Hypercaloric low-carbohydrate high-fat diet protects against the development of nonalcoholic fatty liver disease in obese mice in contrast to isocaloric Western diet.

Objective: Obesity and metabolic complications, such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD), are one of the greatest public health challenges of the 21st century. The major role of high sugar and carbohydrate consumption rather than caloric intake in obesity and NAFLD pathophysiology remains a subject of debate. A low-carbohydrate but high-fat diet (LCHFD) has shown promising results in obesity management, but its effects in preventing NAFLD need to be detailed. This study aims to compare the effects of a LCHFD with a high-fat high-sugar obesogenic Western diet (WD) on the progression of obesity, type 2 diabetes, and nonalcoholic fatty liver disease. Methods: Male C57BL/6J mice were initially fed a WD for 10 weeks. Subsequently, they were either switched to a LCHFD or maintained on the WD for an additional 6 weeks. Hepatic effects of the diet were explored by histological staining and RT-qPCR. Results: After the initial 10 weeks WD feeding, LCHF diet demonstrated effectiveness in halting weight gain, maintaining a normal glucose tolerance and insulin levels, in comparison to the WD-fed mice, which developed obesity, glucose intolerance, increased insulin levels and induced NAFLD. In the liver, LCHFD mitigated the accumulation of hepatic triglycerides and the increase in Fasn relative gene expression compared to the WD mice. Beneficial effects of the LCHFD occurred despite a similar calorie intake compared to the WD mice. Conclusion: Our results emphasize the negative impact of a high sugar/carbohydrate and lipid association for obesity progression and NAFLD development. LCHFD has shown beneficial effects for NAFLD management, notably improving weight management, and maintaining a normal glucose tolerance and liver health.

LSD1 inhibition circumvents glucocorticoid-induced muscle wasting of male mice.

Synthetic glucocorticoids (GC), such as dexamethasone, are extensively used to treat chronic inflammation and autoimmune disorders. However, long-term treatments are limited by various side effects, including muscle atrophy. GC activities are mediated by the glucocorticoid receptor (GR), that regulates target gene expression in various tissues in association with cell-specific co-regulators. Here we show that GR and the lysine-specific demethylase 1 (LSD1) interact in myofibers of male mice, and that LSD1 connects GR-bound enhancers with NRF1-associated promoters to stimulate target gene expression. In addition, we unravel that LSD1 demethylase activity is required for triggering starvation- and dexamethasone-induced skeletal muscle proteolysis in collaboration with GR. Importantly, inhibition of LSD1 circumvents muscle wasting induced by pharmacological levels of dexamethasone, without affecting their anti-inflammatory activities. Thus, our findings provide mechanistic insights into the muscle-specific GC activities, and highlight the therapeutic potential of targeting GR co-regulators to limit corticotherapy-induced side effects.

Changes in Macronutrients during Dieting Lead to Weight Cycling and Metabolic Complications in Mouse Model.

Weight cycling is a major challenge in obesity management. Caloric restriction is known to promote this phenomenon, but the impact of macronutrient changes during dieting remains unclear. This study aimed to determine the role of macronutrient changes in weight maintenance without caloric restriction by alternating between two hypercaloric diets: a high-carbohydrate, high-fat Western diet (WD) and a low-carbohydrate, high-fat diet (LCHDF). Obesity was induced in 8-week-old C57BL/6 male mice by 10 weeks of WD feeding. Then, the mice were subjected to 12 weeks of LCHFD interspersed with WD (I-WD), 3 periods of 2-week LCHFD followed by 2 periods of 3-week WD, or 12 weeks of continuous WD (C-WD). C-WD and I-WD mice were compared to standard diet (SD) mice. In the I-WD group, each LCHFD period decreased weight gain, but mice regained weight after WD resumption. I-WD mice exhibited obesity, dyslipidemia, and glucose intolerance, similarly to the C-WD mice. I-WD mice also developed nonalcoholic steatohepatitis, associated with an increase in type-III collagen gene expression and a decrease in FGF21 protein levels, in comparison with SD. I-WD mice developed weight cycling despite maintaining a high caloric consumption, suggesting that changes in macronutrients during dieting are also a trigger of weight regain.

An overview on androgen-mediated actions in skeletal muscle and adipose tissue.

Androgens are a class of steroid hormones primarily associated with male sexual development and physiology, but exert pleiotropic effects in either sex. They have a crucial role in various physiological processes, including the regulation of skeletal muscle and adipose tissue homeostasis. The effects of androgens are mainly mediated through the androgen receptor (AR), a ligand-activated nuclear receptor expressed in both tissues. In skeletal muscle, androgens via AR exert a multitude of effects, ranging from increased muscle mass and strength, to the regulation of muscle fiber type composition, contraction and metabolic functions. In adipose tissue, androgens influence several processes including proliferation, fat distribution, and metabolism but they display depot-specific and organism-specific effects which differ in certain context. This review further explores the potential mechanisms underlying androgen-AR signaling in skeletal muscle and adipose tissue. Understanding the roles of androgens and their receptor in skeletal muscle and adipose tissue is essential for elucidating their contributions to physiological processes, disease conditions, and potential therapeutic interventions.

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

Genome-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.

Androgen receptor coordinates muscle metabolic and contractile functions.

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 H2 O2 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.

Myod1 and GR coordinate myofiber-specific transcriptional enhancers.

Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.

The histone acetyltransferase inhibitor Nir regulates epidermis development.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

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

Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

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 Spin1M5 mice with ablation of Spin1 in myoblast precursors using the Myf5-Cre deleter strain. Most Spin1M5 mice die shortly after birth displaying severe sarcomere disorganization and necrosis. Surviving Spin1M5 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 Spin1M5 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 Spin1M5 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.

Lsd1, a metabolic sensor of environment requirements that prevents adipose tissue from aging.

Understanding development and maintenance of beige adipocytes provide exciting insights in establishing novel therapies against obesity and obesity-associated disorders. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser required for differentiation and function of adipocytes. Lsd1 is involved in early commitment of preadipocytes, but dispensable for terminal differentiation of white adipose tissue (WAT). In mature adipocytes, Lsd1 responds to different environmental stimuli to alter metabolic function and enable proper thermogenic and oxidative response. Exposure to cold leads to Lsd1 upregulation and subsequent beiging of WAT. Oppositely, Lsd1 levels decline during aging resulting in a conversion of beige into white adipocytes, associated with loss of thermogenic properties of WAT. Lsd1 maintains beige adipocytes by controlling the expression of the nuclear receptor peroxisome proliferator-activated receptor α. In summary, our studies not only provided insights into the mechanism of age-related beige-to-white adipocyte transition, but also established Lsd1 as a sensor that enables thermogenic response in WAT.

Lsd1 prevents age-programed loss of beige adipocytes.

Aging is accompanied by major changes in adipose tissue distribution and function. In particular, with time, thermogenic-competent beige adipocytes progressively gain a white adipocyte morphology. However, the mechanisms controlling the age-related transition of beige adipocytes to white adipocytes remain unclear. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser enzyme positively regulating differentiation and function of adipocytes. Here we show that Lsd1 levels decrease in aging inguinal white adipose tissue concomitantly with beige fat cell decline. Accordingly, adipocyte-specific increase of Lsd1 expression is sufficient to rescue the age-related transition of beige adipocytes to white adipocytes in vivo, whereas loss of Lsd1 precipitates it. Lsd1 maintains beige adipocytes by controlling the expression of peroxisome proliferator-activated receptor α (Ppara), and treatment with a Ppara agonist is sufficient to rescue the loss of beige adipocytes caused by Lsd1 ablation. In summary, our data provide insights into the mechanism controlling the age-related beige-to-white adipocyte transition and identify Lsd1 as a regulator of beige fat cell maintenance.

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.

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 ß3-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.

The transcriptional coregulators TIF2 and SRC-1 regulate energy homeostasis by modulating mitochondrial respiration in skeletal muscles.

The two p160 transcriptional coregulator family members SRC-1 and TIF2 have important metabolic functions in white and brown adipose tissues as well as in the liver. To analyze TIF2 cell-autonomous functions in skeletal muscles, we generated TIF2((i)skm)⁻(/)⁻ mice in which TIF2 was selectively ablated in skeletal muscle myofibers at adulthood. We found 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 3 (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. Thus, modulation of the expression and/or activity of these coregulators represents an attractive way to prevent or treat metabolic disorders.

Myocytic androgen receptor controls the strength but not the mass of limb muscles.

The anabolic effects of androgens on skeletal muscles are thought to be mediated predominantly through the androgen receptor (AR), a member of the ligand-dependent nuclear receptor superfamily. However, despite numerous studies performed in men and in rodents, these effects remain poorly understood. To characterize androgen signaling in skeletal muscles, we generated mice in which the AR is selectively ablated in myofibers. We show that myocytic AR controls androgen-induced insulin-like growth factor IEa (IGF-IEa) expression in the highly androgen-sensitive perineal muscles and that it mediates androgen-stimulated postnatal hypertrophy of these muscles. In contrast, androgen-dependent postnatal hypertrophy of limb muscle fibers is independent of myocytic AR. Thus, androgens control perineal and limb muscle mass in male mice through myocytic AR-dependent and -independent pathways, respectively. Importantly, we also show that AR deficiency in limb myocytes impairs myofibrillar organization of sarcomeres and decreases muscle strength, thus demonstrating that myocytic AR controls key pathways required for maximum force production. These distinct androgen signaling pathways in perineal and limb muscles may allow the design of screens to identify selective androgen modulators of muscle strength.

PGC1alpha expression is controlled in skeletal muscles by PPARbeta, whose ablation results in fiber-type switching, obesity, and type 2 diabetes.

Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.