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Accepted Preprint first posted on 30 April 2010 as Manuscript JME-09-0165
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Both Estrogen and Androgen Receptor Stimulation Maintains Skeletal Muscle Mass
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in Gonadectomized Male Mice but mainly via Different Pathways
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Johan Svensson, Sofia Movérare-Skrtic, Sara Windahl, Charlotte Swanson, and
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Klara Sjögren
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Division of Endocrinology, Institute of Medicine, Sahlgrenska University Hospital, 413
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45 Göteborg, Sweden
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Running head: Effects of ER and AR stimulation in muscle
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Key words: Estrogen receptor, androgen receptor, microarray, muscle
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Corresponding author:
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Klara Sjögren, PhD
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Division of Endocrinology
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Department of Internal Medicine
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Sahlgrenska University Hospital
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SE-41345 Göteborg
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Phone (office) int 46 31 3426311
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Fax int 46 31 821524
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Mobile int 46 735 508064
Copyright © 2010 by the Society for Endocrinology.
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E-mail
[email protected]
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Abstract
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Testosterone (T) is a major regulator of muscle mass. Little is known whether this is due
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to a direct stimulation of the androgen receptor (AR) or mediated by aromatization of T
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to estradiol (E2), the ligand for the estrogen receptors (ERs), in peripheral tissues. In this
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study, we separated AR and ER mediated effects by treating orchidectomized (orx) male
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mice for 5 weeks with E2 or the non aromatizable androgen dihydrotestosterone (DHT).
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Both E2 and DHT increased muscle weight and lean mass although the effect was less
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marked after E2 treatment. Studies of underlying mechanisms were performed using gene
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transcript profiling (microarray and RT-PCR) in skeletal muscle and demonstrated that
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E2 regulated 51 genes and DHT regulated 187 genes, with 13 genes (=25% of E2
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regulated genes) regulated by both treatments. Both E2 and DHT altered the expression
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of Fbxo32, a gene involved in skeletal muscle atrophy, affected the insulin-like growth
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factor–I (IGF-I) system, and regulated genes involved in angiogenesis and the glutathione
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metabolic process. Only E2 affected genes that regulate intermediary glucose and lipid
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metabolism, and only DHT increased the expression of genes involved in synaptic
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transmission and heme and polyamine biosynthesis.
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In summary, ER activation by E2 treatment maintains skeletal muscle mass after orx.
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This effect is less marked than that of AR activation by DHT which completely prevented
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the effect of orx on muscle mass and partly, but not fully, mediated via alternative
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pathways.
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Introduction
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Testosterone (T) is a major regulator of body composition. Androgen deficiency is
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associated with decreased muscle mass and T supplementation increases muscle mass in
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hypogonadal men, HIV-infected men and older men with low T concentrations (Kong
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and Edmonds 2002; Snyder, et al. 2000; Snyder, et al. 1999). Also, in orchidectomized
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(orx) mice, T treatment dose-dependently increases the mass of individual muscles
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(Axell, et al. 2006).
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Both T and the non aromatizable androgen, dihydrotestosterone (DHT) bind to and
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activate the androgen receptor (AR) (MacLean, et al. 1997). The AR gene is expressed
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widely in muscle including myoblasts, myofibers and satellite cells (Chen, et al. 2005).
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The AR is also expressed in motorneurons which may contribute to the regulation of
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muscle mass and function (Yang and Arnold 2000). Several different AR knockout
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(ARKO) mouse models have been reported. In one of these two models, muscle mass
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was unchanged whereas in another model, decreased muscle mass and impaired muscle
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function were observed in male but not female ARKO mice (Lin, et al. 2005; MacLean,
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et al. 2008). Ophoff et al recently reported that a myocyte-specific knockout of the AR in
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male mice resulted in decreased lean mass and a conversion of fast towards slow muscle
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fibers, without affecting muscle strength or fatigue. In their study, similar results were
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obtained in male mice with ubiquitous AR knockout (Ophoff, et al. 2009).
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T can exert its effect either directly by stimulation of the AR or via aromatization in
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target-tissues to estradiol (E2), the ligand for the estrogen receptors (ERs) α and β.
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Muscle from both men and women contains aromatase enzyme activity (Matsumine, et
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al. 1986). The extent to which the actions of T in muscle are a consequence of AR or ER
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activation or both is not clear.
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Skeletal muscle myoblasts, myotubes, and mature fibers all express functional ERs
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indicating a direct effect of estrogen in muscle (Barros, et al. 2006; Kahlert, et al. 1997).
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Women after menopause have decreased lean body mass, which can be reversed by
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estrogen replacement therapy (HRT) (Sorensen, et al. 2001). In animals, estrogen has
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been shown to regulate skeletal muscle mass in developing livestock, rats and mice
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(Kobori and Yamamuro 1989; McCormick, et al. 2004; Moran, et al. 2007; Trenkle
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1976). Furthermore, ovariectomy decreases rat skeletal muscle mass recovery, and
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estrogen replacement benefits atrophied muscle mass recovery (Brown, et al. 2005;
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Sitnick, et al. 2006). Male mice lacking ER β also exhibit altered muscle function
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(Glenmark, et al. 2004).
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By studying the effects on lean mass and weight of individual muscles and comparing
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gene expression after ER and AR mediated stimulation in muscle of gonadectomized
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mice; this study aims to investigate if the effect of T on lean tissue could be due to a
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direct stimulatory effect on the AR or due to an aromatization of T to E2.
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Material and Methods
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Animals and Study Design
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Mice were on a C57BL/6 background and had free access to fresh water and soy-free
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food pellets (R70, Lactamin AB, Stockholm, Sweden or 2016, Harlan Teklad, England).
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The Ethics Committee at University of Gothenburg approved this study.
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At 12 weeks of age, male mice were orchidectomized (orx) and then treated for five
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weeks with DHT (45 µg/day), E2 (0.05 µg/day) or vehicle administered via subcutaneous
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silastic implants (Silclear Tubing; Degania Silicone, Ltd., Jordan Valley, Israel) in the
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cervical region (Vandenput, et al. 2002). Gonadectomy and implantation of pellets were
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performed during the same surgical session for all experimental groups. At the end of the
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treatment period, dual X-ray absorption (DXA) measurements were performed in vivo
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(n=6-8 in each group). Then m. quadriceps and m. gastrocnemius together with various
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organs were dissected and their wet weights determined. Blood was collected for analyses
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of serum IGF-I concentration and distal femur trabecular volumetric bone mineral density
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(vBMD) was determined ex vivo using peripheral Quantitative Computerized
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Tomography (pQCT) (n=6-8 in each group). In addition, total RNA was isolated from
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snap-frozen m. gastrocnemius for analyses using microarray (n=5 in each group).
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To determine the short-term effects of the hormone treatments on the expression of
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selected genes in muscle, a similar experiment as above was performed but with a
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treatment period of only one week. Total RNA was isolated from snap-frozen m.
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gastrocnemius and m. levator ani for analyses using RT-PCR.
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Dual X-ray Absorption (DXA) Analysis
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Body composition in mice was measured by DXA using the Lunar PIXImus Mouse
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Densitometer (Wipro GE Healthcare, Madison, WI) with the mice under inhalation
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anaesthesia with Isoflurane (Forene; Abbot Scandinavia, Solna, Sweden).
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Peripheral Quantitative Computerized Tomography (pQCT)
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Distal femur trabecular volumetric bone mineral density (vBMD) was measured ex vivo
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using the Stratec pQCT XCT Research M (software version 5.4B; Norland Medical
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Systems Inc. White Plains, NY) operating at a resolution of 70 µm (Windahl, et al. 1999).
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The pQCT scan was positioned in the metaphysis at a distance from the distal growth
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plate corresponding to 3% of the total length of the femur and the trabecular bone region
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was defined as the inner 45% of the total cross-sectional area.
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DNA Microarray Analysis
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Total RNA was isolated from snap-frozen m. gastrocnemius using RNeasy Mini Kit
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including an on-column DNase digestion step using the RNase-free DNase set (Qiagen,
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Chatsworth, CA). The mRNA samples derived from each individual mouse were reverse
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transcribed into cDNA, labeled and analyzed using DNA microarray (mouse expression
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set 430; Affymetrix, Santa Clara, CA) (n=5 in each group). Preparation of labelled
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cRNA, hybridization and staining were done according to the Affymetrix Gene Chip
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expression analysis manual. The stained probe array was scanned and the resultant image
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was captured as a data image (.CEL) file. The signal intensities for the β-actin and the
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GAPDH genes were used as internal quality controls. The ratio of fluorescent intensities
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for the 5’ end the 3’ end of those housekeeping genes were
