Cell-specific functions of androgen receptor in skeletal muscles

Abstract

Androgens play a vital role not only in promoting the development of male sexual characteristics but also in exerting diverse physiological effects, including the regulation of skeletal muscle growth and function. Given that the effects of androgens are mediated through androgen receptor (AR) binding, an understanding of AR functionality is crucial for comprehending the mechanisms of androgen action on skeletal muscles. Drawing from insights gained using conditional knockout mouse models facilitated by Cre/loxP technology, we review the cell-specific functions of AR in skeletal muscles. We focus on three specific cell populations expressing AR within skeletal muscles: skeletal muscle cells, responsible for muscle contraction; satellite cells, which are essential stem cells contributing to the growth and regeneration of skeletal muscles; and mesenchymal progenitors, situated in interstitial areas and playing a crucial role in muscle homeostasis. Furthermore, the indirect effects of androgens on skeletal muscle through extra-muscle tissue are essential, especially for the regulation of skeletal muscle mass. The regulation of genes by AR varies across different cell types and contexts, including homeostasis, regeneration and hypertrophy of skeletal muscles. The varied mechanisms orchestrated by AR collectively influence the physiology of skeletal muscles.

Introduction

Androgens, derived from the Greek words “andro” meaning “male” and “gen” meaning “thing that produces,” play a crucial role in promoting the development of male secondary sexual characteristics, as their name suggests. Moreover, androgens exhibit protein anabolic effects, earning them the nickname “anabolic steroids.” Remarkably, just three years after the discovery, isolation, and synthesis of testosterone in 1935 [1-3], the initial observation of androgen administration leading to skeletal muscle hypertrophy was documented [4]. Over the past 80 years, androgen administration has consistently demonstrated its ability to enhance muscle strength and mass in both humans and mice. Despite this extensive body of knowledge, the precise molecular mechanisms of action remain largely elusive.

Androgens are also synthesized in women, although their circulating levels are considerably lower compared to men [5]. Androgens are biosynthesized from cholesterol in various tissues, mainly in Leydig cells of the testis in men. Testosterone (T) and its derivative dihydrotestosterone (DHT) are the main androgens. Androgens perform most of their functions by binding to the androgen receptor (AR), a member of the nuclear receptor superfamily [6]. Androgen-bound ARs translocate into the nucleus and bind to specific DNA sequences called androgen response elements (AREs) [7], that regulate the expression of target genes as a transcription factor. In general, cells expressing AR are the main targets of androgen action.

Skeletal muscle comprises multinucleated skeletal muscle cells, also known as skeletal muscle fibers or myofibers, formed by the fusion of numerous cells. In mammals, skeletal muscle cells have ceased to divide and are incapable of generating new skeletal muscle cells [8]. However, skeletal muscle contains mononuclear stem cells called muscle satellite cells [9], which are the driving force behind the generation of new skeletal muscle cells through proliferation and differentiation [10-12]. Furthermore, skeletal muscle contains mesenchymal progenitors, which are clearly distinct from muscle satellite cells [13, 14]. While mesenchymal progenitors were initially recognized as the cells responsible for abnormal fat accumulation in skeletal muscles [13, 14], recent research has revealed their positive contributions to homeostasis [15], regeneration [16], and hypertrophy [17].

The hypertrophy of skeletal muscle is thought to be attributed to two factors: hypertrophy of skeletal muscle cells and an increase in myonuclei (nuclei within skeletal muscle cells) [18]. Because skeletal muscle cells have stopped dividing and cannot independently increase their nuclei, the fusion of satellite cells is essential for the increase in muscle nuclei. Nonetheless, there are conflicting reports resulting from experiments utilizing satellite cell-depleted mice. Some studies suggest that muscle hypertrophy does not occur in the absence of satellite cells [19], while others indicate that hypertrophy can still occur independently of satellite cells [20, 21]. In any case, both satellite cell-dependent and independent mechanisms are considered necessary for efficient muscle hypertrophy. Furthermore, it has been reported that mesenchymal progenitors sense increased mechanical stimulation resulting from muscle loading and subsequently transmit this signal to satellite cells, thereby promoting their fusion with skeletal muscle cells [17]. Therefore, it is thought that skeletal muscle hypertrophy is achieved through the collaborative interaction of three cell types: skeletal muscle cells, satellite cells, and mesenchymal progenitors.

This review therefore presents and discusses studies on androgens and ARs, especially using knockout model mice in these three cell types within skeletal muscles. As AR expression is not limited to skeletal muscles, we briefly discuss the AR functions in extra-muscle tissues. It should be noted that there are already detailed reviews on muscle hypertrophy and satellite cells [22], molecular mechanisms of muscle hypertrophy [18], or on androgen receptors and exercise [23], respectively, which will not be discussed in detail in this review.

Global AR Knockout Mouse Models

To study the AR mechanism in vivo, it is crucial to develop mouse models with AR knockout. However, given that male mice without a functional AR gene would likely be completely infertile [24], the successful targeted disruption of the AR gene inherently prevents its inheritance in the subsequent generation. Therefore, creating an AR knockout (ARKO) mouse line through conventional breeding or gene targeting approaches was not feasible. To avoid this problem, global ARKO mouse models were created by crossbreeding two distinct transgenic mouse strains: a Cre transgenic mouse strain expressing the recombinase ubiquitously and another mouse strain with loxP sites flanking a portion of the AR gene (Summarized in Table 1). Since the actions of androgens in skeletal muscle are thought to range from gene regulation, metabolism, and muscle contraction to exercise, numerous papers have been published on global and cell-specific AR-deficient mice to elucidate the role of AR in each of these functions.

Table 1

Summary of global or cell-specific AR knockout mouse models

Ref	Targeted cell type	Promoter of Cre	Floxed AR exon	Sex	Body weight	Limb Muscle Mass	Muscle Strength	Other observations
[25]	Systemic	ACTB	2	M	↓	n.d.	n.d.	decreased bone volume
[36]	Systemic	CMV	1	M	↑	n.d.	n.d.	late onset of obesity
[26]	Systemic	CMV	1	M	↓	n.d.	n.d.
[33]	Systemic	PGK	2	M	n.d.	n.d.	n.d.
[34]	Systemic	ACTB	2	M	n.d.	n.d.	n.d.
[37]	Systemic	ACTB	2	M	↑	n.d.	n.d.	enlargement of adipose tissues
[38]	Systemic	CMV	1	M	↑	=	n.d.	increased fat pad
[27]	Systemic	CMV	3	M	↓	n.d.	n.d.
[28]	Systemic	PGK	2	M	↓	n.d.	n.d.
[29]	Systemic	CMV	3	M	↓	↓	↓	no difference in specific force
[29]	Systemic	CMV	3	F	=	=	n.d.
[30]	Systemic	PGK	2	M	↓	↓	n.d.
[31]	Systemic	PGK	2	M	↓	↓	n.d.	decreased cortical bone area and thickness
[32]	Systemic	CMV	3	M	↓	n.d.	n.d.	increased subcutaneous fat mass
[35]	Systemic	PGK	2	M	n.d.	n.d.	=
[44]	Myofibers	MCK	2	M	↓	=	=	decreased intra-abdominal fat tissue
[45]	Myofibers	HSA	1	M	=	=	↓	reduced perineal muscles size and mass
[45]	Myofibers	HSA-CreER	1	M	n.d.	=	↓
[50]	Myofibers	HSA	3	M	n.d.	n.d.	n.d.
[50]	Myofibers	MCK	3	M	n.d.	n.d.	n.d.
[47]	Myofibers	HSA	1	M	n.d.	↓	=	less increased in response to overloading
[46]	Myofibers	MCK	3	M	=	=	n.d.	reduced perineal muscles mass
[46]	Myofibers	HSA	3	M	=	=	n.d.	reduced perineal muscles mass
[59]	Myofibers	MCK	1	M	n.d.	n.d.	n.d.
[48]	Myofibers	HSA	1	M	n.d.	↓	↓
[52]	Myofibers	HSA	1	F	=	=	=
[49]	Myofibers	Mlc1f	1	M	=	↓	↓	reduced bone mineral density
[49]	Myofibers	Mlc1f	1	F	↑	=	=
[51]	Myofibers	HSA	1	M	=	n.d.	n.d.	impaired glycolysis and oxidative metabolism
[51]	Myofibers	HSA-CreER	1	M	=	n.d.	n.d.
[61]	Myofibers/Satellite cells	Myod	2	M	=	=	↓	reduced perineal muscles mass
[73]	Myofibers/Satellite cells	Myod	2	M	=	=	n.d.	reduced perineal muscles mass
[35]	Myofibers/Satellite cells	Myod	2	M	n.d.	=	n.d.	comparable wheel running patterns
[63]	Satellite cells	Pax7 CreERT2	1	M	=	=	n.d.
[72]	Mesenchymal progenitors	Sall1 CreER	1	M	n.d.	n.d.	n.d.	reduced perineal muscles formation
[74]	Mesenchymal progenitors	PDGFRα CreER	1	M	↓	↓	=	reduced perineal muscles mass
[76]	Adipocytes	Fabp4 (aP2)	2	M	=	n.d.	n.d.
[77]	Adipocytes	Fabp4 (aP2)	2	M	↓	n.d.	n.d.
[79]	Hepatocytes	Alb	2	M	=	n.d.	n.d.	obesity after high-fat diet feeding

↑, increased; ↓, decreased; =, unchanged; n.d., not determined; M, male; F, female.

Because skeletal muscles comprise roughly 40% of total body weight, lean body mass can serve as a surrogate indicator for muscle mass. Reduced body mass was reported in almost all studies on systemic ARKO mice [25-32], except in those studies that did not provide information on body weight [33-35]. In contrast, several studies have shown an increase in fat tissue and a subsequent increase in body weight [36-38]. Among the studies involving systemic ARKO mice, several delineate detailed phenotypes associated with skeletal muscle. In ARKO male mice, one group observed downregulation of genes associated with slow-twitch fibers and upregulation of genes related to fast-twitch fibers in the quadriceps muscles, which are predominantly composed of fast-twitch fibers [34]. Also in ARKO male mice, another group reported reduced muscle strength of fast-twitch extensor digitorum longus (EDL) muscles and increased resistance to fatigue of slow-twitch soleus muscles in vitro [29]. They also observed the upregulation of slow-twitch related genes, including calsequestrin 2 (Casq2) and myosin light polypeptide 3 (Myl3) [39], in gastrocnemius muscles from AR knockout mice, leading to the observed phenotype [29]. In addition, Fan et al. reported the upregulation of glucose metabolic related genes in ARKO mice, including solute carrier family 2 member 4 (Slc2a4, also known as GLUT4) and hexokinase 1 (Hk1), suggesting the activation of glucose uptake in skeletal muscle of ARKO mice [38]. Moreover, microarray analysis on gastrocnemius muscles in ARKO male mice showed the upregulation of genes related to metabolic processes, including fatty acid and triacylglycerol synthesis [32]. In summary, AR controls body mass, including skeletal muscle, muscle strength, and glucose and fat metabolism in skeletal muscles.

Androgen Action on Myofibers

Skeletal muscle contracts to facilitate moving and maintain posture. Each skeletal muscle cell, also known as a myofiber, has multiple nuclei. This multinucleated state results from the fusion of individual mono nucleated cells during the development of skeletal muscle [40]. Following fusion, these myofibers no longer undergo division. When examined under electron microscopy, thin sections of myofibers reveal clusters of filaments, or myofibrils, aligned along the length of the fiber. These myofibrils exist in two sizes: thick and thin. Myosin constitutes the thick filaments, while actin constitutes the thin filaments.

Given the high expression of AR in skeletal muscle cell nuclei in both mice and humans [41-43], myofibers are considered direct targets of androgen. Numerous studies have shown that systemic AR gene deficiency reduces skeletal muscle mass, as shown above. However, skeletal muscle cell-specific AR-deficient mice show no [44-46] or limited reduction in limb muscle mass [47-49]. Nonetheless, there is a consistent decrease in the weight of androgen-sensitive perineal muscles, including levator ani (LA) and bulbocavernosus (BC). The absence of AR in myofibers, generated using human α-skeletal actin (HSA)-Cre mice combined with AR floxed mice, reduced maximal tetanic force due to the disruption of myofibrils, as revealed by ultrastructural analyses [45]. On the other hand, in the same mutant mice, the enhancement in muscle performance, including absolute maximal force, specific maximal force, and fatigue resistance, resulting from overload, remains unaffected in myofiber-specific AR-deficient mice [47]. Another myofiber-specific AR-knockout mouse model, using the muscle creatine kinase (MCK)-Cre driver strain, showed a limited impact on muscle fatigue [44]. The varying results seen in these studies may be attributed, at least in part, to the different promoters regulating Cre-recombinase expression and the specific exons floxed in the respective transgenic animal models.

Although the involvement of AR in myofibers is clear, the underlying molecular mechanisms remain largely unknown. A pioneering study showed that the expression of ornithine decarboxylase (Odc1), which is a rate-limiting enzyme in polyamine biosynthesis, is downregulated in myofiber-specific AR-deficient (HSA-Cre or MCK-Cre with AR floxed) mice [50]. We observed a downregulation of Odc1 in HSA-Cre mice with AR floxed, but its expression was upregulated by androgen treatment, regardless of myofiber-specific AR-deficiency [48]. A fast-twitch muscle-specific AR knockout mouse, created utilizing a myosin light chain 1f (Mlc1f)-Cre driver, showed alterations in the expression of polyamine-related genes, including Odc1 [49]. Another study demonstrated that myofiber AR deficiency using HSA-Cre mice led to compromised glycolysis and fatty acid metabolism, reduced oxidative metabolism, and enhanced amino acid catabolism in limb muscles [51]. We also identified myosin light chain kinase family member 4 (Mylk4) as a androgen-responsive gene dependent on AR of myofibers [52]. Because Mylk4-deficient mice showed reduced maximum isometric torque, Mylk4 is likely involved in regulating muscle strength via AR in myofibers [52]. Taken together, these findings suggest that AR in myofibers regulates mainly skeletal muscle quality by orchestrating both the metabolic and contractile functions of skeletal muscle.

Androgen Action on Satellite Cells

Satellite cells are muscle stem cells in skeletal muscle. The term “satellite” was aptly chosen because, under electron microscopy, these cells appear as small, flattened cells positioned just outside the plasma membrane of muscle fibers [9], and appear to be “orbiting” adjacent to the muscle fibers, much like satellites orbiting around a celestial body. In intact muscles, satellite cells expressing paired box 7 (PAX7) [53] or neural cell adhesion molecule 1 (NCAM1) [54] remain in a quiescent and undifferentiated state. The Notch signaling pathway plays a crucial role in maintaining quiescent satellite cells in resting muscles [55]. When myofibers undergo damage and degeneration, satellite cells initiate the expression of myoblast determination protein 1 (MyoD) and subsequently transition into the cell cycle.

The first description of AR expression in satellite cells was reported by Bhasin’s group [42]. In testosterone-treated human skeletal muscle, an elevation in satellite cell count, which was characterized by ultrastructural morphology, was noted, and significantly correlated with changes in testosterone levels [42]. In rats, pre-puberty testosterone treatment led to a substantial surge in the satellite cell count (counted by electron microscopy) in the LA muscle, not only in males but also in females [56]. Conversely, castration in mice reduced the number of proliferating satellite cells (NCAM1+BrdU+) during cardiotoxin-induced muscle regeneration in both young and old mice [57]. Conflicting results were reported that the castration increased the number of proliferating satellite cells (PAX7+BrdU+) in intact muscles without impairing their function during muscle regeneration in young mice [58]. One possible explanation for this inconsistency is that androgen effects on satellite cells differ in the intact, early, and late stages of muscle regeneration. Specifically, at puberty, elevated testosterone/DHT levels trigger the expression of MIB E3 ubiquitin protein ligase 1 (Mib1) in myofibers leading MIB1 to promote the ubiquitylation and trans-endocytosis of Notch ligands, including Delta-like 1 (DLL1) and/or Jagged 1 (JAG1), in myofibers, subsequently initiating Notch signaling in satellite cells [59]. In summary, androgens directly or indirectly regulate the proliferation and differentiation of satellite cells.

There have been only a limited number of experiments specifically targeting AR deficiency in satellite cells (Table 1). Dubois et al. demonstrated that the deletion of AR in both myofibers and satellite cells, using MyoD-iCre mice [60] where MyoD expresses in all developing myogenic cells, resulted in reduced grip strength [61]. This was accompanied by a counterintuitive downregulation of myostatin (Mstn) [61], a potent inhibitor of skeletal muscle growth [62]. The authors hypothesized that Mstn expression may dampen excessive muscle growth induced by androgens [61]. To elucidate the specific role of AR in satellite cells, we generated adult mice deficient in satellite cell-specific AR using two types of Pax7^CreETR2 mice [63]. Inducing muscle regeneration in the skeletal muscles of these mice allowed us to investigate the proliferative and differentiation potential of satellite cells as exerted by AR. We found no significant differences in satellite cell-specific AR-deficient male mice (Pax7^CreERT2-Fan;AR^Flox/Y or Pax7^{CreERT2-Kardon};AR^Flox/Y) compared to Cre control mice (Pax7^CreERT2-Fan or Pax7^{CreERT2-Kardon}) in terms of proliferation and differentiation of satellite cells [63]. Furthermore, another group reported that androgen administration to satellite cell-deficient mice induced muscle fiber hypertrophy without an increase in myonuclear number [64]. These findings suggest that while androgen treatment increases satellite cells, AR in satellite cells has limited involvement in their proliferation and differentiation ability for an increase in muscle nucleus count, i.e., fusion of satellite cells, and in androgens’ effects for skeletal mass regulation.

Androgen Action on Mesenchymal Progenitors

Mesenchymal progenitors, also known as fibro-adipogenic progenitors (FAPs), are a type of multipotent cell residing within the interstitium of skeletal muscle tissue [13, 14]. Mesenchymal progenitors have been recognized as cells expressing platelet-derived growth factor receptor alpha (PDGFRα). While initially characterized in murine muscles, mesenchymal progenitors in humans were found to have similar functions to their mouse counterparts [65, 66]. They are able to differentiate into several different cell types, including fibroblasts, adipocytes, chondrocytes and osteoblasts, leading to fibrosis, ectopic adipocyte infiltration [67], and heterotopic ossification [68]. Although PDGFRα-positive cells are sometimes referred to as fibroblasts, causing nomenclature confusion, they can differentiate into multiple cell lineages, including fibroblasts as previously noted. Therefore, they can be appropriately called mesenchymal progenitors [69]. In addition to their pathological role, mesenchymal progenitors play a crucial role in the maintenance of skeletal muscle under steady-state conditions [15, 16].

The role of AR in mesenchymal progenitors remains unclear. Bhasin’s group conducted a study utilizing C3H 10T1/2 cells, which have the capacity to differentiate into adipocytes, chondrocytes, and osteoblasts, revealing that high concentrations of androgens inhibited adipogenesis in C3H 10T1/2 cells [70]. Mice with forced expression of AR using the collagen type I promoter, which was expressed in mesenchymal progenitors of skeletal muscle, showed a reduction in visceral fat mass accompanied by a decrease in body weight [71]. It has also been reported that the loss of AR in non-myocytic cells during mouse development, using spalt-like transcription factor 1 (Sall1)-Cre mice, reduced the mass of LA/BC muscles with the reduction of proliferating myoblasts through inhibiting p21 expression [72]. Dubois et al. suggested that fibroblasts expressing vimentin (VIM) might be indirectly targeted by androgens in skeletal muscles [73].

We previously reported that PDGFRα cells expressed AR in embryonic BC muscles as “data not shown” in Discussion section [72]. In the current investigation of the role of AR in adult muscle mesenchymal progenitors, we generated PDGFRα-CreER;AR^Flox/Y mice to specifically delete AR in mesenchymal progenitors [74]. Mature male mice (24 weeks old) with targeted ablation of AR in mesenchymal progenitors showed a reduction in limb muscles mass. Moreover, fat infiltration in skeletal muscles was not affected in PDGFRα-CreER;AR^Flox/Y mice. In addition, the absence of AR in mesenchymal progenitors led to perineal muscle hypotrophy, due to abnormal regulation of transcripts related to apoptosis and proteolysis. Furthermore, we demonstrated that AR in mesenchymal progenitors governs the local expression of insulin-like growth factor 1 (Igf1) in skeletal muscles [74]. Taken together, AR in mesenchymal progenitors appears to have a limited effect on adipogenesis in skeletal muscles but plays an important role in maintaining skeletal muscle mass, especially in perineal muscles, through Igf1 expression.

Androgen Actions on Other Cells in Non-muscle Tissues

Given that AR expression is not confined to skeletal muscles, it is plausible that AR in non-muscle tissues plays a role in the indirect regulation of skeletal muscles. Androgens, including testosterone T and DHT, have been identified in non-muscle tissues such as white adipose tissues and the liver [75]. Approximately 60% of the total body T pool and 88% of the DHT pool are found in adipose tissues [75]. Some mouse models with global AR ablation have shown age-dependent development of obesity [36-38]. However, the relationship between AR in adipose tissues and obesity remains unclear, as other global AR ablation models did not exhibit obesity. Intriguingly, a mouse model with selective AR ablation in adipose tissue, using a mouse strain expressing Cre recombinase under the control of the aP2 (fatty acid binding protein 4, FABP4) promoter, displayed similar body weight and adiposity to male control mice [76]. However, when subjected to a high-fat diet (HFD), these mice showed increased susceptibility to visceral obesity [77]. It is noteworthy that the aP2 promoter is active in various cell types in the brain, potentially confounding the effects mediated by AR [78]. In addition, a significant increase in adiposity was reported in a mouse model where AR was selectively ablated in the liver (using a Cre driven by the albumin promoter) after HFD feeding [79]. As these papers did not provide details on skeletal muscles, it is still unclear how AR in non-muscle tissues can regulate skeletal muscles. Our recent findings identified humoral factors secreted from non-muscle tissues that changes serum concentration consistently with androgen levels, potentially influencing skeletal muscle mass. Further studies are required to explore the possibility that AR activity in non-muscle tissues may influence skeletal muscles.

Conclusions

In conclusion, the mechanisms orchestrated by AR vary across different cell types and contexts, influencing skeletal muscle quality and quantity (mass). In myofibers, AR orchestrates both the contractile and metabolic functions of skeletal muscle, contributing to the maintenance of muscle quality. While androgen treatment increases the number of satellite cells, the involvement of AR in satellite cells is limited in their abilities for proliferation and differentiation crucial for increasing muscle nucleus number and for muscle hypertrophy. In addition, AR in mesenchymal progenitors plays a crucial role in maintaining the quantity, or mass of skeletal muscles, possibly through paracrine signaling. We propose that androgens control both the quality and mass of skeletal muscle through different pathways in myofibers, satellite cells, and mesenchymal progenitors, ultimately influencing muscle strength (Fig. 1). Moreover, AR in non-muscle tissues may contribute to this mechanism. A significant challenge for future research is to identify the cell-specific target genes and partners of AR that regulate common and unique regulatory mechanisms in various cellular contexts through androgen/AR signaling in skeletal muscles.

Fig. 1

Overview of the proposed mechanisms controlled by AR for regulation of skeletal muscles. Created with BioRender.com

Conflicts of Interest

Yuuki Imai is a member of Endocrine Journal’s Editorial Board.

Acknowledgments

We acknowledge support from MEXT/JSPS KAKENHI (JP21K17568 to H.S.; JP22H03203 to Y.I.) and HIRAKU-Global Program, which is funded by MEXT’s “Strategic Professional Development Program for Young Researchers” (to H.S.).

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