Circadian Clock Machinery Research Articles

Reciprocal regulation of carbon monoxide metabolism and the circadian clock.

Circadian clocks are cell-autonomous oscillators regulating daily rhythms in a wide range of physiological, metabolic and behavioral processes. Feedback of metabolic signals, such as redox state, NAD+/NADH and AMP/ADP ratios, or heme, modulate circadian rhythms and thereby optimize energy utilization across the 24-h cycle. We show that rhythmic heme degradation, which generates the signaling molecule carbon monoxide (CO), is required for normal circadian rhythms as well as circadian metabolic outputs. CO suppresses circadian transcription by attenuating CLOCK-BMAL1 binding to target promoters. Pharmacological inhibition or genetic depletion of CO-producing heme oxygenases abrogates normal daily cycles in mammalian cells and Drosophila. In mouse hepatocytes, suppression of CO production leads to a global upregulation of CLOCK-BMAL1-dependent circadian gene expression and dysregulated glucose metabolism. Together, our findings show that CO metabolism is an important link between the basic circadian-clock machinery, metabolism and behavior.

Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

Different Roles of Negative and Positive Components of the Circadian Clock in Oncogene-induced Neoplastic Transformation

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptional-translational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras(V12) and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

Genome-wide association analysis identifies novel loci for chronotype in 100,420 individuals from the UK Biobank.

Our sleep timing preference, or chronotype, is a manifestation of our internal biological clock. Variation in chronotype has been linked to sleep disorders, cognitive and physical performance, and chronic disease. Here we perform a genome-wide association study of self-reported chronotype within the UK Biobank cohort (n=100,420). We identify 12 new genetic loci that implicate known components of the circadian clock machinery and point to previously unstudied genetic variants and candidate genes that might modulate core circadian rhythms or light-sensing pathways. Pathway analyses highlight central nervous and ocular systems and fear-response-related processes. Genetic correlation analysis suggests chronotype shares underlying genetic pathways with schizophrenia, educational attainment and possibly BMI. Further, Mendelian randomization suggests that evening chronotype relates to higher educational attainment. These results not only expand our knowledge of the circadian system in humans but also expose the influence of circadian characteristics over human health and life-history variables such as educational attainment.

The Potential of Circadian Realignment in Rheumatoid Arthritis.

In this short review, we discuss evidence supporting the modulation of peripheral circadian systems as a therapeutic strategy for rheumatoid arthritis (RA). We first review the role of proinflammatory cytokines and oxidative stress, two of the primary mediators of chronic inflammation in RA, and their regulation by circadian clock machinery. We further highlight the role of environmental and metabolic signals in regulating the central and peripheral circadian clocks, with an emphasis on seasonal variations in photoperiod and rhythmic metabolic input, respectively. Finally, we hypothesize that the entrainment and realignment of peripheral clock rhythms have the ability to modulate these mediators, improving clinical outcomes in RA patients. Our discussion emphasizes the use of light therapy and time-restricted feeding for entraining peripheral clocks either via the entrainment of the central circadian clock in suprachiasmatic nuclei (SCN) or directly by uncoupling the peripheral circadian clocks from SCN. In doing so, we highlight the use of nonpharmacologic interventions as a potential strategy for improving clinical outcomes in chronic inflammatory conditions such as RA.

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

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

Disruption of Circadian Rhythms and Delirium, Sleep Impairment and Sepsis in Critically ill Patients. Potential Therapeutic Implications for Increased Light-Dark Contrast and Melatonin Therapy in an ICU Environment.

The confinement of critically ill patients in intensive care units (ICU) imposes environmental constancy throughout both day and night (continuous light, noise, caring activities medications, etc.), which has a negative impact on human health by inducing a new syndrome known as circadian misalignment, circadian disruption or chronodisruption (CD). This syndrome contributes to poor sleep quality and delirium, and may impair septic states frequently observed in critically ill patients. However, and although the bidirectional crosstalk between CD with sleep impairment, delirium and inflammation in animal models has been known for years and has been suspected in ICU patients, few changes have been introduced in the environment and management of ICU patients to improve their circadian rhythmicity. Delirium, the most serious condition because it has a severe effect on prognosis and increases mortality, as well as sleep impairment and sepsis, all three of them linked to disorganization of the circadian system in critically ill patients, will be revised considering the functional organization of the circadian system, the main input and output signals that synchronize the clock, including a brief description of the molecular circadian clock machinery, the non-visual effects of light, and the ICU light environment. Finally, the potential usefulness of increased light/dark contrast and melatonin treatment in this context will be analyzed, including some practical countermeasures to minimize circadian disruption and improve circadian system chronoenhancement, helping to make these units optimal healing environments for patients.

Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD

In animal circadian clock machinery, the phosphorylation program of PERIOD (PER) leads to the spatio-temporal regulation of diverse PER functions, which are crucial for the maintenance of ∼24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. Here, we show that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, we demonstrate that Dod associates preferentially with phosphorylated species of PER, which delays the phosphorylation-dependent degradation of PER. Consistent with this, PER protein levels are higher in flies overexpressing dod. Taken together, we suggest that Dod plays a role in the maintenance of circadian period by regulating PER metabolism.

Mathematical Modeling and Validation of Glucose Compensation of the Neurospora Circadian Clock

Autonomous circadian oscillations arise from transcriptional-translational feedback loops of core clock components. The period of a circadian oscillator is relatively insensitive to changes in nutrients (e.g., glucose), which is referred to as “nutrient compensation”. Recently, a transcription repressor, CSP-1, was identified as a component of the circadian system in Neurospora crassa. The transcription of csp-1 is under the circadian regulation. Intriguingly, CSP-1 represses the circadian transcription factor, WC-1, forming a negative feedback loop that can influence the core oscillator. This feedback mechanism is suggested to maintain the circadian period in a wide range of glucose concentrations. In this report, we constructed a mathematical model of the Neurospora circadian clock incorporating the above WC-1/CSP-1 feedback loop, and investigated molecular mechanisms of glucose compensation. Our model shows that glucose compensation exists within a narrow range of parameter space where the activation rates of csp-1 and wc-1 are balanced with each other, and simulates loss of glucose compensation in csp-1 mutants. More importantly, we experimentally validated rhythmic oscillations of the wc-1 gene expression and loss of glucose compensation in the wc-1ov mutant as predicted in the model. Furthermore, our stochastic simulations demonstrate that the CSP-1-dependent negative feedback loop functions in glucose compensation, but does not enhance the overall robustness of oscillations against molecular noise. Our work highlights predictive modeling of circadian clock machinery and experimental validations employing Neurospora and brings a deeper understanding of molecular mechanisms of glucose compensation.

Chromatin landscape and circadian dynamics: Spatial and temporal organization of clock transcription

Circadian rhythms drive the temporal organization of a wide variety of physiological and behavioral functions in ∼24-h cycles. This control is achieved through a complex program of gene expression. In mammals, the molecular clock machinery consists of interconnected transcriptional–translational feedback loops that ultimately ensure the proper oscillation of thousands of genes in a tissue-specific manner. To achieve circadian transcriptional control, chromatin remodelers serve the clock machinery by providing appropriate oscillations to the epigenome. Recent findings have revealed the presence of circadian interactomes, nuclear “hubs” of genome topology where coordinately expressed circadian genes physically interact in a spatial and temporal-specific manner. Thus, a circadian nuclear landscape seems to exist, whose interplay with metabolic pathways and clock regulators translates into specific transcriptional programs. Deciphering the molecular mechanisms that connect the circadian clock machinery with the nuclear landscape will reveal yet unexplored pathways that link cellular metabolism to epigenetic control.

The circadian clock machinery controls adiponectin expression

Adiponectin, an adipokine involved in glucose and lipid metabolism, exhibits a circadian manner of expression. Adiponectin expression is mediated by the helix–loop–helix transcription factor sterol regulatory element binding protein (SREBP)-1c. In this study, we tested whether the circadian clock helix–loop–helix transcription factors CLOCK and BMAL1 regulate adiponectin expression. We found that adiponectin expression is regulated by the clock through the circadian expression of its transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and its co-activator PPARγ co-activator 1α (PGC1α) in mouse white adipose tissue and differentiated adipocytes. In addition, reconstitution of the core clock mechanism and siRNA experiments in cell culture suggest that the clock directly activates the adiponectin promoter and mediates its expression. In summary, adiponectin expression is regulated by the circadian clock and through the circadian expression of its transcription factor PPARγ and its co-activator PGC1α.

Effects of temperature and photoperiod on daily activity rhythms of Lutzomyia longipalpis (Diptera: Psychodidae).

BackgroundInsect vectors have been established as models in Chronobiology for many decades, and recent studies have demonstrated a close relationship between the circadian clock machinery, daily rhythms of activity and vectorial capacity. Lutzomyia longipalpis, the primary vector of Leishmania (Leishmania) infantum in the New World, is reported to have crepuscular/nocturnal activity in the wild. However, most of these studies applied hourly CDC trap captures, which is a good indicative of L. longipalpis behaviour, but has limited accuracy due to the inability to record the daily activity of a single insect during consecutive days. In addition, very little is known about the activity pattern of L. longipalpis under seasonal variations of average temperature and day length in controlled laboratory conditions.MethodsWe recorded the locomotor activity of L. longipalpis males under different artificial regimes of temperature and photoperiod. First, in order to test the effects of temperature on the activity, sandflies were submitted to regimes of light/dark cycles similar to the equinox photoperiod (LD 12:12) combined with different constant temperatures (20°C, 25°C and 30°C). In addition, we recorded sandfly locomotor activity under a mild constant temperature (25°C with different day length regimes: 8 hours, 12 hours and 16 hours).ResultsL. longipalpis exhibited more activity at night, initiating dusk-related activity (onset time) at higher rather than lower temperatures. In parallel, changes of photoperiod affected anticipation as well as all the patterns of activity (onset, peak and offset time). However, under LD 16:08, sandflies presented the earliest values of maximum peak and offset times, contrary to other regimes.ConclusionsHerein, we showed that light and temperature modulate L. longipalpis behaviour under controlled laboratory conditions, suggesting that sandflies might use environmental information to sustain their crepuscular/nocturnal activity, as well as other important aspects as mating and host-seeking at appropriate times in different seasons. Our results depict previously unappreciated aspects of the L. longipalpis daily rhythms of activity that might have important epidemiological implications.

Circadian angiogenesis.

Daily rhythms of light/darkness, activity/rest and feeding/fasting are important in human physiology and their disruption (for example by frequent changes between day and night shifts) increases the risk of disease. Many of the diseases found to be associated with such disrupted circadian lifestyles, including cancer, cardiovascular diseases, metabolic disorders and neurological diseases, depend on pathological de-regulation of angiogenesis, suggesting that disrupting the circadian clock will impair the physiological regulation of angiogenesis leading to development and progression of these diseases. Today there is little known regarding circadian regulation of pathological angiogenesis but there is some evidence that supports both direct and indirect regulation of angiogenic factors by the cellular circadian clock machinery, as well as by circulating circadian factors, important for coordinating circadian rhythms in the organism. Through highlighting recent advances both in pre-clinical and clinical research on various diseases including cancer, cardiovascular disorders and obesity, we will here present an overview of the available knowledge on the importance of circadian regulation of angiogenesis and discuss how the circadian clock may provide alternative targets for pro- or anti-angiogenic therapy in the future.

Regulation of retinal inflammation by rhythmic expression of MiR-146a in diabetic retina.

Chronic inflammation and dysregulation of circadian rhythmicity are involved in the pathogenesis of diabetic retinopathy. MicroRNAs (miRNAs) can regulate inflammation and circadian clock machinery. We tested the hypothesis that altered daily rhythm of miR-146a expression in diabetes contributes to retinal inflammation. Nondiabetic and STZ-induced diabetic rats kept in 12/12 light/dark cycle were killed every 2 hours over a 72-hour period. Human retinal endothelial cells (HRECs) were synchronized with dexamethasone. Expression of miR-146a, IL-1 receptor-associated kinase 1 (IRAK1), IL-1β, VEGF and ICAM-1, as well as clock genes was examined by real-time PCR and Western blot. To modulate expression levels of miR-146a, mimics and inhibitors were used. Diabetes inhibited amplitude of negative arm (per1) and enhanced amplitude of the positive arm (bmal1) of clock machinery in retina. In addition to clock genes, miR-146a and its target gene IRAK1 also exhibited daily oscillations in antiphase; however, these patterns were lost in diabetic retina. This loss of rhythmic pattern was associated with an increase in ICAM-1, IL-β, and VEGF expression. Human retinal endothelial cells had robust miR-146a expression that followed circadian oscillation pattern; however, HRECs isolated from diabetic donors had reduced miR-146a amplitude but increased amplitude of IRAK1 and ICAM-1. In HRECs, miR-146a mimic or inhibitor caused 1.6- and 1.7-fold decrease or 1.5- and 1.6-fold increase, respectively, in mRNA and protein expression levels of ICAM-1 after 48 hours. Diabetes-induced dysregulation of daily rhythms of miR-146a and inflammatory pathways under miR-146a control have potential implications for the development of diabetic retinopathy.

Induction of the CLOCK gene by E2-ERα signaling promotes the proliferation of breast cancer cells.

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells.

Common threads: epigenetics, metabolism and the clock

Circadian rhythms govern a number of fundamental physiological functions in almost all organisms, from prokaryotes to humans. The circadian clocks are intrinsic time-tracking systems with which organisms can anticipate environmental changes and adapt to the appropriate time of day. Disruption of these rhythms can have a profound influence to human health and has been linked to depression, insomnia, jet lag, coronary heart disease, neurodegenerative disorders and cancer. At the heart of circadian regulatory pathways is the clock machinery, a remarkably coordinated transcription-translation system that utilizes also dynamic changes in chromatin transitions and epigenetic control. Recent findings indicate that regulation goes also the other way, since specific elements of the clock are able to sense changes in the cellular metabolism. Understanding in full detail the intimate links between cellular metabolism and the circadian clock machinery will provide not only critical insights into system physiology and endocrinology, but also novel avenues for pharmacological intervention towards metabolic disorders.

Circadian clock mediates ER stress signaling in alcoholic fatty liver (1116.12)

[Purpose] This is the first study to investigate how alcohol regulates ER stress and fatty liver through nuclear receptor SHP mediated liver circadian clock machinery.[Methods] The NIAAA EtOH binge model was established using 8 weeks old, male C57BL/6 (WT) and SHP‐/‐ (SKO) mice (Nat Protoc 2013; 8:627‐637). In brief, the mice were acclimatized to a control liquid diet for 5 days, then fed with 5% Lieber‐DeCarli diet or pair‐fed for 10 days. By day 11, the mice were gavaged maltose (control) or EtOH solutions at zeitgeber time (ZT) 3 (9 am) and sacrificed at ZT12, 18, 0, and 6.[Results] Metabolomics by GC/MS revealed a global alteration by alcohol in the oscillation of serum intermediate metabolites in pathways of glucose, lipid and amino acid metabolism in SKO mice. The rhythmic expression of core clock genes (Clock, Bmal1, Npas2) and nuclear receptors (Rorα, Rorγ, Rev‐erbα) were disturbed by alcohol. Hepatic lipid metabolic genes and ER stress markers exhibited strong oscillations which were perturbed by alcohol. Chop was a clock controlled gene through the Rorα/Rorγ/Rev‐erbα/SHP regulatory cascade. Liver SREBP‐1c protein cleavage and TG content were drastically induced in control SKO livers, which was markedly decreased by alcohol in both WT and SKO mice.[Conclusions] SHP mediated clock machinery is significantly disrupted by alcohol, which in turn perturbs ER stress to control AFL.Grant Funding Source: Supported by AHA and NIDDK

The clock gene cycle plays an important role in the circadian clock of the cricket Gryllus bimaculatus

To dissect the molecular oscillatory mechanism of the circadian clock in the cricket Gryllus bimaculatus, we have cloned a cDNA of the clock gene cycle (Gb’cyc) and analyzed its structure and function. Gb’cyc contains four functional domains, i.e. bHLH, PAS-A, PAS-B and BCTR domains, and is expressed rhythmically in light dark cycles, peaking at mid night. The RNA interference (RNAi) of Clock (Gb’Clk) and period (Gb’per) reduced the Gb’cyc mRNA levels and abolished the rhythmic expression, suggesting that the rhythmic expression of Gb’cyc is regulated by a mechanism including Gb’Clk and Gb’per. These features are more similar to those of mammalian orthologue of cyc (Bmal1) than those of Drosophila cyc. A single treatment with double-stranded RNA (dsRNA) of Gb’cyc effectively knocked down the Gb’cyc mRNA level and abolished its rhythmic expression. The cyc RNAi failed to disrupt the locomotor rhythm, but lengthened its free-running period in constant darkness (DD). It is thus likely that Gb’cyc is involved in the circadian clock machinery of the cricket. The cyc RNAi crickets showed a rhythmic expression of Gb’per and timeless (Gb’tim) in the optic lobe in DD, explaining the persistence of the locomotor rhythm. Surprisingly, cyc RNAi revealed a rhythmic expression of Gb’Clk in DD which is otherwise rather constitutively expressed in the optic lobe. These facts suggest that the cricket might have a unique clock oscillatory mechanism in which both Gb’cyc and Gb’Clk are rhythmically controlled and that under abundant expression of Gb’cyc the rhythmic expression of Gb’Clk may be concealed.

Retinoic acid-related orphan receptors α and γ: key regulators of lipid/glucose metabolism, inflammation, and insulin sensitivity

Retinoic acid-related orphan receptors RORα and RORγ play a regulatory role in lipid/glucose homeostasis and various immune functions, and have been implicated in metabolic syndrome and several inflammatory diseases. RORα-deficient mice are protected against age- and diet-induced obesity, hepatosteatosis, and insulin resistance. The resistance to hepatosteatosis in RORα-deficient mice is related to the reduced expression of several genes regulating lipid synthesis, transport, and storage. Adipose tissue-associated inflammation, which plays a critical role in the development of insulin resistance, is considerably diminished in RORα-deficient mice as indicated by the reduced infiltration of M1 macrophages and decreased expression of many proinflammatory genes. Deficiency in RORγ also protects against diet-induced insulin resistance by a mechanism that appears different from that in RORα deficiency. Recent studies indicated that RORs provide an important link between the circadian clock machinery and its regulation of metabolic genes and metabolic syndrome. As ligand-dependent transcription factors, RORs may provide novel therapeutic targets in the management of obesity and associated metabolic diseases, including hepatosteatosis, adipose tissue-associated inflammation, and insulin resistance.

Circadian clock genes Per1 and Per2 regulate the response of metabolism-associated transcripts to sleep disruption.

Human and animal studies demonstrate that short sleep or poor sleep quality, e.g. in night shift workers, promote the development of obesity and diabetes. Effects of sleep disruption on glucose homeostasis and liver physiology are well documented. However, changes in adipokine levels after sleep disruption suggest that adipocytes might be another important peripheral target of sleep. Circadian clocks regulate metabolic homeostasis and clock disruption can result in obesity and the metabolic syndrome. The finding that sleep and clock disruption have very similar metabolic effects prompted us to ask whether the circadian clock machinery may mediate the metabolic consequences of sleep disruption. To test this we analyzed energy homeostasis and adipocyte transcriptome regulation in a mouse model of shift work, in which we prevented mice from sleeping during the first six hours of their normal inactive phase for five consecutive days (timed sleep restriction – TSR). We compared the effects of TSR between wild-type and Per1/2 double mutant mice with the prediction that the absence of a circadian clock in Per1/2 mutants would result in a blunted metabolic response to TSR. In wild-types, TSR induces significant transcriptional reprogramming of white adipose tissue, suggestive of increased lipogenesis, together with increased secretion of the adipokine leptin and increased food intake, hallmarks of obesity and associated leptin resistance. Some of these changes persist for at least one week after the end of TSR, indicating that even short episodes of sleep disruption can induce prolonged physiological impairments. In contrast, Per1/2 deficient mice show blunted effects of TSR on food intake, leptin levels and adipose transcription. We conclude that the absence of a functional clock in Per1/2 double mutants protects these mice from TSR-induced metabolic reprogramming, suggesting a role of the circadian timing system in regulating the physiological effects of sleep disruption.