NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression.

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase-dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation.

Browning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs.

The LPS/D-Galactosamine-Induced Fulminant Hepatitis Model to Assess the Role of Ligand-Activated Nuclear Receptors on the NLRP3 Inflammasome Pathway In Vivo.

The NLRP3 inflammasome is a cellular sensor of danger signals such as extracellular ATP or abnormally accumulating molecules like crystals. Activation of NLRP3 by such compounds triggers a sterile inflammatory response that may be involved in numerous pathologies including rheumatoid arthritis, atherosclerosis, diabetes, and Alzheimer's disease. A better understanding of the mechanisms that govern NLRP3 inflammasome activation is an important step toward the development of novel therapeutic strategies to dampen over-activation of the immune system. Recent findings demonstrate that ligand-activated nuclear receptors regulate the NLRP3 inflammasome pathway, thus representing possible therapeutic targets. It is therefore important to assess the potential of these putative targets in the regulation of the NLRP3 inflammasome activation in the most appropriate pathophysiological models. Fulminant hepatitis (FH) results from massive hepatocyte apoptosis, hemorrhagic necrosis, and inflammation. Low doses of LPS in combination with the specific hepatotoxic agent D-galactosamine (D-GalN) promote liver injury in mice and induce the production of inflammatory cytokines associated with increased NLRP3 protein and caspase 1 activity, thus recapitulating the clinical picture of FH in humans. We provide a simple method to examine the involvement of nuclear receptors in NLRP3-driven fulminant hepatitis, consisting in the induction of FH, in the isolation of liver macrophages, and in the extraction and analysis of RNA content.

Nuclear Receptor Subfamily 1 Group D Member 1 Regulates Circadian Activity of NLRP3 Inflammasome to Reduce the Severity of Fulminant Hepatitis in Mice.

BACKGROUND & AIMS: The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erbα) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway. METHODS: We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1-/- mice and their Nr1d1+/+ littermates (controls) and analyzed expression NLRP3, interleukin 1ß (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow-derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow-derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B, and IL18; cells were incubated with LPS and adenosine triphosphate to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1-/- mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1-/- mice, control mice, and control mice given injections of SR9009 were given LPS and D-galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (alanine aminotransferase); liver tissues were collected and analyzed by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. RESULTS: In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)-this regulation required NR1D1. Primary macrophages from Nr1d1-/- mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control). CONCLUSIONS: In studies of Nr1d1-/- mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice.

Rev-erb-α regulates atrophy-related genes to control skeletal muscle mass.

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and thermogenesis. We have previously demonstrated that Rev-erb-α is also an important regulator of skeletal muscle mitochondrial biogenesis and function, and autophagy. As such, Rev-erb-α 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-α also controls muscle mass. Rev-erb-α-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-α over-expression results in reduced atrogenes expression and increased fiber size. Finally, Rev-erb-α pharmacological activation blocks dexamethasone-induced upregulation of atrogenes and muscle atrophy. This study identifies Rev-erb-α as a promising pharmacological target to preserve muscle mass.

Circadian control of metabolism and pathological consequences of clock perturbations.

Most organisms have developed an autonomous time-keeping system that generates self-sustained daily fluctuations in behavior and physiological processes. These biological clocks are reset every day by light to adjust physiology to the day/night cycle generated by the rotation of the Earth. Clocks present in organs involved in glucose and lipid metabolism such as the liver, muscle, adipose tissue and pancreas are also reset by feeding cues which permits the local integration of systemic and nutritional signals to switch fuel production and utilization according to the feeding/fasting cycle. However, derangements in this finely tuned system can be induced by extended light exposure, 24/7 food availability and altered food intake patterns, repeated jet-lag and shift-working, promoting metabolic imbalances ranging from body weight gain to the development of insulin resistance and liver diseases. Here, we review recent findings on the link between the clock and metabolic fluxes to maintain whole-body homeostasis, and what clock disruption in mice has revealed about the role of the clock in metabolic regulation.