RESUMO
BACKGROUND: Mitochondrial biogenesis is crucial for myogenic differentiation and regeneration of skeletal muscle tissue and is tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network. In the present study, we hypothesized that inactivation of glycogen synthase kinase (GSK)-3ß, previously suggested to interfere with PGC-1 in non-muscle cells, potentiates PGC-1 signaling and the development of mitochondrial biogenesis during myogenesis, ultimately resulting in an enhanced myotube oxidative capacity. METHODS: GSK-3ß was inactivated genetically or pharmacologically during myogenic differentiation of C2C12 muscle cells. In addition, m. gastrocnemius tissue was collected from wild-type and muscle-specific GSK-3ß knock-out (KO) mice at different time-points during the reloading/regeneration phase following a 14-day hind-limb suspension period. Subsequently, expression levels of constituents of the PGC-1 signaling network as well as key parameters of mitochondrial oxidative metabolism were investigated. RESULTS: In vitro, both knock-down as well as pharmacological inhibition of GSK-3ß not only increased expression levels of important constituents of the PGC-1 signaling network, but also potentiated myogenic differentiation-associated increases in mitochondrial respiration, mitochondrial DNA copy number, oxidative phosphorylation (OXPHOS) protein abundance and the activity of key enzymes involved in the Krebs cycle and fatty acid ß-oxidation. In addition, GSK-3ß KO animals showed augmented reloading-induced increases in skeletal muscle gene expression of constituents of the PGC-1 signaling network as well as sub-units of OXPHOS complexes compared to wild-type animals. CONCLUSION: Inactivation of GSK-3ß stimulates activation of PGC-1 signaling and mitochondrial biogenesis during myogenic differentiation and reloading of the skeletal musculature.
Assuntos
Glicogênio Sintase Quinase 3 beta/fisiologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Biogênese de Organelas , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Elevação dos Membros Posteriores/efeitos adversos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/citologia , Atrofia Muscular/etiologia , Atrofia Muscular/patologia , Mioblastos/citologia , Mioblastos/fisiologia , Fosforilação Oxidativa/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Aberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. Evidence in non-muscle cells suggests that glycogen synthase kinase-3ß (GSK-3ß) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3ß in the regulation of skeletal muscle oxidative metabolism is unknown. AIMS: We hypothesized that inactivation of GSK-3ß stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3ß inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism. METHODS: GSK-3ß was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3ß knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated. RESULTS: In vitro, knock-down of GSK-3ß increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3ß increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3ß KO animals were protected against unloading-induced decrements in expression levels of these constituents. CONCLUSION: Inactivation of GSK-3ß up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3ß KO protects against inactivity-induced reductions in muscle metabolic gene expression.
Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Respiração Celular/fisiologia , Ativação Enzimática , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/enzimologia , Fosforilação Oxidativa , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor. Alternative splicing and enzymatic shedding produce soluble forms that protect against damage by ligands including Advanced Glycation End products (AGEs). A link between RAGE and oxygen levels is evident from studies showing RAGE-mediated injury following hyperoxia. The effect of hypoxia on pulmonary RAGE expression and circulating sRAGE levels is however unknown. Therefore mice were exposed to chronic hypoxia for 21 d and expression of RAGE, sheddases in lungs and circulating sRAGE were determined. In addition, accumulation of AGEs in lungs and expression of the AGE detoxifying enzyme GLO1 and receptors were evaluated. In lung tissue gene expression of total RAGE, variants 1 and 3 were elevated in mice exposed to hypoxia, whereas mRAGE and sRAGE protein levels were decreased. In the hypoxic group plasma sRAGE levels were enhanced. Although the levels of pro-ADAM10 were elevated in lungs of hypoxia exposed mice, the relative amount of the active form was decreased and gelatinase activity unaffected. In the lungs, the RAGE ligand HMGB1 was decreased and of the AGEs, only LW-1 was increased by chronic hypoxia. Gene expression of AGE receptors 2 and 3 was significantly upregulated. Chronic hypoxia is associated with downregulation of pulmonary RAGE protein levels, but a relative increase in sRAGE. These alterations might be part of the adaptive and protective response mechanism to chronic hypoxia and are not associated with AGE formation except for the fluorophore LW-1 which emerges as a novel marker of tissue hypoxia.
Assuntos
Expressão Gênica , Hipóxia/genética , Pulmão/metabolismo , Receptores Imunológicos/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Doença Crônica , Produtos Finais de Glicação Avançada/metabolismo , Proteína HMGB1/metabolismo , Humanos , Hipóxia/sangue , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/sangue , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solubilidade , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Loss of quadriceps muscle oxidative phenotype (OXPHEN) is an evident and debilitating feature of chronic obstructive pulmonary disease (COPD). We recently demonstrated involvement of the inflammatory classical NF-κB pathway in inflammation-induced impairments in muscle OXPHEN. The exact underlying mechanisms however are unclear. Interestingly, IκB kinase α (IKK-α: a key kinase in the alternative NF-κB pathway) was recently identified as a novel positive regulator of skeletal muscle OXPHEN. We hypothesised that inflammation-induced classical NF-κB activation contributes to loss of muscle OXPHEN in COPD by reducing IKK-α expression. METHODS: Classical NF-κB signalling was activated (molecularly or by tumour necrosis factor α: TNF-α) in cultured myotubes and the impact on muscle OXPHEN and IKK-α levels was investigated. Moreover, the alternative NF-κB pathway was modulated to investigate the impact on muscle OXPHEN in absence or presence of an inflammatory stimulus. As a proof of concept, quadriceps muscle biopsies of COPD patients and healthy controls were analysed for expression levels of IKK-α, OXPHEN markers and TNF-α. RESULTS: IKK-α knock-down in cultured myotubes decreased expression of OXPHEN markers and key OXPHEN regulators. Moreover, classical NF-κB activation (both by TNF-α and IKK-ß over-expression) reduced IKK-α levels and IKK-α over-expression prevented TNF-α-induced impairments in muscle OXPHEN. Importantly, muscle IKK-α protein abundance and OXPHEN was reduced in COPD patients compared to controls, which was more pronounced in patients with increased muscle TNF-α mRNA levels. CONCLUSION: Classical NF-κB activation impairs skeletal muscle OXPHEN by reducing IKK-α expression. TNF-α-induced reductions in muscle IKK-α may accelerate muscle OXPHEN deterioration in COPD.
Assuntos
Quinase I-kappa B/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , NF-kappa B/metabolismo , Idoso , Animais , Western Blotting , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , NF-kappa B/genética , Oxirredução/efeitos dos fármacos , Fenótipo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Músculo Quadríceps/metabolismo , Músculo Quadríceps/fisiopatologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Impairments in skeletal muscle oxidative phenotype (OXPHEN) have been linked to the development of insulin resistance, metabolic inflexibility and progression of the metabolic syndrome and have been associated with progressive disability in diseases associated with chronic systemic inflammation. We previously showed that the inflammatory cytokine tumour necrosis factor-α (TNF-α) directly impairs muscle OXPHEN but underlying molecular mechanisms remained unknown. Interestingly, the inflammatory signalling pathway classical nuclear factor-κB (NF-κB) is activated in muscle in abovementioned disorders. Therefore, we hypothesised that muscle activation of classical NF-κB signalling is sufficient and required for inflammation-induced impairment of muscle OXPHEN. METHODS: Myotubes from mouse and human muscle cell lines were subjected to activation or blockade of the classical NF-κB pathway. In addition, wild-type and MISR (muscle-specific inhibition of classical NF-κB) mice were injected intra-muscularly with TNF-α. Markers and key regulators of muscle OXPHEN were investigated. RESULTS: Classical NF-κB activation diminished expression of oxidative phosphorylation (OXPHOS) sub-units, slow myosin heavy chain expression, activity of mitochondrial enzymes and potently reduced intra-cellular ATP levels. Accordingly, PGC-1/PPAR/NRF-1/Tfam signalling, the main pathway controlling muscle OXPHEN, was impaired upon classical NF-κB activation which required intact p65 trans-activation domains and depended on de novo gene transcription. Unlike wild-type myotubes, IκBα-SR myotubes (blocked classical NF-κB signalling) were refractory to TNF-α-induced impairments in OXPHEN and its regulation by the PGC-1/PPAR/NRF-1/Tfam cascade. In line with in vitro data, NF-κB blockade in vivo abrogated TNF-α-induced reductions in PGC-1α expression. CONCLUSION: Classical NF-κB activation impairs skeletal muscle OXPHEN.
Assuntos
Músculo Esquelético/metabolismo , NF-kappa B/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , NF-kappa B/genética , Oxirredução , Fenótipo , Fosforilação , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified, since the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-κB, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. Markers and regulators (including alternative NF-κB signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hindlimb suspension/reloading (HLS/RL) protocol. Expression levels of oxidative phosphorylation subunits and slow myosin heavy chain isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC) molecules PGC-1α and PGC-1ß and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α increased upon RL. PPAR-δ, nuclear respiratory factor 1 (NRF-1), NRF-2α, and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased, and phosphorylation of P100 and subsequent processing to P52 were elevated, reflecting alternative NF-κB activation. We conclude that RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-κB signaling.
Assuntos
Modelos Animais de Doenças , Imobilização/efeitos adversos , Músculo Esquelético/metabolismo , Transtornos Musculares Atróficos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Animais , Biomarcadores/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Elevação dos Membros Posteriores , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transtornos Musculares Atróficos/etiologia , Transtornos Musculares Atróficos/reabilitação , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , NF-kappa B/agonistas , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Distribuição Aleatória , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Suporte de Carga , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND & AIMS: Patients with advanced COPD often have difficulty maintaining sufficient dietary intake. Chemosensory function influences food choice and intake but is often overlooked in dietary assessment and intervention strategies. This study aimed to assess differences in chemosensory function and hedonic evaluation of food between patients with COPD and age- and gender-matched healthy controls. Additionally, a possible association between increased risk of sarcopenia or frailty and chemosensory impairments was explored. METHODS: We recruited 53 COPD patients (34 males, mean age 66.6 ± 7.6 years) and 53 controls (25 males, mean age 68.4 ± 5.7 years). Chemosensory function was assessed using a smell threshold, smell identification (Sniffin' Sticks, Burghart) and taste recognition test (Taste Strips, Burghart) and through self-report. Sensory properties (appearance, smell, taste, mouthfeel) of four standardized food products were evaluated on 9-point hedonic rating scales. Sarcopenia risk was assessed with the SARC-F. RESULTS: The COPD group scored lower on both the smell (p = 0.026 for threshold, p = 0.001 for identification) and taste recognition tests (p < 0.001) and also reported more smell and taste impairments (p < 0.001) compared to controls. Hedonic evaluation of food items' appearance (p = 0.009) and smell (p = 0.033) was lower in COPD patients. Within the COPD group, risk of sarcopenia was not associated with chemosensory function. CONCLUSION: This study demonstrates that COPD patients have poorer chemosensory function and experience more impairments compared to controls. COPD patients also tend to evaluate foods less positive than do their controls but within COPD patients, sarcopenia risk is not associated with chemosensory function.
Assuntos
Doença Pulmonar Obstrutiva Crônica , Sarcopenia , Masculino , Humanos , Pessoa de Meia-Idade , Idoso , Paladar , Sarcopenia/epidemiologia , Sarcopenia/etiologia , Olfato , Percepção , Doença Pulmonar Obstrutiva Crônica/complicaçõesRESUMO
Chronic obstructive pulmonary disease (COPD) is characterized by weight loss, muscle wasting (in advanced disease ultimately resulting in cachexia), and loss of muscle oxidative phenotype (oxphen). This study investigates the effect of inflammation (as a determinant of muscle wasting) on muscle oxphen by using cell studies combined with analyses of muscle biopsies of patients with COPD and control participants. We analyzed markers (citrate synthase, ß-hydroxyacyl-CoA dehydrogenase, and cytochrome c oxidase IV) and regulators (PGC-1α, PPAR-α, and Tfam) of oxphen in vastus lateralis muscle biopsies of patients with advanced COPD and healthy smoking control participants. Here 17 of 73 patients exhibited elevated muscle TNF-α mRNA levels. In these patients, significantly lower mRNA levels of all oxidative markers/regulators were found. Interestingly, these patients also had a significantly lower body mass index and tended to have less muscle mass. In cultured muscle cells, mitochondrial protein content and myosin heavy chain isoform I (but not II) protein and mRNA levels were reduced on chronic TNF-α stimulation. TNF-α also reduced mitochondrial respiration in a nuclear factor kappaB (NF-κB) -dependent manner. Importantly, TNF-α-induced NF-κB activation decreased promoter transactivation and transcriptional activity of regulators of mitochondrial biogenesis and muscle oxphen. In conclusion, these results demonstrate that TNF-α impairs muscle oxphen in a NF-κB-dependent manner.
Assuntos
Caquexia/metabolismo , Músculo Esquelético/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Western Blotting , Linhagem Celular , Citrato (si)-Sintase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico/metabolismo , Humanos , Hidroliases/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Quadriceps strength relates to exercise capacity and prognosis in chronic obstructive pulmonary disease (COPD). We wanted to quantify the prevalence of quadriceps weakness in COPD and hypothesised that it would not be restricted to patients with severe airflow obstruction or dyspnoea. Predicted quadriceps strength was calculated using a regression equation (incorporating age, sex, height and fat-free mass), based on measurements from 212 healthy subjects. The prevalence of weakness (defined as observed values 1.645 standardised residuals below predicted) was related to Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage and Medical Research Council (MRC) dyspnoea score in two cohorts of stable COPD outpatients recruited from the UK (n = 240) and the Netherlands (n = 351). 32% and 33% of UK and Dutch COPD patients had quadriceps weakness. A significant proportion of patients in GOLD stages 1 and 2, or with an MRC dyspnoea score of 1 or 2, had quadriceps weakness (28 and 26%, respectively). These values rose to 38% in GOLD stage 4, and 43% in patients with an MRC Score of 4 or 5. Quadriceps weakness was demonstrable in one-third of COPD patients attending hospital respiratory outpatient services. Quadriceps weakness exists in the absence of severe airflow obstruction or breathlessness.
Assuntos
Dispneia/fisiopatologia , Debilidade Muscular/epidemiologia , Doença Pulmonar Obstrutiva Crônica/complicações , Músculo Quadríceps/fisiopatologia , Índice de Gravidade de Doença , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Tolerância ao Exercício/fisiologia , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Debilidade Muscular/etiologia , Países Baixos , Prevalência , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Estudos Retrospectivos , Reino UnidoRESUMO
OBJECTIVE: In muscle cells, the peroxisome proliferator-activated receptor γ co-activator 1 (PGC-1) signaling network, which has been shown to be disturbed in the skeletal muscle in several chronic diseases, tightly controls mitochondrial biogenesis and oxidative substrate metabolism. Previously, we showed that inactivation of glycogen synthase kinase (GSK)-3ß potently increased Pgc-1α abundance and oxidative metabolism in skeletal muscle cells. The current study aims to unravel the molecular mechanism driving the increase in Pgc-1α mediated by GSK-3ß inactivation. METHODS: GSK-3ß was inactivated genetically or pharmacologically in C2C12 myotubes and the requirement of transcription factors known to be involved in Pgc-1α transcription for increases in Pgc-1α abundance mediated by inactivation of GSK-3ß was examined. RESULTS: Enhanced PGC-1α promoter activation after GSK-3ß inhibition suggested a transcriptionally-controlled mechanism. While myocyte enhancer factor (MEF)2 transcriptional activity was unaltered, GSK-3ß inactivation increased the abundance and activity of the transcription factors estrogen-related receptor (ERR)α and ERRγ. Pharmacological inhibition or knock-down of ERRα and ERRγ however failed to prevent increases in Pgc-1α mRNA mediated by GSK-3ß inactivation. Interestingly, GSK-3ß inactivation activated transcription factor EB (TFEB), evidenced by decreased phosphorylation and enhanced nuclear localization of the TFEB protein. Moreover, knock-down of TFEB completely prevented increases in Pgc-1α gene expression, PGC-1α promoter activity and PGC-1α protein abundance induced by GSK-3ß inactivation. Furthermore, mutation of a specific TFEB binding site on the PGC-1α promoter blocked promoter activation upon inhibition of GSK-3ß. CONCLUSIONS: In skeletal muscle, GSK-3ß inactivation causes dephosphorylation and nuclear translocation of TFEB resulting in TFEB-dependent induction of Pgc-1α expression.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta/genética , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosforilação , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Ativação Transcricional , Regulação para Cima , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Skeletal muscle pathology associated with a chronic inflammatory disease state (e.g., skeletal muscle atrophy and insulin resistance) is a potential consequence of chronic activation of NF-kappaB. It has been demonstrated that peroxisome proliferator-activated receptors (PPARs) can exert anti-inflammatory effects by interfering with transcriptional regulation of inflammatory responses. The goal of the present study, therefore, was to evaluate whether PPAR activation affects cytokine-induced NF-kappaB activity in skeletal muscle. Using C(2)C(12) myotubes as an in vitro model of myofibers, we demonstrate that PPAR, and specifically PPARgamma, activation potently inhibits inflammatory mediator-induced NF-kappaB transcriptional activity in a time- and dose-dependent manner. Furthermore, PPARgamma activation by rosiglitazone strongly suppresses cytokine-induced transcript levels of the NF-kappaB-dependent genes intracellular adhesion molecule 1 (ICAM-1) and CXCL1 (KC), the murine homolog of IL-8, in myotubes. To verify whether muscular NF-kappaB activity in human subjects is suppressed by PPARgamma activation, we examined the effect of 8 wk of rosiglitazone treatment on muscular gene expression of ICAM-1 and IL-8 in type 2 diabetes mellitus patients. In these subjects, we observed a trend toward decreased basal expression of ICAM-1 mRNA levels. Subsequent analyses in cultured myotubes revealed that the anti-inflammatory effect of PPARgamma activation is not due to decreased RelA translocation to the nucleus or reduced RelA DNA binding. These findings demonstrate that muscle-specific inhibition of NF-kappaB activation may be an interesting therapeutic avenue for treatment of several inflammation-associated skeletal muscle abnormalities.
Assuntos
Músculo Esquelético/metabolismo , NF-kappa B/antagonistas & inibidores , PPAR gama/fisiologia , Animais , Células Cultivadas , Citocinas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Mediadores da Inflamação/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , NF-kappa B/metabolismo , NF-kappa B/fisiologia , PPAR gama/agonistas , Pirimidinas/farmacologia , Rosiglitazona , Tiazóis/farmacologia , Tiazolidinedionas/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Systemic proinflammatory cytokines and oxidative stress have been described in association with peripheral muscle wasting and weakness of patients with severe chronic obstructive pulmonary disease (COPD), but their expression in skeletal muscle is unknown. The objectives of the present study were to determine muscle protein levels of selected cytokines in patients with COPD and to study their relationships with protein carbonylation as a marker of oxidative stress, quadriceps function and exercise capacity. METHODS: We conducted a cross sectional study in which 36 cytokines were detected using a human antibody array in quadriceps specimens obtained from 19 patients with severe COPD and seven healthy controls. Subsequently, selected cytokines (tumour necrosis factor (TNF)alpha, TNFalpha receptors I and II, interleukin (IL) 6, interferon gamma, transforming growth factor (TGF) beta and vascular endothelial growth factor (VEGF)), as well as protein carbonylation (oxidative stress index) were determined using an enzyme linked immunosorbent assay (ELISA) in all muscles. RESULTS: Compared with controls, the vastus lateralis of patients with COPD showed significantly lower protein ELISA levels of TNFalpha, which positively correlated with their quadriceps function, TNFalpha receptor II and VEGF. Protein ELISA levels of IL6, interferon gamma and TGFbeta did not differ between patients and controls. Quadriceps protein carbonylation was greater in patients and inversely correlated with quadriceps strength among them. CONCLUSIONS: These findings do not support the presence of a proinflammatory environment within the quadriceps muscles of clinically and weight stable patients with severe COPD, despite evidence for increased oxidative stress and the presence of muscle weakness.
Assuntos
Citocinas/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Músculo Quadríceps/metabolismo , Idoso , Biomarcadores/metabolismo , Biópsia por Agulha , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Debilidade Muscular/metabolismo , Debilidade Muscular/fisiopatologia , Estresse Oxidativo/fisiologiaRESUMO
Extrapulmonary pathology significantly impairs clinical outcome in chronic obstructive pulmonary disease (COPD). The peroxisome proliferator-activated receptors (PPARs) are implicated in the regulation of several hallmarks of systemic COPD pathology, including cachexia, decreased oxidative muscle metabolism, oxidative stress and systemic inflammation. Recently, expression of PPARs and related cofactors was shown to be reduced in peripheral skeletal muscle of patients with moderate-to-severe COPD and muscle weakness. The current authors hypothesise that impaired peroxisome proliferator-activated receptor signalling may underlie some of the muscular disturbances in chronic obstructive pulmonary disease. Proposed mechanisms will be outlined in the present article, as well as the therapeutic potential of peroxisome proliferator-activated receptor modulation in the treatment of skeletal muscle dysfunction.
Assuntos
Debilidade Muscular/fisiopatologia , Receptores Ativados por Proliferador de Peroxissomo/efeitos dos fármacos , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Suplementos Nutricionais , Terapia por Exercício , Ácidos Graxos Insaturados , Humanos , Inflamação/fisiopatologia , Debilidade Muscular/tratamento farmacológico , Músculo Esquelético/fisiologia , Estresse Oxidativo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológicoRESUMO
Loss of skeletal muscle mitochondrial oxidative capacity is well-established in patients with COPD, but the role of mitochondrial breakdown herein is largely unexplored. Currently, we studied if mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients and associates with the loss of mitochondrial content, and whether it is affected in patients with iron deficiency (ID) or systemic inflammation. Therefore, mitophagy, autophagy, mitochondrial dynamics and content markers were analysed in vastus lateralis biopsies of COPD patients (N = 95, FEV1% predicted: 39.0 [31.0-53.6]) and healthy controls (N = 15, FEV1% predicted: 112.8 [107.5-125.5]). Sub-analyses were performed on patients stratified by ID or C-reactive protein (CRP). Compared with controls, COPD patients had lower muscle mitochondrial content, higher BNIP3L and lower FUNDC1 protein, and higher Parkin protein and gene-expression. BNIP3L and Parkin protein levels inversely correlated with mtDNA/gDNA ratio and FEV1% predicted. ID-COPD patients had lower BNIP3L protein and higher BNIP3 gene-expression, while high CRP patients had higher BNIP3 and autophagy-related protein levels. In conclusion, our data indicates that mitochondrial breakdown signalling is increased in skeletal muscle of COPD patients, and is related to disease severity and loss of mitochondrial content. Moreover, systemic inflammation is associated with higher BNIP3 and autophagy-related protein levels.
Assuntos
Inflamação/genética , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Proto-Oncogênicas/genética , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas Supressoras de Tumor/genética , Idoso , Anemia Ferropriva/sangue , Anemia Ferropriva/genética , Anemia Ferropriva/patologia , Autofagia/genética , Proteína C-Reativa/metabolismo , DNA Mitocondrial/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Inflamação/sangue , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mitofagia/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Assessment of quadriceps endurance is of interest to investigators studying human disease. We hypothesized that repetitive magnetic stimulation (rMS) of the intramuscular branches of the femoral nerve could be used to induce and quantify quadriceps endurance. To test this hypothesis, we used a novel stimulating coil to compare the quadriceps endurance properties in eight normal humans and, to confirm that the technique could be used in clinical practice, in eight patients with advanced chronic obstructive pulmonary disease (COPD). To validate the method, we compared in vivo contractile properties of the quadriceps muscle with the fiber-type composition and oxidative enzyme capacity. We used a Magstim Rapid(2) magnetic nerve stimulator with the coil wrapped around the quadriceps. Stimuli were given at 30 Hz, a duty cycle of 0.4 (2 s on, 3 s off), and for 50 trains. Force generation and the surface electromyogram were measured throughout. Quadriceps twitch force, elicited by supramaximal magnetic stimulation of the femoral nerve, was measured before and after the protocol. Quadriceps muscle biopsies were analyzed for oxidative (citrate synthase, CS) and glycolytic (phosphofructokinase, PFK) enzyme activity and myosin heavy chain isoform protein expression. The time for force to fall to 70% of baseline (T(70)) was shorter in the COPD group than the control group: 55.6 +/- 26.0 vs. 121 +/- 38.7 s (P = 0.0014). Considering patients and controls together, positive correlations were observed between T(70) and the proportion of type I fibers (r = 0.68, P = 0.004) and CS-to-PFK ratio (CS/PFK) (r = 0.67, P = 0.005). We conclude that quadriceps endurance assessed using rMS is feasible in clinical studies.
Assuntos
Magnetismo , Fadiga Muscular/fisiologia , Força Muscular/fisiologia , Tono Muscular/fisiologia , Músculo Quadríceps/fisiologia , Idoso , Biópsia , Exercício Físico/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Músculo Quadríceps/patologia , Reprodutibilidade dos TestesRESUMO
Hypoxia as a consequence of acute and chronic respiratory disease has been associated with muscle atrophy. This study investigated the sensitivity of oxidative and glycolytic muscles to hypoxia-induced muscle atrophy. Male mice were exposed to 8% normobaric oxygen for up to 21 days. Oxidative soleus and glycolytic extensor digitorum longus (EDL) muscles were isolated, weighed, and assayed for expression profiles of the ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and glucocorticoid receptor (GR) and hypoxia-inducible factor-1α (HIF1α) signaling. Fiber-type composition and the capillary network were investigated. Hypoxia-induced muscle atrophy was more prominent in the EDL than the soleus muscle. Although increased expression of HIF1α target genes showed that both muscle types sensed hypoxia, their adaptive responses differed. Atrophy consistently involved a hypoxia-specific effect (i.e., not attributable to a hypoxia-mediated reduction of food intake) in the EDL only. Hypoxia-specific activation of the UPS and ALP and increased expression of the glucocorticoid receptor (Gr) and its target genes were also mainly observed in the EDL. In the soleus, stimulation of gene expression of those pathways could be mimicked to a large extent by food restriction alone. Hypoxia increased the number of capillary contacts per fiber cross-sectional area in both muscles. In the EDL, this was due to type II fiber atrophy, whereas in the soleus the absolute number of capillary contacts increased. These responses represent two distinct modes to improve oxygen supply to muscle fibers, but may aggravate muscle atrophy in chronic obstructive pulmonary disease patients who have a predominance of type II fibers.
Assuntos
Hipóxia/patologia , Músculos/patologia , Atrofia Muscular/patologia , Adaptação Fisiológica , Animais , Autofagia , Expressão Gênica , Glucocorticoides/metabolismo , Glicólise , Hipóxia/complicações , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculos/irrigação sanguínea , Músculos/metabolismo , Atrofia Muscular/etiologia , Oxirredução , Distribuição Aleatória , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A shift in quadriceps muscle metabolic profile toward decreased oxidative metabolism and increased glycolysis is a consistent finding in chronic obstructive pulmonary disease (COPD). Chronic inflammation has been proposed as a trigger of this pathological metabolic adaptation. Indeed, the proinflammatory cytokine TNF-α impairs muscle oxidative metabolism through activation of the nuclear factor-κB (NF-κB) pathway. Putative effects on muscle glycolysis, however, are unclear. We hypothesized that TNF-α-induced NF-κB signaling stimulates muscle glycolytic metabolism through activation of the glycolytic regulator hypoxia-inducible factor-1α (HIF-1α). Wild-type C2C12 and C2C12-IκBα-SR (blocked NF-κB signaling) myotubes were stimulated with TNF-α, and its effects on glycolytic metabolism and involvement of the HIF pathway herein were investigated. As proof of principle, expression of HIF signaling constituents was investigated in quadriceps muscle biopsies of a previously well-characterized cohort of clinically stable patients with severe COPD and healthy matched controls. TNF-α increased myotube glucose uptake and lactate production and enhanced the activity and expression levels of multiple effectors of muscle glycolytic metabolism in a NF-κB-dependent manner. In addition, TNF-α activated HIF signaling, which required classical NF-κB activation. Moreover, the knockdown of HIF-1α largely attenuated TNF-α-induced increases in glycolytic metabolism. Accordingly, the mRNA levels of HIF-1α and the HIF-1α target gene, vascular endothelial growth factor (VEGF), were increased in muscle biopsies of COPD patients compared with controls, which was most pronounced in the patients with high levels of muscle TNF-α. In conclusion, these data show that TNF-α-induced classical NF-κB activation enhances muscle glycolytic metabolism in a HIF-1α-dependent manner.
Assuntos
Glicólise/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fibras Musculares Esqueléticas/metabolismo , NF-kappa B/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Estudos de Casos e Controles , Linhagem Celular , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético , NF-kappa B/efeitos dos fármacos , Músculo Quadríceps/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Low exercise tolerance has a large influence on health status in chronic obstructive pulmonary disease and chronic heart failure. In addition to primary organ dysfunction, impaired skeletal muscle performance is a strong predictor of low exercise capacity. There are striking similarities between both disorders with respect to the muscular alterations underlying the impairment. However, different alterations occur in different muscle types. Histologic and metabolic data show that peripheral muscles undergo a shift from oxidative to glycolytic energy metabolism, whereas the opposite is observed in the diaphragm. These findings are in line with the notion that peripheral and diaphragm muscle are limited mainly by endurance and strength capacity, respectively. In both diseases, muscular impairment is multifactorially determined; hypoxia, oxidative stress, disuse, medication, nutritional depletion, and systemic inflammation may contribute to the observed muscle abnormalities and each factor has its own potential for innovative treatment approaches.
Assuntos
Baixo Débito Cardíaco/complicações , Tolerância ao Exercício/fisiologia , Pneumopatias Obstrutivas/complicações , Músculo Esquelético/fisiopatologia , Corticosteroides/efeitos adversos , Baixo Débito Cardíaco/fisiopatologia , Baixo Débito Cardíaco/terapia , Exercício Físico , Humanos , Hipóxia/fisiopatologia , Inflamação/fisiopatologia , Pneumopatias Obstrutivas/fisiopatologia , Pneumopatias Obstrutivas/terapia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Estado Nutricional/fisiologia , Estresse Oxidativo/fisiologia , OxigenoterapiaRESUMO
Skeletal muscle wasting contributes to impaired exercise capacity, reduced health-related quality of life and is an independent determinant of mortality in chronic obstructive pulmonary disease. An imbalance between protein synthesis and myogenesis on the one hand, and muscle proteolysis and apoptosis on the other hand, has been proposed to underlie muscle wasting in this disease. In this review, the current understanding of the state and regulation of these processes governing muscle mass in this condition is presented. In addition, a conceptual mode of action of disease-related determinants of muscle wasting including disuse, hypoxemia, malnutrition, inflammation and glucocorticoids is provided by overlaying the available associative clinical data with causal evidence, mostly derived from experimental models. Significant progression has been made in understanding and managing muscle wasting in chronic obstructive pulmonary disease. Further examination of the time course of muscle wasting and specific disease phenotypes, as well as the application of systems biology and omics approaches in future research will allow the development of tailored strategies to prevent or reverse muscle wasting in chronic obstructive pulmonary disease. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.