Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., "atrogenes"). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss of muscle mass/protein in response to chronic alcohol abuse appears to result primarily from a decrement in muscle protein synthesis, not an increase in degradation.     

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.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.     

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.     

Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle

Adult skeletal muscle undergoes adaptation in response to endurance exercise, including fast-to-slow fiber type transformation and enhanced angiogenesis. The purpose of this study was to determine the temporal and spatial changes in fiber type composition and capillary density in a mouse model of endurance training. Long-term voluntary running (4 wk) in C57BL/6 mice resulted in an approximately twofold increase in capillary density and capillary-to-fiber ratio in plantaris muscle as measured by indirect immunofluorescence with an antibody against the endothelial cell marker CD31 (466 ± 16 capillaries/mm² and 0.95 ± 0.04 capillaries/fiber in sedentary control mice vs. 909 ± 55 capillaries/mm² and 1.70 ± 0.04 capillaries/fiber in trained mice, respectively; P < 0.001). A significant increase in capillary-to-fiber ratio was present at day 7 with increased concentration of vascular endothelial growth factor (VEGF) in the muscle, before a significant increase in percentage of type IIa myofibers, suggesting that exercise-induced angiogenesis occurs first, followed by fiber type transformation. Further analysis with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact manifested transiently in type IIb + IId/x fibers at the time (day 7) of significant increase in total capillary density. These findings suggest that endurance training induces angiogenesis in a subpopulation of type IIb + IId/x fibers before switching to type IIa fibers. adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor     

Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P < 0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P < 0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P < 0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.     

Effects of fasting and refeeding on expression of MAFbx and MuRF1 in chick skeletal muscle

The present study investigated the effects of fasting and refeeding on the expression of proteasome-related genes and their downstream targets in the skeletal muscles of chicks. Seven-day-old chicks were fasted for 24 or 48 h and then refed for 4 h. The expression levels of MAFbx and MuRF1, which function as E3 ligases in the ubiquitin-proteasome system, were investigated at the mRNA and protein levels. MAFbx and MuRF1 expression were increased by fasting and these increases were downregulated by refeeding. The expression of the target proteins of these E3 ligases, MyoD and M-CK, was also analyzed. The levels of these proteins were downregulated by fasting, and these decreases were rescued by refeeding. The results of this study indicate that fasting stimulates MAFbx and MuRF1 expression in chicks, possibly leading to increased degradation of their corresponding target proteins.     

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin‐1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1 but it is not known if atrogin‐1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin‐1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain‐, and caspase‐3‐dependent protein breakdown in addition to proteasome‐dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin‐1 and MuRF1 mRNA levels. The same treatment increased proteasome‐, cathepsin L‐, and calpain‐dependent proteolytic rates by approximately 40% but did not influence caspase‐3‐dependent proteolysis. The expression of atrogin‐1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley‐Liss, Inc.     

mRNA expression signatures of human skeletal muscle atrophy identify a natural compound that increases muscle mass

Skeletal muscle atrophy is a common and debilitating condition that lacks a pharmacologic therapy. To develop a potential therapy, we identified 63 mRNAs that were regulated by fasting in both human and mouse muscle, and 29 mRNAs that were regulated by both fasting and spinal cord injury in human muscle. We used these two unbiased mRNA expression signatures of muscle atrophy to query the Connectivity Map, which singled out ursolic acid as a compound whose signature was opposite to those of atrophy-inducing stresses. A natural compound enriched in apples, ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy in mice. It did so by enhancing skeletal muscle insulin/IGF-I signaling and inhibiting atrophy-associated skeletal muscle mRNA expression. Importantly, ursolic acid's effects on muscle were accompanied by reductions in adiposity, fasting blood glucose, and plasma cholesterol and triglycerides. These findings identify a potential therapy for muscle atrophy and perhaps other metabolic diseases.     

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.     

Myostatin expression in age and denervation-induced skeletal muscle atrophy

Myostatin is hypothesized to regulate skeletal muscle mass and to be a potential target for therapeutic intervention in sarcopenia. To clarify whether myostatin is invariably associated with sarcopenia, this study examined the levels of expression of myostatin mRNA and protein in Sprague Dawley rats during aging- and denervation-induced sarcopenia. The level of myostatin mRNA in the gastrocnemius decreased progressively with age being 9, 34 and 56% lower at 6, 12 and 27 months, respectively, compared with mRNA levels at 1.5 months. In contrast, two low molecular mass isoforms of myostatin protein identified by Western blotting increased progressively with age. With denervation, myostatin mRNA was 31% higher on day 1 but by 14 days after sciatic neurectomy when the muscle had atrophied 50%, myostatin expression decreased 34% relative to the sham operated limb. Western analysis of the denervated gastrocnemius showed that myostatin protein levels varied in parallel with mRNA. These disparate patterns of expression of myostatin during age- and denervation-induced atrophy suggest that the regulation of myostatin is complex and variable depending on whether the atrophy is slowly or rapidly progressive.     

Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Distinct skeletal muscle fiber characteristics and gene expression in diet-sensitive versus diet-resistant obesity

Inter-individual variability in weight gain and loss under energy surfeit and deficit conditions, respectively, are well recognized but poorly understood phenomena. We documented weight loss variability in an intensively supervised clinical weight loss program and assessed skeletal muscle gene expression and phenotypic characteristics related to variable response to a 900 kcal regimen. Matched pairs of healthy, diet-compliant, obese diet-sensitive (ODS) and diet-resistant (ODR) subjects were defined as those in the highest and lowest quintiles for weight loss rate. Physical activity energy expenditure was minimal and comparable. Following program completion and weight stabilization, skeletal muscle biopsies were obtained. Gene expression analysis of rectus femoris and vastus lateralis indicated upregulation of genes and gene sets involved in oxidative phosphorylation and glucose and fatty acid metabolism in ODS compared with ODR. In vastus lateralis, there was a higher proportion of oxidative (type I) fibers in ODS compared with ODR women and lean controls, fiber hypertrophy in ODS compared with ODR women and lean controls, and lower succinate dehydrogenase in oxidative and oxidative-glycolytic fibers in all obese compared with lean subjects. Intramuscular lipid content was generally higher in obese versus lean, and specifically higher in ODS vs. lean women. Altogether, our findings demonstrate differences in muscle gene expression and fiber composition related to clinical weight loss success.