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.

Night eating model shows time-specific depression-like behavior in the forced swimming test.

The circadian clock system is associated with feeding and mood. Patients with night eating syndrome (NES) delay their eating rhythm and their mood declines during the evening and night, manifesting as time-specific depression. Therefore, we hypothesized that the NES feeding pattern might cause time-specific depression. We established new NES model by restricted feeding with high-fat diet during the inactive period under normal-fat diet ad libitum. The FST (forced swimming test) immobility time in the NES model group was prolonged only after lights-on, corresponding to evening and early night for humans. We examined the effect of the NES feeding pattern on peripheral clocks using PER2::LUCIFERASE knock-in mice and an in vivo monitoring system. Caloric intake during the inactive period would shift the peripheral clock, and might be an important factor in causing the time-specific depression-like behavior. In the NES model group, synthesis of serotonin and norepinephrine were increased, but utilization and metabolism of these monoamines were decreased under stress. Desipramine shortened some mice's FST immobility time in the NES model group. The present study suggests that the NES feeding pattern causes phase shift of peripheral clocks and malfunction of the monoamine system, which may contribute to the development of time-specific depression.

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.

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.