Stimulation of both estrogen and androgen receptors maintains skeletal muscle mass in gonadectomized male mice but mainly via different pathways

in Journal of Molecular Endocrinology
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  • Division of Endocrinology, Department of Internal Medicine, Institute of Medicine, Sahlgrenska University Hospital, SE-41345 Göteborg, Sweden

Testosterone is a major regulator of muscle mass. Little is known whether this is due to a direct stimulation of the androgen receptor (AR) or mediated by aromatization of testosterone to estradiol (E2), the ligand for the estrogen receptors (ERs), in peripheral tissues. In this study, we differentiated between the effects mediated by AR and ER by treating orchidectomized (orx) male mice for 5 weeks with E2 or the non-aromatizable androgen dihydrotestosterone (DHT). Both E2 and DHT increased muscle weight and lean mass, although the effect was less marked after E2 treatment. Studies of underlying mechanisms were performed using gene transcript profiling (microarray and real-time PCR) in skeletal muscle, and they demonstrated that E2 regulated 51 genes and DHT regulated 187 genes, with 13 genes (=25% of E2-regulated genes) being regulated by both treatments. Both E2 and DHT altered the expression of Fbxo32, a gene involved in skeletal muscle atrophy, affected the IGF1 system, and regulated genes involved in angiogenesis and the glutathione metabolic process. Only E2 affected genes that regulate intermediary glucose and lipid metabolism, and only DHT increased the expression of genes involved in synaptic transmission and heme and polyamine biosynthesis. In summary, ER activation by E2 treatment maintains skeletal muscle mass after orx. This effect is less marked than that of AR activation by DHT treatment, which completely prevented the effect of orx on muscle mass and was partly, but not fully, mediated via alternative pathways.

Abstract

Testosterone is a major regulator of muscle mass. Little is known whether this is due to a direct stimulation of the androgen receptor (AR) or mediated by aromatization of testosterone to estradiol (E2), the ligand for the estrogen receptors (ERs), in peripheral tissues. In this study, we differentiated between the effects mediated by AR and ER by treating orchidectomized (orx) male mice for 5 weeks with E2 or the non-aromatizable androgen dihydrotestosterone (DHT). Both E2 and DHT increased muscle weight and lean mass, although the effect was less marked after E2 treatment. Studies of underlying mechanisms were performed using gene transcript profiling (microarray and real-time PCR) in skeletal muscle, and they demonstrated that E2 regulated 51 genes and DHT regulated 187 genes, with 13 genes (=25% of E2-regulated genes) being regulated by both treatments. Both E2 and DHT altered the expression of Fbxo32, a gene involved in skeletal muscle atrophy, affected the IGF1 system, and regulated genes involved in angiogenesis and the glutathione metabolic process. Only E2 affected genes that regulate intermediary glucose and lipid metabolism, and only DHT increased the expression of genes involved in synaptic transmission and heme and polyamine biosynthesis. In summary, ER activation by E2 treatment maintains skeletal muscle mass after orx. This effect is less marked than that of AR activation by DHT treatment, which completely prevented the effect of orx on muscle mass and was partly, but not fully, mediated via alternative pathways.

Introduction

Testosterone is a major regulator of body composition. Androgen deficiency is associated with decreased muscle mass, and testosterone supplementation increases muscle mass in hypogonadal men, HIV-infected men, and older men with low testosterone concentrations (Snyder et al. 1999, 2000, Kong & Edmonds 2002). Also, in orchidectomized (orx) mice, testosterone treatment dose dependently increases the mass of individual muscles (Axell et al. 2006).

Both testosterone and the non-aromatizable androgen dihydrotestosterone (DHT) bind to and activate the androgen receptor (AR; MacLean et al. 1997). The AR gene is expressed widely in muscle including myoblasts, myofibers, and satellite cells (Chen et al. 2005). The AR is also expressed in motor neurons which may contribute to the regulation of muscle mass and function (Yang & Arnold 2000). Several different AR knockout (ARKO) mouse models have been reported. In one of these models, muscle mass was unchanged, whereas in another model, decreased muscle mass and impaired muscle function were observed in male ARKO mice but not in female ARKO mice (Lin et al. 2005, MacLean et al. 2008). Ophoff et al. (2009) recently reported that a myocyte-specific knockout of the AR in male mice resulted in decreased lean mass and a conversion of fast towards slow muscle fibers, without affecting muscle strength or fatigue. In their study, similar results were obtained in male mice with ubiquitous ARKO.

Testosterone can exert its effect either directly by stimulation of the AR or via aromatization in target tissues to estradiol (E2), the ligand for the estrogen receptors (ERs) α and β (also known as ESR1 and ESR2 respectively). Muscle from both men and women contains aromatase enzyme activity (Matsumine et al. 1986). The extent to which the actions of testosterone in muscle are a consequence of AR or ER activation or both is not clear.

Skeletal muscle myoblasts, myotubes, and mature fibers all express functional ERs, indicating a direct effect of estrogen in muscle (Kahlert et al. 1997, Barros et al. 2006). Women after menopause have decreased lean body mass, which can be reversed by estrogen replacement therapy (Sorensen et al. 2001). In animals, estrogen has been shown to regulate skeletal muscle mass in developing livestock, rats, and mice (Trenkle 1976, Kobori & Yamamuro 1989, McCormick et al. 2004, Moran et al. 2007). Furthermore, ovariectomy decreases rat skeletal muscle mass recovery, and estrogen replacement benefits atrophied muscle mass recovery (Brown et al. 2005, Sitnick et al. 2006). Male mice lacking ERβ also exhibit altered muscle function (Glenmark et al. 2004).

By studying the effects on lean mass and weight of individual muscles and comparing gene expression after ER- and AR-mediated stimulation in muscle of gonadectomized mice, this study aimed to investigate whether the effect of testosterone on lean tissue could be due to a direct stimulatory effect on the AR or due to aromatization of testosterone to E2.

Materials and methods

Animals and study design

Mice were on a C57BL/6 background, and had free access to fresh water and soy-free food pellets (R70, Lactamin AB, Stockholm, Sweden or 2016, Harlan Teklad, UK). The ethics committee at the University of Gothenburg approved this study.

At 12 weeks of age, male mice were orx, and then treated for 5 weeks with DHT (45 μg/day), E2 (0.05 μg/day), or vehicle (veh) administered via subcutaneous silastic implants (Silclear Tubing; Degania Silicone, Ltd, Jordan Valley, Israel) in the cervical region (Vandenput et al. 2002). Gonadectomy and implantation of pellets were performed during the same surgical session for all experimental groups. At the end of the treatment period, dual X-ray absorption (DXA) measurements were performed in vivo (n=6–8 in each group). Then, m. quadriceps and m. gastrocnemius together with various organs were dissected, and their wet weights were determined. Blood was collected for analyses of serum insulin-like growth factor 1 (IGF1) concentration, and distal femur trabecular volumetric bone mineral density (vBMD) was determined ex vivo using peripheral quantitative computerized tomography (pQCT) (n=6–8 in each group). In addition, total RNA was isolated from snap-frozen m. gastrocnemius for analyses using microarray (n=5 in each group).

To determine the short-term effects of the hormone treatments on the expression of selected genes in muscle, a similar experiment as that described above was performed, but with a treatment period of only 1 week. Total RNA was isolated from snap-frozen m. gastrocnemius and m. levator ani for analyses using real-time PCR (RT-PCR).

DXA analysis

Body composition of mice was measured by DXA using the Lunar PIXImus Mouse Densitometer (Wipro GE Healthcare, Madison, WI, USA) with the mice under inhalation anesthesia with isoflurane (Forane; Abbot Scandinavia).

Peripheral quantitative computerized tomography

Distal femur trabecular vBMD was measured ex vivo using the Stratec pQCT XCT Research M (software version 5.4B; Norland Medical Systems Inc., White Plains, NY, USA) operating at a resolution of 70 μm (Windahl et al. 1999). The pQCT scan was positioned in the metaphysis at a distance from the distal growth plate corresponding to 3% of the total length of the femur, and the trabecular bone region was defined as the inner 45% of the total cross-sectional area.

DNA microarray analysis

Total RNA was isolated from snap-frozen m. gastrocnemius using RNeasy Mini Kit including an on-column DNase digestion step using the RNase-free DNase set (Qiagen). The mRNA samples derived from each individual mouse were reverse transcribed into cDNA, labeled, and analyzed using DNA microarray (mouse expression set 430; Affymetrix, Santa Clara, CA, USA) (n=5 in each group). Preparation of labeled cRNA, hybridization, and staining were done according to the Affymetrix Gene Chip expression analysis manual. The stained probe array was scanned, and the resultant image was captured as a data image (.CEL) file. The signal intensities for the β-actin (Actb) and the Gapdh genes were used as the internal quality controls. The ratio of fluorescent intensities for the 5′ end the 3′ end of these housekeeping genes was <3. The microarray data can be accessed at EMBL-EBI ArrayExpress repository, ArrayExpress accession: E-MEXP-2192.

Bioinformatics

To correct for variation between GeneChips, the signal data of CEL files of Affymetrix mouse expression set 430 chips were quantile normalized, with probe set intensities calculated using the Robust Multiarray Average (Irizarry et al. 2003). For each gene, a t-test was used to estimate the effect of treatment. A gene was considered regulated if it demonstrated a fold change ≥1.5 and P≤0.05 in response to E2 or DHT treatment compared with the veh. The mouse expression set 430 annotation file dated August 2008 was downloaded from Affymetrix, from which the gene title, gene symbol, gene ontology (GO) biological process, GO cellular component, and GO molecular functions of the regulated genes were identified. The genes were then grouped by their unique GO classifications (biological process, cellular component, and molecular function).

Quantitative RT-PCR analysis

Total RNA was isolated from snap-frozen m. gastrocnemius. The RT-PCR analysis was performed using the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Stockholm, Sweden). The mRNA abundance of each gene was calculated using the ‘standard curve method’ (User Bulletin 2; PE Applied Biosystems), and was adjusted for the expression of 18S. Primer and probe sequences are available upon request.

Serum assay

Serum IGF1 level was measured by a double-antibody IGF-binding protein-blocked RIA (Mediagnost, Tubingen, Germany).

Statistical analyses

All the descriptive statistical results are presented as the means±s.e.m. Between-group differences were calculated using unpaired t-tests. Comparisons between multiple groups were done using a one-way ANOVA followed by the Student–Newman–Keuls post hoc test. A two-tailed P≤0.05 was considered significant.

Results

Comparison of the effects of AR and ER stimulation in male mice

To compare the importance of AR versus ER stimulation in muscle in male mice, 12-week-old male orx mice were treated for 5 weeks with either veh, the non-aromatizable androgen DHT, or E2.

Seminal vesicles and bone

To test whether the DHT and E2 doses were physiological, we studied the effects on seminal vesicles and trabecular vBMD. The DHT dose used (45 μg/day) was physiological, since it completely prevented the orx-induced loss of weight of the seminal vesicles (Fig. 1A). As expected, E2 (0.05 μg/day) had no effect on the weight of the seminal vesicles (Fig. 1A). Both E2 and DHT prevented the orx-induced reduction in trabecular vBMD almost completely as measured using pQCT (Fig. 1B).

Figure 1
Figure 1

Seminal vesicle weight (A), trabecular volumetric bone mineral density (B), and serum IGF1 (C) in 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Values are given as means±s.e.m., n=6–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; P values are based on a one-way ANOVA followed by the Student–Newman–Keuls post hoc test.

Citation: Journal of Molecular Endocrinology 45, 1; 10.1677/JME-09-0165

Serum IGF1

Orx had no effect on serum IGF1 levels. E2 treatment increased serum IGF1 by 18.4% (P≤0.05 versus sham, Fig. 1), whereas DHT treatment did not differ significantly from the sham-operated controls (Fig. 1).

Lean tissue and muscle weight

As measured using DXA, there was a loss of whole-body lean tissue mass by 9.4% after orx (P≤0.01 versus sham) that was prevented partly by E2 and completely by DHT (Fig. 2). E2 treatment resulted in an increased lean mass by 3.9% (P≤0.05 versus orx+veh), and DHT treatment resulted in even further increased lean mass (5.6% over orx+E2, P≤0.05, and 9.7% over orx+veh, P≤0.01). These results were confirmed by dissection and weighing of m. quadriceps and m. gastrocnemius (Fig. 2).

Figure 3
Figure 3

To verify the microarray data, the transcript levels of six genes (Igf1, Fbxo32, Grb10, Gpx3, Odc1, and Myl3) were quantified by RT-PCR. Analysis was performed on individual muscle samples from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). 18S was included as an internal control. Data are expressed as percentage of Orx, and are presented as means±s.e.m., n=4–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; #P≤0.01 versus orx+E2; P≤0.05 versus orx+E2. Microarray data are shown for comparison.

Citation: Journal of Molecular Endocrinology 45, 1; 10.1677/JME-09-0165

Figure 2
Figure 2

Lean tissue that was measured by DXA (A) and wet weight of dissected quadriceps muscle (B) and gastrocnemius muscle (C) from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Values are given as means±s.e.m., n=6–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus orx+veh; §P≤0.05 versus orx+E2; #P≤0.01 versus orx+E2. P values are based on a one-way ANOVA followed by the Student–Newman–Keuls post hoc test.

Citation: Journal of Molecular Endocrinology 45, 1; 10.1677/JME-09-0165

The effects of E2 and DHT treatments on gene expression in skeletal muscle

Microarray analyses were performed on gastrocnemius muscle samples from individual mice (n=5 in each group) to compare global gene expression after treating orx mice for 5 weeks with either DHT or E2. Genes regulated by E2 or DHT (fold change ≥1.5 and P≤0.05 versus veh) were assigned to several functional categories (Table 1). A complete catalog of these genes is published as supplementary data (Supplementary Table 1, see section on supplementary data given at the end of this article). DHT regulated 187 (133 up-regulated and 54 down-regulated) genes in muscle (Table 1). Fewer genes (n=51) were regulated after E2 treatment (33 up-regulated and 18 down-regulated, Table 1). Although a treatment period of 5 weeks would mean that several of these genes may reflect secondary effects of hormone treatment, we refer to them as E2/DHT-regulated genes for simplicity.

Table 1

Number of regulated genes in mouse skeletal muscle assigned to functional categories

Regulated genes E2Regulated genes DHT
GO IDTotalUpDownTotalUpDown
Classification
Transcription000635032118126
Signal transduction000716584423176
Metabolic process
 Glucose0006006321
 Lipid0006629211
 Cholesterol000820322
 Ketone body004695011
 Glutathione000674911321
 Others1133
Mitochondrial respiratory chain000574622
Protein metabolism and modification
 Synthesis413
 Degradation00301635411183
 Post-translation modification004368722321
Ion/substrate transport00228923317152
Cell adhesion0007155541
Apoptosis000691522
Cell cycle0007049211422
Microtubule, cytoskeleton organization000022622
Angiogenesis000152521111
Muscle contraction0006936111010
Polyamine biosynthetic process000659633
Extracellular matrix constituent0005201862
Immune response000695521122
Actin binding0003779431
Cellular iron ion homeostasis000687911
Heme biosynthetic process000678311
Cellular calcium ion homeostasis000687411
Synaptic transmission000726833
Multicellular organism development000727533431
Unknown function85335287
Unknown identity5514104
Total responsive genes51331818713354

Microarray analyses of m. gastrocnemius from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). A gene was considered regulated if it demonstrated a fold change ≥1.5 and P≤0.05 (t-test) in response to E2 or DHT treatment compared with the vehicle, n=5 in each group. GO ID, gene ontology biological process identity nos.

Regulated genes in common for DHT and E2

DHT and E2 regulated different sets of genes, except 13 genes (=25% of E2-regulated genes) that were regulated by both treatments (5.8% of the total number of regulated genes, Table 2). Among them was Fbxo32, a gene encoding a muscle-specific F-box protein implicated in muscle atrophy that was up-regulated by both treatments. Five of the regulated genes had unknown function (Table 2).

Table 2

Regulated genes in common for 17β-estradiol (E2) and dihydrotestosterone (DHT) treatments in mouse skeletal muscle

Fold change
SymbolE2DHTFunction
Gene
Up-regulated genes in common after E2 and DHT treatments
 FK506-binding protein 5Fkbp51.8*1.8*Protein post-translational modification
 F-box protein 32Fbxo321.62.2*Protein degradation
 Potassium voltage-gated channel, subfamily Q, member 5Kcnq52.6*2.3*Ion/substrate transport
 Ribosomal protein S6 kinase, polypeptide 5Rps6ka51.5*1.6Transcription
 RIKEN cDNA 5330406M23 gene5330406M23Rik1.6*1.6Unknown
 ARP3 actin-related protein 3 homolog B Actr3b1.5*2.1*Unknown
 Nudix-type motif 18Nudt181.6*1.9*Unknown
 CDKN2A interacting protein N-terminal likeCdkn2aipnl1.6*1.6Unknown
 Phosphodiesterase 4D interacting protein Pde4dip1.6*1.5Unknown
Down-regulated genes in common after E2 and DHT treatments
 Gremlin 2 Grem21.62.4*Signal transduction
 Myosin, light polypeptide 3Myl31.51.8*Muscle contraction
 HtrA serine peptidase 4Htra41.61.5*Protein degradation
 Tumor necrosis factor, α-induced protein 2Tnfaip21.91.5Multicellular organismal development

Microarray analyses of m. gastrocnemius from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and treated for 5 weeks with vehicle (veh), DHT (45 μg/day), or E2 (0.05 μg/day). Values are given as fold change, n=5 in each group. *P≤0.01 versus vehicle; P≤0.05 versus vehicle. t-test.

IGF1 signalling

In muscle, E2 decreased the expression of Igf1 (Table 3). The IGF1 signaling pathway includes many signaling molecules that are also important for signaling through the insulin receptor. E2 up-regulated one of these signaling molecules, Tbc1d1 (Table 3). DHT decreased the expression of Grb10, an inhibitor of IGF1 signaling, and Socs2, a suppressor of cytokine signaling. Furthermore, there was an increased expression of Shp2, a phosphatase important for IGF1 signaling (Table 3).

Table 3

Regulated genes in mouse skeletal muscle after 17β-estradiol (E2) and dihydrotestosterone (DHT) treatments in selected functional categories of importance for skeletal muscle function and development

Gene symbolFold changeDown/upP
Gene title
Genes regulated after E2 treatment
IGF1 signalling
 Insulin-like growth factor 1Igf11.6Down<0.01
 TBC1 domain family, member 1Tbc1d12.3Up<0.01
Fuel metabolism
 Pyruvate carboxylasePcx3.1Up<0.01
 Fructose bisphosphatase 2Fbp21.6Up<0.01
 Carboxylesterase 3Ces31.6Up<0.01
 Stearoyl-coenzyme A desaturase 1Scd11.8Down<0.01
Glutathione metabolic process
 Glutathione peroxidase 3Gpx32.2Up<0.01
Angiogenesis
 Angiopoietin 1Angpt11.6Up0.046
 Thrombospondin 1Thbs11.5Down0.021
Muscle contraction
 Myosin, light polypeptide 3Myl31.5Down0.025
Genes regulated after DHT treatment
IGF1 signalling
 Growth factor receptor-bound protein 10Grb101.5Down<0.01
 Suppressor of cytokine signaling 2Socs21.8Down0.038
 Protein tyrosine phosphatase, non-receptor type 11Ptpn111.5Up<0.01
Fuel metabolism
 3-Oxoacid CoA transferase 1Oxct11.8Down<0.01
 Niemann–Pick type C1Npc11.8Up0.012
 Very low-density lipoprotein receptorVldlr1.6Up<0.01
Mitochondrial ATP synthesis-coupled proton transport
 NADH dehydrogenase (ubiquinone) Fe-S protein 4Ndufs41.6Up<0.01
 Carbonyl reductase 2Cbr22.2Up<0.01
Glutathione metabolic process
 Glutathione S-transferase mu 2 Gstm22.5Down<0.01
 Microsomal glutathione S-transferase 3Mgst31.8Up<0.01
 Microsomal glutathione S-transferase 1Mgst11.9Up<0.01
Angiogenesis
 Angiotensin receptor-like 1Agtrl11.6Up0.017
Synaptic transmission
 Muscle, skeletal, receptor tyrosine kinaseMusk1.6Up0.011
 Ly6/neurotoxin 1Lynx11.6Up<0.01
 Discs, large homolog-associated protein 4Dlgap41.6Up<0.01
Heme biosynthetic process
 Transferrin receptorTfrc1.8Up<0.01
 Aminolevulinic acid synthase 1Alas11.8Up<0.01
Polyamine biosynthesis
 S-Adenosylmethionine decarboxylaseAmd12.3Up<0.01
 Ornithine decarboxylase, structural 1Odc12.8Up<0.01
 Spermine oxidaseSmox2.6Up<0.01
Muscle contraction
 Myosin, light polypeptide 3 Myl31.6Down <0.01
 Myosin, heavy polypeptide 6 Myh61.7Down <0.01
 Troponin ITnni12.1Down<0.01
 Myosin, light polypeptide 2 Myl22.2Down<0.01
 Troponin C Tnnc12.2Down<0.01
 Myosin, heavy polypeptide 7 Myh72.6Down<0.01
 Actin, α, cardiac muscle 1 Actc13.8Down<0.01
 Myosin-binding protein HMybph11.9 Down<0.01
 Troponin T1 Tnnt12.8Down<0.01
 Tropomyosin 3Tpm32.2Down0.017
Extracellular matrix structural constituent
 Collagen, type I, α1Col1a11.6Up0.044
 Collagen, type V, α2Col5a21.7Up0.015
 Collagen, type I, α2Col1a21.7Up0.049
 Collagen, type III, α1Col3a11.8Up0.013
 Collagen, type IV, α2Col4a21.9Up<0.01
 Collagen, type IV, α1Col4a12.2Up<0.01
 LumicanLum1.7Down<0.01
 Collagen, type XIV, α1Col14a11.7Down<0.01

Regulated genes after E2 and DHT treatments in IGF1 signaling, fuel metabolism, mitochondrial ATP synthesis-coupled proton transport, glutathione metabolic process, angiogenesis, synaptic transmission, heme biosynthetic process, polyamine biosynthesis, muscle contraction, and extracellular matrix structural constituents. Microarray analyses of m. gastrocnemius from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and treated for 5 weeks with vehicle (veh), DHT (45 μg/day), or E2 (0.05 μg/day). Values are given as fold change, n=5 in each group. t-test.

Fuel metabolism

In muscle, E2 increased the expression of two genes involved in glucose metabolism: Pcx, which encodes an enzyme converting pyruvate to oxaloacetate, and Fbp2, which encodes an enzyme active in glyconeogenesis. There was an increased expression of Ces3, which encodes a lipase mediating hydrolysis of triglycerides in muscle, and a decreased expression of Scd1, which encodes an enzyme active in triglyceride synthesis (Table 3). After DHT treatment, there was a decreased expression of Oxct1, which encodes an enzyme active in the catabolism of ketone bodies. DHT increased the expression of two genes involved in cholesterol metabolism: Npc1, which encodes a protein that regulates the transport of cholesterol in the cell, and Vldl receptor, which encodes a peripheral lipoprotein receptor. Furthermore, DHT increased the expression of two genes encoding proteins involved in mitochondrial ATP synthesis-coupled proton transport, Ndufs4 and Cbr2 (Table 3).

Both E2 and DHT increased the expression of genes in the glutathione metabolic pathway that functions in the detoxification of hydrogen peroxide, protecting cells from oxidative damage (Table 3).

Angiogenesis

Both E2 and DHT affected the expression of genes involved in angiogenesis. E2 up-regulated Angpt1, which is a promoter of angiogenesis, and down-regulated Thbs1, which is a negative regulator of angiogenesis (Table 3). DHT up-regulated Agtrl1, also known as the apelin receptor, which is active during angiogenesis (Table 3).

Synaptic transmission

Skeletal muscle fibers are innervated by motor neurons, and DHT up-regulated three genes involved in synaptic transmission in muscle: Musk, Lynx1, and Dlgap4 (Table 3).

Heme biosynthetic process

The transferrin receptor (Tfrc), which maintains cellular iron ion homeostasis by importing iron into the cell, and Alas1, a rate-controlling mitochondrial heme biosynthetic enzyme, were both up-regulated after DHT treatment (Table 3).

Muscle contraction and extracellular matrix structural constituent

The gene expression of ten myofibrillar proteins was down-regulated after DHT treatment, and that of one myofibrillar protein was down-regulated after E2 treatment (Table 3). Among the genes that encode the structural proteins that constitute the extracellular matrix including tendons, six different collagen genes were up-regulated after DHT treatment, and one gene was down-regulated. A collagen-binding protein lumican was also down-regulated after DHT treatment (Table 3).

Polyamine biosynthesis

Three rate-limiting polyamine biosynthetic enzymes, Amd1, Odc1, and Smox, were highly up-regulated after DHT treatment (Table 3).

Verification of microarray results

To verify the microarray data, the transcript levels of six genes (Igf1, Fbxo32, Grb10, Gpx3, Odc1, and Myl3) were quantified by RT-PCR. Analysis was performed on individual muscle samples, with 18S included as an internal control. As shown in Fig. 3, the regulation of all genes, except that of Myl3, was confirmed.

For the RT-PCR experiment, we also included the sham-operated control group which added some further information on the regulation of these genes. The expression of Fbxo32 was decreased by 42.3% after orx (P<0.01 versus sham, Fig. 3), and it was normalized by both E2 and DHT treatments to orx mice (Fig. 3). Igf1 expression was decreased in orx mice treated with E2 (−54.8%, P<0.01 versus sham and −48.5%, P<0.05 versus orx), while orx by itself or DHT treatment to orx mice had no effect (Fig. 3). Orx resulted in an increase in Grb10 expression (43.3%, P<0.01 versus sham, Fig. 3), which was normalized by DHT treatment but not by E2 treatment to orx mice. Orx and DHT had no effect on Gpx3 expression, but E2 treatment to orx mice resulted in a dramatic increase (251%, P<0.01 versus sham and 225%, P<0.01 versus orx, Fig. 3). Orx resulted in a decrease in Odc1 expression (−52%, P<0.01 versus sham, Fig. 3), which was normalized by DHT treatment but not by E2 treatment to orx mice.

Time course study of regulated genes

To determine the short-term effects of the hormone treatments on the expression of selected genes in muscle, an experiment with a treatment period of only 1 week was performed. After 1-week treatment, the expression of Fbxo32 was increased in m. gastrocnemius (72%, P<0.01 versus sham, Fig. 4A). There was a tendency to normalization of Fbxo32 expression levels with E2 and DHT, but it did not reach statistical significance (Fig. 4A). For comparison, we analyzed m. levator ani, a fast-twitch muscle known to be testosterone dependent in rodents. The effect on Fbxo32 expression was much more dramatic in m. levator ani, although in the same direction as in m. gastrocnemius, and DHT by itself could completely normalize Fbxo32 expression levels after orx (Fig. 4B). The higher levels of Fbxo32 in m. gastrocnemius after orx were only transient, and after 5-week treatment, the levels were similar for the orx group compared with the orx group after 1-week treatment but had increased for all other groups (Fig. 4C). For the other four genes that were analyzed (Igf1, Grb10, Gpx3, and Odc1), there was no major difference in expression pattern between 1- and 5-week treatments (Table 4). There was no weight difference in m. gastrocnemius between different treatment groups in the 1-week experiment (data not shown).

Figure 4
Figure 4

Time course study for Fbxo32 expression in muscle. RT-PCR analysis was performed on individual muscle samples from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 1 (A and B) or 5 (C) weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Fbxo32 expression in m. gastrocnemius (A) and m. levator ani (B) is presented after 1-week treatment for comparison and in m. gastrocnemius (C) after 5-week treatment. Fbxo32 mRNA levels are corrected for the expression of 18S, and are presented as means±s.e.m., n=4–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; #P≤0.01 versus orx+E2.

Citation: Journal of Molecular Endocrinology 45, 1; 10.1677/JME-09-0165

Table 4

Time course study for the expression of regulated genes in muscle. Data are expressed as percentage of orchidectomized (Orx), and are presented as means±s.e.m., n=4–8 in each group

One-week treatmentFive-week treatment
ShamOrx vehE2DHTShamOrx vehE2DHT
Igf1137±15100±1642±5*,§93±12,114±10100±1452±7*,§82±11
Grb1071±4100±4*70±357±4,,70±6100±7*98±7*55±4,
Gpx3112±11100±10274±11*,75±1292±10100±7325±24*,89±4
Odc1159±12100±5*80±5*193±30,210±29100±6*90±6*226±33,

Time course study for the expression of five genes (Igf1, Fbxo32, Grb10, Gpx3, and Odc1) by RT-PCR. Analysis was performed on individual muscle samples from 12-week-old male mice that were either sham-operated or orx and then treated for 1 or 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). 18S was included as an internal control. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; P≤0.01 versus orx+E2; P≤0.05 versus orx+E2.

Discussion

Testosterone treatment of orx mice has previously been shown to induce a dose-dependent increase in the mass of individual muscles (Axell et al. 2006). However, how much of this effect is through aromatization of testosterone to E2 is unknown. To differentiate between the direct effects of testosterone and those of testosterone aromatized to E2 on muscle mass, we treated orx mice with E2 and the non-aromatizable androgen DHT. Although the effect of DHT was more marked, both treatments prevented loss of muscle mass after gonadectomy, indicating that AR- and ER-mediated signaling has effects on muscle mass that are independent of each other. The doses used were likely in the physiological range as DHT completely prevented the orx-induced loss of seminal vesicle weight, and E2 and DHT almost completely prevented the orx-induced reduction in trabecular vBMD.

To investigate the mechanism underlying the effect on muscle mass, we performed microarray and RT-PCR analyses on skeletal muscle samples. In line with a more profound effect of DHT on muscle mass, the microarray analyses demonstrated that a greater number of genes were regulated after DHT treatment (n=187) than after E2 treatment (n=51). Thirteen genes were regulated by both E2 and DHT, and these could represent mechanisms in common for how activation of AR and ER signaling stimulates skeletal muscle mass. However, the results indicate that most of the effects of E2 and DHT in skeletal muscle of male mice are mediated by different mechanisms. Even when E2 and DHT affected the same pathways, such as IGF1 signaling, increased angiogenesis, and glutathione oxidation, it was done by regulating different sets of genes.

Regulated genes in common for DHT and E2

Among the eight genes with known function that were regulated by both DHT and E2, increased expression was observed for Kcnq5, which encodes a potassium channel that is involved in skeletal muscle cell proliferation by triggering changes in membrane potential and regulating cell volume (Roura-Ferrer et al. 2008). Surprisingly, Fbxo32, a gene encoding a muscle-specific F-box protein implicated in muscle atrophy, was up-regulated by both treatments. The RT-PCR analyses demonstrated that this up-regulation was a normalization of Fbxo32 expression to the same levels as in the sham-operated controls. In multiple models of skeletal muscle atrophy, Fbxo32 is up-regulated, and it appears to be essential for accelerated muscle protein degradation in a variety of experimental models of catabolism, including diabetes, cancer, AIDS, fasting, renal failure, hindlimb suspension, immobilization, oxidative stress, and sepsis (Sacheck et al. 2007). In a recent study done by Pires-Oliveira et al. (2010), Fbxo32 expression was high in rat m. levator ani over a week after castration, and this increase could be reversed by treatment with testosterone. Interestingly, in another recent study done by Rogers et al. (2010), mice showed decreased expression of Fbxo32 12 weeks after castration similar to what was observed in our study. In a short-term experiment of only 1 week, we could show that these seemingly contradictory results depend on the fact that the increase of Fbxo32 with gonadectomy is only transient and could be partly reversed by treatment with E2 and fully reversed by treatment with DHT in m. levator ani. After a 5-week treatment period, the levels of Fbxo32 had increased in all groups but stayed on similar levels in the orx groups. The transient increase of Fbxo32 probably reflects that accelerated muscle loss occurs early and transiently after castration, and that at least in m. levator ani, which is a fast-twitch muscle, this loss can be partly prevented by E2 and fully prevented by DHT.

The size of pre-existing muscle fibers is decreased in skeletal muscle atrophy, and in line with this, one myofibrillar protein was decreased after both DHT and E2 treatments and ten myofibrillar proteins were decreased after DHT treatment alone, despite the observed prevention of loss in muscle mass. Our hypothesis is that a decrease in muscle mass probably occurs very early after orx, and that the biosynthesis and breakdown of myofibrillar proteins have began to enter, or already reached, a new steady state after 4 weeks.

Extracellular matrix structural constituents

Collagens are the main proteins of the extracellular matrix including tendons that surround and support muscle fibers. The expression of six different collagen genes was up-regulated after DHT treatment, indicating that synthesis of more extracellular matrix was an ongoing process. The GH/IGF1 axis exerts, like testosterone and E2, anabolic effects on lean body mass. In a previous study, in which muscle biopsies were analyzed using microarray before and after 2-week GH treatment to hypopituitary men with hypogonadism, we also observed increased synthesis of collagens and a decreased expression of several myofibrillar proteins (Sjogren et al. 2007).

IGF1 signaling

IGF1 is a potent anabolic agent in muscle (Musaro et al. 2001). After E2 treatment, the importance of the observed changes in the IGF1 system for muscle function was not clear as serum levels of IGF1 were increased, whereas the local gene expression of Igf1 in muscle was reduced. However, increased serum IGF1 levels have also been observed in postmenopausal women treated with transdermal E2 (Weissberger et al. 1991, Ho et al. 2003).

DHT treatment decreased the expression of Grb10, a negative regulator of IGF1 signaling (Dufresne & Smith 2005). In line with IGF1 being an anabolic agent in muscle, mice with a disrupted Grb10 gene have increased muscle and lean mass (Smith et al. 2007). The RT-PCR analyses showed that DHT but not E2 could normalize Grb10 expression after orx, indicating that this is an AR-mediated effect. Interestingly, the expression of Grb10 in muscle has been shown to be gender dependent with higher expression in women than in men, suggesting a role for Grb10 in the sexual dimorphism of skeletal muscle mass (Welle et al. 2008). Furthermore, DHT reduced the expression of the Socs2 gene in muscle. SOCS2 reduces GH signaling; therefore, a down-regulation of Socs2 expression in muscle likely increases the effect of GH/IGF1 locally in muscle. Together these indicate that the AR-mediated effects on muscle mass include increased local effect of GH/IGF1 by a down-regulation of Grb10 and Socs2.

Angiogenesis

Increased angiogenesis would result in improved microcirculation in muscle, and hence would provide an increased oxygen and nutrient supply as well as lead to the removal of waste products. E2 up-regulated Angpt1, an angiogenesis promoter during embryonic development, essential to endothelial cell survival, vascular branching, and pericyte recruitment (Carmeliet 2000). Furthermore, E2 down-regulated Thbs1, a potent angiogenesis inhibitor (Lawler 2002). Agtrl1 or the apelin receptor was up-regulated by DHT. Apelin through its receptor AGTRL1 is involved in the regulation of blood vessel diameter during angiogenesis, and apelin-deficient mice showed narrow blood vessels during embryogenesis (Kidoya et al. 2008). Together these results point to increased angiogenesis in muscle after both E2 and DHT treatments.

Glutathione metabolism

Both E2 and DHT increased the expression of genes in the glutathione metabolic pathway that protects cells from oxidative damage. One of these genes, Gpx3, was markedly up-regulated in muscle by E2. Gpx3 has earlier been shown to be directly regulated by E2 in white adipose tissue (Lundholm et al. 2008). Gpx3 improves skeletal muscle insulin sensitivity by mediating the antioxidant effect of PPARγ (PPARG; Chung et al. 2009), and increased glutathione peroxidase activity could mediate the improvement of muscle strength by selenium treatment in selenium-deficient patients (Brown et al. 1986).

Fuel metabolism

E2 had effects on genes regulating intermediary glucose and lipid metabolism. Pyruvate carboxylase, a mitochondrial protein that is important for intermediary metabolism by catalyzing the carboxylation of pyruvate to form oxaloacetate, was strongly up-regulated by E2. Oxaloacetate is an intermediate of the citric acid cycle and glyconeogenesis. Fbp2, expressing an enzyme that catalyzes the hydrolysis of fructose-1,6-bisphosphate into fructose-6-phosphate and is critical in glyconeogenesis pathway, was also up-regulated after E2 treatment, indicating increased glycogen synthesis in muscle. Ces3, which mediates some or all of the lipolysis that is independent of hormone-sensitive lipase (Soni et al. 2004), was up-regulated in muscle by E2, indicating increased utilization of free fatty acids as fuel.

Synaptic transmission

DHT up-regulated several genes involved in synaptic transmission in muscle, among them was Musk, which encodes a receptor tyrosine kinase that is essential for synapse formation and the development of new neuromuscular junctions (Kim & Burden 2008), indicating a role for AR-mediated signaling in this process.

Heme biosynthetic process

DHT also affected the expression in muscle of genes important for the supply of oxygen. Iron, which is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin, is delivered to cells via binding to the Tfrc (Xu et al. 2005). Tfrc together with Alas1, a rate-controlling mitochondrial heme biosynthetic enzyme, was up-regulated after DHT treatment, suggesting increased heme biosynthesis.

Polyamine biosynthesis

Three rate-limiting polyamine biosynthetic enzymes, Amd1, Odc1, and Smox, were highly up-regulated after DHT treatment. In a study measuring the short-term effects of DHT on gene expression in muscle of castrated mice, genes involved in polyamine synthesis were also up-regulated, indicating a direct effect of DHT on this process (Yoshioka et al. 2006). Data suggest that polyamines play a role in muscle hypertrophy, although their mechanisms of action are still unknown (Abukhalaf et al. 2002). Furthermore, male ARKO mice have impaired skeletal muscle development and function, which were associated with decreased expression of polyamine biosynthetic enzymes (MacLean et al. 2008). This indicates a role for these enzymes in the AR-mediated effects on muscle.

In conclusion, activation of both ER and AR preserves muscle and lean mass after gonadectomy in male mice, although the effect was more marked after AR activation. Few genes were regulated by both E2 and DHT, and when E2 and DHT regulated the same pathways (IGF1 signaling, increased angiogenesis, and glutathione oxidation), it was done by affecting different sets of genes. However, short-term treatment with E2 partly prevented and with DHT fully prevented the transient increase in Fbxo32 expression observed after gonadectomy. E2 alone had effects on genes involved in glucose and lipid metabolism, likely increasing glycogen synthesis and utilization of FAs for fuel in muscle. DHT alone regulated the expression of genes influencing synaptic formation and transmission, oxygen transport, and polyamine biosynthesis. Based on these findings, it could be hypothesized that the metabolic effects of testosterone in skeletal muscle of male mice are to a large extent dependent on ER activation, whereas other effects of testosterone in skeletal muscle are more dependent on AR activation.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1677/JME-09-0165.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by the Swedish Research Council, the Swedish Foundation for Strategic Research, the ALF/LUA research grant in Gothenburg, the Lundberg Foundation, the Torsten and Ragnar Söderberg's Foundation, the Novo Nordisk Foundation, the Magnus Bergvall Foundation, the Åke Wiberg Foundation, the Tore Nilsson Foundation, and the Swedish Society for Medical Research.

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Supplementary Materials

 

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    Seminal vesicle weight (A), trabecular volumetric bone mineral density (B), and serum IGF1 (C) in 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Values are given as means±s.e.m., n=6–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; P values are based on a one-way ANOVA followed by the Student–Newman–Keuls post hoc test.

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    To verify the microarray data, the transcript levels of six genes (Igf1, Fbxo32, Grb10, Gpx3, Odc1, and Myl3) were quantified by RT-PCR. Analysis was performed on individual muscle samples from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). 18S was included as an internal control. Data are expressed as percentage of Orx, and are presented as means±s.e.m., n=4–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; #P≤0.01 versus orx+E2; P≤0.05 versus orx+E2. Microarray data are shown for comparison.

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    Lean tissue that was measured by DXA (A) and wet weight of dissected quadriceps muscle (B) and gastrocnemius muscle (C) from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 5 weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Values are given as means±s.e.m., n=6–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus orx+veh; §P≤0.05 versus orx+E2; #P≤0.01 versus orx+E2. P values are based on a one-way ANOVA followed by the Student–Newman–Keuls post hoc test.

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    Time course study for Fbxo32 expression in muscle. RT-PCR analysis was performed on individual muscle samples from 12-week-old male mice that were either sham-operated or orchidectomized (orx) and then treated for 1 (A and B) or 5 (C) weeks with vehicle (veh), dihydrotestosterone (DHT) (45 μg/day), or 17β-estradiol (E2) (0.05 μg/day). Fbxo32 expression in m. gastrocnemius (A) and m. levator ani (B) is presented after 1-week treatment for comparison and in m. gastrocnemius (C) after 5-week treatment. Fbxo32 mRNA levels are corrected for the expression of 18S, and are presented as means±s.e.m., n=4–8 in each group. *P≤0.01 versus sham; P≤0.01 versus orx+veh; P≤0.05 versus sham; §P≤0.05 versus orx+veh; #P≤0.01 versus orx+E2.