Exercise and Ischemia-Activated Pathways in Limb Muscle Angiogenesis and Vascular Regeneration.

Exercise has a profound effect on cardiovascular disease, particularly through vascular remodeling and regeneration. Peripheral artery disease (PAD) is one such cardiovascular condition that benefits from regular exercise or rehabilitative physical therapy in terms of slowing the progression of disease and delaying amputations. Various rodent pre-clinical studies using models of PAD and exercise have shed light on molecular pathways of vascular regeneration. Here, I review key exercise-activated signaling pathways (nuclear receptors, kinases, and hypoxia inducible factors) in the skeletal muscle that drive paracrine regenerative angiogenesis. The rationale for highlighting the skeletal muscle is that it is the largest organ recruited during exercise. During exercise, skeletal muscle releases several myokines, including angiogenic factors and cytokines that drive tissue vascular regeneration via activation of endothelial cells, as well as by recruiting immune and endothelial progenitor cells. Some of these core exercise-activated pathways can be extrapolated to vascular regeneration in other organs. I also highlight future areas of exercise research (including metabolomics, single cell transcriptomics, and extracellular vesicle biology) to advance our understanding of how exercise induces vascular regeneration at the molecular level, and propose the idea of "exercise-mimicking" therapeutics for vascular recovery.

Estrogen-Related Receptor Gamma Gene Therapy Promotes Therapeutic Angiogenesis and Muscle Recovery in Preclinical Model of PAD.

Background Peripheral arterial disease and critical limb ischemia are cardiovascular complications associated with vascular insufficiency, oxidative metabolic dysfunction, and myopathy in the limbs. Estrogen-related receptor gamma (ERRγ) has emerged as a dual regulator of paracrine angiogenesis and oxidative metabolism through transgenic mouse studies. Here our objective was to investigate whether postischemic intramuscular targeting of ERRγ via gene therapy promotes ischemic recovery in a preclinical model of peripheral arterial disease/critical limb ischemia. Methods and Results Adeno-associated virus 9 (AAV9) Esrrg gene delivery vector was developed and first tested via intramuscular injection in murine skeletal muscle. AAV9-Esrrg robustly increased ERRγ protein expression, induced angiogenic and oxidative genes, and boosted capillary density and succinate dehydrogenase oxidative metabolic activity in skeletal muscles of C57Bl/6J mice. Next, hindlimb ischemia was induced via unilateral femoral vessel ligation in mice, followed by intramuscular AAV9-Esrrg (or AAV9-green fluorescent protein) gene delivery 24 hours after injury. ERRγ overexpression increased ischemic neoangiogenesis and markers of endothelial activation, and significantly improved ischemic revascularization measured using laser Doppler flowmetry. Moreover, ERRγ overexpression restored succinate dehydrogenase oxidative metabolic capacity in ischemic muscle, which correlated with increased mitochondrial respiratory complex protein expression. Most importantly, myofiber size to number quantification revealed that AAV9-Esrrg restores myofibrillar size and mitigates ischemia-induced myopathy. Conclusions These results demonstrate that intramuscular AAV9-Esrrg delivery rescues ischemic pathology after hindlimb ischemia, underscoring that Esrrg gene therapy or pharmacological activation could be a promising strategy for the management of peripheral arterial disease/critical limb ischemia.

Novel Potential Targets for Function-Promoting Therapies: Orphan Nuclear Receptors, Anti-inflammatory Drugs, Troponin Activators, Mas Receptor Agonists, and Urolithin A.

In recent years, several new classes of therapies have been investigated with their potential for restoring or improving physical functioning in older adults. These have included Mas receptor agonists, regulators of mitophagy, skeletal muscle troponin activators, anti-inflammatory compounds, and targets of orphan nuclear receptors. The present article summarizes recent developments of the function-promoting effects of these exciting new compounds and shares relevant preclinical and clinical data related to their safety and efficacy. The development of novel compounds in this area is expanding and likely will need the advent of a new treatment paradigm for age-associated mobility loss and disability.

Estrogen-related Receptor Signaling in Skeletal Muscle Fitness.

Skeletal muscle is a highly plastic tissue that can alter its metabolic and contractile features, as well as regenerative potential in response to exercise and other conditions. Multiple signaling factors including metabolites, kinases, receptors, and transcriptional factors have been studied in the regulation of skeletal muscle plasticity. Recently, estrogen-related receptors (ERRs) have emerged as a critical transcriptional hub in control of skeletal muscle homeostasis. ERRα and ERRγ - the two highly expressed ERR sub-types in the muscle respond to various extracellular cues such as exercise, hypoxia, fasting and dietary factors, in turn regulating gene expression in the skeletal muscle. On the other hand, conditions such as diabetes and muscular dystrophy suppress expression of ERRs in the skeletal muscle, likely contributing to disease progression. We highlight key functions of ERRs in the skeletal muscle including the regulation of fiber type, mitochondrial metabolism, vascularization, and regeneration. We also describe how ERRs are regulated in the skeletal muscle, and their interaction with important muscle regulators (e. g. AMPK and PGCs). Finally, we identify critical gaps in our understanding of ERR signaling in the skeletal muscle, and suggest future areas of investigation to advance ERRs as potential targets for function promoting therapeutics in muscle diseases.

Emerging role of TAK1 in the regulation of skeletal muscle mass.

Maintenance of skeletal muscle mass and strength throughout life is crucial for heathy living and longevity. Several signaling pathways have been implicated in the regulation of skeletal muscle mass in adults. TGF-ß-activated kinase 1 (TAK1) is a key protein, which coordinates the activation of multiple signaling pathways. Recently, it was discovered that TAK1 is essential for the maintenance of skeletal muscle mass and myofiber hypertrophy following mechanical overload. Forced activation of TAK1 in skeletal muscle causes hypertrophy and attenuates denervation-induced muscle atrophy. TAK1-mediated signaling in skeletal muscle promotes protein synthesis, redox homeostasis, mitochondrial health, and integrity of neuromuscular junctions. In this article, we have reviewed the role and potential mechanisms through which TAK1 regulates skeletal muscle mass and growth. We have also proposed future areas of research that could be instrumental in exploring TAK1 as therapeutic target for improving muscle mass in various catabolic conditions and diseases.

Innately expressed estrogen-related receptors in the skeletal muscle are indispensable for exercise fitness.

Transcriptional determinants in the skeletal muscle that govern exercise capacity, while poorly defined, could provide molecular insights into how exercise improves fitness. Here, we have elucidated the role of nuclear receptors, estrogen-related receptor alpha and gamma (ERRα/γ) in regulating myofibrillar composition, contractility, and exercise capacity in skeletal muscle. We used muscle-specific single or double (DKO) ERRα/γ knockout mice to investigate the effect of ERRα/γ deletion on muscle and exercise parameters. Individual knockout of ERRα/γ did not have a significant impact on the skeletal muscle. On the other hand, DKO mice exhibit pale muscles compared to wild-type (WT) littermates. RNA-seq analysis revealed a predominant decrease in expression of genes linked to mitochondrial and oxidative metabolism in DKO versus WT muscles. DKO muscles exhibit marked repression of oxidative enzymatic capacity, as well as mitochondrial number and size compared to WT muscles. Mitochondrial function is also impaired in single myofibers isolated from DKO versus WT muscles. In addition, mutant muscles exhibit reduced angiogenic gene expression and decreased capillarity. Consequently, DKO mice have a significantly reduced exercise capacity, further reflected in poor fatigue resistance of DKO mice in in vivo contraction assays. These results show that ERRα and ERRγ together are a critical link between muscle aerobic capacity and exercise tolerance. The ERRα/γ mutant mice could be valuable for understanding the long-term impact of impaired mitochondria and vascular supply on the pathogenesis of muscle-linked disorders.

Nuclear receptors as potential therapeutic targets in peripheral arterial disease and related myopathy.

Peripheral arterial disease (PAD) is a prevalent cardiovascular complication of limb vascular insufficiency, causing ischemic injury, mitochondrial metabolic damage and functional impairment in the skeletal muscle, and ultimately leading to immobility and mortality. While potential therapies have been mostly focussed on revascularization, none of the currently available pharmacological treatments are fully effective in PAD, often leading to amputations, particularly in chronic metabolic diseases. One major limitation of focussed angiogenesis and revascularization as a therapeutic strategy is a limited effect on metabolic restoration and muscle regeneration in the affected limb. Therefore, additional preclinical investigations are needed to discover novel treatment options for PAD preferably targeting multiple aspects of muscle recovery. In this review, we propose nuclear receptors expressed in the skeletal muscle as potential candidates for ischemic muscle repair in PAD. We review classic steroid and orphan receptors that have been reported to be involved in the regulation of paracrine muscle angiogenesis, oxidative metabolism, mitochondrial biogenesis and muscle regeneration, and discuss how these receptors could be critical for recovery from ischemic muscle damage. Furthermore, we identify existing gaps in our understanding of nuclear receptor signalling in the skeletal muscle and propose future areas of research that could be instrumental in exploring nuclear receptors as therapeutic candidates for treating PAD.

Targeted ablation of Fn14 receptor improves exercise capacity and inhibits neurogenic muscle atrophy.

Skeletal muscle atrophy is a prevalent complication in multiple chronic diseases and disuse conditions. Fibroblast growth factor-inducible 14 (Fn14) is a member of the TNF receptor superfamily and a bona fide receptor of the TWEAK cytokine. Accumulating evidence suggests that Fn14 levels are increased in catabolic conditions as well as during exercise. However, the role of Fn14 in the regulation of skeletal muscle mass and function remains poorly understood. In this study, through the generation of novel skeletal muscle-specific Fn14-knockout mice, we have investigated the muscle role of Fn14 in the regulation of exercise capacity and denervation-induced muscle atrophy. Our results demonstrate that there was no difference in skeletal muscle mass between control and muscle-specific Fn14-knockout mice. Nevertheless, the deletion of Fn14 in skeletal muscle significantly improved exercise capacity and resistance to fatigue. This effect of Fn14 deletion is associated with an increased proportion of oxidative myofibers and higher capillaries number per myofiber in skeletal muscle. Furthermore, our results demonstrate that targeted deletion of Fn14 inhibits denervation-induced muscle atrophy in adult mice. Deletion of Fn14 reduced the expression of components of the ubiquitin-proteasome system and non-canonical NF-kappa B signaling in denervated skeletal muscle, as well as increased the phosphorylation of Akt kinase and FoxO3a transcription factor. Collectively, our results demonstrate that targeted inhibition of Fn14 improves exercise tolerance and inhibits denervation-induced muscle atrophy in adult mice.

Estrogen-related receptor alpha is an AMPK-regulated factor that promotes ischemic muscle revascularization and recovery in diet-induced obese mice.

Obesity and type II diabetes are leading causes of peripheral arterial disease (PAD), which is characterized by vascular insufficiency and ischemic damage in the limb skeletal muscle. Glycemic control is not sufficient to prevent progression of PAD, and molecular targets that can promote muscle neo-angiogenesis in obesity and diabetes remain poorly defined. Here, we have investigated whether nuclear receptor estrogen-related receptor alpha (ERRα) can promote ischemic revascularization in the skeletal muscles of diet-induced obese (DIO) mice. Using muscle-specific ERRα transgenic mice, we found that ERRα overexpression promotes revascularization, marked by increased capillary staining and muscle perfusion in DIO mice after hindlimb ischemic injury. Furthermore, ERRα facilitates repair and restoration of skeletal muscle myofiber size after limb ischemia in DIO mice. The ameliorative effects of ERRα overexpression did not involve the prevention of weight gain, hyperglycemia or glucose/insulin intolerance, suggesting a direct role for ERRα in promoting angiogenesis. Interestingly, levels of endogenous ERRα protein are suppressed in the skeletal muscles of DIO mice compared to lean controls, coinciding with the suppression of angiogenic gene expression, and reduced AMPK signaling in the DIO skeletal muscles. Upon further investigating the link between AMPK and ERRα, we found that AMPK activation increases the expression and recruitment of ERRα protein to specific angiogenic gene promoters in muscle cells. Further, the induction of angiogenic factors by AMPK activators in muscle cells is blocked by repressing ERRα. In summary, our results identify an AMPK/ERRα-dependent angiogenic gene program in the skeletal muscle, which is repressed by DIO, and demonstrate that forced ERRα activation can promote ischemic revascularization and muscle recovery in obesity.

Estrogen-related receptor α is involved in angiogenesis and skeletal muscle revascularization in hindlimb ischemia.

Skeletal muscle ischemia is a major consequence of peripheral arterial disease (PAD) or critical limb ischemia (CLI). Although therapeutic options for resolving muscle ischemia in PAD/CLI are limited, the issue is compounded by poor understanding of the mechanisms driving muscle vascularization. We found that nuclear receptor estrogen-related receptor alpha (ERRα) expression is induced in murine skeletal muscle by hindlimb ischemia (HLI), and in cultured myotubes by hypoxia, suggesting a potential role for ERRα in ischemic response. To test this, we generated skeletal muscle-specific ERRα transgenic (TG) mice. In these mice, ERRα drives myofiber type switch from glycolytic type IIB to oxidative type IIA/IIX myofibers, which are typically associated with more vascular supply in muscle. Indeed, RNA sequencing and functional enrichment analysis of TG muscle revealed that "paracrine angiogenesis" is the top-ranked transcriptional program activated by ERRα in the skeletal muscle. Immunohistochemistry and angiography showed that ERRα overexpression increases baseline capillarity, arterioles and non-leaky blood vessel formation in the skeletal muscles. Moreover, ERRα overexpression facilitates ischemic neo-angiogenesis and perfusion recovery in hindlimb musculature of mice subjected to HLI. Therefore, ERRα is a hypoxia inducible nuclear receptor that is involved in skeletal muscle angiogenesis and could be potentially targeted for treating PAD/CLI.

TAK1 preserves skeletal muscle mass and mitochondrial function through redox homeostasis.

Skeletal muscle atrophy is debilitating consequence of a large number of chronic disease states, aging, and disuse conditions. Skeletal muscle mass is regulated through coordinated activation of a number of signaling cascades. Transforming growth factor-ß activated kinase 1 (TAK1) is a central kinase that mediates the activation of multiple signaling pathways in response to various growth factors, cytokines, and microbial products. Accumulating evidence suggests that TAK1 promotes skeletal muscle growth and essential for the maintenance of muscle mass in adults. Targeted inactivation of TAK1 leads to severe muscle wasting and kyphosis in mice. However, the mechanisms by which TAK1 prevents loss of muscle mass remain poorly understood. Through generation of inducible skeletal muscle-specific Tak1-knockout mice, we demonstrate that targeted ablation of TAK1 disrupts redox signaling leading to the accumulation of reactive oxygen species and loss of skeletal muscle mass and contractile function. Suppression of oxidative stress using Trolox improves muscle contractile function and inhibits the activation of catabolic signaling pathways in Tak1-deficient muscle. Moreover, Trolox inhibits the activation of ubiquitin-proteasome system and autophagy markers in skeletal muscle of Tak1-deficient mice. Furthermore, inhibition of oxidative stress using Trolox prevents the slow-to-fast type fiber transition and improves mitochondrial respiration in skeletal muscle of Tak1-deficient mice. Overall, our results demonstrate that TAK1 maintains skeletal muscle mass and health through redox homeostasis.

Regulation of Lin28a-miRNA let-7b-5p pathway in skeletal muscle cells by peroxisome proliferator-activated receptor delta.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

Interleukin-13 drives metabolic conditioning of muscle to endurance exercise.

Repeated bouts of exercise condition muscle mitochondria to meet increased energy demand-an adaptive response associated with improved metabolic fitness. We found that the type 2 cytokine interleukin-13 (IL-13) is induced in exercising muscle, where it orchestrates metabolic reprogramming that preserves glycogen in favor of fatty acid oxidation and mitochondrial respiration. Exercise training-mediated mitochondrial biogenesis, running endurance, and beneficial glycemic effects were lost in Il13-/- mice. By contrast, enhanced muscle IL-13 signaling was sufficient to increase running distance, glucose tolerance, and mitochondrial activity similar to the effects of exercise training. In muscle, IL-13 acts through both its receptor IL-13Rα1 and the transcription factor Stat3. The genetic ablation of either of these downstream effectors reduced running capacity in mice. Thus, coordinated immunological and physiological responses mediate exercise-elicited metabolic adaptations that maximize muscle fuel economy.

A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity.

Ubiquitin-conjugating enzyme E2O (UBE2O) is expressed preferentially in metabolic tissues, but its role in regulating energy homeostasis has yet to be defined. Here we find that UBE2O is markedly upregulated in obese subjects with type 2 diabetes and show that whole-body disruption of Ube2o in mouse models in vivo results in improved metabolic profiles and resistance to high-fat diet-induced (HFD-induced) obesity and metabolic syndrome. With no difference in nutrient intake, Ube2o-/- mice were leaner and expended more energy than WT mice. In addition, hyperinsulinemic-euglycemic clamp studies revealed that Ube2o-/- mice were profoundly insulin sensitive. Through phenotype analysis of HFD mice with muscle-, fat-, or liver-specific knockout of Ube2o, we further identified UBE2O as an essential regulator of glucose and lipid metabolism programs in skeletal muscle, but not in adipose or liver tissue. Mechanistically, UBE2O acted as a ubiquitin ligase and targeted AMPKα2 for ubiquitin-dependent degradation in skeletal muscle; further, muscle-specific heterozygous knockout of Prkaa2 ablated UBE2O-controlled metabolic processes. These results identify the UBE2O/AMPKα2 axis as both a potent regulator of metabolic homeostasis in skeletal muscle and a therapeutic target in the treatment of diabetes and metabolic disorders.

Loss of Estrogen-Related Receptor Alpha Facilitates Angiogenesis in Endothelial Cells.

Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here, we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multifaceted role in vasculature. Of the three ERR subtypes, ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration, and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation but increased migration and tube formation. ERRα depletion increased basal as well as vascular endothelial growth factor A (VEGFA)- and ANG1/2-stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to that in wild-type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets, such as metabolism, mitochondrial biogenesis, and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory, and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, proangiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis and potentially fine-tunes growth factor-mediated angiogenesis.

GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer.

PURPOSE: G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC. METHODS: Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells. RESULTS: Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells. CONCLUSION: Our data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Long-term PGC1ß overexpression leads to apoptosis, autophagy and muscle wasting.

Skeletal muscle wasting is prevalent in many chronic diseases, necessitating inquiries into molecular regulation of muscle mass. Nuclear receptor co-activator peroxisome proliferator-activated receptor co-activator 1 alpha (PGC1α) and its splice variant PGC1α4 increase skeletal muscle mass. However, the effect of the other PGC1 sub-type, PGC1ß, on muscle size is unclear. In transgenic mice selectively over-expressing PGC1ß in the skeletal muscle, we have found that PGC1ß progressively decreases skeletal muscle mass predominantly associated with loss of type 2b fast-twitch myofibers. Paradoxically, PGC1ß represses the ubiquitin-proteolysis degradation pathway genes resulting in ubiquitinated protein accumulation in muscle. However, PGC1ß overexpression triggers up-regulation of apoptosis and autophagy genes, resulting in robust activation of these cell degenerative processes, and a concomitant increase in muscle protein oxidation. Concurrently, PGC1ß up-regulates apoptosis and/or autophagy transcriptional factors such as E2f1, Atf3, Stat1, and Stat3, which may be facilitating myopathy. Therefore, PGC1ß activation negatively affects muscle mass over time, particularly fast-twitch muscles, which should be taken into consideration along with its known aerobic effects in the skeletal muscle.

Muscle Arnt/Hif1ß Is Dispensable in Myofiber Type Determination, Vascularization and Insulin Sensitivity.

Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1ß), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance.