Tissue-specific Clocks Research Articles

Comparison of the Effects of Inappropriate Meal Timing-Induced and Genetic Models of Circadian Clock Disruption on Uterine mRNA Expression Profiles

BackgroundAccumulating evidence reveals that inappropriate meal timing contributes to the development of lifestyle-related diseases. An underlying mechanism is thought to be the disruption of the intracellular circadian clock in various tissues based on observations in both systemic and tissue-specific clock gene-deficient mice. However, whether the effects of conditional clock gene knockout are comparable to those of inappropriate meal timing remains unclear. ObjectivesThis study aimed to compare the effects of a recently developed 28-h feeding cycle model with those of a core clock gene Bmal1 uterine conditional knockout (Bmal1 cKO) model on uterine mRNA expression profiles. MethodsThe models were generated by subjecting C57BL/6J mice to an 8-h/20-h feeding/fasting cycle for 2 wk and crossing Bmal1-floxed mice with PR-Cre mice. Microarray analyses were conducted using uterine samples obtained at the beginning of the dark and light periods. ResultsThe analyses identified 516 and 346, significantly 4-fold and 2-fold, up- or downregulated genes in the 28-h feeding cycle and Bmal1 cKO groups, respectively, compared with each control group. Among these genes, only 7 (1.4%) and 63 (18.2%) were significantly up- or downregulated in the other model. Moreover, most (n = 44, 62.9%) of these genes were oppositely regulated. These findings were confirmed by gene set enrichment analyses. ConclusionsThis study reveals that a 28-h feeding cycle and Bmal1 cKO differently affect gene expression profiles and highlights the need for considering this difference to assess the pathophysiology of diseases associated with inappropriate meal timing.

Where are we in the implementation of tissue-specific epigenetic clocks?

Introduction: DNA methylation clocks presents advantageous characteristics with respect to the ambitious goal of identifying very early markers of disease, based on the concept that accelerated ageing is a reliable predictor in this sense. Methods: Such tools, being epigenomic based, are expected to be conditioned by sex and tissue specificities, and this work is about quantifying this dependency as well as that from the regression model and the size of the training set. Results: Our quantitative results indicate that elastic-net penalization is the best performing strategy, and better so when-unsurprisingly-the data set is bigger; sex does not appear to condition clocks performances and tissue specific clocks appear to perform better than generic blood clocks. Finally, when considering all trained clocks, we identified a subset of genes that, to the best of our knowledge, have not been presented yet and might deserve further investigation: CPT1A, MMP15, SHROOM3, SLIT3, and SYNGR. Conclusion: These factual starting points can be useful for the future medical translation of clocks and in particular in the debate between multi-tissue clocks, generally trained on a large majority of blood samples, and tissue-specific clocks.

AGE-ASSOCIATED DECLINE IN TISSUE CIRCADIAN CLOCK FUNCTION

Abstract Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence has demonstrated age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profile the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in three age groups. We find age-dependent and tissue-specific clock output changes but these changes exhibit tissue specific patterns. In general, aging reduces the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. These REGs are enriched for the hallmarks of aging, adding another dimension to our understanding of aging. We determined there were tissue specific changes in the phase distribution of REGs with the hypothalamus and skeletal muscle tissues of the old mice showing a large reduction with REG peak expression at only one phase of the day. Analyzing all expressed genes within a tissue for differences at four distinct times of day identified unique clusters of differentially expressed genes (DEGs). These outcomes identify non-circadian but temporally distinct age associated changes in gene expression. This analysis extends the landscape for understanding aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression.

Insights into ageing rates comparison across tissues from recalibrating cerebellum DNA methylation clock

DNA methylation (DNAm)-based age clocks have been studied extensively as a biomarker of human ageing and a risk factor for age-related diseases. Despite different tissues having vastly different rates of proliferation, it is still largely unknown whether they age at different rates. It was previously reported that the cerebellum ages slowly; however, this claim was drawn from a single clock using a relatively small sample size and so warrants further investigation. We collected the largest cerebellum DNAm dataset (N = 752) to date. We found the respective epigenetic ages are all severely underestimated by six representative DNAm age clocks, with the underestimation effects more pronounced in the four clocks whose training datasets do not include brain-related tissues. We identified 613 age-associated CpGs in the cerebellum, which accounts for only 14.5% of the number found in the middle temporal gyrus from the same population (N = 404). From the 613 cerebellum age-associated CpGs, we built a highly accurate age prediction model for the cerebellum named CerebellumClockspecific (Pearson correlation=0.941, MAD=3.18 years). Ageing rate comparisons based on the two tissue-specific clocks constructed on the 201 overlapping age-associated CpGs support the cerebellum has younger DNAm age. Nevertheless, we built BrainCortexClock to prove a single DNAm clock is able to unbiasedly estimate DNAm ages of both cerebellum and cerebral cortex, when they are adequately and equally represented in the training dataset. Comparing ageing rates across tissues using DNA methylation multi-tissue clocks is flawed. The large underestimation of age prediction for cerebellums by previous clocks mainly reflects the improper usage of these age clocks. There exist strong and consistent ageing effects on the cerebellar methylome, and we suggest the smaller number of age-associated CpG sites in cerebellum is largely attributed to its extremely low average cell replication rates.

Adrenal-Kidney Clock Crosstalk: Utilizing Tissue-Specific Clock KO Models

Circadian rhythms are 24-hour oscillations that regulate many physiological functions including hormone signaling and electrolyte excretion. These rhythms are coordinated by a molecular clock, comprised of 4 key core clock proteins – BMAL1, CLOCK, CRY, and PER. Our lab investigates the role of the adrenal and kidney clocks on renal function. Previous data from the lab has shown that kidney-specific knockout (KO) of PER1 led to increased renal sodium retention in male mice which was associated with increased serum aldosterone and increased expression of aldosterone synthesis genes in the adrenal gland. Interestingly, these mice also displayed altered adrenal clock gene expression. This led to our hypothesis that adrenal-kidney clock crosstalk exists with implications for the regulation of renal and adrenal function.To test this, we utilized adrenal (AS)- and kidney-specific (KS) Bmal1 KO mouse models to assess clock genes in the adrenal glands and kidneys of these mice. Kidney, separated into cortex and medulla, from AS- Bmal1 KO male and adrenal glands from KS- Bmal1 KO male mice and littermate Cre negative, floxed control mice ( n=7-8/genotype) were collected at 6AM (lights on) and 6PM (lights off). RNA was isolated from tissue for clock gene expression analysis using qPCR, specifically Bmal1, Clock, Cry1, Cry2 and Per1, and analyzed using 2-way ANOVA with Sidak multiple comparisons post hoc analysis.Here, we show significant differences in clock gene expression between the kidney cortex and medulla of AS- Bmal1 KO male mice compared with controls. There was a significant interaction between time and genotype for Bmal1, Clock and Cry1 expression with post hoc analysis showing increases in Bmal1 ( p=0.0070) and Cry1 ( p=0.0235) at 6AM in KO animals vs. controls, whereas Clock was significantly decreased at 6PM ( p=0.0108). A similar change was seen with medulla Bmal1 expression, but there was a significant interaction in medulla Per1 showing significant increases at 6PM ( p=0.0465). Interestingly, KS- Bmal1 KO mice displayed significant genotype effects in both adrenal Bmal1 and Clock with post hoc analysis showing decreases at 6AM ( p=0.0035, and p<0.0001, respectively).Tissue-specific KO of BMAL1 has distinct effects on the clock in different peripheral tissues, challenging the current paradigm of the clock mechanism and suggesting that the clock may function differently across tissues. Furthermore, these data support our hypothesis that adrenal-kidney clock crosstalk exists as knocking out BMAL1 in adrenal gland resulted in changes in clock gene expression in the kidney and vice versa. Future work will utilize these tissue-specific Bmal1 KO models to focus on whether peripheral clock cross talk differs between males and females and whether it contributes to regulation of renal and adrenal function. AHA, NIDDK This is the full abstract presented at the American Physiology Summit 2023 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.

Defining the age-dependent and tissue-specific circadian transcriptome in male mice

Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence has demonstrated age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profile the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in three age groups. We find age-dependent and tissue-specific clock output changes. Aging reduces the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. REGs are enriched for the hallmarks of aging, adding another dimension to our understanding of aging. Analyzing differential gene expression within a tissue at four different times of day identifies distinct clusters of differentially expressed genes (DEGs). Increased variability of gene expression across the day is a common feature of aged tissues. This analysis extends the landscape for understanding aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression.

The interplay between circadian clock and viral infections: A molecular perspective.

The circadian clock influences almost every aspect of mammalian behavioral, physiological and metabolic processes. Being a hierarchical network, the circadian clock is driven by the central clock in the brain and is composed of several peripheral tissue-specific clocks. It orchestrates and synchronizes the daily oscillations of biological processes to the environment. Several pathological events are influenced by time and seasonal variations and as such implicate the clock in pathogenesis mechanisms. In context with viral infections, circadian rhythmicity is closely associated with host susceptibility, disease severity, and pharmacokinetics and efficacies of antivirals and vaccines. Leveraging the circadian molecular mechanism insights has increased our understanding of clock infection biology and proposes new avenues for viral diagnostics and therapeutics. In this chapter, we address the molecular interplay between the circadian clock and viral infections and discuss the importance of chronotherapy as a complementary approach to conventional medicines, emphasizing the significance of virus-clock studies.

Circadian Clock Desynchronization and Insulin Resistance.

The circadian rhythm regulates biological processes that occur within 24 h in living organisms. It plays a fundamental role in maintaining biological functions and responds to several inputs, including food intake, light/dark cycle, sleep/wake cycle, and physical activity. The circadian timing system comprises a central clock located in the suprachiasmatic nucleus (SCN) and tissue-specific clocks in peripheral tissues. Several studies show that the desynchronization of central and peripheral clocks is associated with an increased incidence of insulin resistance (IR) and related diseases. In this review, we discuss the current knowledge of molecular and cellular mechanisms underlying the impact of circadian clock dysregulation on insulin action. We focus our attention on two possible mediators of this interaction: the phosphatases belonging to the pleckstrin homology leucine-rich repeat protein phosphatase family (PHLPP) family and the deacetylase Sirtuin1. We believe that literature data, herein summarized, suggest that a thorough change of life habits, with the return to synchronized food intake, physical activity, and rest, would doubtless halt the vicious cycle linking IR to dysregulated circadian rhythms. However, since such a comprehensive change may be incompatible with the demand of modern society, clarifying the pathways involved may, nonetheless, contribute to the identification of therapeutic targets that may be exploited to cure or prevent IR-related diseases.

Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster.

Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila, which we term Locally Activatable BioLuminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.

Keeping time in the dark: Potato diel and circadian rhythmic gene expression reveals tissue-specific circadian clocks.

The circadian clock is an internal molecular oscillator and coordinates numerous physiological processes through regulation of molecular pathways. Tissue‐specific clocks connected by mobile signals have previously been found to run at different speeds in Arabidopsis thaliana tissues. However, tissue variation in circadian clocks in crop species is unknown. In this study, leaf and tuber global gene expression in cultivated potato under cycling and constant environmental conditions was profiled. In addition, we used a circadian‐regulated luciferase reporter construct to study tuber gene expression rhythms. Diel and circadian expression patterns were present among 17.9% and 5.6% of the expressed genes in the tuber. Over 500 genes displayed differential tissue specific diel phases. Intriguingly, few core circadian clock genes had circadian expression patterns, while all such genes were circadian rhythmic in cultivated tomato leaves. Furthermore, robust diel and circadian transcriptional rhythms were observed among detached tubers. Our results suggest alternative regulatory mechanisms and/or clock composition is present in potato, as well as the presence of tissue‐specific independent circadian clocks. We have provided the first evidence of a functional circadian clock in below‐ground storage organs, holding important implications for other storage root and tuberous crops.

Epigenetic clock and methylation studies in vervet monkeys

DNA methylation-based biomarkers of aging have been developed for many mammals but not yet for the vervet monkey (Chlorocebus sabaeus), which is a valuable non-human primate model for biomedical studies. We generated novel DNA methylation data from vervet cerebral cortex, blood, and liver using highly conserved mammalian CpGs represented on a custom array (HorvathMammalMethylChip40). We present six DNA methylation-based estimators of age: vervet multi-tissue epigenetic clock and tissue-specific clocks for brain cortex, blood, and liver. In addition, we developed two dual species clocks (human-vervet clocks) for measuring chronological age and relative age, respectively. Relative age was defined as ratio of chronological age to maximum lifespan to address the species differences in maximum lifespan. The high accuracy of the human-vervet clocks demonstrates that epigenetic aging processes are evolutionary conserved in primates. When applying these vervet clocks to tissue samples from another primate species, rhesus macaque, we observed high age correlations but strong offsets. We characterized CpGs that correlate significantly with age in the vervet. CpG probes that gain methylation with age across tissues were located near the targets of Polycomb proteins SUZ12 and EED and genes possessing the trimethylated H3K27 mark in their promoters. The epigenetic clocks are expected to be useful for anti-aging studies in vervets.

Estimating breast tissue-specific DNA methylation age using next-generation sequencing data

BackgroundDNA methylation (DNAm) age has been widely accepted as an epigenetic biomarker for biological aging. Emerging evidence suggests that DNAm age can be tissue-specific and female breast tissue ages faster than other parts of the body. The Horvath clock, which estimates DNAm age across multiple tissues, has been shown to be poorly calibrated in breast issue. We aim to develop a model to estimate breast tissue-specific DNAm age.MethodsGenome-wide DNA methylation sequencing data were generated for 459 normal, 107 tumor, and 45 paired adjacent-normal breast tissue samples. We determined a novel set of 286 breast tissue-specific clock CpGs using penalized linear regression and developed a model to estimate breast tissue-specific DNAm age. The model was applied to estimate breast tissue-specific DNAm age in different breast tissue types and in tumors with distinct clinical characteristics to investigate cancer-related aging effects.ResultsOur estimated breast tissue-specific DNAm age was highly correlated with chronological age (r = 0.88; p = 2.9 × 10−31) in normal breast tissue. Breast tumor tissue samples exhibited a positive epigenetic age acceleration, where DNAm age was on average 7 years older than respective chronological age (p = 1.8 × 10−8). In age-matched analyses, tumor breast tissue appeared 12 and 13 years older in DNAm age than adjacent-normal and normal breast tissue (p = 4.0 × 10−6 and 1.0 × 10−6, respectively). Both HER2+ and hormone-receptor positive subtypes demonstrated significant acceleration in DNAm ages (p = 0.04 and 3.8 × 10−6, respectively), while no apparent DNAm age acceleration was observed for triple-negative breast tumors. We observed a non-linear pattern of epigenetic age acceleration with breast tumor grade. In addition, early-staged tumors showed a positive epigenetic age acceleration (p = 0.003) while late-staged tumors exhibited a non-significant negative epigenetic age acceleration (p = 0.10).ConclusionsThe intended applications for this model are wide-spread and have been shown to provide biologically meaningful results for cancer-related aging effects in breast tumor tissue. Future studies are warranted to explore whether breast tissue-specific epigenetic age acceleration is predictive of breast cancer development, treatment response, and survival as well as the clinical utility of whether this model can be extended to blood samples.

DNA METHYLATION CLOCKS IN MOUSE

One of important limiting factors in aging research is the time required to measure the effect of an intervention on lifespan. But this situation is now changing due to a recent discovery of DNA methylation-based markers (DNAm clocks). We developed a whole lifespan multi-tissue DNAm clock for mice with R2 =0.89. We also carried out comparative analyses of the available mouse DNAm clocks (single- or multi-tissue, based on different number of sites, based on one genomic locus or multi-loci). In general, tissue specific clocks are more accurate than muti-tissue clocks. We applied these tools to a variety of experimental systems, ranging from interventions to rejuvenation approaches, and analyzed various mouse tissues and public datasets. We further applied DNAm clocks to newly sequenced sets of blood and liver samples. Multi-loci blood clock outperforms other clocks when applied to blood samples, and the liver and multi-tissue clocks show similar precision on liver.

Distinct Circadian Signatures in Liver and Gut Clocks Revealed by Ketogenic Diet

The circadian clock orchestrates rhythms in physiology and behavior, allowing organismal adaptation to daily environmental changes. While food intake profoundly influences diurnal rhythms in the liver, how nutritional challenges are differentially interpreted by distinct tissue-specific clocks remains poorly explored. Ketogenic diet (KD) is considered to have metabolic and therapeutic value, though its impact on circadian homeostasis is virtually unknown. We show that KD has profound and differential effects on liver and intestine clocks. Specifically, the amplitude of clock-controlled genes and BMAL1 chromatin recruitment are drastically altered by KD in the liver, but not in the intestine. KD induces nuclear accumulation of PPARα in both tissues but with different circadian phase. Also, gut and liver clocks respond differently to carbohydrate supplementation to KD. Importantly, KD induces serum and intestinal β-hydroxyl-butyrate levels to robustly oscillate in a circadian manner, an event coupled to tissue-specific cyclic histone deacetylase (HDAC) activity and histone acetylation.

Circadian rhythm in mRNA expression of the glutathione synthesis gene Gclc is controlled by peripheral glial clocks in Drosophila melanogaster.

Circadian coordination of metabolism, physiology, and behaviour is found in all living kingdoms. Clock genes are transcriptional regulators, and their rhythmic activities generate daily rhythms in clock-controlled genes which result in cellular and organismal rhythms. Insects provide numerous examples of rhythms in behaviour and reproduction, but less is known about control of metabolic processes by circadian clocks in insects. Recent data suggest that several pathways involved in protecting cells from oxidative stress may be modulated by the circadian system, including genes involved in glutathione (GSH) biosynthesis. Specifically, rhythmic expression of the gene encoding the catalytic subunit (Gclc) of the rate-limiting GSH biosynthetic enzyme was detected in Drosophila melanogaster heads. The aim of this study was to determine which clocks in the fly multi-oscillatory circadian system are responsible for Gclc rhythms. Genetic disruption of tissue-specific clocks in D. melanogaster revealed that transcriptional rhythms in Gclc mRNA levels occur independently of the central pacemaker neurons, because these rhythms persisted in heads of behaviourally arrhythmic flies with a disabled central clock but intact peripheral clocks. Disrupting the clock specifically in glial cells abolished rhythmic expression of Gclc, suggesting that glia play an important role in Gclc transcriptional regulation, which may contribute to maintaining homeostasis in the fly nervous system.

Acute Sleep Loss Induces Tissue-Specific Epigenetic and Transcriptional Alterations to Circadian Clock Genes in Men.

Shift workers are at increased risk of metabolic morbidities. Clock genes are known to regulate metabolic processes in peripheral tissues, eg, glucose oxidation. This study aimed to investigate how clock genes are affected at the epigenetic and transcriptional level in peripheral human tissues following acute total sleep deprivation (TSD), mimicking shift work with extended wakefulness. In a randomized, two-period, two-condition, crossover clinical study, 15 healthy men underwent two experimental sessions: x sleep (2230-0700 h) and overnight wakefulness. On the subsequent morning, serum cortisol was measured, followed by skeletal muscle and subcutaneous adipose tissue biopsies for DNA methylation and gene expression analyses of core clock genes (BMAL1, CLOCK, CRY1, PER1). Finally, baseline and 2-h post-oral glucose load plasma glucose concentrations were determined. In adipose tissue, acute sleep deprivation vs sleep increased methylation in the promoter of CRY1 (+4%; P = .026) and in two promoter-interacting enhancer regions of PER1 (+15%; P = .036; +9%; P = .026). In skeletal muscle, TSD vs sleep decreased gene expression of BMAL1 (-18%; P = .033) and CRY1 (-22%; P = .047). Concentrations of serum cortisol, which can reset peripheral tissue clocks, were decreased (2449 ± 932 vs 3178 ± 723 nmol/L; P = .039), whereas postprandial plasma glucose concentrations were elevated after TSD (7.77 ± 1.63 vs 6.59 ± 1.32 mmol/L; P = .011). Our findings demonstrate that a single night of wakefulness can alter the epigenetic and transcriptional profile of core circadian clock genes in key metabolic tissues. Tissue-specific clock alterations could explain why shift work may disrupt metabolic integrity as observed herein.

Tissue-specific clocks in Arabidopsis show asymmetric coupling.

Many organisms rely on a circadian clock system to adapt to daily and seasonal environmental changes. The mammalian circadian clock consists of a central clock in the suprachiasmatic nucleus that has tightly coupled neurons and synchronizes other clocks in peripheral tissues. Plants also have a circadian clock, but plant circadian clock function has long been assumed to be uncoupled. Only a few studies have been able to show weak, local coupling among cells. Here, by implementing two novel techniques, we have performed a comprehensive tissue-specific analysis of leaf tissues, and show that the vasculature and mesophyll clocks asymmetrically regulate each other in Arabidopsis. The circadian clock in the vasculature has characteristics distinct from other tissues, cycles robustly without environmental cues, and affects circadian clock regulation in other tissues. Furthermore, we found that vasculature-enriched genes that are rhythmically expressed are preferentially expressed in the evening, whereas rhythmic mesophyll-enriched genes tend to be expressed in the morning. Our results set the stage for a deeper understanding of how the vasculature circadian clock in plants regulates key physiological responses such as flowering time.

Running on time: the role of circadian clocks in the musculoskeletal system.

The night and day cycle governs the circadian (24 hourly) rhythm of activity and rest in animals and humans. This is reflected in daily changes of the global gene expression pattern and metabolism, but also in the local physiology of various tissues. A central clock in the brain co-ordinates the rhythmic locomotion behaviour, as well as synchronizing various local oscillators, such as those found in the musculoskeletal system. It has become increasingly recognized that the internal molecular clocks in cells allow a tissue to anticipate the rhythmic changes in their local environment and the specific demands of that tissue. Consequently, the majority of the rhythmic clock controlled genes and pathways are tissue specific. The concept of the tissue-specific function of circadian clocks is further supported by the diverse musculoskeletal phenotypes in mice with deletions or mutations of various core clock components, ranging from increased bone mass, dwarfism, arthropathy, reduced muscle strength and tendon calcification. The present review summarizes the current understanding of the circadian clocks in muscle, bone, cartilage and tendon tissues, with particular focus on the evidence of circadian rhythms in tissue physiology, their entrainment mechanisms and disease links, and the tissue-specific clock target genes/pathways. Research in this area holds strong potential to advance our understanding of how circadian rhythms control the health and disease of the musculoskeletal tissues, which has major implications in diseases associated with advancing age. It could also have potential implications in sports performance and sports medicine.

Physiological links between circadian rhythms, metabolism and nutrition.

Circadian rhythms, metabolism and nutrition are closely interlinked. A great deal of recent research has investigated not only how aspects of metabolic physiology are driven by circadian clocks, but also how these circadian clocks are themselves sensitive to metabolic change. At the cellular level, novel feedback loops have been identified that couple circadian 'clock genes' and their proteins to expression of nuclear receptors, regulation of redox state and other major pathways. Using targeted disruption of circadian clocks, mouse models are providing novel insight into the role of tissue-specific clocks in glucose homeostasis and body weight regulation. The relationship between circadian rhythms and obesity appears complex, with variable alteration of rhythms in obese individuals. However, it is clear from animal studies that the timing and nutritional composition of meals can regulate circadian rhythms, particularly in peripheral tissues. Translation of these findings to human physiology now represents an important goal.

Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models

In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.