Circadian Locomotor Output Cycles Kaput Research Articles

The role of CLOCK gene in psychiatric disorders: Evidence from human and animal research.

The circadian clock system drives daily rhythms in physiology, metabolism, and behavior in mammals. Molecular mechanisms of this system consist of multiple clock genes, with Circadian Locomotor Output Cycles Kaput (CLOCK) as a core member that plays an important role in a wide range of behaviors. Alterations in the CLOCK gene are associated with common psychiatric disorders as well as with circadian disturbances comorbidities. This review addresses animal, molecular, and genetic studies evaluating the role of the CLOCK gene on many psychiatric conditions, namely autism spectrum disorder, schizophrenia, attention-deficit/hyperactivity disorder, major depressive disorder, bipolar disorder, anxiety disorder, and substance use disorder. Many animal experiments focusing on the effects of the Clock gene in behavior related to psychiatric conditions have shown consistent biological plausibility and promising findings. In humans, genetic and gene expression studies regarding disorder susceptibility, sleep disturbances related comorbidities, and response to pharmacological treatment, in general, are in agreement with animal studies. However, the number of controversial results is high. Literature suggests that the CLOCK gene exerts important influence on these conditions, and influences the susceptibility to phenotypes of psychiatric disorders.

Agerarin, identified from Ageratum houstonianum, stimulates circadian CLOCK-mediated aquaporin-3 gene expression in HaCaT keratinocytes

The juice of Ageratum houstonianum is used in folk medicine as an external wound healing aid for skin injuries. However, the active component of A. houstonianum and its mode of action in skin wound healing has not been investigated. This study was conducted to investigate the effect of A. houstonianum ethanolnolic extract (AHE) on the expression of aquaporin-3 (AQP3), an integral membrane protein for water and glycerol transport in keratinocytes, and to identify the structure of the A. houstonianum bioactive compound. Here, we show that AHE increased AQP3 gene expression at the transcriptional level through the p38 MAPK pathway in HaCaT cells. Furthermore, AHE ameliorated suppression of AQP3 expression caused by ultraviolet B (UVB) irradiation. Agerarin (6,7-dimethoxy-2,2-dimethyl-2H-chromene) was identified as the bioactive compound responsible for the up-regulation of AQP3 expression by enhancing the expression of the transcription factor circadian locomotor output cycles kaput (CLOCK). In conclusion, agerarin is a bioactive compound in AHE responsible for CLOCK-mediated AQP3 expression in keratinocytes.

Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity

Nuclear hormone receptors (NRs) regulate physiology by sensing lipophilic ligands and adapting cellular transcription appropriately. A growing understanding of the impact of circadian clocks on mammalian transcription has sparked interest in the interregulation of transcriptional programs. Mammalian clocks are based on a transcriptional feedback loop featuring the transcriptional activators circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryptochrome (CRY) and period (PER). CRY1 and CRY2 bind independently of other core clock factors to many genomic sites, which are enriched for NR recognition motifs. Here we report that CRY1/2 serve as corepressors for many NRs, indicating a new facet of circadian control of NR-mediated regulation of metabolism and physiology, and specifically contribute to diurnal modulation of drug metabolism.

CLOCK 3111T/C genetic variant influences the daily rhythm of autonomic nervous function: relevance to body weight control.

Humans carrying the genetic risk variant C at the circadian CLOCK (Circadian Locomotor Output Cycles Kaput) 3111T/C have been shown to have more difficulties to achieve desired weight loss than TT carriers. We tested the hypothesis that the daily rhythm of autonomic nervous function differs in CLOCK 3111C carriers, leading to reduced effectiveness in weight control. We recruited 40 overweight/obese Caucasian women (body mass index>25), 20 carrying CLOCK 3111C (CC and TC) and 20 non-carriers with matched age and body mass index who participated in a dietary obesity treatment program of up to 30 weeks. Following the treatment, ambulatory electrocardiography was continuously monitored for up to 3.5 days when subjects underwent their normal daily activities. To assess autonomic function, heart rate variability analysis (HRV) was performed hourly to obtain mean inter-beat interval between two consecutive R waves (mean RR) and s.d. of normal-to-normal heartbeat intervals (SDNN), and two parasympathetic measures, namely, proportion of differences between adjacent NN intervals that are >50 ms (pNN50), and high-frequency (HF: 0.15-0.4 Hz) power. In the TT carriers, all tested HRV indices showed significant daily rhythms (all P-values <0.0001) with lower mean RR, SDNN, pNN50, and HF during the daytime as compared with the nighttime. The amplitudes of these rhythms except for SDNN were reduced significantly in the C carriers (mean RR: ~19.7%, P=0.001; pNN50: 58.1%, P=0.001; and HF: 41.1%, P=0.001). In addition, subjects with less weight loss during the treatment program had smaller amplitudes in the rhythms of mean RR (P<0.0001), pNN50 (P=0.007) and HF (P=0.003). Furthermore, the rhythmicity-weight loss associations were much stronger in the C carriers as compared to the TT carriers (mean RR: P=0.028, pNN50: P=0.0002; HF: P=0.015). The daily rhythm of parasympathetic modulation may play a role in the influence of the CLOCK variation on body weight control.

Altered GluA1 (Gria1) Function and Accumbal Synaptic Plasticity in the ClockΔ19 Model of Bipolar Mania

BackgroundDisruptions in circadian rhythms are associated with an increased risk for bipolar disorder. Moreover, studies show that the circadian protein CLOCK (circadian locomotor output cycles kaput) is involved in regulating monoaminergic systems and mood-related behavior. However, the molecular and synaptic mechanisms underlying this relationship remain poorly understood. MethodsUsing ex vivo whole-cell patch-clamp electrophysiology in ClockΔ19 mutant and wild-type mice we characterized alterations in excitatory synaptic transmission, strength, and intrinsic excitability of nucleus accumbens (NAc) neurons. We performed protein crosslinking and Western blot analysis to examine surface and intracellular levels and rhythm of the glutamate receptor subunit, GluA1, in the NAc. Viral-mediated overexpression of Gria1 in the NAc and behavioral assays were also used. ResultsCompared with wild-type mice, ClockΔ19 mice display reduced alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor–mediated excitatory synaptic responses at NAc medium spiny neurons. These alterations are likely postsynaptic, as presynaptic release of glutamate onto medium spiny neurons is unaltered in mutant mice. Additionally, NAc surface protein levels and the rhythm of GRIA1 are decreased in ClockΔ19 mice diurnally, consistent with reduced functional synaptic response. Furthermore, we observed a significantly hyperpolarized resting membrane potential of ClockΔ19 medium spiny neurons, suggesting lowered intrinsic excitability. Last, overexpression of functional Gria1 in the NAc of mutant mice was able to normalize increased exploratory drive and reward sensitivity behavior when mice are in a manic-like state. ConclusionsTogether, our findings demonstrate that NAc excitatory signaling via Gria1 expression is integral to the effects of Clock gene disruption on manic-like behaviors.

Passionflower Extract Induces High-amplitude Rhythms without Phase Shifts in the Expression of Several Circadian Clock Genes in Vitro and in Vivo

Circadian rhythms play key roles in the regulation of physiological and behavioral systems including wake-sleep cycles. We evaluated the effects of passionflower (aerial parts of Passiflora incarnata Linnaeus) extract (PFE) on circadian rhythms using NIH3T3 cells and mice. PFE (100 μg/mL) induced high-amplitude rhythms in the expression of period circadian protein (Per) 2, cryptochrome (Cry) 1, superoxide dismutase (SOD) 1, and glutathione peroxidase (GPx) in vitro from 12 h after a treatment with serum-rich medium. Isovitexin 2"-O-glucoside, isoschaftoside, and homoorientin, which were purified from PFE, also significantly enhanced Per2 mRNA expression at 20 h. An oral treatment with PFE (100 mg/kg/day) at zeitgeber time (ZT) 0 h for 15 days improved sleep latencies and sleeping times in the pentobarbital-induced sleep test in mice, similar to muscimol (0.2 mg/kg, i.p.). PFE induced high-amplitude rhythms without obvious phase shifts in serum corticosterone levels and the expression of Per1, Per2, and Cry1 in the liver as well as NIH3T3 cells. However, in the cerebrum, PFE enhanced the circadian expression of brain-muscle ARNT-like protein (Bmal) 1, circadian locomotor output cycles kaput (Clock), and Per1. Regarding this difference, we suggest the involvement of several neurotransmitters that influence the circadian rhythm. Indeed, PFE significantly increased dopamine levels at ZT 18 h, and then affected the mRNA expression of the synthetic and metabolic enzymes such as monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), and glutamic acid decarboxylase (GAD). The results obtained show that PFE positively modulates circadian rhythms by inducing high-amplitude rhythms in the expression of several circadian clock genes.

Melatonin promotes circadian rhythm-induced proliferation through Clock/histone deacetylase 3/c-Myc interaction in mouse adipose tissue.

Melatonin is synthesized in the pineal gland and controls circadian rhythm of peripheral adipose tissue, resulting in changes in body weight. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms of circadian rhythm-mediated proliferation in adipose tissue is still limited. Here, we showed that melatonin (20mg/kg/d) promoted circadian and proliferation processes in white adipose tissue. The circadian amplitudes of brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1, P<.05) and circadian locomotor output cycles kaput (Clock, P<.05), period 2 (Per2, P<.05), cyclin E (P<.05), and c-Myc (P<.05) were directly increased by melatonin in adipose tissue. Melatonin also promoted cell cycle and increased cell numbers (P<.05), which was correlated with the Clock expression (P<.05). Further analysis demonstrated that Clock bound to the E-box elements in the promoter region of c-Myc and then directly stimulated c-Myc transcription. Moreover, Clock physically interacted with histone deacetylase 3 (HDAC3) and formed a complex with c-Myc to promote adipocyte proliferation. Melatonin also attenuated circadian disruption and promoted adipocyte proliferation in chronic jet-lagged mice and obese mice. Thus, our study found that melatonin promoted adipocyte proliferation by forming a Clock/HDAC3/c-Myc complex and subsequently driving the circadian amplitudes of proliferation genes. Our data reveal a novel mechanism that links circadian rhythm to cell proliferation in adipose tissue. These findings also identify a new potential means for melatonin to prevent and treat sleep deprivation-caused obesity.

A genetic CLOCK variant associated with cluster headache causing increased mRNA levels.

Background Cluster headache is characterized by recurrent unilateral headache attacks of severe intensity. One of the main features in a majority of patients is a striking rhythmicity of attacks. The CLOCK ( Circadian Locomotor Output Cycles Kaput) gene encodes a transcription factor that serves as a basic driving force for circadian rhythm in humans and is therefore particularly interesting as a candidate gene for cluster headache. Methods We performed an association study on a large Swedish cluster headache case-control sample (449 patients and 677 controls) screening for three single nucleotide polymorphisms (SNPs) in the CLOCK gene implicated in diurnal preference (rs1801260) or sleep duration (rs11932595 and rs12649507), respectively. We further wanted to investigate the effect of identified associated SNPs on CLOCK gene expression. Results We found a significant association with rs12649507 and cluster headache ( p = 0.0069) and this data was strengthened when stratifying for reported diurnal rhythmicity of attacks ( p = 0.0009). We investigated the effect of rs12649507 on CLOCK gene expression in human primary fibroblast cultures and identified a significant increase in CLOCK mRNA expression ( p = 0.0232). Conclusions Our results strengthen the hypothesis of the involvement of circadian rhythm in cluster headache.

RAI1 Overexpression Promotes Altered Circadian Gene Expression and Dyssomnia in Potocki–Lupski Syndrome

Retinoic acid induced 1 ( RAI1 ) encodes a dosage-sensitive gene that when haploinsufficient results in Smith-Magenis syndrome (SMS) and when overexpressed results in Potocki-Lupski syndrome (PTLS). Phenotypic and molecular evidence illustrates that haploinsufficiency of RAI1 disrupts circadian rhythm through the dysregulation of the master circadian regulator, circadian locomotor output cycles kaput ( CLOCK) , and other core circadian components, contributing to prominent sleep disturbances in SMS. However, the phenotypic and molecular characterization of sleep features in PTLS has not been elucidated. Using the Pittsburgh Sleep Quality Index (PSQI), caregivers of 15 school-aged children with PTLS reported difficulties in initiating sleep. Indeed, more than 70% of individuals manifested moderate to severe sleep latency, as defined by the PSQI. Moreover, these individuals manifested difficulties in sleep maintenance, with middle of the night and early morning awakenings. When assessing daytime sleepiness through the Epworth Sleepiness Scale, approximately 21% of the individuals manifested excessive daytime somnolence. This indicates that mild dyssomnia characterizes the majority of the sleep phenotype, with occasionally problematic daytime somnolence, a phenotype different than that expressed by individuals with SMS, where daytime sleepiness is a chronic problem. Gene expression analysis of the core circadian machinery in the hypothalamus of the PTLS mouse model ( Rai1 -Tg) found significant dysregulation of the transcriptional activators, Clock and Arntl , and the transcriptional repressors, Per1-3 and Cry1/2 , during both light and dark phases. These findings suggest a partial loss of circadian entrainment typically evoked by environmental photic cues. Examination of circadian clock gene expression in the Rai1- Tg mouse heart, liver, and kidney found unchanged expression of Clock and most of its downstream targets during both light and dark phases, suggesting an asynchronized circadian rhythm. Furthermore, examination of circadian gene expression in synchronized PTLS lymphoblasts revealed reduced transcripts of the Period ( PER1-3 ) family and normal expression of CRY1/2 . The finding that central circadian gene expression was altered while many peripheral circadian components were intact suggests a tissue-specific circadian uncoupling of the circadian machinery due to Rai1 overexpression. Overall, our results demonstrate that overexpression of RAI1 results in sleep deficiencies in individuals with PTLS due to a lack of properly regulated circadian machinery gene expression and highlight the importance of evaluating sleep concerns in individuals with PTLS.

Inhibition of G0/G1 Switch 2 Ameliorates Renal Inflammation in Chronic Kidney Disease

Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G0/G1 switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.

GW27-e0343 Association of CLOCK gene polymorphism with insulin resistance in hypertension: A Case-Control Study

Circadian rhythm disruption is shown to be the cause of various health disorders. Circadian Locomotor Output Cycles Kaput (CLOCK) and brain and muscle Arnt-like protein-1 (BMAL1), two core circadian transcriptionfactors, were associated with the regulation of glucose homeostasis. This study

Mammalian Period represses and de-represses transcription by displacing CLOCK–BMAL1 from promoters in a Cryptochrome-dependent manner

The mammalian circadian clock is based on a transcription-translation feedback loop (TTFL) consolidated by secondary loops. In the primary TTFL, the circadian locomotor output cycles kaput (CLOCK)-brain and muscle Arnt-like protein-1 (BMAL1) heterodimer acts as the transcriptional activator, and Cryptochrome (CRY) and Period (PER) proteins function as repressors. PER represses by displacing CLOCK-BMAL1 from promoters in a CRY-dependent manner. Interestingly, genes with complex promoters may either be repressed or de-repressed by PER, depending on the particular promoter regulatory elements. Here, using mouse cell lines with defined knockout mutations in clock genes, RNA-seq, ChIP-seq, and reporter gene assays coupled with measurements of DNA-protein interactions in nuclear extracts, we elucidate the dual functions of PER as repressor and de-repressor in a context-dependent manner.

The effects of cytosine methylation on general transcription factors.

DNA methylation on CpG sites is the most common epigenetic modification. Recently, methylation in a non-CpG context was found to occur widely on genomic DNA. Moreover, methylation of non-CpG sites is a highly controlled process, and its level may vary during cellular development. To study non-CpG methylation effects on DNA/protein interactions, we have chosen three human transcription factors (TFs): glucocorticoid receptor (GR), brain and muscle ARNT-like 1 (BMAL1) - circadian locomotor output cycles kaput (CLOCK) and estrogen receptor (ER) with methylated or unmethylated DNA binding sequences, using single-molecule and isothermal titration calorimetry assays. The results demonstrated that these TFs interact with methylated DNA with different effects compared with their cognate DNA sequences. The effects of non-CpG methylation on transcriptional regulation were validated by cell-based luciferase assay at protein level. The mechanisms of non-CpG methylation influencing DNA-protein interactions were investigated by crystallographic analyses and molecular dynamics simulation. With BisChIP-seq assays in HEK-293T cells, we found that GR can recognize highly methylated sites within chromatin in cells. Therefore, we conclude that non-CpG methylation of DNA can provide a mechanism for regulating gene expression through directly affecting the binding of TFs.

CLOCK promotes 3T3-L1 cell proliferation via Wnt signaling.

Circadian genes control most of the physiological functions including cell cycle. Cell proliferation is a critical factor in the differentiation of progenitor cells. However, the role of Clock gene in the regulation of cell cycle via wingless-type (Wnt) pathway and the relationship between Clock and adipogenesis are unclear. We found that the circadian locomotor output cycles kaput (Clock) regulated the proliferation and the adipogenesis of 3T3-L1 preadipocytes. We found that Clock attenuation inhibited the viability of 3T3-L1 preadipocytes in the cell counting kit 8. The expression of c-Myc and Cyclin D1 decreased dramatically in 3T3-L1 when Clock was silenced with short interfering RNA and was also decreased in fat tissue and adipose tissue-derived stem cells of Clock(Δ19) mice. Clock directly controls the expression of the components of Wnt signal transduction pathway, which was verified by serum shock, chromatin immunoprecipitation, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, IWR-1, a Wnt signal pathway inhibitor, inhibited the cell cycle promotion by CLOCK, which was detected by cell viability assay, flow cytometry, and qRT-PCR. Therefore, CLOCK transcription control of Wnt signaling promotes cell cycle progression in 3T3-L1 preadipocytes. Clock inhibited the adipogenesis on day 2 in 3T3-L1 cells via Oil Red O staining and qRT-PCR detection and probably related to cellular differentiation. These data provide evidence that the circadian gene Clock regulates the proliferation of preadipocytes and affects adipogenesis. © 2016 IUBMB Life, 68(7):557-568, 2016.

Investigations of the CLOCK and BMAL1 Proteins Binding to DNA: A Molecular Dynamics Simulation Study.

The circadian locomotor output cycles kaput (CLOCK), and brain and muscle ARNT-like 1 (BMAL1) proteins are important transcriptional factors of the endogenous circadian clock. The CLOCK and BMAL1 proteins can regulate the transcription-translation activities of the clock-related genes through the DNA binding. The hetero-/homo-dimerization and DNA combination of the CLOCK and BMAL1 proteins play a key role in the positive and negative transcriptional feedback processes. In the present work, we constructed a series of binary and ternary models for the bHLH/bHLH-PAS domains of the CLOCK and BMAL1 proteins, and the DNA molecule, and carried out molecular dynamics simulations, free energy calculations and conformational analysis to explore the interaction properties of the CLOCK and BMAL1 proteins with DNA. The results show that the bHLH domains of CLOCK and BMAL1 can favorably form the heterodimer of the bHLH domains of CLOCK and BMAL1 and the homodimer of the bHLH domains of BMAL1. And both dimers could respectively bind to DNA at its H1-H1 interface. The DNA bindings of the H1 helices in the hetero- and homo-bHLH dimers present the rectangular and diagonal binding modes, respectively. Due to the function of the α-helical forceps in these dimers, the tight gripping of the H1 helices to the major groove of DNA would cause the decrease of interactions at the H1-H2 interfaces in the CLOCK and BMAL1 proteins. The additional PAS domains in the CLOCK and BMAL1 proteins affect insignificantly the interactions of the CLOCK and BMAL1 proteins with the DNA molecule due to the flexible and long loop linkers located at the middle of the PAS and bHLH domains. The present work theoretically explains the interaction mechanisms of the bHLH domains of the CLOCK and BMAL1 proteins with DNA.

CLOCK and BMAL1 Regulate Muscle Insulin Sensitivity via SIRT1 in Male Mice.

Circadian misalignment induces insulin resistance in both human and animal models, and skeletal muscle is the largest organ response to insulin. However, how circadian clock regulates muscle insulin sensitivity and the underlying molecular mechanisms are still largely unknown. Here we show circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, two core circadian transcription factors, are down-regulated in insulin-resistant C2C12 myotubes and mouse skeletal muscle. Furthermore, insulin signaling is attenuated in the skeletal muscle of Clock(Δ19/Δ19) mice, and knockdown of CLOCK or BMAL1 by small interfering RNAs induces insulin resistance in C2C12 myotubes. Consistently, ectopic expression of CLOCK and BMAL1 improves insulin sensitivity in C2C12 myotubes. Moreover, CLOCK and BMAL1 regulate the expression of sirtuin 1 (SIRT1), an important regulator of insulin sensitivity, in C2C12 myotubes and mouse skeletal muscle, and two E-box elements in Sirt1 promoter are responsible for its CLOCK- and BMAL1-dependent transcription in muscle cells. Further studies show that CLOCK and BMAL1 regulate muscle insulin sensitivity through SIRT1. In addition, we find that BMAL1 and SIRT1 are decreased in the muscle of mice maintained in constant darkness, and resveratrol supplementation activates SIRT1 and improves insulin sensitivity. All these data demonstrate that CLOCK and BMAL1 regulate muscle insulin sensitivity via SIRT1, and activation of SIRT1 might be a potential valuable strategy to attenuate muscle insulin resistance related to circadian misalignment.

Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression

Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found thatIrp2mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(Δ19) tumors expressing the clock-mutant protein (CLOCK(Δ19)) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCK(Δ19)expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

Circadian Clock Regulates Bone Resorption in Mice.

The circadian clock controls many behavioral and physiological processes beyond daily rhythms. Circadian dysfunction increases the risk of cancer, obesity, and cardiovascular and metabolic diseases. Although clinical studies have shown that bone resorption is controlled by circadian rhythm, as indicated by diurnal variations in bone resorption, the molecular mechanism of circadian clock-dependent bone resorption remains unknown. To clarify the role of circadian rhythm in bone resorption, aryl hydrocarbon receptor nuclear translocator-like (Bmal1), a prototype circadian gene, was knocked out specifically in osteoclasts. Osteoclast-specific Bmal1-knockout mice showed a high bone mass phenotype due to reduced osteoclast differentiation. A cell-based assay revealed that BMAL1 upregulated nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1 (Nfatc1) transcription through its binding to an E-box element located on the Nfatc1 promoter in cooperation with circadian locomotor output cycles kaput (CLOCK), a heterodimer partner of BMAL1. Moreover, steroid receptor coactivator (SRC) family members were shown to interact with and upregulate BMAL1:CLOCK transcriptional activity. Collectively, these data suggest that bone resorption is controlled by osteoclastic BMAL1 through interactions with the SRC family and binding to the Nfatc1 promoter. © 2016 American Society for Bone and Mineral Research.

Maternal chitosan oligosaccharide supplementation affecting expression of circadian clock genes, and possible association with hepatic cholesterol accumulation in suckling piglets

Chitosan oligosaccharide (COS) has been shown to reduce lipid accumulation in liver in mice and rats. The purpose of this study was to investigate whether maternal COS feeding affects hepatic lipid metabolism via influencing the expression of circadian clock genes in piglets. From day (d) 85 of gestation to d 14 of lactation, sixteen pregnant sows were divided into a control group (basal diet without COS supplementation) and a COS group (30 mg COS/kg basal diet). After farrowing, one piglet per litter in each group was selected for the collection of plasma and liver samples on d 0 and d 14 of age, respectively. Interestingly and significantly, we found that maternal COS supplementation promoted plasma and hepatic cholesterol accumulation and up-regulated the mRNA level of negative-regulated element period 1 (Per1), and reduced the abundance of the positive elements, circadian locomotor output cycles kaput (CLOCK), and brain muscle Arnt-like 1 (BMAL1) in the suckling piglets on d 14. These alterations may promote the hepatic cholesterol accumulation, which, in turn, activates hepatic bile acid metabolism and attenuates the relative expression levels of lipid metabolism-associated genes in the liver. However, the expression of CLOCK and BMAL1 and the lipid profile in the plasma and liver were not affected by COS supplementation on d 0. Collectively, our results indicate that maternal supplementation with COS postpartum up-regulates cholesterol accumulation in suckling piglets at age d 14, in part, by the regulation of circadian clock genes.

Association between genetic variants of the clock gene and obesity and sleep duration.

Obesity is a multifactorial disease caused by the interaction of genetic and environmental factors related to lifestyle aspects. It has been shown that reduced sleep is associated with increased body mass index (BMI). Circadian Locomotor Output Cycles Kaput (CLOCK) gene variants have also been associated with obesity. The objective of this mini-review was to discuss the available literature related to CLOCK gene variants associated with adiposity and sleep duration in humans. In total, 16 articles complied with the terms of the search that reported CLOCK variants associated with sleep duration, energy intake, and BMI. Overall, six CLOCK single nucleotide polymorphisms (SNPs) have been associated with sleep duration, and three variants have been associated with energy intake variables. Overall, the most studied area has been the association of CLOCK gene with obesity; close to eight common variants have been associated with obesity. The most studied CLOCK SNP in different populations is rs1801260, and most of these populations correspond to European populations. Collectively, identifying at risk CLOCK genotypes is a new area of research that may help identify individuals who are more susceptible to overeating and gaining weight when exposed to short sleep durations.