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.
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 1β (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.
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).
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.
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Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute ...induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver‐identity (LIVER‐ID) transcription factor (TF) network, initiated by rapid LIVER‐ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER‐ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co‐recruitment of LIVER‐ID TFs and decommissioning of BRD4 super‐enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER‐ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients.
Synopsis
Functional genomics analyses shows that acute endoplasmic reticulum stress (ERS) in the liver induces a global loss of molecular identity and partial hepatic dedifferentiation, which characterize mouse and human liver upon acute injury.
Acute ERS induces loss of hepatic molecular identity by extensively and preferentially downregulating liver identity (LIVER‐ID) genes.
ERS‐mediated transcriptional repression is an active process involving changes in the transcription factor repertoire with inhibition of the LIVER‐ID network and induction of the NFIL3 repressor.
This translates into a global loss of activity of cis‐regulatory modules densely co‐bound by LIVER‐ID transcription factors and decommissioning of BRD4 at super‐enhancers.
The hepatic and ERS transcriptional programs are in competitive equilibrium with direct relevance towards the liver's ability to recover from injury.
Functional genomics analyses shows that acute endoplasmic reticulum stress (ERS) in the liver induces a global loss of molecular identity and partial hepatic dedifferentiation, which characterize mouse and human liver upon acute injury.
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.
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•Brown adipocytes are functionally differentiated at E17.5 in the mouse embryo•Lipid droplets are formed within glycogen clusters•Glycogen production is crucial for lipid droplet biogenesis during BAT differentiation•Glycophagy-mediated glycogen degradation drives lipid droplet formation
Lipid droplet formation is a major feature of brown adipocyte differentiation. Mayeuf-Louchart et al. characterize the different steps of brown adipocyte differentiation in the mouse embryo and report the essential role of glycogen production and degradation by glycophagy for lipid droplet biogenesis.
Retinoic acid receptor-related orphan receptor-alpha (RORα) is a transcription factor from the nuclear receptor family expressed by immune cells and involved in the development of obesity, insulin ...resistance (IR) and non-alcoholic steatohepatitis (NASH). It was recently reported that mice deficient for RORα in macrophages develop more severe NASH upon high fat diet (HFD) feeding due to altered Kupffer cell function. To better understand the role of RORα in obesity and IR, we independently generated a macrophage RORα-deficient mouse line. We report that RORα deletion in macrophages does not impact on HFD-induced obesity and IR. Surprisingly, we did not confirm an effect on NASH development upon HFD feeding nor in the more severe and obesity-independent choline-deficient, L-amino acid-defined diet model. Our results therefore show that RORα deletion in macrophages does not alter the development of obesity and IR and question its role in NASH.
Aims
Not all people with obesity become glucose intolerant, suggesting differential activation of cellular pathways. The unfolded protein response (UPR) may contribute to the development of insulin ...resistance in several organs, but its role in skeletal muscle remains debated. Therefore, we explored the UPR activation in muscle from non-diabetic glucose tolerant or intolerant patients with obesity and the impact of bariatric procedures.
Methods
Muscle biopsies from 22 normoglycemic (NG, blood glucose measured 120 min after an oral glucose tolerance test, G120 < 7.8 mM) and 22 glucose intolerant (GI, G120 between 7.8 and 11.1 mM) patients with obesity were used to measure UPR activation by RTqPCR and western blot. Then, UPR was studied in biopsies from 7 NG and 7 GI patients before and 1 year after bariatric surgery.
Results
Binding immunoglobulin protein (BIP) protein was ~ 40% higher in the GI compared to NG subjects. Contrastingly, expression of the UPR-related genes
BIP
,
activating transcription factor 6
(
ATF6
) and unspliced
X
-
box binding protein 1
(
XBP1u
) were significantly lower and
C/EBP homologous protein
(
CHOP
) tended to decrease (
p
= 0.08) in GI individuals. While BIP protein positively correlated with fasting blood glucose (
r
= 0.38,
p
= 0.01),
ATF6
and
CHOP
were associated with G120 (
r
= − 0.38 and
r
= − 0.41,
p
< 0.05) and the Matsuda index (
r
= 0.37 and
r
= 0.38,
p
< 0.05). Bariatric surgery improved metabolic parameters, associated with higher
CHOP
expression in GI patients, while
ATF6
tended to increase (
p
= 0.08).
Conclusions
CHOP
and
ATF6
expression decreased in non-diabetic GI patients with obesity and was modified by bariatric surgery. These genes may contribute to glucose homeostasis in human skeletal muscle.
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.
Retinoic acid-related Orphan Receptor alpha (RORα; NR1F1) is a widely distributed nuclear receptor involved in several (patho)physiological functions including lipid metabolism, inflammation, ...angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by RORα in liver cells by generating HepG2 human hepatoma cells stably over-expressing RORα. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which RORα was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by RORα, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by RORα. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by RORα. SPARC was found to be a new putative RORα target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by RORα also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by RORα in HepG2 cells. Therefore these genes must now be considered as direct RORα targets. Our results open new routes on the roles of RORα in glucose metabolism and carcinogenesis within cells of hepatic origin.
The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α 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-α deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1α signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-α, Nr1d1. In complementary experiments, Rev-erb-α overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-α in vivo increased exercise capacity. This study identifies Rev-erb-α as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.
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.
The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator ...of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPARγ action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR−/−) and cells to the PPARγ activator rosiglitazone. Our results show that genetically obese FXR−/−/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR−/− mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR−/− MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPARγ activation. Retroviral-mediated FXR re-expression in FXR−/− MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/β-catenin pathway and target genes was increased in FXR−/− adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR−/− MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPARγ and Wnt/β-catenin pathways.
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