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Physiological root resorption is a common occurrence during the development of deciduous teeth in children. Previous research has shown that the regulation of the inflammatory microenvironment through autophagy in DDPSCs is a significant factor in this process. However, it remains unclear why there are variations in the autophagic status of DDPSCs at different stages of physiological root resorption. To address this gap in knowledge, this study examines the relationship between the circadian clock of DDPSCs, the autophagic status, and the periodicity of masticatory behavior. Samples were collected from deciduous teeth at various stages of physiological root resorption, and DDPSCs were isolated and cultured for analysis. The results indicate that the circadian rhythm of important autophagy genes, such as Beclin-1 and LC3, and the clock gene REV-ERBα in DDPSCs, disappears under mechanical stress. Additionally, the study found that REV-ERBα can regulate Beclin-1 and LC3. Evidence suggests that mechanical stress is a trigger for the regulation of autophagy via REV-ERBα. Overall, this study highlights the importance of mechanical stress in regulating autophagy of DDPSCs via REV-ERBα, which affects the formation of the inflammatory microenvironment and plays a critical role in physiological root resorption in deciduous teeth. [Display omitted] • Deciduous teeth show autophagy control (Beclin-1, LC3) in DDPSCs for inflammation. • Mechanical stress disrupts DDPSCs' circadian rhythm, affecting autophagy. • Mechanical stress links masticatory behavior to resorption of deciduous teeth. [ABSTRACT FROM AUTHOR]

Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration. [ABSTRACT FROM AUTHOR]

The complex progression of type-2 diabetes (T2DM) may result in increased susceptibility to myocardial ischemia-reperfusion (IR) injury. IR injuries in multiple organs involves ferroptosis. Recently, the clock gene Rev-erbα has aroused considerable interest as a novel therapeutic target for metabolic and ischemic heart diseases. Herein, we investigated the roles of Rev-erbα and ferroptosis in myocardial IR injury during T2DM and its potential mechanisms. A T2DM model, myocardial IR and a tissue-specific Rev-erbα−/− mouse in vivo were established, and a high-fat high glucose environment with hypoxia-reoxygenation (HFHG/HR) in H9c2 were also performed. After myocardial IR, glycolipid profiles, creatine kinase-MB, AI, and the expression of Rev-erbα and ferroptosis-related proteins were increased in diabetic rats with impaired cardiac function compared to non-diabetic rats, regardless of the time at which IR was induced. The ferroptosis inhibitor ferrostatin-1 decreased AI in diabetic rats given IR and LPO levels in cells treated with HFHG/HR, as well as the expression of Rev-erbα and ACSL4. The ferroptosis inducer erastin increased AI and LPO levels and ACSL4 expression. Treatment with the circadian regulator nobiletin and genetically targeting Rev-erbα via siRNA or CRISPR/Cas9 technology both protected against severe myocardial injury and decreased Rev-erbα and ACSL4 expression, compared to the respective controls. Taken together, these data suggest that ferroptosis is involved in the susceptibility to myocardial IR injury during T2DM, and that targeting Rev-erbα could alleviate myocardial IR injury by inhibiting ferroptosis. [Display omitted] • The T2DM in rats have a higher susceptibility to myocardial IR, relating to the clock gene Rev-erbα and ferroptosis. • ACSL4 participates in diabetic myocardial IR injury, also characterized by rhythmic expression across the circadian cycle. • Pharmacological or genetic targeting of Rev-erbα attenuated myocardial IR injury during T2DM by inhibiting ferroptosis. [ABSTRACT FROM AUTHOR]

• RKO potentiates neuroinflammation that is exacerbated in females. • Depleting microglia fully rescues RKO-induced neuroinflammation in males. • Depleting microglia partially rescues RKO-induced neuroinflammation in females. • RKO induces hyperlocomotion, mania-like behaviors, and memory impairments. • Microglia depletion improves RKO-induced mood dysregulation. The circadian system is an evolutionarily adaptive system that synchronizes biological and physiological activities within the body to the 24 h oscillations on Earth. At the molecular level, circadian clock proteins are transcriptional factors that regulate the rhythmic expression of genes involved in numerous physiological processes such as sleep, cognition, mood, and immune function. Environmental and genetic disruption of the circadian clock can lead to pathology. For example, global deletion of the circadian clock gene Rev-erbα (RKO) leads to hyperlocomotion, increased anxiety-like behaviors, and cognitive impairments in male mice; however, the mechanisms underlying behavioral changes remain unclear. Here we hypothesized that RKO alters microglia function leading to neuroinflammation and altered mood and cognition, and that microglia depletion can resolve neuroinflammation and restore behavior. We show that microglia depletion (CSF1R inhibitor, PLX5622) in 8-month-old RKO mice ameliorated hyperactivity, memory impairments, and anxiety/risky-like behaviors. RKO mice exhibited striking increases in expression of pro-inflammatory cytokines (e.g., IL-1β and IL-6). Surprisingly, these increases were only fully reversed by microglia depletion in the male but not female RKO hippocampus. In contrast, male RKO mice showed greater alterations in microglial morphology and phagocytic activity than females. In both sexes, microglia depletion reduced microglial branching and decreased CD68 production without altering astrogliosis. Taken together, we show that male and female RKO mice exhibit unique perturbations to the neuroimmune system, but microglia depletion is effective at rescuing aspects of behavioral changes in both sexes. These results demonstrate that microglia are involved in Rev-erbα-mediated changes in behavior and neuroinflammation. [ABSTRACT FROM AUTHOR]

Background: The dysregulation of local circadian clock has been implicated in the pathogenesis of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks Rev-Erbα in ischemia-reperfusion (IR)-induced acute lung injury (ALI) remains unclear. Methods: The IR-ALI model was established by subjecting isolated perfused rat lungs to 40 min of ischemia followed by 60 min of reperfusion. Rats were randomly assigned to one of six groups: control, control + SR9009 (Rev-Erbα agonist, 50 mg/kg), IR, and IR + SR9009 at one of three dosages (12.5, 25, 50 mg/kg). Bronchoalveolar lavage fluids (BALF) and lung tissues were obtained and analyzed. In vitro experiments utilized mouse lung epithelial cells (MLE-12) exposed to hypoxia-reoxygenation (HR) and pretreated with SR9009 (10 µM/L) and Rev-Erbα siRNA. Results: SR9009 exhibited a dose-dependent reduction in lung edema in IR-ALI. It significantly inhibited the production of TNF-α, IL-6, and CINC-1 in BALF. Moreover, SR9009 treatment restored suppressed IκB-α levels and reduced nuclear NF-κB p65 levels in lung tissues. In addition, a SR9009 mitigated IR-induced apoptosis and mitogen-activated protein kinase (MAPK) activation in injured lung tissue. Finally, treatment with Rev-Erbα antagonist SR8278 abolished the protective action of SR9009. In vitro analyses showed that SR9009 attenuated NF-κB activation and KC/CXCL-1 levels in MLE-12 cells exposed to HR, and these effects were significantly abrogated by Rev-Erbα siRNA. Conclusions: The findings suggest that SR9009 exerts protective effects against IR-ALI in a Rev-Erbα-dependent manner. SR9009 may provide a novel adjuvant therapeutic approach for IR-ALI. [ABSTRACT FROM AUTHOR]

The circadian clock is involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), and the target pathways of many NASH candidate drugs are controlled by the circadian clock. However, the application of chronopharmacology in NASH is little considered currently. Here, the time-dependent effect of REV-ERBα agonist SR9009 on diet-induced NASH and microbiota was investigated. C57BL/6J mice were fed a high-cholesterol and high-fat diet (CL) for 12 weeks to induce NASH and then treated with SR9009 either at Zeitgeber time 0 (ZT0) or ZT12 for another 6 weeks. Pharmacological activation of REV-ERBα by SR9009 alleviated hepatic steatosis, insulin resistance, liver inflammation, and fibrosis in CL diet-induced NASH mice. These effects were accompanied by improved gut barrier function and altered microbial composition and function in NASH mice, and the effect tended to be stronger when SR9009 was injected at ZT0. Moreover, SR9009 treatment at different time points resulted in a marked difference in the composition of the microbiota, with a stronger effect on the enrichment of beneficial bacteria and the diminishment of harmful bacteria when SR9009 was administrated at ZT0. Therefore, the time-dependent effect of REV-ERBα agonist on NASH was partly associated with the microbiota, highlighting the potential role of microbiota in the chronopharmacology of NASH and the possibility of discovering new therapeutic strategies for NASH. [ABSTRACT FROM AUTHOR]

Subarachnoid hemorrhage (SAH) is associated with circadian rhythm abnormalities, in which REV-ERBα plays a major regulatory role. Our ambition was to investigate the capacity of REV-ERBα to inhibit neuronal neuroapoptosis induced by early brain injury (EBI) after SAH. The endovascular perforation model was used to produce experimental SAH in Sprague–Dawley rats. Specific small-interfering RNA was used to downregulate the expression REV-ERBα while SR9009 was used to upregulate the expression before assessments. Short- and long-term neurobehavior assessments, immunofluorescence staining, TUNEL staining, Nissl staining, brain water content, and Western blot were performed. The expression level of endogenous REVERBα tended to increase and then decrease after SAH and peaked at 48 h. REV-ERBα upregulation diminished neuronal apoptosis and enhanced neurological function deficits. Meanwhile, REV-ERBα downregulation aggravated the damage. Furthermore, the levels of downstream proteins of REV-ERBα (i.e., brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK)) changed accordingly with REV-ERBα regulation. REV-ERBα may attenuate neuronal apoptosis in EBI after SAH through the BMAL1/CLOCK pathway. [ABSTRACT FROM AUTHOR]

BCRP (breast cancer resistance protein) is a crucial efflux transporter involved in the regulation of the pharmacokinetics and pharmacodynamics of a wide range of drugs. Herein, we aimed to investigate a potential role for the nuclear receptor REV-ERBα in the regulation of BCRP expression and sulfasalazine (a BCRP probe substrate) pharmacokinetics. Regulation of BCRP expression by REV-ERBα was assessed using Rev-erbα-/- mice and AML12 and CT26 cells. Pharmacokinetic analysis was performed with Rev-erbα-/- and wild-type mice after sulfasalazine administration. We found that the expression levels of BCRP mRNA and protein were downregulated in the liver and small intestine of Rev-erbα-dificient mice. In line with this, Rev-erbα ablation increased the systemic exposures of oral sulfasalazine. Positive regulation of BCRP expression and function by REV-ERBα was furtherly confirmed in AML12 and CT26 cells. Moreover, indirect regulation of Bcrp expression by REV-ERBα was potentially mediated by a negative transcription factor DEC2, which is a downstream target of REV-ERBα. In conclusion, REV-ERBα positively regulates BCRP expression in mice, thereby affecting sulfasalazine pharmacokinetics. [ABSTRACT FROM AUTHOR]

Although fluoxetine (FLX) is a commonly used drug in psychiatric disorders, such as major depressive disorder, anxiety disorder, panic disorder, and obsessive–compulsive disorder, the mechanism by which FLX exerts its therapeutic effect is not completely understood. In this study, we aimed to determine the possible mechanism by which FLX focuses on microglial phagocytosis. FLX reduced phagocytic function in BV2 cells and increased REV-ERBα without affecting other microglia-related genes, such as inflammation and phagocytosis. Although FLX did not change BMAL1 protein levels, it restricted the nucleocytoplasmic transport (NCT) of BMAL1, leading to its cytosolic accumulation. REV-ERBα antagonist SR8278 rescued the decreased phagocytic activity and restricted NCT of BMAL1. We also found that REV-ERBα mediates the effect of FLX via the inhibition of phospho-ERK (pERK). The ERK inhibitor FR180204 was sufficient to reduce phagocytic function in BV2 cells and restrict the NCT of BMAL1. These results were recapitulated in the primary microglia. In conclusion, we propose that FLX decreases phagocytic function and restricts BMAL1 NCT via REV-ERBα. In addition, ERK inhibition mimics the effects of FLX on microglia. [ABSTRACT FROM AUTHOR]

Background: Sleep disturbance can cause adverse pregnancy outcomes by changing circadian gene expression. The potential mechanisms remain unclear. Decidualization is critical for the establishment and maintenance of normal pregnancy, which can be regulated by circadian genes. Whether Rev-erbα, a critical circadian gene, affects early pregnancy outcome by regulating decidualization needs to be explored. Methods: QPCR, western blot and artificial decidualization mouse model were used to confirm the effect of sleep disturbance on Rev-erbα expression and decidualization. The regulatory mechanism of Rev-erbα on decidualization was assessed using QPCR, western blot, RNA-Seq, and Chip-PCR. Finally, sleep disturbance mouse model was used to investigate the effect of therapeutic methods targeting Rev-erbα and interleukin 6 (IL-6) on improving adverse pregnancy outcomes induced by sleep disturbance. Results: Dysregulation of circadian rhythm due to sleep disturbance displayed abnormal expression profile of circadian genes in uterine including decreased level of Rev-erbα, accompanied by defective decidualization. Rev-erbα could regulate decidualization by directly repressing IL-6, which reduced the expression of CCAAT/enhancer-binding protein β (C/EBPβ) and its target insulin-like growth factor binding protein 1 (IGFBP1), the marker of decidualization, by inhibiting progesterone receptors (PR) expression. Moreover, deficient decidualization, higher abortion rate and lower implantation number were exhibited in the mouse models with sleep disturbance compared with those in normal mouse. Pharmacological activation of Rev-erbα or neutralization of IL-6 alleviated the adverse effect of sleep disturbance on pregnancy outcomes. Conclusions: Taken together, Rev-erbα regulated decidualization via IL-6-PR-C/EBPβ axis and might be a connector between sleep and pregnancy outcome. Therapies targeting Rev-erbα and IL-6 might help improving adverse pregnancy outcomes induced by sleep disturbance. [ABSTRACT FROM AUTHOR]

Background: Circadian disturbance is a common nonmotor complaint in Parkinson's disease (PD). The molecular basis underlying circadian rhythm in PD is poorly understood. Neuroinflammation has been identified as a key contributor to PD pathology. In this study, we explored the potential link between the core clock molecule Rev-erbα and the microglia-mediated NLR family pyrin domain-containing 3 (NLRP3) inflammasome in PD pathogenesis. Methods: We first examined the diurnal Rev-erbα rhythms and diurnal changes in microglia-mediated inflammatory cytokines expression in the SN of MPTP-induced PD mice. Further, we used BV2 cell to investigate the impacts of Rev-erbα on NLRP3 inflammasome and microglial polarization induced by 1-methyl-4-phenylpyridinium (MPP+) and αsyn pre-formed fibril. The role of Rev-erbα in regulating microglial activation via NF-κB and NLRP3 inflammasome pathway was then explored. Effects of SR9009 against NLRP3 inflammasome activation, microgliosis and nigrostriatal dopaminergic degeneration in the SN and striatum of MPTP-induced PD mice were studied in detail. Results: BV2 cell-based experiments revealed the role of Rev-erbα in regulating microglial activation and polarization through the NF-κB and NLRP3 inflammasome pathways. Circadian oscillation of the core clock gene Rev-erbα in the substantia nigra (SN) disappeared in MPTP-induced PD mice, as well as diurnal changes in microglial morphology. The expression of inflammatory cytokines in SN of the MPTP-induced mice were significantly elevated. Furthermore, dopaminergic neurons loss in the nigrostriatal system were partially reversed by SR9009, a selective Rev-erbα agonist. In addition, SR9009 effectively reduced the MPTP-induced glial activation, microglial polarization and NLRP3 inflammasome activation in the nigrostriatal system. Conclusions: These observations suggest that the circadian clock protein Rev-erbα plays an essential role in attenuating neuroinflammation in PD pathology, and provides a potential therapeutic target for PD treatment. [ABSTRACT FROM AUTHOR]

Background: Circadian disturbance is a common nonmotor complaint in Parkinson's disease (PD). The molecular basis underlying circadian rhythm in PD is poorly understood. Neuroinflammation has been identified as a key contributor to PD pathology. In this study, we explored the potential link between the core clock molecule Rev-erbα and the microglia-mediated NLR family pyrin domain-containing 3 (NLRP3) inflammasome in PD pathogenesis.Methods: We first examined the diurnal Rev-erbα rhythms and diurnal changes in microglia-mediated inflammatory cytokines expression in the SN of MPTP-induced PD mice. Further, we used BV2 cell to investigate the impacts of Rev-erbα on NLRP3 inflammasome and microglial polarization induced by 1-methyl-4-phenylpyridinium (MPP+) and αsyn pre-formed fibril. The role of Rev-erbα in regulating microglial activation via NF-κB and NLRP3 inflammasome pathway was then explored. Effects of SR9009 against NLRP3 inflammasome activation, microgliosis and nigrostriatal dopaminergic degeneration in the SN and striatum of MPTP-induced PD mice were studied in detail.Results: BV2 cell-based experiments revealed the role of Rev-erbα in regulating microglial activation and polarization through the NF-κB and NLRP3 inflammasome pathways. Circadian oscillation of the core clock gene Rev-erbα in the substantia nigra (SN) disappeared in MPTP-induced PD mice, as well as diurnal changes in microglial morphology. The expression of inflammatory cytokines in SN of the MPTP-induced mice were significantly elevated. Furthermore, dopaminergic neurons loss in the nigrostriatal system were partially reversed by SR9009, a selective Rev-erbα agonist. In addition, SR9009 effectively reduced the MPTP-induced glial activation, microglial polarization and NLRP3 inflammasome activation in the nigrostriatal system.Conclusions: These observations suggest that the circadian clock protein Rev-erbα plays an essential role in attenuating neuroinflammation in PD pathology, and provides a potential therapeutic target for PD treatment. [ABSTRACT FROM AUTHOR]

CYP2E1 plays an important role in drug metabolism and drug-induced hepatotoxicity. Here, we aimed to investigate a potential role for the nuclear receptor REV-ERBα in regulation of CYP2E1 expression and acetaminophen (APAP)-induced hepatotoxicity, and to determine the underlying mechanisms. Regulatory effects of REV-ERBα on CYP2E1 expression were assessed in vivo (using Rev-erbα-/- mice) and in vitro (using AML12 and HepG2 cells). In vitro microsomal CYP2E1 activity was probed using its specific substrate p-nitrophenol. Pharmacokinetic and acute toxicity studies were performed with Rev-erbα−/− and wild-type mice after APAP administration. We found that Rev-erbα ablation led to decreases in hepatic CYP2E1 expression and activity in mice. In line with this, APAP-induced hepatotoxicity was attenuated in Rev-erbα-deficient mice. The attenuated toxicity was due to down-regulation of APAP metabolism mediated by CYP2E1, which was evidenced by a decrease in formation of the toxic intermediate metabolite NAPQI (i.e. reduced APAP-cysteine and APAP-N-acetylcysteine levels). Furthermore, positive regulation of CYP2E1 expression by REV-ERBα was confirmed in both AML12 and HepG2 cells. Based on luciferase reporter assays, it was found that REV-ERBα regulated Cyp2e1 transcription and expression through repression of DEC2. In conclusion, REV-ERBα positively regulates CYP2E1 expression in mice, thereby affecting APAP metabolism and hepatotoxicity. [ABSTRACT FROM AUTHOR]

Nuclear receptor subfamily 1 group D member 1 (NR1D1, also known as Rev‐erbα) is a nuclear transcription factor that is part of the molecular clock encoding circadian rhythms and may link daily rhythms with metabolism and inflammation. NR1D1, unlike most nuclear receptors, lacks a ligand‐dependent activation function domain 2 and is a constitutive transcriptional repressor. Amyotrophic lateral sclerosis (ALS) is the most common adult‐onset motor neuron disease, caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Approximately 10%–20% of familial ALS is caused by a toxic gain‐of‐function induced by mutations of the Cu/Zn superoxide dismutase (SOD1). Dysregulated clock and clock‐controlled gene expression occur in multiple tissues from mutant hSOD1‐linked ALS mouse models. Here we explore NR1D1 dysregulation in the spinal cord of ALS mouse models and its consequences on astrocyte–motor neuron interaction. NR1D1 protein and mRNA expression are significantly downregulated in the spinal cord of symptomatic mice expressing mutant hSOD1, while no changes were observed in age‐matched animals overexpressing wild‐type hSOD1. In addition, NR1D1 downregulation in primary astrocyte cultures induces a pro‐inflammatory phenotype and decreases the survival of cocultured motor neurons. NR1D1 orchestrates the cross talk between physiological pathways identified to be disrupted in ALS (e.g., metabolism, inflammation, redox homeostasis, and circadian rhythms) and we observed that downregulation of NR1D1 alters astrocyte–motor neuron interaction. Our results suggest that NR1D1 could be a potential therapeutic target to prevent astrocyte‐mediated motor neuron toxicity in ALS. [ABSTRACT FROM AUTHOR]

Problem: Circadian rhythms are involved not only in the repair and regeneration of the immune system, but may also be associated with regulation of inflammation and immune responses. Rev‐erbα could constitute a link between immunity and circadian rhythms since it is a transcription factor that regulates circadian rhythms and has functions in multiple physiological and pathological processes. Decidual macrophages (dMφs) play crucial roles in immune balance at the maternal‐fetal interface, and abnormal macrophage polarization is related to adverse pregnancy outcomes, such as infertility, recurrent spontaneous abortion, and preterm labor. However, whether Rev‐erbα could modulate the polarization of macrophages is unknown. Methods of study: In this study, we analyzed the phenotype of dMφs and the expression of Rev‐erbα in dMφs from normal pregnancies and miscarriages. The effect of Rev‐erbα on macrophage polarization was evaluated by its knockdown or pharmacological activation. The mechanism by which the Rev‐erbα agonist SR9009 regulates macrophage polarization was also estimated. Results: A type‐1 macrophage (M1)‐like dominance was observed in dMφs from human miscarriages, with a decreased expression of Rev‐erbα compared to that from normal pregnancies. Rev‐erbα knockdown promoted M1 polarization in macrophages differentiated from the THP1 cell line, whereas pharmacological activation of Rev‐erbα by SR9009 induced type‐2 macrophage (M2)‐like polarization in dMφs. Furthermore, we found that SR9009 induced M2 polarization in macrophages differentiated from the U937 cell line via the PI3K/Akt signaling pathway. Conclusion: Rev‐erbα may play an essential role in macrophage polarization. These findings might help elucidate the role of Rev‐erbα in regulating the differentiation and functions of macrophages and suggest a therapeutic target for pregnancy loss and pregnancy complications. [ABSTRACT FROM AUTHOR]

Background: Circadian rhythm is an important player for reproduction. Rev-erbα, a significant clock gene, is involved in regulating cell differentiation, inflammation and metabolism. Macrophage polarization plays crucial roles in immune tolerance at the maternal-fetus interface, which also modulates the initiation and resolution of inflammation. Alteration of macrophage polarization induces adverse pregnancy outcomes such as infertility, recurrent spontaneous abortion and preterm labor. Results: Decidual macrophages from LPS-induced mice abortion model displayed M1-like bias, accompanied by decreased expression of Rev-erbα. SR9009, an agonist of Rev-erbα, may reduce lipopolysaccharide (LPS)-induced M1 polarization of macrophages via activation of PI3K but not NF-κB signaling pathway. Furthermore, SR9009 could reduce M1-like polarization of decidual macrophages induced by LPS and attenuate LPS-induced resorption rates in mice model. Conclusions: Both in vivo and in vitro experiments demonstrated that the pharmacological activation of Rev-erbα using SR9009 could attenuate the effect of LPS on macrophage polarization and protect pregnancy. This study may provide a potential therapeutic strategy for miscarriage induced by inflammation. [ABSTRACT FROM AUTHOR]

Background and aims: Alcoholic fatty liver (AFL) is a liver disease caused by long-term excessive drinking and is characterized by hepatic steatosis. Understanding the regulatory mechanism of steatosis is essential for the treatment of AFL. Rev-erbα is a member of the Rev-erbs family of nuclear receptors, playing an important role in regulating lipid metabolism. However, its functional role in AFL and its underlying mechanism remains unclear. Results: Rev-erbα was upregulated in the liver of EtOH-fed mice and EtOH-treated L-02 cells. Further, Rev-erbα activation exacerbates steatosis in L-02 cells. Inhibition/downexpression of Rev-erbα improved steatosis. Mechanistically, autophagy activity was inhibited in vivo and vitro. Interestingly, inhibition/downexpression of Rev-erbα enhanced autophagy. Furthermore, silencing of Rev-erbα up-regulated the nuclear expression of Bmal1. Autophagy activity was inhibited and steatosis was deteriorated after EtOH-treated L-02 cells were cotransfected with Rev-erbα shRNA and Bmal1 siRNA. Conclusions: Rev-erbα induces liver steatosis, which promotes the progression of AFL. Our study reveals a novel steatosis regulatory mechanism in AFL and suggest that Rev-erbα might be a potential therapeutic target for AFL. [ABSTRACT FROM AUTHOR]

Cooling patients to sub‐physiological temperatures is an integral part of modern medicine. We show that cold exposure induces temperature‐specific changes to the higher‐order chromatin and gene expression profiles of human cells. These changes are particularly dramatic at 18°C, a temperature synonymous with that experienced by patients undergoing controlled deep hypothermia during surgery. Cells exposed to 18°C exhibit largely nuclear‐restricted transcriptome changes. These include the nuclear accumulation of mRNAs encoding components of the negative limbs of the core circadian clock, most notably REV‐ERBα. This response is accompanied by compaction of higher‐order chromatin and hindrance of mRNPs from engaging nuclear pores. Rewarming reverses chromatin compaction and releases the transcripts into the cytoplasm, triggering a pulse of negative limb gene proteins that reset the circadian clock. We show that cold‐induced upregulation of REV‐ERBα is sufficient to trigger this reset. Our findings uncover principles of the cellular cold response that must be considered for current and future applications involving therapeutic deep hypothermia. Synopsis: How human cells respond to cold temperatures such as surgery‐associated deep hypothermia is largely elusive. Here, low temperatures are found to affect chromatin architecture, shape temperature‐specific nuclear and cytoplasmic transcriptomes, and subsequently reset the circadian rhythm in human cells. Exposure of human cells to varying sub‐physiological temperatures induces chromatin compaction in a non‐linear fashion.Chromatin compaction at 18°C correlates with nuclear retention of mRNAs.Incubation at 18°C forces the nuclear‐restricted upregulation of mRNAs encoding circadian clock repressor/co-repressor genes, most notably REV‐ERBα.Rewarming cells to 37°C causes release of nuclear‐accumulated mRNAs into the cytoplasm, resetting the cellular circadian rhythm. [ABSTRACT FROM AUTHOR]

BACKGROUND: The orphan nuclear receptor Rev-erbα has been demonstrated to play important roles during bone metabolism. However, the specific mechanism underlying osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is still unclear. OBJECTIVE: To explore whether the orphan nuclear receptor Rev-erbα participates in the osteogenesis process of BMSCs in mice. METHODS: Mouse BMSCs were isolated and cultured by the whole bone marrow adherence method. And the lentivirus vector carrying the Rev-erbα gene was constructed and then transfected into the BMSCs. Real-time PCR was conducted to detect the mRNA level of Rev-erbα at 48 hours after transfection. There were three groups in the experiment: an experimental group with BMSCs transfected with lentiviral vectors containing overexpressed Rev-erbα gene and EGFP gene, a positive control group with BMSCs transfected with empty lentiviral vectors containing only EGFP gene, and a negative control group. Then the BMSCs were induced to differentiate into osteoblasts after the transfection and we detected the mRNA level of alkaline phosphatase, osteopontin, and osteocalcin using real-time PCR at 0, 7, and 14 days after induction. RESULTS AND CONCLUSION: The lentivirus vector carrying Rev-erbα gene was successfully transfected into BMSCs, and expressed stably. An increase in the mRNA levels of alkaline phosphatase and osteopontin was detected in each group when cultured in osteogenic medium; however, there was no significant difference between groups. The mRNA level of osteocalcin was also increased in each group, and moreover, the mRNA level in the experimental group was significantly lower than that in the two control groups (P < 0.05). These results indicate that Rev-erbα transfection reduces the osteogenic ability of BMSCs, and the expression of osteocalcin, a late-stage osteogenic marker, is inhibited, indicating that the late-stage osteogenesis of BMSCs may be influenced by Rev-erbα. [ABSTRACT FROM AUTHOR]

Hepatic ischemia-reperfusion (I/R) injury is a complex pathophysiological process that often times occurs in liver transplantation, hepatectomy, and ischemic shock. Aberrant activation of inflammatory responses has been implicated in hepatic I/R injury. In this study, we aimed to investigate the role of circadian clock gene Rev-erbα (a well-known regulator of inflammation) in hepatic I/R injury. We first showed that Rev-erbα ablation sensitized mice to hepatic I/R injury as evidenced by higher levels of plasma alanine aminotransferase and aspartate aminotransferase, an increased histological score, as well as enhanced hepatic myeloperoxidase activity in Rev-erbα −/− mice. More severe hepatic I/R injury in Rev-erbα −/− mice was accompanied by higher expression of pro-inflammatory cytokines, exacerbated activation of Nlrp3 inflammasome, and more extensive infiltration of inflammatory cells. Moreover, pharmacological activation of Rev-erbα by SR9009 significantly alleviated the hepatic damage and inflammatory responses. In addition, I/R operation started at ZT18 (corresponding to low Rev-erbα expression) caused more severe liver damage and inflammatory responses in wild-type mice as compared to operation started at ZT6 (corresponding to high Rev-erbα expression), supporting a protective effect of Rev-erbα on hepatic I/R injury. Collectively, Rev-erbα protects hepatic I/R injury probably via repression of inflammatory responses, and targeting Rev-erbα may be a promising approach for management of hepatic I/R injury. • Rev-erbα ablation sensitizes mice to hepatic I/R injury possibly via repressing the inflammatory responses. • Rev-erbα agonist SR9009 alleviates hepatic I/R injury in mice. • Hepatic I/R injury and inflammatory responses depend on the time of I/R operation. [ABSTRACT FROM AUTHOR]

The REV‐ERBα receptor has a recognised role in the regulation of the circadian rhythm system. However, recent evidence suggests that it also contributes to energy balance regulation. Both expression and function of REV‐ERBα can be influenced by the energy status of the body. Considering the possibility of the involvement of REV‐ERBα in the regulation of energy balance, which is critically regulated by the hypothalamus, and based on the impact of intermittent fasting, the present study evaluated the effects of central administration of REV‐ERBα agonist on energy balance in rats exposed to 24 hours of fasting or ad lib. feeding conditions. Initially, 24‐hour fasted rats received an acute i.c.v. administration of agonist at doses of 1, 5, 10 or 15 μg per rat and feed efficiency was evaluated. Because 10 μg was a sufficient dose to affect feed efficiency, subsequent experiments used this dose to assess effects of agonist on the following parameters: energy expenditure induced by physical activity and locomotor activity, time spent in physical activity over 24 hours, and glucose and insulin tolerance. In fasted rats, the agonist promoted increased food intake and feed efficiency, with a greater body weight gain associated with less time spent in locomotor activity, suggesting a reduction in energy expenditure induced by physical activity. Furthermore, a reduction in glucose tolerance was noted. By contrast, free‐fed rats exhibited reduced food intake and feed efficiency with decreased body weight gain along with an increase in locomotor activity and physical activity‐dependent energy expenditure. Thus, i.c.v. administration of REV‐ERBα agonist regulates energy balance depending on the energy status of the organism; that is, it promotes a positive energy balance in the fasted state and a negative energy balance in the fed state. These results may be useful in understanding the underlying mechanisms of energy balance disorders and intermittent fasting for body weight control. [ABSTRACT FROM AUTHOR]

Background: A hallmark of temporal lobe epilepsy (TLE) is brain inflammation accompanied by neuronal demise. Accumulating evidence demonstrates that Rev-Erbα is involved in regulating neuroinflammation and determining the fate of neurons. Therefore, we studied the expression and cellular distribution of Rev-Erbα in the epileptogenic zone of TLE and the effect of treatment with the Rev-Erbα specific agonist SR9009 in the pilocarpine model.Methods: The expression pattern of Rev-Erbα was investigated by western blotting, immunohistochemistry, and immunofluorescence labeling in patients with TLE. Next, the effects of SR9009 on neuroinflammation, neuronal apoptosis, and neuronal loss in the mouse hippocampus 7 days after status epilepticus (SE) were assessed by western blotting, immunofluorescence labeling staining, and TUNEL staining.Results: The western blotting, immunohistochemistry, and immunofluorescence labeling results revealed that Rev-Erbα was downregulated in the epileptogenic zone of TLE patients and mainly localized in neurons, astrocytes, and presumably microglia. Meanwhile, the expression of Rev-Erbα was decreased in the hippocampus and temporal neocortex of mice treated with pilocarpine in the early post-SE and chronic phases. Interestingly, the expression of Rev-Erbα in the normal hippocampus showed a 24-h rhythm; however, the rhythmicity was disturbed in the early phase after SE, and this disturbance was still present in epileptic animals. Our further findings revealed that treatment with SR9009 inhibited NLRP3 inflammasome activation, inflammatory cytokine (IL-1β, IL-18, IL-6, and TNF-α) production, astrocytosis, microgliosis, and neuronal damage in the hippocampus after SE.Conclusions: Taken together, these results suggested that a decrease in Rev-Erbα in the epileptogenic zone may contribute to the process of TLE and that the activation of Rev-Erbα may have anti-inflammatory and neuroprotective effects. [ABSTRACT FROM AUTHOR]

Diabetes is a widespread disease that threatens the life and health of human beings, and diabetic cardiomyopathy (DCM) is one of the major complications of diabetic patients. The pathological mechanisms of DCM are complex, including inflammation, endoplasmic reticulum stress, and oxidative stress that have been reported previously. Although recent studies suggested that ferroptosis is also involved in the progression of DCM, the exact mechanism remains unclear. Rev-erbα cardiac conditional knockout mice were generated and type 2 diabetes were induced by high fat diet (HFD) and intraperitoneal injection of streptozotocin (STZ) in in vivo experiments. In parallel, our in vitro experiments entailed the introduction of elevated levels of glucose (HG) and palmitic acid (PA) to induce glycolipid toxicity in H9c2 cardiomyocytes. Further deterioration of cardiac function was detected by echocardiography after the clock gene rev-erbα was knocked out. This was accompanied by significant elevations in markers of inflammation, myocardial fibrosis, and oxidative stress. In addition, iron content, transmission electron microscopy (TEM), and RT-PCR assays confirmed significantly increased levels of ferroptosis in rev-erbα-deficient DCM. Intriguingly, Co-Immunoprecipitation (Co-IP) data uncovered an interaction between rev-erbα and nuclear factor E2-related factor 2 (NRF2) in diabetic myocardial tissues. It is worth highlighting that ferroptosis within cardiomyocytes witnessed significant mitigation upon the administration of sulforaphane (SFN), an NRF2 agonist, to HG + PA-incubated H9c2 cells. Our study demonstrates for the first time that knockdown of the clock gene rev-erbα exacerbates myocardial injury and ferroptosis in type 2 diabetic mice, which can be reversed by activating NRF2. • Our study is the first study to identify rev-erbα as a potentially promising molecular target for the treatment of DCM. • The deletion of rev-erbα can have deleterious effects by exacerbating myocardial inflammation, oxidative stress, cardiac dysfunction, and ferroptosis. • And we found that rev-erbα could regulate ferroptosis by regulating the antioxidant factor NRF2. [ABSTRACT FROM AUTHOR]

Progressive lung injury and pulmonary inflammation can be induced by an intraperitoneal injection of lipopolysaccharide (LPS). Interleukin-1β (IL-1β) is a key pro-inflammatory cytokine that can further exaggerate inflammation, which is cleaved and activated by the NALP3 inflammasome. Although the nuclear receptor Rev-erbα attenuates the level of LPS-induced pulmonary inflammation, the mechanism remains unclear. In this study, we investigated the influence of LPS-induced production of IL-1β and Rev-erbα on the development of lung inflammation. Herein, we demonstrate that Rev-erbα reduces IL-1β production and lung injury following an intraperitoneal injection of LPS, which is dependent on the NF-κB/NALP3 pathway. Thus, Rev-erbα is able to decrease the extent of acute lung injury by regulating IL-1β production. This mechanism may represent a potential novel therapeutic approach for lung injury. • We firstly demonstrate that Rev-erbα inhibit IL-1β production to reduce lung injury through NF-κB/NALP3 pathway in mice model. Thus, Rev-erbα might be a good target to decrease the extent of acute lung injury. This mechanism may represent a potential novel therapeutic approach for lung injury. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract REV-ERBα (NR1D1) is a nuclear heme receptor that controls many cellular processes including cell differentiation, lipid metabolism, and inflammatory responses. Although REV-ERBα has been also implicated in regulation of glucose homeostasis, the mechanism for this regulation remains unclear. Here we investigate a potential role of REV-ERBα in regulation of PCK1 (phosphoenolpyruvate carboxykinase 1), a rate-limiting enzyme in gluconeogenesis. Hepatoma cells (Hepa-1c1c7 and HepG2 cells), wild-type mice and streptozotocin-induced diabetic mice were treated with SR9009, a specific REV-ERBα agonist. The relative mRNA and protein levels of enzymes in the cells or mouse livers were determined by qPCR and Western blotting, respectively. The fasting plasma glucose test was performed to determine the effects of Rev-erbα on glucose homeostasis. Transcriptional regulation of Pck1 by Rev-erbα was investigated using a combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. SR9009 treatment significantly decreased the mRNA level of Pck1 in mouse hepatoma Hepa-1c1c7 cells, whereas other major enzymes involved in gluconeogenesis (pyruvate carboxylase, glucose-6-phosphatase, fructose bisphosphatase and Pck2) and in glycolysis (phosphofructokinase and hexokinase-1) were unaffected. Consistent with the mRNA change, the protein level of Pck1 was down-regulated. Similarly, a repressive action of REV-ERBα on PCK1 expression was observed in human HepG2 hepatoma cells. SR9009 administration to wild-type or diabetic mice significantly reduced the level of fasting plasma glucose. This coincided with decreased mRNA and protein levels of Pck1 in the liver. In addition, the diabetic mice showed an improvement in glucose tolerability after SR9009 treatment. Promoter analysis, mobility shift, and ChIP assays revealed that Rev-erbα trans-repressed Pck1 through direct binding to -325 to -320 bp region (a RevRE site) in the gene promoter. In conclusion, Rev-erbα activation down-regulates hepatic Pck1 to lower plasma glucose. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Circadian clock is known to modulate phase I metabolism, however whether and how the phase II enzymes UDP-glucuronosyltransferases (UGTs) are regulated by circadian clock are largely unknown. In this study, we aimed to investigate a potential role of the clock gene Rev-erbα in regulation of Ugt2b enzymes. Ugt2b mRNA and protein expression in mouse livers were determined at a 4-h interval around the clock. Ugt2b activity was probed using morphine as a specific substrate. Regulation of Ugt2b by Rev-erbα was investigated using mouse hepatoma Hepa-1c1c7 cells and Rev-erbα knock-out (Rev-erbα−/−) mice. Luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays were performed to identify the Rev-erbα binding site in Ugt2b36 promoter. Circadian variations in hepatic mRNA expression were observed for six Ugt2b genes (Ugt2b1 , Ugt2b5 , Ugt2b35 , Ugt2b36 , Ugt2b37 , and Ugt2b38) in mice. Likewise, the total Ugt2b protein showed a circadian fluctuation. Glucuronidation of morphine (an Ugt2b substrate) both in vitro and in vivo was dosing-time dependent. Morphine glucuronidation was more extensive at the dosing time of ZT2 than at ZT14 consistent with the Ugt2b protein levels. Furthermore, Rev-erbα knockdown significantly increased Ugt2b mRNA and protein in Hepa-1c1c7 cells, whereas Rev-erbα overexpression or activation down-regulated Ugt2b expression. Moreover, Rev-erbα ablation in mice up-regulated the mRNA and protein expression of Ugt2b and blunted Ugt2b rhythmicity in the liver. In addition, Rev-erbα repressed the transcription of Ugt2b36 through specific binding to the −30 to −18 bp of promoter region based on a combination of luciferase reporter, mobility shift and ChIP assays. In summary, the clock gene Rev-erbα negatively regulates the expressions of Ugt2b genes, contributing to their circadian variations. [ABSTRACT FROM AUTHOR]

Most living organisms show circadian rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. To study peripheral clocks in vivo, we developed an original model, the Rev-Luc mouse to follow noninvasively and longitudinally Rev-Luc oscillations in peripheral clocks using in vivo bioluminescence imaging. We found in vitro and in vivo a robust diurnal rhythm of Rev-Luc, mainly in liver, intestine, kidney and adipose tissues. We further confirmed in vivo that Rev-Luc peripheral tissues are food-entrainable oscillators, not affected by age or sex. These data strongly support the relevance of the Rev-Luc model for circadian studies, especially to investigate in vivo the establishment and the entrainment of the rhythm throughout ontogenesis. We then showed that Rev-Luc expression develops dynamically and gradually, both in amplitude and in phase, during fetal and postnatal development. We also demonstrate for the first time that the immature peripheral circadian system of offspring in utero is mainly entrained by maternal cues from feeding regimen. The prenatal entrainment will also differentially determine the Rev-Luc expression in pups before weaning underlining the importance of the maternal chrononutrition on the circadian system entrainment of the offspring. [ABSTRACT FROM AUTHOR]

Brown adipose tissue (BAT) thermogenic functions are primarily mediated by uncoupling protein (UCP)-1. Ucp1 gene expression is highly induced by cold temperature, via sympathetic nervous system and β-adrenergic receptors (βARs). Ucp1 is also repressed by the clock gene Rev-erba, contributing to its circadian rhythmicity. In this study, we investigated mice lacking βARs (β-less mice) to test the relationship between βAR signaling and the BAT molecular clock. We found that in addition to controlling the induction of Ucp1 and other key BAT genes at near freezing temperatures, βARs are essential for the basal expression of BAT Ucp1 at room temperature. Remarkably, although basal Ucp1 expression is low throughout day and night in β-less mice, the circadian rhythmicity of Ucp1 and clock genes in BAT is maintained. Thus, the requirement of βAR signaling for BAT activity is independent of the circadian rhythmicity of Ucp1 expression and circadian oscillation of the molecular clock genes. On the other hand, we found that βARs are essential for the normal circadian rhythms of locomotor activity. Our results demonstrate that in addition to controlling the BAT response to extreme cold, βAR signaling is necessary to maintain basal Ucp1 tone and to couple BAT circadian rhythmicity to the central clock. [ABSTRACT FROM AUTHOR]

In mammals, the suprachiasmatic nucleus (SCN) is the location of a master circadian pacemaker. It receives photic signals from the environment via the retinal hypothalamic tract, which play a key role in synchronizing the body's endogenously generated circadian rhythms with the 24-h rhythm of the environment. Therefore, it is plausible that age-related changes within the SCN contribute to the etiology of perturbed activity- rest cycles that become prevalent in humans during aging. To test this hypothesis, we used gene arrays and quantitative RT-PCR to profile age-related gene expression changes within the SCN of male rhesus macaques - a pragmatic translational animal model of human aging, which similarly displays an age-related attenuation of daytime activity levels. As expected, the SCN showed high expression of arginine vasopressin, vasoactive intestinal polypeptide, calbindin and nuclear receptor subfamily 1, group D, member 1 (NR1D1) (also known as reverse strand of ERBA (REV-ERBα), both at the mRNA and protein level. However, no obvious difference was detected between the SCNs of young (7-12 years) and old animals (21-26 years), in terms of the expression of core clock genes or genes associated with SCN signaling and neurotransmission. These data demonstrate the resilience of the primate SCN to normal aging, at least at the transcriptional level and, at least in males, suggest that age-related disruption of activity-rest cycles in humans may instead stem from changes within other components of the circadian system, such as desynchronization of subordinate oscillators in other parts of the body. [ABSTRACT FROM AUTHOR]

BACKGROUND: The orphan nuclear receptors Rev-erbα and Rorα play important roles in lipid metabolism and glucose metabolism. But it is unclear whether they are involved in bone metabolism. OBJECTIVE: To observe the expression of Rev-erbα and Rorα during the osteoblastogenesis process of bone marrow mesenchymal stem cells (BMSCs). METHODS: BMSCs were isolated and purified from C57BL/6 mice by the whole bone marrow adherence method followed by morphology observations and then BMSCs were induced to differentiate into osteoblasts and adipocytes. We detected their differentiation abilities using oil red O staining and alizarin red staining, respectively. Real-time PCR and western blot assay were conducted to detect the mRNA and protein levels of Rev-erbα and Rorα in BMSCs cultured in the osteogenic medium for 0, 7, 14 days. RESULTS AND CONCLUSION: BMSCs from C57BL/6 mice were mainly spindle-shaped and exhibited the swirl-like pattern of growth. Following induction, oil red O staining and alizarin red staining produced a positive reaction in these cells. Rev-erbα expression at both gene and protein levels decreased at 0, 7, 14 days after osteogenic induction, while Rorα expression increased at both gene and protein levels. These findings indicate that Rev-erbα and Rorα may participate in the osteoblastogenesis of BMSCs. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CES) are a family of phase I enzymes that play an important role in xenobiotic clearance and lipid metabolism. Here, we investigate a potential role of E4 promoter-binding protein 4 (E4bp4) in regulation of Ces and CPT-11 (irinotecan, a first-line drug for treating colorectal cancer) pharmacokinetics in mice. Mouse hepatoma Hepa-1c1c7 cells were transfected with Rev-erbα expression plasmid or siRNA targeting E4bp4. The relative mRNA and protein levels of Ces enzymes in the cells or the livers of wild-type and E4bp4-deficient (E4bp4 −/− ) mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Ces by E4bp4/Rev-erbα were investigated using luciferase reporter, mobility shift, and co-immunoprecipitation (Co-IP) assays. Pharmacokinetic studies were performed with wild-type and E4bp4 −/− mice after intraperitoneal injection of CPT-11. E4bp4 ablation down-regulated an array of hepatic Ces genes in mice. E4bp4 −/− mice also showed reduced Ces-mediated metabolism and elevated systemic exposure of CPT-11, a well-known Ces substrate. Consistently, E4bp4 knockdown reduced the expression of Ces genes ( Ces2b , Ces2e and Ces2f ) in Hepa-1c1c7 cells. Furthermore, Rev-erbα repressed the transcription of Ces2b, whereas E4bp4 antagonized this repressive action. Co-IP experiment confirmed a direct interaction between E4bp4 and Rev-erbα. Through a combination of promoter analysis and mobility shift assays, we demonstrated that Rev-erbα trans-repressed Ces (Ces2b) through its specific binding to the -767 to-754 bp promoter region. In conclusion, E4bp4 regulates Ces enzymes through inhibition of the transrepression activity of Rev-erbα, thereby impacting the metabolism and pharmacokinetics of Ces substrates. [ABSTRACT FROM AUTHOR]

Background/Aims: Angiotensin II (Ang II) regulates the expression of some core clock genes; excess Ang II leads to atherosclerosis advancement. Macrophage Rev-erbα mediates clockwork and inflammation, and plays a role in atherosclerotic lesion progression. However, the role of Ang II in regulating Rev-erbα expression in macrophages remains unclarified. Methods: We induced THP-1 macrophages by phorbol 12-myristate 13-acetate and investigated the effect of Ang II on Rev-erbα expression via real-time polymerase chain reaction, western blotting and small interfering RNA (siRNA) techniques. The cytotoxicity of the Rev-erbα agonist SR9009 was analyzed using a (3-[4,5-dimethylthiazol-2-yl])-2,5- diphenyltetrazolium bromide assay. Results: Ang II suppressed Rev-erbα mRNA and protein expression in THP-1 macrophages in a dose and time dependent manner. This effect was mediated via Ang II type 1 receptor (AT1R), and not Ang II type 2 receptor or peroxisome proliferator-activated receptor γ (PPARγ). Consistent with Rev-erbα expression regulated by Ang II, the liver X receptor α (LXRα) protein expression was downregulated in a time-dependent manner after Ang II treatment. The activation or silence of LXRα significantly increased or decreased Rev-erbα expression regulated by Ang II, respectively. This suggests that LXRα is involved in the effect of Ang II on Rev-erbα expression. MMP-9 mRNA expressions were significantly suppressed by SR9009 in THP-1 and RAW264.7 macrophages; moreover, SR9009-treatment significantly reduced Ang II–induced MMP-9 protein expressions in two types of macrophages. Conclusion: Ang II downregulates Rev-erbα expression in THP-1 macrophages via the AT1R/LXRα pathway. [ABSTRACT FROM AUTHOR]

REV-ERBα is a nuclear heme receptor, transcriptional repressor and critical component of the molecular clock that drives daily rhythms of metabolism. Evidence reveals that REV-ERBα also plays an important regulatory role in clock-dependent lung physiology and inflammatory responses. We hypothesize that cigarette smoke (CS) exposure influences REV-ERBα abundance in the lungs, facilitating a pro-inflammatory phenotype. To determine the impact of REV-ERBα activation in the CS-induced inflammatory response we treated primary human small airway epithelial cells (SAECs) with CS extract (CSE) or lipopolysaccharide (LPS) in the absence or presence of pre-treatment with the REV-ERBα agonist GSK 4112. We also exposed adult C57BL/6J (WT) and Rev-erbα global KO mice to CS (10 and 30 days) and measured pro-inflammatory cytokine release. Our data reveal that pre-treatment with GSK 4112 reduced CSE/LPS induced pro-inflammatory cytokines release from both SAECs and mouse lung fibroblasts (MLFs). Furthermore, REV-ERBα KO mice show a greater inflammatory response to 10 and 30 days of CS, including increased neutrophil lung influx, pro-inflammatory cytokine (IL-6, MCP-1 and KC) release, and pro-senescence marker (p16) when compared to WT mice. These data demonstrate that REV-ERBα is a critical regulator of CS-induced lung inflammatory responses. [ABSTRACT FROM AUTHOR]

• The expression level of NR1D1 in bladder cancer (BC) is correlated with the prognosis. The patients with negative expression of NR1D1 have shorter DFS than those with NR1D1 positive expression. • Both upregulation of NR1D1 and Rev-erbα agonist SR9009 can suppress the proliferation, migration and colony formation of BC cells in vitro. • Upregulation of NR1D1 can inhibit tumorigenesis in vivo, and proliferation, migration, and invasive biological behaviors in BC, and PI3K/AKT/mTOR signaling pathway may be one of the mechanisms of NR1D1 affecting the biological behavior of BC. To explore the role of circadian clock gene NR1D1 (REV-erbα) in bladder cancer (BC). Firstly, the association of NR1D1 level with clinical characteristics and prognosis was investigated among patients diagnosed with BC. Secondly, CCK-8, transwell, and colony formation experiments were performed among BC cells treated with Rev-erbα agonist (SR9009), as well as lentivirus and siRNA, for which NR1D1 were overexpressed (OE) and knocked down (KD), respectively. Thirdly, cell cycle and apoptosis were tested by flowcytometry. PI3K/AKT/mTOR pathway proteins were determined in OE-NR1D1 cells. Finally, OE-NR1D1 and OE-Control BC cells were subcutaneously implanted in BALB/c nude mice. The tumor size and protein levels were compared between groups. A P < 0.05 was considered as statistically significant. Patients with NR1D1 positive status had a longer disease-free survival than those with negative expression. The cell viability, migration, and colony formation of BC cells after treated with SR9009 were significantly suppressed. OE-NR1D1 cells had obviously inhibited cell viability, migration, and colony formation, while those were found strengthened in KD-NR1D1 cells. Besides, KD-NR1D1 cells were observed with a lower proportion of dead cells and G0/G1 cells, but a higher ratio of G2/M. The changes of p-AKT, p-S6, p-4EBP1, and FASN involved in PI3K/AKT/mTOR pathway were detected in OE- and KD-NR1D1 BC cells. Finally, in vivo data demonstrated that overexpression of NR1D1 suppressed the tumorigenicity of BC cells. NR1D1 played a role of tumor suppressor and it might become a novel target for the treatment of BC. [ABSTRACT FROM AUTHOR]

The drive to eat is regulated by two compensatory brain pathways termed as homeostatic and hedonic. Hypothalamic orexinergic (ORX) neurons regulate metabolism, feeding and reward, thus controlling physiological and hedonic appetite. Circadian regulation of feeding, metabolism and rhythmic activity of ORX cells are driven by the brain suprachiasmatic clock. How the circadian clock impacts on ORX signalling and feeding-reward rhythms is, however, unknown. Here we used mice lacking the nuclear receptor REV-ERBα, a transcription repressor and a key component of the molecular clockwork, to study food-reward behaviour. Rev-Erbα mutant mice showed highly motivated behaviours to obtain palatable food, an increase in the intake and preference for tasty diets, and in the expression of the ORX protein in the hypothalamus. Palatable food intake was inhibited in animals treated with the ORX1R antagonist. Analyzing the Orx promoter, we found Retinoic acid-related Orphan receptor Response Element binding sites for Rev-Erbα. Furthermore, Rev-Erbα dampened the activation of Orx in vitro and in vivo. Our data provide evidence for a possible repressive role of Rev-Erbα in the regulation of ORX signalling, highlighting an implication of the circadian clockwork in modulating food-reward behaviours with an important impact for the central regulation of overeating. [ABSTRACT FROM AUTHOR]

Aging can modify the morphology and function of the liver, such as generating a decrease in the mitochondria content, autophagy, and cell senescence. Although exercise training has several beneficial effects on hepatic metabolism, its actions on autophagy processes, mitochondrial function, and cellular senescence need to be more widely explored. The present study verified the effects of aging and exercise on hepatic circadian markers, autophagy, and mitochondria activity in 24-month-old mice with a combined exercise training protocol. In addition, we used public datasets from human livers in several conditions and BMAL1 knockout mice. C57BL/6 mice were distributed into Control (CT, young, 6-month-old mice), sedentary old (Old Sed, sedentary, 24-month-old mice), and exercised old (Old Ex, 24-month-old mice submitted to a combined exercise training protocol). The exercise training protocol consisted of three days of endurance exercise – treadmill running, and two days of resistance exercise – climbing a ladder, for three weeks. At the end of the protocol, the liver was removed and prepared for histological analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunoblotting technique, and oxygen consumption. Heatmaps were built using a human dataset and Bmal1 knockout samples. In summary, the Old Sed had reduced strength, coordination, and balance, as well as a decrease in Bmal1 expression and the presence of degenerated liver cells. Still, this group upregulated the transcription factors related to mitochondrial biogenesis. The Old Ex group had increased strength, coordination, and balance, improved glucose sensitivity, as well as restored Bmal1 expression and the mitochondrial transcription factors. The human datasets indicated that mitochondrial markers and autophagy strongly correlate with specific liver diseases but not aging. We can speculate that mitochondrial and autophagy molecular markers alterations may depend on long-term training. [ABSTRACT FROM AUTHOR]

REV-ERBα (encoded by Nr1d1) is a nuclear receptor that is part of the circadian clock mechanism and regulates metabolism and inflammatory processes. The glucocorticoid receptor (GR, encoded by Nr3c1) influences similar processes, but is not part of the circadian clock, although glucocorticoid signaling affects resetting of the circadian clock in peripheral tissues. Because of their similar impact on physiological processes, we studied the interplay between these two nuclear receptors. We found that REV-ERBα binds to the C-terminal portion and GR to the N-terminal portion of HSP90α and HSP90β, a chaperone responsible for the activation of proteins to ensure survival of a cell. The presence of REV-ERBα influences the stability and nuclear localization of GR by an unknown mechanism, thereby affecting expression of GR target genes, such as IκBα (Nfkbia) and alcohol dehydrogenase 1 (Adh1). Our findings highlight an important interplay between two nuclear receptors that influence the transcriptional potential of each other. This indicates that the transcriptional landscape is strongly dependent on dynamic processes at the protein level. [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor 6 (HNF6) is required for liver development, but its role in adult liver metabolism is not known. Here we show that deletion of HNF6 in livers of adult C57Bl/6 mice leads to hepatic steatosis in mice fed normal laboratory chow. Although HNF6 is known mainly as a transcriptional activator, hepatic loss of HNF6 up-regulated many lipogenic genes bound directly by HNF6. Many of these genes are targets of the circadian nuclear receptor Rev-erbα, and binding of Rev-erbα at these sites was lost when HNF6 was ablated in the liver. While HNF6 and Rev-erbα coordinately regulate hepatic lipid metabolism, each factor also affects additional gene sets independently. These findings highlight a novel mechanism of transcriptional repression by HNF6 and demonstrate how overlapping and distinct mechanisms of transcription factor function contribute to the integrated physiology of the liver. [ABSTRACT FROM AUTHOR]

Circadian rhythms, biological oscillations with a period of about 24 h, are maintained by an innate genetically determined time-keeping system called the molecular circadian clockwork. Despite the physiological and clinical importance of the circadian clock, development of small molecule modulators targeting the core clock machinery has only recently been initiated. BMAL1, a core clock gene, is controlled by a ROR/REV-ERB-response element (RORE)-dependent mechanism, which plays an important role in stabilizing the period of the molecular circadian clock. Therefore, we aimed to identify a novel small molecule modulator that regulates Bmal1 gene expression in RORE-dependency, thereby influencing the molecular feedback loop of the circadian clock. For this purpose, we carried out a cell-based screen of more than 1000 drug-like compounds, using a luciferase reporter driven by the proximal region of the mouse Bmal1 promoter. One compound, designated KK-S6, repressed the RORE-dependent transcriptional activity of the m Bmal1 promoter and reduced endogenous BMAL1 protein expression. More importantly, KK-S6 significantly altered the amplitude of circadian oscillations of Bmal1 and Per2 promoter activities in a dose-dependent manner, but barely affected the period length. KK-S6 effectively decreased mRNA expression of metabolic genes acting downstream of REV-ERBα, Pai-1 and Citrate synthase, that contain RORE cis -element in their promoter. KK-S6 likely acts in a RORE-dependent manner by reinforcing the REV-ERBα activity, though not by the same mechanism as known REV-ERB agonists. In conclusion, the present study demonstrates that KK-S6 functions as a novel modulator of the amplitude of molecular circadian rhythms by influencing RORE-mediated BMAL1 expression. [ABSTRACT FROM AUTHOR]

• Rev-erbα knockout mice exhibit both positive and negative mood phenotypes. • Rev-erbα deficiency leads to dysregulation of the dopaminergic and serotonergic systems. • The Rev-erbα gene is required for the regulation of genes essential for neurogenesis in the murine prefrontal cortex. Mood disorders such as depression, anxiety, and bipolar disorder are highly associated with disrupted daily rhythms of activity, which are often observed in shift work and sleep disturbance in humans. Recent studies have proposed the REV-ERBα protein as a key circadian nuclear receptor that links behavioural rhythms to mood regulation. However, how the Rev-erbα gene participates in the regulation of mood remains poorly understood. Here, we show that the regulation of the serotonergic (5-HTergic) system, which plays a central role in stress-induced mood behaviours, is markedly disrupted in Rev-erbα−/− mice. Rev-erbα−/− mice exhibit both negative and positive behavioural phenotypes, including anxiety-like and mania-like behaviours, when subjected to a stressful environment. Importantly, Rev-erbα−/− mice show a significant decrease in the expression of a gene that encodes the rate-limiting enzyme of serotonin (5-HT) synthesis in the raphe nuclei (RN). In addition, 5-HT levels in Rev-erbα−/− mice are significantly reduced in the prefrontal cortex, which receives strong inputs from the RN and controls stress-related behaviours. Our findings indicate that Rev-erbα plays an important role in controlling the 5-HTergic system and thus regulates mood and behaviour. [ABSTRACT FROM AUTHOR]

Disrupted daily or circadian rhythms of lung function and inflammatory responses are common features of chronic airway diseases. At the molecular level these circadian rhythms depend on the activity of an autoregulatory feedback loop oscillator of clock gene transcription factors, including the BMAL1:CLOCK activator complex and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors and transcription factors REV-ERBα and RORα regulate Bmal1 expression and provide stability to the oscillator. Circadian clock dysfunction is implicated in both immune and inflammatory responses to environmental, inflammatory, and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke, lipopolysaccharide, hyperoxia, allergens, bleomycin, as well as bacterial and viral infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock through acetylation of BMAL1 and PER2 and controls the clock-dependent functions, which can also be affected by environmental stressors. Environmental agents and redox modulation may alter the levels of REV-ERBα and RORα in lung tissue in association with a heightened DNA damage response, cellular senescence, and inflammation. A reciprocal relationship exists between the molecular clock and immune/inflammatory responses in the lungs. Molecular clock function in lung cells may be used as a biomarker of disease severity and exacerbations or for assessing the efficacy of chronotherapy for disease management. Here, we provide a comprehensive overview of clock-controlled cellular and molecular functions in the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Furthermore, we highlight the potential for the molecular clock as a novel chronopharmacological target for the management of lung pathophysiology. [ABSTRACT FROM AUTHOR]

Several hormones are regulated by circadian rhythms to adjust the metabolism to the light/dark cycles and feeding/activity patterns throughout the day. Circadian rhythms are mainly governed by the central clock located in the suprachiasmatic nucleus but also by clocks present in peripheral organs, like the endocrine pancreas. Plasma glucose levels and the main pancreatic hormones insulin and glucagon also exhibit daily variations. Alterations in circadian rhythms are associated with metabolic disturbances and pathologies such as obesity and diabetes. The molecular components of central and peripheral clocks and their regulatory mechanisms are well established. Among the different clock genes, Rev-erbα is considered one of the key links between circadian rhythms and metabolism. Rev-erbα is a critical part of a negative feedback loop in the core circadian clock and modulates the clock oscillatory properties. In addition, Rev-erbα plays an important role in the regulation of lipid and glucose metabolism, thermogenesis, adipocyte and muscle differentiation as well as mitochondrial function. In the endocrine pancreas, Rev-erbα regulates insulin and glucagon secretion and pancreatic β-cell proliferation. In the present review, we discuss all these subjects and, particularly, the role of the clock gene Rev-erbα in the endocrine pancreas. [ABSTRACT FROM AUTHOR]

The circadian clock orchestrates the co-ordinated rhythmicity of numerous metabolic pathways to anticipate daily and seasonal changes in energy demand. This vital physiological function is controlled by a set of individual clock components that are present in each cell of the body, and regulate each other as well as clock output genes. A key factor is the nuclear receptor, Rev-erbα, a transcriptional repressor which functions not only as a clock component but also as a modulator of metabolic programming in an array of tissues. This review explores the role of Rev-erbα in mediating this crosstalk between circadian rhythm and tissue-specific biological networks and its relevance to organismal physiology. [ABSTRACT FROM AUTHOR]

In mammals, the central clock localized in the central nervous system imposes a circadian rhythmicity to all organs. This is achieved thanks to a well-conserved molecular clockwork, involving interactions between several transcription factors, whose pace is conveyed to peripheral tissues through neuronal and humoral signals. The molecular clock plays a key role in the control of numerous physiological processes and takes part in the regulation of metabolism and energy balance. Skeletal muscle is one of the peripheral organs whose function is under the control of the molecular clock. However, although skeletal muscle metabolism and performances display circadian rhythmicity, the role of the molecular clock in the skeletal muscle has remained unappreciated for years. Peripheral organs such as skeletal muscle, and the liver, among others, can be desynchronized from the central clock by external stimuli, such as feeding or exercise, which impose a new rhythm at the organism level. In this review, we discuss our current understanding of the clock in skeletal muscle circadian physiology, focusing on the control of myogenesis and skeletal muscle metabolism. [ABSTRACT FROM AUTHOR]

The nuclear receptor REV-ERBα links circadian rhythms and numerous physiological processes, but its physiological role in ovaries remains largely unknown. The aim of this study was to determine the potential role of REV-ERBα in the regulation of the transcription of its putative target genes in granulosa cells (GCs) prepared from Per2-destablized luciferase ( dLuc) reporter gene transgenic rats. Alas1, Ppargc1a, and Il6 were chosen as representatives for genes analysis. A real-time monitoring system of Per2 promoter activity was performed to detect Per2-dLuc circadian oscillations. Two agonists (GSK4112, heme) and an antagonist (SR8278) of REV-ERBα as well as Rev-erbα siRNA knockdown were used to identify its target genes. Clear Per2-dLuc circadian oscillations were generated in matured GCs after synchronization with GSK4112 or SR8278. GSK4112 treatment lengthened and SR8278 treatment shortened the period of circadian oscillations in matured GCs stimulated with or without luteinizing hormone (LH). GSK4112 showed an inhibitory effect on the amplitude of circadian oscillations and caused an arrhythmic expression of canonical clock genes. SR8278 also had a subtle effect on their daily expression profiles, but the treatment resulted only in the arrhythmic expression of Rev-erbα. These findings indicate the functional biological activity of REV-ERBα in response to its ligands. Its natural ligand heme further elongated the period of circadian oscillations and alleviated their amplitudes in GCs cultured with LH. Heme treatment also repressed the expressions of clock genes, Alas1, Il6, and Ppargc1a. Rev-erbα knockdown up-regulated these transcript levels. Collectively, these data extend the recent finding to rat GCs and demonstrate that REV-ERBα represses the expressions of Alas1, Ppargc1a, and Il6, providing novel insights into the physiological significance of REV-ERBα in ovarian circadian oscillators. [ABSTRACT FROM AUTHOR]

Research spanning nearly four decades has assigned to the translocator protein (18 kDa) (TSPO) a critical role, among others, in the mitochondrial import of cholesterol, the subsequent steps of (neuro)steroid production, and systemic endocrine regulation, with implications for the pathophysiology of immune, inflammatory, neurodegenerative, and psychiatric as well as neoplastic diseases. Recent knockout studies in mice unexpectedly report normal or latent phenotypes, raising doubts about the protein's role in steroidogenesis and other previously postulated functions and challenging the validity of earlier data on the selectivity of TSPO-binding drugs. Here we provide a synthesis of the current debate from a structural and molecular biology perspective, discuss the limits of inference in loss-of-function (gene knockout) studies, and suggest new functions of TSPO. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle is a major contributor to whole-body metabolism as it serves as a depot for both glucose and amino acids, and is a highly metabolically active tissue. Within skeletal muscle exists an intrinsic molecular clock mechanism that regulates the timing of physiological processes. A key function of the clock is to regulate the timing of metabolic processes to anticipate time of day changes in environmental conditions. The purpose of this study was to identify metabolic genes that are expressed in a circadian manner and determine if these genes are regulated downstream of the intrinsic molecular clock by assaying gene expression in an inducible skeletal muscle-specific Bmal1 knockout mouse model (iMS-Bmal1-/-). Methods: We used circadian statistics to analyze a publicly available, high-resolution time-course skeletal muscle expression dataset. Gene ontology analysis was utilized to identify enriched biological processes in the skeletal muscle circadian transcriptome. We generated a tamoxifen-inducible skeletal muscle-specific Bmal1 knockout mouse model and performed a time-course microarray experiment to identify gene expression changes downstream of the molecular clock. Wheel activity monitoring was used to assess circadian behavioral rhythms in iMS-Bmal1-/- and control iMS-Bmal1+/+ mice. Results: The skeletal muscle circadian transcriptome was highly enriched for metabolic processes. Acrophase analysis of circadian metabolic genes revealed a temporal separation of genes involved in substrate utilization and storage over a 24-h period. A number of circadian metabolic genes were differentially expressed in the skeletal muscle of the iMS-Bmal1-/- mice. The iMS-Bmal1-/- mice displayed circadian behavioral rhythms indistinguishable from iMS-Bmal1+/+ mice. We also observed a gene signature indicative of a fast to slow fiber-type shift and a more oxidative skeletal muscle in the iMS-Bmal1-/- model. Conclusions: These data provide evidence that the intrinsic molecular clock in skeletal muscle temporally regulates genes involved in the utilization and storage of substrates independent of circadian activity. Disruption of this mechanism caused by phase shifts (that is, social jetlag) or night eating may ultimately diminish skeletal muscle's ability to efficiently maintain metabolic homeostasis over a 24-h period. [ABSTRACT FROM AUTHOR]

Nuclear receptor REV-ERBα, a key component of an internal timekeeping system, has been considered to be related to disrupted circadian rhythms and mood disorder. In this study, we aimed to evaluate the relationship between the genotype frequencies of single gene polymorphisms (SNPs) of the NR1D1 gene encoding REV-ERBα and circadian typologies. The classification of chronotypes and genotyping of three SNPs (rs2314339, rs2071427, rs12941497) of the NR1D1 gene were conducted in 602 healthy young adults (355 males, 247 females). A significant association was found between the genotypes of rs12941497 and three chronotype categories. These findings support the role of NR1D1 polymorphisms in the regulation of circadian rhythms. [ABSTRACT FROM AUTHOR]

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes (Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2-O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E2 production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE2 production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization. [ABSTRACT FROM AUTHOR]

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states. [ABSTRACT FROM AUTHOR]