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Stimulation of Both Estrogen and Androgen Receptors Maintains Skeletal Muscle Mass in Gonadectomized Male Mice but Mainly Via Different Pathways

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Stimulation of Both Estrogen and Androgen Receptors Maintains Skeletal Muscle Mass in Gonadectomized Male Mice but Mainly Via Different Pathways 45 Stimulation of both estrogen and androgen receptors maintains skeletal muscle mass in gonadectomized male mice but mainly via different pathways Johan Svensson, Sofia Move´rare-Skrtic, Sara Windahl, Charlotte Swanson and Klara Sjo¨ gren Division of Endocrinology, Department of Internal Medicine, Institute of Medicine, Sahlgrenska University Hospital, SE-41345 Go¨teborg, Sweden (Correspondence should be addressed to K Sjo¨gren; Email: [email protected]) 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 (Z25% 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. Journal of Molecular Endocrinology (2010) 45, 45–57 Introduction muscle mass and impaired muscle function were observed in male ARKO mice but not in female Testosterone is a major regulator of body composition. ARKO mice (Lin et al. 2005, MacLean et al. 2008). Androgen deficiency is associated with decreased Ophoff et al. (2009) recently reported that a myocyte- specific knockout of the AR in male mice resulted in muscle mass, and testosterone supplementation decreased lean mass and a conversion of fast towards increases muscle mass in hypogonadal men, HIV- slow muscle fibers, without affecting muscle strength or infected men, and older men with low testosterone fatigue. In their study, similar results were obtained in concentrations (Snyder et al. 1999, 2000, Kong & male mice with ubiquitous ARKO. Edmonds 2002). Also, in orchidectomized (orx) mice, Testosterone can exert its effect either directly by testosterone treatment dose dependently increases the stimulation of the AR or via aromatization in target mass of individual muscles (Axell et al. 2006). tissues to estradiol (E2), the ligand for the estrogen Both testosterone and the non-aromatizable andro- receptors (ERs) a and b (also known as ESR1 and ESR2 gen dihydrotestosterone (DHT) bind to and activate respectively). Muscle from both men and women the androgen receptor (AR; MacLean et al. 1997). contains aromatase enzyme activity (Matsumine et al. The AR gene is expressed widely in muscle including 1986). The extent to which the actions of testosterone myoblasts, myofibers, and satellite cells (Chen et al. in muscle are a consequence of AR or ER activation or 2005). The AR is also expressed in motor neurons both is not clear. which may contribute to the regulation of muscle mass Skeletal muscle myoblasts, myotubes, and mature and function (Yang & Arnold 2000). Several different fibers all express functional ERs, indicating a direct AR knockout (ARKO) mouse models have been effect of estrogen in muscle (Kahlert et al. 1997, Barros reported. In one of these models, muscle mass was et al. 2006). Women after menopause have decreased unchanged, whereas in another model, decreased lean body mass, which can be reversed by estrogen Journal of Molecular Endocrinology (2010) 45, 45–57 DOI: 10.1677/JME-09-0165 0952–5041/10/045–045 q 2010 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 01:48:03PM via free access 46 J SVENSSON and others . Effects of ER and AR stimulation in muscle replacement therapy (Sorensen et al. 2001). In animals, DXA analysis estrogen has been shown to regulate skeletal muscle Body composition of mice was measured by DXA using mass in developing livestock, rats, and mice (Trenkle the Lunar PIXImus Mouse Densitometer (Wipro GE 1976, Kobori & Yamamuro 1989, McCormick et al. 2004, Healthcare, Madison, WI, USA) with the mice under Moran et al. 2007). Furthermore, ovariectomy decreases inhalation anesthesia with isoflurane (Forane; Abbot rat skeletal muscle mass recovery, and estrogen Scandinavia). replacement benefits atrophied muscle mass recovery (Brown et al. 2005, Sitnick et al. 2006). Male mice lacking ERb also exhibit altered muscle function (Glenmark et al. 2004). Peripheral quantitative computerized tomography By studying the effects on lean mass and weight of Distal femur trabecular vBMD was measured ex vivo individual muscles and comparing gene expression using the Stratec pQCT XCT Research M (software after ER- and AR-mediated stimulation in muscle of version 5.4B; Norland Medical Systems Inc., White gonadectomized mice, this study aimed to investigate Plains, NY, USA) operating at a resolution of 70 mm whether the effect of testosterone on lean tissue could (Windahl et al. 1999). The pQCT scan was positioned in be due to a direct stimulatory effect on the AR or due to the metaphysis at a distance from the distal growth plate aromatization of testosterone to E . 2 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. Materials and methods Animals and study design DNA microarray analysis Mice were on a C57BL/6 background, and had free Total RNA was isolated from snap-frozen m. gastro- access to fresh water and soy-free food pellets (R70, cnemius using RNeasy Mini Kit including an on-col- Lactamin AB, Stockholm, Sweden or 2016, Harlan umn DNase digestion step using the RNase-free DNase Teklad, UK). The ethics committee at the University of set (Qiagen). The mRNA samples derived from each Gothenburg approved this study. individual mouse were reverse transcribed into cDNA, At 12 weeks of age, male mice were orx, and then labeled, and analyzed using DNA microarray (mouse treated for 5 weeks with DHT (45 mg/day), E2 expression set 430; Affymetrix, Santa Clara, CA, USA) (0.05 mg/day), or vehicle (veh) administered via (nZ5 in each group). Preparation of labeled cRNA, subcutaneous silastic implants (Silclear Tubing; hybridization, and staining were done according to the Degania Silicone, Ltd, Jordan Valley, Israel) in the Affymetrix Gene Chip expression analysis manual. cervical region (Vandenput et al. 2002). Gonadectomy The stained probe array was scanned, and the and implantation of pellets were performed during the resultant image was captured as a data image (.CEL) same surgical session for all experimental groups. file. The signal intensities for the b-actin (Actb) and At the end of the treatment period, dual X-ray the Gapdh genes were used as the internal quality absorption (DXA) measurements were performed controls. The ratio of fluorescent intensities for the 50 in vivo (nZ6–8 in each group). Then, m. quadriceps end the 30 end of these housekeeping genes was !3. and m. gastrocnemius together with various organs The microarray data can be accessed at EMBL-EBI were dissected, and their wet weights were determined. ArrayExpress repository, ArrayExpress accession: Blood was collected for analyses of serum insulin-like E-MEXP-2192. growth factor 1 (IGF1) concentration, and distal femur trabecular volumetric bone mineral density (vBMD) was determined ex vivo using peripheral quantitative Bioinformatics computerized tomography (pQCT) (nZ6–8 in each group). In addition, total RNA was isolated from To correct for variation between GeneChips, the signal snap-frozen m. gastrocnemius for analyses using data of CEL files of Affymetrix mouse expression set microarray (nZ5 in each group). 430 chips were quantile normalized, with probe set To determine the short-term effects of the hormone intensities calculated using the Robust Multiarray treatments on the expression of selected genes in Average (Irizarry et al. 2003). For each gene, a t-test muscle, a similar experiment as that described above was used to estimate the effect of treatment. A gene was was performed, but with a treatment period of only considered regulated if it demonstrated a fold change R % 1 week. Total RNA was isolated from snap-frozen m. 1.5 and P 0.05 in response to E2 or DHT treatment gastrocnemius and m. levator ani for analyses using compared with the veh. The mouse expression set 430 real-time PCR (RT-PCR). annotation file dated August 2008 was downloaded Journal of Molecular Endocrinology (2010) 45, 45–57 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 01:48:03PM via free access Effects of ER and AR stimulation in muscle . J SVENSSON and others 47 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 Seminal vesicle weight (biological process, cellular component, and molecular Sham function). A 350 † Orx + veh Orx + E2 300 Orx + DHT Quantitative RT-PCR analysis 250 Total RNA was isolated from snap-frozen m. gastro- cnemius. The RT-PCR analysis was performed using 200 the ABI Prism 7000 Sequence Detection System (PE Applied Biosystems, Stockholm, Sweden).
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