Recovery of muscle mass and muscle oxidative phenotype following disuse does not require GSK-3 inactivation.

BACKGROUND: Physical inactivity contributes to muscle wasting and reductions in mitochondrial oxidative phenotype (OXPHEN), reducing physical performance and quality of life during aging and in chronic disease. Previously, it was shown that inactivation of glycogen synthase kinase (GSK)-3ß stimulates muscle protein accretion, myogenesis, and mitochondrial biogenesis. Additionally, GSK-3ß is inactivated during recovery of disuse-induced muscle atrophy. AIM: Therefore, we hypothesize that GSK-3 inhibition is required for reloading-induced recovery of skeletal muscle mass and OXPHEN. METHODS: Wild-type (WT) and whole-body constitutively active (C.A.) Ser21/9 GSK-3α/ß knock-in mice were subjected to a 14-day hind-limb suspension/14-day reloading protocol. Soleus muscle mass, fiber cross-sectional area (CSA), OXPHEN (abundance of sub-units of oxidative phosphorylation (OXPHOS) complexes and fiber-type composition), as well as expression levels of their main regulators (respectively protein synthesis/degradation, myogenesis and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) signaling) were monitored. RESULTS: Subtle but consistent differences suggesting suppression of protein turnover signaling and decreased expression of several OXPHOS sub-units and PGC-1α signaling constituents were observed at baseline in C.A. GSK-3 versus WT mice. Although soleus mass recovery during reloading occurred more rapidly in C.A. GSK-3 mice, this was not accompanied by a parallel increased CSA. The OXPHEN response to reloading was not distinct between C.A. GSK-3 and WT mice. No consistent or significant differences in reloading-induced changes in the regulatory steps of protein turnover, myogenesis or muscle OXPHEN were observed in C.A. GSK-3 compared to WT muscle. CONCLUSION: This study indicates that GSK-3 inactivation is dispensable for reloading-induced recovery of muscle mass and OXPHEN.

Resveratrol and metabolic health in COPD: A proof-of-concept randomized controlled trial.

BACKGROUND: Patients with COPD are often characterized by disturbed metabolic health which is reflected in altered body composition. Current studies in healthy subjects suggest that resveratrol improves metabolic health by enhancing muscle mitochondrial function and adipose tissue morphology. The primary objective was to investigate the effect of four weeks resveratrol supplementation on muscle mitochondrial function in patients with COPD. Secondary objectives were to investigate the effect of resveratrol on adipose tissue inflammatory and metabolic gene expression, systemic inflammation and body composition in patients with COPD. METHODS: In a double-blind randomized placebo-controlled proof-of-concept study, 21 COPD patients (FEV1: 53 ± 15% predicted; age: 67 ± 9 years and BMI: 24.5 ± 3.3 kg/m2) received resveratrol (150 mg/day) or placebo for four weeks. Before and after intervention, blood samples, quadriceps muscle and subcutaneous abdominal fat biopsies were obtained for metabolic and inflammatory profiling. Body composition was assessed by dual energy X-ray absorptiometry. RESULTS: Muscle mitochondrial biogenesis regulators AMPK, SIRT1 and PGC-1α as well as mitochondrial respiration, Oxphos complexes, oxidative enzyme activities and kynurenine aminotransferases were not improved by resveratrol. Plasma high-sensitive C-reactive protein and kynurenine did not change after resveratrol supplementation. Adipose tissue inflammatory markers were unaffected by resveratrol, while markers of glycolysis and lipolysis were significantly increased compared to placebo supplementation. Body weight decreased after resveratrol supplementation (resveratrol -0.95 ± 1.01 kg vs placebo -0.16 ± 0.66 kg, p = 0.049) due to a reduction in lean mass (resveratrol -1.79 ± 1.67 kg vs 0.37 ± 0.86 kg, p = 0.026). CONCLUSION: We do not confirm previously reported positive effects of resveratrol on skeletal muscle mitochondrial function in patients with COPD, but show an unexpected decline in lean mass. CLINICAL TRIAL REGISTRY: Clinicaltrials.gov NCT02245932.

Skeletal muscle unloading results in increased mitophagy and decreased mitochondrial biogenesis regulation.

INTRODUCTION: Physical inactivity significantly contributes to loss of muscle mass and performance in bed-bound patients. Loss of skeletal muscle mitochondrial content has been well-established in muscle unloading models, but the underlying molecular mechanism remains unclear. We hypothesized that apparent unloading-induced loss of muscle mitochondrial content is preceded by increased mitophagy- and decreased mitochondrial biogenesis-signaling during the early stages of unloading. METHODS: We analyzed a comprehensive set of molecular markers involved in mitochondrial-autophagy, -biogenesis, -dynamics, and -content, in the gastrocnemius muscle of C57BL/6J mice subjected to 0- and 3-days hind limb suspension, and in biopsies from human vastus lateralis muscle obtained before and after 7 days of one-leg immobilization. RESULTS: In both mice and men, short-term skeletal muscle unloading results in molecular marker patterns indicative of increased receptor-mediated mitophagy and decreased mitochondrial biogenesis regulation, before apparent loss of mitochondrial content. DISCUSSION: These results emphasize the early-onset of skeletal muscle disuse-induced mitochondrial remodeling.

Impaired Skeletal Muscle Kynurenine Metabolism in Patients with Chronic Obstructive Pulmonary Disease.

BACKGROUND: Loss of peripheral muscle oxidative phenotype, cognitive impairment, and depression are well-recognized systemic manifestations of chronic obstructive pulmonary disease (COPD). Kynurenine (KYN), known to be associated with disturbed mental health, can be metabolized in muscle by kynurenine aminotransferases (KAT) 1-4. These KATs are regulated by peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC1α). We hypothesize that impaired PGC1α signaling in COPD is associated with reduced muscle KAT expression and increased KYN plasma levels. METHODS: Retrospective collected and metabolically phenotyped muscle tissue and blood obtained from 29 well-characterized COPD patients and 15 healthy controls were analyzed. KYN was measured in plasma and KAT1-4 expression and major constituents of PGC1α signaling were assessed in quadriceps muscle biopsies. RESULTS: Circulating KYN levels were increased in COPD. Furthermore, both gene and protein expression levels of KAT4 were reduced in muscle tissue from COPD patients. Finally, in the whole group (even when controlled for airflow obstruction) and in each subgroup separately, KAT4 gene expression correlated significantly with constituents of the PGC1α signaling pathway. CONCLUSIONS: These data support our hypothesis that KYN plasma levels are elevated in COPD through impaired KYN clearance in muscle. Our findings show a pathway via which exercise training and/or nutritional modulation may improve physical and mental health in COPD patients.

Altered protein turnover signaling and myogenesis during impaired recovery of inflammation-induced muscle atrophy in emphysematous mice.

Exacerbations in Chronic obstructive pulmonary disease (COPD) are often accompanied by pulmonary and systemic inflammation, and are associated with an increased susceptibility to weight loss and muscle wasting. As the emphysematous phenotype in COPD appears prone to skeletal muscle wasting, the aims of this study were to evaluate in emphysematous compared to control mice following repetitive exacerbations (1) changes in muscle mass and strength and, (2) whether muscle mass recovery and its underlying processes are impaired. Emphysema was induced by intra-tracheal (IT) elastase instillations, followed by three weekly IT-LPS instillations to mimic repetitive exacerbations. Loss of muscle mass and strength were measured, and related to analyses of muscle protein turnover and myogenesis signaling in tissue collected during and following recovery. Emphysematous mice showed impaired muscle mass recovery in response to pulmonary inflammation-induced muscle atrophy. Proteolysis and protein synthesis signaling remained significantly higher in emphysematous mice during recovery from LPS. Myogenic signaling in skeletal muscle was altered, and fusion capacity of cultured muscle cells treated with plasma derived from LPS-treated emphysematous mice was significantly decreased. In conclusion, repetitive cycles of pulmonary inflammation elicit sustained muscle wasting in emphysematous mice due to impaired muscle mass recovery, which is accompanied by aberrant myogenesis.

Pulmonary inflammation-induced loss and subsequent recovery of skeletal muscle mass require functional poly-ubiquitin conjugation.

BACKGROUND: Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation. METHODS: Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice. RESULTS: Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass. CONCLUSION: Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.

NF-κB-mediated metabolic remodelling in the inflamed heart in acute viral myocarditis.

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.

A novel in vitro model for the assessment of postnatal myonuclear accretion.

BACKGROUND: Due to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis. METHODS: Proliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast-myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content. RESULTS: Live cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion. CONCLUSIONS: We developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.

Glucocorticoid Receptor Signaling Impairs Protein Turnover Regulation in Hypoxia-Induced Muscle Atrophy in Male Mice.

Hypoxemia may contribute to muscle wasting in conditions such as chronic obstructive pulmonary disease. Muscle wasting develops when muscle proteolysis exceeds protein synthesis. Hypoxia induces skeletal muscle atrophy in mice, which can in part be attributed to reduced food intake. We hypothesized that hypoxia elevates circulating corticosterone concentrations by reduced food intake and enhances glucocorticoid receptor (GR) signaling in muscle, which causes elevated protein degradation signaling and dysregulates protein synthesis signaling during hypoxia-induced muscle atrophy. Muscle-specific GR knockout and control mice were subjected to normoxia, normobaric hypoxia (8% oxygen), or pair-feeding to the hypoxia group for 4 days. Plasma corticosterone and muscle GR signaling increased after hypoxia and pair-feeding. GR deficiency prevented muscle atrophy by pair-feeding but not by hypoxia. GR deficiency differentially affected activation of ubiquitin 26S-proteasome and autophagy proteolytic systems by pair-feeding and hypoxia. Reduced food intake suppressed mammalian target of rapamycin complex 1 (mTORC1) activity under normoxic but not hypoxic conditions, and this retained mTORC1 activity was mediated by GR. We conclude that GR signaling is required for muscle atrophy and increased expression of proteolysis-associated genes induced by decreased food intake under normoxic conditions. Under hypoxic conditions, muscle atrophy and elevated gene expression of the ubiquitin proteasomal system-associated E3 ligases Murf1 and Atrogin-1 are mostly independent of GR signaling. Furthermore, impaired inhibition of mTORC1 activity is GR-dependent in hypoxia-induced muscle atrophy.

Increased Myogenic and Protein Turnover Signaling in Skeletal Muscle of Chronic Obstructive Pulmonary Disease Patients With Sarcopenia.

BACKGROUND: Sarcopenia was recently recognized as an independent condition by an International Classification of Diseases, Tenth Revision, Clinical Modification code, and is a frequently observed comorbidity in chronic obstructive pulmonary disease (COPD). Muscle mass is primarily dictated by the balance between protein degradation and synthesis, but their relative contribution to sarcopenia is unclear. OBJECTIVE: We aimed to assess potential differential molecular regulation of protein degradation and synthesis, as well as myogenesis, in the skeletal muscle of COPD patients with and without sarcopenia. METHODS: Muscle biopsies were obtained from the vastus lateralis muscle. Patients with COPD were clustered based on sarcopenia defined by low appendicular skeletal muscle mass index (nonsarcopenic COPD, n = 53; sarcopenic COPD, n = 39), and compared with healthy nonsarcopenic controls (n = 13). The mRNA and protein expression of regulators and mediators of ubiquitin-proteasome system (UPS), autophagy-lysosome system (autophagy), and protein synthesis were analyzed. Furthermore, mRNA expression of myogenesis markers was assessed. RESULTS: UPS signaling was unaltered, whereas indices of UPS regulation (eg, FOXO1 protein; p-FOXO3/FOXO3), autophagy signaling (eg, LC3BII/I; p-ULK1[Ser757]/ULK1), and protein synthesis signaling (eg, AKT1; p-GSK3B/GSK3B; p-4E-BP1/4E-BP1) were increased in COPD. These alterations were even more pronounced in COPD patients with sarcopenia (eg, FOXO1 protein; p-FOXO1/FOXO1; LC3BII/I; p-ULK(Ser555); p-AKT1/AKT1; AKT1; p-4E-BP1). Furthermore, myogenic signaling (eg, MYOG) was increased in COPD despite a concomitant increase of myostatin (MSTN) mRNA expression, with no difference between sarcopenic and nonsarcopenic COPD patients. CONCLUSION: Together with elevated myogenic signaling, the increase in muscle protein turnover signaling in COPD, which is even more prominent in COPD patients with sarcopenia, reflects molecular alterations associated with muscle repair and remodeling.

Differential regulation of muscle protein turnover in response to emphysema and acute pulmonary inflammation.

BACKGROUND: Exacerbations in COPD are often accompanied by pulmonary and systemic inflammation, and associated with increased susceptibility to and prevalence of weight loss and muscle wasting. Muscle mass loss during disease exacerbations may contribute to emphysema-associated muscle atrophy. However, whether pulmonary inflammation in presence of emphysema differentially affects skeletal muscle, including protein synthesis and degradation signaling pathways has not previously been addressed. The aims of this study were to 1) develop a mouse model of disease exacerbation-associated muscle wasting, 2) evaluate whether emphysema and muscle wasting can be monitored non-invasively and 3) assess alterations in muscle protein turnover regulation. METHODS: Emphysema was induced by three, weekly intra-tracheal (IT) elastase (E) or vehicle control (vc) instillations, followed by one single IT-LPS bolus (L) or vc instillation to mimic pulmonary inflammation-driven disease exacerbation. Consequently, four experimental groups were defined: vc/vc ('C'), E/vc ('E'), vc/LPS ('L'), E/LPS ('E + L'). Using micro cone-beam CT-scans, emphysema development and muscle mass changes were monitored, and correlated to muscle weight 48 h after LPS instillation. Protein turnover signaling was assessed in muscle tissue collected 24 h post LPS instillation. RESULTS: Micro-CT imaging correlated strongly with established invasive measurements of emphysema and muscle atrophy. Pulmonary inflammation following LPS instillation developed irrespective of emphysema and body and muscle weight were similarly reduced in the 'L' and 'E + L' groups. Accordingly, mRNA and protein expression levels of genes of the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP) were upregulated in skeletal muscle following IT-LPS ('L' and 'E + L'). In contrast, mTOR signaling, which controls ALP and protein synthesis, was reduced by pulmonary inflammation ('L' and 'E + L') as well as emphysema as a single insult ('E') compared to control. CONCLUSION: Changes in lung tissue density and muscle mass can be monitored non-invasively to evaluate emphysema and muscle atrophy longitudinally. Acute loss of muscle mass evoked by pulmonary inflammation is similar in control and emphysematous mice. Although muscle atrophy cues in response to pulmonary inflammation are not altered by emphysema, emphysema itself affects protein synthesis and ALP signaling, which may interfere with muscle mass recovery and impair maintenance of muscle mass in emphysema.

Alterations in Skeletal Muscle Oxidative Phenotype in Mice Exposed to 3 Weeks of Normobaric Hypoxia.

Skeletal muscle of patients with chronic respiratory failure is prone to loss of muscle mass and oxidative phenotype. Tissue hypoxia has been associated with cachexia and emphysema in humans. Experimental research on the role of hypoxia in loss of muscle oxidative phenotype, however, has yielded inconsistent results. Animal studies are frequently performed in young animals, which may hinder translation to generally older aged patients. Therefore, in this study, we tested the hypothesis that hypoxia induces loss of skeletal muscle oxidative phenotype in a model of aged (52 weeks) mice exposed to 3 weeks of hypoxia. Additional groups of young (4 weeks) and adult (12 weeks) mice were included to examine age effects. To verify hypoxia-induced cachexia, fat pad and muscle weights as well as muscle fiber cross-sectional areas were determined. Muscle oxidative phenotype was assessed by expression and activity of markers of mitochondrial metabolism and fiber-type distribution. A profound loss of muscle and fat was indeed accompanied by a slightly lower expression of markers of muscle oxidative capacity in the aged hypoxic mice. In contrast, hypoxia-associated changes of fiber-type composition were more prominent in the young mice. The differential response of the muscle of young, adult, and aged mice to hypoxia suggests that age matters and that the aged mouse is a better model for translation of findings to elderly patients with chronic respiratory disease. Furthermore, the findings warrant further mechanistic research into putative accelerating effects of hypoxia-induced loss of oxidative phenotype on the cachexia process in chronic respiratory disease.

Muscle-specific GSK-3ß ablation accelerates regeneration of disuse-atrophied skeletal muscle.

Muscle wasting impairs physical performance, increases mortality and reduces medical intervention efficacy in chronic diseases and cancer. Developing proficient intervention strategies requires improved understanding of the molecular mechanisms governing muscle mass wasting and recovery. Involvement of muscle protein- and myonuclear turnover during recovery from muscle atrophy has received limited attention. The insulin-like growth factor (IGF)-I signaling pathway has been implicated in muscle mass regulation. As glycogen synthase kinase 3 (GSK-3) is inhibited by IGF-I signaling, we hypothesized that muscle-specific GSK-3ß deletion facilitates the recovery of disuse-atrophied skeletal muscle. Wild-type mice and mice lacking muscle GSK-3ß (MGSK-3ß KO) were subjected to a hindlimb suspension model of reversible disuse-induced muscle atrophy and followed during recovery. Indices of muscle mass, protein synthesis and proteolysis, and post-natal myogenesis which contribute to myonuclear accretion, were monitored during the reloading of atrophied muscle. Early muscle mass recovery occurred more rapidly in MGSK-3ß KO muscle. Reloading-associated changes in muscle protein turnover were not affected by GSK-3ß ablation. However, coherent effects were observed in the extent and kinetics of satellite cell activation, proliferation and myogenic differentiation observed during reloading, suggestive of increased myonuclear accretion in regenerating skeletal muscle lacking GSK-3ß. This study demonstrates that muscle mass recovery and post-natal myogenesis from disuse-atrophy are accelerated in the absence of GSK-3ß.

Supplementing exposure to hypoxia with a copper depleted diet does not exacerbate right ventricular remodeling in mice.

BACKGROUND: Pulmonary hypertension and subsequent right ventricular (RV) failure are associated with high morbidity and mortality. Prognosis is determined by occurrence of RV failure. Currently, adequate treatment for RV failure is lacking. Further research into the molecular basis for the development of RV failure as well as the development of better murine models of RV failure are therefore imperative. We hypothesize that adding a low-copper diet to chronic hypoxia in mice reinforces their individual effect and that the combination of mild pulmonary vascular remodeling and capillary rarefaction, induces RV failure. METHODS: Six week old mice were subjected to normoxia (N; 21% O2) or hypoxia (H; 10% O2) during a period of 8 weeks and received either a normal diet (Cu+) or a copper depleted diet (Cu-). Cardiac function was assessed by echocardiography and MRI analysis. RESULTS AND CONCLUSION: Here, we characterized a mouse model of chronic hypoxia combined with a copper depleted diet and demonstrate that eight weeks of chronic hypoxia (10%) is sufficient to induce RV hypertrophy and subsequent RV failure. Addition of a low copper diet to hypoxia did not have any further deleterious effects on right ventricular remodeling.

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy. METHODS: Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors. RESULTS: Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors. CONCLUSIONS: In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.

Nuclear transcription factor κ B activation and protein turnover adaptations in skeletal muscle of patients with progressive stages of lung cancer cachexia.

BACKGROUND: Experimental models of cancer cachexia have indicated that systemic inflammation induces muscle-protein breakdown and wasting via muscular nuclear transcription factor κB (NF-κB) activation. This process may limit the efficacy of nutritional intervention. OBJECTIVES: We assessed muscle NF-κB activity and protein turnover signaling in progressive stages of clinical lung cancer cachexia and assessed whether circulating factors can induce muscular NF-κB activity. DESIGN: Patients with lung cancer precachexia (n = 10) and cachexia (n = 16) were cross-sectionally compared with 22 healthy control subjects. mRNA transcripts of muscle proteolytic (ubiquitin proteasome system and autophagy lysosomal pathway) and myogenic markers and protein expression of PI3K/Akt, myostatin, and autophagy signaling were measured. A multiplex analysis showed the systemic inflammatory status, whereas plasma exposure to stable NF-κB-luciferase-reporter muscle cells revealed NF-κB inducibility. RESULTS: Compared with healthy control subjects, cachectic patients had reduced (appendicular) muscle mass (-10%), muscle fiber atrophy (-27%), and decreased quadriceps strength (-31%). Subtle alterations in the muscle morphology were also detectable in precachectic patients, without changes in body composition. Despite increased Akt phosphorylation, downstream phosphosubstrates glycogen synthase kinase 3ß, mammalian target of rapamycin, and Forkhead box protein were unaltered. The expression of autophagy effectors B cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3 and microtubule-associated proteins 1A/1B light chain 3B gradually increased from precachectic to cachectic patients, without differences in E3 ubiquitin ligases. Systemic and local inflammation was evident in cachexia and intermediate in precachexia, but the plasma of both patients groups caused ex vivo muscle NF-κB activation. CONCLUSIONS: In lung cancer, muscular NF-κB activity is induced by factors contained within the circulation. Autophagy may contribute to increased muscle proteolysis in lung cancer cachexia, whereas the absence of downstream changes in phosphosubstrates despite increased Akt phosphorylation suggests impaired anabolic signaling that may require targeted nutritional intervention.

Ablation of Arg1 in hematopoietic cells improves respiratory function of lung parenchyma, but not that of larger airways or inflammation in asthmatic mice.

Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO synthase and because NO modulates bronchial tone and inflammation. Our objective was to investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Arginase 1 expression in the lung was ablated by crossing Arg1(fl/fl) with Tie2Cre(tg/-) mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with quantitative PCR, ELISA, and immunohistochemistry. Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but it improved peripheral lung function (tissue elastance and resistance) and attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL-13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA; lung content of IL-4, IL-5, IL-10, TNF-α, and IFN-γ protein; and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism but had little effect on airway inflammation.

Hepatic adaptation compensates inactivation of intestinal arginine biosynthesis in suckling mice.

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass (fl) and Villin-Cre mice. Unexpectedly, Ass (fl/fl) /VilCre (tg/-) mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass (fl/fl) /VilCre (tg/-) mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass (fl/fl) /VilCre (tg/-) mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.

Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy.

The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food intake in response to hypoxia. Mice were subjected to normoxic, hypoxic (8% oxygen), or pair-fed conditions for 2, 4, and 21 days. Cell-autonomous effects of hypoxia on skeletal muscle were also assessed in differentiated C2C12 myotubes. Hypoxia induced an initial rapid loss of body and muscle weight, which remained decreased during chronic hypoxia and could only in part be explained by the hypoxia-induced reduction of food intake (semistarvation). Regulatory steps of protein synthesis (unfolded protein response and mammal target of rapamycin signaling) remained active in response to acute and sustained hypoxia but not to semistarvation. Activation of regulatory signals for protein degradation, including increased expression of Murf1, Atrogin-1, Bnip3, and Map1lc3b mRNAs, was observed in response to acute hypoxia and to a lesser extent following semistarvation. Conversely, the sustained elevation of Atrogin-1, Bnip3, and Map1lc3b mRNAs and the increased activity of their upstream transcriptional regulator Forkhead box O1 were specific to chronic hypoxia because they were not observed in response to reduced food intake. In conclusion, altered regulation of protein turnover during hypoxia-induced muscle atrophy resulted from an interaction of semistarvation and a hypoxia-specific component. The finding that food restriction but not hypoxia-induced semistarvation inhibited regulatory steps in protein synthesis suggests a hypoxia-specific impairment of the coordination between protein-synthesis signaling and protein-degradation signaling in skeletal muscle.

Characterization of the inflammatory and metabolic profile of adipose tissue in a mouse model of chronic hypoxia.

In both obesity and chronic obstructive pulmonary disease (COPD), altered oxygen tension in adipose tissue (AT) has been suggested to evoke AT dysfunction, subsequently contributing to metabolic complications. Studying the effects of chronic hypoxia on AT function will add to our understanding of the complex pathophysiology of alterations in AT inflammation, metabolism, and mass observed in both obesity and COPD. This study investigated the inflammatory and metabolic profile of AT after chronic hypoxia. Fifty-two-week-old C57Bl/6J mice were exposed to chronic hypoxia (8% O2) or normoxia for 21 days, after which AT and plasma were collected. Adipocyte size, AT gene expression of inflammatory and metabolic genes, AT macrophage density, and circulating adipokine concentrations were measured. Food intake and body weight decreased upon initiation of hypoxia. However, whereas food intake normalized after 10 days, lower body weight persisted. Chronic hypoxia markedly reduced AT mass and adipocyte size. AT macrophage density and expression of Emr1, Ccl2, Lep, and Tnf were decreased, whereas Serpine1 and Adipoq expression levels were increased after chronic hypoxia. Concomitantly, chronic hypoxia increased AT expression of regulators of oxidative metabolism and markers of mitochondrial function and lipolysis. Circulating IL-6 and PAI-1 concentrations were increased, and leptin concentration was decreased after chronic hypoxia. Chronic hypoxia is associated with decreased rather than increased AT inflammation, and markedly decreased fat mass and adipocyte size. Furthermore, our data indicate that chronic hypoxia is accompanied by significant alterations in AT metabolic gene expression, pointing toward an enhanced AT metabolic rate.