Clock Protein Expression Research Articles

Systems Biology Approaches and Precision Oral Health: A Circadian Clock Perspective.

A vast majority of the pathophysiological and metabolic processes in humans are temporally controlled by a master circadian clock located centrally in the hypothalamic suprachiasmatic nucleus of the brain, as well as by specialized peripheral oscillators located in other body tissues. This circadian clock system generates a rhythmical diurnal transcriptional-translational cycle in clock genes and protein expression and activities regulating numerous downstream target genes. Clock genes as key regulators of physiological function and dysfunction of the circadian clock have been linked to various diseases and multiple morbidities. Emerging omics technologies permits largescale multi-dimensional investigations of the molecular landscape of a given disease and the comprehensive characterization of its underlying cellular components (e.g., proteins, genes, lipids, metabolites), their mechanism of actions, functional networks and regulatory systems. Ultimately, they can be used to better understand disease and interpatient heterogeneity, individual profile, identify personalized targetable key molecules and pathways, discover novel biomarkers and genetic alterations, which collectively can allow for a better patient stratification into clinically relevant subgroups to improve disease prediction and prevention, early diagnostic, clinical outcomes, therapeutic benefits, patient's quality of life and survival. The use of “omics” technologies has allowed for recent breakthroughs in several scientific domains, including in the field of circadian clock biology. Although studies have explored the role of clock genes using circadiOmics (which integrates circadian omics, such as genomics, transcriptomics, proteomics and metabolomics) in human disease, no such studies have investigated the implications of circadian disruption in oral, head and neck pathologies using multi-omics approaches and linking the omics data to patient-specific circadian profiles. There is a burgeoning body of evidence that circadian clock controls the development and homeostasis of oral and maxillofacial structures, such as salivary glands, teeth and oral epithelium. Hence, in the current era of precision medicine and dentistry and patient-centered health care, it is becoming evident that a multi-omics approach is needed to improve our understanding of the role of circadian clock-controlled key players in the regulation of head and neck pathologies. This review discusses current knowledge on the role of the circadian clock and the contribution of omics-based approaches toward a novel precision health era for diagnosing and treating head and neck pathologies, with an emphasis on oral, head and neck cancer and Sjögren's syndrome.

MON-227 Roles of Clock Expression Modulated by BMP-7 in Ovarian Steroidogenesis by Human Granulosa Cells

In mammals, the circadian pacemaker is known to control and integrate physiological rhythms via the coordination of oscillating peripheral clocks that reside in various organs, through the biologic actions of various neurochemical and/or hormonal transmitters. The expression of clock genes has been demonstrated in tissues composing the axis of the hypothalamic-pituitary-gonadal system. The roles of hypothalamic oscillators to produce the biological timings and rhythms in the reproductive axis have been recognized. Results of recent studies have shown that clock genes are expressed in the ovary, and accumulated findings have demonstrated a significant interrelationship between clock gene expression and follicular development. It has been reported that the rhythms of clock gene expression are undetectable in primordial and preantral follicles but can be detected at the timing of early antral stages of follicles and then become clearer at the late antral stages to preovulatory follicles. In the ovarian follicles, clock genes are expressed in granulosa cells, theca cells, oocytes and stromal cells composing the developing follicles. Dysfunction of clock gene expression in the ovary and/or desynchronicity between ovarian clocks and extra-ovarian circadian oscillators may cause various reproductive abnormalities. However, the regulatory mechanism and the physiological roles of the intrinsic clock activity in the ovary have remained uncertain. In the preset study, functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between the oscillating patterns of mRNA and protein expression of Bmal1 and Clock and that of Per2 and Cry1 were demonstrated during 24-h culture of KGN cells in the presence of forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Collectively, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.

Interaction of ovarian steroidogenesis and clock gene expression modulated by bone morphogenetic protein-7 in human granulosa cells.

A functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between changes in the mRNA and protein expression levels of Bmal1 and Clock and those of Per2 and Cry1 were found in KGN cells after treatment with forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Thus, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.

Tea polyphenols direct Bmal1-driven ameliorating of the redox imbalance and mitochondrial dysfunction in hepatocytes

Circadian rhythms are intimately linked to cellular redox status homeostasis via the regulation of mitochondrial function. Tea polyphenols (TP) are nutraceuticals that possess powerful antioxidant properties, especially ameliorating oxidative stress. The objective of this study was to investigate whether circadian clock is involved in the protection effect of TP on oxidative stress cell models. TP ameliorate H2O2-triggered relatively shallow daily oscillations and phase shift of circadian clock genes transcription and protein expression. Meanwhile, TP attenuate H2O2-stimulated excessive secretions of reactive oxygen species (ROS) and restore the depletions of mitochondrial function in a Bmal1-dependent manner. Furthermore, TP treatment accelerates nuclear translocation of Nrf2 and modulates the downstream expressions of antioxidant enzymes. Intriguingly, knockdown of Bmal1 notably blocked Nrf2/ARE/HO-1 redox-sensitive transcription pathway. Our study revealed that TP, as a Bmal1-enhancing natural compound, alleviated redox imbalance via strengthening Keap1/Nrf2 antioxidant defense pathway and ameliorating mitochondrial dysfunction in a Bmal1-dependent manner.

Assessment of Proinflammatory Mediators of Beta-Cell Circadian Clock Dysfunction in Diabetes

Both type 1 and type 2 diabetes are characterized by a decline in β-cell function and mass partly attributed to exposure of β-cells to proinflammatory cytokines. The molecular mechanisms underlying this process are not well understood, however several lines of evidence have linked the circadian clock with regulation of the deleterious proinflammatory effects in diabetes. Therefore, in this study, we assessed the effect of known proinflammatory mediators of β-cell failure in diabetes (IL-1β, TNFα, IL-6, and IFNγ) on the β-cell circadian clock. Through real-time bioluminescence tracking of islets from Per2:luciferase (a key circadian clock gene reporter) knock-in mice, our data revealed that IL-1β (2 ng/ml) diminished β-cell circadian rhythmicity via impairment of the amplitude and the phase of oscillations (p<0.vs. vehicle). This was in contrast to TNFα, IL-6, and IFNγ, which showed either mild suppression or no effect on the β-cell clock. We next assessed the ability of proinflammatory cytokines to modulate Bmal1 and Clock (two main transcription factors in the circadian pathway) gene and protein expression. Interestingly, studies using INS-1 832/13 cells and cadaveric human islets revealed that IL-1β addition (0.2-5 ng/ml) for 24 h significantly reduced Bmal1 (but not Clock) at both the mRNA (∼40-50%, p<0.05) and protein (10-65%, p<0.05) level in a dose dependent manner. In contrast, other cytokines (IL-6, TNFα, and IFNγ) only modestly diminished Bmal1 expression after 24 h. In assessing a molecular link between IL-1β and the β-cell clock, Bmal1 promoter activity was determined using a BMAL1:luciferase expressing INS-1 832/13 stable-transfected cell line. Importantly, IL-1β (0.01-5 ng/ml) showed a dose dependent decrease in Bmal1 promoter activation (up to 90%). This body of work provides the first novel connection between diabetogenic inflammatory cytokines, specifically IL-1β, as the dominant mediator of β-cell clock dysfunction in diabetes. Disclosure N. Javeed: None. K. Rakshit: None. A. Matveyenko: None.

Association between circadian gene CLOCK and cisplatin resistance in ovarian cancer cells: A preliminary study.

The present study aimed to observe the expression of circadian gene clock circadian regulator (CLOCK) in ovarian cancer cells and the effects of circadian gene CLOCK on cis-dichlorodiamine platinum (cisplatin) resistance in ovarian cancer cells. The expression of CLOCK mRNA and protein in cisplatin-sensitive A2780 and cisplatin-resistant CP70 cells were detected by quantitative polymerase chain reaction and western blot assay. Cisplatin-sensitive A2780 and cisplatin-resistant CP70 cells were treated with different concentrations of cisplatin for 48 h, and the expression of hCLOCK protein in the two types of cells was detected by western blot assay. RNA interference method was used to knock down the expression of CLOCK in cisplatin-resistant CP70 cells. Subsequently, the cisplatin-resistant CP70 cells were treated with cisplatin. The proliferation of cisplatin-resistant CP70 cells was observed following treatment with cisplatin. The expression of CLOCK mRNA was significantly higher in cisplatin-resistant CP70 cells (1.58±0.49) compared with cisplatin-sensitive A2780 cells (0.44±0.13) (P<0.01). Western blot assay results demonstrated that the expression of CLOCK protein was significantly greater in the cisplatin-resistant CP70 cells (1.47±0.34) compared with the cisplatin-sensitive A2780 cells (0.48±0.15) (P<0.01). Following the treatment of A2780 and CP70 cells with cisplatin, CLOCK protein expression increased with an increased concentration of cisplatin, in a dose-dependent manner (P<0.01). Following the knockdown of CLOCK in cisplatin-resistant CP70 cells by RNA interference, cisplatin treatment was able to significantly inhibit the proliferation of cells and induce apoptosis (P<0.01). The expression of circadian gene CLOCK in ovarian cancer cells was strongly associated with cisplatin resistance. The upregulation of circadian gene CLOCK in ovarian cancer cells may reduce its sensitivity to cisplatin treatment.

Postnatal Ontogenesis of the Islet Circadian Clock Plays a Contributory Role in β-Cell Maturation Process.

Development of cell replacement therapies in diabetes requires understanding of the molecular underpinnings of β-cell maturation. The circadian clock regulates diverse cellular functions important for regulation of β-cell function and turnover. However, postnatal ontogenesis of the islet circadian clock and its potential role in β-cell maturation remain unknown. To address this, we studied wild-type Sprague-Dawley as well as Period1 luciferase transgenic (Per1:LUC) rats to determine circadian clock function, clock protein expression, and diurnal insulin secretion during islet development and maturation process. We additionally studied β-cell-specific Bmal1-deficient mice to elucidate a potential role of this key circadian transcription factor in β-cell functional and transcriptional maturation. We report that emergence of the islet circadian clock 1) occurs during the early postnatal period, 2) depends on the establishment of global behavioral circadian rhythms, and 3) leads to the induction of diurnal insulin secretion and gene expression. Islet cell maturation was also characterized by induction in the expression of circadian transcription factor BMAL1, deletion of which altered postnatal development of glucose-stimulated insulin secretion and the associated transcriptional network. Postnatal development of the islet circadian clock contributes to early-life β-cell maturation and should be considered for optimal design of future β-cell replacement strategies in diabetes.

Circadian Clock Proteins and Melatonin Receptors in Neurons and Glia of the Sapajus apella Cerebellum.

Oscillations of brain proteins in circadian rhythms are important for determining several cellular and physiological processes in anticipation of daily and seasonal environmental rhythms. In addition to the suprachiasmatic nucleus, the primary central oscillator, the cerebellum shows oscillations in gene and protein expression. The variety of local circuit rhythms that the cerebellar cortex contains influences functions such as motivational processes, regulation of feeding, food anticipation, language, and working memory. The molecular basis of the cerebellar oscillator has been demonstrated by “clock gene” expression within cells of the cerebellar layers. Genetic and epidemiological evidence suggests that disruption of circadian rhythms in humans can lead to many pathological conditions. Despite this importance, data about clock gene and protein expression in the cerebellum of diurnal (day-active) species, specifically primates, is currently poorly explored, mainly in regard to cellular identity, as well as the relationship with other molecules also involved in cerebellar functions. These studies could contribute to clarification of the possible mechanisms behind cerebellar rhythmicity. Considering that calcium binding proteins (CaBPs) play crucial roles in preserving and modulating cerebellar functions and that clock gene expression can be controlled by afferent projections or paracrine circadian signals such as the hormone melatonin, the present study aimed to describe cellular identities, distribution patterns and day/night expression changes in PER1, PER2, CaBPs, and MT1 and MT2 melatonin receptors in the cerebellar cortex of a diurnal primate using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. PER1 and PER2 immunoreactive (IR) cells were observed in the Purkinje cells of the cerebellum, and MT1 and MT2 receptors were localized around Purkinje cells in the Pj layer in Bergmann cells. This identity was confirmed by the S100β-IR of these cells. The highest expression of PER seen in the daytime analysis coincided with the highest expression of melatonin receptors. CaBPs showed day/night morphological and density changes in the cerebellar cortex. The presence of the same temporal variations in the expression of PER in the Pj neurons and in MT1 and MT2 receptors in Bergmann cells indicates a possible relation between these cells during the rhythmic processing of the cerebellum, in addition to the CaBP temporal morphological and density changes.

Disruption of circadian clockwork in in vivo reprogramming-induced mouse kidney tumors.

The circadian clock, which regulates cellular physiology, such as energy metabolism, resides in each cell level throughout the body. Recently, it has been elucidated that the cellular circadian clock is closely linked with cellular differentiation. Moreover, the misregulation of cellular differentiation in mouse embryonic stem cells (ESCs) induced abnormally differentiated cells with impaired circadian clock oscillation, concomitant with the post-transcriptional suppression of CLOCK proteins. Here, we show that the circadian molecular oscillation is disrupted in dysdifferentiation-mediated mouse kidney tumors induced by partial in vivo reprogramming, resembling Wilms tumors. The expression of CLOCK protein was dramatically reduced in the tumor cells despite the Clock mRNA expression. We also showed that a similar loss of CLOCK was observed in human Wilms tumors, suggesting that the circadian molecular clockwork may be disrupted in dysdifferentiation-mediated embryonal tumors such as Wilms tumors, similar to the in vivo reprogramming-induced mouse kidney tumors. These results support our previous reports and may provide a novel viewpoint for understanding the pathophysiological nature of cancers through the correlation between cellular differentiation and circadian clock.

(-)-Epigallocatechin-3-gallate Ameliorates Insulin Resistance and Mitochondrial Dysfunction in HepG2 Cells: Involvement of Bmal1.

Normal physiological processes require a robust biological timer called the circadian clock. Dysregulation of circadian rhythms contributes to a variety of metabolic syndrome, including obesity and insulin resistance. (-)-Epigallocatechin-3-gallate (EGCG) has been demonstrated to possess antioxidant, anti-inflammatory, and cardioprotective bioactivities. The objective of this study was to explore whether the circadian clock is involved in the protective effect of EGCG against insulin resistance. The results demonstrated that EGCG reverses the relatively shallow daily oscillations of circadian clock genes transcription and protein expression induced by glucosamine in HepG2 cells. EGCG also alleviates insulin resistance by enhancing tyrosine phosphorylated levels of IRS-1, stimulating the translocation of GLUT2, and activating PI3K/AKT as well as AMPK signaling pathways in a Bmal1-dependent manner both in HepG2 cells and primary hepatocytes. Glucosamine-stimulated excessive secretions of ROS and depletions of mitochondrial membrane potential were notably attenuated in EGCG co-treated HepG2 cells, which consistent with the recovery in expression of mitochondrial respiration complexes. The results demonstrated that EGCG possesses a Bmal1-dependent efficacy against insulin resistance conditions by strengthening the insulin signaling and eliminating oxidative stress, suggesting that EGCG may serve as a promising natural nutraceutical for the regulation of metabolic disorders relevant to circadian clocks.

Tea polyphenols ameliorates neural redox imbalance and mitochondrial dysfunction via mechanisms linking the key circadian regular Bmal1

Circadian rhythms are autonomous anticipatory oscillators that control a large array of physiological and metabolic processes. Compelling evidence points toward an interplay between circadian rhythms and cellular redox metabolism. Dysregulation of circadian rhythms is associated with neurodegenerative diseases and accelerated aging. Tea polyphenols (TP) is one of the most used antioxidants and exerts beneficial effect on neurodegenerative diseases. The aim of this study is to investigate whether circadian clock mechanisms are involved in the protection effect of TP against neural redox imbalance and mitochondrial dysfunction in SH-SY5Y cells. In the current study, our results revealed that TP, as a Bmal1-enhancing natural compound, can reverse the relatively shallow daily oscillations of circadian clock genes transcription and protein expression in SH-SY5Y neuronal cells under oxidative stress conditions. Furthermore, TP pretreatment significantly ameliorated H2O2-elicited mitochondria impairment via manipulating mitochondrial dynamics and mitochondrial membrane potential, which is consistent with the recovery in expression of mitochondrial respiration complex I-IV in Bmal1-dependent efficiency. Furthermore, Bmal1 is involved in TP-stimulated Nrf2/ARE/HO-1 and AKT/CREB/BDNF signaling pathway. Hence, TP may serve as a nutritional preventive strategy in the recovery of oxidative stress-related neurodegenerative disease via modulating circadian clock.

Social Isolation Modulates CLOCK Protein and Beta-Catenin Expression Pattern in Gonadotropin-Inhibitory Hormone Neurons in Male Rats.

Postweaning social isolation reduces the amplitude of the daily variation of CLOCK protein in the brain and induces lower reproductive activity. Gonadotropin-inhibitory hormone (GnIH) acts as an inhibitor in the reproductive system and has been linked to stress. Social isolation has been shown to lower neuronal activity of GnIH-expressing neurons in the dorsomedial hypothalamus (DMH). The exact mechanism by which social isolation may affect GnIH is still unclear. We investigated the impact of social isolation on regulatory cellular mechanisms in GnIH neurons. We examined via immunohistochemistry the expression of CLOCK protein at four different times throughout the day in GnIH cells tagged with enhanced fluorescent green protein (EGFP-GnIH) in 9-week-old adult male rats that have been raised for 6 weeks under postweaning social isolation and compared them with group-raised control rats of the same age. We also studied the expression of β-catenin—which has been shown to be affected by circadian proteins such as Bmal1—in EGFP-GnIH neurons to determine whether it could play a role in linking CLOCK in GnIH neurons. We found that social isolation modifies the pattern of CLOCK expression in GnIH neurons in the DMH. Socially isolated rats displayed greater CLOCK expression in the dark phase, while control rats displayed increased CLOCK expression in the light phase. Furthermore, β-catenin expression pattern in GnIH cells was disrupted by social isolation. This suggests that social isolation triggers changes in CLOCK and GnIH expression, which may be associated with an increase in nuclear β-catenin during the dark phase.

Effects of a free-choice high-fat high-sugar diet on brain PER2 and BMAL1 protein expression in mice

The suprachiasmatic nucleus (SCN) times the daily rhythms of behavioral processes including feeding. Beyond the SCN, the hypothalamic arcuate nucleus (ARC), involved in feeding regulation and metabolism, and the epithalamic lateral habenula (LHb), implicated in reward processing, show circadian rhythmic activity. These brain oscillators are functionally coupled to coordinate the daily rhythm of food intake. In rats, a free choice high-fat high-sugar (fcHFHS) diet leads to a rapid increase of calorie intake and body weight gain. Interestingly, under a fcHFHS condition, rats ingest a similar amount of sugar during day time (rest phase) as during night time (active phase), but keep the rhythmic intake of regular chow-food. The out of phase between feeding patterns of regular (chow) and highly rewarding food (sugar) may involve alterations of brain circadian oscillators regulating feeding. Here, we report that the fcHFHS diet is a successful model to induce calorie intake, body weight gain and fat tissue accumulation in mice, extending its effectiveness as previously reported in rats. Moreover, we observed that whereas in the SCN the day-night difference in the PER2 clock protein expression was similar between chow-fed and fcHFHS-fed animals, in the LHb, this day-night difference was altered in fcHFHS-exposed animals compared to control chow mice. These findings confirm previous observations in rats showing disrupted daily patterns of feeding behavior under a fcHFHS diet exposure, and extend our insights on the effects of the diet on circadian gene expression in brain clocks.

Dietary tea polyphenols ameliorate metabolic syndrome and memory impairment via circadian clock related mechanisms

SCOPECircadian rhythms allow organisms to anticipate and exploit daily environmental changes, and food components can adjust internal rhythms in peripheral tissues or even suprachiasmatic nucleus. Dysregulation of circadian rhythms is associated with many pathological conditions. The aim of the current study is to determine potential effects of tea polyphenols on modulating circadian rhythms regarding ameliorating diabetes, metabolic syndrome, and memory disorders. ResultsResults demonstrated that tea polyphenols can reverse the relatively shallow daily oscillations of circadian clock genes transcription and protein expression in both liver and hypothalamus triggered by constant darkness. These compounds also alleviate insulin resistance and disorder of glucose/lipid metabolism in constant darkness-treated mice. Concomitantly, tea polyphenols prevented constant darkness-induced memory impairment by inhibiting neuron damage and elevating the expression of neurotropic factor related genes. ConclusionTea polyphenols supplementation might serve as future strategies to combat obesity, metabolic syndrome and cognitive disorder induced by circadian dysfunction.

Dopamine: A Modulator of Circadian Rhythms in the Central Nervous System.

Circadian rhythms are daily rhythms that regulate many biological processes – from gene transcription to behavior – and a disruption of these rhythms can lead to a myriad of health risks. Circadian rhythms are entrained by light, and their 24-h oscillation is maintained by a core molecular feedback loop composed of canonical circadian (“clock”) genes and proteins. Different modulators help to maintain the proper rhythmicity of these genes and proteins, and one emerging modulator is dopamine. Dopamine has been shown to have circadian-like activities in the retina, olfactory bulb, striatum, midbrain, and hypothalamus, where it regulates, and is regulated by, clock genes in some of these areas. Thus, it is likely that dopamine is essential to mechanisms that maintain proper rhythmicity of these five brain areas. This review discusses studies that showcase different dopaminergic mechanisms that may be involved with the regulation of these brain areas’ circadian rhythms. Mechanisms include how dopamine and dopamine receptor activity directly and indirectly influence clock genes and proteins, how dopamine’s interactions with gap junctions influence daily neuronal excitability, and how dopamine’s release and effects are gated by low- and high-pass filters. Because the dopamine neurons described in this review also release the inhibitory neurotransmitter GABA which influences clock protein expression in the retina, we discuss articles that explore how GABA may contribute to the actions of dopamine neurons on circadian rhythms. Finally, to understand how the loss of function of dopamine neurons could influence circadian rhythms, we review studies linking the neurodegenerative disease Parkinson’s Disease to disruptions of circadian rhythms in these five brain areas. The purpose of this review is to summarize growing evidence that dopamine is involved in regulating circadian rhythms, either directly or indirectly, in the brain areas discussed here. An appreciation of the growing evidence of dopamine’s influence on circadian rhythms may lead to new treatments including pharmacological agents directed at alleviating the various symptoms of circadian rhythm disruption.

Effects of 72 Hours Sleep Deprivation on Liver Circadian Clock Gene Expression and Oxidative Stress in Rats

Objective: To investigate the effects of 72 hours continuous sleep deprivation (SD) on circadian clock gene expression and oxidative stress in the rat liver. Methods: Twenty healthy male Sprague-Dawley rats were divided into 2 groups (n = 10 each) using a random number table: normal control group (group C), sleep deprivation group (group SD). Group SD was treated with a modified multiple platform water environment method. After 72 hours sleep deprived, the levels of AST (Aspartate transaminase ) and ALT (Alanine aminotransferase) in serum were determined. The contents of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the liver tissue of the rats were examined in both two groups. The expression levels of CLOCK, BMAL1 and CRY1 protein in liver tissue were examined by Western blotting. Results: Compared with group C, the content of MDA, and the levels of AST and ALT in serum were significantly increased (P < 0.05); SOD activity was significantly decreased (P < 0.05); GSH-Px activity was significantly decreased (P < 0.01); and the expression of CLOCK, BMAL1 and CRY1 protein was downregulated in group SD. Conclusion: 72 hours continuous sleep deprivation can downregulate the expression of circadian clock gene and promote oxidative stress in rats.

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect geneexpression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.

Metabolic effect of fluvoxamine in mouse peripheral tissues

Serotonin leads to reduced food intake and satiety. Disrupted circadian rhythms lead to hyperphagia and obesity. The serotonergic and circadian systems are intertwined, as the central brain clock receives direct serotonergic innervation and, in turn, makes polysynaptic output back to serotonergic nuclei. Our objective was to test the hypothesis that peripherally serotonin alters circadian rhythms leading to a shift towards fat synthesis and weight gain. We studied the effect of serotonin and fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), on the circadian clock and metabolic gene and protein expression in mouse liver, muscle and white adipose tissue (WAT) and cell culture. We found that serotonin and/or the SSRI fluvoxamine led to fat accumulation in mouse liver and hepatocytes by shifting metabolism towards fatty acid synthesis mainly through low average levels of phosphorylated acetyl CoA carboxylase (pACC) and phosphorylated protein phosphatase 2A (pPP2A). This shift towards fat synthesis was also observed in adipose tissue. Muscle cells were only slightly affected metabolically by serotonin or fluvoxamine. In conclusion, although centrally it leads to increased satiety, in peripheral tissues, such as the liver and WAT, serotonin induces fat accumulation.

Mining for novel candidate clock genes in the circadian regulatory network.

BackgroundMost physiological processes in mammals are temporally regulated by means of a master circadian clock in the brain and peripheral oscillators in most other tissues. A transcriptional-translation feedback network of clock genes produces near 24 h oscillations in clock gene and protein expression. Here, we aim to identify novel additions to the clock network using a meta-analysis of public chromatin immunoprecipitation sequencing (ChIP-seq), proteomics and protein-protein interaction data starting from a published list of 1000 genes with robust transcriptional rhythms and circadian phenotypes of knockdowns.ResultsWe identified 20 candidate genes including nine known clock genes that received significantly high scores and were also robust to the relative weights assigned to different data types. Our scoring was consistent with the original ranking of the 1000 genes, but also provided novel complementary insights. Candidate genes were enriched for genes expressed in a circadian manner in multiple tissues with regulation driven mainly by transcription factors BMAL1 and REV-ERB α,β. Moreover, peak transcription of candidate genes was remarkably consistent across tissues. While peaks of the 1000 genes were distributed uniformly throughout the day, candidate gene peaks were strongly concentrated around dusk. Finally, we showed that binding of specific transcription factors to a gene promoter was predictive of peak transcription at a certain time of day and discuss combinatorial phase regulation.ConclusionsCombining complementary publicly-available data targeting different levels of regulation within the circadian network, we filtered the original list and found 11 novel robust candidate clock genes. Using the criteria of circadian proteomic expression, circadian expression in multiple tissues and independent gene knockdown data, we propose six genes (Por, Mtss1, Dgat2, Pim3, Ppp1r3b, Upp2) involved in metabolism and cancer for further experimental investigation. The availability of public high-throughput databases makes such meta-analysis a promising approach to test consistency between sources and tap their entire potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0227-2) contains supplementary material, which is available to authorized users.

Circadian phenotyping of obese and diabetic db/db mice.

Growing evidence links metabolic disorders to circadian alterations. Genetically obese db/db mice, lacking the long isoform of leptin receptor, are a recognized model of type 2 diabetes. In this study, we aimed at characterizing the potential circadian alterations of db/db mice in comparison to db/+ control mice. By using telemetry devices, we first reported arrhythmicity in general activity of most db/db mice under both light-dark cycle and constant darkness, while their rhythm of body temperature is less dramatically disrupted. Water access restricted to nighttime restores significant rhythmicity in behaviorally arrhythmic db/db mice, indicating a masking effect of polydipsia when water is available ad libitum. Endogenous period of temperature rhythm under constant dark conditions is significantly increased (+30min) in db/db compared with db/+ mice. Next, we studied the oscillations of clock proteins (PER1, PER2 and BMAL1) in the suprachiasmatic nuclei (SCN), the site of the master clock, and detected no difference according to the genotype. Furthermore, c-FOS and P-ERK1/2 expression in response to a light pulse in late night was significantly increased (+80 and+55%, respectively) in the SCN of these diabetic mice. We previously showed that, in addition to altered activity rhythms, db/db mice exhibit altered feeding rhythm. Therefore, we investigated daily patterns of clock protein expression in medial hypothalamic oscillators involved in feeding behavior (arcuate nucleus, ventro- and dorso-medial hypothalamic nuclei). Compared with db/+ mice, very subtle or no difference in oscillations of PER1 and BMAL1 is found in the medial hypothalamus. Although we did not find a clear link between altered hypothalamic clockwork and behavioral rhythms in db/db mice, our results highlight a lengthened endogenous period and altered photic integration in these genetically obese and diabetic mice.