Brain And Muscle Arnt-like Protein-1 Research Articles

Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Unexplained recurrent spontaneous abortion (URSA) is a serious reproductive issue that affects women of childbearing age. Studies have shown a close association between disrupted circadian rhythm and impaired epithelial-mesenchymal transition (EMT) process in trophoblasts during URSA, though the underlying mechanism has not been elaborated. This study aims to investigate the regulatory relationship between circadian rhythm gene cryptochrome 2 (CRY2) and ferroptosis on the migratory ability of trophoblast cells. Cell proliferation experiments, wound healing assays, and expression of related markers were conducted to verify the EMT efforts. Trophoblastic ferroptosis was confirmed by the expressions of malondialdehyde, glutathione, mitochondrial membrane potential, divalent iron ions, and expression level of related genes. The results showed significant increased expression of CRY2 and decreased expression of brain and muscle ARNT-like protein 1 (BMAL1) in the URSA villous tissues, accompanied by iron-dependent oxidative changes and abnormal expression of ferroptosis related proteins. It was observed that CRY2 and BMAL1 was co-localized and function as a feedback loop, which regulated the dynamic changes of EMT-related markers in trophoblast cells. CRY2 promoted trophoblastic ferroptosis, while BMAL1 had the opposite effect. Particularly, the ferroptosis inhibitor (Ferrostatin-1) effectively reversed the trophoblastic ferroptosis and EMT inhibition caused by CRY2 overexpression. Collectively, these results suggest that CRY2 regulates trophoblastic ferroptosis and hinders cellular EMT and migratory ability by suppressing BMAL1 expression.

The effect of 24-hour sleep deprivation and anaerobic exercise on the expression of BAX, BCL2, BMAL1 and CCAR2 genes in peripheral blood mononuclear cells after L-arginine supplementation

Sleep deprivation disrupt the circadian clock and exercise performance. Defective oxidative stress caused by sleep deprivation may affect the expression of genes involved in cell apoptosis. Since a number of studies have shown the anti-apoptotic effect of L-arginine, so the aim of this study was to evaluate the effect of eight weeks of L-arginine supplementation on the expression of brain and muscle ARNT-like protein 1 (BMAL1), cell cycle and apoptosis regulator 2 (CCAR2), and BAX and BCL2 genes during sleep deprivation and acute anaerobic exercise. Participants included 20 healthy men age 26–35 years, randomized into the L-arginine intervention group (n = 10) and a placebo control (n = 10). The running-based anaerobic sprint test (RAST) was used for anaerobic exercise. Intervention subjects took one 1000 mg L-arginine tablet daily for 8 weeks. The Real-Time PCR method was used to determine apoptosis gene expression in peripheral blood mononuclear cells (PBMCs). Acute anaerobic exercise and sleep deprivation both increased the expression of BAX and CCAR2 genes, and decreased the expression of BCL2 and BMAL1 genes (p < 0.05 for all). L-arginine supplementation increased the expression of BMAL1 and BCL2 genes and decreased the expression of BAX and CCAR2 genes relative to control (p < 0.05). L-Arginine controlled the increase in expression of BAX and CCAR2 genes and the decrease in expression of BCL2 and BMAL1 genes in response to sleep deprivation and acute anaerobic exercise (p < 0.05). Our results showed that 24-hour sleep deprivation and acute anaerobic exercise increased the expression of pro-apoptotic genes (BAX and CCAR2) and decreased the expression of anti-apoptotic genes (BCL2 and BMAL1), although the effect of sleep deprivation is greater. In this situation, L-arginine supplementation may balance the apoptotic state of peripheral blood mononuclear cells. However, any recommendation about this needs further research.

Timing Is Important—Management of Metabolic Syndrome According to the Circadian Rhythm

Physiological processes occur in accordance with a rhythm regulated by the endogenous biological clock. This clock is programmed at the molecular level and synchronized with the daily light-dark cycle, as well as activities such as feeding, exercise, and social interactions. It consists of the core clock genes, Circadian Locomotor Output Cycles Protein Kaput (CLOCK) and Brain and Muscle Arnt-Like protein 1 (BMAL1), and their products, the period (PER) and cryptochrome (CRY) proteins, as well as an interlocked feedback loop which includes reverse-strand avian erythroblastic leukemia (ERBA) oncogene receptors (REV-ERBs) and retinoic acid-related orphan receptors (RORs). These genes are involved in the regulation of metabolic pathways and hormone release. Therefore, circadian rhythm disruption leads to development of metabolic syndrome (MetS). MetS refers to a cluster of risk factors (RFs), which are not only associated with the development of cardiovascular (CV) disease (CVD), but also with increased all-cause mortality. In this review, we consider the importance of the circadian rhythm in the regulation of metabolic processes, the significance of circadian misalignment in the pathogenesis of MetS, and the management of MetS in relation to the cellular molecular clock.

Protective effect of brain and muscle arnt-like protein-1 against ethanol-induced ferroptosis by activating Nrf2 in mice liver and HepG2 cells

Alcohol abuse has recently become a serious health concern worldwide, and the incidence of alcoholic liver disease (ALD) is rapidly increasing with high morbidity and mortality. Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation. Here we showed that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis. Upon exposure to alcohol (5 % Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo, 300 mmol/L for 12 h in vitro, respectively), the content of iron, reactive oxygen species (ROS) and malondialdehyde (MDA) was boosted significantly while glutathione (GSH) was decreased that mainly based on the downregulated protein expression of ferritin heavy chain (FTH), ferroportin (FPN), heme oxygenase1(HO-1) and anti-cystine/glutamate antiporter (SLC7A11), while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1 (5 mg/kg body weight for 10 days in vivo, 10 μmol/L for 2 h in vitro, respectively)]. Further study indicated that the alcohol could activate the protein expression of brain and muscle arnt-like protein-1 (BMAL1) which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into nuclear and subsequently stimulating its downstream proteins FTH, FPN, glutathione peroxidase 4 activity (GPX4), HO-1, SLC7A11, while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol. Collectively, our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.

Major roles of the circadian clock in cancer.

Circadian rhythms are natural rhythms that widely exist in all creatures, and regulate the processes and physiological functions of various biochemical reactions. The circadian clock is critical for cancer occurrence and progression. Its function is regulated by metabolic activities, and the expression and transcription of various genes. This review summarizes the composition of the circadian clock; the biological basis for its function; its relationship with, and mechanisms in, cancer; its various functions in different cancers; the effects of anti-tumor treatment; and potential therapeutic targets. Research in this area is expected to advance understanding of circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) in tumor diseases, and contribute to the development of new anti-tumor treatment strategies.

BMAL1/FOXA2-induced rhythmic fluctuations in IL-6 contribute to nocturnal asthma attacks.

Inflammatory cytokine levels were measured in asthma patients with and without nocturnal symptoms. Allergic airway disease was induced in mice by ovalbumin (OVA), and different periods of light/dark cycles were used to induce circadian rhythm disorders. Serum shock was used to stimulate the rhythmic expression in human bronchial epidermal cells (16HBE). The expression and oscillation of circadian clock genes and inflammatory cytokines in 16HBE cells subjected to brain and muscle ARNT-like protein-1 (BMAL1) and Forkhead Box A2 (FOXA2) knockdown and treatment with a FOXA2 overexpression plasmid were assessed. Serum IL-6 was found to be significantly higher in asthmatic patients with nocturnal symptoms than those without nocturnal symptoms. The OVA-induced asthma model with a circadian rhythm disorder and 16HBE cells treated with serum shock showed an increase in IL-6 levels and a negative correlation with BMAL1 and FOXA2. The knockdown of BMAL1 resulted in a lower correlation between IL-6 and other rhythm clock genes. Furthermore, knockdown of the BMAL1 and FOXA2 in 16HBE cells reduced the expression and rhythmic fluctuations of IL-6. Our findings suggest that there are increased IL-6 levels in nocturnal asthma resulting from inhibition of the BMAL1/FOXA2 signalling pathway in airway epithelial cells.

Sex-specific genetic association of brain and muscle Arnt-like protein-1 (BMAL1) and obesity in Chinese youth

BackgroundThe circadian clock gene brain and muscle Arnt-like protein-1 (BMAL1) regulates energy metabolism, adipocyte proliferation and differentiation, glucose metabolism, and other functions. This study aimed to examine the association of potential polymorphisms in BMAL1 with obesity among Chinese youth. MethodsA total of 2973 participants were included in this study. According to the body mass index obesity standard of China, 208 subjects were defined as experiencing general obesity. According to the waist-to-hip ratio obesity standard, 335 participants were defined as experiencing central obesity. Four single nucleotide polymorphisms (SNPs) (rs9633835, rs6486121, rs7107287, and rs12364562) were genotyped using TaqMan probe techniques. ResultsThere was no significant difference in the either genotypic or allelic frequencies between the non-general and general obesity groups, while a positive association was observed between BMAL1 rs6486121 variant and central obesity risk (CC＋CT vs. TT: OR:2.139, 95% CI:1.164–3.930; P = 0.014) after adjusting for covariates. Stratification analyses revealed significant associations with central obesity risk for rs6486121 polymorphism in women according to the additive model (CC vs. CT vs. TT: OR：1.409; 95 % CI: 1.029–1.930; P = 0.032). Haplotype analysis showed that only paired haplotypes, including rs9633835G with rs6486121T, had a significant effect on central obesity with OR (95%CI) was 1.035 (1.011–1.060) and P = 0.004. Conclusionour findings suggest that BMAL1 polymorphisms are significantly associated with central obesity and sex-specific genetic effects on BMAL1-mediated genetic susceptibility to obesity.

Time-restricted feeding mitigates obesity through adipocyte thermogenesis.

Misalignment of feeding rhythms with the light-dark cycle leads to disrupted peripheral circadian clocks and obesity. Conversely, restricting feeding to the active period mitigates metabolic syndrome through mechanisms that remain unknown. We found that genetic enhancement of adipocyte thermogenesis through ablation of the zinc finger protein 423 (ZFP423) attenuated obesity caused by consumption of a high-fat diet during the inactive (light) period by increasing futile creatine cycling in mice. Circadian control of adipocyte creatine metabolism underlies the timing of diet-induced thermogenesis, and enhancement of adipocyte circadian rhythms through overexpression of the clock activator brain and muscle Arnt-like protein-1 (BMAL1) ameliorated metabolic complications during diet-induced obesity. These findings uncover rhythmic creatine-mediated thermogenesis as an essential mechanism that drives metabolic benefits during time-restricted feeding.

Role of brain and muscle ARNT-like protein 1 in the rat periodontitis-induced liver injury model

Objective: Brain and muscle ARNT-like protein 1 (BMAL1) is a core component of hepatocyte molecular clock and plays an important role in the regulation of other related rhythmic genes in the body through a transcriptional-translational feedback loop in molecular circadian oscillations. Therefore, the aim of this study was to investigate the role of BMAL1 in the rat periodontitis-induced liver injury. Methods: Twelve male Wistar rats were divided into the control group and the periodontitis group according to the random number table method. The rats in the control group were untreated. The periodontitis models were established by ligating the necks of the bilateral maxillary first molars in the periodontitis group rats. After 8 weeks, periodontal clinical indexes of rats in both groups were examined and executed. Micro-CT scans of the maxilla were performed and levels of the alveolar bone resorption were analyzed. Pathological changes in periodontal and liver tissue of rats in two groups were detected by HE and oil red O staining. Biochemical kits were used to detect glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total cholesterol (TC) and triglycerides (TG) in serum. The gene and protein expression levels of BMAL1, nuclear factor kappa-B (NF-κB) and tumor necrosis factor-α (TNF-α) in liver tissue were measured by real time fluorescent quantitative-PCR (qRT-PCR), immunohistochemistry (IHC) and Western blotting (WB) assays. Apoptosis was detected in liver tissues by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) kit staining. Results: The results of HE staining of maxillary first molars and micro-CT results of maxillary bones showed that alveolar bone resorption was significant in the periodontitis group of rats. The liver histopathology results showed infiltrated inflammatory cells in the liver tissue, disorganized liver cords and a large number of lipid droplets formed in the hepatocytes of the periodontitis group compared with the control group. The results of serum biochemical assay showed that the levels of GOT [(62.77±2.59) U/L], GPT [(47.54±1.04) U/L], TC [(3.19±0.23) mmol/L] and TG [(1.11±0.09) mmol/L] in the serum of rats with periodontitis were significantly higher than that in the control group respectively [GOT: (38.66±2.47) U/L, GPT: (31.48±1.57) U/L, TC: (1.60±0.05) mmol/L and TG: (0.61±0.09) mmol/L](P=0.003, P=0.001, P=0.002, P=0.038). qRT-PCR results showed that the mRNA expression level of BMAL1 was significantly decreased in liver tissue of the periodontitis group [(0.60±0.04)%] compared to the control group [(1.01±0.07)%] (t=4.80, P=0.009), while the mRNA expression levels of NF-κB and TNF-α [(1.62±0.12)%, (2.69±0.16)%] were significantly increased compared to the control group [(1.00±0.03)%, (1.03±0.16)%] (P=0.008, P=0.002); IHC results showed that the protein expression level of BMAL1 in liver tissue of the periodontitis group (averaged optical density, AOD) (11.58±2.15) was down-regulated compared to the control group (AOD) (22.66±1.67) (P=0.015), while NF-κB and TNF-α (AOD) (31.77±2.69, 24.31±2.32) were up-regulated compared to the control group (AOD) (19.40±1.82, 11.92±0.94) (P=0.019, P=0.008). WB results showed that the protein expression level of BMAL1 in liver tissue was down-regulated in the periodontitis group [(0.63±0.10)%] compared to the control group [(1.00±0.06)%] (t=3.19, P=0.033), while NF-κB and TNF-α [(1.61±0.12)%, (2.82±0.23)%] were up-regulated compared to the control group [(1.00±0.12)%, (1.00±0.11)%] (P=0.022, P=0.002). TUNEL staining showed increased apoptotic cells in the liver tissue of the periodontitis group of rats compared to the control group. Conclusions: Periodontitis may induce liver injury by down-regulating the BMAL1 expression levels in liver tissue, which in turn activates NF-κB signaling molecules, leading to the elevated levels of inflammation and apoptosis in rat liver.

Nuclear receptor subfamily 1 group D member 1 in the pathology of obesity-induced osteoarthritis progression.

Mechanical overload and chemical factors are both related to obesity-induced progression of knee osteoarthritis. The circadian rhythm is related to the development of metabolic syndrome and the progression of osteoarthritis, and the core clock genes nuclear receptor subfamily 1 group D member 1 (NR1D1) and brain and muscle arnt-like protein 1 (BMAL1) are dysregulated in cartilage from patients with osteoarthritis. Here, we focused on NR1D1 and investigated osteoarthritis-related changes and gene expression in a mouse model of diet-induced obesity. A high-fat diet was provided to C57BL6/J mice, and changes in body weight, blood lipids, and gene expression were investigated. Destabilization of the medial meniscus or sham surgery was performed on mice fed a high-fat diet or normal diet, and histological osteoarthritis-related changes and NR1D1 expression were investigated. The effects of the NR1D1 agonist SR9009 were also assessed. Mice fed a high-fat diet developed significant obesity and dyslipidemia. Nr1d1 and Bmal1 gene expression levels decreased in the liver and knee joints. Moreover, increased osteoarthritis progression and decreased NR1D1 protein expression were observed in high-fat diet-fed mice after surgical osteoarthritis induction. SR9009 decreased the progression of obesity, dyslipidemia, and osteoarthritis. Overall, obesity and dyslipidemia induced by the high-fat diet led to osteoarthritis progression and decreased NR1D1 expression. Thus, NR1D1 may play an important role in obesity-induced osteoarthritis.

BMAL1 promotes colorectal cancer cell migration and invasion through ERK- and JNK-dependent c-Myc expression.

Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT-like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial-to-mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK-8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial-to-mesenchymal transition (EMT) pathway and induce the β-catenin release further promotes the expression of oncogene c-Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK-signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c-Myc in vitro. Taken together, these results suggested that the BMAL1/ c-Myc-signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK-dependent pathway. Our study showed that BMAL1 promotes CRC metastasis through MAPK-c-Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

Bmal1-knockout mice exhibit reduced cocaine-seeking behaviour and cognitive impairments

Brain and Muscle Arnt-like Protein 1 (BMAL1) is an essential component of the molecular clock underlying circadian rhythmicity. Its function has been recently associated with mood and reward processing alterations. We investigated the behavioural and neurobiological impact of Bmal1 gene deletion in mice, and how this could affect rewarding effects of cocaine. Additionally, key clock genes and components of the dopamine system were assessed in several brain areas. Our results evidence behavioural alterations in Bmal1-KO mice, including changes in locomotor activity with impaired habituation to environments, short-term memory and social recognition impairments. In addition, Bmal1-KO mice experienced reduced cocaine-induced sensitisation and rewarding effects of cocaine as well as reduced cocaine-seeking behaviour. Furthermore, Bmal1 deletion influenced the expression of other clock-related genes in the mPFC and striatum, as well as alterations in the expression of dopaminergic elements. Overall, the present article offers a novel and extensive characterisation of Bmal1-KO animals. We suggest that reduced cocaine's rewarding effects in these mutant mice might be related to Bmal1 role as an expression regulator of MAO and TH, two essential enzymes involved in dopamine metabolism.

Restoring the dampened expression of the core clock molecule BMAL1 protects against compression-induced intervertebral disc degeneration

The circadian clock participates in maintaining homeostasis in peripheral tissues, including intervertebral discs (IVDs). Abnormal mechanical loading is a known risk factor for intervertebral disc degeneration (IDD). Based on the rhythmic daily loading pattern of rest and activity, we hypothesized that abnormal mechanical loading could dampen the IVD clock, contributing to IDD. Here, we investigated the effects of abnormal loading on the IVD clock and aimed to inhibit compression-induced IDD by targeting the core clock molecule brain and muscle Arnt-like protein-1 (BMAL1). In this study, we showed that BMAL1 KO mice exhibit radiographic features similar to those of human IDD and that BMAL1 expression was negatively correlated with IDD severity by systematic analysis based on 149 human IVD samples. The intrinsic circadian clock in the IVD was dampened by excessive loading, and BMAL1 overexpression by lentivirus attenuated compression-induced IDD. Inhibition of the RhoA/ROCK pathway by Y-27632 or melatonin attenuated the compression-induced decrease in BMAL1 expression. Finally, the two drugs partially restored BMAL1 expression and alleviated IDD in a diurnal compression model. Our results first show that excessive loading dampens the circadian clock of nucleus pulposus tissues via the RhoA/ROCK pathway, the inhibition of which potentially protects against compression-induced IDD by preserving BMAL1 expression. These findings underline the importance of the circadian clock for IVD homeostasis and provide a potentially effective therapeutic strategy for IDD.

Down-regulation of BMAL1 by MiR-494-3p Promotes Hepatocellular Carcinoma Growth and Metastasis by Increasing GPAM-mediated Lipid Biosynthesis.

The circadian clock confers daily rhythmicity to many crucial biological processes and behaviors and its disruption is closely associated with carcinogenesis in several types of cancer. Brain and muscle arnt-like protein 1 (BMAL1) is a core circadian rhythm component in mammals and its dysregulation has been shown to contribute to aberrant metabolism in human diseases. However, the biological functions of BMAL1, especially its involvement in aberrant lipid metabolism in hepatocellular carcinoma (HCC), remain elusive. In the present study, we found that BMAL1 was frequently down-regulated in HCC cells mainly due to the up-regulation of miR-494-3p. Down-regulation of BMAL1 was significantly associated with poor survival in HCC patients. BMAL1 down-regulation promoted HCC cell growth and metastasis both in vitro and in vivo. Mechanistically, through cooperating with EZH2, BMAL1 transcriptionally suppressed the expression of glycerol-3-phosphate acyltransferase mitochondrial (GPAM), a key enzyme involved in the regulation of lipid biosynthesis, leading to reduced levels lysophosphatidic acid (LPA), which have long been known as mediator of oncogenesis. Particularly, treatment with SR8278, an activator of BMAL1, exhibited a therapeutic effect on HCC in vitro and in vivo. In conclusion, BMAL1 plays a critical anti-oncogenic role in HCC, providing strong research evidence for BMAL1 as a prospective target for HCC therapy.

Melatonin Reduces Apoptosis Resistance of Hepatocellular Carcinoma Cells by Inhibiting BMAL1

Purpose:This study aimed to explore the role and molecular mechanism of brain and muscle ARNT-like protein 1 (BMAL1) in hepatocellular carcinoma (HCC), and the effect of melatonin on BMAL1 expression and apoptosis of HCC cells.Methods:We mainly used immunohistochemistry, western blot, cck-8 assays, flow cytometry, wound-healing assay, transwell assay, and RT-qPCR for this research.Results:The expression of BMAL1 protein was frequently up-regulated in the tissues and cell lines of HCC patients. Its high expression was significantly associated with tumor size, tumor differentiation degree, and shorter survival. In addition, cell functional experiments showed that BMAL1 could promote proliferation and migration, and inhibit apoptosis in HCC cell lines. Furthermore, the expression of BMAL1 was related to the endoplasmic reticulum stress (ERS) level. Knockdown of BMAL1 could inhibit the expression of ERS-related protein, while overexpression of BMAL1 led to the increase of ERS-related protein’s level. Low concentration of ERS led to the increase of BMAL1, and a certain degree of ERS in turn inhibited the expression of BMAL1. Melatonin promoted apoptosis of hepatoma cells by inhibiting the expression of BMAL1.Conclusion:BMAL1 plays a key role in HCC patients’ survival and tumor growth, which may be related to its interaction with ERS-related pathways. Melatonin can regulate ERS-related apoptosis resistance by inhibiting BMAL1 expression, promoting apoptosis of HCC cells.

NADH inhibition of SIRT1 links energy state to transcription during time-restricted feeding

In mammals, circadian rhythms are entrained to the light cycle and drive daily oscillations in levels of NAD+, a cosubstrate of the class III histone deacetylase sirtuin 1 (SIRT1) that associates with clock transcription factors. Although NAD+ also participates in redox reactions, the extent to which NAD(H) couples nutrient state with circadian transcriptional cycles remains unknown. Here we show that nocturnal animals subjected to time-restricted feeding of a calorie-restricted diet (TRF-CR) only during night-time display reduced body temperature and elevated hepatic NADH during daytime. Genetic uncoupling of nutrient state from NADH redox state through transduction of the water-forming NADH oxidase from Lactobacillus brevis (LbNOX) increases daytime body temperature and blood and liver acyl-carnitines. LbNOX expression in TRF-CR mice induces oxidative gene networks controlled by brain and muscle Arnt-like protein 1 (BMAL1) and peroxisome proliferator-activated receptor alpha (PPARα) and suppresses amino acid catabolic pathways. Enzymatic analyses reveal that NADH inhibits SIRT1 in vitro, corresponding with reduced deacetylation of SIRT1 substrates during TRF-CR in vivo. Remarkably, Sirt1 liver nullizygous animals subjected to TRF-CR display persistent hypothermia even when NADH is oxidized by LbNOX. Our findings reveal that the hepatic NADH cycle links nutrient state to whole-body energetics through the rhythmic regulation of SIRT1.

BMAL1 attenuates intracerebral hemorrhage-induced secondary brain injury in rats by regulating the Nrf2 signaling pathway.

BackgroundIntracerebral hemorrhage (ICH) is a severe cerebrovascular disease with high morbidity and mortality rates. Oxidative stress and inflammation are important pathological mechanisms of secondary brain injury (SBI) after ICH. Brain and muscle Arnt-like protein 1 (BMAL1), which forms the core component of the circadian clock, was previously shown to be involved in many diseases and to participate in oxidative stress and inflammatory responses. However, the role of BMAL1 in SBI following ICH is unknown. In addition, treatments targeting miR-155 and its downstream signaling pathway may exert a beneficial effect on SBI after ICH, while miR-155 may regulate Bmal1 mRNA stability and translation. Nevertheless, researchers have not clearly determined whetheantagomir-155 upregulates BMAL1 expression and subsequently attenuates ICH-induced brain injury in rats.MethodsAfter establishing an ICH rat model by injecting autologous blood, the time course of changes in levels of the BMAL1 protein after ICH was analyzed. Subsequently, this study was designed to investigate the potential role and mechanisms of BMAL1 in SBI following ICH using lentiviral overexpression and antagomir-155 treatments.ResultsBMAL1 protein levels were significantly decreased in the ICH group compared to the sham group. Genetic overexpression of BMAL1 alleviated oxidative stress, inflammation, brain edema, blood-brain barrier injury, neuronal death, and neurological dysfunction induced by ICH. On the other hand, we observed that inhibiting miRNA-155 using antagomir-155 promoted the expression of BMAL1 and further activated the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway to attenuate brain injury after ICH.ConclusionsThese results reveal that BMAL1 serves as a neuroprotective agent in ICH and upregulation of BMAL1 attenuates ICH-induced SBI. Therefore, BMAL1 may be a promising therapeutic target for SBI following ICH.

L-Theanine inhibits melanoma cell growth and migration via regulating expression of the clock gene BMAL1

L-Theanine is a unique non-protein amino acid found in green tea, which has been identified as a safe dietary supplement. It has been reported that L-theanine exerts various biological activities. In this study, we explored the anti-cancer effects of L-theanine on melanoma cells. A375, B16-F10, and PIG1 cell lines were used in the present study. EdU labeling, TUNEL and AnnexinV/PI staining, wound-healing, and transwell migration assay were performed to detect the effects of L-theanine on melanoma cell proliferation, apoptosis, and migration. Brain and muscle Arnt-like protein 1 (BMAL1) was knocked down in melanoma cells to evaluate if L-theanine plays the anti-cancer role through regulating circadian rhythm of melanoma cells. The western blot, qRT-PCR, and dual luciferase assay were performed to explore the mechanism involved in the effects of L-theanine on melanoma cells. L-Theanine apparently reduced the viability of melanoma cells. Further experiments showed that L-theanine attenuated the proliferation and migration, and promoted apoptosis of melanoma cells. L-Theanine significantly enhanced the expression of BMAL1, a clock gene in melanoma cells. Down-regulation of BMAL1 suppressed the anti-cancer effects of L-theanine on melanoma cells. Further experiments indicated that the p53 transcriptional activity raised by L-theanine was dependent on BMAL1 expression in melanoma cells. L-Theanine exerts the anti-cancer effect on melanoma cells through attenuating the proliferation and migration, and promoting apoptosis of them, which is dependent on the regulation of the clock gene Bmal1 in melanoma cells.

L-3,4-Dihydroxyphenylalanine Recovers Circadian Rhythm Disturbances in the Rat Models of Parkinson's Disease by Regulating the D1R-ERK1/2-mTOR Pathway.

Objective: Patients with Parkinson's disease (PD) frequently experience disruptions in the 24-h daily profile of both behavioral and biological markers. However, whether L-3,4-dihydroxyphenylalanine (L-dopa) influences these markers associated with circadian rhythm or not is still an open question. This study aims to explore the L-dopa effects on the rhythmic expression of core clock proteins [brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor cycle kaput (CLOCK)], in the striatum of the rat model of PD and its underlying molecular mechanisms.Methods: Unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat models were used in this study. L-dopa administrations were adopted to investigate the changes of circadian rhythm in PD. The behavioral tests and the measurements of the blood pressure (BP) and temperature were evaluated. The striatum was collected at intervals of 4 h. Western blot was used to examine the expressions of clock protein and the molecular protein of the D1R-ERK1/2-mTOR pathway. The rhythmic expressions of symptom parameters and circadian proteins were analyzed using the Cosinor model and/or the coefficient of variability (CV) that was used to describe the variability of the 24-h rhythm.Results: The circadian rhythms of BP and temperature were disrupted in 6-OHDA-lesioned PD rats compared with the sham group, while this process was reversed mildly by L-dopa treatment. The expressions of BMAL1 and CLOCK protein were rhythmic fluctuated without significant phase alterations when 6-OHDA or L-dopa was applied. Furthermore, the expressions of striatal BMAL1 protein in the 6-OHDA-lesioned group were significantly lower than those in the sham group at 04:00, 08:00, and 12:00, and the CLOCK protein was decreased at 04:00, 08:00, 12:00, 16:00, and 20:00 (all p < 0.05). The CV of the expressions of both BMAL1 and CLOCK was decreased in the 6-OHDA group; this process was reversed by L-dopa. Moreover, the CV of BMAL1 and CLOCK was elevated in the L-dopa rats. The phosphorylation levels of ERK1/2, S6K1, and 4E-BP1 in 6-OHDA-lesioned striatum were increased by L-dopa or D1 receptor agonist SKF38393 (p < 0.05, respectively), not by the combination of L-dopa and D1 receptor antagonist SCH23390, which was similar to the expressions of BMAL1 and CLOCK.Conclusion: L-dopa recovers the circadian rhythm disturbances in PD rats by regulating the D1R-ERK1/2-mTOR pathway.

Roles of NPAS2 in circadian rhythm and disease.

NPAS2, a circadian rhythm gene encoding the neuronal PAS domain protein 2 (NPAS2), has received widespread attention because of its complex functions in cells and diverse roles in disease progression, especially tumorigenesis. NPAS2 binds with DNA at E-box sequences and forms heterodimers with another circadian protein, brain and muscle ARNT-like protein 1 (BMAL1). Nucleotide variations of the NPAS2 gene have been shown to influence the overall survival and risk of death of cancer patients, and differential expression of NPAS2 has been linked to patient outcomes in breast cancer, lung cancer, non-Hodgkin's lymphoma, and other diseases. Here, we review the latest advances in our understanding of NPAS2 with the aim of drawing attention to its potential clinical applications and prospects.