Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver.

The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.     

Effect of glucocorticoids on contractile apparatus of rat skeletal muscle

Skeletal muscles which have a high oxidative potential are less sensitive to the catabolic action of dexamethasone. In fast-twitch white muscles, where the oxidative capacity is low, the alkaline proteinase activity as well as the rise in the number of lysosomes was more pronounced. It seems that the glucocorticoid-caused myopathy is a result of elevated degradation of contractile proteins. This process of degradation of contractile proteins begins in the myosine filaments and then spreads to the thin filaments and the z-line.     

USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.     

Activation of rat liver and rabbit skeletal muscle glycogen synthases by rat liver cytosolic protein phosphatases

To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.     

Limited resistance of hypertrophied skeletal muscle to glucocorticoids

Male hypophysectomized rats were initially assigned to a control or an overloaded group that underwent compensatory hypertrophy of plantaris muscles to steady-state levels following removal of synergistic musculature. Plantaris muscle mass of overloaded animals was higher than that of controls by 38% (391 +/- 8 vs 284 +/- 7 mg) and glucocorticoid cytosol specific binding concentrations, using [3H]triamcinolone acetonide (TA) as the labeled steroid, was also significantly higher in hypertrophied muscles (83.3 +/- 3.9 fmol . mg protein-1) than in control muscles 56.3 +/- 3.9 fmol . mg protein-1). Cortisone acetate (CA) was then administered daily subcutaneously in high, 100 mg; intermediate, 10 mg; or low, 1.0 mg . kg-1 body wt doses. Groups of rats were killed after 1/4, 2 days and 7 days. Absolute muscle mass losses after 7 days of CA treatment were approx 80 mg with high doses and 60 mg with intermediate doses in both hypertrophied and control muscles. The low CA dose did not produce atrophy. The absolute depletion of [3H]TA binding activity with CA treatment was always greater in hypertrophied muscles of high and intermediate dose treated than those of their respective controls, but TA binding capacities remained higher in hypertrophied muscles than in controls at almost all time points in all treatment groups. Unlike previous findings in which the simultaneous initiation of overload prevented glucocorticoid induced muscle wasting, no resistance to the effect of CA treatment was observed when treatment was begun after hypertrophy had occurred.     

The gene expression of ubiquitin ligase E3alpha is upregulated in skeletal muscle during sepsis in rats-potential role of glucocorticoids

Muscle protein breakdown during sepsis is associated with upregulated expression and activity of the ubiquitin-proteasome proteolytic pathway. Previous studies suggest that ubiquitination of proteins in skeletal muscle is regulated by the ubiquitin ligase E3alpha together with the 14 kDa ubiquitin-conjugating enzyme E2(14k). The E3alpha gene was cloned only recently. The influence of sepsis on the gene expression of E3alpha in skeletal muscle has not been reported. In the present study, induction of sepsis in rats by cecal ligation and puncture resulted in increased mRNA levels for E3alpha in white, fast-twitch but not in red slow-twitch muscle. Treatment with the glucocorticoid receptor antagonist RU38486 (10 mg/kg) prevented the sepsis-induced increase in E3alpha and E2(14k) mRNA levels. The present study is the first report of increased E3alpha expression in skeletal muscle during sepsis. The results lend further support to the concept that glucocorticoid-mediated upregulation of the ubiquitin-proteasome proteolytic pathway is involved in sepsis-induced muscle cachexia. Increased expression of both E3alpha and E2(14k) suggests that muscle proteins are degraded in the N-end rule pathway during sepsis.     

Activation of transgene expression in skeletal muscle by focused ultrasound

To correlate thermal dose from focused ultrasound (FUS) with gene expression and tissue injury, a temperature plateau strategy was employed. Plasmids encoding luciferase gene under the control of hsp70B promoter were transfected into the right gastrocnemius muscle in a rat via electroporation. One day after transfection, hind limbs were treated with 3.3-MHz focused ultrasound, using one of four different temperature plateaus with spatial-peak time-average focal temperatures (T_SPTA) of 46 °C, 48 °C, 51 °C and 62 °C. The treatment duration at the plateau temperature was varied from 0 to 30 s. Gene expression was analyzed in vivo one day following FUS treatment, and H&E staining was employed to assess tissue injury. Gene activation and tissue damage correlated closely with thermal dose. The highest level of gene activation was induced by FUS at T_SPTA = 51 °C for 20 s, which was found to be statistically equivalent to that produced by water-bath hyperthermia.     

Induction of MafBx and Murf ubiquitin ligase mRNAs in rat skeletal muscle after LPS injection

MafBx and Murf are two new rat E3 ubiquitin ligases induced in muscle atrophy. Our goal was to investigate whether lipopolysaccharide (LPS) injection, a model of muscle catabolism, is associated with increased expression of MafBx and Murf. LPS (750 microg/100 g body weight) induces MafBx and Murf mRNA (respectively, 23-fold and 33-fold after 12 h; P<0.001). A transient induction of tumor necrosis factor-alpha mRNA (21-fold; P<0.001 at 3 h) and a decrease of insulin like growth factor-I mRNA (50%; P<0.001 at 6 h), two potential regulators of the ubiquitin-proteasome system were also demonstrated. In summary, MafBx and Murf mRNA are up-regulated in response to LPS and might play a role in the muscle proteolysis observed.     

Acute attenuation of translation initiation and protein synthesis by glucocorticoids in skeletal muscle

Glucocorticoids are diabetogenic factors that not only antagonize the action of insulin in target tissues but also render these tissues catabolic. Therefore, in rats, we endeavored to characterize the effects in skeletal muscle of glucocorticoids on translation initiation, a regulated process that, in part, governs overall protein synthesis through the modulated activities of eukaryotic initiation factors (eIFs). Four hours after intraperitoneal administration of dexamethasone (100 microg/100 g body wt), protein synthesis in skeletal muscle was reduced to 59% of the value recorded in untreated control animals. Furthermore, translation initiation factor eIF4E preferred association with its endogenous inhibitor 4E-BP1 rather than eIF4G. Dexamethasone treatment resulted in dephosphorylation of both 4E-BP1 and the 40S ribosomal protein S6 kinase concomitant with enhanced phosphorylation of eIF4E. Moreover, the guanine nucleotide exchange activity of eIF2B was unaffected as was phosphorylation of the alpha-subunit of eIF2. Hence glucocorticoids negatively modulate the activation of a subset of the protein synthetic machinery, thereby contributing to the catabolic properties of this class of hormones in vivo.     

Antagonism by glucocorticoids and exercise on expression of glutamine synthetase in skeletal muscle

Chronic glucocorticoid treatment results in skeletal muscle wasting. However, if the contractile activity of muscle is increased, this effect is abated. Because the gene encoding glutamine synthetase is known to be glucocorticoid inducible, it represents an appropriate model for testing whether glucocorticoids and endurance training can exert antagonistic effects on the expression of specific genes in muscle tissue. Our data confirm that administration of hydrocortisone 21-acetate to rats produces 2.4- and 5.9-fold increases in plantaris muscle glutamine synthetase enzyme activity and mRNA, respectively. Moreover, subjecting rats to a 12- to 16-wk exercise program diminishes the basal levels of these indices of glutamine synthetase expression to approximately 60% of the values observed in sedentary controls. Endurance training produces a similar effect on plantaris muscle glutamine synthetase expression in glucocorticoid-treated animals. These data demonstrate that the therapeutic effects of exercise in counteracting muscle atrophy are associated with attenuation of expression of a glucocorticoid-inducible gene in skeletal muscle.     

Activation of ubiquitin-proteasome pathway is involved in skeletal muscle wasting in a rat model with biliary cirrhosis: potential role of TNF-alpha

Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-alpha can stimulate muscle proteolysis; therefore, a potential relationship between TNF-alpha and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 (P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated (P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-alpha levels were increased and correlated positively with free and conjugated ubiquitin (P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-alpha might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.     

Insulin action on skeletal muscle protein metabolism during catabolic states

Insulin plays a major role in the regulation of skeletal muscle protein turnover but its mechanism of action is not fully understood, especially in vivo during catabolic states. These aspects are presently reviewed. Insulin inhibits the ATP-ubiquitin proteasome proteolytic pathway which is presumably the predominant pathway involved in the breakdown of muscle protein. Evidence of the ability of insulin to stimulate muscle protein synthesis in vivo was also presented. Many catabolic states in rats, e.g. streptozotocin diabetes, glucocorticoid excess or sepsis-induced cytokines, resulted in a decrease in insulin action on protein synthesis or degradation. The effect of catabolic factors would therefore be facilitated. In contrast, the antiproteolytic action of insulin was improved during hyperthyroidism in man and early lactation in goats. Excessive muscle protein breakdown should therefore be prevented. In other words, the anabolic hormone insulin partly controlled the 'catabolic drive'. Advances in the understanding of insulin signalling pathways and targets should provide information on the interactions between insulin action, muscle protein turnover and catabolic factors.     

Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle

Muscle wasting during sepsis reflects increased expression and activity of the ubiquitin-proteasome proteolytic pathway and is at least in part mediated by glucocorticoids. The ubiquitination of proteins destined to be degraded by the proteasome is regulated by multiple enzymes, including ubiquitin ligases. We tested the hypothesis that sepsis upregulates the gene expression of the newly described ubiquitin ligases, MuRF1 and atrogin-1/MAFbx. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. In some experiments, rats were treated with the glucocorticoid receptor antagonist RU 38486 before induction of sepsis. At various time points after induction of sepsis, mRNA levels for MuRF1 and atrogin-1/MAFbx were determined in extensor digitorum longus muscles by real-time PCR. Sepsis resulted in a 10-16-fold increase in gene expression of the ubiquitin ligases studied here. These changes were much greater than those observed previously for another ubiquitin ligase, E3alpha, in muscle during sepsis. Treatment of rats with RU 38486 prevented the sepsis-induced increase in mRNA levels for MuRF1 and atrogin-1/MAFbx, suggesting that glucocorticoids participate in the upregulation of these genes in muscle during sepsis. The present results lend further support to the concept that the ubiquitin-proteasome pathway plays an important role in sepsis-induced muscle proteolysis and suggest that multiple ubiquitin ligases may participate in the development of muscle wasting during sepsis.     

Regulation of the ubiquitin proteasome system in mechanically injured human skeletal muscle

Metabolic consequences of direct muscle trauma are insufficiently defined. Their effects on the ubiquitin-proteasome pathway (UPP) of protein degradation in human skeletal muscles are as yet unknown. Thus, we investigated whether the UPP is involved in the metabolic response evoked in directly traumatized human skeletal muscles. Biopsies were obtained from contused muscles after fractures and from normal muscles during elective implant removal (control). As estimated by western blot analyses, concentrations of free ubiquitin and ubiquitin protein conjugates were similar in extracts from injured and uninjured muscles. Ubiquitin protein ligation rates were reduced after injury (1.5+/-0.2 vs. 1.0+/-0.15 fkat/microg; p=0.04). Chymotryptic-, tryptic- and caspase-like proteasome peptidase activities (total activity minus activity in the presence of proteasome inhibitors) increased significantly after trauma (p=0.04 - 0.001). Significant increases in total chymotryptic- and caspase-like activities were attributable to proteasome activation. Our results extend the possible role of the UPP in muscle wasting to direct muscle trauma. They further suggest that the effects of direct mechanical trauma are not limited to the proteasome and imply that ubiquitin protein ligase systems are also involved. Based on the potential role of the UPP in systemic diseases, it might also be a therapeutic target to influence muscle loss in critically ill blunt trauma patients, in which large proportions of muscle are exposed to direct trauma.     

Activation of a UBC4-dependent pathway of ubiquitin conjugation during postnatal development of the rat testis.

During spermatogenesis, germ cells undergo mitotic and meiotic divisions to form haploid round spermatids which mature to functional elongated spermatozoa. During this process there occurs remodeling of cell structure and loss of most of the cytoplasm and a large fraction of cellular proteins. To evaluate the role of the ubiquitin proteolytic system in this protein loss, we measured levels of ubiquitinated proteins and rates of ubiquitin conjugation in extracts of testes from rats of different ages. Endogenous ubiquitin-protein conjugates increased till day 30 and then reached a plateau. In parallel, there was a progressive increase in the rate of conjugation of ubiquitin to proteins in testis extracts from these animals. To test the importance of two major ubiquitin conjugating enzyme families in the conjugation, immunoprecipitation of UBC2 or UBC4 from 10- and 30-day-old testis extracts was carried out and the remaining conjugation activity in supernatants was assayed. Depletion of either enzyme family resulted in decreased conjugation. However, most of the conjugation activity and, more importantly, the increased conjugation during development were UBC4-dependent. Immunocytochemistry demonstrated a marked increase in expression of UBC4 in spermatids, consistent with the UBC4-dependent activation of conjugation seen in vitro. In situ hybridization studies evaluated the contribution of various UBC4 isoforms to this induction. UBC4-1 mRNA was expressed in most cells. UBC4-2 mRNA was restricted to germ cells with high levels of expression in round and elongated spermatids. UBC4-testis had previously been shown to be expressed only in spermatids. Our data suggest that induction of various UBC4 isoforms activates overall conjugation and plays an important role in the cellular remodeling and protein loss occurring during spermatogenesis.