Impaired exercise training-induced muscle fiber hypertrophy and Akt/mTOR pathway activation in hypoxemic patients with COPD

Exercise training (ExTr) is largely used to improve functional capacity of chronic obstructive pulmonary disease (COPD) patients. However, ExTr partially restores muscle function in COPD patients, suggesting that confounding factors may limit the efficiency of ExTr. In the present study, we hypothesized that skeletal muscle adaptations triggered by ExTr could be compromised in hypoxemic COPD patients. Vastus lateralis muscle biopsies were obtained from normoxemic (n = 15; resting arterial PO2 = 68.5 +/- 1.5 mm Hg) and hypoxemic (n = 8; resting arterial PO2 = 57.0 +/- 1.0 mm Hg) COPD patients before and after a 2 month-ExTr program. ExTr induced a significant increase in exercise capacity both in normoxemic and hypoxemic COPD patients. However, ExTr increased citrate synthase and lactate dehydrogenase enzyme activities only in skeletal muscle of normoxemic patients. Similarly, muscle fiber cross-sectional area and capillary-to-fiber ratio were only increased in normoxemic patients. Expression of atrogenes (MuRF1, MAFbx/Atrogin-1) and autophagy-related genes (Beclin, LC3, Bnip, Gabarapl) remained unchanged in both groups. The phosphorylation level of Akt (Ser473), GSK-3beta (Ser9) and p70S6k (Thr389), which was non-significantly increased in normoxemic patients in response to ExTr, was significantly decreased in hypoxemic patients. We further showed on C2C12 myotubes that hypoxia completely prevented IGF-1-induced phosphorylation of Akt, GSK-3beta and p70S6K. Together, our observations suggest a role for hypoxemia in the adaptive response of skeletal muscle of COPD patients to ExTr

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.

AbstractCachexia 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 (V˙O2 peak) 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

Glycogen Synthase Kinase 3 Suppresses Myogenic Differentiation through Negative Regulation of NFATc3.

Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth could be an important therapeutic strategy. Glycogen Synthase Kinase 3beta (GSK-3beta) has been implicated in the negative regulation of skeletal muscle growth. Since myogenic differentiation is an essential part of muscle growth we investigated if inhibition of GSK-3beta is sufficient to stimulate myogenic differentiation, and whether this depended on regulation of the transcription factor Nuclear Factor of Activated T-cells (NFAT). In both, myogenically converted Mouse Embryonic Fibroblasts (MEFs) or C2C12 myoblasts, deficiency of GSK-3beta protein (activity) resulted in enhanced myotube formation and muscle specific gene expression during differentiation, which was reversed by reintroduction of wt, but not kinase-inactive (K85R) GSK-3beta. In addition, GSK-3beta inhibition restored myogenic differentiation following Calcineurin blockade, which suggested the involvement of NFAT. GSK-3beta deficient MEFs or myoblasts displayed enhanced nuclear translocation of NFATc3, and elevated NFAT-sensitive promoter transactivation, which was reduced by re-introducing wt-, but not K85R-GSK-3beta. Over-expression of NFATc3 increased muscle gene promoter transactivation, which was abolished by co-expression of wt-GSK-3beta. Finally, stimulation of muscle gene expression observed following GSK-3beta inhibition was strongly attenuated in NFATc3-deficient myoblasts, indicating that this response requires NFATc3. Collectively, our data demonstrate negative regulation of myogenic differentiation by GSK-3beta through a transcriptional mechanism which depends on NFATc3. Inhibition of GSK-3beta may be a potential strategy in prevention or treatment of muscle atrophy

Muscle Wasting and Impaired Muscle Regeneration in a Murine Model of Chronic Pulmonary Inflammation

Muscle wasting and increased circulating levels of inflammatory cytokines, including tumor necrosis factor alpha (TNFalpha) are common features of COPD. To investigate if inflammation of the lung is responsible for systemic inflammation and muscle wasting, we adopted a mouse model of pulmonary inflammation resulting from directed over-expression of a TNFalpha transgene controlled by the surfactant protein C (SP-C) promoter. Compared to wild type, SP-C/TNFalpha mice exhibited increased levels of TNFalpha in the circulation and increased endogenous TNFalpha expression in skeletal muscle, potentially reflecting an amplificatory response to circulating TNFalpha. Decreased muscle and body weights observed in SP-C/TNFalpha mice were indicative of muscle wasting. Further evaluation of the SP-C/TNFalpha mouse musculature revealed a decreased muscle regenerative capacity, evidenced by attenuated myoblast proliferation and differentiation in response to reloading of disuse-atrophied muscle, which may contribute to skeletal muscle wasting. Importantly, incubation of cultured myoblasts with TNFalpha also resulted in elevated TNFalpha mRNA levels and inhibition of myoblast differentiation. Collectively, our results demonstrate that chronic pulmonary inflammation results in muscle wasting and impaired muscle regeneration in SP-C/TNFalpha mice, possibly as a consequence of an amplificatory TNFalpha expression circuit extending from the lung to skeletal muscle

Inhibition of Glycogen Synthase Kinase 3{beta} activity is sufficient to stimulate myogenic differentiation

Skeletal muscle atrophy is a prominent and disabling feature of chronic wasting diseases. Prevention or reversal of muscle atrophy by administration of skeletal muscle growth (hypertrophy) stimulating agents such as Insulin Like Growth Factor I (IGF-I) could be an important therapeutic strategy in these diseases. To elucidate the IGF-I signal transduction responsible for muscle formation (myogenesis) during muscle growth and regeneration, IGF-I was applied to differentiating C2C12 myoblasts, and the effects on phosphatidyl-inositol 3-kinase (PI-(3)K)/Akt /Glycogen Synthase Kinase 3beta (GSK-3beta) signaling and myogenesis were evaluated. IGF-I caused phosphorylation and inactivation of GSK-3beta activity via signaling through the PI-(3)K/Akt pathway. We assessed whether pharmacological inhibition of GSK-3beta using lithium chloride (LiCl) was sufficient to stimulate myogenesis. Addition of IGF-I or LiCl stimulated myogenesis evidenced by increased myotube formation, Muscle Creatine Kinase (MCK) activity and Troponin I (TnI) promoter transactivation during differentiation. Moreover, mRNA's encoding MyoD, Myf-5, myogenin, TnI-slow, TnI-fast, MCK and myoglobin were upregulated in myoblasts differentiated in the presence of IGF-I or LiCl. Importantly, blockade of GSK-3beta inhibition abrogated IGF-I, but not LiCl dependent stimulation of myogenic mRNA accumulation, suggesting that the pro-myogenic effects of IGF-I require GSK-3beta inactivation, and revealing an important negative regulatory role for GSK-3beta in myogenesis. Therefore, this study identifies GSK-3beta as a potential target for pharmacological stimulation of muscle growth

Glycogen synthase kinase-3ß is required for the induction of skeletal muscle atrophy.

Verhees KJ, Schols AM, Kelders MC, Op den Kamp CM, van der Velden JL, Langen RC. Glycogen synthase kinase-3 beta is required for the induction of skeletal muscle atrophy. Am J Physiol Cell Physiol 301: C995-C1007, 2011. First published August 10, 2011; doi:10.1152/ajpcell.00520.2010.-Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3 beta (GSK-3 beta), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C2C12 skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3 beta using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3 beta suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3 beta inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3 beta dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3 beta enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3 beta

Trans-10, cis-12 conjugated linoleic acid inhibits skeletal muscle differentiation and GLUT4 expression independently from NF-'B activation.

Scope: The capacity of skeletal muscle to contribute to glucose homeostasis depends on muscular insulin sensitivity. The expression of glucose transporter (GLUT)-4 is increased during myoblast differentiation, a process essential in maintenance of adult muscle. Therefore, processes that affect muscle differentiation may influence insulin dependent glucose homeostasis. Conjugated linoleic acids, and in particular trans-10, cis-12 CLA (t10, c12-CLA), are potent inducers of NF-kB in cultured skeletal myotubes, and NF-kB activation inhibits muscle differentiation. The aims of this study were to evaluate whether CLAs inhibit myogenic differentiation and lower GLUT4 mRNA expression and to address the involvement of NF-kB activation in potential effects of CLA on these processes.Methods and results: Incubation of C2C12 cells with t10, c12-CLA blocked the formation of myotubes, which was accompanied by reduced expression of the muscle specific genes creatine kinase, myogenin, myosin heavy chain perinatal and myosin heavy chain IIB, as well as decreased GLUT4 mRNA levels. However, genetic blockade of NF-kB was not sufficient to restore reduced myosin heavy chain protein expression following t10, c12-CLA treatment. Surprisingly, in contrast to myotubes, t10, c12-CLA was not able to activate NF-kB transcriptional activity in myoblasts. Conclusion: In conclusion, t10, c12-CLA inhibits myogenic differentiation and GLUT4 expression, independently from NF-kB activation

Palmitate-induced skeletal muscle insulin resistance does not require NF-κB activation

Palmitate activates the NF-kappaB pathway, and induces accumulation of lipid metabolites and insulin resistance in skeletal muscle cells. Little information is available whether and how these processes are causally related. Therefore, the objectives were to investigate whether intra-cellular lipid metabolites are involved in FA-induced NF-kappaB activation and/or insulin resistance in skeletal muscle and to investigate whether FA-induced insulin resistance and NF-kappaB activation are causally related. Inhibiting DGAT or CPT-1 by using, respectively, amidepsine or etomoxir increased DAG accumulation and sensitized myotubes to palmitate-induced insulin resistance. While co-incubation of palmitate with etomoxir increased NF-kappaB transactivation, co-incubation with amidepsine did not, indicating that DAG accumulation is associated with insulin resistance but not with NF-kappaB activation. Furthermore, pharmacological or genetic inhibition of the NF-kappaB pathway could not prevent palmitate-induced insulin resistance. In conclusion, we have demonstrated that activation of the NF-kappaB pathway is not required for palmitate-induced insulin resistance in skeletal muscle cells