Alternating mealtimes during pregnancy and weaning triggers behavioral changes in adult offspring.

In brief: Mealtime changes in pregnant mice revealed impaired neurobehavioral development in mouse offspring. This study is the basis for investigating diseases associated with neurobehavioral development of adult offspring of pregnant shift-working women. Abstract: Most organisms on Earth have a biological clock, and their physiological processes are regulated by a 1-day cycle. In modern society, several factors can disturb these biological clocks in humans; in particular, individuals working in shifts are exposed to stark environmental changes that interfere with their biological clock. They have a high risk of various diseases. However, there are scarce experimental approaches to address the reproductive and health consequences of shift work in the offspring of exposed individuals. In this study, considering the fact that shift workers usually have their meals during their adjusted working time, we aimed to examine the effects of a 12-h shift with usual mealtime as a plausible night work model on the neurobehavioral development of adult mouse offspring. In these offspring, early exposure to this mealtime shift differentially affected circadian rhythmic variables and total locomotor activity depending on the timing and duration of restrictive feeding. Moreover, neurobehavioral alterations such as declined short-term memory and depressive-like behavior were observed in adulthood. These results have implications for the health concerns of shift-working women and their children.

Daily injection of melatonin inhibits insulin resistance induced by chronic mealtime shift.

Shift work disorders have become an emerging concern worldwide. Shift disorders encompass a wide range of illnesses that have yet to be identified. The study focused on the relationship between shift work disorders and insulin resistance. Previously, it was reported that advancing the usual mealtime of mice triggered insulin resistance. Here, the hypothesis that chronic mealtime shifts induce oxidative damage leading to chronic diseases such as type 2 diabetes was tested. It was found that mealtime shift causes imbalances between anti-oxidative capacity and reactive oxygen species (ROS) levels, indicating increased oxidative damage during the light/rest phase. This study further demonstrated that daily supplementation of antioxidants at the appropriate time of day inhibited insulin resistance caused by chronic mealtime shifts, suggesting significant and chronic health implications for shift workers. In conclusion, it was confirmed that increased ROS levels caused by mealtime shift induce insulin resistance, which is inhibited by the antioxidant melatonin.

Mealtime shift delays conception in mice.

The physiological processes of organisms in this rotating planet can adjust according to the time of day via built-in circadian clocks. However, more people are having different shift works, which can increase the risk of pathological conditions including altered reproductive function. Thus, circadian rhythm disturbance has become prevalent in the modern society. Specifically, epidemiological evidence has shown that shift-working women are at high risk of spontaneous abortions, irregular menstrual cycles, and low-birth-weight babies. The current study aimed to investigate the effects of circadian rhythm disturbances on the reproductive function of mice caused by dietary time shift, which is common among night-shift workers. According to the schedule of restricted feeding, the mice were classified into the free feeding, daytime feeding, and night feeding groups. The fertility indices of each group were then evaluated. Activity monitoring was performed to determine whether pregnancy delay might be attributed to mealtime shift. Moreover, the estrous cycle of female mice and the reproductive phenotype of male mice were investigated. Results showed that a 12-h mealtime shift significantly delayed successful conception, which could be attributed to a disrupted estrous cycle, in adult female mice.

Disrupted-in-schizophrenia 1 enhances the quality of circadian rhythm by stabilizing BMAL1.

Disrupted-in-schizophrenia 1 (DISC1) is a scaffold protein that has been implicated in multiple mental disorders. DISC1 is known to regulate neuronal proliferation, signaling, and intracellular calcium homeostasis, as well as neurodevelopment. Although DISC1 was linked to sleep-associated behaviors, whether DISC1 functions in the circadian rhythm has not been determined yet. In this work, we revealed that Disc1 expression exhibits daily oscillating pattern and is regulated by binding of circadian locomotor output cycles kaput (CLOCK) and Brain and muscle Arnt-like protein-1 (BMAL1) heterodimer to E-box sequences in its promoter. Interestingly, Disc1 deficiency increases the ubiquitination of BMAL1 and de-stabilizes it, thereby reducing its protein levels. DISC1 inhibits the activity of GSK3ß, which promotes BMAL1 ubiquitination, suggesting that DISC1 regulates BMAL1 stability by inhibiting its ubiquitination. Moreover, Disc1-deficient cells and mice show reduced expression of other circadian genes. Finally, Disc1-LI (Disc1 knockout) mice exhibit damped circadian physiology and behaviors. Collectively, these findings demonstrate that the oscillation of DISC1 expression is under the control of CLOCK and BMAL1, and that DISC1 contributes to the core circadian system by regulating BMAL1 stability.

Treadmill Exercise Alleviates Circadian Rhythm Disruption-Induced Memory Deficits by Activation of Glucocorticoid Receptor and Brain-Derived Neurotrophic Factor-Dependent Pathway.

PURPOSE: Circadian rhythm affects learning process, memory consolidation, and long-term memory. In this study, the alleviating effect of exercise on circadian rhythm disruption-induced memory deficits was investigated. METHODS: BMAL1 knockdown transgenic mice (BMAL1 TG) were used as the BMAL1-TG group and the BMAL1-TG with treadmill exercise group. Female C57BL/6J mice of the same age were used as the wildtype group and the wildtype with treadmill exercise group. The mice in the treadmill exercise groups performed running on a motorized treadmill under the dark-dark conditions for 8 weeks. Short-term memory, nonspatial object memory, and spatial learning memory were determined using stepdown avoidance test, novel object-recognition test, and radial 8-arm maze test. Immunohistochemistry for doublecortin and 5-bromo-2'-deoxyuridine was conducted for the determination of hippocampal neurogenesis. Using the western blot analysis, we determined the expressions of glucocorticoid receptor (GR) and factors related to the neurogenesis and memory consolidation, such as brain-derived neurotrophic factor, tyrosine kinase B, p44/42 mitogen-activated protein kinase, cyclic AMP-responsive element binding protein, phosphatidylinositol 3-kinase, protein kinas B, protein kinase C alpha, early-growth-response gene 1. RESULTS: Circadian rhythm disruption impaired memory function through inhibiting the expressions of GR and the factors related to neurogenesis and memory consolidation. Treadmill exercise improved memory function via enhancing the expressions of GR and above-mentioned factors. CONCLUSION: Treadmill exercise acts as the zeitgeber that improves memory function under the circadian rhythm disrupted conditions.

Peroxiredoxin 6 Inhibits Osteogenic Differentiation and Bone Formation Through Human Dental Pulp Stem Cells and Induces Delayed Bone Development.

Aims: Peroxiredoxins (PRDXs) are thiol-specific antioxidant enzymes that regulate redox balance that are critical for maintaining the cellular potential for self-renewal and stemness. Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. However, to obtain functional cells for use in clinical applications, stem cell technology still requires improvements. Results: In this study, we found that PRDX6 levels were decreased during osteogenic differentiation in human dental pulp stem cells (hDPSCs). hDPSCs stably expressing Myc-PRDX6 (hDPSC/myc-PRDX6) inhibited cell growth in hDPSCs during osteogenic differentiation and impaired osteogenic phenotypes such as alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenic marker genes [ALP and osteocalcin (OCN)]. hDPSC cell lines stably expressing mutant glutathione peroxidase (PRDX6(C47S)) and independent phospholipase A2 (PRDX6(S32A)) were also generated. Each mutant form of PRDX6 abolished the impaired osteogenic phenotypes, the transforming growth factor-ß-mediated Smad2 and p38 pathways, and RUNX2 expression. Furthermore, in vivo experiments revealed that hDPSC/myc-PRDX6 suppressed hDPSC-based bone regeneration in calvarial defect mice, and newborn PRDX6 transgenic mice exhibited delayed bone development and reduced RUNX2 expression. Innovation and Conclusion: These findings illuminate the effects of PRDX6 during osteogenic differentiation of hDPSCs, and also suggest that regulating PRDX6 may improve the clinical utility of stem cell-based regenerative medicine for the treatment of bone diseases. Antioxid. Redox Signal. 30, 1969-1982.

Astrometrically registered maps of H2O and SiO masers toward VX Sagittarii.

The supergiant VX Sagittarii is a strong emitter of both H2O and SiO masers. However, previous VLBI observations have been performed separately, which makes it difficult to spatially trace the outward transfer of the material consecutively. Here we present the astrometrically registered, simultaneous maps of 22.2 GHz H2O and 43.1/42.8/86.2/129.3 GHz SiO masers toward VX Sagittarii. The H2O masers detected above the dust-forming layers have an asymmetric distribution. The multi-transition SiO masers are nearly circular ring, suggesting spherically symmetric wind within a few stellar radii. These results provide the clear evidence that the asymmetry in the outflow is enhanced after the smaller molecular gas clump transform into the inhomogeneous dust layers. The 129.3 GHz maser arises from the outermost region compared to that of 43.1/42.8/86.2 GHz SiO masers. The ring size of the 129.3 GHz maser is maximized around the optical maximum, suggesting that radiative pumping is dominant.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2.

There has been a growing interest in circadian regulation of the expression and function of drug transporters. In this study, we investigated circadian rhythm in the expression and function of multidrug resistance-associated protein 2 (Mrp2) in mouse liver and involvement of circadian clock in their regulations by using the circadian clock genes (period 1 and period 2) knockout mice. The mRNA and protein expression of Mrp2, P-glycoprotein, and breast cancer resistance protein was measured in the mouse liver at different times of the day. Circadian variation of hepatobiliary excretion of phenolsulfonphthalein, a model substrate of Mrp2, was also investigated in mice. Circadian oscillation of Mrp2 protein expression was clearly observed in the mouse liver with levels down at the light phase and up at the dark phase. The cumulative biliary excretion and biliary clearance of phenolsulfonphthalein from the liver to the bile was 2.37- and 1.74-fold greater in mice administered during the dark phase than in those administered during the light phase, respectively. The circadian oscillation in mRNA expression of Mrp2 disappeared in period 1 and period 2 double knockout mice. These results suggest that the expression and function of Mrp2 show the circadian rhythm, controlled by circadian clock genes.

HL271, a novel chemical compound derived from metformin, differs from metformin in its effects on the circadian clock and metabolism.

Metformin is a treatment of choice for patients with type 2 diabetes. Its action involves the phosphorylation of 5'-adenosine monophosphate activated protein kinase (AMPK), leading to inhibition of liver gluconeogenesis. The effects of a novel chemical compound derived from metformin, HL271, on molecular and physiological actions involving AMPK and rhythmically-expressed circadian clock genes were investigated. HL271 potently activated AMPK in a dose-dependent manner, and produced shortening of the circadian period and enhanced degradation of the clock genes PER2 and CRY1. Although the molecular effects of HL271 resembled those of metformin, it produced different physiological effects in mice with diet-induced obesity. HL271 did not elicit glucose-lowering or insulin-sensitizing effects, possibly because of altered regulation of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1. This indicated that, although HL271 acted on circadian clock machinery through a similar molecular mechanism to metformin, it differed in its systemic effect on glucose and lipid metabolite regulations.

A Novel Bmal1 Mutant Mouse Reveals Essential Roles of the C-Terminal Domain on Circadian Rhythms.

The mammalian circadian clock is an endogenous biological timer comprised of transcriptional/translational feedback loops of clock genes. Bmal1 encodes an indispensable transcription factor for the generation of circadian rhythms. Here, we report a new circadian mutant mouse from gene-trapped embryonic stem cells harboring a C-terminus truncated Bmal1 (Bmal1GTΔC) allele. The homozygous mutant (Bmal1GTΔC/GTΔC) mice immediately lost circadian behavioral rhythms under constant darkness. The heterozygous (Bmal1+/GTΔC) mice displayed a gradual loss of rhythms, in contrast to Bmal1+/- mice where rhythms were sustained. Bmal1GTΔC/GTΔC mice also showed arrhythmic mRNA and protein expression in the SCN and liver. Lack of circadian reporter oscillation was also observed in cultured fibroblast cells, indicating that the arrhythmicity of Bmal1GTΔC/GTΔC mice resulted from impaired molecular clock machinery. Expression of clock genes exhibited distinct responses to the mutant allele in Bmal1+/GTΔC and Bmal1GTΔC/GTΔC mice. Despite normal cellular localization and heterodimerization with CLOCK, overexpressed BMAL1GTΔC was unable to activate transcription of Per1 promoter and BMAL1-dependent CLOCK degradation. These results indicate that the C-terminal region of Bmal1 has pivotal roles in the regulation of circadian rhythms and the Bmal1GTΔC mice constitute a novel model system to evaluate circadian functional mechanism of BMAL1.

Aß-induced degradation of BMAL1 and CBP leads to circadian rhythm disruption in Alzheimer's disease.

BACKGROUND: Patients with Alzheimer's disease (AD) frequently experience disruption of their circadian rhythms, but whether and how circadian clock molecules are perturbed by AD remains unknown. AD is an age-related neurological disorder and amyloid-ß (Aß) is one of major causative molecules in the pathogenesis of AD. RESULTS: In this study, we investigated the role of Aß in the regulation of clock molecules and circadian rhythm using an AD mouse model. These mice exhibited altered circadian behavior, and altered expression patterns of the circadian clock genes, Bmal1 and Per2. Using cultured cells, we showed that Aß induces post-translational degradation of the circadian clock regulator CBP, as well as the transcription factor BMAL1, which forms a complex with the master circadian transcription factor CLOCK. Aß-induced degradation of BMAL1 and CBP correlated with the reduced binding of transcription factors to the Per2 promoter, which in turn resulted in disruptions to PER2 protein expression and the oscillation of Per2 mRNA levels. CONCLUSIONS: Our results elucidate the underlying mechanisms for disrupted circadian rhythm in AD.

Presence of multiple peripheral circadian oscillators in the tissues controlling voiding function in mice.

Circadian clocks are the endogenous oscillators that harmonize a variety of physiological processes within the body. Although many urinary functions exhibit clear daily or circadian variation in diurnal humans and nocturnal rodents, the precise mechanisms of these variations are as yet unclear. In the present study, we demonstrate that Per2 promoter activity clearly oscillates in neonate and adult bladders cultured ex vivo from Per2::Luc knock-in mice. In subsequent experiments, we show that multiple local oscillators are operating in all the bladder tissues (detrusor, sphincter and urothelim) and the lumbar spinal cord (L4-5) but not in the pontine micturition center or the ventrolateral periaqueductal gray of the brain. Accordingly, the water intake and urine volume exhibited daily and circadian variations in young adult wild-type mice but not in Per1(-/-)Per2(-/-) mice, suggesting a functional clock-dependent nature of the micturition rhythm. Particularly in PDK mice, the water intake and urinary excretion displayed an arrhythmic pattern under constant darkness, and the amount of water consumed and excreted significantly increased compared with those of WT mice. These results suggest that local circadian clocks reside in three types of bladder tissue and the lumbar spinal cord and may have important roles in the circadian control of micturition function.

Phosphorylation of LSD1 by PKCα is crucial for circadian rhythmicity and phase resetting.

The circadian clock is a self-sustaining oscillator that controls daily rhythms. For the proper circadian gene expression, dynamic changes in chromatin structure are important. Although chromatin modifiers have been shown to play a role in circadian gene expression, the in vivo role of circadian signal-modulated chromatin modifiers at an organism level remains to be elucidated. Here, we provide evidence that the lysine-specific demethylase 1 (LSD1) is phosphorylated by protein kinase Cα (PKCα) in a circadian manner and the phosphorylated LSD1 forms a complex with CLOCK:BMAL1 to facilitate E-box-mediated transcriptional activation. Knockin mice bearing phosphorylation-defective Lsd1(SA/SA) alleles exhibited altered circadian rhythms in locomotor behavior with attenuation of rhythmic expression of core clock genes and impaired phase resetting of circadian clock. These data demonstrate that LSD1 is a key component of the molecular circadian oscillator, which plays a pivotal role in rhythmicity and phase resetting of the circadian clock.

Modulation of glucocorticoid receptor induction properties by core circadian clock proteins.

Glucocorticoid (GC) plays important roles in diverse physiological processes including metabolism and immune functions. While circadian control of GC synthesis and secretion is relatively well appreciated, circadian control of GC action within target tissues remains poorly understood. Here, we demonstrate that CLOCK/BMAL1, the core circadian clock components, reduces maximal GR transactivation (A(max)) as well as efficacy (EC50) by a novel mechanism that requires binding to DNA and transactivation of target genes. Accordingly, we observe that PER1 and CRY1, the primary targets of CLOCK/BMAL1 action, reduce maximal GR transactivation while not affecting the efficacy. Moreover, we observe hyper-activations of GRE-dependent transcription in BMAL1- or PERs-deficient MEFs. In addition, endogenous GC target genes expression negatively correlates with the CLOCK/BMAL1 activity. Considering that GC sensitivity is widely implicated in human health and diseases, these results provide valuable insights into plethora of GC-related physiology and pathology.

Do rotational shifts affect micturition patterns in real practice? A pilot study in healthy, young female nurses.

PURPOSE: Healthy, young individuals are known to exhibit circadian variation in urinary functions. However, the effects of chronic circadian disturbance on voiding functions are largely unknown. The present work compared the effects of rotational shifts on the micturition patterns of female nurses to that in female nurses with routine daytime shifts. METHODS: A total of 19 nurses without lower urinary tract symptoms who worked rotational shifts for an average duration of 2 years were recruited. A voiding diary was kept for 9 consecutive days, and the overactive bladder symptom score (OABSS) questionnaire was completed three times, starting 3 days before their night duties until 3 days after completion of their night duties. For comparison, seven nurses with regular shifts completed a 3-day voiding diary and the OABSS questionnaire. RESULTS: Female nurses working rotational shifts had lower overall urine production and had decreased urination frequency and nocturia than female nurses working regular shifts, even when the nurses who worked rotational shifts had a regular night's sleep for at least 7 days. Upon reinitiation of night duty, overall urine production increased significantly, with no significant changes in urgency and frequency. When these nurses returned to daytime duty, the volume of urine decreased but nocturnal urine production remained high, and the incidence of nocturia also increased significantly. However, the effects on OABSS score were not significant under the study design used. CONCLUSIONS: Long-term rotational shifts resulted in adaptive changes such as decreased urine production and frequency in healthy, young female nurses. In addition, their micturition patterns were significantly affected by abrupt changes in their work schedules. Although working in shifts did not increase urgency or frequency of urination in healthy, young female nurses working rotational shifts for an average 2 years, large-scale studies are needed to systematically analyze the influence of shift work timings on micturition in humans.

Cyclin-dependent kinase 5 (Cdk5) regulates the function of CLOCK protein by direct phosphorylation.

Circadian rhythm is a biological rhythm governing physiology and behavior with a period of ∼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

Glucocorticoid-mediated Period2 induction delays the phase of circadian rhythm.

Glucocorticoid (GC) signaling synchronizes the circadian rhythm of individual peripheral cells and induces the expression of circadian genes, including Period1 (Per1) and Period2 (Per2). However, no GC response element (GRE) has been reported in the Per2 promoter region. Here we report the molecular mechanisms of Per2 induction by GC signaling and its relevance to the regulation of circadian timing. We found that GC prominently induced Per2 expression and delayed the circadian phase. The overlapping GRE and E-box (GE2) region in the proximal Per2 promoter was responsible for GC-mediated Per2 induction. The GRE in the Per2 promoter was unique in that brain and muscle ARNT-like protein-1 (BMAL1) was essential for GC-induced Per2 expression, whereas other GRE-containing promoters, such as Per1 and mouse mammary tumor virus, responded to dexamethasone in the absence of BMAL1. This specialized regulatory mechanism was mediated by BMAL1-dependent binding of the GC receptor to GRE in Per2 promoter. When Per2 induction was abrogated by the mutation of the GRE or E-box, the circadian oscillation phase failed to be delayed compared with that of the wild-type. Therefore, the current study demonstrates that the rapid Per2 induction mediated by GC is crucial for delaying the circadian rhythm.

Role of type II protein arginine methyltransferase 5 in the regulation of Circadian Per1 gene.

Circadian clocks are the endogenous oscillators that regulate rhythmic physiological and behavioral changes to correspond to daily light-dark cycles. Molecular dissections have revealed that transcriptional feedback loops of the circadian clock genes drive the molecular oscillation, in which PER/CRY complexes inhibit the transcriptional activity of the CLOCK/BMAL1 heterodimer to constitute a negative feedback loop. In this study, we identified the type II protein arginine methyltransferase 5 (PRMT5) as an interacting molecule of CRY1. Although the Prmt5 gene was constitutively expressed, increased interaction of PRMT5 with CRY1 was observed when the Per1 gene was repressed both in synchronized mouse liver and NIH3T3 cells. Moreover, rhythmic recruitment of PRMT5 and CRY1 to the Per1 gene promoter was found to be associated with an increased level of histone H4R3 dimethylation and Per1 gene repression. Consistently, decreased histone H4R3 dimethylation and altered rhythmic Per1 gene expression were observed in Prmt5-depleted cells. Taken together, these findings provide an insight into the link between histone arginine methylation by PRMT5 and transcriptional regulation of the circadian Per1 gene.

Meal time shift disturbs circadian rhythmicity along with metabolic and behavioral alterations in mice.

In modern society, growing numbers of people are engaged in various forms of shift works or trans-meridian travels. Such circadian misalignment is known to disturb endogenous diurnal rhythms, which may lead to harmful physiological consequences including metabolic syndrome, obesity, cancer, cardiovascular disorders, and gastric disorders as well as other physical and mental disorders. However, the precise mechanism(s) underlying these changes are yet unclear. The present work, therefore examined the effects of 6 h advance or delay of usual meal time on diurnal rhythmicities in home cage activity (HCA), body temperature (BT), blood metabolic markers, glucose homeostasis, and expression of genes that are involved in cholesterol homeostasis by feeding young adult male mice in a time-restrictive manner. Delay of meal time caused locomotive hyperactivity in a significant portion (42%) of subjects, while 6 h advance caused a torpor-like symptom during the late scotophase. Accordingly, daily rhythms of blood glucose and triglyceride were differentially affected by time-restrictive feeding regimen with concurrent metabolic alterations. Along with these physiological changes, time-restrictive feeding also influenced the circadian expression patterns of low density lipoprotein receptor (LDLR) as well as most LDLR regulatory factors. Strikingly, chronic advance of meal time induced insulin resistance, while chronic delay significantly elevated blood glucose levels. Taken together, our findings indicate that persistent shifts in usual meal time impact the diurnal rhythms of carbohydrate and lipid metabolisms in addition to HCA and BT, thereby posing critical implications for the health and diseases of shift workers.

Chronic circadian disturbance by a shortened light-dark cycle increases mortality.

Chronic circadian disturbance, a condition of desynchronization between endogenous clock and environmental light-dark (LD) cycle, is known to cause adverse physiological changes including mortality. However, it is yet unclear whether these consequences result from disturbance of endogenous clock or condition of the LD cycle per se. To address this issue, we imposed 3 different periods of LD cycle (T) on wild type and functional clock-defective (Per1(-/-)Per2(-/-)) mice. We found that the disturbed rhythms of locomotor activity and body temperature resulted from interaction of endogenous clock and T cycle and the chronic state of the disturbance suppressed the endogenous circadian rhythm. Interestingly, the endogenous clock and the T cycles affected body weight and food intake independently, while their interaction affected the life span resulting increased mortality of wild type mice in a shortened T cycle. These results strongly indicate the presence of both separate and combined effects of the endogenous clock and T cycle on different physiological variables implying that shift work scheduling can be an important influence on health parameters.