BMAL1 as a central chronobiological integrator of intestinal homeostasis, inflammation, and tumorigenesis

A link between circadian rhythm and metabolism has long been discussed. Circadian rhythm is controlled by positive and negative transcriptional and translational feedback loops composed of several clock genes. Among clock genes, the brain and muscle Arnt-like protein-1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) play important roles in the regulation of the positive rhythmic transcription. In addition to control of circadian rhythm, we have previously shown that BMAL1 regulates adipogenesis. In metabolic syndrome patients, the function of BMAL1 is dysregulated in visceral adipose tissue. In addition, analysis of SNPs has revealed that BMAL1 is associated with susceptibility to hypertension and type II diabetes. Furthermore, the significant roles of BMAL1 in pancreatic β cells proliferation and maturation were recently reported. These results suggest that BMAL1 regulates energy homeostasis. Therefore, in this study, we examined whether loss of BMAL1 function is capable of inducing metabolic syndrome. Deficient of the Bmal1 gene in mice resulted in elevation of the respiratory quotient value, indicating that BMAL1 is involved in the utilization of fat as an energy source. Indeed, lack of Bmal1 reduced the capacity of fat storage in adipose tissue, resulting in an increase in the levels of circulating fatty acids, including triglycerides, free fatty acids, and cholesterol. Elevation of the circulating fatty acids level induced the formation of ectopic fat in the liver and skeletal muscle in Bmal1 -/- mice. Interestingly, ectopic fat formation was not observed in tissue-specific (liver or skeletal muscle) Bmal1 -/- mice even under high fat diet feeding condition. Therefore, we were led to conclude that BMAL1 is a crucial factor in the regulation of energy homeostasis, and disorders of the functions of BMAL1 lead to the development of metabolic syndrome.

Brain and muscle Arnt-like protein-1 (BMAL1) plays an important role in circadian rhythm, which is involved in daily behaviours and physiological activities. However, little is known about the molecular function of BMAL1 in the Pigeon ( Columba livia). In our study, the full-length cDNA of Bmal1 was cloned and sequenced from the Pigeon for the first time, and submitted to the GenBank to obtain the accession number (KF906247). The full-length cDNA of Bmal1 consists of 2,488 nucleotides, and encodes 634 amino acids. Phylogenetic analysis showed that it bore the greatest similarity to Bmal1 from the Chicken ( Gallus gallus) and Barn Owl ( Tyto alba). The amino acid sequence of the Pigeon BMAL1 contained a HLH domain and two PAS domains, which are involved in forming hetero-homodimers with the CLOCK as the positive element of the circadian rhythm. The results of real-time quantitative PCR of Bmal1 under different light regimes showed that the amplitude and expression pattern of Bmal1 were strongly affected by day length. Bmal1 was most highly expressed in the pancreas. Relative to Bmal1 expression level under 12 h of light exposure, it was increased significantly in the pituitary gland, ovary and uterus under 15 h of light exposure ( P < 0.05). However, other tissues, including the hypothalamus, heart, liver, spleen, kidney, intestines, crureus, and pectorals exhibited no significant difference ( P < 0.05) under the two light regimes. This is the first study to investigate Bmal1 mRNA levels in various tissues under different light cycles, and thereby provide data for further study of the molecular and regulatory mechanisms of Bmal1 and circadian clock genes of the Pigeon.

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes.

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1(-/-) and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1(-/-) primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1(-/-) hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.

Cardiac function is markedly impaired as a result of myocardial fibrosis, a major pathological consequence that develops after myocardial infarction (MI). While BMAL1 (Brain and Muscle ARNT-like protein 1), a core circadian rhythm regulator, has been implicated in various cardiovascular pathologies, its role in post-MI cardiac fibrosis remains unclear. This study aimed to elucidate the role and underlying molecular mechanisms of BMAL1 in cardiac fibrosis. MI was induced in mice by permanent ligation of the left anterior descending coronary artery, and TGF-β1 was used to induce fibroblast activation in vitro. BMAL1 expression was manipulated through adeno-associated virus 9 (AAV9) overexpression and small interfering RNA (siRNA) knockdown. Our findings revealed a downregulation of BMAL1 expression in both infarcted myocardial tissue and TGF-β1–treated cardiac fibroblasts. In vivo, AAV9-mediated BMAL1 overexpression in MI mice significantly improved cardiac function and reduced myocardial fibrotic area. At the cellular level, BMAL1 overexpression effectively inhibited TGF-β1-induced fibroblast activation and extracellular matrix (ECM) deposition. Conversely, BMAL1 knockdown exacerbated fibroblast activation. Mechanistically, we demonstrated that BMAL1 suppresses the TGF-β1/SMAD3 signaling cascade by enhancing SMAD7 expression, reducing the expression of fibrosis-related genes. Collectively, our findings reveal BMAL1 as a critical negative regulator of post-MI myocardial fibrosis by inhibiting the TGF-β1/SMAD3 pathway mediated by SMAD7. Targeting BMAL1 may offer a novel therapeutic approach for improving cardiac remodeling following MI.

Brain and muscle ARNT-like protein 1 (BMAL1) is necessary for fertility and has been found to be essential to follicle growth and steroidogenesis. Sirtuin1 (SIRT1) has been reported to interact with BMAL1 and function in a circadian manner. Evidence has shown that SIRT1 regulates aromatase expression in estrogen-producing cells. We aimed to ascertain if there is a relationship between polycystic ovary syndrome (PCOS) and BMAL1, and whether and how BMAL1 takes part in estrogen synthesis in human granulosa cells (hGCs). Twenty-four women diagnosed with PCOS and 24 healthy individuals undergoing assisted reproduction were studied. BMAL1 expression in their granulosa cells (GCs) was observed by quantitative real-time polymerase chain reaction (qRT-PCR). The level of expression in the PCOS group was lower than that of the group without PCOS (p<0.05). We also analyzed estrogen synthesis and aromatase expression in KGN cell lines. Both were downregulated after BMAL1 and SIRT1 knock-down and, conversely, upregulated after overexpression treatments of these two genes in KGN cells. Both BMAL1 and SIRT1 had a mutually positive regulation, as did the phosphorylation of JNK. Furthermore, JNK overexpression increased estrogen synthesis activity and the expression levels of aromatase, BMAL1, and SIRT1. In KGN and hGCs, estrogen synthesis and aromatase expression were downregulated after treatment with JNK and SIRT1 inhibitors. In addition, BMAL1, SIRT1, and JNK expression levels were all downregulated. Our results demonstrate the effects of BMAL1 on estrogen synthesis in hGCs and suggest a BMAL1-SIRT1-JNK positive feedback cycle in this process, which points out an important role of BMAL1 in the development of PCOS.

Circadian disruptions appear at the presymptomatic stage of Alzheimer's disease (AD) and may exacerbate mental dysfunction in AD. The downregulation of brain and muscle ARNT-like protein 1 (BMAL1), a key clock element for the maintenance of circadian rhythms, has been linked to epigenetic mechanisms. Our previous study revealed that the mRNA level of DNA demethylase ten-eleven translocation ( Tet) 3 was reduced in the hippocampi of APPswe/PS1dE9 (APP/PS1) mice. However, the effects of TET3 on BMAL1 downregulation and circadian dysregulation in AD are still unclear. Our investigation first confirms that Tet3 mRNA and protein levels are decreased in both APP/PS1 mice and APPswe cells. In addition, decreased levels of 5hmC are observed in HT22 cells after TET3 knockdown, whereas TET3 overexpression reverses the reduction in 5hmC. Critically, we report that TET3 knockdown remethylates the Bmal1 promoter, thus downregulating BMAL1 expression in HT22 cells. In contrast, TET3 overexpression could upregulate BMAL1 by decreasing its methylation level. These results indicate that reduced TET3 is responsible for BMAL1 downregulation through decreased TET3 demethylation. Additionally, TET3 knockdown could lead to circadian disruption of BMAL1 in U2OS cells, whereas overexpression of TET3 alleviates the dysregulated biological rhythm in Aβ-treated U2OS cells. Our data suggest that TET3 plays a vital role in modulating the circadian rhythm at the epigenetic level through DNA demethylation.

The transcription factor brain and muscle Arnt-like protein-1 (BMAL1) is a clock protein involved in various diseases, including atherosclerosis and cancer. However, BMAL1's involvement in kidney fibrosis and the underlying mechanisms remain largely unknown, a gap addressed in this study. Analysis through Masson's trichrome and Sirius red staining revealed that all groups exposed to unilateral ureteral obstruction showed increased BMAL1 protein expression accompanied by increased TGF-β1 expression and elevated key fibrosis markers, including α-SMA, compared with sham groups. Although TGF-β1 induced BMAL1 protein expression accompanied by increased α-SMA expression in NRK-49F cells, the REV-ERBα agonist GSK4112, a transcriptional repressor of BMAL1, or siRNA targeting BMAL1 significantly inhibited TGF-β1-induced α-SMA expression. Furthermore, BMAL1 knockdown significantly suppressed TGF-β1-induced NOX4/ROS/p38 pathways in NRK-49F cells. Thus, BMAL1 positively regulates TGF-β1-induced signaling associated with fibrotic responses via the NOX4/ROS/p38 pathway. Overall, this study uncovers BMAL1 as a promising therapeutic target for preventing and treating kidney fibrosis, potentially preventing renal failure.

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.

The role of circadian clock gene BMAL1 in vascular proliferation

Deficiency of Bmal1 disrupts the diurnal rhythm of haemostasis.

Disrupted-in-schizophrenia 1 (DISC1) is a scaffold protein that has been implicated in multiple mental disorders. DISC1 is known to regulate neuronal proliferation, signaling, and intracellular calcium homeostasis, as well as neurodevelopment. Although DISC1 was linked to sleep-associated behaviors, whether DISC1 functions in the circadian rhythm has not been determined yet. In this work, we revealed that Disc1 expression exhibits daily oscillating pattern and is regulated by binding of circadian locomotor output cycles kaput (CLOCK) and Brain and muscle Arnt-like protein-1 (BMAL1) heterodimer to E-box sequences in its promoter. Interestingly, Disc1 deficiency increases the ubiquitination of BMAL1 and de-stabilizes it, thereby reducing its protein levels. DISC1 inhibits the activity of GSK3β, which promotes BMAL1 ubiquitination, suggesting that DISC1 regulates BMAL1 stability by inhibiting its ubiquitination. Moreover, Disc1-deficient cells and mice show reduced expression of other circadian genes. Finally, Disc1-LI (Disc1 knockout) mice exhibit damped circadian physiology and behaviors. Collectively, these findings demonstrate that the oscillation of DISC1 expression is under the control of CLOCK and BMAL1, and that DISC1 contributes to the core circadian system by regulating BMAL1 stability.

It is known that baldness caused by androgenetic alopecia is involved with androgen and the androgen receptor. Furthermore, it has been reported that testosterone secretion follows a circadian rhythm. Therefore, we hypothesized that a relationship exists between androgen-induced alopecia and biological rhythm. The mammalian circadian rhythm is controlled by several clock genes. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1), one of the clock genes, is a transcription factor that plays central roles in the regulation of circadian rhythms. In this study, we investigated the influence of BMAL1 on hair follicle functions and hair growth. Mice deficient in BMAL1 expression exhibited a delay in hair regrowth after shaving. In hair follicles of mouse vibrissa, expression of Bmal1 and other clock genes was found to be rhythmic. Knockdown of BMAL1 in human follicle dermal papilla cells resulted in modulation of expression of several hair growth-related genes. Therefore, we concluded that expression of clock genes in hair follicles is linked to the circadian rhythm and that BMAL1 can regulate hair growth.

Acute kidney injury (AKI) is a common and severe complication of sepsis and represents an independent risk factor for mortality in septic patients. Despite its clinical significance, the mechanisms of sepsis-induced AKI (Sepsis-AKI) remain incompletely understood. This study investigates the role of methyltransferase like 14 (METTL14)-mediated m6A modification in regulating brain and muscle ARNT-like protein-1 (BMAL1) stability and its effect on tubular epithelial cell injury and ferroptosis. Human renal proximal tubular epithelial (HK-2) cells were treated with lipopolysaccharide (LPS) to establish an invitro model of Sepsis-AKI. Cell proliferation and viability were assessed using EdU and CCK-8 assays; apoptosis was evaluated by TUNEL staining, and inflammatory cytokines Interleukin-6 (IL-6) and IL-1β were measured by ELISA. Ferroptosis indices were detected using corresponding kits. RT-qPCR and Western blotting were used to detect mRNA and protein expression. MeRIP and RIP assays were used to evaluate BMAL1 m6A modification and RNA-protein interaction. The stability of BMAL1 mRNA was determined using an Actinomycin D chase assay. A Sepsis-AKI model was established to examine the effect of METTL14 silencing on renal injury. BMAL1 overexpression significantly alleviated LPS-induced apoptosis, inflammatory responses, and ferroptosis in HK-2 cells. Furthermore, METTL14 silencing reduced BMAL1 m6A modification, stabilized BMAL1 mRNA, and consequently improved HK-2 cell injury. In addition, YTHN6-methyladenosine RNA binding protein 1 (YTHDF1) was identified as the critical m6A reader mediating BMAL1 mRNA degradation. Consistently, invivo experiments demonstrated that METTL14 knockdown mitigated Sepsis-AKI and ferroptosis in mice. METTL14 enhanced BMAL1 m6A modification and promoted YTHDF1-mediated BMAL1 degradation, thereby facilitating ferroptosis and aggravating Sepsis-AKI.

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.