The renin-angiotensin-aldosterone system (RAAS) is crucial for regulating blood pressure and maintaining fluid balance, while clock genes are essential for sustaining biological rhythms and regulating metabolism. There exists a complex interplay between RAAS and clock genes that may significantly contribute to the development of various cardiovascular and metabolic diseases. Although current literature has identified correlations between these two systems, the specific mechanisms of their interaction remain unclear. Moreover, the interaction patterns under different physiological and pathological conditions need further investigation. This review summarizes the synergistic roles of the RAAS and clock genes in cardiovascular diseases, explores their molecular mechanisms and pathophysiological connections, discusses the application of chronotherapy, and highlights potential future research directions, aiming to provide novel insights for the prevention and treatment of related diseases.
Humans ; Renin-Angiotensin System/genetics* ; Cardiovascular Diseases/genetics* ; CLOCK Proteins/physiology* ; Animals

Recently, male reproductive health has attracted extensive attention, with the adverse effects of circadian disruption on male fertility gradually gaining recognition. However, the mechanism by which circadian disruption leads to damage to male reproductive system remains unclear. In this review, we first summarized the dual regulatory roles of circadian clock genes on the male reproductive system: (1) circadian regulation of testosterone synthesis via the hypothalamic-pituitary-testicular (HPT) and hypothalamic-pituitary-adrenal (HPA) axes; (2) non-circadian regulation of spermatogenesis. Next, we further listed the possible mechanisms by which circadian disruption impairs male fertility, including interference with the oscillatory function of the reproductive system, i.e., synchronization of the HPT axis, crosstalk between the HPT axis and the HPA axis, as well as direct damage to germ cells by disturbing the non-oscillatory function of the reproductive system. Future research using spatiotemporal omics, epigenomic assays, and neural circuit mapping in studying the male reproductive system may provide new clues to systematically unravel the mechanisms by which circadian disruption affects male reproductive system through circadian clock genes.
Male ; Humans ; Animals ; Circadian Clocks/physiology* ; Hypothalamo-Hypophyseal System/physiology* ; Circadian Rhythm/genetics* ; Spermatogenesis/physiology* ; Pituitary-Adrenal System/physiology* ; Testis/physiology* ; Testosterone/biosynthesis* ; CLOCK Proteins ; Infertility, Male/physiopathology*

The circadian clock is a highly conserved timekeeping system in organisms, which maintains physiological homeostasis by precisely regulating periodic fluctuations in gene expression. Substantial clinical and experimental evidence has established a close association between circadian rhythm disruption and the development of various malignancies. Research has revealed characteristic alterations in the circadian gene expression profiles in tumor tissues, primarily manifested as a dysfunction of core clock components (particularly circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1)) and the widespread dysregulation of their downstream target genes. Notably, CLOCK demonstrates non-canonical oncogenic functions, including epigenetic regulation via histone acetyltransferase activity and the circadian-independent modulation of cancer pathways. This review systematically elaborates on the oncogenic mechanisms mediated by CLOCK/BMAL1, encompassing multidimensional effects such as cell cycle control, DNA damage response, metabolic reprogramming, and tumor microenvironment (TME) remodeling. Regarding the therapeutic strategies, we focus on cutting-edge approaches such as chrononutritional interventions, chronopharmacological modulation, and treatment regimen optimization, along with a discussion of future perspectives. The research breakthroughs highlighted in this work not only deepen our understanding of the crucial role of circadian regulation in cancer biology but also provide novel insights for the development of chronotherapeutic oncology, particularly through targeting the non-canonical functions of circadian proteins to develop innovative anti-cancer strategies.
Humans ; ARNTL Transcription Factors/physiology* ; Neoplasms/therapy* ; CLOCK Proteins/physiology* ; Circadian Clocks/genetics* ; Animals ; Circadian Rhythm/genetics* ; Tumor Microenvironment ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic

This study aims to investigate the mechanism of berberine in regulating the metabolism network via clock-controlled genes represented by brain and muscle arnt-like 1(BMAL1) to ameliorate insulin resistance(IR) of hepatocytes in vitro. The HepG2 cell model of dexamethasone-induced IR(IR-HepG2) was established and treated with 5, 10, and 20 μmol·L~(-1) berberine, respectively, for 24 h. The glucose oxidase method and cell counting kit-8(CCK-8) assay were employed to measure extracellular glucose concentration and cell viability, respectively. Periodic acid-Schiff(PAS) staining and lipid fluorescence method were used to detect glycogen and lipids. The immunofluorescence(IF) assay was employed to detect the nuclear localization of BMAL1 and circadian locomotor output cycles kaput(CLOCK) in IR-HepG2 cells. Western blot was employed to determine the protein levels of BMAL1, CLOCK, period circadian clock 2(PER2), cryptochrome circadian regulator 1(CRY1), Rev-Erbα, carbohydrate response element-binding protein(ChREBP), peroxisome proliferator-activated receptors alpha and gamma(PPARα/γ), sterol regulatory element-binding protein 1C(SREBP-1C), mammalian target of rapamycin(mTOR), protein kinase B(Akt), glycogen synthase kinase-3β(GSK3β), acetyl coenzyme A carboxylase 1(ACC1), fatty acid synthase(FASN), carnitine palmitoyltransferase 1α(CPT1α), nicotinamide phosphoribosyltransferase(NAMPT), silent information regulator 1(SIRT1), adiponectin(ADPN), insulin receptor substrate 2(IRS2), and phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85). In addition, the levels of phosphorylated adenosine monophosphate-activated protein kinase alpha(AMPKα), Akt, GSK3β, BMAL1, and mTOR were determined. Furthermore, 20 μmol·L~(-1) CLK8 was added to measure the glucose consumption as well as the protein levels of ChREBP, PPARα, and mTOR in IR-HepG2 cells. The results showed that berberine increased the glucose consumption, lowered the lipid levels, increased the expression and nuclear localization of BMAL1 and CLOCK, and up-regulated the level of BMAL1 in IR-HepG2 cells. Furthermore, berberine up-regulated the levels of ADPN, IRS2, PI3Kp85, p-Akt(Ser473)/Akt, p-mTOR(Ser2448)/mTOR, PPARα, and CPT1α, and down-regulated the levels of p-GSK3β(Ser9)/GSK3β, ChREBP, SREBP-1C, ACC1, and FASN. The addition of CLK8 reduced glucose consumption in IR-HepG2 cells, up-regulated the ChREBP level, and down-regulated PPARα and mTOR levels by inhibiting the BMAL1 and CLOCK interaction. In summary, berberine regulated glucose and lipid metabolism via clock-controlled genes with BMAL1 at the core to ameliorate IR of hepatocytes.
Humans ; Hepatocytes/drug effects* ; Lipid Metabolism/drug effects* ; Glucose/metabolism* ; Berberine/pharmacology* ; Insulin Resistance ; Hep G2 Cells ; CLOCK Proteins/genetics* ; ARNTL Transcription Factors/genetics*

Objective: To investigate the correlation between CLOCK and BMAL1 genes and MEN2 medullary thyroid carcinoma (MTC). Methods: Thirteen cases with MEN2 MTC and thirteen cases with non-MEN2 MTC were selected who were treated in the Yantai Yuhuangding Hospital between January 2013 and September 2021. Clinical indicators such as blood calcitonin level, tumor diameter and metastatic lymph node of patients were collected. The expression differences of CLOCK and BMAL1 between MEN2 MTC and para-carcinoma tissue as well as between MEN2 MTC and non-MEN2 MTC were detected by immunohistochemistry and qPCR. The correlation between lymph node metastasis and CLOCK or BMAL1 expression was analyzed. Protein-protein interaction (PPI) network analysis combined with qPCR and correlation analysis was used to explore the expression regulation relationship between RET and circadian clock genes. The rhythm disorder of MEN2 cells was verified by lipopolysaccharide cell stimulation experiment after dexamethasone rhythm synchronization. Results: MEN2 MTC exhibited typical RET gene mutation. The mean blood calcitonin level, the tumor diameter and the number of metastatic lymph nodes of patients with MEN2 MTC were higher than those of patients with non-MEN2 MTC (t value was 2.76, 2.53, 2.26, all P<0.05). Immunohistochemical results showed that the expression levels of CLOCK and BMAL1 in MEN2 MTC were higher than those in non-MEN2 MTC, while negatively expressed in para-cancerous thyroid follicle. qPCR displayed that the expression of CLOCK gene in cancer tissues was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.68 and 2.86, all P<0.05); the expression of BMAL1 gene in MEN2 MTC was higher than that in non-MEN2 MTC and para-cancerous tissues (t value was 2.21 and 2.35, all P<0.05). Correlation analysis showed that the expression levels of CLOCK and BMAL1 genes were positively correlated with the number of lymph node metastases in patients with MEN2 MTC (r=0.65, P<0.001; r=0.52, P=0.005). PPI network analysis indicated that the expression of CLOCK gene was positively correlated with the abnormal expression of RET gene (r=0.96, P<0.001). With lipopolysaccharide to stimulate cultured cells in vitro after dexamethasone rhythm synchronization, the expressions of CLOCK and BMAL1 in MEN2 MTC cells (0.47±0.22 and 2.60±1.48) at 12 hours of synchronization were significantly lower than those in para-cancerous tissues (1.70±1.62 and 8.23±2.52), the difference was statistically significant(t=5.04, P=0.007; t=3.34, P=0.029). Conclusion: CLOCK and BMAL1 are correlated with the occurrence and development of MEN2 MTC, and may be potential targets for the development of new therapeutic strategies for MEN2 MTC.
ARNTL Transcription Factors/genetics* ; CLOCK Proteins/genetics* ; Calcitonin ; Carcinoma, Neuroendocrine/genetics* ; Dexamethasone ; Humans ; Lipopolysaccharides ; Lymphatic Metastasis ; Multiple Endocrine Neoplasia Type 2a/genetics* ; Thyroid Neoplasms/surgery*

Circadian clock is an internal autonomous time-keeping system, including central clocks located in the suprachiasmatic nucleus (SCN) and peripheral clocks. The molecular circadian clock consists of a set of interlocking transcriptional-translational feedback loops that take the clock-controlled genes 24 h to oscillate. The core mechanism of molecular circadian clock is that CLOCK/BMAL1 dimer activates the transcription of cryptochromes (CRYs) and Periods (PERs), which act as transcriptional repressors of further CLOCK/BMAL1-mediated transcription. In addition to this basic clock, there is an additional sub-loop of REV-ERBα and RORα regulating the transcription of BMAL1. Approximately 80% protein-coding genes demonstrate significant rhythmicity. The earth rotation is responsible for the generation of the daily circadian rhythms. To coordinate metabolic balance and energy availability, almost all organisms adapt to the rhythm. Studies have shown that circadian clock integrating with metabolic homeostasis increases the efficiency of energy usage and coordinates with different organs in order to adapt to internal physiology and external environment soon. As the central organ of metabolism, the liver performs various physiological activities nearly all controlled by the circadian clock. There are multiple interactive regulation mechanisms between the circadian clock and the regulation of liver metabolism. The misalignment of metabolism with tissue circadian is identified as a high-risk factor of metabolic diseases. This article reviews the recent studies on circadian physiological regulation of liver glucose, lipid and protein metabolism and emphasizes oscillation of mitochondrial function. We also take an outlook for new methods and application of circadian clock research in the future.
CLOCK Proteins ; Circadian Clocks/genetics* ; Circadian Rhythm ; Liver ; Suprachiasmatic Nucleus

BACKGROUND: Exposure to the ionizing radiation (IR) encountered outside the magnetic field of the Earth poses a persistent threat to the reproductive functions of astronauts. The potential effects of space IR on the circadian rhythms of male reproductive functions have not been well characterized so far. METHODS: Here, we investigated the circadian effects of IR exposure (3 Gy X-rays) on reproductive functional markers in mouse testicular tissue and epididymis at regular intervals over a 24-h day. For each animal, epididymis was tested for sperm motility, and the testis tissue was used for daily sperm production (DSP), testosterone levels, and activities of testicular enzymes (glucose-6-phosphate dehydrogenase (G6PDH), sorbitol dehydrogenase (SDH), lactic dehydrogenase (LDH), and acid phosphatase (ACP)), and the clock genes mRNA expression such as Clock, Bmal1, Ror-α, Ror-β, or Ror-γ. RESULTS: Mice exposed to IR exhibited a disruption in circadian rhythms of reproductive markers, as indicated by decreased sperm motility, increased daily sperm production (DSP), and reduced activities of testis enzymes such as G6PDH, SDH, LDH, and ACP. Moreover, IR exposure also decreased mRNA expression of five clock genes (Clock, Bmal1, Ror-α, Ror-β, or Ror-γ) in testis, with alteration in the rhythm parameters. CONCLUSION These findings suggested potential health effects of IR exposure on reproductive functions of male astronauts, in terms of both the daily overall level as well as the circadian rhythmicity.
ARNTL Transcription Factors/genetics* ; Acid Phosphatase ; Animals ; CLOCK Proteins/genetics* ; Circadian Rhythm/radiation effects* ; Epididymis/radiation effects* ; Gene Expression/radiation effects* ; Genitalia, Male/radiation effects* ; Glucosephosphate Dehydrogenase ; L-Iditol 2-Dehydrogenase ; L-Lactate Dehydrogenase ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Nuclear Receptor Subfamily 1, Group F, Member 1/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 2/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; RNA, Messenger/genetics* ; Radiation Exposure ; Radiation, Ionizing ; Reproductive Physiological Phenomena/radiation effects* ; Sperm Motility/radiation effects* ; Spermatozoa/radiation effects* ; Testis/radiation effects*

OBJECTIVETo study the changes of miRNA expression in the pineal gland of neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible roles of miRNA in the pathogenesis of circadian rhythm disturbance after HIBD.

METHODSSeven-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups: HIBD and sham-operated. HIBD was induced according to the Rice-Vannucci method. The pineal glands were obtained 24 hours after the HIBD event. The expression profiles of miRNAs were determined using GeneChip technigue and quantitative real-time PCR (RT-PCR). Then the miRNA which was highly expressed was selected. The expression levels of the chosen miRNA were detected in different tissues (lungs, intestines, stomach, kidneys, cerebral cortex, pineal gland). RT-PCR analysis was performed to measure the expression profiles of the chosen miRNA and the targeted gene Clock mRNA in the pineal gland at 0, 24, 48 and 72 hours after HIBD.

RESULTSmiRNA-182 that met the criteria was selected by GeneChip and RT-PCR. miRNA-182 was highly expressed in the pineal gland. Compared with the sham-operated group, the expression of miRNA-182 was significantly up-regulated in the pineal gland at 24 and 48 hours after HIBD (P<0.05). Compared with the sham-operated group, Clock mRNA expression in the HIBD group increased at 0 hour after HIBD, decreased at 48 hours after HIBD and increased at 72 hours after HIBD (P<0.05).

CONCLUSIONSmiRNA-182 may be involved in the pathogenesis of circadian rhythm disturbance after HIBD.

Animals ; Animals, Newborn ; CLOCK Proteins ; genetics ; Circadian Rhythm ; physiology ; Female ; Gene Expression Regulation ; Hypoxia-Ischemia, Brain ; physiopathology ; Male ; MicroRNAs ; analysis ; physiology ; Pineal Gland ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells (ESCs). However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells (iPSCs). These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any difference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before (DeBruyne et al., 2007b). After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.
Animals ; Apoptosis ; Base Sequence ; CLOCK Proteins ; genetics ; metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Cellular Reprogramming ; Circadian Clocks ; genetics ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Hepatocyte Nuclear Factor 3-beta ; genetics ; metabolism ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

OBJECTIVETo investigate the relationship between genetic variantions of circadian clock genes and risk of breast cancer.

METHODSA case-control study including 406 breast cancer patients and 412 controls was conducted and genes Clock (rs2070062) and Per2 (rs2304672, rs2304669, rs934945) were genotyped by TaqMan real-time PCR. Unconditional logistic regression model was used to analyze the association between the genetic polymorphisms and breast cancer.

RESULTSIndividuals with the rs2304669-TT genotype showed significantly increased breast cancer risk with the OR of 2.33 when compared with the individuals with rs2304669-CC and CT genotypes (P = 0.001). In addition, the three haplotypes containing the risk T allele of rs2304669 were identified to be associated with increased breast cancer risk. However, it was found that rs2304672, rs2070062 and rs934945 polymorphisms were not related with breast cancer risk.

CONCLUSIONSThe locus rs2304669 on Per2 gene is associated with breast cancer risk. Genetic variation of circadian clock genes may increase the susceptibility to breast cancer. Therefore, it may become an important biomarker of susceptibility to breast cancer.

Adult ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; CLOCK Proteins ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Case-Control Studies ; Female ; Genetic Variation ; Humans ; Period Circadian Proteins ; genetics ; Polymorphism, Single Nucleotide ; Risk Factors