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.     

Circadian organization is governed by extra-SCN pacemakers

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.     

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.     

Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators.

The circadian timing system in mammals is composed of a master pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus and slave clocks in most peripheral cell types. The phase of peripheral clocks can be completely uncoupled from the SCN pacemaker by restricted feeding. Thus, feeding time, while not affecting the phase of the SCN pacemaker, is a dominant Zeitgeber for peripheral circadian oscillators. Here we show that the phase resetting in peripheral clocks of nocturnal mice is slow when feeding time is changed from night to day and rapid when switched back from day to night. Unexpectedly, the inertia in daytime feeding-induced phase resetting of circadian gene expression in liver and kidney is not an intrinsic property of peripheral oscillators, but is caused by glucocorticoid signaling. Thus, glucocorticoid hormones inhibit the uncoupling of peripheral and central circadian oscillators by altered 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.     

FEEDING ENTRAINMENT OF FOOD-ANTICIPATORY ACTIVITY AND per1 EXPRESSION IN THE BRAIN AND LIVER OF ZEBRAFISH UNDER DIFFERENT LIGHTING AND FEEDING CONDITIONS

Food provided on a periodic basis can act as a potent synchronizer, being a stronger zeitgeber than light for peripheral oscillators in mammals. In fish, however, little is known about the influence of feeding time on the circadian pacemaker and the relationship between central and peripheral oscillators. The aim of this research was to investigate the influence of mealtime on the activity rhythms, and on central (brain) and peripheral (liver) oscillators in zebrafish. The authors tested different feeding times under a light-dark (LD) cycle and the endogenous origin of food-anticipatory activity (FAA) by feeding zebrafish at a fixed time under constant bright-light conditions (LL). The authors then measured locomotor activity and the expression of the clock gene per1 in animals under a LD cycle and fed at random times during the light phase, with restricted feeding at the mid-light phase (ML) or with restricted feeding during the mid-dark phase (MD). Finally, the authors measured locomotor activity and per1 expression in fish maintained under LL under either random feeding or scheduled feeding. Zebrafish displayed FAA in all the groups fed at a fixed time but not when feeding was randomly scheduled. Under LL, fish entrainment persisted, and when released under fasting conditions FAA free-ran with a circa-24-h period. The expression of per1 in the brain of fish under LD showed a daily rhythm with the acrophase (peak time) at the end of the dark phase regardless of feeding schedule. This brain rhythm disappeared in LL fish under both random feeding and scheduled feeding. Feeding at MD advanced the phase of per1 in the liver by 7?h compared with the ML-fed group phase (23:54 versus 07:23?h, respectively). In addition, under LL scheduled feeding entrained the rhythms of per1 expression in the liver. This study reveals for the first time that scheduled feeding entrains peripheral oscillators in a fish species, zebrafish, which is a powerful model widely used for molecular genetics and for the study of basic clock mechanisms of the vertebrate circadian system. (Author correspondence: jflopez@um.es)     

Feeding and circadian clocks

The mammalian genome encodes at least a dozen of genes directly involved in the regulation of the feedback loops constituting the circadian clock. The circadian system is built up on a multitude of oscillators organized according to a hierarchical model in which neurons of the suprachiasmatic nuclei of the hypothalamus may drive the central circadian clock and all the other somatic cells may possess the molecular components allowing tissues and organs to constitute peripheral clocks. Suprachiasmatic neurons are driving the central circadian clock which is reset by lighting cues captured and integrated by the melanopsin cells of the retina and define the daily rhythms of locomotor activity and associated physiological regulatory pathways like feeding and metabolism. This central clock entrains peripheral clocks which can be synchronized by non-photic environmental cues and uncoupled from the central one depending on the nature and the strength of the circadian signal. The human circadian clock and its functioning in central or peripheral tissues are currently being explored to increase the therapeutic efficacy of timed administration of drugs or radiation, and to offer better advice on lighting and meal timing useful for frequent travelers suffering from jet lag and for night workers' comfort. However, the molecular mechanism driving and coordinating the central and peripheral clocks through a wide range of synchronizers (lighting, feeding, physical or social activities) remains a mystery.     

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.     

"Feeding time" for the brain: a matter of clocks.

Circadian clocks are autonomous time-keeping mechanisms that allow living organisms to predict and adapt to environmental rhythms of light, temperature and food availability. At the molecular level, circadian clocks use clock and clock-controlled genes to generate rhythmicity and distribute temporal signals. In mammals, synchronization of the master circadian clock located in the suprachiasmatic nuclei of the hypothalamus is accomplished mainly by light stimuli. Meal time, that can be experimentally modulated by temporal restricted feeding, is a potent synchronizer for peripheral oscillators with no clear synchronizing influence on the suprachiasmatic clock. Furthermore, food-restricted animals are able to predict meal time, as revealed by anticipatory bouts of locomotor activity, body temperature and plasma corticosterone. These food anticipatory rhythms have long been thought to be under the control of a food-entrainable clock (FEC). Analysis of clock mutant mice has highlighted the relevance of some, but not all of the clock genes for food-entrainable clockwork. Mutations of Clock or Per1 do not impair expression of food anticipatory components, suggesting that these clock genes are not essential for food-entrainable oscillations. By contrast, mice mutant for Npas2 or deficient for Cry1 and Cry2 show more or less altered responses to restricted feeding conditions. Moreover, a lack of food anticipation is specifically associated with a mutation of Per2, demonstrating the critical involvement of this gene in the anticipation of meal time. The actual location of the FEC is not yet clearly defined. Nevertheless, current knowledge of the putative brain regions involved in food-entrainable oscillations is discussed. We also describe several neurochemical pathways, including orexinergic and noradrenergic, likely to participate in conveying inputs to and outputs from the FEC to control anticipatory processes.     

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.     

Interactions between light, mealtime and calorie restriction to control daily timing in mammals

Daily variations in behaviour and physiology are controlled by a circadian timing system consisting of a network of oscillatory structures. In mammals, a master clock, located in the suprachiasmatic nuclei (SCN) of the hypothalamus, adjusts timing of other self-sustained oscillators in the brain and peripheral organs. Synchronisation to external cues is mainly achieved by ambient light, which resets the SCN clock. Other environmental factors, in particular food availability and time of feeding, also influence internal timing. Timed feeding can reset the phase of the peripheral oscillators whilst having almost no effect in shifting the phase of the SCN clockwork when animals are exposed (synchronised) to a light–dark cycle. Food deprivation and calorie restriction lead not only to loss of body mass (>15%) and increased motor activity, but also affect the timing of daily activity, nocturnal animals becoming partially diurnal (i.e. they are active during their usual sleep period). This change in behavioural timing is due in part to the fact that metabolic cues associated with calorie restriction affect the SCN clock and its synchronisation to light.     

Disruption of gene expression rhythms in mice lacking secretory vesicle proteins IA-2 and IA-2β

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.     

“Feeding time” for the brain: A matter of clocks

Circadian clocks are autonomous time-keeping mechanisms that allow living organisms to predict and adapt to environmental rhythms of light, temperature and food availability. At the molecular level, circadian clocks use clock and clock-controlled genes to generate rhythmicity and distribute temporal signals. In mammals, synchronization of the master circadian clock located in the suprachiasmatic nuclei of the hypothalamus is accomplished mainly by light stimuli. Meal time, that can be experimentally modulated by temporal restricted feeding, is a potent synchronizer for peripheral oscillators with no clear synchronizing influence on the suprachiasmatic clock. Furthermore, food-restricted animals are able to predict meal time, as revealed by anticipatory bouts of locomotor activity, body temperature and plasma corticosterone. These food anticipatory rhythms have long been thought to be under the control of a food-entrainable clock (FEC). Analysis of clock mutant mice has highlighted the relevance of some, but not all of the clock genes for food-entrainable clockwork. Mutations of Clock or Per1 do not impair expression of food anticipatory components, suggesting that these clock genes are not essential for food-entrainable oscillations. By contrast, mice mutant for Npas2 or deficient for Cry1 and Cry2 show more or less altered responses to restricted feeding conditions. Moreover, a lack of food anticipation is specifically associated with a mutation of Per2, demonstrating the critical involvement of this gene in the anticipation of meal time. The actual location of the FEC is not yet clearly defined. Nevertheless, current knowledge of the putative brain regions involved in food-entrainable oscillations is discussed. We also describe several neurochemical pathways, including orexinergic and noradrenergic, likely to participate in conveying inputs to and outputs from the FEC to control anticipatory processes.     

Is the food‐entrainable circadian oscillator in the digestive system?

Food-anticipatory activity (FAA) is the increase in locomotion and core body temperature that precedes a daily scheduled meal. It is driven by a circadian oscillator but is independent of the suprachiasmatic nuclei. Recent results that reveal meal-entrained clock gene expression in rat and mouse peripheral organs raise the intriguing possibility that the digestive system is the site of the feeding-entrained oscillator (FEO) that underlies FAA. We tested this possibility by comparing FAA and Per1 rhythmicity in the digestive system of the Per 1-luciferase transgenic rat. First, rats were entrained to daytime restricted feeding (RF, 10 days), then fed ad libitum (AL, 10 days), then food deprived (FD, 2 days). As expected FAA was evident during RF and disappeared during subsequent AL feeding, but returned at the correct phase during deprivation. The phase of Per1 in liver, stomach and colon shifted from a nocturnal to a diurnal peak during RF, but shifted back to nocturnal phase during the subsequent AL and remained nocturnal during food deprivation periods. Second, rats were entrained to two daily meals at zeitgeber time (ZT) 0400 and ZT 1600. FAA to both meals emerged after about 10 days of dual RF. However, all tissues studied (all five liver lobes, esophagus, antral stomach, body of stomach, colon) showed entrainment consistent with only the night-time meal. These two results are inconsistent with the hypothesis that FAA arises as an output of rhythms in the gastrointestinal (GI) system. The results also highlight an interesting diversity among peripheral oscillators in their ability to entrain to meals and the direction of the phase shift after RF ends.