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BackgroundThe MTNR1B gene encodes a receptor for melatonin, a hormone regulating biorhythms. Disruptions in biorhythms contribute to the development of type 2 diabetes mellitus (T2DM). Genetic studies suggest that variability in the MTNR1B gene affects T2DM development. Our aim was to compare the distribution of the genetic variant rs10830963 between persons differing in glucose tolerance in a sample of the Czech population (N=1206). We also evaluated possible associations of the polymorphism with insulin sensitivity, beta cell function, with the shape of glucose, insulin and C-peptide trajectories measured 7 times during a 3-hour oral glucose tolerance test (OGTT) and with glucagon response. In a subgroup of 268 volunteers we also evaluated sleep patterns and biorhythm.Results13 persons were diagnosed with T2DM, 119 had impaired fasting blood glucose (IFG) and/or impaired glucose tolerance (IGT). 1074 participants showed normal results and formed a control group. A higher frequency of minor allele G was found in the IFG/IGT group in comparison with controls. The GG constellation was present in 23% of diabetics, in 17% of IFG/IGT probands and in 11% of controls. Compared to CC and CG genotypes, GG homozygotes showed higher stimulated glycemia levels during the OGTT. Homozygous as well as heterozygous carriers of the G allele showed lower very early phase of insulin and C-peptide secretion with unchanged insulin sensitivity. These differences remained significant after excluding diabetics and the IFG/IGT group from the analysis. No associations of the genotype with the shape of OGTT-based trajectories, with glucagon or with chronobiological patterns were observed. However, the shape of the trajectories differed significantly between men and women.ConclusionIn a representative sample of the Czech population, the G allele of the rs10830963 polymorphism is associated with impaired early phase of beta cell function, and this is evident even in healthy individuals.

Normal neurodevelopment requires precise expression of the key ubiquitin ligase gene Ube3a. Comparing newly generated mouse models for Ube3a downregulation (models of Angelman syndrome) vs. Ube3a upregulation (models for autism), we find reciprocal effects of Ube3a gene dosage on phenotypes associated with circadian rhythmicity, including the amount of locomotor activity. Consistent with results from neurons in general, we find that Ube3a is imprinted in neurons of the suprachiasmatic nuclei (SCN), the pacemaking circadian brain locus, despite other claims that SCN neurons were somehow exceptional to these imprinting rules. In addition, Ube3a-deficient mice lack the typical drop in wake late in the dark period and have blunted responses to sleep deprivation. Suppression of physical activity by light in Ube3a-deficient mice is not due to anxiety as measured by behavioral tests and stress hormones; quantification of stress hormones may provide a mechanistic link to sleep alteration and memory deficits caused by Ube3a deficiency, and serve as an easily measurable biomarker for evaluating potential therapeutic treatments for Angelman syndrome. We conclude that reduced Ube3a gene dosage affects not only neurodevelopment but also sleep patterns and circadian rhythms.

The McGill-R-Thy1-APP transgenic rat is an animal model of the familial form of Alzheimer's disease (AD). This model mirrors several neuropathological hallmarks of the disease, including the accumulation of beta-amyloid and the formation of amyloid plaques (in homozygous animals only), neuroinflammation and the gradual deterioration of cognitive functions even prior to plaque formation, although it lacks the tauopathy observed in human victims of AD. The goal of the present study was a thorough characterization of the homozygous model with emphasis on its face validity in several domains of behavior known to be affected in AD patients, including cognitive functions, motor coordination, emotionality, sociability, and circadian activity patterns. On the behavioral level, we found normal locomotor activity in spontaneous exploration, but problems with balance and gait coordination, increased anxiety and severely impaired spatial cognition in 4–7 month old homozygous animals. The profile of social behavior and ultrasonic communication was altered in the McGill rats, without a general social withdrawal. McGill rats also exhibited changes in circadian profile, with a shorter free-running period and increased total activity during the subjective night, without signs of sleep disturbances during the inactive phase. Expression of circadian clock gene Bmal1 was found to be increased in the parietal cortex and cerebellum, while Nr1d1 expression was not changed. The clock-controlled gene Prok2 expression was found to be elevated in the parietal cortex and hippocampus, which might have contributed to the observed changes in circadian phenotype. We conclude that the phenotype in the McGill rat model is not restricted to the cognitive domain, but also includes gait problems, changes in emotionality, social behavior, and circadian profiles. Our findings show that the model should be useful for the development of new therapeutic approaches targeting not only memory decline but also other symptoms decreasing the quality of life of AD patients.

Abstract Choroid plexus (ChP), the brain structure primarily responsible for cerebrospinal fluid production, contains a robust circadian clock, whose role remains to be elucidated. The aim of our study was to [1] identify rhythmically controlled cellular processes in the mouse ChP and [2] assess the role and nature of signals derived from the master clock in the suprachiasmatic nuclei (SCN) that control ChP rhythms. To accomplish this goal, we used various mouse models (WT, mPer2 Luc , ChP-specific Bmal1 knockout) and combined multiple experimental approaches, including surgical lesion of the SCN (SCNx), time-resolved transcriptomics, and single cell luminescence microscopy. In ChP of control (Ctrl) mice collected every 4 h over 2 circadian cycles in darkness, we found that the ChP clock regulates many processes, including the cerebrospinal fluid circadian secretome, precisely times endoplasmic reticulum stress response, and controls genes involved in neurodegenerative diseases (Alzheimer’s disease, Huntington’s disease, and frontotemporal dementia). In ChP of SCNx mice, the rhythmicity detected in vivo and ex vivo was severely dampened to a comparable extent as in mice with ChP-specific Bmal1 knockout, and the dampened cellular rhythms were restored by daily injections of dexamethasone in mice. Our data demonstrate that the ChP clock controls tissue-specific gene expression and is strongly dependent on the presence of a functional connection with the SCN. The results may contribute to the search for a novel link between ChP clock disruption and impaired brain health.

The topic of human circadian rhythms is not only attracting the attention of clinical researchers from various fields but also sparking a growing public interest. The circadian system comprises the central clock, located in the suprachiasmatic nucleus of the hypothalamus, and the peripheral clocks in various tissues that are interconnected; together they coordinate many daily activities, including sleep and wakefulness, physical activity, food intake, glucose sensitivity and cardiovascular functions. Disruption of circadian regulation seems to be associated with metabolic disorders (particularly impaired glucose tolerance) and cardiovascular disease. Previous clinical trials revealed that disturbance of the circadian system, specifically due to shift work, is associated with an increased risk of type 2 diabetes mellitus. This review is intended to provide clinicians who wish to implement knowledge of circadian disruption in diagnosis and strategies to avoid cardio-metabolic disease with a general overview of this topic.

Lithium is an effective mood stabilizer, but the mechanism of its therapeutic action is not well understood. We investigated the effect of lithium on the circadian clock located in the ventricle barrier complex containing the choroid plexus (CP), a part of the glymphatic system that influences gross brain function via the production of cerebrospinal fluid. The mPer2Luc mice were injected with lithium chloride (LiCl) or vehicle, and their effects on the clock gene Nr1d1 in CP were detected by RT qPCR. CP organotypic explants were prepared to monitor bioluminescence rhythms in real time and examine the responses of the CP clock to LiCl and inhibitors of glycogen synthase kinase-3 (CHIR-99021) and protein kinase C (chelerythrine). LiCl affected Nr1d1 expression levels in CP in vivo and dose-dependently delayed the phase and prolonged the period of the CP clock in vitro. LiCl and CHIR-99021 had different effects on 1] CP clock parameters (amplitude, period, phase), 2] dexamethasone-induced phase shifts of the CP clock, and 3] dynamics of PER2 degradation and de novo accumulation. LiCl-induced phase delays were significantly reduced by chelerythrine, suggesting the involvement of PKC activity. The effects on the CP clock may be involved in the therapeutic effects of lithium and hypothetically improve brain function in psychiatric patients by aligning the function of the CP clock-related glymphatic system with the sleep-wake cycle. Importantly, our data argue for personalized timing of lithium treatment in BD patients.

The suprachiasmatic nuclei (SCN) of the hypothalamus harbor the central clock of the circadian system, which gradually matures during the perinatal period. In this study, time-resolved transcriptomic and proteomic approaches were used to describe fetal SCN tissue-level rhythms before rhythms in clock gene expression develop. Pregnant rats were maintained in constant darkness and had intact SCN, or their SCN were lesioned and behavioral rhythm was imposed by temporal restriction of food availability. Model-selecting tools dryR and CompareRhythms identified sets of genes in the fetal SCN that were rhythmic in the absence of the fetal canonical clock. Subsets of rhythmically expressed genes were assigned to groups of fetuses from mothers with either intact or lesioned SCN, or both groups. Enrichment analysis for GO terms and signaling pathways revealed that neurodevelopment and cell-to-cell signaling were significantly enriched within the subsets of genes that were rhythmic in response to distinct maternal signals. The findings discovered a previously unexpected breadth of rhythmicity in the fetal SCN at a developmental stage when the canonical clock has not yet developed at the tissue level and thus likely represents responses to rhythmic maternal signals. Circadian gene and protein expression profiling reveals that fetal rat suprachiasmatic nuclei respond rhythmically to distinct maternal signals before rhythmicity develops in the expression of canonical clock genes.

Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

During fetal stage, maternal circadian system sets the phase of the developing clock in the suprachiasmatic nuclei (SCN) via complex pathways. We addressed the issue of how impaired maternal signaling due to a disturbed environmental light/dark (LD) cycle affects the fetal SCN. We exposed pregnant Wistar rats to two different challenges – a 6-h phase shift in the LD cycle on gestational day 14, or exposure to constant light (LL) throughout pregnancy – and detected the impact on gene expression profiles in 19-day-old fetuses. The LD phase shift, which changed the maternal SCN into a transient state, caused robust downregulation of expression profiles of clock genes (Per1, Per2, and Nr1d1), clock-controlled (Dbp) genes, as well as genes involved in sensing various signals, such as c-fos and Nr3c1. Removal of the rhythmic maternal signals via exposure of pregnant rats to LL abolished the rhythms in expression of c-fos and Nr3c1 in the fetal SCN. We identified c-fos as the gene primarily responsible for sensing rhythmic maternal signals because its expression profile tracked the shifted or arrhythmic maternal SCN clock. Pathways related to the maternal rhythmic behavioral state were likely not involved in driving the c-fos expression rhythm. Instead, introduction of a behavioral rhythm to LL-exposed mothers via restricted feeding regime strengthened rhythm in Vip expression in the fetal SCN. Our results revealed for the first time that the fetal SCN is highly sensitive in a gene-specific manner to various changes in maternal signaling due to disturbances of environmental cycles related to the modern lifestyle in humans.

Light entrains the master circadian clock in the suprachiasmatic nucleus (SCN) predominantly through glutamatergic signaling via NMDA receptors. The magnitude and the direction of resulting phase shifts depend on timing of the photic stimulus. Previous reports based on behavioral and electrophysiological data suggested that endocannabinoids (EC) might reduce the ability of the SCN clock to respond to light. However, there is little direct evidence for the involvement of EC in entrainment of the rhythmic clock gene expression in the SCN. We have used luminescence recording of cultured SCN slices from mPer2Luc mice to construct a complete phase response curve (PRC) for NMDA receptor activation. The results demonstrated that NMDA administration phase-shifts the PER2 rhythm in a time-specific manner. A stable “singularity,” in the course of which the clock seemingly stops while the overall phase is caught between delays and advances, can occur in response to NMDA at a narrow interval during the PER2 level decrease. NMDA-induced phase delays were affected neither by the agonist (WIN 55,212-2 mesylate) nor by the antagonist (rimonabant hydrochloride) of EC receptors. However, the agonist significantly reduced the NMDA-induced phase advance of the clock, while the antagonist enhanced the phase advance, causing a shift in the sensitivity window of the SCN to NMDA. The modulation of EC signaling in the SCN had no effect by itself on the phase of the PER2 rhythm. The results provide evidence for a modulatory role of EC in photic entrainment of the circadian clock in the SCN.

Alzheimer's disease (AD) is a neurodegenerative disease often accompanied with disruption of sleep-wake cycle. The sleep-wake cycle is controlled by mechanisms involving internal timekeeping (circadian) regulation. The aim of our present pilot study was to assess the circadian system in patients with mild form of AD in their home environment. In the study, 13 elderly AD patients and 13 age-matched healthy control subjects (the patient's spouses) were enrolled. Sleep was recorded for 21 days by sleep diaries in all participants and checked by actigraphy in 4 of the AD patient/control couples. The samples of saliva and buccal mucosa were collected every 4 hours during the same 24 h-interval to detect melatonin and clock gene (PER1 and BMAL1) mRNA levels, respectively. The AD patients exhibited significantly longer inactivity interval during the 24 h and significantly higher number of daytime naps than controls. Daily profiles of melatonin levels exhibited circadian rhythms in both groups. Compared with controls, decline in amplitude of the melatonin rhythm in AD patients was not significant, however, in AD patients more melatonin profiles were dampened or had atypical waveforms. The clock genes PER1 and BMAL1 were expressed rhythmically with high amplitudes in both groups and no significant differences in phases between both groups were detected. Our results suggest moderate differences in functional state of the circadian system in patients with mild form of AD compared with healthy controls which are present in conditions of their home dwelling.

The mammalian suprachiasmatic nuclei (SCN) and their intrinsic rhythmicity develop gradually during ontogenesis. In the rat, the SCN forms between embryonic day (E) 14 and E17, with gestation terminating at E21-22. Overt SCN rhythmicity is already present in the late embryonic stage. The aim of the present study was to determine when the fetal SCN clock develops in vivo and whether overt rhythmicity results from a functional fetal clock. To achieve this goal, the prenatal development of rhythmic expression of clock genes was measured with a more sensitive method for detection of the clock gene expression than previously. Fetal SCN were collected at 3 h intervals during the 24 h period on E19 and E21 by laser dissection and expression of clock genes (Per2, Nr1d1 and Bmal1) and genes related to cellular activity (c-fos, Avp and Vip) was measured by qRT PCR. At E19, the expression of canonical clock genes Per2 and Bmal1 was not rhythmic; however, the expression of all other studied genes followed clear circadian rhythms. At E21, Per2 and Bmal1 expression exhibited low amplitude but significant rhythmicity. From E19 to E21, the levels of the non-rhythmic transcripts (Per2 and Bmal1) decreased; however, the levels of the rhythmic transcripts (Nr1d1, c-fos, Avp and Vip) increased. In summary, these data demonstrate that at E19, rhythms in Per2 and Bmal1 expression were absent in the fetal SCN; however, the expression of Nr1d1 and other genes related to cellular activity was driven rhythmically. Therefore, at the early stage in vivo, the developing fetal SCN clock could theoretically be entrained by oscillation of Nr1d1 which may be driven by the maternal rather than fetal circadian system.

The functional state of the circadian system of spontaneously hypertensive rats (SHR) differs in several characteristics from the functional state of normotensive Wistar rats. Some of these changes might be due to the compromised ability of the central pacemaker to entrain the peripheral clocks. Daily body temperature cycles represent one of the important cues responsible for the integrity of the circadian system, because these cycles are driven by the central pacemaker and are able to entrain the peripheral clocks. This study tested the hypothesis that the aberrant peripheral clock entrainment of SHR results from a compromised peripheral clock sensitivity to the daily temperature cycle resetting. Using cultured Wistar rat and SHR fibroblasts transfected with the circadian luminescence reporter Bmal1-dLuc, we demonstrated that two consecutive square-wave temperature cycles with amplitudes of 2.5 °C are necessary and sufficient to restart the dampened oscillations and entrain the circadian clocks in both Wistar rat and SHR fibroblasts. We also generated a phase response curve to temperature cycles for fibroblasts of both rat strains. Although some of the data suggested a slight resistance of SHR fibroblasts to temperature entrainment, we concluded that the overall effect it too weak to be responsible for the differences between the SHR and Wistar in vivo circadian phenotype.

The mammalian timekeeping system generates circadian oscillations that rhythmically drive various functions in the body, including metabolic processes. In the liver, circadian clocks may respond both to actual feeding conditions and to the metabolic state. The temporal restriction of food availability to improper times of day (restricted feeding, RF) leads to the development of food anticipatory activity (FAA) and resets the hepatic clock accordingly. The aim of this study was to assess this response in a rat strain exhibiting complex pathophysiological symptoms involving spontaneous hypertension, an abnormal metabolic state and changes in the circadian system, i.e., in spontaneously hypertensive rats (SHR). The results revealed that SHR were more sensitive to RF compared with control rats, developing earlier and more pronounced FAA. Whereas in control rats, the RF only redistributed the activity profiles into two bouts (one corresponding to FAA and the other corresponding to the dark phase), in SHR the RF completely phase-advanced the locomotor activity according to the time of food presentation. The higher behavioral sensitivity to RF was correlated with larger phase advances of the hepatic clock in response to RF in SHR. Moreover, in contrast to the controls, RF did not suppress the amplitude of the hepatic clock oscillation in SHR. In the colon, no significant differences in response to RF between the two rat strains were detected. The results suggested the possible involvement of the Bmal2 gene in the higher sensitivity of the hepatic clock to RF in SHR because, in contrast to the Wistar rats, the rhythm of Bmal2 expression was advanced similarly to that of Bmal1 under RF. Altogether, the data demonstrate a higher behavioral and circadian responsiveness to RF in the rat strain with a cardiovascular and metabolic pathology and suggest a likely functional role for the Bmal2 gene within the circadian clock.

Malfunction of the circadian timing system may result in cardiovascular and metabolic diseases, and conversely, these diseases can impair the circadian system. The aim of this study was to reveal whether the functional state of the circadian system of spontaneously hypertensive rats (SHR) differs from that of control Wistar rat. This study is the first to analyze the function of the circadian system of SHR in its complexity, i.e., of the central clock in the suprachiasmatic nuclei (SCN) as well as of the peripheral clocks. The functional properties of the SCN clock were estimated by behavioral output rhythm in locomotor activity and daily profiles of clock gene expression in the SCN determined by in situ hybridization. The function of the peripheral clocks was assessed by daily profiles of clock gene expression in the liver and colon by RT-PCR and in vitro using real time recording of Bmal1-dLuc reporter. The potential impact of the SHR phenotype on circadian control of the metabolic pathways was estimated by daily profiles of metabolism-relevant gene expression in the liver and colon. The results revealed that SHR exhibited an early chronotype, because the central SCN clock was phase advanced relative to light/dark cycle and the SCN driven output rhythm ran faster compared to Wistar rats. Moreover, the output rhythm was dampened. The SHR peripheral clock reacted to the dampened SCN output with tissue-specific consequences. In the colon of SHR the clock function was severely altered, whereas the differences are only marginal in the liver. These changes may likely result in a mutual desynchrony of circadian oscillators within the circadian system of SHR, thereby potentially contributing to metabolic pathology of the strain. The SHR may thus serve as a valuable model of human circadian disorders originating in poor synchrony of the circadian system with external light/dark regime.