Peripheral circadian oscillators in mammals: time and food

Peripheral cells from mammalian tissues, while perfectly capable of circadian rhythm generation, are not light sensitive and thus have to be entrained by nonphotic cues. Feeding time is the dominant zeitgeber for peripheral mammalian clocks: Daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in many tissues, including liver, heart, kidney, and pancreas, but it has no effect on the SCN pacemaker. It is thus plausible that in intact animals, the SCN synchronizes peripheral docks primarily through temporal feeding patterns that are imposed through behavioral rest-activity cycles. In addition, body temperature rhythms, which are themselves dependent on both feeding patterns and rest-activity cycles, can sustain circadian, clock gene activity in vivo and in vitro. The SCN may also influence the phase of rhythmic gene expression in peripheral tissues through direct chemical pathways. In fact, many chemical signals induce circadian gene expression in tissue culture cells. Some of these have been shown to elicit phase shifts when injected into intact animals and are thus candidates for physiologically relevant timing cues. While the response of the SCN to light is strictly gated to respond only during the night, peripheral oscillators can be chemically phase shifted throughout the day. For example, injection of dexamethasone, a glucocorticoid receptor agonist, resets the phase of circadian liver gene expression during the entire 24-h day. Given the bewildering array of agents capable of influencing peripheral clocks, the identification of physiologically relevant agents used by the SCN to synchronize peripheral clocks will clearly be an arduous undertaking. Nevertheless, we feel that experimental systems by which this enticing problem can be tackled are now at hand.     

Mammalian cultured cells as a model system of peripheral circadian clocks

The mammalian circadian system consists of multiple oscillators with basically hierarchical relationship, in which the hypothalamic suprachiasmatic nucleus (SCN) is the master pacemaker and the other oscillators in the periphery are subordinate. Although peripheral oscillators have been preceded by the SCN in circadian studies, accumulating data have revealed the importance and characteristics of peripheral oscillators. Cultured cell lines have also provided valuable information about intracellular mechanisms of circadian rhythms. This review outlines the properties of peripheral clocks in several perspectives such as the mechanisms of autonomous oscillations, the clock resetting, and the clock outputs, and describes the usefulness of immortalized cultured cells as a model system of mammalian circadian clocks by introducing some fruits of related works.     

Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus (SCN) of the brain and subsidiary oscillators in most peripheral cell types. While oscillators in SCN neurons are known to function in a self-sustained fashion, peripheral oscillators have been thought to damp rapidly when disconnected from the control exerted by the SCN. Using two reporter systems, we monitored circadian gene expression in NIH3T3 mouse fibroblasts in real time and in individual cells. In conjunction with mathematical modeling and cell co-culture experiments, these data demonstrated that in vitro cultured fibroblasts harbor self-sustained and cell-autonomous circadian clocks similar to those operative in SCN neurons. Circadian gene expression in fibroblasts continues during cell division, and our experiments unveiled unexpected interactions between the circadian clock and the cell division clock. Specifically, the circadian oscillator gates cytokinesis to defined time windows, and mitosis elicits phase shifts in circadian cycles.     

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.     

Synchronization of the molecular clockwork by light- and food-related cues in mammals

The molecular clockwork in mammals involves various clock genes with specific temporal expression patterns. Synchronization of the master circadian clock located in the suprachiasmatic nucleus (SCN) is accomplished mainly via daily resetting of the phase of the clock by light stimuli. Phase shifting responses to light are correlated with induction of Per1, Per2 and Dec1 expression and a possible reduction of Cry2 expression within SCN cells. The timing of peripheral oscillators is controlled by the SCN when food is available ad libitum. Time of feeding, as modulated by temporal restricted feeding, is a potent 'Zeitgeber' (synchronizer) for peripheral oscillators with only weak synchronizing influence on the SCN clockwork. When restricted feeding is coupled with caloric restriction, however, timing of clock gene expression is altered within the SCN, indicating that the SCN function is sensitive to metabolic cues. The components of the circadian timing system can be differentially synchronized according to distinct, sometimes conflicting, temporal (time of light exposure and feeding) and homeostatic (metabolic) cues.     

Potent synchronization of peripheral circadian clocks by glucocorticoid injections in PER2::LUC-Clock/Clock mice

In mammals, the central clock (the suprachiasmatic nuclei, SCN) is entrained mainly by the light-dark cycle, whereas peripheral clocks in the peripheral tissues are entrained/synchronized by multiple factors, including feeding patterns and endocrine hormones such as glucocorticoids. Clock-mutant mice (Clock/Clock), which have a mutation in a core clock gene, show potent phase resetting in response to light pulses compared with wild-type (WT) mice, owing to the damped and flexible oscillator in the SCN. However, the phase resetting of the peripheral clocks in Clock/Clock mice has not been elucidated. Here, we characterized the peripheral clock gene synchronization in Clock/Clock mice by daily injections of a synthetic glucocorticoid (dexamethasone, DEX) by monitoring in vivo PER2::LUCIFERASE bioluminescence. Compared with WT mice, the Clock/Clock mice showed significantly decreased bioluminescence and peripheral clock rhythms with decreased amplitudes and delayed phases. In addition, the DEX injections increased the amplitudes and advanced the phases. In order to examine the robustness of the internal oscillator, T-cycle experiments involving DEX stimulations with 24- or 30-h intervals were performed. The Clock/Clock mice synchronized to the 30-h T-cycle stimulation, which suggested that the peripheral clocks in the Clock/Clock mice had increased synchronizing ability upon DEX stimulation, to that of circadian and hour-glass type oscillations, because of weak internal clock oscillators.     

Hormones, subjective night and season of the year

Production and release of many mammalian hormones exhibit circadian rhythms controlled by a pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Under conditions when the circadian pacemaker free-runs with a period close to, but not equal to 24 h, subjective day and night may not be identical with the environmental day and night. The present study was aimed to define the phase and state of the circadian pacemaker when the circadian system is experiencing subjective night and to ascertain whether and how such a defined subjective night depends on the photoperiod. The results indicate that the subjective night may be defined as the time interval when i) light stimuli can reset the circadian system, ii) pineal melatonin production and photic induction of the c-Fos gene in the ventrolateral SCN are high, and iii) the spontaneous c-Fos protein production in the dorsomedial SCN is low. Such a defined subjective night and, logically, the whole circadian pacemaking system depend on the photoperiod and hence on the season of the year which the animals are experiencing.     

Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.     

Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) clock, which is the main oscillator and peripheral clock. SCN clock can be entrained by both photic and non-photic stimuli, and an interaction exists between photic and non-photic entrainment. Moreover, peripheral circadian clocks can be entrained not only by scheduled restricted feeding, but also by scheduled exercise. Thus, the entrainment of peripheral circadian clocks may be the result of an interaction between the entrainment caused by feeding and exercise. In this study, we examined the effect of wheel-running exercise on the phase of the peripheral clocks (kidney, liver and submandibular gland) in PER2::LUC mice under various feeding schedules. Phase and waveforms of the peripheral clocks were not affected by voluntary wheel-running exercise. Exercise for a period of 4 h during the early dark period (morning) delayed the peripheral clocks, while exercise for the same duration during the late dark period (evening) advanced the peripheral clocks. The feeding phase was advanced and delayed by evening and morning exercise, respectively, suggesting that the feeding pattern elicited by the scheduled exercise may entrain the peripheral clocks. Exercise did not affect the phase of the peripheral clock under the 1 meal per day schedule. When the phase of the peripheral clocks was advanced by the feeding schedule of 2 or 4 meals per day during light and/or dark periods, wheel-running exercise during the morning period significantly and equally shifted the phase of all organs back to the original positions observed in mice maintained under free-feeding conditions and with no exercise. When the schedule of 2 meals per day during the dark period failed to affect the phase of peripheral clock, morning exercise did not affect the phase. Wheel-running exercise increased the levels of serum corticosterone, and the injection of dexamethasone/corticosterone instead of exercise shifted a phase that had advanced under the feeding schedule of 2 meals per day, back to the normal position. The liver and submandibular glands exhibit higher sensitivity to dexamethasone than the kidneys. In adrenalectomized mice, treadmill-induced normalization of the advanced phase under a feeding schedule of 2 meals per day was not observed. In summary, scheduled exercise-induced phase shifts were weaker compared to scheduled feeding-induced phase shifts. The phase advance caused by the feeding schedule of 2 or 4 meals per day was suppressed by wheel-running, treadmill exercise or dexamethasone/corticosterone injection in the early dark period (morning). Corticosterone release may be involved in exercise-induced phase shift of peripheral clocks. These results suggest that there is an interaction between the phase shifts caused by feeding and exercise schedules in peripheral clocks.     

The comparison between circadian oscillators in mouse liver and pituitary gland reveals different integration of feeding and light schedules

The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues.     

Sympathetic input modulates, but does not determine, phase of peripheral circadian oscillators

The circadian clock in the suprachiasmatic nucleus (SCN) maintains phase synchrony among circadian oscillators throughout the organism. Environmental light signals entrain the SCN, but timed, limited meal access acts as an overriding time cue for several peripheral tissues. We present data from a peripheral oscillator, the submaxillary salivary gland, in which temporal restriction of meals fails to entrain gene expression. In day-fed rats, submaxillary gland rhythms in expression of the clock gene Period1 (Per1) stay entrained to the light cycle (peaking at night) or become arrhythmic. This result suggests that feeding cues compete weakly with light cycle cues to set the phase of clock genes in this tissue. Since the submaxillary glands receive sympathetic innervation originating in the SCN, which relays light cycle cues to other oscillators, we attempted to assess the role of this neural input in phase control of submaxillary Per1 expression. We sympathetically denervated the submaxillary glands before subjecting rats to daytime-restricted feeding. After denervation, Per1 rhythms in all submaxillary glands shifted phase 180 degrees and entrained to daytime feeding. These results support the hypothesis that peripheral oscillators may receive multiple signals contributing to their phase of entrainment. Sympathetic efferents from the SCN can relay light cycle information, while other external cues may reach tissues through other efferents or nonneural pathways. In an abnormal, disruptive regimen such as daytime-restricted feeding, these different signals compete. Arrhythmicity may result if one signal is not clearly dominant. Elimination of the dominant signal (e.g., surgical sympathectomy) may allow a secondary signal to control phase.     

Daily restricted feeding resets the circadian clock in the suprachiasmatic nucleus of CS mice

Circadian rhythms in clock gene expressions in the suprachiasmatic nucleus (SCN) of CS mice and C57BL/6J mice were measured under a daily restricted feeding (RF) schedule in continuous darkness (DD), and entrainment of the SCN circadian pacemaker to RF was examined. After 2-3 wk under a light-dark cycle with free access to food, animals were released into DD and fed for 3 h at a fixed time of day for 3-4 wk. Subsequently, they returned to having free access to food for 2-3 wk. In CS mice, wheel-running rhythms entrained to RF with a stable phase relationship between the activity onset and feeding time, and the rhythms started to free run from the feeding time after the termination of RF. mPer1, mPer2, and mBMAL1 mRNA rhythms in the SCN showed a fixed phase relationship with feeding time, indicating that the circadian pacemaker in the SCN entrained to RF. On the other hand, in C57BL/6J mice, wheel-running rhythms free ran under RF, and clock gene expression rhythms in the SCN showed a stable phase relation not to feeding time but to the behavioral rhythms, indicating that the circadian pacemaker in the SCN did not entrain. These results indicate that the SCN circadian pacemaker of CS mice is entrainable to RF under DD and suggest that CS mice have a circadian clock system that can be reset by a signal associated with feeding time.     

Circadian molecular clocks tick along ontogenesis

The circadian system controls the timing of behavioral and physiological functions in most organisms studied. The review addresses the question of when and how the molecular clockwork underlying circadian oscillations within the central circadian clock in the suprachiasmatic nuclei of the hypothalamus (SCN) and the peripheral circadian clocks develops during ontogenesis. The current model of the molecular clockwork is summarized. The central SCN clock is viewed as a complex structure composed of a web of mutually synchronized individual oscillators. The importance of development of both the intracellular molecular clockwork as well as intercellular coupling for development of the formal properties of the circadian SCN clock is also highlighted. Recently, data has accumulated to demonstrate that synchronized molecular oscillations in the central and peripheral clocks develop gradually during ontogenesis and development extends into postnatal period. Synchronized molecular oscillations develop earlier in the SCN than in the peripheral clocks. A hypothesis is suggested that the immature clocks might be first driven by external entraining cues, and therefore, serve as "slave" oscillators. During ontogenesis, the clocks may gradually develop a complete set of molecular interlocked oscillations, i.e., the molecular clockwork, and become self-sustained clocks.     

肾上腺糖皮质激素与生物钟基因表达调控的相关研究进展

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。哺乳动物中的昼夜节律系统由位于下丘脑SCN核内的主钟和位于多数外周细胞中的子钟组成。生物钟基因及其编码的蛋白质组成反馈回路,维持振荡系统持续进行并与环境周期保持同步。光照和食物是生物钟重要的授时因子, 光照刺激能引起肾上腺中基因表达变化以及糖皮质激素的分泌, 而肾上腺糖皮质激素能减缓由食物因子引起的外周生物钟时相的移动。可见, 肾上腺糖皮质激素与生物钟有着非常密切的关系。文章综述了两者的相互影响并对今后的研究方向做了展望。