leg strength to testosterone in eugonadal young and elderly men (3
, 4)
. Anecdotal reports from anabolic steroid use in female athletes are highly suggestive that androgens also increase muscle strength in women (5)
. However, it is yet to be determined whether the low testosterone levels in females play a physiological role in developing normal peak muscle mass, and the mechanisms of androgen action in male muscle are still unknown.
Androgens act predominantly through the androgen receptor (AR), a member of the ligand-dependent nuclear transcription factor family. Both testosterone and dihydrotestosterone bind and activate the AR to regulate target gene expression (6)
. Although both naturally occurring AR mutant mice (Tfm) and AR knockout (ARKO) mouse models have been generated (7)
, no systematic analysis of AR-null skeletal muscle phenotype has been performed, and thus the AR-mediated actions of androgens in muscle remain undefined. In addition, testosterone in males can also be aromatized to estradiol, to act via the estrogen receptor (ER) (8)
. Male muscle contains aromatase enzyme activity (9)
, suggesting the potential for testosterone action via estradiol in muscle. Male mice lacking ERβ also show altered muscle function (10)
; however, the relative importance of AR- vs. ER-mediated pathways in muscle is unknown.
The AR gene is expressed widely, including in myoblasts, myofibers and satellite cells of males and females (11)
. The AR is also expressed in motor neurons, which are also a direct target for androgen action (12)
and may contribute to regulation of muscle mass. Androgens may also modulate levels of circulating insulin-like growth factor 1 (IGF1) (13)
, which is also a potent anabolic agent in muscle. Further, androgen-dependent behavioral modifications, such as modulation of activity levels, could potentially impact on muscle mass.
The major unanswered questions regarding the anabolic actions of androgens in skeletal muscle are the following. 1) Are these actions mediated predominantly via the AR? 2) What is their role in development of normal muscle mass in females vs. males? 3) What are the functional consequences of androgen action in skeletal muscle? 4) What are the target genes that mediate these actions in muscle? To answer these questions, we have used the cre/loxP system to create male and female global ARKO mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, with exon 3 flanked by loxP sites. ARlox heterozygous mice were back-crossed onto a C57BL/6 background for >6 generations prior to experimental analysis. CMV-cre transgenic mice were obtained with permission from Dr. Ursula Lichtenberg (Institute for Genetics, University of Cologne, Cologne, Germany) and were maintained on a C57BL/6 background. Control littermates were used for both males [wild type (WT)] and females (WT or CMV-cre heterozygotes), as described. Mice were housed in a conventional facility, and standard chow and water were provided ad libitum. Studies were performed with the approval of the Austin Health Animal Ethics Committee, Melbourne Health Animal Ethics Committee, and the University of Melbourne Animal Ethics Committee.
Tissue collection
Wet weight of tissues was determined to an accuracy of 0.1 mg, from the mean mass bilaterally. Body and muscle mass were measured in 9 and 12 wk ARKO and WT male littermates (n
24/group at 9 wk, n12/group at 12 wk), and in 9 wk ARKO females, and WT and CMV-cre heterozygote female littermates (n12/group). Data from WT and CMV-cre females were pooled, as there was no statistical difference between all parameters in these groups (data not shown).
IGF1 assay
Serum IGF1 was assayed in 9- and 12-wk WT and ARKO males (n=12/group), using the IGF1 RIA (Bioclone; Marrickville, NSW, Australia) according to manufacturer’s instructions, except the assay was scaled down 2-fold to 50 µl serum/mouse.
cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), and PCR
Total RNA isolation and cDNA synthesis were performed as described previously (15)
. To detect expression of the normal and exon 3-deleted AR genes, RT-PCR was performed on 500 ng cDNA using primers flanking exon 3 (14)
.
To detect the ARlox allele in the spiking experiment, genomic DNA from gastrocnemius muscle of an ARKO female and an ARlox heterozygote female was combined (100 ng/reaction), and PCR primers within exon 3 of the AR gene and the neo cassette were used (14)
. The amount of control ARlox female DNA varied from 100% to 0.1%. For the 100% ARKO female samples, DNA from two independent ARKO females was used in separate reactions. The GAPDH gene was amplified to control for amount of DNA.
Quantitative real-time PCR
Quantitative real-time PCR (Q-PCR) was performed in duplicate using 500 ng cDNA in a 25-µl reaction, using TaqMan gene expression assays and the 7500 real-time PCR system (Applied Biosystems, Scoresby, VIC, Australia). Relative expression was determined using the 
CT method, as previously described (15)
. To quantitate AR gene expression, up to 7 samples/group were analyzed using the mouse AR gene expression assay (assay ID: Mm00442688_m1), which amplifies between exons 2 and 3 with a probe against the exon2/exon 3 boundary. To compare gene expression in WT and ARKO gastrocnemius muscle, Q-PCR was performed on 12 samples/group, and 9 samples/group were used for the orchidectomy vs. orchidectomy plus testosterone groups, using gastrocnemius muscle collected in our previous study (16)
. Preoptimized or custom TaqMan assays were used as listed in Supplemental Data.
Microarray analysis
Microarray analysis was performed by the Australian Genome Research Facility using the Affymetrix system (Millennium Sciences, Surrey Hills, VIC, Australia). Gastrocnemius muscle RNA from WT and ARKO males (n=2/group) was analyzed, using Affymetrix mouse genome 430 2.0 array. This array contains >45,000 probe sets representing >34,000 mouse genes, with 11 pairs of oligonucleotide probes for each sequence. Androgen-responsive genes were identified using normalized signal intensity to identify genes with 1.6-fold-change in duplicate samples. For genes with more than one probe set in the array, data from all probes were combined in the statistical analyses. GO mining was performed using the online Affymetrix NetAffx analysis center (https://www.affymetrix.com/analysis/index.affx).
In vitro physiology
In vitro physiological analyses were performed on muscles from 9 wk WT males, WT females, and ARKO males (n=6/group), using two hind limb muscles: the fast-twitch extensor digitorum longus and the slow-twitch soleus. Contractile properties, including maximum tetanic force, specific force, fatigue, time-to-peak tension and half-relaxation time were assessed as described previously (16)
.
Statistical analysis
For comparison of means for more than 2 groups, data were analyzed by 1-way ANOVA with Tukey’s post hoc analysis; for 2 groups, unpaired Student’s t test was used. Fatigue was analyzed by general linear model univariate analysis with Tamhane’s post hoc test, as Levene’s test of equality of error indicated unequal variance across genotypes. GO distributions were analyzed by 
2 analysis. All analyses were performed using SPSS 11, except the GO 2 analysis, which was calculated using the Affymetrix online GO tool.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Exon 3 encodes the second zinc finger of the DNA binding domain, and deletion maintains the mRNA reading frame, leading to AR protein that lacks DNA binding and has ablation of genomic actions (14)
. To generate global ARKO males, the AR line was created (Fig. 1B
). Because hemizygous ARKO males are androgen insensitive and infertile, the cre/loxP system was required to generate global ARKO females (Fig. 1B
).
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To confirm the cre-mediated deletion of the ARlox allele in ARKO females, we used primers within exon 3 to amplify DNA from a number of tissues including tail, kidney, heart, and skeletal muscle, and no band was detectable (data not shown). To further quantitate the degree of ARlox deletion in the ARKO females, we spiked genomic DNA from ARKO female muscle with varying concentrations of control ARlox heterozygous female muscle DNA, and carried out PCR using primers specific for the ARlox allele, amplifying both exon 3 (data not shown) and the neo cDNA sequence (Fig. 1C
). In each case, the ARlox allele could be amplified from ARKO female DNA containing down to 0.1% control ARlox DNA, but not from the 100% ARKO female DNA, suggesting a very high efficiency of ARlox deletion.
AR mRNA expression in muscle from 9-wk-old WT and ARKO males was measured by Q-PCR, using a probe homologous to exon 3. The level of AR gene expression in different muscles varied in different WT muscles, with the highest expression in the known androgen-responsive levator ani muscle (Fig. 1D
). As expected, no WT AR mRNA was detectable in muscle from ARKO males using this exon 3-specific probe (Fig. 1D
). However, reverse transcriptase PCR using primers flanking exon 3 amplified the AR mRNA in ARKO male muscle samples (Fig. 1E
), confirming that the mutant gene is still expressed.
We previously showed that ARKO males have decreased serum testosterone levels (14)
. In the present study, we measured serum IGF1 levels in 9- and 12-wk-old WT and ARKO males and showed no difference in IGF1 levels between the two groups (Table 1
).
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ARKO males have reduced muscle mass but ARKO females are normal
Body mass and muscle mass were examined in ARKO males at 9 and 12 wk of age, and in ARKO females at 9 wk. At both ages, body mass in ARKO males was intermediate between that of WT males and females (Fig. 2
A, Table 2
and data not shown), with a 12–13% reduction in body mass compared with WT males (P<0.001). In contrast, there was no difference in body mass between ARKO females and control females (WT and CMV-cre heterozygotes) (Fig. 2A
).
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We measured the mass of a number of hind limb muscles, including the fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL), the slow-twitch soleus (SOL), and the mixed-fiber gastrocnemius (GAST) muscles. At 9 wk of age, the mass of all hind limb muscles was significantly reduced in ARKO males compared with WT males (Fig. 2B-E
), with up to a 20% reduction in mass of individual muscles. This change in absolute mass reflects both direct (muscle-specific) and indirect effects of AR deletion, but even when adjusted for total body mass, which is itself androgen-dependent (14
, 16)
, TA mass was still significantly reduced in 9 wk ARKO males (Fig. 2F
). At 12 wk, a similar significant decrease in absolute muscle mass was observed in all muscles of the ARKO vs. WT males (Table 2)
. The percentage decrease in muscle mass in ARKO males was smaller when adjusted for total body mass (Table 2)
; however, when adjusted for heart mass, which is not androgen-dependent and controls for differences in total body size, muscle mass was still significantly reduced in ARKO males, by 7–16% (Table 2)
. The highly androgen-dependent levator ani muscle, a perineal muscle that is present in males but fails to develop in female rodents (17)
, did not develop in ARKO males (data not shown).
ARKO females were examined at 9 wk of age. In contrast to the ARKO males, there was no difference in muscle mass between ARKO females and control WT and CMV-cre heterozygote female littermates (Fig. 2B-E
).
ARKO male muscles have reduced strength and increased resistance to fatigue
To determine the AR-mediated actions of androgens on muscle function, we analyzed muscle contractile properties in vitro from fast-twitch EDL and slow-twitch SOL muscles in ARKO males and WT males and females. As no effect of AR deletion was observed on ARKO female muscle mass, ARKO females were not included in the functional studies. Maximum tetanic force, a measure of muscle contractile strength, was significantly decreased in the EDL from ARKO males vs. WT males (P=0.001), down to the level of WT females (Fig. 3
A); however, there was no difference in the SOL muscle between ARKO and WT males (Fig. 3A
). To determine whether the changes in maximum tetanic force were due to changes in intrinsic contractile function, specific force was calculated (16)
. There was no difference in EDL-specific force between the ARKO males, WT males, and WT females (Fig. 3B
), suggesting the decrease in strength in the ARKO males is caused by the decreased muscle mass.
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To measure the ability of muscles to resist fatigue, they were stimulated to contract with maximum force every 4 s for 4 min, with tetanic force produced during this period determined. For the EDL muscle, there was no difference in the fatigue resistance of ARKO males, WT males, and WT females (Fig. 3C
). In contrast, for the SOL muscle, WT females were more fatigue resistant than WT males, evidenced by a higher force maintained over the contraction period (P<0.001) (Fig. 3C
). Furthermore, ARKO males were equivalent to WT females and more fatigue resistant than WT males (P<0.05) (Fig. 3C
).
Altered gene expression in ARKO male muscle
To identify target genes mediating the structural and functional differences in the ARKO male muscle, microarray analysis was performed on WT and ARKO male gastrocnemius muscle. A suite of 93 genes was identified that showed 1.6-fold differences in expression, comprising 46 genes down-regulated in the ARKO and 47 genes up-regulated (Table 3
and data not shown). Genes in a number of functional categories were identified, including contractile/structural, transcriptional regulation, immune/inflammatory response, growth factors/signal transduction, energy metabolism, and cell cycle and transport. Gene ontology (GO) mining performed on the androgen-responsive genes identified significantly overrepresented functional processes, with the most highly overrepresented being polyamine biosynthesis (2 analysis, P<0.001). S-adenosylmethionine decarboxylase 1 (Amd1), ornithine decarboxylase 1 (Odc1) and spermine oxidase (Smox) were all down-regulated in the ARKO. A cluster of genes with a smaller magnitude up-regulation in the ARKO male muscle was characteristic of a fast-to-slow-twitch phenotype change (Fig. 4
). Other genes previously demonstrated to play a role in regulation of muscle atrophy or hypertrophy were unchanged (Table 4
).
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Microarray results were confirmed by Q-PCR on a subset of genes, chosen based on their overrepresentation from GO mining, their putative role in regulation of proliferation/differentiation or signaling pathways, or their potential role in skeletal muscle. Q-PCR demonstrated that expression of Amd1 was decreased 10-fold in ARKO muscle and Odc1 was decreased 3-fold (Fig. 5
A). Expression of other regulatory genes, Wnt4, frizzled 4 (Fzd4), cyclin dependent kinase inhibitor 1c (Cdkn1c) (p57Kip2) and protein phosphatase 3, catalytic subunit 
(Ppp3ca) (calcineurin A) was significantly increased in ARKO muscle, and integrin β1 binding protein 3 (Itgb1bp3) was significantly decreased (Fig. 5B
). Expression of transcription elongation factor A (SII)-like 7 (Tceal7) was also increased in ARKO muscle (Fig. 5C
). Growth factor genes insulin-like growth factor 2 (Igf2) and transforming growth factor β2 (Tgfb2) were up-regulated in ARKO muscle; however, there was no change in the expression of Igf1 in muscle (Fig. 5C
).
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Altered gene expression in orchidectomized male muscle
We previously showed that 10 wk postorchidectomy, WT male mice have decreased muscle mass and strength, and this effect is prevented by testosterone supplementation (16)
. We therefore examined the expression of the androgen-responsive genes identified above in gastrocnemius from control orchidectomized (orx+C) and testosterone-treated orchidectomized (orx+T) males. The majority of genes showed a similar pattern of expression in the orx+C vs. orx+T groups to that observed in the ARKO males (Fig. 6
). We also confirmed that Igf1 expression was not altered by orchidectomy and testosterone treatment (data not shown). These results indicate that many of the same androgen-dependent pathways are regulated during androgen deprivation-dependent muscle atrophy as during androgen/AR-mediated muscle growth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lack of effect of AR deletion on muscle mass in females is strongly suggestive that androgens are not required to achieve peak muscle mass in sedentary females. Thus, although female muscle expresses the AR and is capable of an anabolic response when exogenous androgens are administered (5)
, our data suggest that androgens play little or no role in regulating normal female muscle mass. It remains to be demonstrated whether treatment of females with supraphysiological doses of androgens would activate the same pathways that we identified in the ARKO male muscle.
In males, androgens have been proposed to promote muscle hypertrophy through a number of different cellular pathways, including stimulating mesenchymal commitment into the myogenic lineage (18)
, promoting satellite cell or myoblast proliferation (11
, 19)
, and increasing muscle protein synthesis (20)
. The LA muscle is one of the most androgen-dependent muscles (21)
, with satellite cells in the female undergoing apoptosis during perinatal development causing failure of the LA to develop in adult females (22
, 23)
. The complete lack of development of the LA muscle in adult ARKO males indicates that these actions of androgens occur through the AR, during muscle development. Previous studies have suggested that androgens also regulate myofiber protein turnover (20)
. In microarray analysis of ARKO muscle, there was no change in expression of genes encoding the major muscle contractile proteins, nor was there any change in the expression of ubiquitin ligase genes up-regulated during muscle atrophy. However, there was a significant reduction in the expression of genes encoding cytoskeletal proteins. Therefore, our data suggest that androgens regulate muscle mass both through control of muscle cell commitment or proliferation and through regulation of muscle protein balance.
In ARKO males, fast-twitch muscle mass and force production were reduced proportionally, down to the level of WT females, indicating muscle strength is dependent on muscle mass, which is controlled by androgens in males. This decrease in force production from the EDL muscle was similar to the decrease that we previously observed in WT C57BL/6 males 10 wk postorchidectomy, compared to testosterone-treated orchidectomized mice (16)
. In contrast, the slow-twitch soleus muscle showed a significant increase in fatigue resistance in ARKO males, also equivalent to WT female soleus. Previous studies have shown that female muscles are more fatigue resistant than male (24)
, and our results indicate that this occurs through an AR-dependent effect. In ARKO male muscle, there was increased expression of genes characteristic of slow-twitch phenotype (Fig. 4)
. These data suggest that androgens promote the fast-twitch phenotype, favoring type II glycolytic fibers that produce higher force but are highly fatigable. Our previous orchidectomy study showed that testosterone treatment of orchidectomized males also paradoxically increased the fatigue resistance of the soleus muscle compared to control orchidectomized mice (16)
. This difference in findings could be caused by the effect of aromatization of testosterone to estradiol in our previous study, as supraphysiological doses of testosterone were used, or may reflect differences in the effect of loss of androgen action throughout development in the ARKO muscle vs. androgen deprivation in adult muscle in the orchidectomy study.
Microarray analysis ruled out known myogenic regulatory factors as being targets of the AR, and there was no change in the expression of other factors implicated in regulation of muscle atrophy and hypertrophy (25)
. Q-PCR showed that expression of Ppp3ca (calcineurin A) was increased in muscle from both ARKO and orchidectomized males, despite the fact that up-regulation of calcineurin signaling pathways has been implicated in IGF1-dependent muscle hypertrophy (26)
. Other studies do not support a role for calcineurin in muscle hypertrophy (27)
, and our data are more consistent with the demonstrated function of calcineurin A as an inducer of slow-twitch fiber phenotype (27)
than as a mediator of hypertrophy.
Our results suggest that one of the major regulatory mechanisms of androgens in skeletal muscle is regulation of polyamine biosynthesis. Q-PCR confirmed that there was a significant decrease in the expression of the genes encoding the rate-limiting polyamine biosynthetic enzymes Odc1 and Amd1 in ARKO males. Polyamines have been implicated in numerous cellular processes, including transcription, cell proliferation, and apoptosis (reviewed in ref. 28
). Data suggest that polyamines play a role in muscle hypertrophy (29)
, although their mechanisms of action are still unknown. Odc1 is androgen responsive in the murine kidney and is directly regulated by the AR (30)
. Expression of Odc1 and Amd1 was also decreased in muscle from orchidectomized males compared to orchidectomized males treated with testosterone. Thus, it is likely that one of the major pathways via which androgens increase or maintain muscle mass is through increased polyamine synthesis, via up-regulation of Amd1 and Odc1 expression.
Expression of the cell cycle regulatory gene Cdkn1c (p57Kip2), which couples differentiation and cell cycle exit in myoblasts (31)
, was increased in ARKO muscle. This may limit the number of cycles of myoblast proliferation and cause premature differentiation, contributing to reduced muscle mass in ARKO muscle. Testosterone has been shown to have no effect on proliferation of porcine primary myoblasts but suppresses differentiation into myotubes (32)
. Therefore, the anabolic actions of androgens in muscle may occur in part via the AR repressing p57Kip2 expression, thus maintaining myoblasts in the proliferative state and allowing prolonged muscle growth. The expression pattern of other genes in ARKO muscle is also consistent with the proposed mechanism that androgens maintain myoblasts in the proliferative state and delay differentiation. Itgb1bp3 is a negative regulator of muscle differentiation (33)
. Expression of Itgb1bp3 is high in proliferating C2C12 myoblasts in vitro, and overexpression of Itgb1bp3 prevents terminal myogenic differentiation (33)
. The expression of Itgb1bp3 is decreased in ARKO muscle, consistent with a smaller, more differentiated muscle. Similarly, Igf2 expression, which is increased in ARKO muscle compared with WT, is up-regulated in differentiated myotubes in vitro (34)
, and may drive terminal myogenic differentiation (35)
.
Wnt signaling through both canonical and noncanonical pathways occurs during embryonic muscle determination, and muscle regeneration and hypertrophy (36)
. Although the expression of Wnt4 is up-regulated in muscles from myostatin-null mice, which exhibit fiber hyperplasia (37)
; Wnt4 and one of its receptors, Fzd4, were also up-regulated in ARKO and orchidectomized males, and the negative regulator Dkk3 was down-regulated. This finding suggests that up-regulation of components of the Wnt signaling pathway is not sufficient to induce hypertrophy, but instead can also be associated with reduced muscle mass.
IGF1 has been a proposed mediator of the anabolic actions of androgens in muscle, as IGF1 is a potent muscle growth factor, and data suggest that androgens can regulate circulating or local muscle IGF1 levels (13
, 38)
. However, serum IGF1 levels did not differ between WT and ARKO males, Q-PCR demonstrated that Igf1 gene expression in ARKO muscle was normal, and expression was also not affected by orchidectomy. From microarray analysis, there was also no difference in the expression of the genes encoding the IGF binding proteins, or the IGF1 receptor. Therefore, it is likely that the anabolic actions of androgens in skeletal muscle are not mediated via modulation of IGF1 production or action.
The ARKO male muscle phenotype is likely to be caused by absence of androgen action during both embryonic development and in the peri- to postpubertal period, when androgen levels are high in males. We and others have previously investigated muscle responses to androgen deprivation or supplementation in adult males (3
, 16)
. Orchidectomy causes androgen withdrawal-dependent muscle atrophy, whereas in our ARKO model, WT males have androgen-dependent muscle growth compared to the androgen-insensitive ARKO males. Therefore, different androgen-dependent pathways could potentially be regulated during skeletal muscle atrophy following orchidectomy, compared to ARKO muscle, which fails to develop peak muscle mass but has not undergone atrophy. However, our data demonstrate that a number of androgen-responsive genes show the same pattern of expression following orchidectomy as in ARKO muscle, suggesting conservation of gene pathways during androgen deprivation-dependent atrophy and androgen-stimulated growth.
Only one previous study has investigated AR-mediated actions in muscle (39)
, using an ARKO mouse model in which no AR protein is detectable (40)
. In contrast to our findings, these ARKO males showed down-regulation of slow-twitch markers and increased fast troponin T1. Muscle mass was not investigated (39)
; however, in another study the group reported that muscle mass is unchanged in these ARKO mice (41)
. These differing results may reflect the effects of genetic background (42)
, with the mice in the previous studies on a mixed genetic background, compared to the homogenous C57BL/6 background in our study. Alternatively, this could potentially reflect a difference between total ablation of AR-dependent signaling vs. loss of genomic signaling. Our genomic ARKO model retains the potential for nonclassical AR signaling, as the second zinc finger of the DNA binding domain is deleted inframe, and AR protein is still produced (14)
. In vitro studies have shown effects of testosterone on intracellular Ca2+ and ERK phosphorylation in myotubes, which occur too rapidly to involve DNA binding (reviewed in ref. 11
). Although the physiological relevance of these nonclassical signaling pathways is unknown, our genomic ARKO model can be used to address this question further in the future. Given the consistency of our findings with the known actions of androgens on muscle in humans, we also believe our study provides a good model for elucidating the mechanisms of anabolic actions of androgens in skeletal muscle.
Our data provide the first direct evidence that androgens acting via the AR are not required for development of normal female muscle mass, but are required for normal male muscle mass, and contractile strength. Differences in fatigue resistance between male and female muscles are also controlled via the AR. Our results indicate that IGF1 does not mediate the anabolic actions of androgens in skeletal muscle. This study suggests that two of the major mechanisms mediating the anabolic actions of androgens are the regulation of genes encoding the polyamine biosynthetic enzymes and genes controlling progression of myoblast proliferation to differentiation.
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ACKNOWLEDGMENTS |
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Received for publication January 16, 2008. Accepted for publication March 13, 2008.
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) flanking exon 3 and neo cassette in intron 3, and AR
, ARlox heterozygote female; ARKO 
0.001 vs. control male; 
P < 0.05, 
