Lsd1 ablation triggers metabolic reprogramming of brown adipose tissue

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT- selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1- deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT

The histone code reader Spin1 controls skeletal muscle development

While several studies correlated increased expression of the histone code reader Spin1 with tumor formation or growth, little is known about physiological functions of the protein. We generated Spin1(M5) mice with ablation of Spin1 in myoblast precursors using the Myf5-Cre deleter strain. Most Spin1(M5) mice die shortly after birth displaying severe sarcomere disorganization and necrosis. Surviving Spin1(M5) mice are growth-retarded and exhibit the most prominent defects in soleus, tibialis anterior, and diaphragm muscle. Transcriptome analyses of limb muscle at embryonic day (E) 15.5, E16.5, and at three weeks of age provided evidence for aberrant fetal myogenesis and identified deregulated skeletal muscle (SkM) functional networks. Determination of genome-wide chromatin occupancy in primary myoblast revealed direct Spin1 target genes and suggested that deregulated basic helix-loop-helix transcription factor networks account for developmental defects in Spin1(M5) fetuses. Furthermore, correlating histological and transcriptome analyses, we show that aberrant expression of titin-associated proteins, abnormal glycogen metabolism, and neuromuscular junction defects contribute to SkM pathology in Spin1(M5) mice. Together, we describe the first example of a histone code reader controlling SkM development in mice, which hints at Spin1 as a potential player in human SkM disease

Role of glucocorticoid receptor (GR) and transcription intermediairy factor (TIF2) in mouse skeletal muscle at adulte stage

Le muscle squelettique est un tissu dynamique ayant la capacité de réguler sa taille et son activité en réponse à différents stimuli extérieurs. Mon travail de thèse a principalement porté sur l'étude d'un récepteur nucléaire, le récepteur des glucocorticoïdes (GR) et de l'un de ces co-régulateur, le facteur intermédiaire de transcription TIF2, dans ce tissu. Ainsi afin de mieux comprendre le rôle de GR et de TIF2 dans les myofibres, nous avons généré des souris dans lesquelles ils sont sélectivement invalidés dans le muscle squelettique de souris adultes (souris GR(i)skm-/- et TIF2(i)skm-/-). La première partie de ma thèse a démontré que l'augmentation du découplage mitochondrial dans les myofibres protège les souris TIF2(i)skm-/- de la diminution des capacités oxydatives induite par la sédentarité, retarde le développement du diabète de type 2 et atténue la prise de poids induite par un régime hypercalorique. De plus, nos résultats démontrent que SRC-1 et TIF2 peuvent moduler l'expression de la protéine découplante UCP3 de manière antagoniste, et que l'augmentation des niveaux de SRC-1 dans les myofibres des souris TIF2(i)skm-/- est impliquée de manière critique dans la mise en place des changements métaboliques de ces souris. La seconde partie de ma thèse a montré que les souris GR(i)skm-/- ont une masse et une force musculaire plus importantes que des souris contrôles, du fait d'une hyperactivation des voies anaboliques. Par ailleurs, ces animaux ne subissent pas l'atrophie musculaire induite par un traitement à la dexaméthasone, un glucocorticoïde de synthèse, ou par une mise à jeun prolongée, montrant que la fonte musculaire est strictement contrôlée par GR dans les myofibres. Cette étude nous a permis d'éclaircir les mécanismes moléculaires et cellulaires régulant l'homéostasie musculaire et ouvriront de nouvelles voies dans le traitement des myopathies.Skeletal muscle is a dynamic tissue that has the capacity to regulate its size in response to a variety of external cues. My thesis work focused on the role of a nuclear receptor, the glucocorticoid receptor (GR) and of one of it co-regulator, the transcriptional intermediary factor TIF2, in this tissue. To improve our knowledge concerning the role of GR and TIF2 in myofibers, we generated mice in which GR or TIF2 are selectively ablated in skeletal muscle myofibers at adult stage (GR(i)skm-/- and TIF2(i)skm-/- mice). The first part of this work demonstrated that increased mitochondrial uncoupling in skeletal muscle myocytes protected these mice from decreased muscle oxidative capacities induced by sedentariness, delayed the development of type 2 diabetes and attenuated high caloric diet-induced obesity. Moreover, our results demonstrate that SRC-1 and TIF2 can modulate the expression of the uncoupling protein UCP3 in an antagonistic manner, and that enhanced SRC-1 levels in TIF2-deficient myofibers are critically involved in the metabolic changes of TIF2(i)skm-/- mice. The second part of this work demonstrated that GR(i)skm-/- mice skeletal muscle mass and strength were increased, due to anabolic pathway enhancement. Moreover, such mice are protected against dexamethasone-induced muscle catabolism and partially resistant to fasting-induced muscle atrophy, thus demonstrating that myofiber GR plays a major role in coordinating degradation of muscle proteins. This work highlighted molecular mechanisms regulating muscle homeostasis and should provide new insights in treatment of muscular disorders

Role of glucocorticoid receptor (GR) and transcription intermediairy factor (TIF2) in mouse skeletal muscle at adulte stage

Le muscle squelettique est un tissu dynamique ayant la capacité de réguler sa taille et son activité en réponse à différents stimuli extérieurs. Mon travail de thèse a principalement porté sur l'étude d'un récepteur nucléaire, le récepteur des glucocorticSkeletal muscle is a dynamic tissue that has the capacity to regulate its size in response to a variety of external cues. My thesis work focused on the role of a nuclear receptor, the glucocorticoid receptor (GR) and of one of it co-regulator, the transc

Rôle du récepteur des glucocorticoïdes (GR) et du facteur intermédiaire de transcription -TIF2) dans le muscle squelettique chez la souris adulte

Le muscle squelettique est un tissu dynamique ayant la capacité de réguler sa taille et son activité en réponse à différents stimuli extérieurs. Mon travail de thèse a principalement porté sur l étude d un récepteur nucléaire, le récepteur des glucocorticoïdes (GR) et de l un de ces co-régulateur, le facteur intermédiaire de transcription TIF2, dans ce tissu. Ainsi afin de mieux comprendre le rôle de GR et de TIF2 dans les myofibres, nous avons généré des souris dans lesquelles ils sont sélectivement invalidés dans le muscle squelettique de souris adultes (souris GR(i)skm-/- et TIF2(i)skm-/-).La première partie de ma thèse a démontré que l augmentation du découplage mitochondrial dans les myofibres protège les souris TIF2(i)skm-/- de la diminution des capacités oxydatives induite par la sédentarité, retarde le développement du diabète de type 2 et atténue la prise de poids induite par un régime hypercalorique. De plus, nos résultats démontrent que SRC-1 et TIF2 peuvent moduler l expression de la protéine découplante UCP3 de manière antagoniste, et que l augmentation des niveaux de SRC-1 dans les myofibres des souris TIF2(i)skm-/- est impliquée de manière critique dans la mise en place des changements métaboliques de ces souris.La seconde partie de ma thèse a montré que les souris GR(i)skm-/- ont une masse et une force musculaire plus importantes que des souris contrôles, du fait d une hyperactivation des voies anaboliques. Par ailleurs, ces animaux ne subissent pas l atrophie musculaire induite par un traitement à la dexaméthasone, un glucocorticoïde de synthèse, ou par une mise à jeun prolongée, montrant que la fonte musculaire est strictement contrôlée par GR dans les myofibres.Cette étude nous a permis d éclaircir les mécanismes moléculaires et cellulaires régulant l homéostasie musculaire et ouvriront de nouvelles voies dans le traitement des myopathies.Skeletal muscle is a dynamic tissue that has the capacity to regulate its size in response to a variety of external cues. My thesis work focused on the role of a nuclear receptor, the glucocorticoid receptor (GR) and of one of it co-regulator, the transcriptional intermediary factor TIF2, in this tissue. To improve our knowledge concerning the role of GR and TIF2 in myofibers, we generated mice in which GR or TIF2 are selectively ablated in skeletal muscle myofibers at adult stage (GR(i)skm-/- and TIF2(i)skm-/- mice).The first part of this work demonstrated that increased mitochondrial uncoupling in skeletal muscle myocytes protected these mice from decreased muscle oxidative capacities induced by sedentariness, delayed the development of type 2 diabetes and attenuated high caloric diet-induced obesity. Moreover, our results demonstrate that SRC-1 and TIF2 can modulate the expression of the uncoupling protein UCP3 in an antagonistic manner, and that enhanced SRC-1 levels in TIF2-deficient myofibers are critically involved in the metabolic changes of TIF2(i)skm-/- mice.The second part of this work demonstrated that GR(i)skm-/- mice skeletal muscle mass and strength were increased, due to anabolic pathway enhancement. Moreover, such mice are protected against dexamethasone-induced muscle catabolism and partially resistant to fasting-induced muscle atrophy, thus demonstrating that myofiber GR plays a major role in coordinating degradation of muscle proteins.This work highlighted molecular mechanisms regulating muscle homeostasis and should provide new insights in treatment of muscular disorders.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells

Satellite cells can differentiate both into myocytes and brown adipocytes. Here, the authors show that the histone demethylase Lsd1 prevents adipogenic differentiation of satellite cells by repressing expression of Glis1, and that its ablation changes satellite cell fate towards brown adipocytes and delays muscle regeneration in mice

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells

International audienceGenome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion, the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells

LSD1 promotes oxidative metabolism of white adipose tissue

Exposure to environmental cues such as cold or nutritional imbalance requires white adipose tissue (WAT) to adapt its metabolism to ensure survival. Metabolic plasticity is prominently exemplified by the enhancement of mitochondrial biogenesis in WAT in response to cold exposure or b3-adrenergic stimulation. Here we show that these stimuli increase the levels of lysine-specific demethylase 1 (LSD1) in WAT of mice and that elevated LSD1 levels induce mitochondrial activity. Genome-wide binding and transcriptome analyses demonstrate that LSD1 directly stimulates the expression of genes involved in oxidative phosphorylation (OXPHOS) in cooperation with nuclear respiratory factor 1 (Nrf1). In transgenic (Tg) mice, increased levels of LSD1 promote in a cell-autonomous manner the formation of islets of metabolically active brown-like adipocytes in WAT. Notably, Tg mice show limited weight gain when fed a high-fat diet. Taken together, our data establish LSD1 as a key regulator of OXPHOS and metabolic adaptation in WAT

Androgen receptor coordinates muscle metabolic and contractile functions

Abstract Background Androgens are anabolic steroid hormones that exert their function by binding to the androgen receptor (AR). We have previously established that AR deficiency in limb muscles impairs sarcomere myofibrillar organization and decreases muscle strength in male mice. However, despite numerous studies performed in men and rodents, the signalling pathways controlled by androgens via their receptor in skeletal muscles remain poorly understood. Methods Male ARskm−/y (n = 7–12) and female ARskm−/− mice (n = 9), in which AR is selectively ablated in myofibres of musculoskeletal tissue, and male AR(i)skm−/y, in which AR is selectively ablated in post‐mitotic skeletal muscle myofibres (n = 6), were generated. Longitudinal monitoring of body weight, blood glucose, insulin, lipids and lipoproteins was performed, alongside metabolomic analyses. Glucose metabolism was evaluated in C2C12 cells treated with 5α‐dihydrotestosterone (DHT) and the anti‐androgen flutamide (n = 6). Histological analyses on macroscopic and ultrastructural levels of longitudinal and transversal muscle sections were conducted. The transcriptome of gastrocnemius muscles from control and ARskm−/y mice was analysed at the age of 9 weeks (P < 0.05, 2138 differentially expressed genes) and validated by RT‐qPCR analysis. The AR (4691 peaks with false discovery rate [FDR] < 0.1) and H3K4me2 (47 225 peaks with FDR < 0.05) cistromes in limb muscles were determined in 11‐week‐old wild‐type mice. Results We show that disrupting the androgen/AR axis impairs in vivo glycolytic activity and fastens the development of type 2 diabetes in male, but not in female mice. In agreement, treatment with DHT increases glycolysis in C2C12 myotubes by 30%, whereas flutamide has an opposite effect. Fatty acids are less efficiently metabolized in skeletal muscles of ARskm−/y mice and accumulate in cytoplasm, despite increased transcript levels of genes encoding key enzymes of beta‐oxidation and mitochondrial content. Impaired glucose and fatty acid metabolism in AR‐deficient muscle fibres is associated with 30% increased lysine and branched‐chain amino acid catabolism, decreased polyamine biosynthesis and disrupted glutamate transamination. This metabolic switch generates ammonia (2‐fold increase) and oxidative stress (30% increased H2O2 levels), which impacts mitochondrial functions and causes necrosis in <1% fibres. We unravel that AR directly activates the transcription of genes involved in glycolysis, oxidative metabolism and muscle contraction. Conclusions Our study provides important insights into diseases caused by impaired AR function in musculoskeletal system and delivers a deeper understanding of skeletal muscle pathophysiological dynamics that is instrumental to develop effective treatment for muscle disorders