Yu Tahara

In Vivo Monitoring of Peripheral Circadian Clocks in the Mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.

Refeeding after Fasting Elicits Insulin-Dependent Regulation of Per2 and Rev-erbα with Shifts in the Liver Clock

The mammalian circadian clock is known to be entrained by both a daily light-dark cycle and daily feeding cycle. However, the mechanisms of feeding-induced entrainment are not as fully understood as those of light entrainment. To elucidate the first step of entrainment of the liver clock, we identified the circadian clock gene(s) that show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice. The expressions of liver Per2 and Rev-erbα genes were phase-advanced within 1 day of refeeding. Additionally, the upregulation of Per2 mRNA and down-regulation of Rev-erbα mRNA were induced within 2 hours, not only by food intake but also by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-treated insulin-deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-erbα gene expression. Furthermore, we demonstrated using an ex vivo luciferase monitoring system that insulin injection during the daytime causes a phase advance of liver Per2 expression rhythm in Per2::luciferase knock-in mice. In embryonic fibroblasts from Per2::luciferase knock-in mice, insulin infusion caused an acute increase of Per2 gene expression and a similar phase advance of Per2 expression rhythm. Our results indicate that an acute change of Per2 and Rev-erbα gene expression mediated by refeeding-induced insulin secretion is a critical step mediating the early phase of feeding-induced entrainment of the liver clock.

A balanced diet is necessary for proper entrainment signals of the mouse liver clock.

Background The peripheral circadian clock in mice is entrained not only by light-dark cycles but also by daily restricted feeding schedules. Behavioral and cell culture experiments suggest an increase in glucose level as a factor in such feeding-induced entrainment. For application of feeding-induced entrainment in humans, nutrient content and dietary variations should be considered. Principal Finding To elucidate the food composition necessary for dietary entrainment, we examined whether complete or partial substitution of dietary nutrients affected phase shifts in liver clocks of mice. Compared with fasting mice or ad libitum fed mice, the liver bioluminescence rhythm advanced by 3–4 h on the middle day in Per2::luciferase knock-in mice that were administered a standard mouse diet, i.e. AIN-93M formula [0.6–0.85 g/10 g mouse BW] (composition: 14% casein, 47% cornstarch, 15% gelatinized cornstarch, 10% sugar, 4% soybean oil, and 10% other [fiber, vitamins, minerals, etc.]), for 2 days. When each nutrient was tested alone (100% nutrient), an insignificant weak phase advance was found to be induced by cornstarch and soybean oil, but almost no phase advance was induced by gelatinized cornstarch, high-amylose cornstarch, glucose, sucrose, or casein. A combination of glucose and casein without oil, vitamin, or fiber caused a significant phase advance. When cornstarch in AIN-93M was substituted with glucose, sucrose, fructose, polydextrose, high-amylose cornstarch, or gelatinized cornstarch, the amplitude of phase advance paralleled the increase in blood glucose concentration. Conclusions Our results strongly suggest the following: (1) balanced diets containing carbohydrates/sugars and proteins are good for restricted feeding-induced entrainment of the peripheral circadian clock and (2) a balanced diet that increases blood glucose, but not by sugar alone, is suitable for entrainment. These findings may assist in the development of dietary recommendations for on-board meals served to air travelers and shift workers to reduce jet lag-like symptoms.

Meal frequency patterns determine the phase of mouse peripheral circadian clocks

Peripheral circadian clocks in mammals are strongly entrained by light-dark and eating cycles. Their physiological functions are maintained by the synchronization of the phase of organs via clock gene expression patterns. However, little is known about the adaptation of peripheral clocks to the timing of multiple daily meals. Here, we investigated the effect of irregular eating patterns, in terms of timing and volume, on their peripheral clocks in vivo. We found that the phase of the peripheral clocks was altered by the amount of food and the interval between feeding time points but was unaffected by the frequency of feeding, as long as the interval remained fixed. Moreover, our results suggest that a late dinner should be separated into 2 half-dinners in order to alleviate the effect of irregular phases of peripheral clocks.

The adjustment and manipulation of biological rhythms by light, nutrition, and abused drugs

Daily restricted feeding entrains the circadian rhythm of mouse clock gene expression in the central nervous system, excluding the suprachiasmatic nucleus (SCN), as well as in the peripheral tissues such as the liver, lung, and heart. In addition to entrainment of the clock genes, daily restricted feeding induces a locomotor activity increase 2-3h before the restricted feeding time initiates. The increase in activity is called the food-anticipatory activity (FAA). In addition to FAA, daily restricted feeding can also entrain peripheral circadian clocks in other organs such as liver, lung, and heart. This type of oscillator is called the food-entrainable peripheral oscillator (FEPO). At present, the mechanisms for restricted feeding-induced entrainment of locomotor activity (FAA) and/or peripheral clock (FEPO) are still unknown. In this review, we describe the role of the central nervous system and peripheral tissues in FAA performance and also in the entrainment of clock gene expression. In addition, the mechanism for entrainment of circadian oscillators by the abuse of drugs, such as methamphetamine, is discussed.

Combination of starvation interval and food volume determines the phase of liver circadian rhythm in Per2::Luc knock-in mice under two meals per day feeding

Although the circadian liver clock is entrained by the feeding cycle, factors such as food volume and starvation interval are poorly understood. Per2::Luc knock-in mice were given two meals per day with different food volume sizes and/or with different intervals of starvation between two mealtimes with the total food volume per day fixed at 3.6 g (80 food pellets, ∼75% of free feeding) per mouse. The bioluminescence rhythm in the liver produced a unimodal peak but not bimodal peak under the regimen of two meals per day over 14-15 days. Peak Per2 expression occurred concurrently with the mealtime of the larger food volume, when the first and second meal were given as different food volume ratios under a 12 h feeding interval. When an equal volume of food was given under different starvation interval (8 h:16 h), the peak of the Per2 rhythm was close to peak by mealtime after long starvation (16 h). When food volumes for each mealtime were changed under 8 h:16 h, the peak rhythm was influenced by combined factors of food volume and starvation interval. Food intake after the 16-h starvation caused a significant increase in liver Per2, Dec1, and Bmal1 gene expression compared with food intake after the 8-h starvation with 8 h:16 h feeding intervals. In conclusion, the present results clearly demonstrate that food-induced entrainment of the liver clock is dependent on both food volume and the starvation interval between two meals. Therefore, normal feeding habits may help to maintain normal clock function in the liver organ.

Circadian rhythms of liver physiology and disease: experimental and clinical evidence

The circadian clock system consists of a central clock located in the suprachiasmatic nucleus in the hypothalamus and peripheral clocks in peripheral tissues. Peripheral clocks in the liver have fundamental roles in maintaining liver homeostasis, including the regulation of energy metabolism and the expression of enzymes controlling the absorption and metabolism of xenobiotics. Over the past two decades, research has investigated the molecular mechanisms linking circadian clock genes with the regulation of hepatic physiological functions, using global clock-gene-knockout mice, or mice with liver-specific knockout of clock genes or clock-controlled genes. Clock dysfunction accelerates the development of liver diseases such as fatty liver diseases, cirrhosis, hepatitis and liver cancer, and these disorders also disrupt clock function. Food is an important regulator of circadian clocks in peripheral tissues. Thus, controlling the timing of food consumption and food composition, a concept known as chrononutrition, is one area of active research to aid recovery from many physiological dysfunctions. In this Review, we focus on the molecular mechanisms of hepatic circadian gene regulation and the relationships between hepatic circadian clock systems and liver physiology and disease. We concentrate on experimental data obtained from cell or mice and rat models and discuss how these findings translate into clinical research, and we highlight the latest developments in chrononutritional studies.

Circadian clock gene Period2 regulates a time-of-day–dependent variation in cutaneous anaphylactic reaction

BACKGROUND IgE-mediated immediate-type skin reaction shows a diurnal rhythm, although the precise mechanisms remain uncertain. Period2 (Per2) is a key circadian gene that is essential for endogenous clockworks in mammals. OBJECTIVE This study investigated whether Per2 regulates a time-of-day-dependent variation in IgE-mediated immediate-type skin reaction. METHODS The kinetics of a passive cutaneous anaphylactic reaction were compared between wild-type mice and mice with a loss-of-function mutation of Per2 (mPer2(m/m) mice). The effects of adrenalectomy, aging, and dexamethasone on the kinetics of a passive cutaneous anaphylactic reaction were also examined. In addition, the extent of IgE-mediated degranulation in bone marrow-derived mast cells (BMMCs) was compared between wild-type and mPer2(m/m) mice. RESULTS A time-of-day-dependent variation in a passive cutaneous anaphylactic reaction observed in wild-type mice was absent in mPer2(m/m) mice and in adrenalectomized and aged mice associated with the loss of rhythmic secretion of corticosterone. In addition, mPer2(m/m) mice showed decreased sensitivity to the inhibitory effects of dexamethasone on the passive cutaneous anaphylactic reactions. IgE-mediated degranulation in BMMCs was comparable between wild-type and mPer2(m/m) mice, but Per2 mutation decreased sensitivity to the inhibitory effects of dexamethasone on IgE-mediated degranulation in BMMCs. CONCLUSION A circadian oscillator, Per2, regulates a time-of-day-dependent variation in a passive cutaneous anaphylactic reaction in mice. Per2 may do so by controlling the rhythmic secretion of glucocorticoid from adrenal glands and/or by gating the glucocorticoid responses of mast cells to certain times of the day (possibly when Per2 levels are high in mast cells).

Differential roles of breakfast only (one meal per day) and a bigger breakfast with a small dinner (two meals per day) in mice fed a high-fat diet with regard to induced obesity and lipid metabolism

Background Recent studies on humans and rodents have suggested that the timing of food intake plays an important role in circadian regulation and metabolic health. Consumption of high-fat foods during the inactive period or at the end of the awake period results in weight gain and metabolic syndrome in rodents. However, the distinct effects of breakfast size and the breakfast/dinner size ratio on metabolic health have not yet been fully examined in mice. Methods We examined whether the parameters of metabolic syndrome were differentially affected in mice that consumed a large meal at the beginning of the awake period (breakfast; one meal group) and a relatively smaller meal at end of the awake period (dinner; two meals group). The mice of each group were provided equal food volume per day. Results Mice on one meal exhibited an increase in body weight gain, hyperinsulinemia, hyperleptinemia, and a decrease of gene expression associated with β-oxidation in adipose tissue and liver compared with those on two meals. The circadian expression pattern of the Clock gene in mice on one meal was disturbed compared with those on two meals. Conclusions In conclusion, a bigger breakfast with a smaller dinner (two meals per day) but not breakfast only (one meal per day) helps control body weight and fat accumulation in mice on a high-fat meals schedule. The findings of this study suggest that dietary recommendations for weight reduction and/or maintenance should include information on the timing and quantity of dietary intake.

Enantioselective syntheses of various chiral multicyclic compounds with quaternary carbon stereocenters by catalytic intramolecular cycloaddition.

The intramolecular cycloaddition of 1,n-diene-ynes (n = 4-6), where alkyne and alkene moieties are connected by a 1,1-disubstituted alkene, was examined using a chiral rhodium catalyst, and various types of cycloadducts with quaternary carbon stereocenter(s) were obtained in high to excellent enantiomeric excess. In the case of 1,4-diene-ynes, tricyclic, bicyclic, and spirocyclic compounds were obtained depending upon the substituents at the 2-position of the 1,4-diene moiety and those at their alkyne termini. On the other hand, 1,5- and 1,6-diene-ynes gave tricyclic and bicyclic compounds, which included medium-sized ring systems. The mechanistic considerations for different reaction pathways and the synthetic transformation of tricyclic products into functionalized spirocyclic compounds are also described. The reaction of enediynes, where two alkyne moieties are connected by a 1,1-disubstituted alkene, was also examined, and sterically strained tricyclic compounds with two carbon stereocenters were obtained.