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.

Long-term intake of 9-PAHPA or 9-OAHPA modulates favorably the basal metabolism and exerts an insulin sensitizing effect in obesogenic diet-fed mice.

PURPOSE: Fatty acid esters of hydroxy fatty acids (FAHFAs) are a large family of endogenous bioactive lipids. To date, most of the studied FAHFAs are branched regioisomers of Palmitic Acid Hydroxyl Stearic Acid (PAHSA) that were reported to possess anti-diabetic and anti-inflammatory activity in humans and rodents. Recently, we have demonstrated that 9-PAHPA or 9-OAHPA intake increased basal metabolism and enhanced insulin sensitivity in healthy control diet-fed mice but induced liver damage in some mice. The present work aims to explore whether a long-term intake of 9-PAHPA or 9-OAHPA may have similar effects in obesogenic diet-fed mice. METHODS: C57Bl6 mice were fed with a control or high fat-high sugar (HFHS) diets for 12 weeks. The HFHS diet was supplemented or not with 9-PAHPA or 9-OAHPA. Whole-body metabolism was explored. Glucose and lipid metabolism as well as mitochondrial activity and oxidative stress status were analyzed. RESULTS: As expected, the intake of HFHS diet led to obesity and lower insulin sensitivity with minor effects on liver parameters. The long-term intake of 9-PAHPA or 9-OAHPA modulated favorably the basal metabolism and improved insulin sensitivity as measured by insulin tolerance test. On the contrary to what we have reported previously in healthy mice, no marked effect for these FAHFAs was observed on liver metabolism of obese diabetic mice. CONCLUSION: This study indicates that both 9-PAHPA and 9-OAHPA may have interesting insulin-sensitizing effects in obese mice with lower insulin sensitivity.

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.

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.

FAHFAs Regulate the Proliferation of C2C12 Myoblasts and Induce a Shift toward a More Oxidative Phenotype in Mouse Skeletal Muscle.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are endogenous lipids reported to have antidiabetic and anti-inflammatory effects. Since skeletal muscle is a major target for insulin, the aim of this study is to explore for the first time the influence of several FAHFAs in C2C12 myoblasts and in skeletal muscle phenotype in mice. Here, we show that eleven FAHFAs belonging to different families inhibit C2C12 myoblast proliferation. In addition, all FAHFAs decreased mitochondrial cytochrome c oxidase activity without affecting reactive oxygen species production and the mitochondrial network. During C2C12 myoblasts differentiation, we found that two of the most active lipids, 9-PAHPA and 9-OAHPA, did not significantly affect the fusion index and the expression of myosin heavy chains. However, we found that three months' intake of 9-PAHPA or 9-OAHPA in mice increased the expression of more oxidative myosin in skeletal muscle without affecting skeletal muscle mass, number, and mean fiber area, mitochondrial activity, and oxidative stress parameters. In conclusion, our study indicated that the eleven FAHFAs tested decreased the proliferation rate of C2C12 myoblasts, probably through the inhibition of mitochondrial activity. In addition, we found that 9-PAHPA or 9-OAHPA supplementation in mice induced a switch toward a more oxidative contractile phenotype of skeletal muscle. These data suggest that the increase in insulin sensitivity previously described for these two FAHFAs is of muscular origin.

E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity.

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

Skeletal muscle expression of p43, a truncated thyroid hormone receptor α, affects lipid composition and metabolism.

Thyroid hormone is a major regulator of metabolism and mitochondrial function. Thyroid hormone also affects reactions in almost all pathways of lipids metabolism and as such is considered as the main hormonal regulator of lipid biogenesis. The aim of this study was to explore the possible involvement of p43, a 43 Kda truncated form of the nuclear thyroid hormone receptor TRα1 which stimulates mitochondrial activity. Therefore, using mouse models overexpressing p43 in skeletal muscle (p43-Tg) or lacking p43 (p43-/-), we have investigated the lipid composition in quadriceps muscle and in mitochondria. Here, we reported in the quadriceps muscle of p43-/- mice, a fall in triglycerides, an inhibition of monounsaturated fatty acids (MUFA) synthesis, an increase in elongase index and an decrease in desaturase index. However, in mitochondria from p43-/- mice, fatty acid profile was barely modified. In the quadriceps muscle of p43-Tg mice, MUFA content was decreased whereas the unsaturation index was increased. In addition, in quadriceps mitochondria of p43-Tg mice, we found an increase of linoleic acid level and unsaturation index. Last, we showed that cardiolipin content, a key phospholipid for mitochondrial function, remained unchanged both in quadriceps muscle and in its mitochondria whatever the mice genotype. In conclusion, this study shows that muscle lipid content and fatty acid profile are strongly affected in skeletal muscle by p43 levels. We also demonstrate that regulation of cardiolipin biosynthesis by the thyroid hormone does not imply p43.

Skeletal muscle overexpression of short isoform Sirt3 altered mitochondrial cardiolipin content and fatty acid composition.

Cardiolipin (CL) is a phospholipid at the heart of mitochondrial metabolism, which plays a key role in mitochondrial function and bioenergetics. Among mitochondrial activity regulators, SIRT3 plays a crucial role in controlling the acetylation status of many enzymes participating in the energy metabolism in particular concerning lipid metabolism and fatty acid oxidation. Data suggest that possible connection may exist between SIRT3 and CL status that has not been evaluated in skeletal muscle. In the present study, we have characterized skeletal muscle lipids as well as mitochondrial lipids composition in mice overexpressing long (SIRT3-M1) and short (SIRT3-M3) isoforms of SIRT3. Particular attention has been paid for CL. We reported no alteration in muscle lipids content and fatty acids composition between the two mice SIRT3 strains and the control mice. However, mitochondrial CL content was significantly decreased in SIRT3-M3 mice and associated to an upregulation of tafazzin gene expression. In addition, mitochondrial phospholipids and fatty acids composition was altered with an increase in the PC/PE ratio and arachidonic acid content and a reduction in the MUFA/SFA ratio. These modifications in mitochondrial membrane composition are associated with a reduction in the enzymatic activities of mitochondrial respiratory chain complexes I and IV. In spite of these mitochondrial enzymatic alterations, skeletal muscle mitochondrial respiration remained similar in SIRT3-M3 and control mice. Surprisingly, none of those metabolic alterations were detected in mitochondria from SIRT3-M1 mice. In conclusion, our data indicate a specific action of the shorter SIRT3 isoform on lipid mitochondrial membrane biosynthesis and functioning.

Early structural and functional signature of 3-day human skeletal muscle disuse using the dry immersion model.

KEY POINTS: Our study contributes to the characterization of muscle loss and weakness processes induced by a sedentary life style, chronic hypoactivity, clinical bed rest, immobilization and microgravity. This study, by bringing together integrated and cellular evaluation of muscle structure and function, identifies the early functional markers and biomarkers of muscle deconditioning. Three days of muscle disuse in healthy adult subjects is sufficient to significantly decrease muscle mass, tone and force, and to induce changes in function relating to a weakness in aerobic metabolism and muscle fibre denervation. The outcomes of this study should be considered in the development of an early muscle loss prevention programme and/or the development of pre-conditioning programmes required before clinical bed rest, immobilization and spaceflight travel. ABSTRACT: Microgravity and hypoactivity are associated with skeletal muscle deconditioning. The decrease of muscle mass follows an exponential decay, with major changes in the first days. The purpose of the study was to dissect out the effects of a short-term 3-day dry immersion (DI) on human quadriceps muscle function and structure. The DI model, by suppressing all support zones, accurately reproduces the effects of microgravity. Twelve healthy volunteers (32 ± 5 years) completed 3 days of DI. Muscle function was investigated through maximal voluntary contraction (MVC) tests and muscle viscoelasticity. Structural experiments were performed using MRI analysis and invasive experiments on muscle fibres. Our results indicated a significant 9.1% decrease of the normalized MVC constant (P = 0.048). Contraction and relaxation modelization kinetics reported modifications related to torque generation (kACT  = -29%; P = 0.014) and to the relaxation phase (kREL  = +34%; P = 0.040) after 3 days of DI. Muscle viscoelasticity was also altered. From day one, rectus femoris stiffness and tone decreased by, respectively, 7.3% (P = 0.002) and 10.2% (P = 0.002), and rectus femoris elasticity decreased by 31.5% (P = 0.004) after 3 days of DI. At the cellular level, 3 days of DI translated into a significant atrophy of type I muscle fibres (-10.6 ± 12.1%, P = 0.027) and an increased proportion of hybrid, type I/IIX fibre co-expression. Finally, we report an increase (6-fold; P = 0.002) in NCAM+ muscle fibres, showing an early denervation process. This study is the first to report experiments performed in Europe investigating human short-term DI-induced muscle adaptations, and contributes to deciphering the early changes and biomarkers of skeletal muscle deconditioning.

Depletion of the p43 mitochondrial T3 receptor in mice affects skeletal muscle development and activity.

In vertebrates, skeletal muscle myofibers display different contractile and metabolic properties associated with different mitochondrial content and activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) regulating mitochondrial transcription and mitochondrial biogenesis. When overexpressed in skeletal muscle, it increases mitochondrial DNA content, stimulates mitochondrial respiration, and induces a shift in the metabolic and contractile features of muscle fibers toward a slower and more oxidative phenotype. Here we show that a p43 depletion in mice decreases mitochondrial DNA replication and respiratory chain activity in skeletal muscle in association with the induction of a more glycolytic muscle phenotype and a decrease of capillary density. In addition, p43(-/-) mice displayed a significant increase in muscle mass relative to control animals and had an improved ability to use lipids. Our findings establish that the p43 mitochondrial receptor strongly affects muscle mass and the metabolic and contractile features of myofibers and provides evidence that this receptor mediates, in part, the influence of thyroid hormone in skeletal muscle.

Mitochondrial T3 receptor p43 regulates insulin secretion and glucose homeostasis.

Thyroid hormone is a major determinant of energy expenditure and a key regulator of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) that acts as a mitochondrial transcription factor of the organelle genome, which leads, in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Here we generated mice specifically lacking p43 to address its physiological influence. We found that p43 is required for normal glucose homeostasis. The p43(-/-) mice had a major defect in insulin secretion both in vivo and in isolated pancreatic islets and a loss of glucose-stimulated insulin secretion. Moreover, a high-fat/high-sucrose diet elicited more severe glucose intolerance than that recorded in normal animals. In addition, we observed in p43(-/-) mice both a decrease in pancreatic islet density and in the activity of complexes of the respiratory chain in isolated pancreatic islets. These dysfunctions were associated with a down-regulation of the expression of the glucose transporter Glut2 and of Kir6.2, a key component of the K(ATP) channel. Our findings establish that p43 is an important regulator of glucose homeostasis and pancreatic ß-cell function and provide evidence for the first time of a physiological role for a mitochondrial endocrine receptor.

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.

Protein sequences involved in the mitochondrial import of the 3,5,3'-L-triiodothyronine receptor p43.

The major effect of T3 on mitochondrial activity has been partly explained by the discovery of p43, a T3-dependent transcription factor of the mitochondrial genome. P43 is imported into mitochondria in an atypical manner which is not yet fully understood. Our aim was to characterize the p43 sequences inducing its mitochondrial import, using in organello import experiments with wild-type or mutated proteins and validation in CV1 cells. We find that several sequences define the mitochondrial addressing. Two alpha helices in the C-terminal part of p43 are actual mitochondrial import sequences as fusion to a cytosolic protein induces its mitochondrial translocation. Helix 5 drives the atypical mitochondrial import process, whereas helices 10/11 induce a classical import process. However, despite its inability to drive a mitochondrial import, the N-terminal region of p43 also plays a permissive role as in the presence of the C-terminal import sequences different N-terminal regions determine whether the protein is imported or not. These results can be extrapolated to other mitochondrial proteins related to the nuclear receptor superfamily, devoid of classical mitochondrial import sequences.

P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression.

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.

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.

Long-term high intake of 9-PAHPA or 9-OAHPA increases basal metabolism and insulin sensitivity but disrupts liver homeostasis in healthy mice.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a new family of endogenous lipids recently discovered. Several studies reported that some FAHFAs have antidiabetic and anti-inflammatory effects. The objective of this study was to explore the impact of two FAHFAs, 9-PAHPA or 9-OAHPA, on the metabolism of mice. C57Bl/6J male mice, 6 weeks old, were divided into 3 groups of 10 mice each. One group received a control diet and the two others groups received the control diet supplemented with 9-PAHPA or 9-OAHPA for 12 weeks. Mouse weight and body composition were monitored throughout the study. Some days before euthanasia, energy expenditure, glucose tolerance and insulin sensitivity were also determined. After sacrifice, blood and organs were collected for relevant molecular, biochemical and histological analyses. Although high intake of 9-PAHPA or 9-OAHPA increased basal metabolism, it had no direct effect on body weight. Interestingly, the 9-PAHPA or 9-OAHPA intake increased insulin sensitivity but without modifying glucose tolerance. Nevertheless, 9-PAHPA intake induced a loss of glucose-stimulated insulin secretion. Surprisingly, both studied FAHFAs induced hepatic steatosis and fibrosis in some mice, which were more marked with 9-PAHPA. Finally, a slight remodeling of white adipose tissue was also observed with 9-PAHPA intake. In conclusion, the long-term high intake of 9-PAHPA or 9-OAHPA increased basal metabolism and insulin sensitivity in healthy mice. However, this effect, highly likely beneficial in a diabetic state, was accompanied by manifest liver damage in certain mice that should deserve special attention in both healthy and pathological studies.

Regulation of mitochondrial activity controls the duration of skeletal muscle regeneration in response to injury.

Thyroid hormone is a major regulator of skeletal muscle development and repair, and also a key regulator of mitochondrial activity. We have previously identified a 43 kDa truncated form of the nuclear T3 receptor TRα1 (p43) which stimulates mitochondrial activity and regulates skeletal muscle features. However, its role in skeletal muscle regeneration remains to be addressed. To this end, we performed acute muscle injury induced by cardiotoxin in mouse tibialis in two mouse models where p43 is overexpressed in or depleted from skeletal muscle. The measurement of muscle fiber size distribution at different time point (up to 70 days) upon injury lead us to unravel requirement of the p43 signaling pathway for satellite cells dependent muscle regeneration; strongly delayed in the absence of p43; whereas the overexpression of the receptor enhances of the regeneration process. In addition, we found that satellite cells derived from p43-Tg mice display higher proliferation rates when cultured in vitro when compared to control myoblasts, whereas p43-/- satellites shows reduced proliferation capacity. These finding strongly support that p43 plays an important role in vivo by controling the duration of skeletal muscle regeneration after acute injury, possibly through the regulation of mitochondrial activity and myoblasts proliferation.

Characterization of a novel thyroid hormone receptor alpha variant involved in the regulation of myoblast differentiation.

The regulation of gene expression by thyroid hormone (T3) involves binding of the hormone to nuclear receptors [thyroid hormone receptor (TR)] acting as T3-dependent transcription factors encoded by TRalpha (NR1A1) and TRbeta (NR1A2) genes. Several TRalpha variants have already been characterized, but only some of them display T3 binding activity. In this study, we have identified another transcript, TRalpha-DeltaE6, produced by alternative splicing with microexon 6b instead of exon 6. This splicing leads to the synthesis of a protein devoid of a hinge domain. The TRalpha-DeltaE6 transcript is detected in all mouse tissues tested. Although TRalpha-DeltaE6 did not bind DNA, its expression induced a TRalpha1 sequestration in the cytoplasm. Functional studies demonstrated that TRalpha-DeltaE6 inhibits the transcriptional activity of TRalpha1 and retinoic X receptor-alpha, but not of retinoic acid receptor-alpha. We also found that TRalpha-DeltaE6 efficiently decreased the ability of TRalpha to inhibit MyoD transcriptional activity during myoblast proliferation. Consequently, when overexpressed in myoblasts, it stimulated terminal differentiation. We suggest that this novel TRalpha variant may act as down regulator of overall T3 receptor activity, including its ability to repress MyoD transcriptional activity during myoblast proliferation.