Ā@Ā@ILSI JapanĀ@110, 24 (2012)

Hiroaki Oda, Nagoya University

1. Introduction

Well-regulated eating habits are said to be important for health in both the East and the West. The importance of breakfast is emphasized in ancient Japanese books (1,2). We seem to have recognized the importance of the timing of meals empirically. It is generally understood that people who work at night suffer from coronary disease and obesity more frequently (3). Furthermore, there is a relationship between shift work and cancer (4). Nowadays, very active people in the modern society tend to have erratic schedules. Many cannot lead well-regulated lives due to their jobs, making it difficult to fix such lifestyles. However, the vast majority of these people are unaware that irregular eating habits are a major factor leading to health problems. People have inadequate knowledge of the importance of well-regulated eating habits because the mechanisms leading to the effects of the timing of meals on health are unclear. Therefore, clarifying these mechanisms may lead to the development of foods and drugs that normalize body clock.

2. Why is chrononutrition receiving attention now?

According to the National Health and Nutrition Survey, Japan, 2007, one-sixth to one-fifth of the Japanese are suspected to have diabetes (5). This issue is becoming more common worldwide. Obesity is particularly increasing in men. However, caloric intake is decreasing slightly in Japan. Although it is thought that fat intake and decreasing physical activity are contributing to this problem, many people are health conscious and do exercise regularly. Then, what factor(s) are causing this problem? Meals are important. People generally think of what they should eat when they hear the word “meal.” However, it is better to think about the way to eat (“Meal Style”). Nutritional sciences have traditionally prioritized what we eat. The “5 W’s and 1 H (5W1H) of meals” should be considered (i.e., what, when, where, who, why, and how); in other words, the way meals are eaten should be considered to better understand nutrition. Furukawa states that wealth and information are the factors controlling mean lifespan (6) and points out that slightly improving environment further extends mean lifespan. Increasing wealth makes desired foodstuffs more available. Therefore, information regarding ways to eat may be the key to increasing mean and healthy lifespans in the future.

Among the 5W1H of meals, why one eats is excluded because eating itself is the meaning and nature of life. Considering the second law of thermodynamics, metabolic turnover and dynamic equilibrium indicate that the definition of life is “eating and metabolism” in a sense (7). This importantly reflects the fundamental significance of meals.

The most commonly performed molecular biological analyses involve the relationship between time and life. A field called “chronobiology,” which has been widely recognized as one of the basic phenomena of life for a long time, includes the clinically serious subject of sleep disorders. In addition to sleep, many biochemical phenomena involving rhythms were examined extensively in the 1980s. In 1971, an abnormal rhythmicity in Drosophila mutants was discovered (8), indicating that a biological clock is coded in its genes. In 1984, the clock gene of Drosophila, called Period, was discovered (9). However, this was not a breakthrough, and further research on clock genes was performed. Meanwhile, the Japanese Society for Chronobiology was established in 1995. Mouse Period and Clock genes were cloned in 1997 (10). A major breakthrough was the discovery of the negative regulatory feedback for transcription via the binding of CLOCK/BMAL1 to E-box, which forms the basis of biological clocks (11). Since then, research in chronobiology has boomed. However, it was established only approximately 10 years ago and is a relatively immature discipline. We began to study DBP (albumin D-element binding protein), a transcription factor involved in circadian rhythms, as a part of a study on hepatocyte differentiation. This represented a chance for us to launch chronobiology. Just after the start of the study, we became aware of nutritional importance and subsequently progressed towards an experiment aiming to regulate meal timing, which is discussed below. In 2005, the first instance of the term “chrononutrition” appeared in a nutrition textbook edited by us (12). The world’s first book on the subject, entitled “Chrononutrition,” was published in 2009 (13). Chrononutrition, a relatively new field of nutritional sciences, is gaining recognition because it is regarded as the key that will help in understanding why there is an increase in the number of patients with obesity or metabolic syndromes in spite of a decrease in the energy intake.

3. Intrinsic circadian clocks

The human body has a diurnal rhythm (a phenomenon with daily periodicity) (14). Children who sleep well are said to grow up strong, maybe because growth hormone is secreted at night. On the other hand, there are periods called danger times in which sudden death occurs frequently, such as morning (14). Physiological phenomena leading to myocardial and cerebral infarction often occur in the morning. Diurnal rhythms are observed not only with respect to when diseases occur (e.g., stomach ulcers worsen early in the morning), but also when death occurs, which varies depending on the cause of death. The Olympic finals are not held in the morning because that is not when performance is increased both psychologically and physically. Most of these time periods are controlled by intrinsic clocks. Human body is regulated by an internal clock more than we expected.

An internal clock with an approximately 24-hour cycle is called a circadian clock that is responsible for the circadian rhythm, which is different from the passive diurnal rhythm. Organisms are thought to need a circadian clock because of the predictability and functional division of labor. Predictability is essential for obtaining food and escaping from predators. Many mammals are still nocturnal because those that coexisted with dinosaurs were active at night i.e., when the dinosaurs rested. Some mammals are believed to have started becoming active in the daytime to obtain food more easily. Functional division of labor enables temporal division of labor for many complicated cell functions. In general, cell growth occurs at night, while differentiation functions required for activity occurs in the daytime. Many biochemical metabolic pathways overlap. Metabolism cannot occur smoothly unless temporal division of labor occurs. As gluconeogenesis and glycolysis systems cannot work simultaneously, temporal functional division of labor is essential to avoid metabolic contradiction.

We here discuss “time” again. In our minds, we are governed by “absolute time” as suggested by Newton. Time appears to progress linearly. However, modern physics recognizes that time is more complicated. Aristotle thought that movement existed, and time was derived from movement. This concept seems to approximate the “time” of life shown in circadian rhythms. Considering that we feel time by periodic movement, rhythmic biochemical reactions are important for time of life. Although pacemakers are required for life phenomena, human beings do not have precise clock-like watches. Naturally, it is thought that periodic biochemical reactions are used as a clock, which perceives the time.

During evolution, circadian clocks began with the rhythm of simple biochemical reactions (15). Cyanobacteria possess a 24-hour clock that functions via enzymatic reactions (16). However, these cases are exceptions. Circadian clocks mainly function through the negative regulatory feedback of transcription via the clock genes (17). However, there are significant differences in clock genes between plants and animals. Although circadian clocks functioning with respect to the Earth’s rotation have been conserved throughout evolution, the mechanisms and genes themselves have not been conserved. Considering these conditions, it is better to think that periodic biochemical reactions function as clocks rather than to consider biochemical reactions to be coordinated by clocks. The Earth’s rotation is slowing due to the influence of the moon. Therefore, hundreds of millions of years later, days are thought to be 30 or more hours longer; thus, different internal clocks may be involved then.

4. Clock genes

As mentioned above, circadian rhythms involve a clock regulated by transcriptional negative feedback. CLOCK/BMAL1, transcription factors, binds to E-box hexanucleotides to activate the transcription of Per and Cry, which are the clock genes. The complex consisting of Per and Cry inhibits transcriptional activation by CLOCK/BMAL1. Subsequently, decreased Per and Cry activation in turn causes transcriptional activation. This cycle takes about 24 hours. Other clock genes adjust the clock by affecting the feedback system via E-box. Although small gaps among cells occur, it is surprising that this relatively precise clock is regulated by such a simple system.

Biological clock studies originally focused on the clock of the brain. They found that there is a master clock in the suprachiasmatic nucleus (SCN) of the brain, which exhibits strong autonomous oscillations over 24 hours. Consequently, it was understood that the brain clock, which is stimulated by light, controls the entire body. However, this understanding was revised because it was discovered that all peripheral cells have a clock. At present, it is understood that all cells have their own 24-hour clocks that function together as organ clocks, which collectively form an integrated clock through factors that synchronize organs. Therefore, synchronizers are important. The brain clock is generally synchronized by sunlight, which controls the peripheral tissues via the autonomic nervous system and endocrine system, which controls the clock of the entire body. In other words, light is the strongest synchronizer. This makes sense considering that circadian rhythms are based on the Earth’s rotation.

However, the rhythm of the digestive system is reversed when the meal timing is reversed, indicating that the synchronizer of the digestive system clock is stronger than light. Meals have come to be understood as the strongest synchronizer of all organs. Therefore, meals synchronize the clocks of all organs present below the neck. This thought is rational considering that circadian clocks exist to help organisms obtain food in a timely manner. Thus, sunlight is used as a pacemaker to adjust the timing of meals. Furthermore, a study reports that meals synchronize the brain clock, too (18). Meals may facilitate early recovery from jetlag in conjunction with light by adjusting the brain clock.

Examination at the gene level showed that approximately 10% of genes maintain a rhythm in most organs including the liver, heart, and large intestine (19-21). The peak time of gene expression varies by organ even for the same gene, which further indicates that each organ has its own clock. The clocks of the organs cooperate to control the functions of the entire body, which can be defined as good health. Although meals are a strong synchronizer of the organ clocks as mentioned above, the body clock is generally synchronized by sunlight as long as people keep eating at regular hours according to sunlight. However, it has become hard for many people to lead such lives in modern society. Many people have inverted meal timings similar to those in the animal experiment mentioned above. What should such people do? This is a problem of chrononutrition.

5. Clock gene abnormalities and lifestyle-related diseases

Experiments using clock gene knockout mice shows that the loss of clock gene causes not only behavioral disorders, but also metabolic disorders. A report which showed Clock gene knockout mice exhibited obesity and metabolic syndrome published in 2005 received much attention (22). Furthermore, Bmal1 plays an important role in obesity (23, 24). In addition to dysrhythmia, which was originally predicted, metabolic disorders in knockout mice revealed that the circadian clock is strongly linked to peripheral metabolism. Moreover, there is a report on familial advanced sleep phase syndrome due to mutations of Per2 in humans (25). However, no associations between mutations of clock genes and metabolic disorders in human have been reported. Mutations in human clock genes are uncommon, indicating that clock genes may be essential for survival.

6. Irregular meal timing and lipid metabolism abnormalities

As mentioned above, lifestyle-related diseases are common in people who have irregular eating habits, such as shift workers. However, there is no evidence at the molecular level that meal timing itself causes metabolic disorders. Therefore, the influence of meal timing on lipid metabolism is not considered as significant, while the importance of well-regulated eating habits is recognized. Therefore, we examined the influence of meal timing using non-genetically modified animals. We developed a feeding protocol, in which the animals ate continually irrespective of time; although restricted feeding (e.g., feeding at noon only) causes day/night inversion in nocturnal rats, they get used to it. In 2009, we reported for the first time that irregular meals cause abnormalities in the circadian clock of the liver and increase blood cholesterol (26). It was the first study demonstrating experimentally that irregular meal timing leads to metabolic disorders. It indicated that differences in meal timing cause cholesterol metabolism abnormalities even if the same quantity of food is provided. In the study, in order to make the rats lead irregular meal habits, a quarter of their daily feed was given 4 times a day, irrespective of night or day. We named this style “irregular eating.” Such timing can be used clinically for total parenteral nutrition. Although the weight of rats did not change as a result of irregular eating, levels of blood cholesterol, particularly that of VLDL (very low density lipoproteins)-cholesterol, increased significantly. Blood cholesterol levels increased to about 50 mg/dL due to the irregular meal timings. This was caused by the advanced shift of the circadian rhythm of the gene expression of CYP7A1, a rate-limiting enzyme involved in bile acid synthesis. Thus, orchestrated cholesterol metabolism did not occur and bile acid excreted in the feces decreased. Moreover, at that time, the rhythmicity of the clock gene DBP in the liver was advanced, which might be a major cause. In addition, clock genes, particularly Dec1 and Dec2, were susceptible to meal timings. These results indicate that well-regulated eating habits normalize the liver clock gene, normalize the rhythm of CYP7A1 (which facilitates bile acid excretion), and that blood cholesterol levels are normalized due to normalized secretion of VLDL. In addition, apolipoprotein A-I, the main constituent protein of HDL (high density lipoprotein), is also under the control of DBP. This result indicates that irregular eating habits may reduce HDL. In other words, regular eating habits decrease “bad” cholesterol and increase “good” cholesterol.

The mice and rats with ad lib feeding eat 80% of the food during their active period in dark phase and the other 20% during their rest period. This raises the question of how mice and rats will react if they are forced to have tightly regulated eating habits, in which they eat no food during their rest period. A recent report indicates that diet-induced obesity is reduced only by tightly regulated eating habits (27). This report importantly demonstrates that well-regulated eating habits actively contribute to good health rather than demonstrating that irregular eating habits are unhealthy.

7. Factors synchronizing peripheral clocks

The peripheral clocks, including the liver clock, maintain rhythmicity as a integrated clock of the whole body via the control of the SCN. As discussed earlier, the peripheral clocks are synchronized by meals independent of the brain clock, which is synchronized by light. In other words, there are presumably several factors that synchronize the peripheral clocks, including the nervous system, endocrine system (i.e., hormones), exercise (i.e., activity), body temperature, and eating behavior. To determine which factors synchronize the liver clock, rat primary cultured hepatocytes were treated with various hormonal factors.

Primary cultured hepatocytes obtained from rats kept on a 24-hour rhythm because the hepatocytes recognized the time even if the organism was dead. However, although spread monolayer cultures of hepatocytes, which were different from their original morphology, lost rhythmicity immediately, 3-dimensional cultures maintained circadian clock for long time (28). These spherical hepatocytes were used for subsequent studies. Glucocorticoids (29), cAMP and cytokines which activate tyrosine phosphorylation changed gene expression of the clock genes in the liver and hepatocytes. Since it is already known that nutrients themselves, such as glucose, are synchronizers, we treated cells with single types of amino acids in high concentrations and found that many of them triggered various clock genes. We then focused on insulin, which is though to be the most closely involved in the synchronization associated with meal timing. Insulin is a well-known hormone that fluctuates according to the meal timing (30-32). However, experimental conditions and results vary widely among studies. Therefore, we aimed to resolve these discrepancies by performing a considerably large-scale experiment. The results show that insulin is a strong factor that synchronizes the liver clock (28). The procedures of this experiment are described. First, insulin was added to hepatocytes with desynchronized clock gene expression, and synchronization by insulin was confirmed to ensure they had the same rhythm. In order to demonstrate that insulin is a synchronizer, we observed in real time hepatoyctes obtained from transgenic rats that had a gene that linked luciferase to genes downstream of the clock gene promoter. The hepatocytes exhibited an obvious phase response curve to insulin. The phase response curve is a schematization to show that a given stimulus has a particular effect on clock resetting. If a phase response curve is found, it will demonstrate that the agent in question is a synchronizer. For example, in light therapy for treating sleep disturbances in “night people,” although patients will become “morning people” by being exposed to strong light early in the morning, this therapy will be adverse if the patient is exposed to strong light at night. The effect of insulin in individual animals was examined using rats with streptozotocin-induced type I diabetes mellitus. The results revealed that the liver clocks advanced in those diabetic rats due to insulin deficiency. Insulin administration exhibited a phase response curve, which presumably suggests that the abnormalities of the liver clocks in diabetic rats might be improved if treatment was performed during the active period when insulin was secreted. Although insulin administration during the active period (i.e., eating period) normalized the liver clock, insulin administration during the rest period hastened and worsened the liver clock. Considering these findings, we concluded that insulin is the synchronizer of the liver clock. Insulin does not synchronize the clocks of the cells or organs that do not generally respond to insulin, such as fibroblasts, the brain, and the lung, although it synchronizes the clocks of adipose tissue (28). In other words, the clocks of the organs contributing to metabolic syndrome are entrained by insulin.

8. Eating itself as a synchronizer

The synchronization of the peripheral clocks, including those in the liver, are thought to be associated with the effects of meals when nutrients enter the body. However, synchronization also occurs via eating behaviors that stimulate the digestive system. Serum concentrations of glucocorticoid hormone (cortisol in humans and corticosterone in rats) secreted by the adrenal cortex shows circadian rhythm, and it is high just before the active period. Although the diurnal rhythm of insulin disappears when animals are starved, while glucocorticoid horomone continues to exhibit circadian oscillation., However, the circadian rhythm of glucocorticoid hormone is maintained when meals are administered orally, while the oscillations disappear when nutrients are administered parenterally (i.e., not via the intestinal tract). On the other hand, the liver clock maintains its rhythmicity regardless of the administration route. In other words, the rhythmicity of glucocorticoid hormone from adrenal gland is entrained through nutrients entering the body orally (33). Furthermore, resection of the jejunum abolishes the rhythmicity of the glucocorticoid, but not changes the that of the liver clock (34). These results show that food passing through the digestive organs or that getting absorbed per se synchronizes the clocks of some organs.

9. Food factors as synchronizers

As mentioned above, some nutrients act as synchronizers. Glucose is most important energy source that synchronizes circadian rhythms (35, 36). It is reasonable that an energy source essential for survival has a rhythm-synchronizing function. Glucose also synchronizes the rhythm of cultured cells. These facts suggested that clocks of cultured cells could be synchronized by medium exchange alone. Actually, clocks can only be reset by medium exchange. These results unexpectedly gave us a problem. Although most researchers do not usually consider when culture media are exchanged, the timing of medium exchange may affect the results when the study subject possesses a rhythm. However, if we change the viewpoint, this problem may suggest a new concept of culture method, because metabolic contradiction is inefficient even in cultured cells. Cells cultured in media with fluctuated nutrient concentrations (“Rhythm culture”) must be effective when cultured cells are used for industrial purpose.

Amino acids alone can also exert synchronizing functions. It is reported that the rhythms of not only the liver, but also the SCN are synchronized when glucose and amino acids are administered to rats (18). Meals may be a strong synchronizer for the brain. Another study also demonstrates that carbohydrate and protein act as strong synchronizers of the liver (37).

On the other hand, lipids have not been though to be synchronizers, although high-fat diets seem to change the length (i.e., frequency) of one period (i.e., day) (38). In mice, one period is generally approximately 23.5 hours; this increases to approximately 24 hours when they are fed a high-fat diet. On the contrary, a drug called clofibrate, which promotes lipid catabolism, counteracts this effect (39). A pathway through PPARa seems to control the period of the rhythm.

Salt (40) and vitamin A (41) also synchronize the clocks. Resveratrol, a non-nutrient, affects the clocks (42). Thus, there are many ingredients in food that control circadian clocks. Our everyday meals include many synchronizers. Consequently, daily meals provide synchronization stimuli.

10. Smart meal styles from an aspect of chrononutrition

Considering the findings mentioned above, we must determine what kinds of meal styles are good for our health, e.g., well-regulated eating habits that differ between daytime and nighttime (i.e., only eating during the active period but not during the rest period). Insulin will be secreted 3 times if 3 meals are eaten; the first insulin secretion is the most important (i.e., after long fasting). Eating breakfast after insufficient fasting may provide a weak reset effect. Midnight snacks alter the liver clock, making metabolism to function suboptimally.

Considering our biological clock, what should we eat? As mentioned above, meals act as bundles of synchronizers. Therefore, it may be sufficient if daily meals include carbohydrate and protein. Thus, it can be thought that we may take meals we usually eat without any specific limitation. However, it is important is to eat breakfast.

Is it acceptable to eat only at night due to daytime and nighttime inversion if eating habits are well-regulated in a sense? In humans, night eating syndrome, which causes lipid metabolism abnormality, has already become a problem. We performed restricted feeding such that rats were fed only at noon during the rest period and found that blood cholesterol levels increased remarkably. This experiment is still under investigation. Even if the timing of meals includes the accents (i.e., meal time and non-meal time) for 24 hours, metabolic disorders start occurring when the brain and peripheral clocks such as the liver clock are not orchestrated. A lack of coordination among the organ clocks also seems to lead to unhealthy condition, independent of the effects due to disturbances in the liver clock.

So therefore, should we strictly have 3 meals a day? The number of meals is thought to have increased from 2 to 3 after modernization. Eating more meals is reported to be better for health (43). Although overeating is not good, having 4 or 5 meals a day is better than 3 or less because the index of obesity and blood lipid levels remain within normal range. Additionally, midnight snacking spoils the beneficial effect of the increased meal number.

We next think about “who” eats. Inactive lifestyles are now becoming a concern. Disuse syndrome and inactivity syndrome exist. Inactivity itself seems to cause poor health. Although inactivity syndrome is again attracting attention due to the Great East Japan Earthquake, the mechanisms of metabolic disorders derived from inactivity are not well understood. In Japan, bedridden elderly people so-called “Netakri” have been a social and medical problem. We created bedridden “Netakiri” animal models for molecular biological study in order let bedridden people recover and to examine the biological reactions within their bodies. We found that this “Netakiri” rats showed various metabolic disorders, gene expression changes, and changes in the liver clock. It is possible that inactivity contributes to poor health by causing abnormalities in circadian rhythms including that in the liver. Furthermore, this result shows that physical activity itself may synchronize the organ clocks, e.g., that of the liver. “Netakiri” bedridden or inactive people may have disturbances in their body clocks. Therefore, if these disturbances are corrected, those people may be able to regain their health.

Because the timing of meals is usually linked to the sleep cycle, in order to correct disorderly eating habits, it is necessary to improve the basic life rhythm. In any case, people should refrain from midnight snacking and have regular scheduled mealtimes; breakfast is especially important even if it is light. However, shift workers are forced to have irregular mealtimes. Hopefully, foods and drugs that exploit the known molecular mechanisms of biological clocks will be developed; such products would help shift workers. Considering that sleep disturbance is already treated with drugs, the prescription of drugs may be a practical method for treating metabolic disorders caused by disturbances in body clocks. It was recently found that some ligands of nuclear receptors (NR) can regulate the clocks (43), since some clock gene are NR such as Rev-erb and ROR.

A major problem faced when discussing eating habits from the perspective of chrononutrition is that details of one’s own body clock cannot be understood. As long as we have no way to measure the biological clock, we cannot evaluate it, making it difficult to use in clinical settings. It is possible to collect blood every few hours, but this is impractical. The body clock was recently measured using hair follicle cells (44); however, this is also impractical for use with the public and for use in clinical practice. Therefore, it is necessary to develop a method for measuring body clocks in a non-invasive and practical way. The oral mucosa is thought to be a substitute for hair follicle cells, but it is still impractical because mRNA of clock genes has to be extracted and measured. A simpler and easier method is required to utilize existing knowledge of chrononutrition completely. We are currently developing a smartphone application that estimates a person’s body clock called “chrononutrition clock.”

11. Conclusion and perspective

The first issue to be addressed in the future focuses on dysrhythmia underlying various diseases. Unless we clarify the usefulness and quantify the extent of how rhythm normalization prevents diseases, rhythm normalization cannot be used for prevention or treatment. Reduction of serum albumin secreted from the liver in elderly people is the second issue that must be overcome. Both albumin protein and mRNA have long half-lives but albumin gene exhibit circadian rhythms at the transcriptional level. The reasons for this apparently needless and hectic work remain unknown. Meal timing should help maintain normal albumin level because abnormalities occur in its transcriptional rhythm in the case of irregular eating. The third issue to be addressed is that the liver is a central organ of drug metabolism; disturbances to the liver clocks induced by irregular eating habits may cause abnormalities in drug metabolism, inhibiting expected efficacy and increasing unexpected side effects. Although it is known that drugs are affected by the timing of administration in chronopharmacology, drugs may not be effective unless the patient lives a regular life. It may be possible to improve the efficacy of drugs by having patients have regular meal timings, which would normalize the liver clock. We expect that the importance of chrononutrition as the basis for treatment timing, including chronotherapy and chronopharmacology, will become more important.

Nutritional sciences have traditionally focused on what a person eats. However, our future goal is to establish a well-regulated smart meal style called the “Smart Nutri Style” (SNS), which considers the way people eat or the meal style used (i.e., “5W1H of meals”). The French painter Delacroix stated that, “we work not only to produce but to give value to time.” “To give value to time” means to give a meaning to life and to enrich human life. This wise remark should be revised to state that eating and metabolisim not only to produce or maintain the body, but also to give value to time via circadian rhythms. Regular meal timings can help maintain our health and enrich our lives. By comprehensively considering molecular biological analyses, we would like to advance nutritional sciences by increasing the understanding of living entities as integrated systems.

Oda's HP
Updated 4/28/13