β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB.     

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.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

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.     

Heart failure increases atrogin-1 and MuRF1 gene expression in skeletal muscle with fiber type-specific atrophy

Heart failure (HF) is characterized by a reduced tolerance to exercise due to early fatigue and dyspnea; this may be due in part to skeletal muscle myopathy with a shift from slow to fast fibers and loss of muscle mass. Muscle wasting does not occur similarly in all types of muscle fiber, thus we tested the hypothesis that HF induces skeletal muscle atrophy in a fiber type-specific manner altering the expression of atrogin-1 and MuRF1 in a fast muscle of rats with monocrotaline-induced heart failure. We studied extensor digitorum longus (EDL) muscle from both HF and control Wistar rats. Atrogin-1 and MuRF1 mRNA content were determined using Real-Time RT-qPCR while muscle fiber cross-sectional area (CSA) from sections stained histochemically for myofibrillar ATPase were used as an index of type-specific fiber atrophy. The measurement of gene expression by RT-qPCR revealed that EDL muscle mRNA expression of MuRF1 and atrogin-1 was significantly increased in the HF group. Muscle fiber type IIB CSA decreased in the HF group compared to the CT group; there was no significant difference in muscle fiber types I and IIA/D CSA between the HF and CT groups. In conclusion, we showed that HF induces fiber type IIB specific atrophy, up-regulating atrogin-1 and MuRF1 mRNA expression in EDL muscle of monocrotaline treated rats.     

Hormone, cytokine, and nutritional regulation of sepsis-induced increases in atrogin-1 and MuRF1 in skeletal muscle

Various atrophic stimuli increase two muscle-specific E3 ligases, muscle RING finger 1 (MuRF1) and atrogin-1, and knockout mice for these "atrogenes" display resistance to denervation-induced atrophy. The present study determined whether increased atrogin-1 and MuRF1 mRNA are mediated by overproduction of endogenous glucocorticoids or inflammatory cytokines in adult rats and whether atrogene expression can be downregulated by anabolic agents such as insulin-like growth factor (IGF)-I and the nutrient-signaling amino acid leucine. Both atrogin-1 and MuRF1 mRNA in gastrocnemius was upregulated dose and time dependently by endotoxin. Additionally, peritonitis produced by cecal ligation and puncture increased atrogin-1 and MuRF1 mRNA in gastrocnemius (but not soleus or heart) by 8 h, which was sustained for 72 and 24 h, respectively. Whereas the sepsis-induced increase in atrogin-1 expression was completely prevented by IGF-I, the increased MuRF1 was not altered. In contrast to the IGF-I effect, the sepsis-induced increased mRNA of both atrogenes was unresponsive to either acute or repetitive administration of leucine. Whereas exogenous infusion of TNF-alpha increased atrogin-1 and MuRF1 in gastrocnemius, pretreatment of septic rats with the TNF antagonist TNF-binding protein did not prevent increased expression of either atrogene. Similarly, whereas dexamethasone increased atrogene expression, pretreatment with the glucocorticoid receptor antagonist RU-486 failed to ameliorate the sepsis-induced increase in atrogin-1 and MuRF1. Thus, under in vivo conditions in mature adult rats, the sepsis-induced increase in muscle atrogin-1 and MuRF1 mRNA appears both glucocorticoid and TNF independent and is unresponsive to leucine.     

Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.     

Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.     

GHRP-2, a GHS-R agonist, directly acts on myocytes to attenuate the dexamethasone-induced expressions of muscle-specific ubiquitin ligases, Atrogin-1 and MuRF1

Recent reports suggest that Atrogin-1 and MuRF1, E3 ubiquitin ligases, play a pivotal role in muscle atrophy. In the present study, effect of Growth Hormone Releasing Peptide-2 (GHRP-2), a GH secretagogue receptor (GHS-R) agonist, on the expressions of Atrogin-1 and MuRF1 in vivo rat muscles was examined. Dexamethasone administration increased Atrogin-1 mRNA level in rat soleus muscle. The increased mRNA level of Atrogin-1 was significantly attenuated by GHRP-2. In addition, GHRP-2 decreased MuRF1 mRNA level irrespective of the presence of dexamethasone. Although IGF-I is a well-known protective factor for muscle atrophy, GHRP-2 did not influence plasma IGF-I levels and IGF-I mRNA levels in muscles. To clarify a direct effect of GHRP-2, differentiated C2C12 myocytes were used. Ten micrometer dexamethasone increased both Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 attenuated dexamethasone-induced expression of them dose-dependently and decreased the basal level of MuRF1 mRNA. The suppressive effect on the expressions of Atrogin-1 and MuRF1 by GHRP-2 was blocked by [D-Lys(3)]-GHRP-6, a GHS-R1a blocker, suggesting the effect of GHRP-2 was mediated through GHS-R1a. Taken together, GHRP-2 directly attenuates Atrogin-1 and MuRF1 mRNA levels through ghrelin receptors in myocytes.     

Acupuncture ameliorated skeletal muscle atrophy induced by hindlimb suspension in mice

Preventing skeletal muscle atrophy is critical for maintaining quality of life, but it is often a challenging goal for the elderly and patients with severe conditions. We hypothesized that acupuncture in place of exercise training is an alternative non-pharmacological intervention that can help to prevent muscle atrophy. To elucidate the effects of acupuncture on skeletal muscle atrophy caused by hindlimb suspension (HS), we performed acupuncture on mice according to two different methods: acupuncture with electrical stimulation (EA: electroacupuncture) and without electrical stimulation (MA: manual acupuncture). A needle was retained in the gastrocnemius muscle for 30 min every day for 2 weeks in the EA and MA groups. In the EA group, 30 min of repetitive electrical stimulation (1 Hz, 1 ms pulse width, 6.5 mA intensity) was also applied. HS significantly reduced muscle mass and the cross-sectional area of the soleus muscles. This HS-induced reduction was significantly improved in the EA group, although the level of improvement remained insufficient when compared with the control group. We found that the mRNA expression levels of atrogin-1 and MuRF1, which play a principal role in muscle-specific degradation as E3 ubiquitin ligases, were significantly increased in the HS group compared to the control group. EA and MA reduced the HS-induced upregulation of atrogin-1 (p < 0.01 in EA and MA) and MuRF1 (p < 0.01 in EA) mRNAs. We also found that the expression levels of PI3K, Akt1, TRPV4, adenosine A1 receptor, myostatin, and SIRT1 mRNAs tended to be increased by HS. EA and MA further increased the HS-induced upregulation of Akt1 (p < 0.05 in MA) and TRPV4 (p < 0.05 in MA) mRNAs. We concluded that acupuncture partially prevented skeletal muscle atrophy. This effect might be due to an increase in protein synthesis and a decrease in protein degradation.     

Curcumin prevents lipopolysaccharide-induced atrogin-1/MAFbx upregulation and muscle mass loss

Because elevated ubiquitin ligase atrogin-1/MAFbx and MuRF1 mediate skeletal muscle wasting associated with various catabolic conditions, the signaling pathways involved in the upregulation of these genes under pathological conditions are considered therapeutic targets. AKT and NF-kappaB have been previously shown to regulate the expression of atrogin-1/MAFbx or MuRF1, respectively. In addition, we recently found that p38 MAPK mediates TNF-alpha upregulation of atrogin-1/MAFbx expression, suggesting that multiple signaling pathways mediate muscle wasting in inflammatory diseases. To date, however, these advances have not resulted in a practical clinical intervention for disease-induced muscle wasting. In the present study, we tested the effect of curcumin--a non-toxic anti-inflammatory reagent that inhibits p38 and NF-kappaB--on lipopolysaccharide (LPS)-induced muscle wasting in mice. Daily intraperitoneal (i.p.) injection of curcumin (10-60 micro g/kg) for 4 days inhibited, in a dose-dependent manner, the LPS-stimulated (1 mg/kg, i.p.) increase of atrogin-1/MAFbx expression in gastrocnemius and extensor digitorum longus (EDL) muscles, resulting in the attenuation of muscle protein loss. It should also be noted that curcumin administration did not alter the basal expression of atrogin-1/MAFbx, nor did it affect LPS-stimulated MuRF1 and polyubiquitin expression. LPS activated p38 and NF-kappaB, while inhibiting AKT; whereas, curcumin administration inhibited LPS-stimulated p38 activation, without altering the effect of LPS on NF-kappaB and AKT. These results indicate that curcumin is effective in blocking LPS-induced loss of muscle mass through the inhibition of p38-mediated upregulation of atrogin-1/MAFbx.