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Circadian rhythm has been involved in the regulation of many physiological activities. Autophagy is the metabolic process that transports substances in the cytoplasm to the lysosomes for degradation, and involved in the process of many diseases, including cancer. Studies have shown that autophagy activity has a circadian rhythm feature. As the core gene of the circadian rhythm system, clock has participated in the occurrence and development of cancer. The expression of clock whether regulates tumor development by autophagy has not been illustrated at present. In this study, we established a stable clock-knockdown strain of mouse breast cancer cell 4T1 to explore the changes in autophagy activity. The results showed that autophagy-related proteins ATG9A, ATG7, LC3B and Beclin1 (ATG6) were down-regulated with clock-knockdown, and the p62, the key factors of autophagy, was up-regulated. Moreover, clock-knockdown caused significant up-regulation of p65, and inhibition of IκBα and p-AKT，which were the core factors of several signaling pathways involved in autophagy. Serum rhythm-inducing experiments behaved that the expression of LC3B and Beclin1 had rhythmic characteristics，differed from LC3A. The regulative gene clock was initially established to regulate the autophagy-related genes, and the suggested direction of its regulatory pathways. [ABSTRACT FROM AUTHOR]

Social isolation usually affects the physical and mental health of the human and produces depression, whether it may relate with circadian rhythm still remains to be investigated. Two groups of mice were employed to this experiment to continuously monitoring their activities in a dark environment. Immunofluorescence was used to show the expression of the circadian clock gene in two groups of mice. The results demonstrate that the social isolation leads to decrease of mouse activities, phase advance of rhythm relatively and down-regulated of clock gene expression. We conclude that social isolation can affect the physiological function and the normal physiological rhythm of mice, which may be related to depression. [ABSTRACT FROM AUTHOR]

Transactivator (Tat), a regulatory protein of HIV-1, plays a very important role in HIV-1 infection by promoting the rapid replication of HIV. Research surrounding Tat protein function has mainly focused on inhibition of the immune system, promotion of growth of vascular endothelial cells, and nervous system damage. To date, very little research has addressed the role of Tat in circadian rhythms. Previous studies in our lab have found that the concentration of Tat protein in HIV patients’ blood was positively correlated with patients’ sleep quality and melatonin concentrations. In this study, we applied a Tat expression plasmid in mice. Result demonstrated that the locomotor activities of mice and the concentration of melatonin were significantly increased. Alternatively, the expression ofClockgene was markedly decreased. On the other hand, the expression of theCry1gene was significantly increased, while the expression ofBmal1andPer1genes exhibited no significant difference. [ABSTRACT FROM PUBLISHER]

The circadian system regulates many important aspects of physiology, including the immune response to infectious agents, which is mediated by the activation of the transcription factor NF-κB. Thus, understanding the mechanisms by which circadian clocks regulate NF-κB is a necessary step toward the development of improved therapies underwritten by NF-κB manipulation. Previous reports have identified OTUD7B a deubiquitinase, as a novel regulator of noncanonical NF-κB signaling, largely through the maintenance of TRAF3, a negative regulator of NF-κB. In investigations in our laboratory, when theClockgene was repressed by shRNA, the results of microarray analysis demonstrated thatOtud7bwas down-regulated. Further research by real-time PCR and western blot revealed that thatOtud7bexhibits rhythmic mRNA expression. These findings confirmOtud7bas a novelclock-regulated gene. Additionally,Otud7bmay be an important bridge between the circadian system and noncanonical NF-κB pathway regulation. [ABSTRACT FROM PUBLISHER]

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm ofPer1andPer2synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation ofKras,Raf1and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm ofPer1andPer2synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation. [ABSTRACT FROM PUBLISHER]

Traumatic brain injury (TBI) generally influences circadian rhythms and has been implicated changes in circadian rhythm. Whether TBI-induced changes in circadian rhythm may affect the prognosis or recovery from TBI remains to be investigated. Sixty-two patients with TBI were continuously monitored for intracranial pressure (ICP) during the first 24 hours after the implantation of ICP monitor. The data from each patient were analyzed using the least squares fit of a 24-h cosine function by single cosinor method. Parameters of circadian A (Amplitude)/M (MESOR) were used to evaluate the circadian rhythm of the patients. Student’st-test and Pearson’s chi-squared test were utilized to analyze the differences between good prognosis group and poor prognosis. A linear regression analysis was then applied to calculate the correlation between circadian A/M of ICP and Glasgow Coma Scale (GCS) before discharge, the Extended Glasgow Outcome Scale (GOS-E), the dosage of mannitol, and time spent in the intensive care unit (ICU), respectively. The results demonstrated that circadian A/M of patients’ ICP exhibited a positive correlation with GCS scores taken before discharge, GOS-E scores, and was negatively correlated with the amount of mannitol, and time spent in the ICU. We conclude that changes in the ICP circadian rhythm in TBI patients could reflect an internal signal of brain damage and, therefore, may be useful to predict a patient’s prognosis and recovery from TBI. [ABSTRACT FROM PUBLISHER]

Circadian clocks in organisms drive physiology and behavior, helping with adjustment to earth’s environment and human society. The generation and regulation mechanisms of circadian rhythms are starting to be understood. The transcriptional/post-transcriptional delayed feedback loop has been widely studied as the main regulation mechanism. But it cannot fully account for all circadian rhythms in cells. It has been reported that the “proto-clock” may have been a cytosolic metabolic oscillation and that the transcriptional translational regulation system, which provides robustness and amplifies circadian outputs, was developed during evolution. Research on circadian rhythms in cyanobacteriaSynechococcus elongatus, algaeOstreococcus tauri, and human red blood cells in the absence of transcription and translation indicates the existence of a non-transcriptional/translational regulation mechanism, in addition to the classical transcriptional/translational regulation mechanism. It revealed a close link between the two regulation systems of circadian rhythms, in which they might complement each other. And NAD, involved in energy metabolism, may be involved in their interaction. [ABSTRACT FROM PUBLISHER]

Circadian locomotor output cycles kaput (Clock) gene is a core gene in the circadian rhythm system that is involved in cancer cell proliferation. However, the molecular mechanism ofClockgene participate in the cancer cell proliferation is unclear. Previous studies demonstrated that cell proliferation could be regulated by the canonical Wnt pathway (also known as the Wnt/β-catenin pathway), and the Wnt/β-catenin pathway had a relation with the circadian system. To investigate whether theClockgene affects the proliferation of breast cancer cell by regulating the expression ofβ-catenin, we knocked down theClockexpression of mouse breast cancer cells (4T1) by RNA interference. Then detected their proliferation rates using CCK8 assay and the expression of theβ-cateningene by real-time PCR and Western blot. The results showed that the proliferation of theClockknocked down 4T1 cells is slower than the control. The expression level ofβ-cateninof these 4T1 cells is reduced. Our study showed thatClockgene knocked down inhibiting the proliferation of the 4T1 cells, probably by suppressing the expression of β-catenin. [ABSTRACT FROM PUBLISHER]

Emerging evidences have shown that circadian rhythm can also be regulated by a nontranscriptional mechanism. In this study, we investigated thein vitrorhythmic characters of peroxiredoxin2 (PRX2) and nicotinamide adenine dinucleotide (NAD) in red blood cells (RBCs) and/or in heparin anticoagulated plasma under different light conditions. Our results showed that NAD had a free-running rhythm in accordance with that of PRX2 in RBCs, and both rhythms were liable to light entrainment. A decreased expression of PRX2 in RBCs and a notable phase delay of locomotor activity were found in mice intravenously injected with NAD, suggesting that NAD in nontranscriptional oscillating systems could receive the light zeitgeber signal and influence the expression of PRX2. NAD may also act as a nonphotic zeitgeber, which could make an advanced phase shift ofper2, entraining the transcriptional oscillating system to make these two oscillating systems synchronized in a very short time. This zeitgeber might be of great significance as a mediator between these two oscillating systems. [ABSTRACT FROM AUTHOR]

Mice homozygous for a dominant-negative allele of the Clock gene (ClockΔ19/Δ19) have slightly but significantly decreased male fertility. The molecular mechanism for this reduction in fertility is unknown. In the present study, we used a small hairpin RNA (shRNA) strategy to specifically knock down the Clock gene expression in the testes of male mice and determined its effect on male fertility. Clock knockdown led to smaller litter size, a lower in vitro fertility rate, lower blastula formation rate, and lower acrosin activity of the knockdown sperm. Locomotor activity analysis of the Clock knockdown mice revealed that Clock knockdown in testes did not alter their circadian rhythm. Taken together, these results provide the first evidence that Clock gene expression in round spermatids is essential for maintaining male reproductivity and suggest that acrosin may be a novel regulatory target of the Clock gene that would regulate the fertilization and early embryonic development to blastula. These findings may provide new clues for development of novel male contraceptive strategies. [ABSTRACT FROM PUBLISHER]

It has been widely studied and demonstrated that circadian rhythm, as well as circadian clock genes, plays an important role in many physiological and pathological processes.Period1 (Per1)gene is a core gene in the circadian clock system that is involved in cell differentiation and apoptosis. To verify the role of PER1 on the proliferation of normal cells, we chose NIH3T3 cells to have their PER1 up-regulated by transfecting aPer1expression plasmid and determining their proliferation rates. Previous studies demonstrated that cell migration could be regulated by matrix metallopeptidase 2 (MMP2), and this MMP2 protein had a relation with the circadian system. To investigate the function of PER1 on tumor cell migration, Lewis lung carcinoma (LLC) cells were chosen to have their PER1 up-regulated, and the MMP2 level and the migration of LLC cells were tested. Our study showed that PER1 overexpression led to MMP2 down regulation in both NIH3T3 and CCL cells and inhibited the proliferation rate in NIH3T3. Migration of LLC cells could also be suppressed by the overexpression of PER1, probably by the down-regulation effect of PER1 on MMP2. [ABSTRACT FROM AUTHOR]

Circadian gene, Clock and Clif, not only maintain the circadian rhythm of cells but also regulate lipid and glucose metabolism in peripheral organs. The purpose of this study was to determine whether genetic variations (single nucleotide polymorphisms – SNPs) in the Clock and/or Clif genes were associated with hyperlipidemia in Chinese Han descent. The Clock variants (rs3840267, rs3749474, rs1052925) and the Clif variant (rs2289709) were analyzed in 205 patients with hyperlipidemia and 281 healthy controls by sequence-specific primer polymerase chain reaction (SSP-PCR). The Clock variant (rs1052925) showed significant association to hyperlipidemia (P alleo = 0.003, ORalleo =  0.529). We also found correlations between the Clock (rs1052925), the Clif (rs2289709), and types of hyperlipidemia (P alleo-IV = 0.043, ORalleo-IV = 0.525; P alleo-IIb = 4.738 × 10−4, OR alleo-IIb = 2.959). In addition, the Clock (rs1052925), the level of triglycerides in carriers of A/- heterozygote was significantly different from carriers of A/A heterozygote (1.27 ± 0.69 vs. 1.76 ± 1.22; P = 0.01). The Clif (rs2289709), the level of triglycerides and LDL cholesterol in carriers of G/A heterozygote wassignificantly different from carriers of G/G heterozygote (2.31 ± 1.81 vs. 1.11 ± 0.32; P = 2.33 × 10−8; 2.83 ± 0.98 vs. 1.63 ± 0.69; P = 5.83 × 10−15, respectively). Our findings indicate that Clock/Clif genetic variations might be associated with hyperlipidemia. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 115, 353-362. Abstract Protracted opiate withdrawal can extend for months of disrupted hormonal circadian rhythms. We examined rodent behaviors and these circadian disturbances in hormone and peptide levels as well as brain clock gene expression during 60 days of protracted withdrawal. Our behavioral tests included open field, elevated plus maze, and sucrose preference tests at 36 h, 10, 30, and 60 days after stopping chronic morphine. At these four assessment points, we collected samples every 4 h for 24 h to examine circadian rhythms in blood hormone and peptide levels and brain expression of rPER1, rPER2, and rPER3 clock genes. Decreased locomotor activity and elevated adrenocorticotropic hormone and melatonin levels persisted for 2 months after morphine withdrawal, but corticosterone was elevated only at 36 h and 10 days after withdrawal. Orexin levels were high at 36 h after withdrawal, but then reversed during protracted withdrawal to abnormally low levels. Beta-endorphin (β-EP) levels showed no differences from normal. However, circadian rhythms were blunted for all of these hormones. Corticosterone, adrenocorticotropic hormone, and orexin blunting persisted at least for 60 days. The blunted circadian rhythm of β-EP and melatonin recovered by day 60, but the peak phase of β-EP was delayed about 8 h. Blunted circadian rhythms and reduced expression of rPER1, rPER2, and rPER3 persisted at least for 60 days in the suprachiasmatic nucleus, prefrontal cortex, nucleus accumbens core, central nucleus of the amygdala, Hippocampus, and ventral tegmental area. Circadian rhythms of rPER1 in the nucleus accumbens shell and basolateral nucleus of the amygdala and of rPER2 in the central nucleus of the amygdala were reversed. Disrupted circadian rhythms of rPER1, rPER 2, and rPER3 expression in reward-related brain circuits and blunted circadian rhythms in peripheral hormones and peptides may play a role in protracted opiate withdrawal and contribute to relapse. [ABSTRACT FROM AUTHOR]

Objective. This study investigates how the shortening or lengthening of the circadian period of the environmental lighting schedule affects the circadian and extra-circadian structure of locomotor activity in mice, with particular focus on the circasemiseptan, circaseptan, and circadecadian components with anticipated periods of about 3.5, 7, and 10 days, respectively. Methods. The locomotor activity of mice kept on different light - dark (LD) cycles was automatically monitored around the clock for about five weeks. The data were analyzed by linear - nonlinear rhythmometry. Results. As anticipated, in LD12:12, a prominent circadian rhythm was observed, with a marked 12-hour component qualifying the circadian waveform. By comparison, in LD10:10 and in LD14:14, the circadian rhythm had a reduced amplitude and a period close to that of the environmental synchronizer. Extra-circadian variation was then also demonstrated. In conclusion, circadian lighting schedules affect a broader-than-circadian time structure of locomotor activity in mice. [ABSTRACT FROM AUTHOR]

PER1, an important component of circadian clock systems, plays a critical role in regulating the period length and maintaining the precision and stability of the period of circadian rhythms. RACK1 (receptor for activated protein kinase C-1), a member of the WD-40 family of proteins, can interact with numerous signaling proteins and is regarded as a scaffolding, anchor, or adaptor protein in multiple intracellular signal transduction pathways. In the present study, we identified and confirmed RACK1 as a novel protein interacting with human clock protein, hPER1, using the yeast two-hybrid system and co-immunoprecipitation experiment. Further study by RT-PCR showed that RACK1 was expressed widely in tissues and there was no obvious expressional rhythmicity. However, RNA interfering plasmid inhibiting, hPER1 (pTER/hPER1-II) could not interfere expression of RACK1. These results together suggested that RACK1 might act as a novel signal molecule to mediate or regulate the functions of PER1 through, protein interaction. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is a potent anti-neoplastic agent with cumulative cardiotoxicity. DOX-induced cardiotoxicity has been shown to depend on the different dosing times. However, the basis for determining the dosing time to minimize DOX-induced cardiotoxicity and the underlying mechanisms remain incompletely understood. Here we first showed that SIRT3, the major mitochondrial deacetylase, is negatively correlated to DOX-induced cardiotoxicity through the regulation of ATP production, mitochondrial membrane potential (MMP) level and ROS level in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Then, we used in vivo experiments to demonstrate that DOX significantly reduced the SIRT3 expression and the SIRT3 activity as reflected by the increased AcK68MnSOD/MnSOD ratio in rats after six weeks of treatment. Notably, the activity of SIRT3 had an obvious diurnal rhythm pattern in the myocardium of healthy rats. More importantly, an obvious lower AcK68MnSOD/MnSOD ratio was observed in rat hearts with DOX administrated at Zeitgeber time (ZT) 9 (ZT 0 was the time lights were turned on) than ZT1, which represent the peak and trough of SIRT3 activity. Moreover, DOX ZT9 reduced the body weight loss, extended the survival period, improved the heart function and alleviated the myocardial lesions compared to DOX ZT1. Mechanistic investigations demonstrated that DOX ZT1 significantly reduced ATP production, oxygen consumption rate (OCR) at various respiration states, MMP level and MnSOD activity and enhanced the H 2 O 2 level compared with CON ZT1, whereas there was no significant effect for DOX ZT9 compared with CON ZT9. Taken together, dosing at the peak time of SIRT3 activity reduced DOX-induced cardiotoxicity, which may be related to the increased endogenous tolerance against the mitochondrial dysfunction and oxidative stress caused by DOX. • SIRT3 plays a critical role in doxorubicin-induced cardiotoxicity in hiPSC-CMs. • Dosing depending on SIRT3 activity attenuates doxorubicin-induced cardiotoxicity. • Dosing depending on SIRT3 activity renders tolerance against mitochondrial dysfunction and oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Alterations in metabolism could be due to cell-autonomous effects associated with altered expression of Clock in central nervous system feeding centers and/or peripheral tissues involved in metabolism. Clock mutant mice are hyperphagic and obese, which indicates that Clock is related to obesity. In the present study, we used intracerebroventricular injection of recombinant adenoviral vector harboring Clock genes to explore the role of Clock on diet induced obesity and the mechanisms involved in leptin resistance and leptin signaling in mice. The results demonstrated that expression of Clock in the arcuate nucleus of diet induced obesity mice was down-regulated. The recombinant adenoviral vector harboring Clock genes could reduce obesity indexes of diet induced obesity mice including body weight, BMI and total fat mass, attenuate hyperleptinemia, increase leptin sensitivity and decrease accumulated suppressor of cytokine signaling-3 in the arcuate nucleus. These results indicate that Clock plays an important role on obesity, which may be involved in leptin resistance and regulation of suppressor of cytokine signaling-3 in arcuate nucleus. [Copyright &y& Elsevier]