Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice.

Polymethoxyflavonoids, such as nobiletin (abundant in Citrus depressa), have been reported to have antioxidant, anti-inflammatory, anticancer, and anti-dementia effects, and are also a circadian clock modulator through retinoic acid receptor-related orphan receptor (ROR) α/γ. However, the optimal timing of nobiletin intake has not yet been determined. Here, we explored the time-dependent treatment effects of nobiletin and a possible novel mechanistic idea for nobiletin-induced circadian clock regulation in mice. In vivo imaging showed that the PER2::LUC rhythm in the peripheral organs was altered in accordance with the timing of nobiletin administration (100 mg/kg). Administration at ZT4 (middle of the light period) caused an advance in the peripheral clock, whereas administration at ZT16 (middle of the dark period) caused an increase in amplitude. In addition, the intraperitoneal injection of nobiletin significantly and potently stimulated corticosterone and adrenaline secretion and caused an increase in Per1 expression in the peripheral tissues. Nobiletin inhibited phosphodiesterase (PDE) 4A1A, 4B1, and 10A2. Nobiletin or rolipram (PDE4 inhibitor) injection, but not SR1078 (RORα/γ agonist), caused acute Per1 expression in the peripheral tissues. Thus, the present study demonstrated a novel function of nobiletin and the regulation of the peripheral circadian clock.

Euglena gracilis-derived ß-glucan paramylon entrains the peripheral circadian clocks in mice.

Paramylon, a ß-1,3-glucan storage polysaccharide derived from Euglena gracilis, has various health benefits, such as anti-obesity effects and modulation of immune function. However, whether paramylon intake affects the circadian clock remains unknown. In this study, we examined the effect of paramylon intake on the circadian clock. The results showed that the paramylon intake regulated peripheral clocks in mice. Furthermore, cecal pH and short-chain fatty acid concentrations after paramylon intake were measured. The correlation between changes in the expression of clock-related genes and alterations in the intestinal environment was confirmed. In addition, peripheral clock entrainment by paramylon intake was not observed in antibiotic-treated mice whose gut microbiota was weakened. These findings suggest that the regulation of the circadian clock by paramylon intake was mediated by changes in gut microbiota. In addition, the entraining effect of paramylon intake was also confirmed in mice bred under conditions mimicking social jetlag, which implies that paramylon intake may contribute to recovery from social jetlag. Thus, the appropriate consumption of paramylon may have a beneficial effect on health from a chrono-nutritional perspective.

Effect of circadian clock and claudin regulations on inulin-induced calcium absorption in the mouse intestinal tract.

Dietary calcium supplementation has been shown to be an effective adjunct therapy in an inflammatory bowel disease model. Soluble dietary fiber reduces intestinal pH and is known to enhance calcium absorption. Although many circadian clock regulations of nutrient absorption in the intestinal tract have been reported, the effects of clock regulation on calcium absorption have yet to be understood. In this study, we investigated the timing of efficient calcium intake by measuring urinary calcium excretion in mice. The diurnal variations in channel-forming tight junctions (claudins) were detected in both the jejunum and ileum. Following 2 days of feeding with a Ca2+-free diet, Ca2+-containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hr. There was an evident increase in urinary calcium concentration when the inulin diet was fed at the beginning of the resting period. The Claudin 2 (Cldn2) expression level also showed a significant day-night change, which seemed to be a mechanism for the increased calcium excretion after inulin intake. This diurnal rhythm and enhanced Cldn2 expression were abolished by disruption of the suprachiasmatic nucleus, the central clock in the hypothalamus. This study suggests that intestinal calcium absorption might be modulated by the circadian clock and that the intake of inulin is more effective at the beginning of the resting period in mice.

Wheel-Running Facilitates Phase Advances in Locomotor and Peripheral Circadian Rhythm in Social Jet Lag Model Mice.

The circadian clock maintains our health by controlling physiological functions. Social jet lag is one factor that can disrupt the body clock. This is caused by the difference in sleeping hours between weekdays when we live according to social time and holidays when we live according to our body clock. The body clock can be altered by exercise, nutrition, and stress, and several studies have reported that these factors can be used to improve a disturbed body clock. Here we focused on exercise and examined whether continuous wheel-running could improve the disordered body clock in a mouse model that mimics social jet lag. The results showed that the wheel-running exercise group showed faster synchronization of the onset of activities on weekdays which had been delayed by social jet lag and the results were even more pronounced in the high-fat diet feeding condition. Also, when the expression rhythms of the clock genes were examined, they experienced a sudden time shift in the advance light condition or social jet lag condition, it was found that the wheel-running group had a higher ability to adapt to the advance direction. Thus, it is possible that the effective inclusion of exercise in human, especially those who eat high-fat foods, life can improve the disordered body clock in terms of social jet lag.

Polygalae Radix shortens the circadian period through activation of the CaMKII pathway.

CONTEXT: The mammalian circadian clock system regulates physiological function. Crude drugs, containing Polygalae Radix, and Kampo, combining multiple crude drugs, have been used to treat various diseases, but few studies have focussed on the circadian clock. OBJECTIVE: We examine effective crude drugs, which cover at least one or two of Kampo, for the shortening effects on period length of clock gene expression rhythm, and reveal the mechanism of shortening effects. MATERIALS AND METHODS: We prepared 40 crude drugs. In the in vitro experiments, we used mouse embryonic fibroblasts from PERIOD2::LUCIFERASE knock-in mice (background; C57BL/6J mice) to evaluate the effect of crude drugs on the period length of core clock gene, Per2, expression rhythm by chronic treatment (six days) with distilled water or crude drugs (100 µg/mL). In the in vivo experiments, we evaluated the free-running period length of C57BL/6J mice fed AIN-93M or AIN-93M supplemented with 1% crude drug (6 weeks) that shortened the period length of the PERIOD2::LUCIFERASE expression rhythm in the in vitro experiments. RESULTS: We found that Polygalae Radix (ED50: 24.01 µg/mL) had the most shortened PERIOD2::LUCIFERASE rhythm period length in 40 crude drugs and that the CaMKII pathway was involved in this effect. Moreover, long-term feeding with AIN-93M+Polygalae Radix slightly shortened the free-running period of the mouse locomotor activity rhythm. DISCUSSION AND CONCLUSIONS: Our results indicate that Polygalae Radix may be regarded as a new therapy for circadian rhythm disorder and that the CaMKII pathway may be regarded as a target pathway for circadian rhythm disorders.

Oak extracts modulate circadian rhythms of clock gene expression in vitro and wheel-running activity in mice.

Introduction: In mammals, the central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which coordinates the circadian rhythm and controls locomotor activity rhythms. In addition to SCN cells, the peripheral tissues and embryonic fibroblasts also have clock genes, such as Per1/2 and Bmal1, which generate the transcriptional-translational feedback loop to produce an approximately 24-h cycle. Aging adversely affects the circadian clock system and locomotor functions. Oak extract has been reported to improve age-related physiological changes. However, no study has examined the effect of oak extract on the circadian clock system. Methods: We examined the effects of oak extract and its metabolites (urolithin A [ULT] and ellagic acid [EA]) on clock gene expression rhythms in mouse embryonic fibroblasts (MEFs) and SCN. Furthermore, locomotor activity rhythm was assessed in young and aged mice. Results: Chronic treatment with EA and ULT delayed the phase of PER2::LUC rhythms in SCN explants, and ULT prolonged the period of PER2::LUC rhythms in MEFs in a dose-dependent manner and increased the amplitude of PER2::LUC rhythms in MEFs, though only at low concentrations. Acute treatment with ULT affected the phase of PER2::LUC rhythms in MEFs depending on the concentration and timing of the treatment. In addition, oak extract prolonged the activity time of behavioral rhythms in old mice and tended to increase daily wheel-running revolutions in both young and old mice. Conclusions: These results suggest that oak extract is a novel modulator of the circadian clock in vitro and in vivo. Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-021-00365-2.

Use of a social jetlag-mimicking mouse model to determine the effects of a two-day delayed light- and/or feeding-shift on central and peripheral clock rhythms plus cognitive functioning.

Social jetlag (SJL) is defined as the discrepancy between social and biological rhythms and calculated by the difference between the midpoint of sleep time on working-days and free-days. Previous human and mouse studies showed SJL is positively related to evening chronotype and significantly related to smoking habit, cardiovascular risk, cognitive ability, and that SJL-mimicking conditions, simulating the real lifestyle situation of SJL in many humans, disrupt the regularity of estrous cycles of female animals. The effects of SJL-mimicking conditions on circadian rhythms and cognitive function and the reasons why the discrepancy between social and biological rhythms is involved in SJL have not yet been investigated. Therefore, in this study, we utilized a mouse model of SJL-mimicking conditions - 6-hour delayed-light/dark (LD) conditions for 2 days and normal-LD conditions for the following 5 days - applied for several weeks during which biological rhythms were monitored. Circadian rhythms of central and peripheral clocks and metabolism of the mice under the SJL-mimicking condition were always delayed for 2-3 hours compared with those under the normal-LD condition. Moreover, SJL-mimicking conditions impaired their cognitive function using a novel object recognition test. Only the delayed timing of either the light phase of the LD or of feeding for 2 days, comparable to the free-days situation of humans, delayed the circadian staging of rhythms the following 5 days. Furthermore, sleep deprivation during the early mornings for 5 days, which is comparable to early rise times experienced by humans on working-days and does affect the staging of circadian rhythms (circadian misalignment schedule), delayed the locomotor activity rhythms the next 2 days, comparable to free-days in humans, which is similar to the lifestyle rhythm of the evening chronotype. Our results demonstrated that the circadian misalignment schedule for 5 days changed the locomotor activity rhythms the following 2 days to the evening chronotype, that light- and/or feeding-shift conditions for 2 days exacerbate SJL, and that SJL-mimicking conditions delay the metabolic rhythm and cause cognitive impairment.

Crosstalk Among Circadian Rhythm, Obesity and Allergy.

The circadian clock system works not only as a cellular time-keeper but also as a coordinator for almost all physiological functions essential to maintaining human health. Therefore, disruptions or malfunctions of this system can cause many diseases and pre-symptomatic conditions. Indeed, previous studies have indicated that disrupted clock gene expression rhythm is closely related to obesity, and that allergic diseases can be regulated by controlling peripheral clocks in organs and tissues. Moreover, recent studies have found that obesity can lead to immune disorders. Accordingly, in this review, we assess the connection between obesity and allergy from the point of view of the circadian clock system anew and summarize the relationships among the circadian clock system, obesity, and allergy.

The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.

Chronic Kidney Disease (CKD) is increasing in incidence and has become a worldwide health problem. Sleep disorders are prevalent in patients with CKD raising the possibility that these patients have a disorganized circadian timing system. Here, we examined the effect of adenine-induced tubulointerstitial nephropathy on the circadian system in mice. Compared to controls, adenine-treated mice showed serum biochemistry evidence of CKD as well as increased kidney expression of inflammation and fibrosis markers. Mice with CKD exhibited fragmented sleep behavior and locomotor activity, with lower degrees of cage activity compared to mice without CKD. On a molecular level, mice with CKD exhibited low amplitude rhythms in their central circadian clock as measured by bioluminescence in slices of the suprachiasmatic nucleus of PERIOD 2::LUCIFERASE mice. Whole animal imaging indicated that adenine treated mice also exhibited dampened oscillations in intact kidney, liver, and submandibular gland. Consistently, dampened circadian oscillations were observed in several circadian clock genes and clock-controlled genes in the kidney of the mice with CKD. Finally, mice with a genetically disrupted circadian clock (Clock mutants) were treated with adenine and compared to wild type control mice. The treatment evoked worse kidney damage as indicated by higher deposition of gelatinases (matrix metalloproteinase-2 and 9) and adenine metabolites in the kidney. Adenine also caused non-dipping hypertension and lower heart rate. Thus, our data indicate that central and peripheral circadian clocks are disrupted in the adenine-treated mice, and suggest that the disruption of the circadian clock accelerates CKD progression.

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.

Correlation among clock gene expression rhythms, sleep quality, and meal conditions in delayed sleep-wake phase disorder and night eating syndrome.

Clock genes that comprise the circadian clock system control various physiological functions. Delayed sleep-wake phase disorder (DSWPD) and night eating syndrome (NES) are characterized by delayed sleep and meal timing, respectively. We estimated that clock gene expression rhythms in DSWPD patients may be delayed in comparison with the healthy subjects due to delayed melatonin secretion rhythms, producing eveningness chronotype in these individuals. However, it was difficult to estimate which clock gene expression rhythms were delayed or not in NES patients, because previous studies revealed that melatonin secretion rhythm was a little delayed compared with healthy individuals and that chronotype of NES patients depended on the individuals. Therefore, we examined expression rhythms of clock genes such as Period3 (Per3), nuclear receptor subfamily 1, group D, member 1 (Nr1d1) and Nr1d2 in these patients. Further, we expected sleep and meal patterns in DSWPD and NES patients may be more diverse than patterns observed in healthy subjects, and thus analyzed relationships among clock gene expression rhythms, sleep quality, sleep midpoint time, and meal times. We enrolled healthy male participants along with DSWPD and NES male patients, and asked all participants to answer questionnaires and to keep diaries to record information on their sleep and meals. Further, we asked them to collect 5-10 beard follicle samples, 6 times every 4 h. We measured clock gene expression rhythms using total RNA extracted from beard follicle cells. Peak time of clock gene expression in the NES group showed more diversity than the other groups, and that in the DSWPD group was delayed compared with the control group. In addition, the peak time of clock gene expression was negatively correlated with sleep quality and positively correlated with meal time after a long fast. Amplitudes of clock gene expression, especially Per3, positively responded to better mental and physical conditions as well as with better sleep quality. Results of this study suggest that peak times of clock gene expression in NES patients depended on the individuals; some patients with NES showed similar clock gene expression rhythm to healthy subjects, and other patients with NES showed similar to DSWPD patients. Moreover, this study suggests that meal time after a long fast may influence more determination in clock gene expression rhythms than the time of breakfast. Therefore, this study also indicates that Per3 clock gene may be one of the parameters that will help us understand sleep and meal rhythm disturbances.

A low-protein diet eliminates the circadian rhythm of serum insulin and hepatic lipid metabolism in mice.

Insulin is a key molecule that synchronizes peripheral clocks, such as that in the liver. Although we previously reported that mice fed a low-protein diet showed altered expression of lipid-related genes in the liver and induction of hepatic steatosis, it is unknown whether a low-protein diet impairs insulin secretion and modifies the hepatic circadian rhythm. Therefore, we investigated the effects of the intake of a low-protein diet on the circadian rhythm of insulin secretion and hepatic lipid metabolism in mice. Under 12-h light/12-h dark cycle, mice fed a low-protein diet for 7 days displayed enhanced food intake at the end of the light phase, although central and peripheral PER2 expression rhythm was maintained. Serum insulin levels in mice fed a low-protein diet remained low during the day, and the insulin secretion in OGTT was also markedly lower than in normal mice. In mice fed low-protein diet, hepatic TG accumulation was observed during the nighttime, with relatively high levels of ACC1 mRNA and total ACC proteins. Although there were no differences in the activity rhythm of hepatic mTOR between mice fed a normal or low-protein diet, hepatic IRS-2 expression in mice fed a low-protein diet remained low during the day, with no increase at the beginning of the light period. These results suggested that the low-protein diet eliminated the circadian rhythm of serum insulin and hepatic lipid metabolism in mice, providing insights into our understanding of the mechanisms of hepatic disorders of lipid metabolism.

Day-Night Oscillation of Atrogin1 and Timing-Dependent Preventive Effect of Weight-Bearing on Muscle Atrophy.

BACKGROUND: Atrogin1, which is one of the key genes for the promotion of muscle atrophy, exhibits day-night variation. However, its mechanism and the role of its day-night variation are largely unknown in a muscle atrophic context. METHODS: The mice were induced a muscle atrophy by hindlimb-unloading (HU). To examine a role of circadian clock, Wild-type (WT) and Clock mutant mice were used. To test the effects of a neuronal effects, an unilateral ablation of sciatic nerve was performed in HU mice. To test a timing-dependent effects of weight-bearing, mice were released from HU for 4 h in a day at early or late active phase (W-EAP and W-LAP groups, respectively). FINDINGS: We found that the day-night oscillation of Atrogin1 expression was not observed in Clock mutant mice or in the sciatic denervated muscle. In addition, the therapeutic effects of weight-bearing were dependent on its timing with a better effect in the early active phase. INTERPRETATION: These findings suggest that the circadian clock controls the day-night oscillation of Atrogin1 expression and the therapeutic effects of weight-bearing are dependent on its timing. FUND: Council for Science, Technology, and Innovation, SIP, "Technologies for creating next-generation agriculture, forestry, and fisheries".

A randomized, double-blind and placebo-controlled crossover trial on the effect of l-ornithine ingestion on the human circadian clock.

In mammals, daily physiological events are regulated by the circadian rhythm, which comprises two types of internal clocks: the central clock and peripheral clocks. Circadian rhythm plays an important role in maintaining physiological functions including the sleep-wake cycle, body temperature, metabolism and organ functions. Circadian rhythm disorder, which is caused, for example, by an irregular lifestyle or long-haul travel, increases the risk of developing disease; therefore, it is important to properly maintain the rhythm of the circadian clock. Food and the circadian clock system are known to be closely linked. Studies on rodents suggest that ingesting specific food ingredients, such as the flavonoid nobiletin, fish oil, the polyphenol resveratrol and the amino acid L-ornithine affects the circadian clock. However, there are few reports on the foods that affect these circadian clocks in humans. In this study, therefore, we examined whether L-ornithine affects the human central clock in a crossover design placebo-controlled human trial. In total, 28 healthy adults (i.e. ≥20 years) were randomly divided into two groups and completed the study protocol. In the 1st intake period, participants were asked to take either L-ornithine (400 mg) capsules or placebo capsules for 7 days. After 7 days' interval, they then took the alternative test capsules for 7 days in the 2nd intake period. On the final day of each intake period, saliva was sampled at various time points in the dim light condition, and the concentration of melatonin was quantified to evaluate the phase of the central clock. The results revealed that dim light melatonin onset, a recognized marker of central circadian phase, was delayed by 15 min after ingestion of L-ornithine. Not only is this finding an indication that L-ornithine affects the human central clock, but it also demonstrates that the human central clock can be regulated by food ingredients.

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.

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue.

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

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.

Polyporus and Bupleuri radix effectively alter peripheral circadian clock phase acutely in male mice.

In mammals, daily physiological events are precisely regulated by an internal circadian clock system. An important function of this system is to readjust the phase of the clock daily. In Japan, traditional herb medicines, so-called crude drugs (Shoyaku), are widely used for many diseases, and some are reported to affect circadian clock impairment, suggesting that some of them might have an ability to modify clock gene expression rhythms. Therefore, from selected 40 crude drugs, finding candidates that control the circadian clock phases was the first purpose of this study. As there are several crude drugs used for liver- and/or kidney-related diseases, the second aim of the present study was to find some crude drugs affecting liver/kidney circadian clock in vivo. To assess phase changes in the daily circadian rhythm, bioluminescence from the core clock gene product Period 2 was continuously monitored in mouse embryonic fibroblasts in vitro and in some peripheral tissues (kidney, liver, and submandibular gland) of PERIOD2::LUCIFERASE knock-in mice in vivo. In our screening, Polyporus and Bupleuri radix were found to be good candidates to effectively manipulate the peripheral circadian clock phase acutely, with stimulation time-of-day dependency in vitro as well as in vivo. Interestingly, Polyporus and Bupleuri radix are traditional herb medicines use for treating edema and promoting diuresis, and for chronic hepatitis, respectively. These crude drugs may be therefore good modulators of the circadian peripheral clocks including liver and kidney, and circadian clock genes become new molecular targets for these crude drugs.

Age-related circadian disorganization caused by sympathetic dysfunction in peripheral clock regulation.

The ability of the circadian clock to adapt to environmental changes is critical for maintaining homeostasis, preventing disease, and limiting the detrimental effects of aging. To date, little is known about age-related changes in the entrainment of peripheral clocks to external cues. We therefore evaluated the ability of the peripheral clocks of the kidney, liver, and submandibular gland to be entrained by external stimuli including light, food, stress, and exercise in young versus aged mice using in vivo bioluminescence monitoring. Despite a decline in locomotor activity, peripheral clocks in aged mice exhibited normal oscillation amplitudes under light-dark, constant darkness, and simulated jet lag conditions, with some abnormal phase alterations. However, age-related impairments were observed in peripheral clock entrainment to stress and exercise stimuli. Conversely, age-related enhancements were observed in peripheral clock entrainment to food stimuli and in the display of food anticipatory behaviors. Finally, we evaluated the hypothesis that deficits in sympathetic input from the central clock located in the suprachiasmatic nucleus of the hypothalamus were in part responsible for age-related differences in the entrainment. Aged animals showed an attenuated entrainment response to noradrenergic stimulation as well as decreased adrenergic receptor mRNA expression in target peripheral organs. Taken together, the present findings indicate that age-related circadian disorganization in entrainment to light, stress, and exercise is due to sympathetic dysfunctions in peripheral organs, while meal timing produces effective entrainment of aged peripheral circadian clocks.

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.