Insight into the Role of Gut Microbiota in Duchenne Muscular Dystrophy: An Age-Related Study in Mdx Mice.

Dystrophin deficiency alters the sarcolemma structure, leading to muscle dystrophy, muscle disuse, and ultimately death. Beyond limb muscle deficits, patients with Duchenne muscular dystrophy have numerous transit disorders. Many studies have highlighted the strong relationship between gut microbiota and skeletal muscle. The aims of this study were: i) to characterize the gut microbiota composition over time up to 1 year in dystrophin-deficient mdx mice, and ii) to analyze the intestine structure and function and expression of genes linked to bacterial-derived metabolites in ileum, blood, and skeletal muscles to study interorgan interactions. Mdx mice displayed a significant reduction in the overall number of different operational taxonomic units and their abundance (α-diversity). Mdx genotype predicted 20% of ß-diversity divergence, with a large taxonomic modification of Actinobacteria, Proteobacteria, Tenericutes, and Deferribacteres phyla and the included genera. Interestingly, mdx intestinal motility and gene expressions of tight junction and Ffar2 receptor were down-regulated in the ileum. Concomitantly, circulating inflammatory markers related to gut microbiota (tumor necrosis factor, IL-6, monocyte chemoattractant protein-1) and muscle inflammation Tlr4/Myd88 pathway (Toll-like receptor 4, which recognizes pathogen-associated molecular patterns) were up-regulated. Finally, in mdx mice, adiponectin was reduced in blood and its receptor modulated in muscles. This study highlights a specific gut microbiota composition and highlights interorgan interactions in mdx physiopathology with gut microbiota as the potential central metabolic organ.

Furan fatty acid extracted from Hevea brasiliensis latex increases muscle mass in mice.

Skeletal muscle is essential for locomotion and plays a crucial role in energy homeostasis. It is regulated by nutrition, genetic factors, physical activity and hormones. Furan fatty acids (FuFAs) are minor fatty acids present in small quantities in food from plants and animals origin. Recently, we showed that a preventive nutritional supplementation with furan fatty acid in a DIO mouse model reduces metabolic disorders. The present study was designed to determine the influence of FuFA-F2 extracted from Hevea brasiliensis latex on skeletal muscle phenotype. In C2C12 myotubes we found that FuFA-F2 whatever the concentration used increased protein content. We revealed that in C2C12 myotubes FuFA-F2 (10 µM) increases protein synthesis as shown by the stimulation of mTOR phosphorylation. Next, to confirm in vivo our results C57Bl6 mice were supplemented by oral gavage with vehicle or FuFA-F2 (20 mg/kg) for 3 and a half weeks. We found that mice supplemented with FuFA-F2 had a greater lean mass than the control mice. In line with this observation, we revealed that FuFA-F2 increased muscle mass and promoted more oxidative muscle metabolism in mice as attested by cytochrome c oxidase activity. In conclusion, we demonstrated that FuFA-F2 stimulates muscle anabolism in mice in vitro and in vivo, mimicking in part physical activity. This study highlights that in vivo FuFA-F2 may have health benefits by increasing muscle mass and oxidative metabolism.

Preventive nutritional supplementation with furan fatty acid in a DIO mouse model increases muscle mass and reduces metabolic disorders.

The increase in obesity has become a major global health problem and is associated with numerous metabolic dysfunctions. Furan fatty acids (FuFAs) are minor lipids present in our diet. Recently we showed that FuFA-F2 extracted from Hevea brasiliensis latex stimulates muscle anabolism in mice in vitro and in vivo, mimicking in part physical activity. While skeletal muscle is essential for energy metabolism and is the predominant site of insulin-mediated glucose uptake in the post prandial state, our results suggested that FuFA-F2 could have favorable effects against obesity. The aim of this work was therefore to study whether a preventive nutritional supplementation with FuFA-F2 (40 mg or 110 mg/day/kg of body weight) in a diet-induced obesity (DIO) mouse model may have beneficial effects against obesity and liver and skeletal muscle metabolic dysfunction. We showed that 12 weeks of FuFA-F2 supplementation in DIO mice decreased fat mass, increased lean mass and restored normal energy expenditure. In addition, we found that FuFA-F2 improved insulin sensitivity. We revealed that FuFA-F2 increased muscle mass but had no effect on mitochondrial function and oxidative stress in skeletal muscle. Furthermore, we observed that FuFA-F2 supplementation reduced liver steatosis without impact on mitochondrial function and oxidative stress in liver. Our findings demonstrated for the first time that a preventive nutritional supplementation with a furan fatty acid in DIO mice reduced metabolic disorders and was able to mimic partly the positive effects of physical activity. This study highlights that nutritional FuFA-F2 supplementation could be an effective approach to treat obesity and metabolic syndrome.

9-PAHPA long term intake in DIO and db/db mice ameliorates insulin sensitivity but has few effects on obesity and associated metabolic disorders.

Branched fatty acid esters of hydroxy fatty acids are endogenous lipids reported to have antidiabetic and anti-inflammatory effects. Recently, we showed that 9-palmitic acid esters of hydroxypalmitic acid (9-PAHPA) and 9-oleic acid esters of hydroxypalmitic acid increased insulin sensitivity in mice when incorporated to a chow diet or to a high fat and high sucrose diet. However, preventive supplementation with 9-PAHPA and 9-oleic acid esters of hydroxypalmitic acid in high fat and high sucrose diet mice did not impair significant weight gain or the development of hyperglycemia. The aim of this work was therefore to study whether in two animal models of obesity, namely the classical diet-induced obesity (DIO) and the db/db mice, 9-PAHPA may have beneficial effects against obesity and liver and skeletal muscle metabolic dysfunction. In DIO mice, we observed that 9-PAHPA increased body weight and fat mass. In line with this observation, we found that 9-PAHPA supplementation decreased energy expenditure. In liver and in skeletal muscle, mitochondrial activities and oxidative stress parameters were not modified by 9-PAHPA supplementation. In db/db mice, 9-PAHPA had no effect on the dramatic weight gain and hyperglycemia. In addition, 9-PAHPA supplementation did not correct either the hepatomegaly and hepatic steatosis or the severe muscle atrophy recorded compared with db/+ animals. Likewise, supplementation with 9-PAHPA did not impact the different metabolic parameters analyzed, either in the liver or in the skeletal muscles. However, it decreased insulin resistance in DIO and db/db mice. In conclusion, our study indicated that a long-term intake of 9-PAHPA in DIO and db/db mice improved insulin sensitivity but had only few effects on obesity and associated metabolic disorders.

A single short reprogramming early in life initiates and propagates an epigenetically related mechanism improving fitness and promoting an increased healthy lifespan.

Recent advances in cell reprogramming showed that OSKM induction is able to improve cell physiology in vitro and in vivo. Here, we show that a single short reprogramming induction is sufficient to prevent musculoskeletal functions deterioration of mice, when applied in early life. In addition, in old age, treated mice have improved tissue structures in kidney, spleen, skin, and lung, with an increased lifespan of 15% associated with organ-specific differential age-related DNA methylation signatures rejuvenated by the treatment. Altogether, our results indicate that a single short reprogramming early in life might initiate and propagate an epigenetically related mechanism to promote a healthy lifespan.

Myostatin gene inactivation increases post-mortem calpain-dependent muscle proteolysis in mice.

Myostatin deficiency leads to extensive skeletal muscle hypertrophy, but its consequence on post-mortem muscle proteolysis is unknown. Here, we compared muscle myofibrillar protein degradation, and autophagy, ubiquitin-proteasome and Ca2+-dependent proteolysis relative to the energetic and redox status in wild-type (WT) and myostatin knock-out mice (KO) during early post-mortem storage. KO muscles showed higher degradation of myofibrillar proteins in the first 24 h after death, associated with preserved antioxidant status, compared with WT muscles. Analysis of key autophagy and ubiquitin-proteasome system markers indicated that these two pathways were not upregulated in post-mortem muscle (both genotypes), but basal autophagic flux and ATP content were lower in KO muscles. Proteasome and caspase activities were not different between WT and KO mice. Conversely, calpain activity was higher in KO muscles, concomitantly with higher troponin T and desmin degradation. Altogether, these results suggest that calpains but not the autophagy, proteasome and caspase systems, explain the difference in post-mortem muscle protein proteolysis between both genotypes.

p43, a Truncated Form of Thyroid Hormone Receptor α, Regulates Maturation of Pancreatic ß Cells.

P43 is a truncated form of thyroid hormone receptor α localized in mitochondria, which stimulates mitochondrial respiratory chain activity. Previously, we showed that deletion of p43 led to reduction of pancreatic islet density and a loss of glucose-stimulated insulin secretion in adult mice. The present study was designed to determine whether p43 was involved in the processes of ß cell development and maturation. We used neonatal, juvenile, and adult p43-/- mice, and we analyzed the development of ß cells in the pancreas. Here, we show that p43 deletion affected only slightly ß cell proliferation during the postnatal period. However, we found a dramatic fall in p43-/- mice of MafA expression (V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog A), a key transcription factor of beta-cell maturation. Analysis of the expression of antioxidant enzymes in pancreatic islet and 4-hydroxynonenal (4-HNE) (a specific marker of lipid peroxidation) staining revealed that oxidative stress occurred in mice lacking p43. Lastly, administration of antioxidants cocktail to p43-/- pregnant mice restored a normal islet density but failed to ensure an insulin secretion in response to glucose. Our findings demonstrated that p43 drives the maturation of ß cells via its induction of transcription factor MafA during the critical postnatal window.

Acute and chronic effects of Rhaponticum carthamoides and Rhodiola rosea extracts supplementation coupled to resistance exercise on muscle protein synthesis and mechanical power in rats.

BACKGROUND: Owing to its strength-building and adaptogenic properties, Rhaponticum carthamoides (Rha) has been commonly used by elite Soviet and Russian athletes. Rhodiola rosea (Rho) is known to reduce physical and mental fatigue and improve endurance performance. However, the association of these two nutritional supplements with resistance exercise performance has never been tested. Resistance exercise is still the best way to stimulate protein synthesis and induce chronic muscle adaptations. The aim of this study was to investigate the acute and chronic effects of resistance exercise coupled with Rha and Rho supplementation on protein synthesis, muscle phenotype, and physical performance. METHODS: For the acute study, fifty-six rats were assigned to either a trained control group or one of the groups treated with specific doses of Rha and/or Rho. Each rats performed a single bout of climbing resistance exercise. The supplements were administered immediately after exercise by oral gavage. Protein synthesis was measured via puromycin incorporation. For the chronic study, forty rats were assigned to either the control group or one of the groups treated with doses adjusted from the acute study results. The rats were trained five times per week for 4 weeks with the same bout of climbing resistance exercise with additionals loads. Rha + Rho supplement was administered immediately after each training by oral gavage. RESULTS: The findings of the acute study indicated that Rha and Rha + Rho supplementation after resistance exercise stimulated protein synthesis more than resistance exercise alone (p < 0.05). After 4 weeks of training, the mean power performance was increased in the Rha + Rho and Rha-alone groups (p < 0.05) without any significant supplementation effect on muscle weight or fiber cross-sectional area. A tendency towards an increase in type I/ type II fiber ratio was observed in Rha/Rho-treated groups compared to that in the trained control group. CONCLUSION: Rhodiola and Rhaponticum supplementation after resistance exercise could synergistically improve protein synthesis, muscle phenotype and physical performance.

Transient Changes of Metabolism at the Pronuclear Stage in Mice Influences Skeletal Muscle Phenotype in Adulthood.

Skeletal muscle has a remarkable plasticity, and its phenotype is strongly influenced by hormones, transcription factors, and physical activity. However, whether skeletal phenotype can be oriented or not during early embryonic stages has never been investigated. Here, we report that pyruvate as the only source of carbohydrate in the culture medium of mouse one cell stage embryo influenced the establishment of the muscular phenotype in adulthood. We found that pyruvate alone induced changes in the contractile phenotype of the skeletal muscle in a sexually dependent manner. For male mice, a switch to a more glycolytic phenotype was recorded, whereas, in females, the pyruvate induced a switch to a more oxidative phenotype. In addition, the influence of pyruvate on the contractile phenotypes was confirmed in two mouse models of muscle hypertrophy: the well-known myostatin deficient mouse (Mstn-/-) and a mouse carrying a specific deletion of p43, a mitochondrial triiodothyronine receptor. Finally, to understand the link between these adult phenotypes and the early embryonic period, we assessed the levels of two histone H3 post-translational modifications in presence of pyruvate alone just after the wave of chromatin reprogramming specific of the first cell cycle. We showed that H3K4 acetylation level was decreased in Mstn-/- 2-cell embryos, whereas no difference was found for H3K27 trimethylation level, whatever the genotype. These findings demonstrate for the first time that changes in the access of energy substrate during the very first embryonic stage can induce a precocious orientation of skeletal muscle phenotype in adulthood.

New evidence of exercise training benefits in myostatin-deficient mice: Effect on lipidomic abnormalities.

Myostatin (Mstn) inactivation or inhibition is considered as a promising treatment for various muscle-wasting disorders because it promotes muscle growth. However, myostatin-deficient hypertrophic muscles show strong fatigability associated with abnormal mitochondria and lipid metabolism. Here, we investigated whether endurance training could improve lipid metabolism and mitochondrial membrane lipid composition in mice where the Mstn gene was genetically ablated (Mstn-/- mice). In Mstn-/- mice, 4 weeks of daily running exercise sessions (65-70% of the maximal aerobic speed for 1 h) improved significantly aerobic performance, particularly the endurance capacity (up to +280% compared with untrained Mstn-/- mice), to levels comparable to those of trained wild type (WT) littermates. The expression of oxidative and lipid metabolism markers also was increased, as indicated by the upregulation of the Cpt1, Ppar-δ and Fasn genes. Moreover, endurance training also increased, but far less than WT, citrate synthase level and mitochondrial protein content. Interestingly endurance training normalized the cardiolipin fraction in the mitochondrial membrane of Mstn-/- muscle compared with WT. These results suggest that the combination of myostatin inhibition and endurance training could increase the muscle mass while preserving the physical performance with specific effects on cardiolipin and lipid-related pathways.

eIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse.

KEY POINTS: In muscular cells, eukaryotic initiation factor subunit f (eIF3f) activates protein synthesis by allowing physical interaction between mechanistic target of rapamycin complex 1 (MTORC1) and ribosomal protein S6 kinase 1 (S6K1), although its physiological role in animals is unknown. A knockout approach suggests that homozygous mice carrying a null mutation of the eIF3f gene fail to develop and consequently die at early embryonic stage, whereas heterozygous mice associated with a partial depletion of eIF3f gene grow normally and are phenotypically indistinguishable from wild-type mice. Heterozygous mice express reduced eIF3f mRNA and protein levels in skeletal muscles and show diminished muscle mass associated with a decrease in the protein synthesis rate and an inhibition of the MTORC1 pathway. During hindlimb immobilization, heterozygous eIF3f mice display an exacerbated immobilization-induced muscle atrophy associated with reduced protein synthesis. These results highlight the essential role of eIF3f during embryonic development and its involvement in muscular homeostasis via protein synthesis regulation. ABSTRACT: Eukaryotic translation initiation factor 3, subunit F (eIF3f), a component of eIF3 complex, plays an important role in protein synthesis regulation, although its physiological functions are unknown. We generated and analysed mice carrying a null mutation in the eIF3f gene. We showed that homozygous eIF3f knockout fail to develop and that eIF3f-/- embryos die at an early stage of development but after the pre-implantation stage. However, disrupting one eIF3f allele does not affect growth, viability and fertility of heterozygous mice but, instead, reduces eIF3f mRNA and protein levels in all tissues examined. Although heterozygous mice are phenotypically indistinguishable from wild-type mice, they present a diminished body weight and a lean mass reduction associated with normal body size. Interestingly, skeletal muscles are mainly affected and display an altered cell size without modification of fibre number. Skeletal muscles of heterozygous mice show a deficiency in polysome content, a decrease in protein synthesis rate and an inhibition of the mechanistic target of rapamycin (MTOR) pathway. We then studied the effects of hindlimb immobilization that mimic muscle disuse on heterozygous mice aiming to further explore the involvement of eIF3f in protein synthesis. We found that eIF3f partial depletion amplifies muscle atrophy compared to wild-type mice. Mass and cross-sectional area decreases were associated with reduced MTOR pathway activation and protein synthesis rate. Taken together, our data indicate that eIF3f is essential for mice embryonic development and controls adult skeletal muscle mass via protein synthesis regulation in a MTOR-dependent manner.

Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53.

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

Temperature homeostasis in mice lacking the p43 mitochondrial T3 receptor.

Thyroid hormones and Thra gene play a key role in energy expenditure regulation, temperature homeostasis, and mitochondrial function. To decipher the function of the mitochondrial TRα receptor in these phenomena, we used mice lacking specifically the p43 mitochondrial T3 receptor. We found that these animals were hypermetabolic, hyperphagic, and displayed a down setting of the core body temperature. However, p43-/- animals do not present cold intolerance or defect of facultative thermogenesis. In addition, the mitochondrial function of BAT is slightly affected in the absence of p43. Our study, therefore, suggests a complementarity of action between the mitochondrial receptor and other proteins encoded by the Thra gene in the control of basal metabolism, facultative thermogenesis, and determination of the set point of temperature regulation.

Muscle Regeneration with Intermuscular Adipose Tissue (IMAT) Accumulation Is Modulated by Mechanical Constraints.

Sports trauma are able to induce muscle injury with fibrosis and accumulation of intermuscular adipose tissue (IMAT), which affect muscle function. This study was designed to investigate whether hypoactivity would influence IMAT accumulation in regenerating mouse skeletal muscle using the glycerol model of muscle regeneration. The animals were immediately hindlimb unloaded for 21 days after glycerol injection into the tibialis anterior (TA) muscle. Muscle fiber and adipocyte cross-sectional area (CSA) and IMAT accumulation were determined by histomorphometric analysis. Adipogenesis during regenerative processes was examined using RT-qPCR and Western blot quantification. Twenty-one days of hindlimb unloading resulted in decreases of 38% and 50.6% in the muscle weight/body weight ratio and CSA, respectively, in soleus muscle. Glycerol injection into TA induced IMAT accumulation, reaching 3% of control normal-loading muscle area. This IMAT accumulation was largely inhibited in unloading conditions (0.09%) and concomitant with a marked reduction in perilipin and FABP4 protein content, two key markers of mature adipocytes. Induction of PPARγ and C/EBPα mRNA, two markers of adipogenesis, was also decreased. Furthermore, the protein expression of PDGFRα, a cell surface marker of fibro/adipogenic progenitors, was much lower in regenerating TA from the unloaded group. Exposure of regenerating muscle to hypoactivity severely reduces IMAT development and accumulation. These results provide new insight into the mechanisms regulating IMAT development in skeletal muscle and highlight the importance of taking into account the level of mechanical constraint imposed on skeletal muscle during the regeneration processes.