RESUMO
Although there are reports that polyphenol resveratrol (Rsv) may cause muscle hypertrophy in basal conditions and attenuate muscle wasting in catabolic situations, its mechanism of action is still unclear. Our study evaluated the ex vivo effects of Rsv on protein metabolism and intracellular signaling in innervated (sham-operated; Sham) and 3-day sciatic denervated (Den) rat skeletal muscles. Rsv (10-4 M) reduced total proteolysis (40%) in sham muscles. Den increased total proteolysis (~40%) in muscle, which was accompanied by an increase in the activities of ubiquitin-proteasome (~3-fold) and lysosomal (100%) proteolytic systems. Rsv reduced total proteolysis (59%) in Den muscles by inhibiting the hyperactivation of ubiquitin-proteasome (50%) and lysosomal (~70%) systems. Neither Rsv nor Den altered calcium-dependent proteolysis in muscles. Mechanistically, Rsv stimulated PKA/CREB signaling in Den muscles, and PKA blockage by H89 (50µM) abolished the antiproteolytic action of the polyphenol. Rsv reduced FoxO4 phosphorylation (~60%) in both Sham and Den muscles and Akt phosphorylation (36%) in Den muscles. Rsv also caused a homeostatic effect in Den muscles by returning their protein synthesis rates to levels similar to Sham muscles. These data indicate that Rsv directly inhibits the proteolytic activity of lysosomal and ubiquitin-proteasome systems, mainly in Den muscles through, at least in part, the activation of PKA/CREB signaling.
Assuntos
Músculo Esquelético , Complexo de Endopeptidases do Proteassoma , Ratos , Animais , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Resveratrol/farmacologia , Músculo Esquelético/metabolismo , Ratos Wistar , Ubiquitinas/metabolismo , Ubiquitinas/farmacologiaRESUMO
OBJECTIVE: Although it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive. METHODS: To elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro. RESULTS: Here, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt). CONCLUSIONS: These data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Proteínas Proto-Oncogênicas c-akt , Urocortinas/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Animais , Hipertrofia/metabolismo , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Urocortinas/farmacologiaRESUMO
Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 µg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation.
Assuntos
Autofagia/efeitos dos fármacos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Lisossomos/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Denervação , Lisossomos/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/inervação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos WistarRESUMO
Although it is well known that administration of the selective ß(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 µM), a PKA activator. The in vitro addition of triciribine (10 µM), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.
Assuntos
Agonistas Adrenérgicos beta/uso terapêutico , Clembuterol/uso terapêutico , Lisossomos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Agonistas Adrenérgicos beta/farmacologia , Animais , Catepsina L/metabolismo , Clembuterol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/química , Ativadores de Enzimas/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Lisossomos/enzimologia , Masculino , Denervação Muscular/efeitos adversos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/enzimologia , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin-proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Inibidores da Fosfodiesterase 4/farmacologia , Proteólise/efeitos dos fármacos , Rolipram/farmacologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Expressão Gênica/fisiologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Animais , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas com Motivo Tripartido , Tirosina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.