Circadian clocks for all meal-times: anticipation of 2 daily meals in rats

Anticipation of a daily meal in rats has been conceptualized as a rest-activity rhythm driven by a food-entrained circadian oscillator separate from the pacemaker generating light-dark (LD) entrained rhythms. Rats can also anticipate two daily mealtimes, but whether this involves independently entrained oscillators, one 'continuously consulted' clock, cue-dependent non-circadian interval timing or a combination of processes, is unclear. Rats received two daily meals, beginning 3-h (meal 1) and 13-h (meal 2) after lights-on (LD 14:10). Anticipatory wheel running began 68±8 min prior to meal 1 and 101±9 min prior to meal 2 but neither the duration nor the variability of anticipation bout lengths exhibited the scalar property, a hallmark of interval timing. Meal omission tests in LD and constant dark (DD) did not alter the timing of either bout of anticipation, and anticipation of meal 2 was not altered by a 3-h advance of meal 1. Food anticipatory running in this 2-meal protocol thus does not exhibit properties of interval timing despite the availability of external time cues in LD. Across all days, the two bouts of anticipation were uncorrelated, a result more consistent with two independently entrained oscillators than a single consulted clock. Similar results were obtained for meals scheduled 3-h and 10-h after lights-on, and for a food-bin measure of anticipation. Most rats that showed weak or no anticipation to one or both meals exhibited elevated activity at mealtime during 1 or 2 day food deprivation tests in DD, suggesting covert operation of circadian timing in the absence of anticipatory behavior. A control experiment confirmed that daytime feeding did not shift LD-entrained rhythms, ruling out displaced nocturnal activity as an explanation for daytime activity. The results favor a multiple oscillator basis for 2-meal anticipatory rhythms and provide no evidence for involvement of cue-dependent interval timing.     

Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata)

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD?+?RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD?+?RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture. (Author correspondence: javisan@um.es).     

Hormonal and metabolic rhythms associated with the daily scheduled nursing in rabbit pups

Young rabbits are nursed every 24 h for a period of 3-5 min. As a consequence, pups are synchronized to this nursing event; this synchronization is characterized by increased locomotor activity and a peaking of core temperature and plasma corticosterone in anticipation of the daily meal. Ghrelin is a hormone suggested to play a role in meal initiation and to promote food intake. The present study explored the role of ghrelin in food-entrained conditions. Newborn rabbits were maintained in constant darkness and nursed once daily at 1000 by the lactating dam. On postnatal day 7, rabbits were killed at six different time points to complete a 24-h cycle. All pups developed locomotor rhythms entrained by mealtime and exhibited anticipatory activity. Food-entrained rhythms in plasma corticosterone and free fatty acids were observed even if two meals were omitted. In contrast, daily food-driven rhythms in stomach weight, plasma glucose, liver glycogen, and ghrelin did not persist when two meals were omitted. Peak ghrelin levels were observed at the moment in the cycle when the stomach weight was lowest, i.e., before initiation of anticipation. The present data are in agreement with previous data from rabbit pups maintained in light-dark conditions and provide evidence that 7- to 9-day-old rabbits in constant darkness can exhibit metabolic and hormonal rhythms mainly driven by the restricted daily nursing.     

Chronobiology in mammalian health

Circadian rhythms are daily cycles of physiology and behavior that are driven by an endogenous oscillator with a period of approximately one day. In mammals, the hypothalamic suprachiasmatic nuclei are our principal circadian oscillators which influences peripheral tissue clocks via endocrine, autonomic and behavioral cues, and other brain regions and most peripheral tissues contain circadian clocks as well. The circadian molecular machinery comprises a group of circadian genes, namely Clock, Bmal1, Per1, Per2, Per3, Cry1 and Cry2. These circadian genes drive endogenous oscillations which promote rhythmically expression of downstream genes and thereby physiological and behavioral processes. Disruptions in circadian homeostasis have pronounced impact on physiological functioning, overall health and disease susceptibility. This review introduces the general profile of circadian gene expression and tissue-specific circadian regulation, highlights the connection between the circadian rhythms and physiological processes, and discusses the role of circadian rhythms in human disease.     

Food-anticipatory rhythms under 24-hour schedules of limited access to single macronutrients

Food-restricted rats anticipate a fixed daily mealtime by entrainment of a circadian timekeeping mechanism separate from that which generates daily light-entrainable activity rhythms. The entrainment pathways and rhythm-generating substrates for food-anticipatory rhythms are unknown. In this study, we attempted to define minimal food-related stimuli necessary or sufficient for food anticipation by employing schedules of restricted macronutrient availability, with or without free access to a complementary diet. Rats did not anticipate a daily meal of protein, carbohydrate, or fat, as measured by tilt-cage, running-wheel, or food-bin activity, when they had free access to other nutrients. However, rats did anticipate single-macronutrient meals when they were limited to only two, larger, complementary meals each day (protein-fat, protein-carbohydrate) providing a reduced total number of calories. Previous work has shown that caloric restriction per se is not a prerequisite for food anticipation. In combination with that study, the present results indicate that the size of a nutrient meal, in absolute terms or relative to total daily nutrient intake, is of pre-eminent importance in determining its value as a synchronizer of anticipatory rhythms. The results further suggest that physiological responses unique to the ingestion and absorption of any particular macronutrient are not necessary components of the entrainment pathway.     

Differential roles of breakfast and supper in rats of a daily three-meal schedule upon circadian regulation and physiology

The timing of meals has been suggested to play an important role in circadian regulation and metabolic health. Three meals a day is a well-established human feeding habit, which in today's lifestyle may or may not be followed. The aim of this study was to test whether the absence of breakfast or supper significantly affects the circadian system and physiological function. The authors developed a rat model for their daily three meals study, whereby animals were divided into three groups (three meals, TM; no first meal, NF; no last meal, NL) all fed with the same amount of food every day. Rats in the NF group displayed significantly decreased levels of plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose in the activity phase, accompanied by delayed circadian phases of hepatic peripheral clock and downstream metabolic genes. Rats in the NL group showed lower concentration of plasma TC, HDL-C, and glucose in the rest phase, plus reduced adipose tissue accumulation and body weight gain. Real-time polymerase chain reaction (PCR) analysis indicated an attenuated rhythm in the food-entraining pathway, including down-regulated expression of the clock genes Per2, Bmal1, and Rev-erbα, which may further contribute to the delayed and decreased expression of FAS in lipogenesis in this group. Our findings are consistent with the conclusion that the daily first meal determines the circadian phasing of peripheral clocks, such as in the liver, whereas the daily last meal tightly couples to lipid metabolism and adipose tissue accumulation, which suggests differential physiological effects and function of the respective meal timings.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Differential Roles of Breakfast and Supper in Rats of a Daily Three-Meal Schedule Upon Circadian Regulation and Physiology

The timing of meals has been suggested to play an important role in circadian regulation and metabolic health. Three meals a day is a well-established human feeding habit, which in today's lifestyle may or may not be followed. The aim of this study was to test whether the absence of breakfast or supper significantly affects the circadian system and physiological function. The authors developed a rat model for their daily three meals study, whereby animals were divided into three groups (three meals, TM; no first meal, NF; no last meal, NL) all fed with the same amount of food every day. Rats in the NF group displayed significantly decreased levels of plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and glucose in the activity phase, accompanied by delayed circadian phases of hepatic peripheral clock and downstream metabolic genes. Rats in the NL group showed lower concentration of plasma TC, HDL-C, and glucose in the rest phase, plus reduced adipose tissue accumulation and body weight gain. Real-time polymerase chain reaction (PCR) analysis indicated an attenuated rhythm in the food-entraining pathway, including down-regulated expression of the clock genes Per2, Bmal1, and Rev-erbα, which may further contribute to the delayed and decreased expression of FAS in lipogenesis in this group. Our findings are consistent with the conclusion that the daily first meal determines the circadian phasing of peripheral clocks, such as in the liver, whereas the daily last meal tightly couples to lipid metabolism and adipose tissue accumulation, which suggests differential physiological effects and function of the respective meal timings. (Author correspondence: azwfu2003@yahoo.com.cn)     

Dopaminergic Regulation of Circadian Food Anticipatory Activity Rhythms in the Rat

Circadian activity rhythms are jointly controlled by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) and by food-entrainable circadian oscillators (FEOs) located elsewhere. The SCN mediates synchrony to daily light-dark cycles, whereas FEOs generate activity rhythms synchronized with regular daily mealtimes. The location of FEOs generating food anticipation rhythms, and the pathways that entrain these FEOs, remain to be clarified. To gain insight into entrainment pathways, we developed a protocol for measuring phase shifts of anticipatory activity rhythms in response to pharmacological probes. We used this protocol to examine a role for dopamine signaling in the timing of circadian food anticipation. To generate a stable food anticipation rhythm, rats were fed 3h/day beginning 6-h after lights-on or in constant light for at least 3 weeks. Rats then received the D2 agonist quinpirole (1 mg/kg IP) alone or after pretreatment with the dopamine synthesis inhibitor α-methylparatyrosine (AMPT). By comparison with vehicle injections, quinpirole administered 1-h before lights-off (19h before mealtime) induced a phase delay of activity onset prior to the next meal. Delay shifts were larger in rats pretreated with AMPT, and smaller following quinpirole administered 4-h after lights-on. A significant shift was not observed in response to the D1 agonist SKF81297. These results provide evidence that signaling at D2 receptors is involved in phase control of FEOs responsible for circadian food anticipatory rhythms in rats.     

Scheduled feeding results in adipogenesis and increased acylated ghrelin

Ghrelin, known to stimulate adipogenesis, displays an endogenous secretory rhythmicity closely related to meal patterns. Therefore, a chronic imposed feeding schedule might induce modified ghrelin levels and consequently adiposity. Growing Wistar rats were schedule-fed by imposing a particular fixed feeding schedule of 3 meals/day without caloric restriction compared with total daily control intake. After 14 days, their body composition was measured by DEXA and compared with ad libitum-fed controls and to rats daily intraperitoneal injection with ghrelin. Feeding patterns, circadian activity, and pulsatile acylated ghrelin variations were monitored. After 14 days, rats on the imposed feeding schedule displayed, despite an equal daily calorie intake, a slower growth rate compared with ad libitum-fed controls. Moreover, schedule-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared with ad libitum-fed controls. Interestingly, ghrelin-treated rats also showed an increase in fat mass, but the fat/lean ratio was not significantly increased compared with controls. In the schedule-fed rats, spontaneous activity and acylated ghrelin levels were increased and associated with the scheduled meals, indicating anticipatory effects. Our results suggest that scheduled feeding, associated with intermittent fasting periods, even without nutrient/calorie restriction on a daily basis, results in adipogenesis. This repartitioning effect is associated with increased endogenous acylated ghrelin levels. This schedule-fed model points out the delicate role of meal frequency in adipogenesis and provides an investigative tool to clarify any effects of endogenous ghrelin without the need for ghrelin administration.     

Review of the Dual-Clock Control of Tidal Rhythms and the Hypothesis that the Same Clock Governs Both Circatidal and Circadian Rhythms

Discoveries first published in 1986 did not fit the de rigueur working hypothesis that the clocks governing tide-associated rhythms had a fundamental period of 12.4 h, a value equal to the average interval between successive tides on most coastlines of the world. To explain the results a dual-clock schema was fashioned that envisioned two clocks, strongly coupled together 180° antiphase, each running at a basic rate of 24.8 h (the interval of a lunar day), as the driving agents of tide-associated rhythms (details are given in the text). This elaboration has been named the circalunidian-clock hypothesis, a hypocorism used in some armchair ruminations back in 1973. In the decade since 1986, a goodly amount of evidence has been garnered that is consistent with this hypothesis—suggesting that first-call divination appears to have been visionary. Acceptance of this hypothesis leads to further cerebration that a 24.8-h clock, its circa periods in constant conditions, and other properties—which fully overlap with our perception of the circadian clock that drives daily rhythms—may indicate that circadian and circalunidan timepieces are not different entities. The known properties of both daily and lunar clock-types are compared and contrasted, and, with the exception of one feature (for which there is at least a philosophical explanation), it is concluded that the same clock that drives tidal rhythms could also motor daily rhythms, i.e., there may be no such thing as a 12.4-h horologue.     

Review of the Dual-Clock Control of Tidal Rhythms and the Hypothesis that the Same Clock Governs Both Circatidal and Circadian Rhythms

Discoveries first published in 1986 did not fit the de rigueur working hypothesis that the clocks governing tide-associated rhythms had a fundamental period of 12.4 h, a value equal to the average interval between successive tides on most coastlines of the world. To explain the results a dual-clock schema was fashioned that envisioned two clocks, strongly coupled together 180° antiphase, each running at a basic rate of 24.8 h (the interval of a lunar day), as the driving agents of tide-associated rhythms (details are given in the text). This elaboration has been named the circalunidian-clock hypothesis, a hypocorism used in some armchair ruminations back in 1973. In the decade since 1986, a goodly amount of evidence has been garnered that is consistent with this hypothesis—suggesting that first-call divination appears to have been visionary. Acceptance of this hypothesis leads to further cerebration that a 24.8-h clock, its circa periods in constant conditions, and other properties—which fully overlap with our perception of the circadian clock that drives daily rhythms—may indicate that circadian and circalunidan timepieces are not different entities. The known properties of both daily and lunar clock-types are compared and contrasted, and, with the exception of one feature (for which there is at least a philosophical explanation), it is concluded that the same clock that drives tidal rhythms could also motor daily rhythms, i.e., there may be no such thing as a 12.4-h horologue.     

Modelling and Analysis of the Feeding Regimen Induced Entrainment of Hepatocyte Circadian Oscillators Using Petri Nets

Circadian rhythms are certain periodic behaviours exhibited by living organism at different levels, including cellular and system-wide scales. Recent studies have found that the circadian rhythms of several peripheral organs in mammals, such as the liver, are able to entrain their clocks to received signals independent of other system level clocks, in particular when responding to signals generated during feeding. These studies have found SIRT1, PARP1, and HSF1 proteins to be the major influencers of the core CLOCKBMAL1:PER-CRY circadian clock. These entities, along with abstracted feeding induced signals were modelled collectively in this study using Petri Nets. The properties of the model show that the circadian system itself is strongly robust, and is able to continually evolve. The modelled feeding regimens suggest that the usual 3 meals/day and 2 meals/day feeding regimens are beneficial with any more or less meals/day negatively affecting the system.     

Food anticipatory activity behavior of mice across a wide range of circadian and non-circadian intervals

When rodents are fed in a limited amount during the daytime, they rapidly redistribute some of their nocturnal activity to the time preceding the delivery of food. In rats, anticipation of a daily meal has been interpreted as a circadian rhythm controlled by a food-entrained oscillator (FEO) with circadian limits to entrainment. Lesion experiments place this FEO outside of the light-entrainable circadian pacemaker in the suprachiasmatic nucleus. Mice also anticipate a fixed daily meal, but circadian limits to entrainment and anticipation of more than 2 daily meals, have not been assessed. We used a video-based behavior recognition system to quantify food anticipatory activity in mice receiving 2, 3, or 6 daily meals at intervals of 12, 8, or 4-hours (h). Individual mice were able to anticipate as many as 4 of 6 daily meals, and anticipation persisted during meal omission tests. On the 6 meal schedule, pre-prandial activity and body temperature were poorly correlated, suggesting independent regulation. Mice showed a limited ability to anticipate an 18 h feeding schedule. Finally, mice showed concurrent circadian and sub-hourly anticipation when provided with 6 small meals, at 30 minute intervals, at a fixed time of day. These results indicate that mice can anticipate feeding opportunities at a fixed time of day across a wide range of intervals not previously associated with anticipatory behavior in studies of rats. The methods described here can be exploited to determine the extent to which timing of different intervals in mice relies on common or distinct neural and molecular mechanisms.     

Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

Resveratrol Regulates Circadian Clock Genes in Rat-1 Fibroblast Cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 μM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

Insect circadian clock outputs

Insects display an impressive variety of daily rhythms, which are most evident in their behaviour. Circadian timekeeping systems that generate these daily rhythms of physiology and behaviour all involve three interacting elements: the timekeeper itself (i.e. the clock), inputs to the clock through which it entrains and otherwise responds to environmental cues such as light and temperature, and outputs from the clock through which it imposes daily rhythms on various physiological and behavioural parameters. In insects, as in other animals, cellular clocks are embodied in clock neurons capable of sustained autonomous circadian rhythmicity, and those clock neurons are organized into clock circuits. Drosophila flies spend their entire lives in small areas near the ground, and use their circadian brain clock to regulate daily rhythms of rest and activity, so as to organize their behaviour appropriately to the daily rhythms of their local environment. Migratory locusts and butterflies, on the other hand, spend substantial portions of their lives high up in the air migrating long distances (sometimes thousands of miles) and use their circadian brain clocks to provide time-compensation to their sun-compass navigational systems. Interestingly, however, there appear to be substantial similarities in the cellular and network mechanisms that underlie circadian outputs in all insects.