Testosterone regulation of Akt/mTORC1/FoxO3a signaling in skeletal muscle

Abstract

Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C₂C₁₂ myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C₂C₁₂ myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24 h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt/mTOR signaling. In summary, androgen withdrawal decreases muscle myofibrillar protein synthesis through Akt/mTORC1 signaling, which is independent of AMPK activation, and readily reversible by anabolic steroid administration. Acute Akt activation in C₂C₁₂ myotubes is sensitive to a high concentration of testosterone, and low concentrations of testosterone can activate mTOR signaling independent of Akt.

Introduction

A reduction in circulating testosterone levels, referred to as hypogonadism is associated with a reduction in lean body mass and increased percentage of fat mass (Katznelson et al., 1996). These effects are rescued with supplementation of testosterone or pharmacological derivatives (Bhasin et al., 1997, Brodsky et al., 1996). In addition, there is a high prevalence of hypogonadism in wasting diseases including human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) (Dobs et al., 1988), end-stage renal disease (Johansen, 2004), COPD (Van Vliet et al., 2005), cancer (Vigano et al., 2010), and type 2 diabetes (Dhindsa et al., 2004). Testosterone replacement therapy has shown to be effective in rescuing the loss of muscle mass in several conditions of muscle wasting (Bhasin et al., 2000, Snyder et al., 1999, Kenny et al., 2001, Morley et al., 1993, Tenover, 1992, Bhasin et al., 1998). Despite hypogonadism underlying several conditions of muscle wasting, the mechanism/s of action and the sensitivity of these processes to androgens are still unclear. Further investigation is warranted to elucidate androgen-induced regulation of anabolic/catabolic signaling in muscle.

Castration is an effective technique for the elimination of endogenous testosterone production in rodents (Rogozkin, 1979). Testosterone loss results in a reduction in body weight gain, muscle atrophy and increased fat stores (Antonio et al., 1999, Axell et al., 2006). As little as 2-weeks of castration can result in muscle atrophy and suppression of muscle androgen receptor expression, which can be rescued by Dihydrotestosterone (DHT) treatment (Antonio et al., 1999). Castration induced muscle mass loss is associated with reduced myofiber cross sectional area in both fast and slow muscles, and reduced contractile strength (Axell et al., 2006). Castration has also been shown to induce alteration in the morphology of the soleus muscle that includes irregular Z lines, loss of the lamina externa and glycogen clusters under the sarcomere, which were prevented by testosterone administration (Oner et al., 2008). Although muscle mass and morphology with androgen loss has been well documented, the androgen sensitive mechanisms regulating muscle protein turnover require further investigation.

The IGF/Akt/mTORC1 signaling pathway is an essential regulator of skeletal muscle’s capacity for protein synthesis and degradation (Frost and Lang, 2007). In humans, testosterone deficiency is associated with a reduction in both circulating IGF-1 (Grinspoon et al., 1996) and intramuscular IGF-1 expression (Mauras et al., 1998). Similar effects were observed in rats as five weeks of castration reduced muscle IGF-1 staining while testosterone administration reversed these effects (Oner et al., 2008). In the canonical IGF-1/Akt/mTORC1 pathway, Akt activation of mTOR and subsequent phosphorylation of p70S6K and 4E-BP1 will induce protein synthesis. Akt can also enhance protein synthesis through inhibition of proteins that impede protein synthesis such as glycogen syntheses kinase-3 beta (GSK3β). Testosterone administration to female rats increased the IGF-1 signaling pathway by increasing muscle IGF-1 mRNA expression and phosphorylation of Akt and GSK3β (Yin et al., 2009). The effect of androgen loss and subsequent administration on muscle Akt signaling is currently equivocal, and in need of further clarification. Androgen loss has demonstrated either a reduced or increased phosphorylation of Akt with castration (Ibebunjo et al., 2011, Haren et al., 2011). The response to testosterone supplementation after castration is also equivocal on the activation of Akt (Hourde et al., 2009). These differences may be related to the muscle examined, dose and type of androgen, and the length of time the rodent was castrated. However, it is clear that further investigation is necessary to understand the sensitivity of muscle IGF-1/Akt/mTORC1 signaling to androgen.

mTORC1 complex activity has emerged as a critical regulator of protein synthesis, as well as several other cellular processes, which can occur independent of upstream Akt activation (Potier et al., 2009, Sarbassov et al., 2005). Although administration of mTORC1 inhibitor rapamycin to L6 myotubes can block testosterone induced increases in protein content (Wu et al., 2010), mTORC1 regulation by anabolic steroid administration is not well understood. While regulation of mTORC1 through PI3K/Akt signaling has been extensively documented (Inoki et al., 2003, Vander Haar et al., 2007), the potential for testosterone to regulate mTOR through other signaling pathways has not been as well defined. mTORC1 consists of raptor (regulated associated protein of mTOR), mLST8, PRAS40 (proline-rich Akt substrate-40) and mTOR. PRAS40, a known repressor of mTOR signaling, can be phosphorylated by Akt and thus relieving inhibition of PRAS40 on mTOR (Sancak et al., 2007). An additional well-described negative regulator of mTORC1 activity is the energy sensitive 5′-adenosine monophosphate-activated protein kinase (AMPK) (Bolster et al., 2002, Horman et al., 2002, Chan et al., 2004). AMPK can inhibit mTOR signaling by phosphorylation of the tuberous sclerosis complex 2 (TSC2) gene product Tuberin on Thr1227 and Ser1345 (Inoki et al., 2003) and phosphorylation of raptor at Ser792 (Gwinn et al., 2008). The phosphorylation of raptor prevents binding of and eventual phosphorylation of p70S6K and 4E-BP1. C₂C₁₂ cells treated with AICAR to activate AMPK have reduced protein synthesis, polysome aggregation and down steam mTOR signaling proteins 4E-BP1, S6K1 and eEF2 (Williamson et al., 2006). Although IGF-1 expression and Akt activation have been shown to be sensitive to circulating anabolic steroids, regulation of the mTORC1 complex beyond the canonical IGF-1 pathways need further examination.

In addition to protein synthesis regulation, Akt can inhibit protein degradation through phosphorylation and inhibition of the forkhead box O (FoxO) family of transcription factors. In skeletal muscle, the activation of FoxO1 and 3 have been identified in conditions of muscle atrophy such as starvation (Lecker et al., 2004), diabetes (Lee et al., 2004) and cachexia (Lecker et al., 2004, White et al., 2011). The FoxO family of proteins translocates from the cytosol to the nucleus to promote transcription of atrophy related genes, in particular the muscle atrophy F-box (MAFbx; also called atrogin1) (Sandri et al., 2004), muscle ring Finger-1 also called MuRF1 (Bodine et al., 2001) and regulated in development and DNA damage response 1 (REDD1, also referred to as Rtp801 and DDIT4) (Harvey et al., 2008). Akt can phosphorylate and inhibit the FoxO proteins from entering the nucleus and prevents gene transcription (Latres et al., 2005). REDD1 inhibits mTORC1 signaling through activation of upstream TSC2 (Brugarolas et al., 2004, Reiling and Hafen, 2004, Sofer et al., 2005). REDD1 protein and mRNA expression are increased in muscle during starvation (McGhee et al., 2009), dexamethasone-induced atrophy (McGhee et al., 2009, Wang et al., 2006) and hypoxia (Favier et al., 2010). Androgen withdrawal has been reported to cause muscle atrophy (Antonio et al., 1999, Axell et al., 2006, Oner et al., 2008) through ubiquitin-proteasome-dependent proteolysis pathways (Glass, 2005). A purposed mechanism for androgen-induced inhibition of muscle catabolism is through the down regulation of FoxO and FoxO-related genes transcription (Ibebunjo et al., 2011, Pires-Oliveira et al., 2010). Testosterone and other pharmacological derivatives prevent dexamethasone-induced muscle atrophy and block gene expression of FoxO1 (Qin et al., 2010a) and atrogin1/MAFbx (Van Balkom et al., 1998). In addition, anabolic steroid treatment can reduce REDD1/2 gene expression during dexamethasone (Wu et al., 2010b) and denervation-induced atrophy (Qin et al., 2010b). Although FoxO targets atrogin1/MAFbx and MuRF1 are increased with castration (Ibebunjo et al., 2011), the role of REDD1 expression during castration-induced muscle atrophy has not been explored.

Hypogonadism is common during several conditions associated with muscle wasting, and while acknowledged, its physiological significance in the regulation of muscle wasting is just emerging. The Ak/mTORC1 pathway has been extensively investigated as a regulator of muscle protein turnover, controlling both muscle protein synthesis through mTORC1 and degradation processes through FoxO. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C₂C₁₂ myotubes. We hypothesized that castration-induced androgen withdrawal would disrupt the regulation of muscle protein turnover through Akt dependent regulation of mTORC1 signaling and FoxO transcriptional targets.