Phase resetting of circadian peripheral clocks using human and rodent diets in mouse models of type 2 diabetes and chronic kidney disease.

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.

Glucagon and/or IGF-1 Production Regulates Resetting of the Liver Circadian Clock in Response to a Protein or Amino Acid-only Diet.

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment.

Circadian clock-dependent increase in salivary IgA secretion modulated by sympathetic receptor activation in mice.

The salivary gland is rhythmically controlled by sympathetic nerve activation from the suprachiasmatic nucleus (SCN), which functions as the main oscillator of circadian rhythms. In humans, salivary IgA concentrations reflect circadian rhythmicity, which peak during sleep. However, the mechanisms controlling this rhythmicity are not well understood. Therefore, we examined whether the timing of parasympathetic (pilocarpine) or sympathetic (norepinephrine; NE) activation affects IgA secretion in the saliva. The concentrations of saliva IgA modulated by pilocarpine activation or by a combination of pilocarpine and NE activation were the highest in the middle of the light period, independent of saliva flow rate. The circadian rhythm of IgA secretion was weakened by an SCN lesion and Clock gene mutation, suggesting the importance of the SCN and Clock gene on this rhythm. Adrenoceptor antagonists blocked both NE- and pilocarpine-induced basal secretion of IgA. Dimeric IgA binds to the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of epithelial cells and forms the IgA-pIgR complex. The circadian rhythm of Pigr abundance peaked during the light period, suggesting pIgR expression upon rhythmic secretion of IgA. We speculate that activation of sympathetic nerves during sleep may protect from bacterial access to the epithelial surface through enhanced secretion of IgA.

Potent synchronization of peripheral circadian clocks by glucocorticoid injections in PER2::LUC-Clock/Clock mice.

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.

Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts.

Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer's have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag.

Forced rather than voluntary exercise entrains peripheral clocks via a corticosterone/noradrenaline increase in PER2::LUC mice.

Exercise during the inactive period can entrain locomotor activity and peripheral circadian clock rhythm in mice; however, mechanisms underlying this entrainment are yet to be elucidated. Here, we showed that the bioluminescence rhythm of peripheral clocks in PER2::LUC mice was strongly entrained by forced treadmill and forced wheel-running exercise rather than by voluntary wheel-running exercise at middle time during the inactivity period. Exercise-induced entrainment was accompanied by increased levels of serum corticosterone and norepinephrine in peripheral tissues, similar to the physical stress-induced response. Adrenalectomy with norepinephrine receptor blockers completely blocked the treadmill exercise-induced entrainment. The entrainment of the peripheral clock by exercise is independent of the suprachiasmatic nucleus clock, the main oscillator in mammals. The present results suggest that the response of forced exercise, but not voluntary exercise, may be similar to that of stress, and possesses the entrainment ability of peripheral clocks through the activation of the adrenal gland and the sympathetic nervous system.

Phase shifts in circadian peripheral clocks caused by exercise are dependent on the feeding schedule in PER2::LUC mice.

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN) clock, which is the main oscillator and peripheral clock. SCN clock can be entrained by both photic and non-photic stimuli, and an interaction exists between photic and non-photic entrainment. Moreover, peripheral circadian clocks can be entrained not only by scheduled restricted feeding, but also by scheduled exercise. Thus, the entrainment of peripheral circadian clocks may be the result of an interaction between the entrainment caused by feeding and exercise. In this study, we examined the effect of wheel-running exercise on the phase of the peripheral clocks (kidney, liver and submandibular gland) in PER2::LUC mice under various feeding schedules. Phase and waveforms of the peripheral clocks were not affected by voluntary wheel-running exercise. Exercise for a period of 4 h during the early dark period (morning) delayed the peripheral clocks, while exercise for the same duration during the late dark period (evening) advanced the peripheral clocks. The feeding phase was advanced and delayed by evening and morning exercise, respectively, suggesting that the feeding pattern elicited by the scheduled exercise may entrain the peripheral clocks. Exercise did not affect the phase of the peripheral clock under the 1 meal per day schedule. When the phase of the peripheral clocks was advanced by the feeding schedule of 2 or 4 meals per day during light and/or dark periods, wheel-running exercise during the morning period significantly and equally shifted the phase of all organs back to the original positions observed in mice maintained under free-feeding conditions and with no exercise. When the schedule of 2 meals per day during the dark period failed to affect the phase of peripheral clock, morning exercise did not affect the phase. Wheel-running exercise increased the levels of serum corticosterone, and the injection of dexamethasone/corticosterone instead of exercise shifted a phase that had advanced under the feeding schedule of 2 meals per day, back to the normal position. The liver and submandibular glands exhibit higher sensitivity to dexamethasone than the kidneys. In adrenalectomized mice, treadmill-induced normalization of the advanced phase under a feeding schedule of 2 meals per day was not observed. In summary, scheduled exercise-induced phase shifts were weaker compared to scheduled feeding-induced phase shifts. The phase advance caused by the feeding schedule of 2 or 4 meals per day was suppressed by wheel-running, treadmill exercise or dexamethasone/corticosterone injection in the early dark period (morning). Corticosterone release may be involved in exercise-induced phase shift of peripheral clocks. These results suggest that there is an interaction between the phase shifts caused by feeding and exercise schedules in peripheral clocks.

Fish Oil Accelerates Diet-Induced Entrainment of the Mouse Peripheral Clock via GPR120.

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock.

Eating meals before wheel-running exercise attenuate high fat diet-driven obesity in mice under two meals per day schedule.

Mice that exercise after meals gain less body weight and visceral fat compared to those that exercised before meals under a one meal/exercise time per day schedule. Humans generally eat two or three meals per day, and rarely have only one meal. To extend our previous observations, we examined here whether a "two meals, two exercise sessions per day" schedule was optimal in terms of maintaining a healthy body weight. In this experiment, "morning" refers to the beginning of the active phase (the "morning" for nocturnal animals). We found that 2-h feeding before 2-h exercise in the morning and evening (F-Ex/F-Ex) resulted in greater attenuation of high fat diet (HFD)-induced weight gain compared to other combinations of feeding and exercise under two daily meals and two daily exercise periods. There were no significant differences in total food intake and total wheel counts, but feeding before exercise in the morning groups (F-Ex/F-Ex and F-Ex/Ex-F) increased the morning wheel counts. These results suggest that habitual exercise after feeding in the morning and evening is more effective for preventing HFD-induced weight gain. We also determined whether there were any correlations between food intake, wheel rotation, visceral fat volume and skeletal muscle volumes. We found positive associations between gastrocnemius muscle volumes and morning wheel counts, as well as negative associations between morning food intake volumes/body weight and morning wheel counts. These results suggest that morning exercise-induced increase of muscle volume may refer to anti-obesity. Evening exercise is negatively associated with fat volume increases, suggesting that this practice may counteract fat deposition. Our multifactorial analysis revealed that morning food intake helps to increase exercise, and that evening exercise reduced fat volumes. Thus, exercise in the morning or evening is important for preventing the onset of obesity.

Effects of caffeine on circadian phase, amplitude and period evaluated in cells in vitro and peripheral organs in vivo in PER2::LUCIFERASE mice.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.