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研究科: 千葉大学大学院医学薬学府（先端医学薬学専攻）, 学位記番号: 千大院医薬博甲第医1785号

第一章 近代工芸運動

The expression rhythms of clock genes, such as Per1, Per2, Bmal1, and Rev-erb α, in mouse peripheral clocks, are entrained by a scheduled feeding paradigm. In terms of food composition, a carbohydrate-containing diet is reported to cause strong entrainment through insulin secretion. However, it is unknown whether human diets entrain peripheral circadian clocks. In this study, we used freeze-dried diets for type 2 diabetes (DB) and chronic kidney disease (CKD), as well as low-carbohydrate diets. After 24 h of fasting, PER2::LUC knock-in mice were given access to food for 2 days during inactive periods, and bioluminescence rhythm was then measured using an in vivo imaging system. AIN-93M, the control mouse diet with a protein:fat:carbohydrate (PFC) ratio of 14.7:9.5:75.8, caused a significant phase advance (7.3 h) in the liver clock compared with that in 24 h fasted mice, whereas human diets caused significant but smaller phase advances (4.7–6.2 h). Compared with healthy and high fat/sucrose-induced DB mice, adenine-induced CKD mice showed attenuation of a phase-advance with a normal diet. There were no significant differences in phase-advance values between human diets (normal, DB, and CKD). In addition, a normal-carbohydrate diet (PFC ratio of 20.3:23.3:56.4) and a low-carbohydrate diet (PFC ratio of 36.4:42.9:20.7) caused similar phase advances in peripheral clocks. The present results strongly suggest that scheduled feeding with human diets can cause phase advances in the peripheral clocks of not only healthy, but also DB and CKD mice. This discovery provides support to the food-induced entrainment of peripheral clocks in human clinical trials.

In mammals, the central clock (the suprachiasmatic nuclei, SCN) is entrained mainly by the light-dark cycle, whereas peripheral clocks in the peripheral tissues are entrained/synchronized by multiple factors, including feeding patterns and endocrine hormones such as glucocorticoids. Clock-mutant mice (Clock/Clock), which have a mutation in a core clock gene, show potent phase resetting in response to light pulses compared with wild-type (WT) mice, owing to the damped and flexible oscillator in the SCN. However, the phase resetting of the peripheral clocks in Clock/Clock mice has not been elucidated. Here, we characterized the peripheral clock gene synchronization in Clock/Clock mice by daily injections of a synthetic glucocorticoid (dexamethasone, DEX) by monitoring in vivo PER2::LUCIFERASE bioluminescence. Compared with WT mice, the Clock/Clock mice showed significantly decreased bioluminescence and peripheral clock rhythms with decreased amplitudes and delayed phases. In addition, the DEX injections increased the amplitudes and advanced the phases. In order to examine the robustness of the internal oscillator, T-cycle experiments involving DEX stimulations with 24- or 30-h intervals were performed. The Clock/Clock mice synchronized to the 30-h T-cycle stimulation, which suggested that the peripheral clocks in the Clock/Clock mice had increased synchronizing ability upon DEX stimulation, to that of circadian and hour-glass type oscillations, because of weak internal clock oscillators.

早大学位記番号:新7822

Caffeine is one of the most commonly used psychoactive substances. Circadian rhythms consist of the main suprachiasmatic nucleus (SCN) clocks and peripheral clocks. Although caffeine lengthens circadian rhythms and modifies phase changes in SCN-operated rhythms, the effects on caffeine on the phase, period and amplitude of peripheral organ clocks are not known. In addition, the role of cAMP/Ca(2+) signalling in effects of caffeine on rhythm has not been fully elucidated.We examined whether chronic or transient application of caffeine affects circadian period/amplitude and phase by evaluating bioluminescence rhythm in PER2::LUCIFERASE knock-in mice. Circadian rhythms were monitored in vitro using fibroblasts and ex vivo and in vivo for monitoring of peripheral clocks.Chronic application of caffeine (0.1-10 mM) increased period and amplitude in vitro. Transient application of caffeine (10 mM) near the bottom of the decreasing phase of bioluminescence rhythm caused phase advance in vitro. Caffeine (0.1%) intake caused a phase delay under light-dark or constant dark conditions, suggesting a period-lengthening effect in vivo. Caffeine (20 mg·kg(-1) ) at daytime or at late night-time caused phase advance or delay in bioluminescence rhythm in the liver and kidney respectively. The complicated roles of cAMP/Ca(2+) signalling may be involved in the caffeine-induced increase of period and amplitude in vitro.Caffeine affects circadian rhythm in mice by lengthening the period and causing a phase shift of peripheral clocks. These results suggest that caffeine intake with food/drink may help with food-induced resetting of peripheral circadian clocks.