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In almost all living organisms, there is a sequence of biochemical, physiological, and behavioral processes that repeat roughly every 24 h. The timing of such processes is governed by an internal clock that ensures that these processes occur in a manner that optimizes the utilization of external resources such as the sunlight. In humans, peak cognitive and physical performance occurs during the daytime and is coupled with metabolic functions associated with catabolism, while the nighttime is associated with anabolic functions that take place during sleep. The daily rhythms of mental, physiological, and metabolic changes are collectively referred to as circadian cycles, and the internal clock that governs the timing of these processes lies in the suprachiasmatic nuclei (SCN).

The food-entrainable oscillator, which underlies the prefeeding activity peak developed by restricted daily feeding (RF) in rodents, does not depend on the circadian pacemaker in the suprachiasmatic nucleus (SCN) or on the known clock genes. In the present study, to clarify the roles of SCN circadian pacemaker and nutrient conditions on the development of prefeeding activity peak, RF of 3-h daily feeding was imposed on four groups of adult male mice for 10 cycles at different circadian times, zeitgeber time (ZT)2, ZT8, ZT14, and ZT20, where ZT0 is the time of lights-on in LD12:12. Seven days after the termination of RF session with ad libitum feeding in between, total food deprivation (FD) for 72 h was imposed. Wheel-running activity and core body temperature were measured throughout the experiment. Immediately after the RF or FD session, the PER2::LUC rhythms were measured in the cultured SCN slices and peripheral tissues. Not only the buildup process and magnitude of the prefeeding activity peak, but also the percentages of nocturnal activity and hypothermia developed under RF were significantly different among the four groups, indicating the involvement of light entrained circadian pacemaker. The buildup of prefeeding activity peak was accomplished by either phase-advance or phase-delay shifts (or both) of activity bouts comprising a nocturnal band. Hypothermia under FD was less prominent in RF-exposed mice than in naïve counterparts, indicating that restricted feeding increases tolerance to caloric restriction as well as to the heat loss mechanism. RF phase-shifted the peripheral clocks but FD did not affect the clocks in any tissue examined. These findings are better understood by assuming multiple bout oscillators, which are located outside the SCN and directly drive activity bouts uncoupled from the circadian pacemaker by RF or hypothermia.

Purpose of review Glucose metabolism is under circadian regulation, with insulin secretion and sensitivity being highest in the morning as compared to the evening. The present review will discuss the existing evidence for the role of meal and macronutrient timing to improve glucose metabolism and reset circadian clocks, with a focus on the evidence in humans. Recent findings Shortening the daily eating window (also known as time-restricted eating), or skewing food intake towards breakfast and away from the evening meal both improve glucose control in people with impaired glucose metabolism. Insulin is recently purported to be a zeitgeber and thus an important reset signal for peripheral circadian clocks in vitro and in mice. Although few studies have tested the impact of macronutrient timing in humans, eating a greater proportion of carbohydrates earlier, rather than later, in the day is associated with better glucose control. Summary The impact of carbohydrate intake timing on endogenous central and peripheral clocks, and its potential to optimize circadian regulation and improve glycaemic control, are not well understood but are currently under intense exploration.

Circadian disruptions precede high-fat diet (HFD)-induced obesity (DIO). Deviation of the endogenous circadian rhythm period length (tau) from 24 hours correlates with mice inter-strain DIO under t...

In the dromedary camel, a well-adapted desert mammal, daily ambient temperature (Ta)-cycles have been shown to synchronize the central circadian clock. Such entrainment has been demonstrated by examining two circadian outputs, body temperature and melatonin rhythms. Locomotor activity (LA), another circadian output not yet investigated in the camel, may provide further information on such specific entrainment. To verify if daily LA is an endogenous rhythm and whether the desert Ta-cycle can entrain it, six dromedaries were first kept under total darkness and constant-Ta. Results showed that the LA rhythm free runs with a period of 24.8–24.9 h. After having verified that the light–dark cycle synchronizes LA, camels were subjected to a Ta-cycle with warmer temperatures during subjective days and cooler temperatures during subjective nights. Results showed that the free-running LA rhythm was entrained by the Ta-cycle with a period of exactly 24.0 h, while a 12 h Ta-cycle phase advance induced an inversion of the LA rhythm and advanced the acrophase by 9 h. Similarly, activity onset and offset were significantly advanced. All together, these results demonstrate that the Ta-cycle is a strong zeitgeber, able to entrain the camel LA rhythm, hence corroborating previous results concerning the Ta non-photic synchronization of the circadian master clock.

Environmental temperature remarkably impacts on metabolic homeostasis, raising a serious concern about the optimum housing temperature for translational studies. Recent studies suggested that mice should be housed slightly below their thermoneutral temperature (26°C). On the other hand, the external temperature, also known as a zeitgeber, can reset the circadian rhythm. However, whether housing temperature affects the circadian oscillators of the liver remains unknown. Therefore, we have compared the effect of two housing temperatures, namely 21°C (conventional; TC) and 26°C (thermoneutral; TN), on the circadian rhythms in mice. We found that the rhythmicity of food intake showed an advanced phase at TC, while the activity was more robust at TN, with a prolonged period onset. The serum levels of norepinephrine were remarkably induced at TC, but failed to oscillate rhythmically at both temperatures. Likewise, circulating glucose levels were increased but were non-rhythmic under TC. Both total cholesterol and triglycerides levels were induced at TN, but showed an advanced phase under TC. Additionally, the expression of hepatic metabolic genes and clock genes remained rhythmic at both temperatures, with the exception of G6Pase, Fasn, Cpt1a and Cry2, at TN. Nevertheless, the liver histology examination did not show any significant changes in response to housing temperature. Although the non-consistent trends of phase changes in each temperature, our results suggest a non-reductant role of temperature in mouse internal rhythmicity resetting. Thus, the temperature-controlled internal circadian synchronization within organs should be taken into consideration when optimizing housing temperature for mice.

Growing evidence demonstrates circadian rhythms of pain hypersensitivity in various chronic disorders. In chemotherapy-induced peripheral neuropathy (CIPN), agents such as paclitaxel are known to elicit chronic neuropathic pain in cancer patients and seriously compromise their quality of life. Here, we report that the mechanical threshold for allodynia in paclitaxel-treated rats exhibited a robust circadian oscillation, reaching the nadir during the daytime (inactive phase). Using Per2::LucSV circadian reporter mice expressing a PER2::LUC fusion protein, we isolated dorsal root ganglia (DRG), the primary sensory cell body for peripheral nerve injury generated hypersensitivity, and monitored ex vivo reporter bioluminescence. We observed strong circadian reporter rhythms in DRG neurons which are highly entrainable by external cues. Paclitaxel treatment significantly lengthened DRG circadian periods, with little effects on the amplitude of oscillation. We further observed the core protein BMAL1 and PER2 in DRG neurons and satellite cells. Using DRG and dorsal horn (DH; another key structure for CIPN pain response) tissues from vehicle and paclitaxel treated rats, we performed RNA-sequencing and identified diurnal expression of core clock genes as well as clock-controlled genes in both sites. Interestingly, 20.1% and 30.4% of diurnal differentially expressed genes (DEGs) overlapped with paclitaxel-induced DEGs in the DRG and the DH respectively. In contrast, paclitaxel-induced DEGs displayed only a modest overlap between daytime and nighttime (Zeitgeber Time 8 and 20). Furthermore, paclitaxel treatment induced de novo diurnal DEGs, suggesting reciprocal interaction of circadian rhythms and chemotherapy. Our study therefore demonstrates a circadian oscillation of CIPN and its underlying transcriptomic landscape.

International audience; Objective: Intrinsically photosensitive retinal ganglion cells are most sensitive to short wavelengths and reach brain regions that modulate biological rhythms and energy metabolism. The increased exposure nowadays to artificial light at night (ALAN), especially short wavelengths, perturbs our synchronization with the 24-hour solar cycle. Here, the time-and wavelength dependence of the metabolic effects of ALAN are investigated. Methods: Male Wistar rats were exposed to white, blue, or green light at different time points during the dark phase. Locomotor activity, energy expenditure, respiratory exchange ratio (RER), and food intake were recorded. Brains, livers, and blood were collected. Results: All wavelengths decreased locomotor activity regardless of time of exposure, but changes in energy expenditure were dependent on the time of exposure. Blue and green light reduced RER at Zeitgeber time 16-18 without changing food intake. Blue light increased period 1 (Per1) gene expression in the liver, while green and white light increased Per2. Blue light decreased plasma glucose and phosphoenolpyruvate carboxykinase (Pepck) expression in the liver. All wavelengths increased c-Fos activity in the suprachiasmatic nucleus, but blue and green light decreased c-Fos activity in the paraventricular nucleus. Conclusions: ALAN affects locomotor activity, energy expenditure, RER, hypothalamic c-Fos expression, and expression of clock and metabolic genes in the liver depending on the time of day and wavelength.

The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR −/− mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

The known crosstalk between short-chain fatty acids (SCFAs) and the circadian clock is tightly intertwined with feeding time. We aimed to investigate the role of the core clock gene Bmal1 and feeding time in the diurnal rhythms in plasma and caecal SCFA levels and in their effect on the release of the hunger hormone ghrelin in the stomach and colon. WT, Bmal1-/- (ad libitum fed) and night-time-restricted-fed (RF)-Bmal1-/- littermates were killed at zeitgeber time (ZT) 4 and 16. SCFA concentrations were measured by gas chromatography. To investigate the effect of SCFAs on ghrelin release, stomach and colonic full-thickness strips were incubated with Krebs or a SCFA mix mimicking plasma or caecal concentrations, after which octanoyl ghrelin release was measured by RIA. Diurnal rhythms in caecal and plasma SCFAs oscillated in phase but rhythmic changes were abolished in Bmal1-/- mice. RF of Bmal1-/- mice restored fluctuations in caecal SCFAs. Plasma SCFA concentrations failed to affect gastric ghrelin release. The effect of caecal SCFA concentrations on colonic ghrelin release was rhythmic (inhibition at ZT 4, no effect at ZT 16). In Bmal1-/- mice, the inhibitory effect of SCFAs at ZT 4 was abolished. RF Bmal1-/- mice restored the inhibitory effect and increased colonic Clock expression. To conclude, diurnal fluctuations in caecal SCFAs and the effect of SCFAs on colonic ghrelin release are regulated by feeding time, independent of the core clock gene Bmal1. However, local entrainment of other clock genes might contribute to the observed effects.

We investigated the effects of sexual arousal induced by olfactory stimuli on the expression of neuromodulators, neurotransmitters and sexual steroid receptors in the suprachiasmatic nucleus (SCN, the circadian pacemaker of mammals) and other cerebral entities of Syrian hamsters (Mesocricetus auratus) compared to manual sleep deprivation and immobilization stress. The hamsters kept under a 12:12 hours (h) light:dark cycle were deprived of sleep by sexual stimulation, gentle manual handling or immobilization stress for 1 h at the beginning of the light phase and subsequently sacrificed at zeitgeber time 01:00, respectively, for comparison, hamsters were manually sleep deprived for 6 or 20 h or sacrificed after completing a full sleep phase. As demonstrated by immunohistochemistry, apart from various alterations after manual sleep deprivation, sexual stimulation caused down-regulation of arginine-vasopressin (AVP), vasointestinal peptide (VIP), serotonin (5-HT), substance P (SP), and met-enkephalin (ME) in the SCN. Somatostatin (SOM) was diminished in the medial periventricular nucleus (MPVN). In contrast, an increase in AVP was observed in the PVN, that of oxytocin (OXY) in the supraoptic nucleus (SON), of tyrosine-hydroxylase (TH) in the infundibular nucleus (IN), and dopamine beta-hydroxylase (DBH) in the A7 neuron population of the brain stem (A7), respectively. Testosterone in plasma was increased. The results indicate that sexual arousal extensively influences the neuropeptide systems of the SCN, suggesting an involvement of the SCN in reproductive behavior.

Sleep disturbance is one of the neurobehavioral complications of lead neurotoxicity. The present study evaluated the impacts of chronic lead exposure on alteration of the sleep–wake cycle in association with changes of clock gene expression in the hypothalamus. Sprague–Dawley rats with chronic lead exposure consumed drinking water that contained 250 ppm of lead acetate for five weeks. Electroencephalography and electromyography were recorded for scoring the architecture of the sleep–wake cycle in animals. At six Zeitgeber time (ZT) points (ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22), three clock genes, including rPer1, rPer2, and rBmal1b, were analyzed. The rats with chronic lead exposure showed decreased slow wave sleep and increased wakefulness in the whole light period (ZT1 to ZT12) and the early dark period (ZT13 to ZT15) that was followed with a rebound of rapid-eye-movement sleep at the end of the dark period (ZT22 to ZT24). The disturbance of the sleep–wake cycle was associated with changes in clock gene expression that was characterized by the upregulation of rPer1 and rPer2 and the feedback repression of rBmal1b. We concluded that chronic lead exposure has a negative impact on the sleep–wake cycle in rats that predominantly disrupts sleep homeostasis. The disruption of sleep homeostasis was associated with a toxic effect of lead on the clock gene expression in the hypothalamus.

In the freshwater catfish, Mystus cavasius, locally known as gulsha, ovarian maturation is triggered by long-day conditions. Using dopaminergic neuronal activity in the brain, the purpose of this study was to identify the brain’s detection of a nocturnal light pulse that induced ovarian development. Since direct inhibition of pituitary gonadotropin release is exerted by dopamine (DA), it may serve as a neuromodulator of photoperiodic stimulation in teleosts. We studied functional effects of photoperiodicity on dopaminergic rhythmicity in gulsha brain. Nocturnal illumination and Nanda-Hamner photocycles revealed that ovarian development is induced by a 1 h light pulse between zeitgeber time (ZT) 12 and 13. Daily fluctuations in DA, 3, 4-dihydroxyphenylacetic acid (DOPAC) and DOPAC/DA were observed under a 12L:12D photoperiod. Fish exhibited increased levels during the daytime and decreased levels at night. Rhythmic patterns of dopaminergic activity also showed clear circadian oscillations under constant light, but not constant dark conditions. After 7 days of exposure to long photoperiod (14L:10D), DA, DOPAC and DOPAC/DA in the brain at ZT12 and ZT16 were significantly higher than during a short photoperiod (10L:14D). Melatonin-containing water inhibited the release of DA and DOPAC 6 h and 24 h after treatment, respectively, and DOPAC/DA 6 h after treatment. This inhibition was blocked by the melatonin receptor antagonist, luzindole. These results suggest that a 1 h nocturnal light pulse induces ovarian development through alteration of dopaminergic neuronal excitability in the brain, via oscillation in melatonin triggered by photic stimuli, which may interfere with the reproductive endocrine axis in gulsha.

Background: To investigate whether shifted timing of eating, breakfast skipping, induces alterations in the circadian clock and abnormal lipid metabolism, we have established a delayed meal timing (DMT) protocol for rats, which started eating food 4 h delay. In the present study, control and DMT rats were fed a high-cholesterol diet during zeitgeber time (ZT) 12-24 and ZT 16-4, respectively. The DMT protocol increased the hepatic lipids and epididymal adipose tissue weight without changes in food intake and body weight. The surge in body temperature was delayed by 4 h in the DMT group, suggesting that energy expenditure was decreased in response to DMT. The peaks of the diurnal rhythm of serum non-esterified fatty acids and insulin were delayed by 2 and 4 h due to DMT, respectively. The oscillation peaks of hepatic de novo fatty acid synthesis gene expression was delayed by 4 h in response to DMT, whereas the peak of hepatic clock genes were 2 h delayed or not by DMT. Although metabolic oscillation is considered to be controlled by clock genes, the disintegration rhythms between the clock genes and lipid metabolism-related genes were not observed in rats fed a high-fat diet in our previous study. These data suggest that the circadian rhythm of de novo fatty acid metabolism is regulated by timing of eating, but is not directly controlled by clock genes. The present study suggests that breakfast skipping would complicate fatty liver and body fat accumulation.

Adipose tissue (AT) is a key metabolic organ which functions are rhythmically regulated by an endogenous circadian clock. Feeding is a zeitgeber aligning the clock in AT with the external time but mechanisms of this regulation remain largely unclear. We tested the hypothesis that postprandial changes of the hormone insulin directly entrain circadian clocks in AT and investigated transcriptional-dependent mechanism of this regulation. We analysed gene expression in subcutaneous AT (SAT) of obese subjects collected before and after the hyperinsulinemic-euglycemic clamp (EC) or control saline infusion (SC). The expression of core clock gene PER2, PER3 and NR1D1 in SAT were differentially changed upon insulin and saline infusion suggesting insulin-dependent clock regulation. In human stem cell-derived adipocytes, mouse 3T3-L1 cells and AT explants from mPer2Luc knockin mice, insulin induced a transient increase of the Per2 mRNA and protein expression leading to the phase shift of circadian oscillations and showing similar effects for Per1. Insulin effects were dependent on the region between the -64 and -43 in the Per2 promoter, but not on CRE and E-box elements. Our results demonstrate that insulin directly regulates circadian clocks in AT and isolated adipocytes and thus represent a primary mechanism of feeding-induced AT clock entrainment.

Noise trauma involves a plethora of mechanisms including reactive oxygen species, apoptosis, tissue damage, and inflammation. Recently, circadian mechanisms were also found to contribute to the vulnerability to noise trauma in mice, with greater damage occurring during their active phase (nighttime), when compared to similar noise exposures during their inactive phase (daytime). These effects seem to be regulated by mechanisms involving Bdnf responses to noise trauma and circulating levels of corticosterone (CORT). However, recent studies using different noise paradigms show contradicting results and it remains unclear how universal these findings are. Here we show that these findings differ even between substrains of mice and are restricted to a narrow window of noise intensity. We found that CBA/Sca mice exposed to 103 dB SPL display differential day/night noise sensitivity as measured by auditory brainstem responses (ABRs), but not at 100 (where full recovery is observed in day or night exposed mice) or 105 dB SPL (where permanent damage is found in both groups). In contrast, neither CBA/CaJ or CBA/JRj displayed such differences in day/night noise sensitivity, whatever noise intensity used. These effects appeared to be independent from outer hair cell function, as distortion product otoacoustic emissions appeared equally affected by day or night noise exposure, in all strains and in all noise conditions. Minor differences in ribbon counts or synaptic pairing were found in CBA/Sca mice, which were inconsistent with ABR wave 1 amplitude changes. Interestingly, CORT levels peaked in CBA/Sca mice at the onset of darkness at zeitgeber time 12 reaching levels of 43.8 ng/ml, while in the CBA/CaJ and the CBA/JRj, levels were 11.9 and 15.6 ng/ml respectively and peaking 4 hours earlier (zeitgeber time 8). These findings were consistent with higher period of daily rhythm in CBA/Sca mice when measured in complete darkness using running wheels (23.7h), than in CBA/CaJ (23.45h) or CBA/JRj (23.13h). In conclusion, our study suggests that the differential vulnerability to noise trauma between inactive and active phase is not universal and is as sensitive as substrain differences that might be governed by the circadian amplitude of the circulating CORT profiles.

Physical forces are critical for successful function of many organs including bone. Interestingly, the timing of exercise during the day alters physiology and gene expression in many organs due to circadian rhythms. Circadian clocks in tissues, such as bone, express circadian clock genes that target tissue-specific genes, resulting in tissue-specific rhythmic gene expression (clock-controlled genes). We hypothesized that the adaptive response of bone to mechanical loading is regulated by circadian rhythms. First, mice were sham loaded and sacrificed 8 h later, which amounted to tissues being collected at zeitgeber time (ZT)2, 6, 10, 14, 18, and 22. Cortical bone of the tibiae collected from these mice displayed diurnal expression of core clock genes and key osteocyte and osteoblast-related genes, such as the Wnt-signaling inhibitors Sost and Dkk1, indicating these are clock-controlled genes. Serum bone turnover markers did not display rhythmicity. Second, mice underwent a single bout of in vivo loading at either ZT2 or ZT14 and were sacrificed 1, 8, or 24 h after loading. Loading at ZT2 resulted in Sost upregulation, while loading at ZT14 led to Sost and Dkk1 downregulation. Third, mice underwent daily in vivo tibial loading over 2 weeks administered either in the morning, (ZT2, resting phase) or evening (ZT14, active phase). In vivo microCT was performed at days 0, 5, 10, and 15 and conventional histomorphometry was performed at day 15. All outcome measures indicated a robust response to loading, but only microCT-based time-lapse morphometry showed that loading at ZT14 resulted in a greater endocortical bone formation response compared to mice loaded at ZT2. The decreased Sost and Dkk1 expression coincident with the modest, but significant time-of-day specific increase in adaptive bone formation, suggests that circadian clocks influence bone mechanoresponse.

Synchronization between biologic clocks and metabolism is crucial for most species. Here, we examined the ability of leptin, important in the control of energy metabolism, to induce leptin signaling at the molecular as well as the behavioral level throughout the 24-h day in mice fed either a control or a high-fat diet (HFD). Furthermore, we investigated the effects of time-restricted feeding (TRF; a limitation of HFD access to 6 h each day) on energy metabolism during different periods throughout the 24-h day. In control mice, molecular leptin sensitivity was highest at zeitgeber time (ZT)0 (lights on), declining during the light phase, and increasing during the dark phase. Surprisingly, leptin resistance in HFD-fed mice was only present from the middle of the dark to the middle of the light period. Specifically, when TRF occurred from ZT21 to ZT3 (when leptin resistance in HFD-fed mice was most profound), it resulted in a disruption of the daily rhythms of locomotor activity and energy expenditure and in increased plasma insulin levels compared with other TRF periods. These data provide evidence that leptin sensitivity is controlled by the circadian rhythm and that TRF periods may be most efficient when aligned with the leptin-sensitive period.—Boucsein, A., Rizwan, M. Z., Tups, A. Hypothalamic leptin sensitivity and health benefits of time-restricted feeding are dependent on the time of day in male mice.

Carbohydrate and lipid metabolism in the liver has been reported to follow a certain circadian rhythm. Moreover, uridine supplementation also affected glucose and lipid homeostasis in previous studies; however, the mechanisms remain unclear. Therefore, this study was conducted to investigate whether uridine supplementation at an appropriate time during the day can alleviate obesity in mice. C57BL/6J mice were randomly assigned to four groups (n = 24 per group) with different diets for 2 weeks: (1) HFD group, fed a diet containing 60% fat calories; (2) DUR group, fed with HFD supplemented with 400 mg/kg uridine during zeitgeber time (ZT0) 0 - zeitgeber time 12 ZT12; (3) NUR group, fed HFD supplemented with 400 mg/kg uridine during ZT12 - ZT24. Starting at ZT4, liver samples were collected every 6 h for 24 h. Results showed that uridine supplementation, independent of the time of administration during the day, significantly reduced body weight gain (P < .05). Furthermore, liver weight and ratio showed a strong time dependence (P < .001). Additionally, oral administration of uridine during daytime or nighttime changed the expression levels of genes involved in the metabolism of uridine (SLC29A1, UMPS, UPP, UGT1A1, and DHODH; P < .05). Furthermore, uridine affected the levels of 10 fatty acids, lipid and glucose gene (FASN, LCAT, PC, PEPCK, GSK3β, and GLUT2; P < .05) depending on the timing of administration (P < .05). In conclusion, oral supplementation with uridine affected the diurnal variations in liver nucleotide and lipid metabolism, which contributed to the weight loss in HFD-fed mice.

Our previous studies have found that fish oil rich in ω-3 polyunsaturated fatty acids (ω-3 PUFA) protects against non-alcoholic fatty liver disease (NAFLD) in mice. This study was aimed to explore the effects of fish oil on high fat diet (HFD)-induced circadian bile composition chaos. Male C57BL/6 mice were randomly divided into three groups, a control group (CON), a HFD group and a fish oil (FO) group, which were fed a normal chow diet, a HFD, and a HFD supplemented with FO, respectively for 12 weeks. At the end of the experiment, liver tissue, blood and bile samples were processed at 12-h intervals with the first one at zeitgeber time 0 (ZT0) and the second at zeitgeber time 12 (ZT12). Metabolites in bile were determined using UPLC-QTOF-MS, screened using multivariate statistical analysis, and analyzed using KEGG database and Metaboanalyst. The expression levels of key proteins in bile acid metabolism were examined using western blot. Results of biochemical analysis and H&E staining illustrated that feeding of HFD induced NAFLD, which was ameliorated in FO group. The bile content of each group at ZT0 (CON, HFD, or FO group) was respectively higher than that at ZT12 (P

Rodent studies often must be conducted during an animal's active phase (that is, in darkness) yet also during a typical day shift for staff. Low-pressure sodium lighting (LPSL), to which human retinas are more sensitive than rodents' at low intensity, has been used to facilitate study conduct in dark phase. The assumption was that LPSL would be equivalent to total darkness due to low rodent retinal sensitivity but provide enough lighting for safe technical manipulations due to higher human retinal sensitivity. Unlike other light sources, LPSL has been tested for effects on circadian rhythm specific to locomotive activities in albino mice. Whether LPSL affects circadian rhythms in rats is unknown. In this study, circadian endpoints were derived from body temperature and locomotor activity via telemeters in 8 adult male Wistar rats. When moved from a 12:12-h white-light (that is, cold white fluorescent light):dark (LD) cycle to a 12:12-h white-light:sodium-light cycle, rats demonstrated free-running and disrupted circadian rhythms (that is, lengthened circadian period and reduced circadian robustness and amplitude). Body temperature and locomotor activity were significantly lower in the LPSL phase as compared with dark phase under the baseline condition. When exposed to a 12:12 h sodium-light:dark (SD) cycle, rats entrained with a circadian period similar to 12:12-h white-light:dark (LD), but significantly different from the period under constant darkness (DD). Circadian onset and acrophase were delayed under SD compared with LD. When illuminated with a LPSL pulse under DD, rats showed phase shifts similar to white-light pulse effects, consistent with the phase response curve. To determine whether the image-forming photoreceptors are involved in this process, we used electroretinography. Compared with white light, 589-nm light generated during electroretinography elicited rod photoreceptors responses with longer latency and cone photoreceptor responses with lower amplitude. These results indicate that LPSL is a weaker zeitgeber than white light and may alter the circadian system in rats. Furthermore, because LPSL appeared to be visible to rats, it may not be an appropriate substitute for actual darkness.

Adult rabbits show robust circadian rhythms of: nursing, food and water intake, hard faeces excretion, locomotion, body temperature, blood and intraocular pressure, corticosteroid secretion, and sleep. Control of several circadian rhythms involves a light-entrained circadian clock and a food-entrained oscillator. Nursing periodicity, however, relies on a suckling stimulation threshold. Brain structures regulating this activity include the paraventricular nucleus and preoptic area, as determined by lesions and quantification of cFOS- and PER1 clock gene-immunoreactive proteins. Melatonin synthesis in the rabbit pineal gland shows a diurnal rhythm, with highest values at night and lowest ones during the day. In kits the main zeitgeber is milk intake, which synchronizes locomotor activity, body temperature, and corticosterone secretion. Brain regions involved in these effects include the median preoptic nucleus and several olfactory structures. As models for particular human illnesses rabbits have been valuable for studying glaucoma and cardiovascular disease. Circadian variations in intraocular pressure (main risk factor for glaucoma) have been found, with highest values at night, which depend on sympathetic innervation. Rabbits fed a high fat diet develop cholesterol plaques and high blood pressure, as do humans, and such increased fat intake directly modulates cardiovascular homeostasis and circadian patterns, independently of white adipose tissue accumulation. Rabbits have also been useful to investigate the characteristics of sleep across the day and its modulation by infections, cytokines and other endogenous humoral factors. Rabbit circadian biology warrants deeper investigation of the role of the suprachiasmatic nucleus in regulating most behavioral and physiological rhythms described above.

The purpose of this study was to analyze the rest-activity rhythm of type 2 diabetics mellitus (T2DM) and compare it to healthy controls using the nonparametric analytic approach. Twentyone diabetics and 21 healthy subjects matched for gender and age were recruited to participate in the study. Data were analyzed using the Independent t-test, Mann-Whitney U test, and Spearmans correlation. T2DM subjects demonstrate lower interdaily stability (IS) (p=.03), higher intradaily variability (p=.046) and lower rhythm amplitude (p=.02) when compared to healthy controls. Also, there was a positive correlation between IS and most active 10 hr (M10) in the average of 24 hours pattern (r =.44; p=.046) in the diabetes group and a negative correlation between IV and M10 in the healthy group (r =-.57; p=.007). These data together suggest that T2DM exhibits a dysfunction in the rest-activity rhythm due to alterations in the circadian function as well as in the homeostatic capacity to maintain sleep; mainly characterized by less consistency across days of the daily circadian signal, higher rhythm fragmentation and lower rhythm amplitude. Future approaches may be developed considering the influence of circadian glucose variations throughout the day on the coupling of the rest-activity rhythm to zeitgeber and rhythm fragmentation.

Objective— Evening but not morning administration of low-dose aspirin has been reported to lower blood pressure in hypertensive patients. The present study was designed to determine whether this phenomenon could be replicated in mice, and if so, whether a time-dependent effect of aspirin on blood pressure was because of alteration of circadian clock function. Approach and Results— We recapitulated the protective effect of aspirin (50 μg/d for 7 days) at zeitgeber time 0 (active-to-rest transit), but not at zeitgeber time 12, on a high-salt diet–induced increase of blood pressure. However, the time of aspirin administration did not influence expression of canonical clock genes or their acetylation. We used mouse Bmal1 and Per2-luciferase reporters expressed in U2OS cells to determine the real-time effect of aspirin on circadian function but found that the oscillation of bioluminescence was unaltered. Timing of aspirin administration also failed to alter urinary prostaglandin metabolites or catecholamines, or the acetylation of its COX-1 (cyclooxygenase-1) target in platelets. Conclusions— The time-dependent hypotensive effect of aspirin in humans has been recapitulated in hypertensive mice. However, this does not seem to reflect a direct impact of aspirin on circadian clocks or on acetylation of platelet COX-1.

Light is the key regulator of circadian clock, the time-keeping system synchronizing organism physiology and behavior with environmental day and night conditions. In its natural habitat, the strictly subterranean naked mole-rat (Heterocephalus glaber) has lived in a light-free environment for millennia. We questioned if this species retains a circadian clock and if the patterns of this clock and concomitant rhythms differed in liver tissue from mice and naked mole-rats. As expected, in mice, the various circadian clock genes peaked at different times of the day; the Period gene (Per) group peaked in the evening, whereas Brain and Muscle ARNT-like1 (Bmal1) gene peaked in the morning; this phase shift is considered to be fundamental for circadian clock function. In sharp contrast, in the naked mole-rat both Per1 and Per2, as well as Bmal1, peaked at the same time in the morning—around ZT2—suggesting the organization of the molecular circadian oscillator was different. Moreover, gene expression rhythms associated with glucose metabolism and mTOR signaling also differed between the species. Although the activity of mTORC1 was lower, while that of mTORC2 was higher in naked mole-rat livers compared to mice, unlike that of mice where the expression profiles of glucose metabolism genes were not synchronized, these were highly synchronized in naked mole-rats and likely linked to their use of feeding times at zeitgebers.

Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects.

Temperature rhythms can act as potent signals for the modulation of the amplitude and phase of clock gene expression in peripheral organs in vitro, but the relevance of the circadian rhythm of core body temperature (Tc) as a modulating signal in vivo has not yet been investigated. Using calorie restriction and cafeteria feeding, we induced a larger and a dampened Tc amplitude, respectively, in male Wistar rats, and investigated the circadian expression profile of the core clock genes Bmal1, Per2, Cry1, and Rev-erbα, the heat-responsive genes heat shock protein 90 (Hsp90) and cold-inducible RNA binding protein (Cirbp), and Pgc1α, Pparα/γ/δ, Glut1/4, and Chop10 in the liver, skeletal muscle, white adipose tissue (WAT), and adrenal glands. Diet-altered Tc rhythms differentially affected the profiles of clock genes, Hsp90, and Cirbp expression in peripheral tissues. Greater Tc amplitudes elicited by calorie restriction were associated with large amplitudes of Hsp90 and Cirbp expression in the liver and WAT, in which larger amplitudes of clock gene expression were also observed. The amplitudes of metabolic gene expression were greater in the WAT, but not in the liver, in calorie-restricted rats. Conversely, diet-altered Tc rhythms were not translated to distinct changes in the amplitude of Hsp90, Cirbp, or clock or metabolic genes in the skeletal muscle or adrenal glands. While it was not possible to disentangle the effects of diet and temperature in this model, taken together with previous in vitro studies, our study presents novel data consistent with the notion that the circadian Tc rhythm can modulate the amplitude of circadian gene expression in vivo. The different responses of Hsp90 and Cirbp in peripheral tissues may be linked to the tissue-specific responses of peripheral clocks to diet and/or body temperature rhythms, but the association with the amplitude of metabolic gene expression is limited to the WAT.

Objectives Nocturia is a major problem in geriatric patients. Clock genes regulate circadian bladder function and Piezo type mechanosensitive ion channel component 1 (Piezo1) that senses bladder fullness. We utilized WT and Clock mutant (ClockΔ19/Δ19: nocturia phenotype) mice to determine if the effects of GsMTx4, a Piezo1 inhibitor, is dependent on circadian Piezo1 expression in the bladder. Methods We compared voiding behavior in mice after the administration of vehicle, low dose, or high dose of GsMTx4. Intraperitoneal injections (IP) were performed at Zeitgeber time (ZT) 0, lower Piezo1 expression phase (ZT0-IP) and ZT12, higher Piezo1 expression phase (ZT12-IP). Urine volume (Uvol), voiding frequency (VF), and urine volume per void (Uvol/v) were measured using metabolic cages. Results VF decreased at ZT12-IP in WT mice only with high dose of GsMTx4 but showed no effects in ClockΔ19/Δ19 mice. VF decreased significantly at ZT0-IP in WT mice after both doses, but only decreased after high dose in ClockΔ19/Δ19 mice. Uvol/v increased in WT mice at ZT0-IP after both doses and at ZT12-IP after high dose. Uvol/v increased in ClockΔ19/Δ19 mice only at ZT0-IP after high dose. GsMTx4 did not affect Uvol in both mice at ZT12-IP. A decrease in Uvol was observed in both mice at ZT0-IP; however, it was unrelated to GsMTx4-IP. Conclusions The effects of GsMTx4 changed associated with the circadian clock and Piezo1 expression level. The maximum effect occurred during sleep phase in WT. These results may lead to new therapeutic strategies against nocturia.

Cardiac surgery-associated acute kidney injury (AKI) is a serious and frequent complication with poor prognosis, and disruption in circadian rhythm shall adversely influence cardiovascular and renal functions via oxidative stress mechanisms. However, the role of circadian clock genes (circadian locomotor output cycle kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1)) and its interaction with nuclear factor erythroid 2-related factor 2 (Nrf2) in AKI following myocardial ischemia-reperfusion (MIR) in the diabetic rat has not yet been explored. In this study, rats were divided into the sham (S) group, MIR (M) group, diabetic (D) group, and diabetic+MIR (DM) group. At light (zeitgeber time (ZT) 0) and dark time points (ZT12), rat MIR model was established by occlusion of the left anterior descending coronary artery for 30 min followed by 2 -hour reperfusion, and then renal injury was evaluated. The renal histological changes in the DM group were significantly high compared to other groups; serum creatinine, blood urea nitrogen, and neutrophil gelatinase-associated lipocalin levels, as well as malondialdehyde and 8-iso-prostaglandin-F2α levels in renal tissues of M ZT12 and DM ZT12 subgroups, were significantly higher than those of M ZT0 and DM ZT0 subgroups, individually indicating increased oxidative stress at a dark cycle. Further, Nrf2 protein accumulated in a circadian manner with decreasing levels at night in the DM and M groups. In conclusion, renal injury following MIR was exacerbated in the diabetic rat at night through molecular mechanisms involving transcriptional control of the circadian clock on light-dark activation of Nrf2.

Timed physical activity might potentiate the health benefits of training. The underlying signaling events triggered by exercise at different times of the day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naïve mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of the day resulted in a vastly different activation of signaling pathways, e.g., related to stress response, vesicular trafficking, repair, and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed minimal Zeitgeber (i.e., synchronizing) activity of training. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. These results are of high importance to understand circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.HighlightsMaximal endurance performance is greater in the early morningTimed exercise differentially alters the muscle transcriptome and (phospho)-proteomeMorning exercise triggers energy provisioning and tissue regenerationEvening exercise activates stress-related and catabolic pathwaysTraining exerts poor Zeitgeber activity on the muscle and liver clocks

Time-restricted feeding/eating (TRF/TRE) – limiting not the amount of food but the daily time window of food intake – is a dietary intervention that has been shown to improve health markers in model organisms and humans, but whether these benefits translate into positive effects on aging and longevity is not clear. We demonstrate here that TRF robustly prolongs lifespan in the short-lived genetically tractable model organismDrosophila melanogaster. Median TRF lifespan extensions range between ∼10% and ∼50% dependent on sex, reproductive status, TRF duration, and genotype. TRF’s positive effect on longevity is independent of food intake and at least in part relies on a functioning circadian clock: TRF benefits on longevity are abolished in arrhythmicper0andtim01mutants as well as in constant light, suggesting that timed feeding acts as a zeitgeber partitioning eating and associated metabolic processes into certain phases of day and night. TRF-mediated longevity extension is unaffected in flies whose neural circadian clocks have been abolished genetically, pointing towards peripheral clocks as the target of TRF mediating lifespan extension.

A number of studies implicate biogenic amines in regulating circadian rhythms. In particular, dopamine and serotonin influence the entrainment of circadian rhythms to daily food availability. To study circadian entrainment to feeding, food availability is typically restricted to a short period within the light cycle daily. This results in a notable increase in pre-meal activity, termed “food anticipatory activity” (FAA), which typically develops within about 1 week of scheduled feeding. Several studies have implicated serotonin as a negative regulator of FAA: (1) aged rats treated with serotonin 5-HT2 and 3 receptor antagonists showed enhanced FAA, (2) mice lacking for the 2C serotonin receptor demonstrate enhanced FAA, and (3) pharmacologically increased serotonin levels suppressed FAA while decreased serotonin levels enhanced FAA in mice. We sought to confirm and extend these findings using genetic models with impairments in central serotonin production or re-uptake, but were surprised to find that both serotonin transporter (Slc6a4) and tryptophan hydroxylase-2 knockout mice demonstrated a normal behavioral response to timed, calorie restricted feeding. Our data suggest that FAA is largely independent of central serotonin and/or serotonin reuptake and that serotonin may not be a robust negative regulator of FAA.

Animals regulate a variety of aspects of physiology according to environmental light conditions via nonvisual opsins such as melanopsin. In order to study photic regulation of fish physiology, expression changes of the genes for melanopsin (opn4xa and opn4xb) and effects of light on them were examined in juvenile grass puffer Takifugu alboplumbeus using quantitative real-time PCR. In the brain of juvenile fish, no significant diurnal nor circadian changes were observed in opn4x mRNA levels. On the other hand, in the eyes, the mRNA level of opn4xa showed a significant diurnal rhythm with a peak at Zeitgeber time (ZT) 4, while no apparent circadian changes were observed. The mRNA level of opn4xb in the eyes showed a diurnal change similar to that of opn4xa, while it showed a significant circadian change. Furthermore, continuous exposure to light during a subjective night significantly increased the mRNA levels of opn4xa in the eyes at ZT24, suggesting that light induces gene expression of opn4xa in the eyes and that the induction occurs only during the night-day transition period. These results suggest that Opn4xa and Opn4xb play differential roles in the eyes of juvenile grass puffer to mediate the physiological effects of environmental light information.

Circadian rhythms play a critical role in the physiological processes involved in energy metabolism and energy balance (EB). A large array of metabolic processes, including the expression of many energy-regulating endocrine hormones, display temporal rhythms that are driven by both the circadian clock and food intake. Mealtime has been shown to be a compelling zeitgeber in peripheral tissue rhythms. Inconsistent signalling to the periphery, because of mismatched input from the central clock vs time of eating, results in circadian disruption in which central and/or peripheral rhythms are asynchronously time shifted or their amplitudes reduced. A growing body of evidence supports the negative health effects of circadian disruption, with strong evidence in murine models that mealtime-induced circadian disruption results in various metabolic consequences, including energy imbalance and weight gain. Increased weight gain has been reported to occur even without differences in energy intake, indicating an effect of circadian disruption on energy expenditure. However, the translation of these findings to humans is not well established because the ability to undertake rigorously controlled dietary studies that explore the chronic effects on energy regulation is challenging. Establishing the neuroendocrine changes in response to both acute and chronic variations in mealtime, along with observations in populations with routinely abnormal mealtimes, may provide greater insight into underlying mechanisms that influence long-term weight management under different meal patterns. Human studies should explore mechanisms through relevant biomarkers; for example, cortisol, leptin, ghrelin and other energy-regulating neuroendocrine factors. Mistiming between aggregate hormonal signals, or between hormones with their receptors, may cause reduced signalling intensity and hormonal resistance. Understanding how mealtimes may impact on the coordination of endocrine factors is essential for untangling the complex regulation of EB. Here a review is provided on current evidence of the impacts of mealtime on energy metabolism and the underlying neuroendocrine mechanisms, with a specific focus on human research.

Childhood obesity in girls is associated with early puberty and menarche. Breast tissue exhibits circadian rhythms. These rhythms may be altered by environmental factors. We hypothesized that a high-fat diet (HFD) disrupts circadian rhythms in pubertal mammary glands. Weanling female C57BL/6 mice were fed the standard AIN93G diet or a HFD (providing 16% or 45% of energy from soybean oil) for 3 weeks. Mammary glands were harvested from 6-week-old mice every 4 h on Zeitgeber time over a 48-h period; rhythmic expressions of circadian genes and genes encoding estrogen receptor and progesterone receptor were analyzed by using the Cosinor model. HFD, compared to AIN93G diet, altered diurnal oscillations of circadian genes in pubertal mammary glands. These included changes in amplitude of Per2, Cry1 (reduced), Clock, Rev-erbα, and Per1 (elevated), a delay in acrophase (the hour at which the rhythm peaks) of Bmal1 by 2.2 h, and changes in mesor (the mean of the rhythm from peak to trough) of Bmal1, Per2, Cry1 (reduced), Rev-rebα, and Per1 (elevated). Furthermore, HFD altered diurnal expression of estrogen receptor and progesterone receptor at both mRNA and protein levels. These findings indicate that HFD alters circadian regulation in pubertal mammary glands, which may contribute to the disturbance of hormonal homeostasis and lead to early development and growth of mammary glands in pubertal mice.

Objective: Several studies have shown that the time of day regulates the reinforcing effects of cocaine. Additionally, melatonin and its MT1 and MT2 receptors have been found to participate in modulation of the reinforcing effects of such addictive drugs as cocaine. Loss of the diurnal variation in cocaine-induced locomotor sensitization and cocaine-induced place preference has been identified in pinealectomized mice. In addition, several studies in rodents have shown that administration of melatonin decreased the reinforcing effects of cocaine. The objective of this study was to evaluate the effect of melatonin on cocaine-induced locomotor activity in pinealectomized rats at different times of day (zeitgeber time [ZT]4, ZT10, ZT16, and ZT22). Methods: Naïve, pinealectomized Wistar rats received cocaine at different times of day. Melatonin was administered 30 min before cocaine; luzindole was administered 15 min prior to melatonin and 45 min before cocaine. After administration of each treatment, locomotor activity for each animal was recorded for a total of 30 min. Pinealectomy was confirmed at the end of the experiment through melatonin quantitation by ELISA. Results: Cocaine-induced locomotor activity varied according to the time of day. Continuous lighting and pinealectomy increased cocaine-induced locomotor activity. Melatonin administration decreased cocaine-induced locomotor activity in naïve and pinealectomized rats at different times of day. Luzindole blocked the melatonin-induced reduction in cocaine-induced locomotor activity in pinealectomized rats. Conclusion: Given its ability to mitigate various reinforcing effects of cocaine, melatonin could be a useful therapy for cocaine abuse.

Disruptions to circadian rhythm have been associated with an increased risk of nonalcoholic fatty liver disease (NAFLD). DHA has been found to affect both circadian rhythm and lipid metabolism. In this study, the relationship between DHA substitution and improvements in lipid metabolism and circadian clock regulation was studied. Male C57BL/6 mice were fed a control, a high fat or a DHA substituted diet for 12 weeks. Biochemical analysis and H&E staining showed that the high-fat diet (HFD) could induce NAFLD, and DHA substitution (AOH) could attenuate NAFLD. The qPCR results showed that the expressions of core clock genes Clock and Bmal1 were significantly higher at zeitgeber (ZT) 0 (7:00 am) than those at ZT12 (7:00 pm) in the control group, while this difference in day and night disappeared in the HFD group, but was observed in the AOH group. Western blotting results indicated that the expressions of rhythm output molecules (RORα and REV-ERBα) and their downstream protein INSIG2 all showed the corresponding circadian changes. SREBP-regulated proteins were significantly increased in the HFD group at both ZT0 and ZT12, but decreased in the AOH group accompanied by the corresponding changes in the protein expressions of HMGCR, LXR, CYP7A1 and CYP27A1. Altogether, HFD can decrease or disrupt circadian rhythm fluctuation by up-regulating the expression of core circadian rhythm genes Clock and Bmal1 at ZT12, and induce metabolic abnormalities through the INSIG2-SREBP pathway regulated by RORα and REV-ERBα. DHA substitution seems to restore circadian rhythm similar to the normal circadian rhythm of "night-high, day-low" through the metabolic pathway regulated by rhythmic nuclear receptors, improving the lipid metabolism rhythm and reducing liver fat.

Key points Disruptions in circadian rhythms across an organism are associated with negative health outcomes, such as cardiometabolic and neurodegenerative diseases.Exercise has been proposed as a time cue for the circadian clock in rodents and humans.In this study, we assessed the effect of a single bout of endurance exercise on the skeletal muscle clock in vivo and a bout of muscle contractions in vitro.Timing of exercise or contractions influences the directional response of the muscle clock phase in vivo and in vitro.Our findings demonstrate that muscle contractions, as a component of exercise, can directly modulate the expression of muscle clock components in a time‐of‐day dependent manner. Abstract Exercise has been proposed to be a zeitgeber for the muscle circadian clock mechanism. However, this is not well defined and it is unknown if exercise timing induces directional shifts of the muscle clock. Our purpose herein was to assess the effect of one bout of treadmill exercise on skeletal muscle clock phase changes. We subjected PERIOD2::LUCIFERASE mice (n = 30F) to one 60 min treadmill exercise bout at three times of day. Exercise at ZT5, 5 h after lights on, induced a phase advance (100.2 ± 25.8 min; P = 0.0002), whereas exercise at ZT11, 1 h before lights off, induced a phase delay (62.1 ± 21.1 min; P = 0.0003). Exercise at ZT17, middle of the dark phase, did not alter the muscle clock phase. Exercise induces diverse systemic changes so we developed an in vitro model system to examine the effects of contractile activity on muscle clock phase. Contractions applied at peak or trough Bmal1 expression induced significant phase delays (applied at peak: 27.2 ± 10.2 min; P = 0.0017; applied at trough: 64.6 ± 6.5 min, P, Key points Disruptions in circadian rhythms across an organism are associated with negative health outcomes, such as cardiometabolic and neurodegenerative diseases.Exercise has been proposed as a time cue for the circadian clock in rodents and humans.In this study, we assessed the effect of a single bout of endurance exercise on the skeletal muscle clock in vivo and a bout of muscle contractions in vitro.Timing of exercise or contractions influences the directional response of the muscle clock phase in vivo and in vitro.Our findings demonstrate that muscle contractions, as a component of exercise, can directly modulate the expression of muscle clock components in a time‐of‐day dependent manner.

Exercise has been proposed to be a zeitgeber for the muscle circadian clock mechanism. However, this is not well defined and it is unknown if exercise timing induces directional shifts of the muscle clock. Our purpose herein was to assess the effect of one bout of treadmill exercise on skeletal muscle clock phase changes. We subjected PERIOD2::LUCIFERASE mice (n=30F) to one 60-minute treadmill exercise bout at three times of day. Exercise at ZT5, 5h after lights on, induced a phase advance (1.4±0.53h; p=0.038), whereas exercise at ZT11, 1h before lights off, induced a phase delay (−0.95±0.43h; p=0.0315). Exercise at ZT17, middle of the dark phase, did not alter muscle clock phase. Exercise induces diverse systemic changes so we developed an in-vitro model system to examine effects of contractile activity on muscle clock phase. Contractions applied at peak or trough Bmal1 expression induced significant phase delays (applied at peak: 1.3±0.02h; p=0.0425; applied at trough: 1.8±0.02h, p=0.0074). Contractions applied during the transition from peak to trough Bmal1 expression induced a phase advance (1.8±0.03h; p=0.0265). Lastly, contractions at different times of day resulted in differential changes of core-clock gene expression demonstrating an exercise and clock interaction, providing insight into potential mechanisms exercise-induced phase shifts. These data demonstrate that muscle contractions, as part of exercise, are sufficient to shift muscle circadian clock phase, likely through changes in core-clock gene expression. Additionally, our findings that exercise induces directional muscle clock phase changes confirms exercise is a bone fide environmental time cue for skeletal muscle.

Western diets that are high in saturated fat and sugar disrupt circadian rhythms, induce weight gain, and lead to metabolic diseases including obesity. However, the mechanistic link between altered circadian rhythms and energy homeostasis remains poorly understood. In C57BL/6J mice, consuming a Western diet for 16 weeks significantly reduced food intake (at zeitgeber 12–16), in association with decreases in hypothalamic expression of the orexigenic neuropeptides, neuropeptide Y (Npy) and agouti-related peptide (AgRP). To examine the acute effects of the most prevalent saturated fatty acid in a Western diet, palmitate, and the role of the core clock gene, Bmal1, in the regulation of hypothalamic feeding neuropeptides, we used heterogeneous and clonal BMAL1 knockout (KO) immortalized hypothalamic cell lines, expressing specific neuropeptides, derived from male (M) and female (F) mice. Both mHypoA-BMAL1-KO/F and mHypoA-BMAL1-KO/M cells demonstrated a loss of circadian rhythmicity in expression of the clock gene, Per2, as compared to wild-type (control) cultures. Loss of BMAL1 also altered the time-dependent expression of Npy and proopiomelanocortin, and disrupted AgRP rhythmicity. Furthermore, palmitate increased BMAL1 binding to the Npy promotor region, and palmitate treatment (50 μM for 24 h) stimulated Npy expression in a BMAL1-dependent manner in both heterogeneous and clonal NPY-expressing female-derived cell models. The results of this study demonstrate that circadian expression of Bmal1 serves as a mechanistic link between Western diet- and palmitate-induced disruptions of the normal rhythmic patterns in hypothalamic feeding-related neuropeptides.

Background: Circadian clock has been closely related to the development of diabetes mellitus and cardiovascular disease; the disruption of circadian clock exacerbates myocardial ischemia/reperfusion injury (MI/RI). HDAC3 is a key component of the circadian negative feedback loop by recruitmenting the expression pattern of circadian nuclear receptor Rev-erbα, then to maintain the stability of circadian gene such as BMAL1. In this research, we explored the mechanism of HDAC3-orchestrated Rev-erbα/BMAL1 pathway in increasing MI/RI vulnerability of diabetes, and its relationship with mitophagy. Methods: Streptozocin (STZ) was used to establish type 1 diabetes by intraperitoneal injection. After 8 weeks, the diabetic and non-diabetic rats were exposed to MI/RI by ligating the left anterior descending coronary artery (LDA) for 30 minutes and reperfusion for 120 minutes at four time points of zeitgeber (ZT) 0, 6, 12 and 18. In vitro experiments, primary cardiomyocytes with or without HDAC3 siRNA and Rev-erbα siRNA exposed to hypoxia/reoxygenation (H/R). Findings: Circadian clock gene oscillations were rapidly attenuated in diabetic I/RI hearts, versus sham and/or the non-diabetic I/RI hearts, in accord with circadian mitophagy. By utilizing AAV-HDAC3 to knockdown HDAC3 expression, HDAC3 deficiency significantly attenuated diabetic MI/RI associated with increased autophagy activation. The expression of HDAC3 and Rev-erbα in cardiomyocytes was increased under high glucose condition with decreased BMAL1 expression and autophagy level. After H/R stimulation, the cell injury was obviously increased with upregulated HDAC3 and Rev-erbα expression and decreased BMAL1 level. Moreover, high glucose aggravated H/R injury compared with low glucose by reducing autophagy level, increasing the levels of HDAC3 and Rev-erbα and down-regulating BMAL1 expression. HDAC3 and Rev-erbα siRNA can alleviate high glucose and H/R-induced injury by up-regulating BMAL1 expression and mitophagy levels. Interpretation: These findings suggest that in the MI/RI of diabetic rat, the circadian clocks were rapidly impaired then to inhibit mitophagy; cardiac-targeted manipulation of HDAC3-orchestrated Rev-erbα/BMAL1 pathway may be the mechanism of the decreased toleration to ischemia with a circadian dependent variability. Funding Statement: This study was supported by grants from the National Natural Science Foundation of China 81970722, 81671891 and 81701891. Declaration of Interests: The authors declare that there is no conflict of interests regarding the publication of this paper. Ethics Approval Statement: Experimental protocols were implemented after being reviewed and approved by the Laboratory Animal Welfare & Ethics Committee (IACUC) of Renmin Hospital of Wuhan University. All researchers have obtained the certificate of professional laboratory animal technology in Hubei Province.

High-calorie diet, circadian rhythms and metabolic features are intimately linked. However, the mediator(s) between nutritional status, circadian rhythms and metabolism remain largely unknown. This article aims to clarify the key metabolic pathways bridging nutritional status and circadian rhythms based on a combination of metabolomics and molecular biological techniques. A mouse model of high-fat diet-induced obesity was established and serum samples were collected in obese and normal mice at different zeitgeber times. Gas chromatography/mass spectrometry, multivariate/univariate data analyses and metabolic pathway analysis were used to reveal changes in metabolism. Metabolites involved in the metabolism of purines, carbohydrates, fatty acids and amino acids were markedly perturbed in accordance with circadian related variations, among which purine catabolism showed a typical oscillation. What&rsquo, s more, the rhythmicity of purine catabolism dampened in the high-fat diet group. The expressions of clock genes and metabolic enzymes in the liver were measured. The mRNA expression of Xanthine oxidase (Xor) was highly correlated with the rhythmicity of Clock, Rev-erb&alpha, and Bmal1, as well as the metabolites involved in purine catabolism. These data showed that a high-fat diet altered the circadian rhythm of metabolic pathways, especially purine catabolism. It had an obvious circadian oscillation and a high-fat diet dampened its circadian rhythmicity. It was suggested that circadian rhythmicity of purine catabolism is related to circadian oscillations of expression of Xor, Uox and corresponding clock genes.

Circadian rhythms coordinate an organism’s activities and biological processes to the optimal time in the 24-h daylight cycle. We previously demonstrated that male C57BL/6 mice develop sepsis more rapidly when the disease is induced in the nighttime versus the daytime. In this report, we elucidate the mechanism of this diurnal difference. Sepsis was induced via cecal ligation and puncture (CLP) at zeitgeber time (ZT)–19 (2 am) or ZT-7 (2 pm). Like the males used in our prior study, female C57BL/6 mice had a worse outcome when CLP was induced at ZT-19 versus ZT-7, and these effects persisted when we pooled the data from both sexes. In contrast, mice with a mutated Period 2 (Per2) gene had a similar outcome when CLP was induced at ZT-19 versus ZT-7. Bone marrow chimeras reconstituted with C57BL/6 immune cells exhibited a worse outcome when sepsis was induced at ZT-19 versus ZT-7, whereas chimeras with Per2-mutated immune cells did not. Next, murine macrophages were subjected to serum shock to synchronize circadian rhythms and exposed to bacteria cultured from the mouse cecum at 4-h intervals for 48 h. We observed that IL-6 production oscillated with a 24-h period in C57BL/6 cells exposed to cecal bacteria. Interestingly, we observed a similar pattern when cells were exposed to the TLR2 agonist lipoteichoic acid. Furthermore, TLR2-knockout mice exhibited a similar sepsis phenotype when CLP was induced at ZT-19 versus ZT-7. Together, these data suggest that circadian rhythms in immune cells mediate diurnal variations in murine sepsis severity via a TLR2-dependent mechanism.

Type 2 diabetes mellitus is characterized by the loss of β‐cell function and mass, resulting from interactions between genetic predisposition and various environmental factors1. One environmental condition identified as a risk factor for type 2 diabetes mellitus is circadian rhythm disruption, which is induced by shift work or sleep disturbance. However, the mechanism whereby circadian disruption leads to impaired glucose metabolism is not well understood. A circadian rhythm is an approximately 24‐h cycle in the physiological processes of living beings, including cyanobacteria. The circadian clock system is based on transcriptional–translational regulation of ‘core clock genes’ in mammals. Clock genes are expressed throughout mammalian organ systems, and they play an important role by generating both behavioral and physiological rhythms, and synchronizing metabolism in anticipation of the sleep/wake and fasting/feeding cycle. The circadian clock is encoded by the heterodimeric basic helix‐loop‐helix Per‐Arnt‐Sim (bHLH‐PAS) transcription factors CLOCK, and brain and muscle Arnt‐like 1 (BMAL1), which trigger the expressions of period (PER)/cryptochrome (CRY) repressors that inhibit CLOCK/BMAL1 in a cycle that repeats itself every 24 h. According to genetic studies, Clock mutant mice show altered expressions of genes known to regulate islet growth, survival, maturation and proliferation2. Bmal1 deletion in β‐cells also results in failed metabolic adaptation to a high‐fat diet, characterized by hyperglycemia, glucose intolerance and loss of glucose‐stimulated insulin secretion3. These results suggest that circadian disruption‐induced metabolic disorder is, at least in part, attributable to β‐cell failure, though the mechanisms remain largely unknown. The Wfs1 −/− A y/a mouse is an animal model of Wolfram syndrome with mild obesity4. These mice develop diabetes with severe insulin deficiency as a result of endoplasmic reticulum (ER) stress‐induced β‐cell failure. The precise mechanism whereby Wolfram syndrome 1 (WFS1) mutation induces ER stress, followed by β‐cell failure, is not fully understood. However, various studies have shown that the WFS1 protein localizes to the ER membrane, and exerts a protective effect against ER stress5. In gene expression studies, we showed alteration of clock‐related gene expressions in Wfs1 −/− A y/a mice islets, as compared with those in A y/a mice; D site of albumin promoter (Dbp) messenger ribonucleic acid (RNA) levels of Wfs1 −/− A y/a mice islets were decreased by 50%, whereas their E4 promoter‐binding protein 4 (E4bp4) messenger RNA levels were increased by 50% at Zeitgeber Time 12. Notably, similar alterations were observed with chemically‐induced ER stress in in vitro experiments using MIN6 cells.

Biological rhythms are thought to be related to the pathogenesis and therapy of various diseases including depression. Here we investigated the influence of circadian rhythms on the antidepressant activity of the dual-action serotonin-noradrenaline reuptake inhibitor (SNRI) milnacipran. Rats administered milnacipran in the morning (8:00 a.m.; zeitgeber time [ZT]1) or in the evening (8:00 p.m.; ZT13) were analyzed in a forced swim test (FST). At ZT1, the rats’ immobility was reduced and the swimming was increased, whereas at ZT13, their climbing was increased. These results suggest that the serotonergic and noradrenergic systems are preferentially affected at ZT1 and ZT13, respectively by milnacipran. We analyzed the plasma and brain levels of milnacipran after administration, and there were no differences between ZT1 and ZT13. The circadian rhythm of monoamine neurotransmitters was analyzed in several brain regions. The serotonin turnover showed rhythms with a peak during ZT18–ZT22 in hippocampus. The noradrenaline turnover showed rhythms with a peak during ZT22–ZT2. There was a difference of approx. 4 h between the serotonergic and noradrenergic systems. This time difference might be one of the factors that affect the action of milnacipran and contribute to the dosing time-dependent behavioral pattern in the FST.

Activation of 5-HT7 receptors influences memory as well as circadian rhythms and other processes. This study investigated the regulation of the 5-HT7 receptors in the hippocampus, a likely substrate for the effects of 5-HT7 receptor compounds on memory. Because endogenous serotonin release is higher during the active phase, and chronic treatment with a serotonin-selective reuptake inhibitor down-regulates 5-HT7 receptors, we hypothesized that 5-HT7 receptors exhibit 24-h variations. We also hypothesized that aging decreases 5-HT7 receptors in the hippocampus, as it does in the dorsal raphe nucleus, a brain site for serotonergic resetting of circadian rhythms. Male hamsters (young, 3-5 mos; old, 17-21 mos) exposed to a light:dark cycle were euthanized at 4 times of day (zeitgeber times [ZT]1, 6, 13, & 19; ZT12=time of lights:off). 5-HT7 receptor autoradiography was conducted on hippocampal sections using [3H]8-OH-DPAT [2nM] as the radioligand and SB-269970 [1μM] to define nonspecific binding. Slide-mounted sections and radioactive standards were apposed to X-ray films; the resultant autoradiograms were assessed by computer-assisted microdensitometry. Specific 5-HT7 receptor binding was robustly expressed in the dentate gyrus (DG) and CA1 but not in the CA2 or CA3. In the CA1 and DG, specific 5-HT7 receptor binding exhibited 24-h rhythms with troughs at night (P

Neurological and neuropsychiatric disorders, including addiction, schizophrenia, and Parkinson's disease (PD), involve dysfunction in midbrain dopamine (DA) neurotransmission with severity of disease symptoms and progression associated with disrupted circadian rhythms. The nuclear transcription factor Nurr1, essential for DA neuron (DAN) development, survival, and maintenance, is also known to interact with circadian rhythm regulating clock proteins. In the Nurr1-null heterozygous (+/-) mice, a Nurr1 deficient model which reproduces some of the alterations in DA function found in schizophrenia and PD, we measured, using wheel-running activity, the free running period (tau) and photoperiod entrainment. Because Nurr1 has a role in regulating the DA phenotype, we also measured the circadian fluctuations in the number of DANs using tyrosine hydroxylase (TH) immunofluorescence. In Nurr1 +/- mice, tau was significantly shorter and entrainment to a 6 h earlier shift in the dark cycle was accelerated. The Nurr1 wild-type (+/+) mice cycled DAN numbers across time, with a significantly greater number (∼2-fold increase) of DANs at zeitgeber time (ZT) 0 than ZT12. The +/- mice, however, did not cycle the DA phenotype, as no differences in DAN numbers were observed between ZT0 and ZT12. Additionally, the +/- mice had significantly fewer DANs at ZT0 but not at ZT12 as compared to +/+ mice. Based these data, circadian rhythms and fluctuations in the DA phenotype requires normal Nurr1 function. A better understanding is needed of the mechanisms regulating the DA phenotype and subsequent neurotransmission across the circadian cycle and how this is altered in circadian rhythm and DA neurotransmission-associated disorders.

Background Skipping breakfast is associated with dysmenorrhea in young women. This suggests that the delay of food intake in the active phase impairs uterine functions by interfering with circadian rhythms. Objectives To examine the relation between the delay of feeding and uterine circadian rhythms, we investigated the effects of the first meal occasion in the active phase on the uterine clock. Methods Zeitgeber time (ZT) was defined as ZT0 (08:45) with lights on and ZT12 (20:45) with lights off. Young female mice (8 wk of age) were divided into 3 groups: group I (ad libitum consumption), group II (time-restricted feeding during ZT12–16, initial 4 h of the active period), and group III (time-restricted feeding during ZT20–24, last 4 h of the active period, a breakfast-skipping model). After 2 wk of dietary restriction, mice in each group were killed at 4-h intervals and the expression profiles of uterine clock genes, Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1), Per1 (period circadian clock 1), Per2, and Cry1 (cryptochrome 1), were examined. Results qPCR and western blot analyses demonstrated synchronized circadian clock gene expression within the uterus. Immunohistochemical analysis confirmed that BMAL1 protein expression was synchronized among the endometrium and myometrium. In groups I and II, mRNA expression of Bmal1 was elevated after ZT12 at the start of the active phase. In contrast, Bmal1 expression was elevated just after ZT20 in group III, showing that the uterine clock rhythm had shifted 8 h backward. The changes in BMAL1 protein expression were confirmed by western blot analysis. Conclusions This study is the first to indicate that time-restricted feeding regulates a circadian rhythm of the uterine clock that is synchronized throughout the uterine body. These findings suggest that the uterine clock system is a new candidate to explain the etiology of breakfast skipping–induced uterine dysfunction., This study indicates that time-restricted feeding regulates circadian clock of the murine uterus, suggesting the involvement of a uterine clock system in the relation between inadequate diets and uterine dysfunction.

Molecular oscillators exist in peripheral tissues such as in skeletal muscles and diet is a dominant factor to affect Zeitgeber for peripheral clocks. MicroRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of starvation effects on miRNA rhythmic expression remains limited in teleost. In this study, the circadian expression pattern of miRNAs was investigated in goldfish muscle upon restricting feeding treatment. The data showed that 15 miRNAs exhibited a daily rhythmicity among the 70 miRNAs assayed in muscles of normally fed goldfish. While after 7-day and 15-day fasting treatment, 23 and 18 miRNAs showed circadian rhythmicity in goldfish muscles respectively. Only 4 miRNAs (miR-23a, miR-29, miR-199a-3p and miR-455) exhibited a daily rhythmicity in all three groups of different nutrient treatments. Correlation analysis of the circadian-miRNAs indicates different feeding stimulation could alter the circadian-miRNA components and their expression profile in skeletal muscle upon short-term feed deprivation. These miRNAs may play important roles in regulating circadian expression of genes involved in muscle cell differentiation and growth by nutritional alteration, thus having a potential application in fish aquaculture.

Objective: The aim of this study is to investigate the effects of light and administration time of isoflurane on circadian gene expression in the brains and liver tissues of rats kept in light-dark cycle. Methods: Seventy tw o 15-days-old rats pups were divided into four groups. All animals were exposed to 1.5% concentration of isoflurane or to 6 L min(-1) O-2 for six hours between Zeitgeber Time (ZT) 0-ZT06 ( day-time administration) or ZT12-ZT18 ( night-time administration). Rats were sacrificed after six hours of anaesthesia with four-hour time intervals. Total RNA was isolated from brains and liver tissues. Circadian gene expression was examined using quantitative real-time Reverse transcription polymerase chain reaction (RT-PCR). Results: BMAL1, CLOCK, PER2 and CRY2 gene expression levels were markedly suppressed after day-time anaesthesia in the both brain and liver, but night-time administration caused only temporary suppression of gene expression. Conclusion: The effect of isoflurane on the circadian clock is time-dependent, and administered isoflurane anaesthesia at night had minimal effect on clock gene expression. Additionally, when the treated animals were kept in a regular light-dark cycle, isoflurane-induced phase shift was not observed, possibly because of the light., NA