Disassociation between Preprandial Gut Peptide Release and Food-Anticipatory Activity

Abstract

Animals learn to anticipate a meal as evidenced by increases in premeal activity. This learned response appears to be independent of the nutrient status of an animal because food-anticipatory activity (FAA) can be seen after entrainment by a highly palatable food when rats remain ad libitum on chow. Mealtime feeding not only induces an increase in activity but also appears to entrain the secretion of various peptides prior to a meal including insulin, ghrelin, and glucagon-like peptide-1 (GLP-1). It is not clear whether these meal-anticipatory changes in peptides are causally associated with FAA. To assess whether FAA and preprandial peptide changes co-occur with meal entrainment using different diets, rats were conditioned to receive a 6-h chow meal, 6-h high-fat meal, or 2 h access of chocolate while ad libitum on chow in the middle of the light cycle. FAA was measured for 4 h prior to mealtime. Rats were then killed at 90, 60, and 30 min prior to mealtime and plasma was collected. Although the chocolate-entrained rats showed comparable FAA with the nonchocolate-entrained animals, they did not show anticipatory increases in the ghrelin or GLP-1. All entrainment conditions induced a decrease in insulin and an increase in glucose prior to mealtime. These data suggest that separate mechanisms may underlie the preprandial increases in ghrelin and GLP-1 and changes in FAA, insulin, and glucose.

Organisms have evolved to predict the 24-h light, 24-h dark cycle and have developed an endogenous biological clock to ensure that physiological processes are performed at the optimal time of day or night. These biological rhythms influence nearly all aspects of physiology and behavior, including feeding behavior and metabolism. Disruptions in the normal timing system of the body are associated with disturbances of these energy-related processes. For example, night-shift workers, whose day/night cycle activity patterns are reversed, are more likely to develop metabolic syndrome (1). Individuals who get a limited amount of sleep also have reduced circulating levels of the anorectic hormone leptin, increased levels of the orexigenic hormone ghrelin, and increased hunger and appetite (2, 3). These findings suggest an association between circadian cycles and physiological factors involved in energy homeostasis. A disruption in the normal biological rhythms may have a negative effect on body weight in humans, and resetting the circadian cycle may be a key to alleviating many metabolic disturbances and obesity.

Disruptions in biological rhythms can occur because of altered sleep/wake cycles or changes in scheduled mealtimes. The biological significance of food makes the temporal availability of meals critical for many different behavioral or physiological functions. Humans and other animal species learn to associate food availability with cues in the environment or properties intrinsic to the food (4, 5). We associate times of the day with eating so that we are normally eating meals at breakfast, lunch, and dinner. Humans and other animal species have learned to expect food at certain times of the day as indicated by preprandial increases in both locomotor activity and peptides/hormones. When animals, including humans, eat at mealtimes, there are increases in anticipatory activity in the hours before meal delivery [food anticipatory activity (FAA)]. FAA occurs even when light cues are omitted, which suggests that other signals are able to entrain these behavioral rhythms (6, 7). The nutritional content or the palatability of a food or the energy status of the animal may all play roles in meal entrainment. Thus, FAA is induced by a chocolate meal when animals are fed ad libitum on chow (8), and high-fat meal feeding has been shown to attenuate FAA compared with chow meal feeding (9). The rhythms of many physiological parameters are also affected by meal entrainment [e.g. body temperature (10), gastrointestinal motility (11)]. Plasma levels of insulin, ghrelin, and glucagon-like peptide (GLP-1) have been shown to be altered before a meal at times that parallel the increases in FAA (12–14). It is not known, though, whether the preprandial changes in gut peptides also occur under the variety of meal entrainment conditions that induce FAA. Evaluating whether FAA and gut peptide changes always occur in tandem under similar conditions or whether there is a differential expression of the behavioral and physiological meal entrainment rhythms that is diet dependent will help to elucidate the underlying mechanism responsible for setting these biological rhythms. To assess whether these preprandial peptide changes are associated with the FAA that occurs under chow or high-fat meal entrainment, rats were conditioned to a 6-h meal of each diet in the middle of the light cycle. The peptide responses were also evaluated after palatable meal entrainment that occurred by conditioning rats to a 2-h access of chocolate in the middle of the light cycle while remaining ad libitum on chow. FAA was measured under all entrainment conditions and plasma levels of insulin, glucose, ghrelin, and GLP-1 were assessed at 90, 60, and 30 min before the normal mealtime.

Materials and Methods

Animals

Two replicates of male Sprague Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) with an initial weight range of 275–300 g were used for all experiments (n = 42 for each meal entrainment condition). The rats were individually housed in tub cages and maintained in a 12-h light, 12-h dark schedule (lights on at 0700 h). Rats were acclimated to housing conditions and ad libitum fed either standard laboratory chow (Global Diet-2018; Harlan Teklad, Indianapolis, IN; 3.3 kcal/g; 5% fat, 18% protein, 77% carbohydrate) or high-fat diet (Research Diets, New Brunswick, NJ; D12492; 5.24 kcal/g; 60% fat, 20% protein, 20% carbohydrate). Body weights were taken weekly. After there was a significant difference in body weight between the rats on the high-fat compared with the chow diet (first replicate: chow diet group = 465.6 ± 9.13, high fat group = 489.3 ± 12.0628; second replicate: chow diet group = 409.99 ± 5.26, high fat group = 444.31 ± 6.47), meal entrainment began for all animals. Water was available at all times during the experiment. All procedures were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University.

Chow and high-fat meal entrainment

Half of the 42 rats in each diet condition were meal entrained (n = 21 for each diet condition) and the other half received food ad libitum (n = 21 for each diet condition). The meal-entrained animals were fed chow or high-fat diet for 6 h in the middle of the light cycle (1000–1600 h). The food was given in excess and taken away at the end of the 6-h period and weighed. All animals were meal entrained or remained ad libitum on the diet for 3 wk. FAA and peptide changes were then evaluated as stated below.

Chocolate entrainment

In a separate group, half of the 42 rats were chocolate entrained (n = 21) and the other half did not receive a chocolate meal (n = 21). The chocolate-entrained animals were fed 5 g of Hershey's chocolate (4.9 kcal/g; 50% fat, 5% protein, 45% carbohydrate; Hershey, PA) for 2 h in the middle of the light cycle (1200–1400 h), while remaining ab libitum on chow. Any excess chocolate was taken away at the end of the 2-h period and weighed. All animals were fed under each condition for 3 wk. FAA and peptide changes were then evaluated as stated below.

Food-anticipatory activity

FAA was measured by manually scoring behaviors of videotaped animals for 4 h before meal delivery (n = 21 for each of the six groups of animals). Sleeping vs. activity was scored as minutes with the activity behaviors subdivided into the following: grooming, sitting, exploring, eating, drinking, and repositioning. An observer that was blind to the animal groups observed the videotape for the animals and recorded the time when the animal initiated each behavior to the time when the behavior was terminated. The behaviors were defined as follows: sleeping as curled up or stretched out, eyes closed; exploring as locomoting around the cage; grooming as cleaning self; sitting as alert, eyes open but not moving; eating as holding food, eating or mouth in food hopper eating; drinking as licking end of water spout; repositioning as adjusting the position of the body that occurred less than 10 sec. Behaviors were scored as the length of time for each behavior over the 4-h observation period, and then the percentage of total time (4 h) was calculated for each group.

Plasma hormone assays

Subsets of rats from each group (d 22–25 of entrainment) were killed by decapitation at 90, 60, and 30 min before meal delivery (n = 7 for each time point from each of the six groups of animals). These time points were chosen based on the time period of greatest increase in FAA as indicated by scoring of videotaped animals. Trunk blood from each rat was collected. Blood glucose levels were determined with a glucometer (Freestyle Lite; Abbott Laboratories, Abbott Park, IL) using a small sample of the trunk blood. The remaining blood was collected into an EDTA-coated tube with the addition of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride to the samples to be processed for ghrelin (active) and dipeptidyl peptidase IV inhibitor to the GLP-1 samples as described by the manufacturer's instructions (see below). All samples were maintained on ice until centrifuged at 3000 rpm for 10 min. Samples were then stored at −80 C until processed using standard RIA kits (Millipore, St. Charles, MO) according to the manufacturers' protocol to determine plasma ghrelin (active) and insulin. Plasma GLP-1 (active) was determined using an ELISA kit (Millipore) and processed according to the manufacturers' instructions.

Data analysis

Data are presented as mean ± sem. FAA was analyzed using a one-way ANOVA for each feeding condition. Body weight, food intake, and plasma peptide levels were analyzed using separate two-way, repeated-measures ANOVA with group (meal fed or non-meal fed) as the between-subject factors and time as the within-subject factor using Number Crunching statistical software (NCSS version 2000; Kaysville, UT). Newman-Keuls post hoc tests were used when appropriate. Differences among groups were considered statistically significant if P < 0.05.

Results

Food-anticipatory activity

FAA was increased in the chow, high-fat, and chocolate entrainment conditions compared with the non-meal-fed control animals for each group (P < 0.05; Fig. 1, A–C).

Fig. 1.

Activity of non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions. The dotted oval highlights the percentage of total time during the 4 h before meal feeding that the animals in each group spent being active. Explore, groom, sit, eat, drink, and reposition are the specific behaviors that were scored and added to formulate the activity for each group. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Body weight

There was a significant decrease in body weight in the chow and high-fat meal fed animals compared with non-meal-fed animals in each condition (P < 0.05; Fig. 2, A and B). Specifically, significant decreases in body weight on wk 3 for the chow-entrained animals and wk 2 and 3 for the high-fat-entrained animals were seen compared with the non-meal-entrained animals in each condition (P < 0.05; Fig. 2, A and B). There was no significant difference in the body weight of the chocolate-entrained animals compared with the non-chocolate-entrained group (Fig. 2C).

Fig. 2.

Body weight for the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions across the 3 wk of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Food intake

Food intake during the last 2 wk of meal entrainment was lower relative to that of the non-meal-fed rats (P < 0.05; Fig. 3, A and B). Post hoc tests revealed that intake was significantly lower each day for the last 2 wk of chow or high-fat meal-fed animals compared with non-meal-fed controls (P < 0.05; Fig. 3, A and B). Although there was also a significant decrease in chow intake in the chocolate-entrained animals compared with the non-chocolate-fed animals (P < 0.05), there was no difference in the total kilocalories consumed (kilocalories from chocolate + kilocalories from chow vs. kilocalories from chow in the chow only group) (Fig. 3C).

Fig. 3.

Daily caloric intake of the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions across the last 2 wk of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Plasma hormones

Plasma ghrelin levels were significantly increased in the chow- and high-fat-entrained animals compared with the non-meal-fed controls in each condition (P < 0.05; Fig. 4, A and B). Plasma ghrelin was specifically increased in the chow entrained animals at 90, 60, and 30 before meal feeding and in the high-fat-entrained animals at 60 and 30 min before meal feeding (P < 0.05; Fig. 4, A and B). There was no significant difference in plasma ghrelin between the chocolate-entrained and non-chocolate-entrained animals (Fig. 4C). Plasma GLP-1 levels were significantly increased in the chow- and high-fat-entrained animals compared with the non-meal-fed animals in each condition (P < 0.05; Fig. 5, A and B). Specifically, GLP-1 was significantly increased in the chow-entrained animals at 90 and 60 min before meal feeding and in the high-fat-entrained animals at 60 min before meal feeding (P < 0.05; Fig. 5, A and B). There was no significant difference in plasma GLP-1 between the chocolate-entrained and non-chocolate-entrained animals (Fig. 5C). Blood glucose levels were significantly increased in the chow-, high-fat, and chocolate-entrained animals compared with the non-meal-fed animals in each condition (P < 0.05; Fig. 6, A–C). Specifically, glucose was significantly increased in the chow- and high-fat entrained animals at 90 min before meal feeding and in the chocolate-entrained animals at 30 min before meal feeding (P < 0.05; Fig. 6, A–C). Plasma insulin levels were significantly decreased in the chow-, high-fat-, and chocolate-entrained animals compared with the non-meal-fed animals in each condition (P < 0.05; Fig. 7, A–C). Specifically, insulin was significantly decreased in the chow-entrained animals at 90 and 30 min before meal feeding (P < 0.05; Fig. 7A). Insulin levels in the high-fat and chocolate-entrained animals were significantly decreased at 90 and 60 min before meal feeding (P < 0.05; Fig. 7, B and C).

Fig. 4.

Plasma ghrelin levels of the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions for animals killed at 90, 60, and 30 min before the normal time of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Fig. 5.

Plasma GLP-1 levels of the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions for animals killed at 90, 60, and 30 min before the normal time of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Fig. 6.

Blood glucose levels of the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions for animals killed at 90, 60, and 30 min before the normal time of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Fig. 7.

Plasma insulin levels of the non-meal-fed and meal-fed animals in the chow-entrainment (A), high-fat-entrainment (B), and chocolate-entrainment (C) conditions for animals killed at 90, 60, and 30 min before the normal time of meal feeding. Values are mean ± sem. *, Significant difference from the non-meal-fed group for each condition (P < 0.05).

Discussion

The aim of the present study was to investigate whether preprandial peptide changes and FAA co-occur under a variety of meal entrainment conditions. Although FAA was induced by both meal (chow and high fat) and chocolate entrainment, chocolate-entrained animals did not show the same anticipatory increases in ghrelin and GLP-1 seen in the chow- or high-fat-entrained animals. By contrast, an increase in glucose and a decrease in insulin occurred before mealtime in all entrainment conditions. Thus, elevations in ghrelin and GLP-1 plasma levels are not necessary for FAA to occur, suggesting that separate mechanisms may underlie these meal-entrained changes in peptide levels vs. those in control of the anticipatory behavior seen in both meal and chocolate entrainment conditions. The similar changes in FAA and insulin and glucose levels under all entrainment conditions may be driven by a common mechanism.

Anticipatory increases in ghrelin and GLP-1 were evident only in the chow- and high-fat-entrained animals, the two meal-feeding conditions in which the food intake and body weight were significantly decreased compared with the ad libitum control animals in the same diet condition. The chocolate-entrained animals that did not show preprandial increases in ghrelin and GLP-1 consumed equal kilocalories and had a similar body weight as the non-chocolate-fed controls. Thus, the anticipatory changes in peptides with meal feeding may be driven by food restriction or decreased body weight compared with the ad libitum control animals and not by the meal entrainment. Ghrelin is known to be an orexigenic peptide that promotes hunger and increases food intake (15, 16). The increase in ghrelin levels in the present study follows the preprandial rhythm of expression that has been shown in a variety of species, including humans (15, 17, 18). On the other hand, the increase in GLP-1 before meal feeding is in opposition of the traditionally defined role of GLP-1 as a satiety peptide. These data, though, replicate previous findings by Vahl et al. (13) that show GLP-1 does increase before meal feeding in rats. Vahl et al. (13) proposed that the preprandial increases in GLP-1 play a role in preparing the gastrointestinal tract for a large amount of food in a limited amount of time. When a GLP-1 receptor antagonist was administered 1 h before the normal preprandial increase in GLP-1, a significant reduction in food intake occurred once meal feeding began (13). Taken together, it appears that the amount of food eaten or energy status of the animal may be more important than the feeding pattern in driving the preprandial rhythm in ghrelin and GLP-1.

Even though increases in ghrelin and GLP-1 are not necessary for FAA to occur, these peripheral hormonal responses may still be linked to the anticipatory activity observed before meals. The meal and chocolate-entrained animals may have a graded level of anticipation for the meal based on the caloric load and/or energy status of the animal. The meal-entrained rats were entrained to expect all of their calories for a given day (chow entrained ate ∼60 kcal and high fat entrained ate ∼70 kcal), whereas the chocolate-entrained animals were receiving only a portion of their overall intake from the chocolate meal (∼20 kcal). The daily food intake of the chow- and high-fat meal-fed animals also was significantly lower than their ad libitum control animals (chow entrained ate ∼30 kcal less and high fat entrained ate ∼40 kcal less than control animals), whereas the chocolate meal-fed animals ate equal daily kilocalorie amounts as their non-meal-fed counterparts. This difference in caloric load of the meal may be reflected in the time point of greatest activity before meal between the groups. The level of activity was the greatest in the meal-fed animals 2 h before the meal and continued until the meal began, whereas the chocolate-fed animals had the greatest amount of activity 1.5–1 h before the meal. The increase in preprandial glucose occurs at 90 min before the meal vs. 30 min prior the chocolate meal. This earlier increase in glucose in the animals that are expecting a greater caloric load may be correlated with the earlier onset and longer-lasting FAA response. This might suggest that ghrelin and GLP-1 are responsible for an enhanced FAA effect after meal entrainment. Meal entrained mice that lack the ghrelin receptor (GH secretagogue receptor) still show FAA, but the response is attenuated compared with meal entrained wild-type mice (18). In particular, the onset of FAA occurs later in the GH secretagogue receptor knockout animals, but the peaks are still similar to the wild types. Thus, it is possible that there is a graded effect of peripheral peptide changes and FAA that could co-occur: the greater the caloric load or energy deprivation status of the animal, the greater the peptide response (i.e. a greater cephalic response) and the greater the FAA.

The decrease in body weight in the chow- and high-fat-entrained animals makes it difficult to tease apart the separate contribution of meal entrainment from the general effects of food deprivation. We had designed the experiment using a 6-h meal, instead of a 2- or 4-h meal used in most other meal-feeding experiments, to alleviate the confound of a decrease in body weight. We found that even a longer mealtime does not give the animal enough time to consume the amount of kilocalories that the ad libitum animals eat. Using the chocolate meal-entrained condition while the animals are ad libitum on chow is another way to test meal entrainment-induced effects on FAA and plasma peptide levels without differences in body weight between the chocolate-fed and control animals. It should also be pointed out that there is a difference in body between all three meal-entrained groups when the animals were killed at wk 3. Interestingly, the difference in body weight is greater between the chow- and high-fat-entrained animals than the chow- and chocolate-entrained animals (chow = 438, high-fat = 488, and chocolate = 454), but the chow- and high-fat-entrained animals had the same peptide changes and the chow- and chocolate-entrained animals did not. This suggests that it is not a body weight difference that is responsible for the peptide changes.

It is not clear whether the decreased consumption of food or body weight in our 6-h chow or high-fat diet conditions is consistent with previous meal entrainment models because these are not usual measures in studies of entrainment. We had previously found that a 4-h chow meal in male Sprague Dawley rats, the same type of animals used in the present experiments, also resulted in significant decreases in food intake and body weight after 3 wk of meal entrainment (Dailey M.J., K.C. Stingl, and T.H. Moran, unpublished observations). Previous experiments investigating ghrelin and GLP-1 after a 4-h meal entrainment reported that the meal-fed Long-Evans rats eventually consumed the same amount as they had before the beginning of the meal feeding, but there was no comparison of the food intake to non-meal-fed animals at the time of peptide analysis (13, 14). Another study using male Wistar rats reports that after 3 wk of meal entrainment, animals are able to reach similar food intake amounts as controls, but the body weight of meal entrained animals was significantly reduced compared with freely feeding rats (19). Differences in food intake and body weight need to be taken into consideration when trying to understand the underlying mechanisms driving changes in behavioral and peptide rhythms. In the present study, it appears that the preprandial increases in ghrelin and GLP-1 may be driven by food restriction and not solely by mealtime cues.

The increase in glucose and decrease in insulin occurred in all entrainment conditions and may be driven by mealtime cues. La Fleur et al. (20) showed that blood glucose concentrations start to rise before rats start to eat, indicating the existence of an endogenous glucose rhythm. This rhythm, though, may be independent of the time of feeding and rely more on light cues (20). Multiple studies have shown insulin levels in meal-entrained animals to be decreased compared with freely feeding animals (12, 20, 21). The main physiological stimulus for insulin secretion from the pancreas is an elevation in blood glucose concentration due to food intake, and insulin levels closely match food intake in meal entrained animals (20). Insulin secretion also is stimulated by increases in GLP-1, a well-known incretin (22). When you compare the timing of the increases in glucose and GLP-1 with the decreases in insulin, there is no indication that there may be a causal effect of one on the other. Thus, the decrease in insulin may be entrained by another mechanism. Insulin not only promotes glucose uptake into tissues but has also been proposed to be a satiety peptide (23) It may be physiologically adaptive to decrease levels before the only meal period in a given day or before a high-calorie palatable meal to ensure the greatest amount of food can be eaten in this limited time. By contrast, a cephalic phase increase in insulin has been described to prepare the animal for a bolus of food that it has to digest and incorporate into tissue to use for energy (12). Having a greater amount of insulin release would allow glucose to immediately be taken up into tissues for use. This cephalic phase increase in the level of insulin is compared with other time points throughout the day/night cycle within the same meal entrained animal, not compared with ad libitum counterparts at the same time point (12). In the present study, we did not measure insulin levels at times throughout a 24-h period in the meal- or chocolate-entrained animals, only in the 90 min before meal feeding. Others, though, have taken multiple blood samples throughout the 24-h period and still have not replicated this cephalic insulin response in meal entrainment (20, 21). It is not clear why there is this discrepancy in the literature, but it may have something to do with the timing or method of blood sampling.

FAA itself may drive the changes in glucose and insulin seen in all entrainment conditions in the present study. Increased locomotor activity decreases insulin secretion through the increased release of noradrenaline by the sympathetic nervous system (24). Increased sympathetic input to the liver also enhances glucose output, thus promoting hyperglycemia (25). A daily bout of activity can also restore hormone synthesis in animals that have lost normal biological rhythms caused by a lesion of the suprachiasmatic nucleus, the master clock considered to drive the rhythmic release of many peptides and hormones (26). Thus, it is possible that the changes in activity may drive the premeal changes in glucose and insulin present in the meal and chocolate entrained animals.

Meal entrainment is able to alter biological rhythms for behavior and physiological indices important in energy homeostasis. The mechanism underlying this entrainment appears to involve autoregulatory transcriptional/translational feedback loops of circadian genes located centrally and peripherally (27). We know the importance of these circadian gene feedback loops because disruption of expression of these genes leads to metabolic disorders. Circadian locomotor output cycles (Clock) mutants have altered feeding rhythm, overeat, have increased fat, and are hyperglycemic (28). Brain and muscle aryl hydrocarbon receptor nuclear translocator-like (Bmal) mutants have type 2 diabetic symptoms (29). Period 2 (Per2) mutants develop obesity and lose rhythmic expression of feeding hormones (30). Therefore, understanding the interaction of circadian gene expression and feeding/metabolism may reveal how to optimize circadian rhythms and correct metabolic disorders.

Under freely feeding conditions, there is a pattern of hormone secretions that synchronize the light/dark cycle and normal feeding time (light in humans and dark in nocturnal animals). When alterations occur in the normal sleep/wake cycle, mealtime, or both, these rhythms are desynchronized and appear to entrain to the new mealtime schedule (31, 32). It is not clear, though, how or what mealtime cues set these new biological rhythms. Intrinsic properties of the food (food metabolites or hormones/peptides released as a result of digestion) may be able to set the circadian gene feedback loops to drive further changes in behavior and physiology. A direct mechanism of action has been suggested by experiments that show glucose decreases Per1 and Per2 mRNA levels in cultured Rat-1 fibroblasts (33). The ability of insulin to increase the mRNA expression of Per1 in cultured Rat-1 fibroblasts suggests an indirect mechanism by which a meal can affect circadian rhythms (34).

The biological rhythms of metabolic enzymes, hormones, and peptides contribute to energy homeostasis. Teasing apart the separate contributions of organ-specific rhythmic factors may help in identifying important components in a pathway responsible for determining when we eat, how much we eat, and how the food we eat is metabolized. Meal entrainment may serve as a useful model for understanding how food interacts with circadian gene oscillators that are responsible for the timing of expression/release of factors important in metabolism and the development of metabolic disorders.