Testosterone protects against dexamethasone-induced muscle atrophy, protein degradation and MAFbx upregulation

Administration of glucocorticoids in pharmacological amounts results in muscle atrophy due, in part, to accelerated degradation of muscle proteins by the ubiquitin-proteasome pathway. The ubiquitin ligase MAFbx is upregulated during muscle loss including that caused by glucocorticoids and has been implicated in accelerated muscle protein catabolism during such loss. Testosterone has been found to reverse glucocorticoid-induced muscle loss due to prolonged glucocorticoid administration. Here, we tested the possibility that testosterone would block muscle loss, upregulation of MAFbx, and protein catabolism when begun at the time of glucocorticoid administration. Coadministration of testosterone to male rats blocked dexamethasone-induced reduction in gastrocnemius muscle mass and upregulation of MAFbx mRNA levels. Administration of testosterone together with dexamethasone also prevented glucocorticoid-induced upregulation of MAFbx mRNA levels and protein catabolism in C2C12 myotube expressing the androgen receptor. Half-life of MAFbx was not altered by testosterone, dexamethasone or the combination. Testosterone blocked dexamethasone-induced increases in activity of the human MAFbx promotor. The findings indicate that administration testosterone prevents glucocorticoid-induced muscle atrophy and suggest that this results, in part at least, from reductions in muscle protein catabolism and expression of MAFbx.     

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.     

Insulin down-regulates the expression of ubiquitin E3 ligases partially by inhibiting the activity and expression of AMP-activated protein kinase in L6 myotubes

While insulin is an anabolic hormone, AMP-activated protein kinase (AMPK) is not only a key energy regulator, but it can also control substrate metabolism directly by inducing skeletal muscle protein degradation. The hypothesis of the present study was that insulin inhibits AMPK and thus down-regulates the expression of the ubiquitin E3 ligases, muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in skeletal muscle cells. Differentiated L6 myotubes were treated with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and/or compound C to stimulate and/or block AMPK respectively. These treatments were also conducted in the presence or absence of insulin and the cells were analysed by western blot and quantitative real-time PCR. In addition, nuleotide levels were determined using HPLC. The activation of AMPK with AICAR enhanced the mRNA levels of MAFbx and MuRF1. Insulin reduced the phosphorylation and activity AMPK, which was accompanied by reduced MAFbx and MuRF1 mRNA levels. Using a protein kinase B (PKB/Akt) inhibitor, we found that insulin regulates AMPK through the activation of Akt. Furthermore, insulin down-regulated AMPK α2 mRNA. We conclude that insulin inhibits AMPK through Akt phosphorylation in L6 myotubes, which may serve as a possible signalling pathway for the down-regulation of protein degradation. In addition, decreased expression of AMPK α2 may partially participate in inhibiting the activity of AMPK.     

Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases.     

推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制*

目的:探讨推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制。方法:48只雄性SD大鼠随机分为模型组(n=24)和推拿组(n=24),通过切断右侧胫神经制备腓肠肌萎缩大鼠模型。术后第2日开始给推拿组大鼠手术侧腓肠肌给予手法干预,模型组不予干预。两组分别在0 d、7 d、14 d、21 d四个时间点各处死6只大鼠,取大鼠双侧腓肠肌,称重后计算各组大鼠腓肠肌湿重比;HE染色测定肌纤维截面积和直径,实时荧光定量PCR检测腓肠肌中miR-23a、Akt、MuRF1、MAFbx基因相对表达量。结果:与0 d比较,模型组和推拿组大鼠腓肠肌湿重比、肌纤维截面积和直径呈现进行性下降的趋势,其中7 d、14 d、21 d推拿组腓肠肌湿重比、肌纤维截面积和直径均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组MuRF1、MAFbx、Akt mRNA表达均呈现先升后降的趋势,其中7 d、21 d推拿组MuRF1 mRNA表达均显著低于模型组(P＜0.05,P＜0.01),7 d、14 d、21 d推拿组MAFbx mRNA表达均显著低于模型组(P＜0.01,P＜0.05,P＜0.01),7 d、14 d、21 d推拿组Akt mRNA表达均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组21 d时miR-23a mRNA表达升高,推拿组miR-23a mRNA表达显著高于模型组(P＜0.05)。结论:推拿能延缓失神经骨骼肌的萎缩,其机制可能与上调miR-23a、Akt基因的表达,下调 MuRF1、MAFbx基因的表达,使蛋白降解速度受到抑制,从而减轻骨骼肌蛋白的降解程度有关。     

Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes

Dexamethasone-treated L6 (a rat cell line) and C2C12 (a mouse cell line) myotubes are frequently used as in vitro models of muscle wasting. We compared the effects of different concentrations of dexamethasone and corticosterone (the naturally occurring glucocorticoid in rodents) on protein breakdown rates, myotube size, and atrogin-1 and MuRF1 mRNA levels in the two cell lines. In addition, the expression of the glucocorticoid receptor (GR) and its role in glucocorticoid-induced metabolic changes were determined. Treatment with dexamethasone or corticosterone resulted in dose-dependent increases in protein degradation rates in both L6 and C2C12 myotubes accompanied by 25-30% reduction of myotube diameter. The same treatments increased atrogin-1 mRNA levels in L6 and C2C12 myotubes but, surprisingly, upregulated the expression of MuRF1 in L6 myotubes only. Both cell types expressed the GR and treatment with dexamethasone or corticosterone downregulated total cellular GR levels while increasing nuclear translocation of the GR in both L6 and C2C12 myotubes. The GR antagonist RU38486 inhibited the dexamethasone- and corticosterone-induced increases in atrogin-1 and MuRF1 expression in L6 myotubes but not in C2C12 myotubes. Interestingly, RU38486 exerted agonist effects in the C2C12, but not in the L6 myotubes. The present results suggest that muscle wasting-related responses to dexamethasone and corticosterone are similar, but not identical, in L6 and C2C12 myotubes. Most notably, the regulation by glucocorticoids of MuRF1 and the role of the GR may be different in the two cell lines. These differences need to be taken into account when cultured myotubes are used in future studies to further explore mechanisms of muscle wasting.     

A cell-autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by systemic glucocorticoid exposure

Glucocorticoids (GCs) are important regulators of skeletal muscle mass, and prolonged exposure will induce significant muscle atrophy. To better understand the mechanism of skeletal muscle atrophy induced by elevated GC levels, we examined three different models: exogenous synthetic GC treatment [dexamethasone (DEX)], nutritional deprivation, and denervation. Specifically, we tested the direct contribution of the glucocorticoid receptor (GR) in skeletal muscle atrophy by creating a muscle-specific GR-knockout mouse line (MGR(e3)KO) using Cre-lox technology. In MGR(e3)KO mice, we found that the GR is essential for muscle atrophy in response to high-dose DEX treatment. In addition, DEX regulation of multiple genes, including two important atrophy markers, MuRF1 and MAFbx, is eliminated completely in the MGR(e3)KO mice. In a condition where endogenous GCs are elevated, such as nutritional deprivation, induction of MuRF1 and MAFbx was inhibited, but not completely blocked, in MGR(e3)KO mice. In response to sciatic nerve lesion and hindlimb muscle denervation, muscle atrophy and upregulation of MuRF1 and MAFbx occurred to the same extent in both wild-type and MGR(e3)KO mice, indicating that a functional GR is not required to induce atrophy under these conditions. Therefore, we demonstrate conclusively that the GR is an important mediator of skeletal muscle atrophy and associated gene expression in response to exogenous synthetic GCs in vivo and that the MGR(e3)KO mouse is a useful model for studying the role of the GR and its target genes in multiple skeletal muscle atrophy models.     

Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.