Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans

This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48?h (sleep vs. no-sleep nights) in 12 young males (mean?±?SD: 23?±?5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation.     

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

Influence of light and food on the circadian clock in liver of rainbow trout,Oncorhynchus mykiss

Several reports support the existence of multiple peripheral oscillators in fish, which may be able to modulate the rhythmic functions developed by those tissues hosting them. Thus, a circadian oscillator has been proposed to be located within fish liver. In this vertebrate group, the role played by the circadian system in regulating metabolic processes in liver is mostly unknown. We, therefore investigated the liver of rainbow trout (Oncorhynchus mykiss) as a potential element participating in the regulation of circadian rhythms in fish by hosting a functional circadian oscillator. The presence and expression pattern of main components of the circadian molecular machinery (clock1a, bmal1, per1 and rev-erbβ-like) were assessed. Furthermore, the role of environmental cues such as light and food, and their interaction in order to modulate the circadian oscillator was also assessed by exposing animals to constant conditions (absence of light for 48 h, and/or a 4 days fasting period). Our results demonstrate the existence of a functional circadian oscillator within trout liver, as demonstrated by significant rhythms of all clock genes assessed, independently of the environmental conditions studied. In addition, the daily profile of mRNA abundance of clock genes is influenced by both light (mainly clock1a and per1) and food (rev-erbβ-like), which is indicative of an interaction between both synchronizers. Our results point to rev-erbβ-like as possible mediator between the influence of light and food on the circadian oscillator within trout liver, since its daily profile is influenced by both light and food, thus affecting that of bmal1.     

Temperature cycle amplitude alters the adult eclosion time and expression pattern of the circadian clock gene period in the onion fly

Soil temperature cycles are considered to play an important role in the entrainment of circadian clocks of underground insects. However, because of the low conductivity of soil, temperature cycles are gradually dampened and the phase of the temperature cycle is delayed with increasing soil depth. The onion fly, Delia antiqua, pupates at various soil depths, and its eclosion is timed by a circadian clock. This fly is able to compensate for the depth-dependent phase delay of temperature change by advancing the eclosion time with decreasing amplitude of the temperature cycle. Therefore, pupae can eclose at the appropriate time irrespective of their location at any depth. However, the mechanism that regulates eclosion time in response to temperature amplitude is still unknown. To understand whether this mechanism involves the circadian clock or further downstream physiological processes, we examined the expression patterns of period (per), a circadian clock gene, of D. antiqua under temperature cycles that were square wave cycles of 12-h warm phase (W) and 12-h cool phase (C) with the temperature difference of 8 °C (WC 29:21 °C) and 1 °C (WC 25.5:24.5 °C). The phase of oscillation in per expression was found to commence 3.5 h earlier under WC 25.5:24.5 °C as compared to WC 29:21 °C. This difference was in close agreement with the eclosion time difference between the two temperature cycles, suggesting that the mechanism that responds to the temperature amplitude involves the circadian clock.     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.     

Impaired expression of the mPer2 circadian clock gene in the suprachiasmatic nuclei of aging mice

The expression of circadian clock genes was investigated in the suprachiasmatic nuclei (SCN) of young adult and old laboratory mice. Samples were taken at two time points, which corresponded to the expected maximum (circadian time 7 [CT7]) or minimum (CT21) of mPer mRNA expression. Whereas the young mice had a stable and well-synchronized circadian activity/rest cycle, the rhythms of old animals were less stable and were phase advanced. The expression of mPer1 mRNA and mPer2 mRNA was rhythmic in both groups, with peak values at CT7. The levels of mClock and mCry1 mRNA were not different depending on the time of day and did not vary with age. In contrast, an age-dependent difference was found in the case of mPer2 (but not mPer1) mRNA expression, with the maximum at CT7 significantly lower in old mice. The decreased expression of mPer2 may be relevant for the observed differences in the overt activity rhythm of aged mice. (Chronobiology International, 18(3), 559-565, 2001)     

Mechanisms of hormonal regulation of the peripheral circadian clock in the colon

Colonic function is controlled by an endogenous clock that allows the colon to optimize its function on the daytime basis. For the first time, this study provided evidence that the clock is synchronized by rhythmic hormonal signals. In rat colon, adrenalectomy decreased and repeated applications of dexamethasone selectively rescued circadian rhythm in the expression of the clock gene Per1. Dexamethasone entrained the colonic clock in explants from mPer2^Luc mice in vitro. In contrast, pinealectomy had no effect on the rat colonic clock, and repeated melatonin injections were not able to rescue the clock in animals maintained in constant light. Additionally, melatonin did not entrain the clock in colonic explants from mPer2^Luc mice in vitro. However, melatonin affected rhythmic regulation of Nr1d1 gene expression in vivo. The findings provide novel insight into possible beneficial effects of glucocorticoids in the treatment of digestive tract-related diseases, greatly exceeding their anti-inflammatory action.     

Crosstalk between xenobiotics metabolism and circadian clock

Many aspects of physiology and behavior in organisms from bacteria to man are subjected to circadian regulation. Indeed, the major function of the circadian clock consists in the adaptation of physiology to daily environmental change and the accompanying stresses such as exposition to UV-light and food-contained toxic compounds. In this way, most aspects of xenobiotic detoxification are subjected to circadian regulation. These phenomena are now considered as the molecular basis for the time-dependence of drug toxicities and efficacy. However, there is now evidences that these toxic compounds can, in turn, regulate circadian gene expression and thus influence circadian rhythms. As food seems to be the major regulator of peripheral clock, the possibility that food-contained toxic compounds participate in the entrainment of the clock will be discussed.     

Circadian clock gene expression regulates cancer cell growth through glutaminase

Glutamine is an essential amino acid for malignant tumor cells. Glutaminase that metabolizes glutamine reaches a maximum expression in tumors immediately before the maximum proliferation rate. Tumor cells grow at different rates during the day. We postulated that the activity of glutaminase in tumor cells is subject to the regulation of circadian clock gene. We measured glutaminase by western blot analysis and circadian clock gene expression by real-time polymerase chain reaction in the liver and tumor cells at six equispaced time points of the day in individual mice of a 12/12 h light/dark schedule. The results showed that the tumor-bearing mice, under normal diurnal conditions, are circadianly entrained, as reflected by the normal host locomotor activity rhythms and rhythmic liver clock gene expression. The tumors within these mice are also circadianly organized, as reflected by circadian clock gene （Bmall） expression. What is most remarkable is that kidney-type glutaminase also showed circadian rhythms in the same pattern with tumor circadian clock gene expression in liver cancer xenograft model, indicating that conditionally inhibiting glutaminase activity may provide a new target for cancer therapy.     

生物钟机制研究进展

由生物体内源性生物钟所产生的昼夜节律是近年来生命科学的研究热点之一。几种模型生物（蓝细菌、脉孢菌、拟南芥、果蝇、小鼠）的生物钟相关基因相继被克隆和鉴定,为理解昼夜节律的分子机制奠定了基础。振荡器蛋白对其编码基因的负反馈调控可能是不同生物的生物运作普遍机制,在此基础上,不同生物有不尽相同的调控方式;隐色素可能是高等生物的共同生物钟光受体。     

Interactions of polymorphisms in different clock genes associated with circadian phenotypes in humans

Several studies have shown that mutations and polymorphisms in clock genes are associated with abnormal circadian parameters in humans and also with more subtle non-pathological phenotypes like chronotypes. However, there have been conflicting results, and none of these studies analyzed the combined effects of more than one clock gene. Up to date, association studies in humans have focused on the analysis of only one clock gene per study. Since these genes encode proteins that physically interact with each other, combinations of polymorphisms in different clock genes could have a synergistic or an inhibitory effect upon circadian phenotypes. In the present study, we analyzed the combined effects of four polymorphisms in four clock genes (Per2, Per3, Clock and Bmal1) in people with extreme diurnal preferences (morning or evening). We found that a specific combination of polymorphisms in these genes is more frequent in people who have a morning preference for activity and there is a different combination in individuals with an evening preference for activity. Taken together, these results show that it is possible to detect clock gene interactions associated with human circadian phenotypes and bring an innovative idea of building a clock gene variation map that may be applied to human circadian biology.     

The human circadian clock and aging

The suprachiasmatic nucleus (SCN) of the hypothalamus is implicated in the timing of a wide variety of circadian processes. Since the environmental light-dark cycle is the main zeitgeber for many of the rhythms, photic information may have a synchronizing effect on the endogenous clock of the SCN by inducing periodic changes in the biological activity of certain groups of neurons. By studying the brains obtained at autopsy of human subjects, marked diurnal oscillations were observed in the neuropeptide content of the SCN. Vasopressin, for example, one of the most abundant peptides in the human SCN, exhibited a diurnal rhythm, with low values at night and peak values during the early morning. However, with advancing age, these diurnal fluctuations deteriorated, leading to a disrupted cycle with a reduced amplitude in elderly people. These findings suggest that the synthesis of some peptides in the human SCN exhibits an endogenous circadian rhythmicity, and that the temporal organization of these rhythms becomes progressively disturbed in senescence. (Chronobiology International, 17(3), 245-259, 2000)     

Circadian oscillations of neuropeptide expression in the human biological clock

The mammalian suprachiasmatic nucleus is the principal component of a neural timing system implicated in the temporal organization of circadian and seasonal processes. The present study was performed to analyze the circadian profiles of two major neuropeptidergic cell groups in the human suprachiasmatic nucleus. To that end the brains of 40 human subjects collected at autopsy were investigated. The populations of arginine vasopressin- and vasoactive intestinal polypeptide-expressing neurons, located in the shell and core of the suprachiasmatic nucleus, respectively, showed marked circadian rhythms with an asymmetrical, bimodal waveform. Time series analysis revealed that these circadian cycles in neuronal activity could be described by a composite model consisting of a nonlinear periodic function, with mono- and diphasic cycles. The findings suggest that the 24-h biosynthesis of neuropeptides in the human suprachiasmatic nucleus, being part of the neural output pathway of the clock, is driven by a complex pacemaker system consisting of coupled nonlinear oscillators, in accordance with a multioscillator model of circadian timekeeping.Abbreviations AIC Akaikie's information criterion - ARMA autoregressive moving average - AVP arginine vasopressin - c-fos immediate early gene - Per period gene - SCN suprachiasmatic nucleus - VIP vasoactive intestinal polypeptide