The circadian clock plays a critical role in regulating various physiological processes, including gene expression, metabolic regulation, immune response, and the sleep-wake cycle in living organisms. Post-translational modifications (PTMs) are crucial regulatory mechanisms to maintain the precise oscillation of the circadian clock. By modulating the stability, activity, cell localization and protein-protein interactions of core clock proteins, PTMs enable these proteins to respond dynamically to environmental and intracellular changes, thereby sustaining the periodic oscillations of the circadian clock. Different types of PTMs exert their effects through distincting molecular mechanisms, collectively ensuring the proper function of the circadian system. This review systematically summarized several major types of PTMs, including phosphorylation, acetylation, ubiquitination, SUMOylation and oxidative modification, and overviewed their roles in regulating the core clock proteins and the associated pathways, with the goals of providing a theoretical foundation for the deeper understanding of clock mechanisms and the treatment of diseases associated with circadian disruption.
Protein Processing, Post-Translational/physiology* ; Circadian Rhythm/physiology* ; Humans ; Animals ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Phosphorylation ; Acetylation ; Ubiquitination ; Sumoylation

The circadian rhythm regulates the 24-hour physiological and behavioral cycles through endogenous molecular clocks governed by core clock genes via the transcription-translation feedback loop (TTFL). In mammals, the suprachiasmatic nucleus (SCN) serves as the central pacemaker, coordinating the timing of physiological processes throughout the body by regulating clock genes such as CLOCK, BMAL1, PER, and CRY. The molecular clocks of peripheral tissues and cells are synchronized by the SCN through TTFLs to regulate metabolism, immunity, and energy homeostasis. Numerous studies indicate that circadian rhythm disruption is closely related to obesity, type 2 diabetes, metabolic syndrome and other diseases, and the mechanism involves the dysregulation of glucose and lipid metabolism, abnormal insulin signaling and low-grade inflammation. In recent years, small-molecule compounds targeting the core clock components such as CRY, REV-ERB, and ROR have been identified and shown potential to modulate metabolic diseases by stabilizing or inhibiting the activity of key clock proteins. This review summarizes the mechanisms and advances in these compounds, and explores the challenges and future directions for their clinical translation, providing insights for chronotherapy-based metabolic disease interventions.
Humans ; Metabolic Diseases/physiopathology* ; Animals ; Circadian Rhythm/physiology* ; Biological Clocks/drug effects* ; CLOCK Proteins/physiology* ; Circadian Clocks/physiology* ; Suprachiasmatic Nucleus/physiology*

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