RESUMO
The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Canais de Cálcio Tipo L/genética , Complexos Multiproteicos/genética , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Serina-Treonina Quinases TOR/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Potenciais Evocados/fisiologia , Acoplamento Excitação-Contração/fisiologia , Regulação da Expressão Gênica , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Contração Isométrica , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Proteína Regulatória Associada a mTOR , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mammalian target of rapamycin complex 1 (mTORC1) is central to the control of cell, organ, and body size. Skeletal muscle-specific inactivation of mTORC1 in mice results in smaller muscle fibers, fewer mitochondria, increased glycogen stores, and a progressive myopathy that causes premature death. In mTORC1-deficient muscles, peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), which regulates mitochondrial biogenesis and glucose homeostasis, is strongly down-regulated. Here we tested whether induction of mitochondrial biogenesis pharmacologically or by the overexpression of PGC-1α is sufficient to reverse the phenotype of mice deficient for mTORC1. We show that both approaches normalize mitochondrial function, such as oxidative capacity and expression of mitochondrial genes. However, they do not prevent or delay the progressive myopathy. In addition, we find that mTORC1 has a much stronger effect than PGC-1α on the glycogen content in muscle. This effect is based on the strong activation of PKB/Akt in mTORC1-deficient mice. We also show that activation of PKB/Akt not only affects glycogen synthesis but also diminishes glycogen degradation. Thus, our work provides strong functional evidence that mitochondrial dysfunction in mice with inactivated mTORC1 signaling is caused by the down-regulation of PGC-1α. However, our data also show that the impairment of mitochondria does not lead directly to the lethal myopathy.
Assuntos
Bezafibrato/farmacologia , Regulação da Expressão Gênica , Doenças Musculares/metabolismo , Proteínas/metabolismo , Animais , Glicogênio/química , Glicogênio/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Modelos Genéticos , Complexos Multiproteicos , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Serina-Treonina Quinases TOR , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: The mammalian target of rapamycin complex 1 (mTORC1) is a central node in a network of signaling pathways controlling cell growth and survival. This multiprotein complex integrates external signals and affects different nutrient pathways in various organs. However, it is not clear how alterations of mTORC1 signaling in skeletal muscle affect whole-body metabolism. RESULTS: We characterized the metabolic phenotype of young and old raptor muscle knock-out (RAmKO) and TSC1 muscle knock-out (TSCmKO) mice, where mTORC1 activity in skeletal muscle is inhibited or constitutively activated, respectively. Ten-week-old RAmKO mice are lean and insulin resistant with increased energy expenditure, and they are resistant to a high-fat diet (HFD). This correlates with an increased expression of histone deacetylases (HDACs) and a downregulation of genes involved in glucose and fatty acid metabolism. Ten-week-old TSCmKO mice are also lean, glucose intolerant with a decreased activation of protein kinase B (Akt/PKB) targets that regulate glucose transporters in the muscle. The mice are resistant to a HFD and show reduced accumulation of glycogen and lipids in the liver. Both mouse models suffer from a myopathy with age, with reduced fat and lean mass, and both RAmKO and TSCmKO mice develop insulin resistance and increased intramyocellular lipid content. CONCLUSIONS: Our study shows that alterations of mTORC1 signaling in the skeletal muscle differentially affect whole-body metabolism. While both inhibition and constitutive activation of mTORC1 induce leanness and resistance to obesity, changes in the metabolism of muscle and peripheral organs are distinct. These results indicate that a balanced mTORC1 signaling in the muscle is required for proper metabolic homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Metabolismo Energético , Complexos Multiproteicos/metabolismo , Músculo Esquelético/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores Etários , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Composição Corporal , Dieta Hiperlipídica , Genótipo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Insulina/sangue , Resistência à Insulina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Doenças Musculares/enzimologia , Doenças Musculares/genética , Doenças Musculares/fisiopatologia , Obesidade/enzimologia , Obesidade/genética , Obesidade/prevenção & controle , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Regulatória Associada a mTOR , Transdução de Sinais , Magreza/enzimologia , Magreza/genética , Fatores de Tempo , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice, constitutive and starvation-induced autophagy is blocked at the induction steps via mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation. Rapamycin is sufficient to restore autophagy in TSCmKO mice and improves the muscle phenotype of old mutant mice. Inversely, abrogation of mTORC1 signaling by depletion of raptor induces autophagy regardless of FoxO inhibition. Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and ensures a tight coordination of metabolic pathways. These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.
Assuntos
Autofagia/fisiologia , Complexos Multiproteicos/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares/fisiopatologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Doenças Musculares/metabolismo , Inanição/fisiopatologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Skeletal muscle mass is determined by the balance between protein synthesis and degradation. Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of protein translation and has been implicated in the control of muscle mass. Inactivation of mTORC1 by skeletal muscle-specific deletion of its obligatory component raptor results in smaller muscles and a lethal dystrophy. Moreover, raptor-deficient muscles are less oxidative through changes in the expression PGC-1α, a critical determinant of mitochondrial biogenesis. These results suggest that activation of mTORC1 might be beneficial to skeletal muscle by providing resistance to muscle atrophy and increasing oxidative function. Here, we tested this hypothesis by deletion of the mTORC1 inhibitor tuberous sclerosis complex (TSC) in muscle fibers. METHOD: Skeletal muscles of mice with an acute or a permanent deletion of raptor or TSC1 were examined using histological, biochemical and molecular biological methods. Response of the muscles to changes in mechanical load and nerve input was investigated by ablation of synergistic muscles or by denervation . RESULTS: Genetic deletion or knockdown of raptor, causing inactivation of mTORC1, was sufficient to prevent muscle growth and enhance muscle atrophy. Conversely, short-term activation of mTORC1 by knockdown of TSC induced muscle fiber hypertrophy and atrophy-resistance upon denervation, in both fast tibialis anterior (TA) and slow soleus muscles. Surprisingly, however, sustained activation of mTORC1 by genetic deletion of Tsc1 caused muscle atrophy in all but soleus muscles. In contrast, oxidative capacity was increased in all muscles examined. Consistently, TSC1-deficient soleus muscle was atrophy-resistant whereas TA underwent normal atrophy upon denervation. Moreover, upon overloading, plantaris muscle did not display enhanced hypertrophy compared to controls. Biochemical analysis indicated that the atrophy response of muscles was based on the suppressed phosphorylation of PKB/Akt via feedback inhibition by mTORC1 and subsequent increased expression of the E3 ubiquitin ligases MuRF1 and atrogin-1/MAFbx. In contrast, expression of both E3 ligases was not increased in soleus muscle suggesting the presence of compensatory mechanisms in this muscle. CONCLUSIONS: Our study shows that the mTORC1- and the PKB/Akt-FoxO pathways are tightly interconnected and differentially regulated depending on the muscle type. These results indicate that long-term activation of the mTORC1 signaling axis is not a therapeutic option to promote muscle growth because of its strong feedback induction of the E3 ubiquitin ligases involved in protein degradation.
RESUMO
Rapamycin and its derivatives are mTOR inhibitors used in tissue transplantation and cancer therapy. A percentage of patients treated with these inhibitors develop diabetic-like symptoms, but the molecular mechanisms are unknown. We show here that chronic rapamycin treatment in mice led to insulin resistance with suppression of insulin/IGF signaling and genes associated within this pathway, such as Igf1-2, Irs1-2, and Akt1-3. Importantly, skeletal muscle-specific YY1 knockout mice were protected from rapamycin-induced diabetic-like symptoms. This protection was caused by hyperactivation of insulin/IGF signaling with increased gene expression in this cascade that, in contrast to wild-type mice, was not suppressed by rapamycin. Mechanistically, rapamycin induced YY1 dephosphorylation and recruitment to promoters of insulin/IGF genes, which promoted interaction with the polycomb protein-2 corepressor. This was associated with H3K27 trimethylation leading to decreased gene expression and insulin signaling. These results have implications for rapamycin action in human diseases and biological processes such as longevity.
Assuntos
Diabetes Mellitus Experimental/prevenção & controle , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Fator de Transcrição YY1/deficiência , Animais , Diabetes Mellitus Experimental/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Resistência à Insulina/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisina/metabolismo , Metilação/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Fator de Transcrição YY1/metabolismoRESUMO
The formation, distribution, and maintenance of functional mitochondria are achieved through dynamic processes that depend strictly on the transcription of nuclear genes encoding mitochondrial proteins. A large number of these mitochondrial genes contain binding sites for the transcription factor Yin Yang 1 (YY1) in their proximal promoters, but the physiological relevance is unknown. We report here that skeletal-muscle-specific YY1 knockout (YY1mKO) mice have severely defective mitochondrial morphology and oxidative function associated with exercise intolerance, signs of mitochondrial myopathy, and short stature. Gene set enrichment analysis (GSEA) revealed that the top pathways downregulated in YY1mKO mice were assigned to key metabolic and regulatory mitochondrial genes. This analysis was consistent with a profound decrease in the level of mitochondrial proteins and oxidative phosphorylation (OXPHOS) bioenergetic function in these mice. In contrast to the finding for wild-type mice, inactivation of the mammalian target of rapamycin (mTOR) did not suppress mitochondrial genes in YY1mKO mice. Mechanistically, mTOR-dependent phosphorylation of YY1 resulted in a strong interaction between YY1 and the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), a major regulator of mitochondrial function. These results underscore the important role of YY1 in the maintenance of mitochondrial function and explain how its inactivation might contribute to exercise intolerance and mitochondrial myopathies.
Assuntos
Mitocôndrias/metabolismo , Fator de Transcrição YY1/genética , Alelos , Animais , Metabolismo Energético/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Fosforilação Oxidativa , Fenótipo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/fisiologiaRESUMO
Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.
Assuntos
Proteínas de Transporte/metabolismo , Distrofina/metabolismo , Músculo Esquelético/enzimologia , Distrofia Muscular Animal/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fatores Etários , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Distrofina/genética , Eletroporação , Metabolismo Energético , Ativação Enzimática , Feminino , Glucose/metabolismo , Glicogênio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/enzimologia , Contração Muscular , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/fisiopatologia , Mutação , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Ratos , Proteína Regulatória Associada a mTOR , Índice de Gravidade de Doença , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , Transdução Genética , Utrofina/metabolismoRESUMO
Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.