Circadian disruption reduces MUC4 expression via the clock molecule BMAL1 during dry eye development.

Circadian disruption, as a result of shiftwork, jet lag, and other lifestyle factors, is a common public health problem associated with a wide range of diseases, such as metabolic disorders, neurodegenerative diseases, and cancer. In the present study, we established a chronic jet lag model using a time shift method every 3 days and assessed the effects of circadian disruption on ocular surface homeostasis. Our results indicated that jet lag increased corneal epithelial defects, cell apoptosis, and proinflammatory cytokine expression. However, the volume of tear secretion and the number of conjunctival goblet cells did not significantly change after 30 days of jet lag. Moreover, further analysis of the pathogenic mechanism using RNA sequencing revealed that jet lag caused corneal transmembrane mucin deficiency, specifically MUC4 deficiency. The crucial role of MUC4 in pathogenic progression was demonstrated by the protection of corneal epithelial cells and the inhibition of inflammatory activation following MUC4 replenishment. Unexpectedly, genetic ablation of BMAL1 in mice caused MUC4 deficiency and dry eye disease. The underlying mechanism was revealed in cultured human corneal epithelial cells in vitro, where BMAL1 silencing reduced MUC4 expression, and BMAL1 overexpression increased MUC4 expression. Furthermore, melatonin, a circadian rhythm restorer, had a therapeutic effect on jet lag-induced dry eye by restoring the expression of BMAL1, which upregulated MUC4. Thus, we generated a novel dry eye mouse model induced by circadian disruption, elucidated the underlying mechanism, and identified a potential clinical treatment.

Social jet lag impairs exercise volume and attenuates physiological and metabolic adaptations to voluntary exercise training.

Social jet lag (SJL) is a misalignment between sleep and wake times on workdays and free days. SJL leads to chronic circadian rhythm disruption and may affect nearly 70% of the general population, leading to increased risk for cardiometabolic diseases. This study investigated the effects of SJL on metabolic health, exercise performance, and exercise-induced skeletal muscle adaptations in mice. Ten-week-old C57BL/6J mice (n = 40) were allocated to four groups: control sedentary (CON-SED), control exercise (CON-EX), social jet lag sedentary (SJL-SED), and social jet lag exercise (SJL-EX). CON mice were housed under a 12:12-h light-dark cycle. SJL was simulated by implementing a 4-h phase delay for 3 days to simulate "weekends," followed by a 4-h phase advance back to "weekdays," for 6 wk. EX mice had free access to a running wheel. Graded exercise tests (GXTs) and glucose tolerance tests (GTTs) were performed at baseline and after intervention to monitor the effects of exercise and social jet lag on cardiorespiratory and metabolic health, respectively. SJL led to alterations in activity and running patterns and clock gene expression in skeletal muscle and decreased average running distance (P < 0.05). SJL-SED mice gained significantly more weight compared with CON-SED and SJL-EX mice (P < 0.01). SJL impaired fasting blood glucose and glucose tolerance compared with CON mice (P < 0.05), which was partially restored by exercise in SJL-EX mice. SJL also blunted improvements in exercise performance and mitochondrial content in the quadriceps. These data suggest that SJL blunted some cardiometabolic adaptations to exercise and that proper circadian hygiene is necessary for maintaining health and performance.NEW & NOTEWORTHY In mice, disrupting circadian rhythms with social jet lag for 6 wk caused significant weight gain, higher fasting blood glucose, and impaired glucose tolerance compared with control. Voluntary exercise in mice experiencing social jet lag prevented weight gain, though the mice still experienced increased fasting blood glucose and impaired exercise performance compared with trained mice not experiencing social jet lag. Social jet lag seems to be a potent circadian rhythm disruptor that impacts exercise-induced training adaptations.

Chronic jetlag reprograms gene expression in the colonic smooth muscle layer inducing diurnal rhythmicity in the effect of bile acids on colonic contractility.

BACKGROUND: Secondary bile acids entrain peripheral circadian clocks and inhibit colonic motility via the bile acid receptor GPBAR1. We aimed to investigate whether chronodisruption affected the rhythm in serum bile acid levels and whether this was associated with alterations in clock gene and Gpbar1 mRNA expression in the colonic smooth muscle layer. We hypothesized that this in turn may affect the rhythm in the inhibitory effect of secondary bile acids on colonic contractility. METHODS: Mice were exposed to 4 weeks of chronic jetlag induction. The expression of Gpbar1 and clock genes was measured in colonic smooth muscle tissue using RT-qPCR over 24 h (4 h time interval). The effect of secondary bile acids on electrical field-induced neural contractions was measured isometrically in colonic smooth muscle strips. KEY RESULTS: Chronic jetlag abolished the rhythmicity in serum bile acid levels. This was associated with a phase-shift in diurnal clock gene mRNA fluctuations in smooth muscle tissue. Chronic jetlag induced a rhythm in Gpbar1 expression in the colonic smooth muscle layer. In parallel, a rhythm was induced in the inhibitory effect of taurodeoxycholic acid (TDCA), but not deoxycholic acid, on neural colonic contractions that peaked together with Gpbar1 expression. CONCLUSIONS & INFERENCES: Chronodisruption abolished the rhythm in bile acid levels which might contribute to a shift in smooth muscle clock gene expression. Our findings suggest that chronodisruption caused a transcriptional reprogramming in the colonic smooth layer thereby inducing a rhythm in the expression of Gpbar1 and in the inhibitory effect of TDCA on colonic contractility.

The duper mutation reveals previously unsuspected functions of Cryptochrome 1 in circadian entrainment and heart disease.

The Cryptochrome 1 (Cry1)-deficient duper mutant hamster has a short free-running period in constant darkness (τDD) and shows large phase shifts in response to brief light pulses. We tested whether this measure of the lability of the circadian phase is a general characteristic of Cry1-null animals and whether it indicates resistance to jet lag. Upon advance of the light:dark (LD) cycle, both duper hamsters and Cry1-/- mice re-entrained locomotor rhythms three times as fast as wild types. However, accelerated re-entrainment was dissociated from the amplified phase-response curve (PRC): unlike duper hamsters, Cry1-/- mice show no amplification of the phase response to 15' light pulses. Neither the amplified acute shifts nor the increased rate of re-entrainment in duper mutants is due to acceleration of the circadian clock: when mutants drank heavy water to lengthen the period, these aspects of the phenotype persisted. In light of the health consequences of circadian misalignment, we examined effects of duper and phase shifts on a hamster model of heart disease previously shown to be aggravated by repeated phase shifts. The mutation shortened the lifespan of cardiomyopathic hamsters relative to wild types, but this effect was eliminated when mutants experienced 8-h phase shifts every second week, to which they rapidly re-entrained. Our results reveal previously unsuspected roles of Cry1 in phase shifting and longevity in the face of heart disease. The duper mutant offers new opportunities to understand the basis of circadian disruption and jet lag.

Time-Restricted Feeding in Mice Prevents the Disruption of the Peripheral Circadian Clocks and Its Metabolic Impact during Chronic Jetlag.

We used time-restricted feeding (TRF) to investigate whether microbial metabolites and the hunger hormone ghrelin can become the dominant entraining factor during chronic jetlag to prevent disruption of the master and peripheral clocks, in order to promote health. Therefore, hypothalamic clock gene and Agrp/Npy mRNA expression were measured in mice that were either chronically jetlagged and fed ad libitum, jetlagged and fed a TRF diet, or not jetlagged and fed a TRF diet. Fecal short-chain fatty acid (SCFA) concentrations, plasma ghrelin and corticosterone levels, and colonic clock gene mRNA expression were measured. Preventing the disruption of the food intake pattern during chronic jetlag using TRF restored the rhythmicity in hypothalamic clock gene mRNA expression of Reverbα but not of Arntl. TRF countered the changes in plasma ghrelin levels and in hypothalamic Npy mRNA expression induced by chronic jetlag, thereby reestablishing the food intake pattern. Increase in body mass induced by chronic jetlag was prevented. Alterations in diurnal fluctuations in fecal SCFAs during chronic jetlag were prevented thereby re-entraining the rhythmic expression of peripheral clock genes. In conclusion, TRF during chronodisruption re-entrains the rhythms in clock gene expression and signals from the gut that regulate food intake to normalize body homeostasis.

Adverse Effect of Circadian Rhythm Disorder on Reparative Angiogenesis in Hind Limb Ischemia.

Background Circadian rhythm disorders, often seen in modern lifestyles, are a major social health concern. The aim of this study was to examine whether circadian rhythm disorders would influence angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia. Methods and Results A jet-lag model was established in C57BL/6J mice using a light-controlled isolation box. Control mice were kept at a light/dark 12:12 (12-hour light and 12-hour dark) condition. Concentrations of plasma vascular endothelial growth factor and circulating endothelial progenitor cells in control mice formed a circadian rhythm, which was diminished in the jet-lag model (P<0.05). The jet-lag condition deteriorated tissue capillary formation (P<0.001) and tissue blood perfusion recovery (P<0.01) in hind limb ischemia, which was associated with downregulation of vascular endothelial growth factor expression in local ischemic tissue and in the plasma. Although the expression of clock genes (ie, Clock, Bmal1, and Cry) in local tissues was upregulated after ischemic injury, the expression levels of cryptochrome (Cry) 1 and Cry2 were inhibited by the jet-lag condition. Next, Cry1 and Cry2 double-knockout mice were examined for blood perfusion recoveries and a reparative angiogenesis. Cry1 and Cry2 double-knockout mice revealed suppressed capillary density (P<0.001) and suppressed tissue blood perfusion recovery (P<0.05) in the hind limb ischemia model. Moreover, knockdown of CRY1/2 in human umbilical vein endothelial cells was accompanied by increased expression of WEE1 and decreased expression of HOXC5. This was associated with decreased proliferative capacity, migration ability, and tube formation ability of human umbilical vein endothelial cells, respectively, leading to impairment of angiogenesis. Conclusions Our data suggest that circadian rhythm disorder deteriorates reparative ischemia-induced angiogenesis and that maintenance of circadian rhythm plays an important role in angiogenesis.

[Research progress of circadian rhythm].

Circadian rhythm disorder is a common society issue caused by jet lag,shift work,sleep disruption and changes in food consumption. Light is the major factor affecting the circadian rhythm system. Disruption of the circadian rhythm system can cause damage to the body,leading to some diseases. Maintaining a normal circadian system is of great importance for good health. Ideal therapeutic effect can not only alleviate symptoms of the diseases,but also recovery the disturbed circadian rhythm to normal. The paper summarizes the modeling methods of animal circadian rhythm disorder,diseases of circadian rhythm abnormality,regulation of circadian clock genes and medicine which are related to circadian rhythm to diseases of circadian rhythm disorder.

Chronic jetlag-induced alterations in pancreatic diurnal gene expression.

Cell-autonomous circadian clocks exist in nearly every organ and function to maintain homeostasis through a complex series of transcriptional-translational feedback loops. The response of these peripheral clocks to external perturbations, such as chronic jetlag and shift work, has been extensively investigated. However, an evaluation of the effects of chronic jetlag on the mouse pancreatic transcriptome is still lacking. Herein, we report an evaluation of the diurnal variations encountered in the pancreatic transcriptome following exposure to an established chronic jetlag protocol. We found approximately 5.4% of the pancreatic transcriptome was rhythmic. Following chronic jetlag, we found the number of rhythmic transcripts decreased to approximately 3.6% of the transcriptome. Analysis of the core clock genes, which orchestrate circadian physiology, revealed that nearly all exhibited a shift in the timing of peak gene expression-known as a phase shift. Similarly, over 95% of the rhythmically expressed genes in the pancreatic transcriptome exhibited a phase shift, many of which were found to be important for metabolism. Evaluation of the genes involved in pancreatic exocrine secretion and insulin signaling revealed many pancreas-specific genes were also rhythmically expressed and several displayed a concomitant phase shift with chronic jetlag. Phase differences were found 9 days after normalization, indicating a persistent failure to reentrain to the new light-dark cycle. This study is the first to evaluate the endogenous pancreatic clock and rhythmic gene expression in whole pancreas over 48 h, and how the external perturbation of chronic jetlag affects the rhythmic expression of genes in the pancreatic transcriptome.

Research progress of circadian rhythm / 中国中药杂志

Circadian rhythm disorder is a common society issue caused by jet lag,shift work,sleep disruption and changes in food consumption. Light is the major factor affecting the circadian rhythm system. Disruption of the circadian rhythm system can cause damage to the body,leading to some diseases. Maintaining a normal circadian system is of great importance for good health. Ideal therapeutic effect can not only alleviate symptoms of the diseases,but also recovery the disturbed circadian rhythm to normal. The paper summarizes the modeling methods of animal circadian rhythm disorder,diseases of circadian rhythm abnormality,regulation of circadian clock genes and medicine which are related to circadian rhythm to diseases of circadian rhythm disorder.

Circadian regulation of sleep in a pre-clinical model of Dravet syndrome: dynamics of sleep stage and siesta re-entrainment.

STUDY OBJECTIVES: Sleep disturbances are common co-morbidities of epileptic disorders. Dravet syndrome (DS) is an intractable epilepsy accompanied by disturbed sleep. While there is evidence that daily sleep timing is disrupted in DS, the difficulty of chronically recording polysomnographic sleep from patients has left our understanding of the effect of DS on circadian sleep regulation incomplete. We aim to characterize circadian sleep regulation in a mouse model of DS. METHODS: Here we exploit long-term electrocorticographic recordings of sleep in a mouse model of DS in which one copy of the Scn1a gene is deleted. This model both genocopies and phenocopies the disease in humans. We test the hypothesis that the deletion of Scn1a in DS mice is associated with impaired circadian regulation of sleep. RESULTS: We find that DS mice show impairments in circadian sleep regulation, including a fragmented rhythm of non-rapid eye movement (NREM) sleep and an elongated circadian period of sleep. Next, we characterize re-entrainment of sleep stages and siesta following jet lag in the mouse. Strikingly, we find that re-entrainment of sleep following jet lag is normal in DS mice, in contrast to previous demonstrations of slowed re-entrainment of wheel-running activity. Finally, we report that DS mice are more likely to have an absent or altered daily "siesta". CONCLUSIONS: Our findings support the hypothesis that the circadian regulation of sleep is altered in DS and highlight the value of long-term chronic polysomnographic recording in studying the role of the circadian clock on sleep/wake cycles in pre-clinical models of disease.

PER3 gene regulation of sleep-wake behavior as a function of latitude.

OBJECTIVES: We searched for interactions between PER3 gene VNTR polymorphism, latitude, sleep duration, diurnal sleepiness, and social jetlag. DESIGN: We selected samples from 3 distinct cities along the latitudinal range of Brazil and comprising the same time zone. SETTING: Undergraduate universities located in 3 major cities of Brazil. PARTICIPANTS: A total of 980 undergraduate students: 276 from Maceio (latitude 9°), 358 from Campinas (latitude 22°), and 346 from Porto Alegre (latitude 30°). MEASUREMENTS: PER3 variable number of tandem repeats genotyping, diurnal sleepiness, sleep duration (weekdays and weekend), chronotype, and social jetlag. RESULTS: Latitude is associated with a differential expression of circadian and sleep profiles. We observed a shift toward eveningness with increased latitude and increased social jetlag and diurnal sleepiness at latitude 30°. Moreover, our results suggest that the PER3 variable number of tandem repeats polymorphism has a modulatory effect on these circadian and sleep profiles: the variant PER34/4 is associated with a smaller difference in the sleep duration on weekdays among different latitudes and is associated with longer sleep duration on weekends just at latitude 30°, even when compared to both other genotypes at the same latitude. On the other hand, irrespective of the genotype, volunteers from latitude 30° expressed increased social jetlag and diurnal sleepiness. CONCLUSIONS: The seasonal variation in the light/dark cycle, tied to latitude, together with the tight social time constraints that young adults are subjected to during weekdays, generates differences in the sleep phenotypes. Volunteers with the PER34/4 variant who live farther from the equator have a greater increase in their weekend sleep duration.

Circadian rhythms of micturition during jet lag.

Micturition behavior follows regular day/night fluctuations, and unwanted increase in micturition could occur during night in jet lag condition. To clarify the effect of jet lag on micturition behavior, we simultaneously detected circadian micturition patterns and locomotor activity rhythms of mice under experimental jet lag conditions, by applying the improved automated Voided Stain on Paper (aVSOP) method. When wild-type (WT) mice were phase-advanced for 8 hours, day-night variation of micturition was disrupted suddenly, and this irregular daily micturition continued until 8 days, although their activity rhythms entrained gradually day by day until 8 days. We also examined how jet lag induced changes of micturition in Per-null mice lacking Per1, Per2 and Per3 genes, whose endogenous clock is completely disrupted. We found both micturition and locomotor activity of Per-null mice promptly entrained to the new LD cycle. These findings suggest that the irregular micturition during jet lag is caused along with the gradual shift of the endogenous clock, and paradoxically, jet lag-associated abnormality was absent when endogenous circadian oscillations were genetically disrupted.

Preventive effect of L-carnitine on the disorder of lipid metabolism and circadian clock of mice subjected to chronic jet-lag.

Circadian clock plays an essential role in orchestrating daily physiology, and its disruption can evoke metabolic diseases such as obesity. L-Carnitine can reduce blood lipid levels, and ameliorate fatty liver through regulating lipid metabolism. However, whether L-Carnitine administration may affect the disturbance of lipid metabolism and circadian rhythm of mice induced by prolonged circadian disruption is still unknown. Herein, we investigated the effects of L-Carnitine on conditions of circadian clock and lipid metabolism through a chronic jet-lag mice model which was developed by reversing 12 h light/12 h dark cycle every 4 days for a continuous 12 weeks. Results showed that L-Carnitine administration significantly decreased levels of serum glutamic-oxaloacetic transaminase (GOT) and triglycerides (TG), which were remarkably elevated by chronic jet-lag. More importantly, quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that L-Carnitine supplementation would effectively counteract the negative alterations in gene expression which related to lipid metabolism (Srebp1, Acaca, Fasn, and Scd1), metabolic regulator (mTOR) and circadian rhythm (Bmal1, Per1, Cry1 and Dec1) in the liver of mice subjected to the chronic jet-lag. As a conclusion, L-Carnitine was partly effective in preventing the disruption of circadian clock and lipid metabolic disorders induced by the chronic jet-lag.

Uncoupling genotoxic stress responses from circadian control increases susceptibility to mammary carcinogenesis.

We previously demonstrated that chemopreventive methylselenocysteine (MSC) prevents N-Nitroso-N-methylurea (NMU)-induced mammary carcinogenesis in the susceptible Fischer 344 (F344) rats by enhancing NAD+-dependent SIRT1 activity, restoring circadian expression of Period 2 (Per2) and circadian controlled genes. Here, we show that compared to the genetically resistant Copenhagen (COP) rat strain, mammary glands of the F344 rats have a 4-hour phase delay in circadian expression of Per2. Consequently, F344 rats failed to increase SIRT1 activity and circadian expression of Per2 and DDRR genes after exposure to NMU. Exposure of COP rats to NMU had the opposite effect, enhancing SIRT1 activity, increasing circadian expression of Per2 and DDRR genes. Significantly, SIRT1 activity and circadian expression of Per2 and DDRR genes in NMU-treated F344 rats on a chemopreventive regimen of MSC approximated those in NMU-treated COP rats. These results indicated that COP rats have an increased capacity to maintain NAD+-dependent SIRT1 activity under genotoxic stress. This contention was supported by increased stability of the period and phase of circadian locomotor activity in COP vs F344 rats exposed to changing light conditions. The increased sensitivity and rapid response of COP to changing light were correlated with the enhanced circadian response of this strain to carcinogen. Disturbance of circadian rhythm by jet lag also disrupted circadian expression of Per2 and DDRR genes, and accelerated mammary tumorigenesis in rodent models. These results suggested that uncoupling of DDRR responses from circadian control by environmental stresses and endogenous factors increases susceptibility to mammary carcinogenesis, possibly by inducing a promutagenic state.

Rhythmic Oxygen Levels Reset Circadian Clocks through HIF1α.

The mammalian circadian system consists of a master clock in the brain that synchronizes subsidiary oscillators in peripheral tissues. The master clock maintains phase coherence in peripheral cells through systemic cues such as feeding-fasting and temperature cycles. Here, we examined the role of oxygen as a resetting cue for circadian clocks. We continuously measured oxygen levels in living animals and detected daily rhythms in tissue oxygenation. Oxygen cycles, within the physiological range, were sufficient to synchronize cellular clocks in a HIF1α-dependent manner. Furthermore, several clock genes responded to changes in oxygen levels through HIF1α. Finally, we found that a moderate reduction in oxygen levels for a short period accelerates the adaptation of wild-type but not of HIF1α-deficient mice to the new time in a jet lag protocol. We conclude that oxygen, via HIF1α activation, is a resetting cue for circadian clocks and propose oxygen modulation as therapy for jet lag.

Chronotype and stability of spontaneous locomotor activity rhythm in BMAL1-deficient mice.

Behavior, physiological functions and cognitive performance change over the time of the day. These daily rhythms are either externally driven by rhythmic environmental cues such as the light/dark cycle (masking) or controlled by an internal circadian clock, the suprachiasmatic nucleus (SCN), which can be entrained to the light/dark cycle. Within a given species, there is genetically determined variability in the temporal preference for the onset of the active phase, the chronotype. The chronotype is the phase of entrainment between external and internal time and is largely regulated by the circadian clock. Genetic variations in clock genes and environmental influences contribute to the distribution of chronotypes in a given population. However, little is known about the determination of the chronotype, the stability of the locomotor rhythm and the re-synchronization capacity to jet lag in an animal without a functional endogenous clock. Therefore, we analyzed the chronotype of BMAL1-deficient mice (BMAL1-/-) as well as the effects of repeated experimental jet lag on locomotor activity rhythms. Moreover, light-induced period expression in the retina was analyzed to assess the responsiveness of the circadian light input system. In contrast to wild-type mice, BMAL1-/- showed a significantly later chronotype, adapted more rapidly to both phase advance and delay but showed reduced robustness of rhythmic locomotor activity after repeated phase shifts. However, photic induction of Period in the retina was not different between the two genotypes. Our findings suggest that a disturbed clockwork is associated with a late chronotype, reduced rhythm stability and higher vulnerability to repeated external desynchronization.

The effect of L-carnosine on the circadian resetting of clock genes in the heart of rats.

It is reported that the circadian timing system may be included in the mechanism by which L-carnosine (Car) affects multiple physiological alterations including blood glucose, cardiovascular functions etc. However, it is not clear whether Car would affect the circadian rhythm of clock genes in the heart and what is the possible mechanism underlying. To clarify these issues, we compared the effects of Car on the expression of circadian genes in the heart of normal and vagotomized rats under control and jet lag conditions. The normal and vagotomized (va) male Wistar rats were divided into three groups respectively. The control and va-Control groups (fed with regular chow) were sampled before the reversal of LD cycle and feeding schedule (day 0). The normal and va-Normal resetting groups (fed with regular chow) as well as the Car and va-Car resetting groups (fed with Car-containing diet) were sampled on day 3 and day 5 after the experimental jet lag. Car-feeding obviously enhanced the resetting rates of clock genes (Bmal1, Dec1, Cry1) in the heart of normal rats after the experimental jet lag. The unilateral surgical vagotomy didn't alter the diurnal expression patterns and resetting rates of the examined clock genes in normal diet feeding rats. In contrast, it abolished the Car-induced rapid resetting of the clock genes in the heart. Therefore, Car feeding plays a positive role in the circadian resynchronization of the heart clock, which is underlied by the autonomic nervous system.

The making of the master clock.

A genetic basis for the anatomic master circadian clock in mammals has been found.

Lhx1 maintains synchrony among circadian oscillator neurons of the SCN.

The robustness and limited plasticity of the master circadian clock in the suprachiasmatic nucleus (SCN) is attributed to strong intercellular communication among its constituent neurons. However, factors that specify this characteristic feature of the SCN are unknown. Here, we identified Lhx1 as a regulator of SCN coupling. A phase-shifting light pulse causes acute reduction in Lhx1 expression and of its target genes that participate in SCN coupling. Mice lacking Lhx1 in the SCN have intact circadian oscillators, but reduced levels of coupling factors. Consequently, the mice rapidly phase shift under a jet lag paradigm and their behavior rhythms gradually deteriorate under constant condition. Ex vivo recordings of the SCN from these mice showed rapid desynchronization of unit oscillators. Therefore, by regulating expression of genes mediating intercellular communication, Lhx1 imparts synchrony among SCN neurons and ensures consolidated rhythms of activity and rest that is resistant to photic noise.