Rev-erb-? modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy.
ABSTRACT: The nuclear receptor Rev-erb-? modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-? is highly expressed in oxidative skeletal muscle and that its deficiency in muscle leads to reduced mitochondrial content and oxidative function, as well as upregulation of autophagy. These cellular effects resulted in both impaired mitochondrial biogenesis and increased clearance of this organelle, leading to compromised exercise capacity. On a molecular level, Rev-erb-? deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1? signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-?, Nr1d1. In complementary experiments, Rev-erb-? overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-? in vivo increased exercise capacity. This study identifies Rev-erb-? as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.
Project description:The nuclear receptor Rev-erb-? modulates hepatic lipid and glucose metabolism, adipogenesis and thermogenesis. We have previously demonstrated that Rev-erb-? is also an important regulator of skeletal muscle mitochondrial biogenesis and function, and autophagy. As such, Rev-erb-? over-expression in skeletal muscle or its pharmacological activation improved mitochondrial respiration and enhanced exercise capacity. Here, in gain- and loss-of function studies, we show that Rev-erb-? also controls muscle mass. Rev-erb-?-deficiency in skeletal muscle leads to increased expression of the atrophy-related genes (atrogenes), associated with reduced muscle mass and decreased fiber size. By contrast, in vivo and in vitro Rev-erb-? over-expression results in reduced atrogenes expression and increased fiber size. Finally, Rev-erb-? pharmacological activation blocks dexamethasone-induced upregulation of atrogenes and muscle atrophy. This study identifies Rev-erb-? as a promising pharmacological target to preserve muscle mass.
Project description:The nuclear receptors REV-ERB? and REV-ERB? have been demonstrated to be core members of the circadian clock and participate in the regulation of a diverse set of metabolic functions. Due to their overlapping tissue expression patterns and gene expression profiles, REV-ERB? is thought to be redundant to REV-ERB?. Recent work has highlighted REV-ERB?'s role in the regulation of skeletal muscle oxidative capacity and mitochondrial biogenesis. Considering the similarity between the REV-ERBs and the hypothesized overlap in function, we sought to determine whether REV-ERB?-deficiency presented with a similar skeletal muscle phenotype as REV-ERB?-deficiency. Ectopic overexpression in C2C12 cells demonstrated that REV-ERB? drives mitochondrial biogenesis and the expression of genes involved in fatty acid oxidation. Intriguingly, knock down of REV-ERB? in C2C12 cultures also resulted in mitochondrial biogenesis and increased expression of genes involved in fatty acid ?-oxidation. To determine whether these effects occurred in vivo, we examined REV-ERB?-deficient mice and observed a similar increase in expression of genes involved in mitochondrial biogenesis and fatty acid ?-oxidation. Consistent with these results, REV-ERB?-deficient mice exhibited an altered metabolic phenotype compared to wild-type littermate controls when measured by indirect calorimetry. This likely compensated for the increased food consumption that occurred, possibly aiding in the maintenance of their weight over time. Since feeding behaviors are a direct circadian output, this study suggests that REV-ERB? may have more subtle effects on circadian behaviors than originally identified. Furthermore, these data implicate REV-ERB? in the control of skeletal muscle metabolism and energy expenditure and suggest that development of REV-ERB? versus REV-ERB? selective ligands may have therapeutic utility in the treatment of metabolic syndrome.
Project description:Numerous mutational studies have demonstrated that circadian clock proteins regulate behavior and metabolism. Nr1d1(Rev-erb?) is a key regulator of circadian gene expression and a pleiotropic regulator of skeletal muscle homeostasis and lipid metabolism. Loss of Rev-erb? expression induces muscular atrophy, high adiposity, and metabolic syndrome in mice. Here we show that, unlike knockout mice, Nr1d1 heterozygous mice are not susceptible to muscular atrophy and in fact paradoxically possess larger myofiber diameters and improved neuromuscular function, compared to wildtype mice. Heterozygous mice lacked dyslipidemia, a characteristic of Nr1d1 knockout mice and displayed increased whole-body fatty-acid oxidation during periods of inactivity (light cycle). Heterozygous mice also exhibited higher rates of glucose uptake when fasted, and had elevated basal rates of gluconeogenesis compared to wildtype and knockout littermates. Rev-erb? ablation suppressed glycolysis and fatty acid-oxidation in white-adipose tissue (WAT), whereas partial Rev-erb? loss, curiously stimulated these processes. Our investigations revealed that Rev-erb? dose-dependently regulates glucose metabolism and fatty acid oxidation in WAT and muscle.
Project description:Diurnal oscillations in the expression of antioxidant genes imply that protection against oxidative stress is circadian-gated. We hypothesized that stabilization of the core circadian gene Rev-erb? (Nr1d1) improves cellular bioenergetics and protects against nutrient deprivation and oxidative stress. Compared to WT, mouse lung fibroblasts (MLG) stably transfected with a degradation resistant Rev-erb? (Ser(55/59) to Asp; hence referred to as SD) had 40% higher protein content, 1.5-fold higher mitochondrial area (confocal microscopy), doubled oxidative phosphorylation by high-resolution respirometry (Oroboros) and were resistant to glucose deprivation for 24h. This resulted from a 4-fold reduction in mitophagy (L3CB co-localized with MitoTracker Red) versus WT. Although PGC1? protein expression was comparable between SD and WT MLG cells, the role of mitochondrial biogenesis in explaining increased mitochondrial mass in SD cells was less clear. Embryonic fibroblasts (MEF) from C57Bl/6-SD transgenic mice, had a 9-fold induction of FoxO1 mRNA and increased mRNA of downstream antioxidant targets heme oxygenase-1 (HO-1), Mn superoxide dismutase and catalase (1.5, 2 fold and 2 fold respectively) versus WT. This allowed the SD cells to survive 1h incubation with 500 µM H2O2 as well as 24h of exposure to 95% O2 and remain attached whereas most WT cells did not. These observations establish a mechanistic link between the metabolic functions of Rev-erb? with mitochondrial homeostasis and protection against oxidative stress.
Project description:Rev-erb? is a ligand-dependent nuclear receptor and a key repressor of the molecular clock transcription network. Accumulating evidence indicate that the circadian clock machinery governs diverse biological processes in skeletal muscle, including muscle growth, repair and mass maintenance. The physiological function of Rev-erb? in myogenic regulation remains largely unknown. Here we show that Rev-erb? exerts cell-autonomous inhibitory effects on proliferation and differentiation of myogenic precursor cells, and these actions concertedly inhibit muscle regeneration in vivo. Mechanistic studies reveal Rev-erb? direct transcriptional control of two major myogenic mechanisms, proliferative pathway and the Wnt signaling cascade. Consistent with this finding, primary myoblasts lacking Rev-erb? display significantly enhanced proliferative growth and myogenic progression. Furthermore, pharmacological activation of Rev-erb? activity attenuates, whereas its inhibition by an antagonist promotes these processes. Notably, upon muscle injury, the loss-of-function of Rev-erb? in vivo augmented satellite cell proliferative expansion and regenerative progression during regeneration. Collectively, our study identifies Rev-erb? as a novel inhibitory regulator of myogenic progenitor cell properties that suppresses postnatal myogenesis. Pharmacological interventions to dampen Rev-erb? activity may have potential utilities to enhance regenerative capacity in muscle diseases.
Project description:Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERB? (NR1D1) and REV-ERB? (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERB? ligand-binding domain (LBD). We solved two crystal structures of REV-ERB? LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.
Project description:Several investigations suggested abnormalities in circadian rhythms are related to the pathophysiology of psychiatric disorders, including drug addiction. Recently, orphan nuclear receptor rev-erb alpha and glycogen synthase kinase-3 ? (GSK-3?) were shown to be important circadian components. In addition, the orphan nuclear receptor rev-erb alpha is a key negative feedback regulator of the circadian clock. These evidences indicate that rev-erb alpha gene (NR1D1) is a good candidate gene for the pathogenesis of methamphetamine dependence. To evaluate the association between NR1D1 and methamphetamine dependence, we conducted a case-control study of Japanese samples (215 methamphetamine dependence and 232 controls) with three tagging SNPs selected by HapMap database. Written informed consent was obtained from each subject. This study was approved by the ethics committees at Fujita Health University, Nagoya University Graduate School of Medicine and each participating member of the Institute of the Japanese Genetics Initiative for Drug Abuse (JGIDA). We did not detect an association between NR1D1 and Japanese methamphetamine dependence patients in allele/genotype-wise analysis, or the haplotype analysis. Our findings suggest that NR1D1 does not play a major role in the pathophysiology of methamphetamine dependence in the Japanese population.
Project description:Circadian oscillation of body temperature is a basic, evolutionarily conserved feature of mammalian biology. In addition, homeostatic pathways allow organisms to protect their core temperatures in response to cold exposure. However, the mechanism responsible for coordinating daily body temperature rhythm and adaptability to environmental challenges is unknown. Here we show that the nuclear receptor Rev-erb? (also known as Nr1d1), a powerful transcriptional repressor, links circadian and thermogenic networks through the regulation of brown adipose tissue (BAT) function. Mice exposed to cold fare considerably better at 05:00 (Zeitgeber time?22) when Rev-erb? is barely expressed than at 17:00 (Zeitgeber time?10) when Rev-erb? is abundant. Deletion of Rev-erb? markedly improves cold tolerance at 17:00, indicating that overcoming Rev-erb?-dependent repression is a fundamental feature of the thermogenic response to cold. Physiological induction of uncoupling protein 1 (Ucp1) by cold temperatures is preceded by rapid downregulation of Rev-erb? in BAT. Rev-erb? represses Ucp1 in a brown-adipose-cell-autonomous manner and BAT Ucp1 levels are high in Rev-erb?-null mice, even at thermoneutrality. Genetic loss of Rev-erb? also abolishes normal rhythms of body temperature and BAT activity. Thus, Rev-erb? acts as a thermogenic focal point required for establishing and maintaining body temperature rhythm in a manner that is adaptable to environmental demands.
Project description:T helper 17 (Th17) cells produce interleukin-17 (IL-17) cytokines and drive inflammatory responses in autoimmune diseases such as multiple sclerosis. The differentiation of Th17 cells is dependent on the retinoic acid receptor-related orphan nuclear receptor ROR?t. Here, we identify REV-ERB? (encoded by Nr1d1), a member of the nuclear hormone receptor family, as a transcriptional repressor that antagonizes ROR?t function in Th17 cells. REV-ERB? binds to ROR response elements (RORE) in Th17 cells and inhibits the expression of ROR?t-dependent genes including Il17a and Il17f Furthermore, elevated REV-ERB? expression or treatment with a synthetic REV-ERB agonist significantly delays the onset and impedes the progression of experimental autoimmune encephalomyelitis (EAE). These results suggest that modulating REV-ERB? activity may be used to manipulate Th17 cells in autoimmune diseases.
Project description:REV-ERB? (NR1D1) is a circadian clock component that functions as a transcriptional repressor. Due to its role in direct modulation of metabolic genes, REV-ERB? is regarded as an integrator of cell metabolism with circadian clock. Accordingly, REV-ERB? is first proposed as a drug target for treating sleep disorders and metabolic syndromes (e.g., dyslipidaemia, hyperglycaemia and obesity). Recent years of studies uncover a rather broad role of REV-ERB? in pathological conditions including local inflammatory diseases, heart failure and cancers. Moreover, REV-ERB? is involved in regulation of circadian drug metabolism that has implications in chronopharmacology. In the meantime, recent years have witnessed discovery of an array of new REV-ERB? ligands most of which have pharmacological activities in vivo. In this article, we review the regulatory role of REV-ERB? in various types of diseases and discuss the underlying mechanisms. We also describe the newly discovered ligands and the old ones together with their targeting potential. Despite well-established pharmacological effects of REV-ERB? ligands in animals (preclinical studies), no progress has been made regarding their translation to clinical trials. This implies certain challenges associated with drug development of REV-ERB? ligands. In particular, we discuss the potential challenges related to drug safety (or adverse effects) and bioavailability. For new drug development, it is advocated that REV-ERB? should be targeted to treat local diseases and a targeting drug should be locally distributed, avoiding the adverse effects on other tissues.