Clock-dependent Manner Research Articles

Nobiletin promotes lipolysis of white adipose tissue in a circadian clock-dependent manner

Nobiletin has been reported to protect against obesity-related metabolic disorders by enhancing the circadian rhythm; however its effects on lipid metabolism in adipose tissue are unclear. In this study, mice were fed with high-fat diet (HFD) for four weeks firstly and gavaged with 50 or 200 mg/kg bodyweight/day nobiletin at Zeitgeber time (ZT) 4 for another four weeks while still receiving HFD. At the end of the 8-week experimental period, the mice were sacrificed at ZT4 or ZT8 on the same day. Mature 3T3-L1 adipocytes were treated with nobiletin in the presence or absence of siBmal1, siRora, siRorc, SR8278 or SR9009. Nobiletin reduced the weight of white adipose tissue (WAT) and the size of adipocytes in WAT. At ZT4, nobiletin decreased the TG, TC and LDL-c levels and increased serum FFA level and glucose tolerance. Nobiletin triggered the lipolysis of mesenteric and epididymal WAT at both ZT4 and ZT16. Nobiletin increased the level of RORγ at ZT16, that of BMAL1 and PPARγ at ZT4, and that of ATGL at both ZT4 and ZT16. Nobiletin increased lipolysis and ATGL levels in 3T3-L1 adipocytes in Bmal1- or Rora/c- dependent manner. Dual luciferase assay indicated that nobiletin enhanced the transcriptional activation of RORα/γ on Atgl promoter and decreased the repression of RORα/γ on PPARγ-binding PPRE. Promoter deletion analysis indicated that nobiletin inhibited the suppression of PPARγ-mediated Atgl transcription by RORα/γ. Taken together, nobiletin elevated lipolysis in WAT by increasing ATGL levels through activating the transcriptional activity of RORα/γ and decreasing the repression of RORα/γ on PPARγ-binding PPRE.

Identification of novel pharmacological activators of circadian clock with anti-adipogenic properties

The circadian clock drives rhythmic oscillations of metabolic processes to orchestrate metabolic homeostasis, and disruption of this mechanism predisposes to obesity and insulin resistance. Preserving or augmenting clock function could be a potential therapeutic target for metabolic diseases, particularly with the wide-spread of circadian misalignment in a modern lifestyle. To date, targeting the clock for metabolic disease prevention or treatment remains to be explored. Adipocyte possesses cell-autonomous clocks that exert transcriptional control of the Wnt pathway to inhibit adipocyte development. Using a high through-put screening pipeline, we recently identified Chlorhexidine as a novel clock-activating molecule that targets the key driver of the circadian clock transcriptional feedback loop, the CLOCK protein. Based on clock function in suppressing adipogenesis, we hypothesize that Chlorhexidine and related compounds may possess adipogenic-inhibitory properties suitable for anti-obesity drug development. In distinct adipogenic progenitor models, we demonstrate the activity of Chlorhexidine in inhibiting adipogenic lineage commitment and terminal differentiation. Furthermore, we report the structural optimization of Chlorhexidine chemical scaffold that led to the discovery of new analogs with improved anti-adipogenic effcacy. Consistent with its activity for clock activation, in adipogenic progenitors containing a Period2::dLuc luciferase reporter, Chlorhexidine induced significant shortening of clock period length with induction of core clock components. Chlorhexidine treatment of adipogenic mesenchymal precursors robustly suppressed their adipogenic maturation, with a comparable effect observed on inhibiting terminal differentiation of primary preadipocytes in a clock-dependent manner. Mechanistically, Chlorhexidine stimulates clock-controlled Wnt signaling that mediates its anti-adipogenic effect. Through medicinal chemistry modification of its chemical scaffold, we generated a panel of Chlorhexidine analogs with validation for their clock-modulatory activities. Structure activity relationship analysis of these analogs led to the identification of CM002 as a new clock activator with improved clock-dependent anti-adipogenic activity. Collectively, our findings uncovered a new class of clock-modulatory compounds that inhibit adipocyte development for potential anti-obesity drug discovery and provide clock-targeting chemical probes for dissecting clock function in metabolic physiology. This project was supported by grants from National Institute of Aging R56AG080294 and Arthur Riggs-Diabetes & Metabolism Research Institute T2D and T1D Innovative Awards to KM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tob Regulates the Timing of Sleep Onset at Night in Drosophila.

Sleep is regulated by homeostatic sleep drive and the circadian clock. While tremendous progress has been made in elucidating the molecular components of the core circadian oscillator, the output mechanisms by which this robust oscillator generates rhythmic sleep behavior remain poorly understood. At the cellular level, growing evidence suggests that subcircuits in the master circadian pacemaker suprachiasmatic nucleus (SCN) in mammals and in the clock network in Drosophila regulate distinct aspects of sleep. Thus, to identify novel molecules regulating the circadian timing of sleep, we conducted a large-scale screen of mouse SCN-enriched genes in Drosophila Here, we show that Tob (Transducer of ERB-B2) regulates the timing of sleep onset at night in female fruit flies. Knockdown of Tob pan-neuronally, either constitutively or conditionally, advances sleep onset at night. We show that Tob is specifically required in "evening neurons" (the LNds and the fifth s-LNv) of the clock network for proper timing of sleep onset. Tob levels cycle in a clock-dependent manner in these neurons. Silencing of these "evening" clock neurons results in an advanced sleep onset at night, similar to that seen with Tob knockdown. Finally, sharp intracellular recordings demonstrate that the amplitude and kinetics of LNd postsynaptic potentials (PSPs) cycle between day and night, and this cycling is attenuated with Tob knockdown in these cells. Our data suggest that Tob acts as a clock output molecule in a subset of clock neurons to potentiate their activity in the evening and enable the proper timing of sleep onset at night.

The Clock-modulatory Activity of Nobiletin Suppresses Adipogenesis Via Wnt Signaling.

The circadian clock machinery exerts transcriptional control to modulate adipogenesis and its disruption leads to the development of obesity. Here, we report that Nobiletin, a circadian clock amplitude-enhancing molecule, displays antiadipogenic properties via activation of Wnt signaling pathway that is dependent on its clock modulation. Nobiletin augmented clock oscillatory amplitude with period lengthening in the adipogenic mesenchymal precursor cells and preadipocytes, accompanied by an induction of Bmal1 and clock components within the negative feedback arm. Consistent with its clock-modulatory activity, Nobiletin strongly inhibited the lineage commitment and terminal differentiation of adipogenic progenitors. Mechanistically, we show that Nobiletin induced the reactivation of Wnt signaling during adipogenesis via transcriptional up-regulation of key components within this pathway. Furthermore, Nobiletin administration in mice markedly reduced adipocyte hypertrophy, leading to a significant loss of fat mass and reduction of body weight. Last, Nobiletin inhibited the differentiation of primary preadipocytes, and this effect was dependent on a functional clock regulation. Collectively, our findings uncover a novel activity of Nobiletin in suppressing adipocyte development in a clock-dependent manner, implicating its potential application in countering obesity and associated metabolic consequences.

The biological clock enhancer nobiletin ameliorates steatosis in genetically obese mice by restoring aberrant hepatic circadian rhythm.

Nonalcoholic fatty liver disease (NAFLD) is associated with disruption of homeostatic lipid metabolism, but underlying processes are poorly understood. One possible mechanism is impairment in hepatic circadian rhythm, which regulates key lipogenic mediators in the liver and whose circadian oscillation is diminished in obesity. Nobiletin enhances biological rhythms by activating RAR-related orphan receptor nuclear receptor, protecting against metabolic syndrome in a clock-dependent manner. The effect of nobiletin in NAFLD is unclear. In this study, we investigate the clock-enhancing effects of nobiletin in genetically obese (db/db) PER2::LUCIFERASE reporter mice with fatty liver. We report microarray expression data suggesting hepatic circadian signaling is impaired in db/db mice with profound hepatic steatosis. Circadian PER2 activity, as assessed by mRNA and luciferase assay, was significantly diminished in liver of db/db PER2::LUCIFERASE reporter mice. Continuous animal monitoring systems and constant dark studies suggest the primary circadian defect in db/db mice lies within peripheral hepatic oscillators and not behavioral rhythms or the master clock. In vitro, nobiletin restored PER2 amplitude in lipid-laden PER2::LUCIFERASE reporter macrophages. In vivo, nobiletin dramatically upregulated core clock gene expression, hepatic PER2 activity, and ameliorated steatosis in db/db PER2::LUCIFERASE reporter mice. Mechanistically, nobiletin reduced serum insulin levels, decreased hepatic Srebp1c, Acaca1, Tnfα, and Fgf21 expression, but did not improve Plin2, Plin5, or Cpt1, suggesting nobiletin attenuates steatosis in db/db mice via downregulation of hepatic lipid accumulation. These data suggest restoring endogenous rhythm with nobiletin resolves steatosis in obesity, proposing that hypothesis that targeting the biological clock may be an attractive therapeutic strategy for NAFLD.NEW & NOTEWORTHY NAFLD is the most common chronic liver disease, but underlying mechanisms are unclear. We show here that genetically obese (db/db) mice with fatty liver have impaired hepatic circadian rhythm. Hepatic Per2 expression and PER2 reporter activity are diminished in db/db PER2::LUCIFERASE mice. The biological clock-enhancer nobiletin restores hepatic PER2 in db/db PER2::LUCIFERASE mice, resolving steatosis via downregulation of Srebp1c. These studies suggest targeting the circadian clock may be beneficial strategy in NAFLD.

Rheostatic Balance of Circadian Rhythm and Autophagy in Metabolism and Disease.

Circadian rhythms are physical, behavioral and environmental cycles that respond primarily to light and dark, with a period of time of approximately 24 h. The most essential physiological functions of mammals are manifested in circadian rhythm patterns, including the sleep-wake cycle and nutrient and energy metabolism. Autophagy is a conserved biological process contributing to nutrient and cellular homeostasis. The factors affecting autophagy are numerous, such as diet, drugs, and aging. Recent studies have indicated that autophagy is activated rhythmically in a clock-dependent manner whether the organism is healthy or has certain diseases. In addition, autophagy can affect circadian rhythm by degrading circadian proteins. This review discusses the interaction and mechanisms between autophagy and circadian rhythm. Moreover, we introduce the molecules influencing both autophagy and circadian rhythm. We then discuss the drugs affecting the circadian rhythm of autophagy. Finally, we present the role of rhythmic autophagy in nutrient and energy metabolism and its significance in physiology and metabolic disease.

COLD-REGULATED GENE27 Integrates Signals from Light and the Circadian Clock to Promote Hypocotyl Growth in Arabidopsis.

Light and the circadian clock are two essential external and internal cues affecting seedling development. COLD-REGULATED GENE27 (COR27), which is regulated by cold temperatures and light signals, functions as a key regulator of the circadian clock. Here, we report that COR27 acts as a negative regulator of light signaling. COR27 physically interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-SUPPRESSOR OF PHYTOCHROME A1 (SPA1) E3 ubiquitin ligase complex and undergoes COP1-mediated degradation via the 26S proteasome system in the dark. cor27 mutant seedlings exhibit shorter hypocotyls, while transgenic lines overexpressing COR27 show elongated hypocotyls in the light. In addition, light induces the accumulation of COR27. On one hand, accumulated COR27 interacts with ELONGATED HYPOCOTYL5 (HY5) to repress HY5 DNA binding activity. On the other hand, COR27 associates with the chromatin at the PHYTOCHROME INTERACTING FACTOR4 (PIF4) promoter region and upregulates PIF4 expression in a circadian clock-dependent manner. Together, our findings reveal a mechanistic framework whereby COR27 represses photomorphogenesis in the light and provide insights toward how light and the circadian clock synergistically control hypocotyl growth.

Control of Sleep Onset by Shal/Kv4 Channels in Drosophila Circadian Neurons.

Sleep is highly conserved across animal species. Both wake- and sleep-promoting neurons are implicated in the regulation of wake-sleep transition at dusk in Drosophila However, little is known about how they cooperate and whether they act via different mechanisms. Here, we demonstrated that in female Drosophila, sleep onset was specifically delayed by blocking the Shaker cognate L channels [Shal; also known as voltage-gated K+ channel 4 (Kv4)] in wake-promoting cells, including large ventral lateral neurons (l-LNvs) and pars intercerebralis (PI), but not in sleep-promoting dorsal neurons (DN1s). Delayed sleep onset was also observed in males by blocking Kv4 activity in wake-promoting neurons. Electrophysiological recordings show that Kv4 channels contribute A-type currents in LNvs and PI cells, but are much less conspicuous in DN1s. Interestingly, blocking Kv4 in wake-promoting neurons preferentially increased firing rates at dusk ∼ZT13, when the resting membrane potentials and firing rates were at lower levels. Furthermore, pigment-dispersing factor (PDF) is essential for the regulation of sleep onset by Kv4 in l-LNvs, and downregulation of PDF receptor (PDFR) in PI neurons advanced sleep onset, indicating Kv4 controls sleep onset via regulating PDF/PDFR signaling in wake-promoting neurons. We propose that Kv4 acts as a sleep onset controller by suppressing membrane excitability in a clock-dependent manner to balance the wake-sleep transition at dusk. Our results have important implications for the understanding and treatment of sleep disorders such as insomnia.SIGNIFICANCE STATEMENT The mechanisms by which our brains reversibly switch from waking to sleep state remain an unanswered and intriguing question in biological research. In this study, we identified that Shal/Kv4, a well known voltage-gated K+ channel, acts as a controller of wake-sleep transition at dusk in Drosophila circadian neurons. We find that interference of Kv4 function with a dominant-negative form (DNKv4) in subsets of circadian neurons specifically disrupts sleep onset at dusk, although Kv4 itself does not exhibit circadian oscillation. Kv4 preferentially downregulates neuronal firings at ZT9-ZT17, supporting that it plays an essential role in wake-sleep transition at dusk. Our findings may help understand and eventually treat sleep disorders such as insomnia.

TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

As the outermost physical barrier of an organism, the skin is diurnally exposed to UV radiation (UVR). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVR. However, a molecular link between circadian perturbation by UVR and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase (TIMP)-3 as a novel circadian locomotor output cycles kaput (CLOCK)–dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8 via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK. As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVR and UVR-induced inflammation.—Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.

Haeme oxygenase protects against UV light DNA damages in the retina in clock-dependent manner

In the present study, we showed that in the retina of Drosophila, the expression of the ho gene, encoding haeme oxygenase (HO), is regulated by light but only at the beginning of the day. This timing must be set by the circadian clock as light pulses applied at other time points during the day do not increase the ho mRNA level. Moreover, light-induced activation of HO does not depend on the canonical phototransduction pathway but instead involves cryptochrome and is enhanced by ultraviolet (UV) light. Interestingly, the level of DNA damage in the retina after UV exposure was inversely related to the circadian oscillation of the ho mRNA level during the night, being the highest when the HO level was low and reversed during the day. Accordingly, induction of HO by hemin was associated with low DNA damage, while inhibition of HO activity by SnPPIX aggravated the damage. Our data suggest that HO acts in the retina to decrease oxidative DNA damage in photoreceptors caused by UV-rich light in the morning.

Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey.

The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets.

Abstract 15771: Genetic and Pharmacological Regulation of Circadian Energy Metabolism

The circadian clock governs essential metabolic and physiological processes. For example, the sharp rise of blood pressure in the morning coincides with peak incidence rate for cardiovascular episodes such as heart attacks. The objective of the current studies is to elucidate novel mechanisms of genetic and pharmacological regulation of circadian clock and clock-related energy metabolism important for cardiovascular health. On the one hand, we investigated the functional mechanism of the Clockdelta19 mutation. Homozygous mutant mice (Clk/Clk) for this mutation have been shown to exhibit a wide spectrum of metabolic and cardiovascular disorders, correlated with a severe circadian misalignment or loss of rhythmicity in these mice. In comparison, Clockdelta19/+ heterozygous (Clk/+) mice showed sustained circadian rhythms, yet with significantly lengthened periodicity and dampened amplitude. We showed that Clk/+ mice exhibited improved glycemic control and resistance to circadian behavioral period lengthening under high-fat diet (HFD). Molecular analysis revealed that BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD conditions. Mechanistic studies further demonstrated that CLOCKdelta19 stabilized BMAL1 proteins. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT. This study reveals an unforeseen plasticity of the circadian and metabolic networks, providing important insight into potential therapeutic intervention of clocks under disease conditions. On the other hand, we also identified small molecule modulators of the circadian clock. These small molecules, termed C lock amplitude- E nhancing small M olecules (CEMs), were able to enhance circadian amplitude of reporter rhythms in cardiomyocyte cells in vitro and improve energy expenditure and reduce cholesterol levels in vivo in a clock-dependent manner. Our studies together elucidate novel regulatory mechanisms for the circadian clock, and suggest novel therapeutic targets and drug candidates against clock-associated metabolic and cardiovascular diseases.

The MAP kinase p38 is part of Drosophila melanogaster's circadian clock.

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.

Mucosal immunology: killing time in the lungs.

Pulmonary epithelial cells drive rhythmic neutrophil recruitment by secreting chemokines in a clock-dependent manner.

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

A Mechanism for Circadian Control of Pacemaker Neuron Excitability

Although the intracellular molecular clocks that regulate circadian (~24 h) behavioral rhythms are well understood, it remains unclear how molecular clock information is transduced into rhythmic neuronal activity that in turn drives behavioral rhythms. To identify potential clock outputs, the authors generated expression profiles from a homogeneous population of purified pacemaker neurons (LN(v)s) from wild-type and clock mutant Drosophila. They identified a group of genes with enriched expression in LN(v)s and a second group of genes rhythmically expressed in LN(v)s in a clock-dependent manner. Only 10 genes fell into both groups: 4 core clock genes, including period (per) and timeless (tim), and 6 genes previously unstudied in circadian rhythms. The authors focused on one of these 6 genes, Ir, which encodes an inward rectifier K(+) channel likely to regulate resting membrane potential, whose expression peaks around dusk. Reducing Ir expression in LN(v)s increased larval light avoidance and lengthened the period of adult locomotor rhythms, consistent with increased LN(v) excitability. In contrast, increased Ir expression made many adult flies arrhythmic and dampened PER protein oscillations. The authors propose that rhythmic Ir expression contributes to daily rhythms in LN(v) neuronal activity, which in turn feed back to regulate molecular clock oscillations.

Coordination of the transcriptome and metabolome by the circadian clock

The circadian clock governs a large array of physiological functions through the transcriptional control of a significant fraction of the genome. Disruption of the clock leads to metabolic disorders, including obesity and diabetes. As food is a potent zeitgeber (ZT) for peripheral clocks, metabolites are implicated as cellular transducers of circadian time for tissues such as the liver. From a comprehensive dataset of over 500 metabolites identified by mass spectrometry, we reveal the coordinate clock-controlled oscillation of many metabolites, including those within the amino acid and carbohydrate metabolic pathways as well as the lipid, nucleotide, and xenobiotic metabolic pathways. Using computational modeling, we present evidence of synergistic nodes between the circadian transcriptome and specific metabolic pathways. Validation of these nodes reveals that diverse metabolic pathways, including the uracil salvage pathway, oscillate in a circadian fashion and in a CLOCK-dependent manner. This integrated map illustrates the coherence within the circadian metabolome, transcriptome, and proteome and how these are connected through specific nodes that operate in concert to achieve metabolic homeostasis.

Circadian regulation of low density lipoprotein receptor promoter activity by CLOCK/BMAL1, Hes1 and Hes6

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.

The Circadian Clock, Light, and Cryptochrome Regulate Feeding and Metabolism in Drosophila

Recent studies in mammals have demonstrated a central role for the circadian clock in maintaining metabolic homeostasis. In spite of these advances, however, little is known about how these complex pathways are coordinated. Here, we show that fundamental aspects of the circadian control of metabolism are conserved in the fruit fly Drosophila. We assay feeding behavior and basic metabolite levels in individual flies and show that, like mammals, Drosophila display a rapid increase in circulating sugar following a meal, which is subsequently stored in the form of glycogen. These daily rhythms in carbohydrate levels are disrupted in clock mutants, demonstrating a critical role for the circadian clock in the postprandial response to feeding. We also show that basic metabolite levels are coordinated in a clock-dependent manner and that clock function is required to maintain lipid homeostasis. By examining feeding behavior, we show that flies feed primarily during the first 4 hours of the day and that light suppresses a late day feeding bout through the cryptochrome photoreceptor. These studies demonstrate that central aspects of feeding and metabolism are dependent on the circadian clock in Drosophila. Our work also uncovers novel roles for light and cryptochrome on both feeding behavior and metabolism.

Bezafibrate Induces Plasminogen Activator Inhibitor-1 Gene Expression in a CLOCK-Dependent Circadian Manner

A functional interaction between peroxisome proliferator-activated receptor alpha (PPARalpha) and components of the circadian clock has been suggested, but whether these transcriptional factors interact to regulate the expression of their target genes remains obscure. Here we used a PPARalpha ligand, bezafibrate, to search for PPARalpha-regulated genes that are expressed in a CLOCK-dependent circadian manner. Microarray analyses using hepatic RNA isolated from bezafibrate treated-wild type, Clock mutant (Clk/Clk), and PPARalpha-null mice revealed that 136 genes are transcriptionally regulated by PPARalpha in a CLOCK-dependent manner. Among them, we focused on the plasminogen activator inhibitor-1 (PAI-1) gene, because its expression typically shows circadian variation, and it has transcriptional response elements for both PPAR and CLOCK. The bezafibrate-induced expression of PAI-1 mRNA was attenuated in Clk/Clk mice and in PPARalpha-null mice. The protein levels of PPARalpha were reduced in Clk/Clk hepatocytes. However, the overexpression of PPARalpha could not rescue bezafibrate-induced PAI-1 expression in Clk/Clk hepatocytes, suggesting that impaired bezafibrate-induced PAI-1 expression in Clk/Clk mice is not due to reduced PPARalpha expression. Luciferase reporter and chromatin immunoprecipitation analyses using primary hepatocytes demonstrated that DNA binding of both PPARalpha and CLOCK is essential for bezafibrate-induced PAI-1 gene expression. Pull-down assays in vitro showed that both PPARalpha and its heterodimerized partner retinoic acid receptor-alpha can serve as potential interaction targets of CLOCK. The present findings revealed that molecular interaction between the circadian clock and the lipid metabolism regulator affects the bezafibrate-induced gene expression.