Regulation of PGC-1α expression by a GSK-3ß-TFEB signaling axis in skeletal muscle.

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.

TNF-α-induced NF-κB activation stimulates skeletal muscle glycolytic metabolism through activation of HIF-1α.

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.

Activation of alternative NF-κB signaling during recovery of disuse-induced loss of muscle oxidative phenotype.

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.

Classical NF-κB activation impairs skeletal muscle oxidative phenotype by reducing IKK-α expression.

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.

Triggers and mechanisms of skeletal muscle wasting in chronic obstructive pulmonary disease.

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.

Regulation of skeletal muscle oxidative phenotype by classical NF-κB signalling.

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.

The mechanisms of cachexia underlying muscle dysfunction in COPD.

Pulmonary cachexia is a prevalent, debilitating, and well-recognized feature of COPD associated with increased mortality and loss of peripheral and respiratory muscle function. The exact cause and underlying mechanisms of cachexia in COPD are still poorly understood. Increasing evidence, however, shows that pathological changes in intracellular mechanisms of muscle mass maintenance (i.e., protein turnover and myonuclear turnover) are likely involved. Potential factors triggering alterations in these mechanisms in COPD include oxidative stress, myostatin, and inflammation. In addition to muscle wasting, peripheral muscle in COPD is characterized by a fiber-type shift toward a more type II, glycolytic phenotype and an impaired oxidative capacity (collectively referred to as an impaired oxidative phenotype). Atrophied diaphragm muscle in COPD, however, displays an enhanced oxidative phenotype. Interestingly, intrinsic abnormalities in (lower limb) peripheral muscle seem more pronounced in either cachectic patients or weight loss-susceptible emphysema patients, suggesting that muscle wasting and intrinsic changes in peripheral muscle's oxidative phenotype are somehow intertwined. In this manuscript, we will review alterations in mechanisms of muscle mass maintenance in COPD and discuss the involvement of oxidative stress, inflammation, and myostatin as potential triggers of cachexia. Moreover, we postulate that an impaired muscle oxidative phenotype in COPD can accelerate the process of cachexia, as it renders muscle in COPD less energy efficient, thereby contributing to an energy deficit and weight loss when not dietary compensated. Furthermore, loss of peripheral muscle oxidative phenotype may increase the muscle's susceptibility to inflammation- and oxidative stress-induced muscle damage and wasting.

Pre-cachexia in patients with stages I-III non-small cell lung cancer: systemic inflammation and functional impairment without activation of skeletal muscle ubiquitin proteasome system.

Cachexia is a prevalent phenomenon of non-small cell lung cancer (NSCLC) which is responsible for increased mortality and deterioration of physical performance. Preclinical research indicates that systemic inflammation induces cachexia-related muscle wasting through muscular Nuclear Factor-kappa B (NF-κB) signaling and subsequent ubiquitin proteasome system (UPS)-mediated proteolysis. As these pathways could be a target for early intervention strategies, it needs to be elucidated whether increased activation of these pathways is already present in early stage NSCLC cachexia. The aim of the present study was therefore to assess muscular NF-κB and UPS activation in patients with NSCLC pre-cachexia. Sixteen patients with newly diagnosed stages I-III NSCLC having <10% weight loss and ten healthy controls were studied. Body composition, systemic inflammation and exercise capacity were assessed in all subjects and NF-κB and UPS activity in vastus lateralis muscle biopsies in a subset. Patients showed increased plasma levels of C-reactive protein (CRP) (P<0.001), soluble Tumor Necrosis Factor receptor 1 (sTNF-R1) (P<0.05), fibrinogen (P<0.001) and decreased levels of albumin (P<0.001). No changes in fat free body mass or skeletal muscle NF-κB and UPS activity were observed, while peak oxygen consumption ( [Formula: see text] ) was significantly decreased in patients compared with healthy controls. In conclusion, this exploratory study demonstrates significantly reduced exercise capacity in NSCLC pre-cachexia despite maintenance of muscle mass and unaltered indices of UPS activation. The absence of muscular NF-κB-dependent inflammatory signaling supports the notion that transition of systemic to local inflammation is required to initiate UPS-dependent muscle wasting characteristic for (experimental) cachexia.

TNF-alpha impairs regulation of muscle oxidative phenotype: implications for cachexia?

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.

Peroxisome proliferator-activated receptors: a therapeutic target in COPD?

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.

Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB.

Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor alpha (TNF-alpha) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF-alpha activated nuclear factor-kappaB (NF-kappaB) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein kappaBalpha (IkappaBalpha) restored myogenic differentiation in myoblasts treated with TNF-alpha or interleukin 1beta. Conversely, activation of NF-kappaB by overexpression of IkappaB kinase was sufficient to block myogenesis, illustrating the causal link between NF-kappaB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF-alpha on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF-alpha did not result in a striking loss of muscle-specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF-alpha. An important finding was that NF-kappaB was activated to the same extent in differentiating and differentiated cells, illustrating that once myocytes have differentiated they become refractory to the effects of NF-kappaB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF-kappaB-dependent pathway.