Alterations of lipid homeostasis in morbid obese patients are partly reversed by bariatric surgery.

Besides its beneficial effect on weight loss, gastric bypass surgery (GBS) may impact the circulating levels of phospho- and sphingolipids. However, long-term effects have not been explored. To investigate alterations in lipidomic signatures associated with massive weight loss following GBS, we conducted direct infusion tandem mass spectrometry on serum and subcutaneous adipose tissue (SAT) samples collected in a longitudinal cohort of morbid obese patients prior to GBS and 1 year following the surgery. A tissue-specific rearrangement of 13% among over 400 phospholipid and sphingolipid species quantified in serum and SAT was observed 1 year following GBS, with a substantial reduction of ceramide levels and increased amount of hexosylceramides detected in both tissues. The comparison of these new lipidomic profiles with the serum and SAT lipidomes established from an independent cohort of lean and morbid obese subjects revealed that GBS partly restored the lipid alterations associated with morbid obesity.

Multi-omics correlates of insulin resistance and circadian parameters mapped directly from human serum.

While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome-wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi-omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells.

Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis.

Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.

Circulating 1,5-Anhydroglucitol as a Biomarker of ß-cell Mass Independent of a Diabetes Phenotype in Human Subjects.

CONTEXT: During an asymptomatic prediabetic state, the functional ß-cell mass decreases to a critical threshold, triggering diabetes and related symptoms. To date, there are no reliable readouts able to capture in vivo a potential drop of the ß-cell mass. OBJECTIVE: Beside its use as a short-term marker of glycemic control, the deoxyhexose 1,5-anhydroglucitol was identified in rodents as a circulating biomarker of the functional ß-cell mass already in the asymptomatic prediabetic stage. The present study investigated the putative corresponding relevance of circulating 1,5-anhydroglucitol in different human cohorts. METHODS: We analyzed clinical and blood parameters in patients with established type 2 diabetes and subjects considered at high risk of developing diabetes, as well as patients with no history of diabetes scheduled for pancreaticoduodenectomy. RESULTS: Circulating 1,5-anhydroglucitol was reduced in type 2 diabetic patients, negatively correlating with fasting plasma glucose (P < 0.0001) and hemoglobin A1c (P < 0.0001). In healthy subjects, 1,5-AG levels positively correlated with body mass index (P = 0.004) and Homeostatic Model Assessment of Insulin Resistance %S (P < 0.03) and was particularly high in nondiabetic obese individuals, potentially accounting for compensatory ß-cell expansion. Patients with no history of diabetes undergoing pancreaticoduodenectomy exhibited a 50% reduction of circulating 1,5-anhydroglucitol levels following surgery leading to an acute loss of their ß-cell mass (P = 0.002), regardless their glucose tolerance status. CONCLUSION: In summary, plasma concentration of 1,5-anhydroglucitol follows the ß-cell mass and its noninvasive monitoring may alert about the loss of ß cells in subjects at risk for diabetes, an event that cannot be captured by other clinical parameters of glycemic control.

Ether lipids, sphingolipids and toxic 1-deoxyceramides as hallmarks for lean and obese type 2 diabetic patients.

AIM: The worldwide increase in obesity and type 2 diabetes (T2D) represents a major health challenge. Chronically altered lipids induced by obesity further promote the development of T2D, and the accumulation of toxic lipid metabolites in serum and peripheral organs may contribute to the diabetic phenotype. METHODS: To better understand the complex metabolic pattern of lean and obese T2D and non-T2D individuals, we analysed the lipid profile of human serum, skeletal muscle and visceral adipose tissue of two cohorts by systematic mass spectrometry-based lipid analysis. RESULTS: Lipid homeostasis was strongly altered in a disease- and tissue-specific manner, allowing us to define T2D signatures associated with obesity from those that were obesity independent. Lipid changes encompassed lyso-, diacyl- and ether-phospholipids. Moreover, strong changes in sphingolipids included cytotoxic 1-deoxyceramide accumulation in a disease-specific manner in serum and visceral adipose tissue. The high amounts of non-canonical 1-deoxyceramide present in human adipose tissue most likely come from cell-autonomous synthesis because 1-deoxyceramide production increased upon differentiation to adipocytes in mouse cell culture experiments. CONCLUSION: Taken together, the observed lipidome changes in obesity and T2D will facilitate the identification of T2D patient subgroups and represent an important step towards personalized medicine in diabetes.

Proinflammatory Cytokines Perturb Mouse and Human Pancreatic Islet Circadian Rhythmicity and Induce Uncoordinated ß-Cell Clock Gene Expression via Nitric Oxide, Lysine Deacetylases, and Immunoproteasomal Activity.

Pancreatic ß-cell-specific clock knockout mice develop ß-cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine interleukin-1ß (IL-1ß) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1ß and interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/ß agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast, low (<5,0 µM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1ß mediated ß-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3, and immunoproteasome activity.

The importance of being rhythmic: Living in harmony with your body clocks.

Circadian rhythms have developed in all light-sensitive organisms, including humans, as a fundamental anticipatory mechanism that enables proactive adaptation to environmental changes. The circadian system is organized in a highly hierarchical manner, with clocks operative in most cells of the body ensuring the temporal coordination of physiological processes. Circadian misalignment, stemming from modern life style, draws increasing attention due to its tight association with the development of metabolic, cardiovascular, inflammatory and mental diseases as well as cancer. This review highlights recent findings emphasizing the role of the circadian system in the temporal orchestration of physiology, with a particular focus on implications of circadian misalignment in human pathologies.

Validation of molecular biomarkers for preoperative diagnostics of human papillary thyroid carcinoma in fine needle aspirates.

BACKGROUND: Despite substantial efforts, reliable preoperative diagnostic for human thyroid malignancies in case of cytologically indeterminate nodules is still missing, resulting in high number of unnecessary thyroidectomies. In an attempt to increase precision of existing preoperative diagnostics, we aimed at validating the panel of molecular biomarkers predictive for papillary thyroid carcinoma (PTC) in preoperative fine needle aspirate (FNA) samples. METHODS: In this prospective study conducted in preoperative thyroid FNA from 44 thyroid nodules, expression levels of 11 molecular biomarkers previously validated on the postoperative samples of PTCs were measured by Cell-to-CT and QuantiGene Plex methods and correlated with final diagnosis. RESULTS: The QuantiGene Plex resulted in reliable gene expression measurements for FNA and core-needle biopsy (CNB) samples, however this method was less sensitive than pre-amplification based Cell-to-CT. Measurements conducted on the same samples by the two methods significantly correlated for most of the genes. Expression levels of TIMP1, c-MET and ARNTL were upregulated in PTC nodules as compared to benign counterparts, supporting previous post-operative studies. Strong correlation was observed between these biomarker alterations in the same samples. Within the sub-group of 15 indeterminate nodules (Bethesda II-V), TIMP1 had 100% specificity and 83% sensitivity for PTC cases. CONCLUSIONS: Assessment of TIMP1, c-MET and core-clock gene ARNTL expression levels by QuantiGene Plex assay in FNA samples holds promise as an ancillary method to the cytological preoperative diagnostics.

Cellular circadian period length inversely correlates with HbA1c levels in individuals with type 2 diabetes.

AIMS/HYPOTHESIS: The circadian system plays an essential role in regulating the timing of human metabolism. Indeed, circadian misalignment is strongly associated with high rates of metabolic disorders. The properties of the circadian oscillator can be measured in cells cultured in vitro and these cellular rhythms are highly informative of the physiological circadian rhythm in vivo. We aimed to discover whether molecular properties of the circadian oscillator are altered as a result of type 2 diabetes. METHODS: We assessed molecular clock properties in dermal fibroblasts established from skin biopsies taken from nine obese and eight non-obese individuals with type 2 diabetes and 11 non-diabetic control individuals. Following in vitro synchronisation, primary fibroblast cultures were subjected to continuous assessment of circadian bioluminescence profiles based on lentiviral luciferase reporters. RESULTS: We observed a significant inverse correlation (ρ = -0.592; p < 0.05) between HbA1c values and circadian period length within cells from the type 2 diabetes group. RNA sequencing analysis conducted on samples from this group revealed that ICAM1, encoding the endothelial adhesion protein, was differentially expressed in fibroblasts from individuals with poorly controlled vs well-controlled type 2 diabetes and its levels correlated with cellular period length. Consistent with this circadian link, the ICAM1 gene also displayed rhythmic binding of the circadian locomotor output cycles kaput (CLOCK) protein that correlated with gene expression. CONCLUSIONS/INTERPRETATION: We provide for the first time a potential molecular link between glycaemic control in individuals with type 2 diabetes and circadian clock machinery. This paves the way for further mechanistic understanding of circadian oscillator changes upon type 2 diabetes development in humans. DATA AVAILABILITY: RNA sequencing data and clinical phenotypic data have been deposited at the European Genome-phenome Archive (EGA), which is hosted by the European Bioinformatics Institute (EBI) and the Centre for Genomic Regulation (CRG), ega-box-1210, under accession no. EGAS00001003622.

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism.

Context: Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective: To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design: Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results: The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions: The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology.

The search for preoperative biomarkers for thyroid carcinoma: application of the thyroid circadian clock properties.

Accumulating evidence suggests that alterations in the molecular clocks underlying the circadian time-keeping system might be connected to changes in cell cycle, resulting in oncogenic transformation. The hypothalamic-pituitary-thyroid axis is driven by a circadian clock at several levels, with an endocrine feedback loop regulating thyroid-stimulating hormone. Changes in the expression levels of circadian and cell cycle markers may correlate with clinic-pathological characteristics in differentiated follicular thyroid carcinomas. Here we summarize recent advances in exploring complex regulation of the thyroid gland transcriptome and function by the circadian oscillator. We particularly focus on clinical implications of the parallel assessment of the circadian clock, cell-cycle and cell functionality markers in human thyroid tissue, which might help improving preoperative diagnostics of thyroid malignancies.

Identification of CHEK1, SLC26A4, c-KIT, TPO and TG as new biomarkers for human follicular thyroid carcinoma.

The search for preoperative biomarkers for thyroid malignancies, in particular for follicular thyroid carcinoma (FTC) diagnostics, is of utmost clinical importance. We thus aimed at screening for potential biomarker candidates for FTC. To evaluate dynamic alterations in molecular patterns as a function of thyroid malignancy progression, a comparative analysis was conducted in clinically distinct subgroups of FTC and poorly differentiated thyroid carcinoma (PDTC) nodules. NanoString analysis of FFPE samples was performed in 22 follicular adenomas, 56 FTC and 25 PDTC nodules, including oncocytic and non-oncocytic subgroups. The expression levels of CHEK1, c-KIT, SLC26A4, TG and TPO were significantly altered in all types of thyroid carcinomas. Based on collective changes of these biomarkers which correlating among each other, a predictive score has been established, allowing for discrimination between benign and FTC samples with high sensitivity and specificity. Additional transcripts related to thyroid function, cell cycle, circadian clock, and apoptosis regulation were altered in the more aggressive oncocytic subgroups only, with expression levels correlating with disease progression. Distinct molecular patterns were observed for oncocytic and non-oncocytic FTCs and PDTCs. A predictive score correlation coefficient based on collective alterations of identified here biomarkers might help to improve the preoperative diagnosis of FTC nodules.

Human peripheral clocks: applications for studying circadian phenotypes in physiology and pathophysiology.

Most light-sensitive organisms on earth have acquired an internal system of circadian clocks allowing the anticipation of light or darkness. In humans, the circadian system governs nearly all aspects of physiology and behavior. Circadian phenotypes, including chronotype, vary dramatically among individuals and over individual lifespan. Recent studies have revealed that the characteristics of human skin fibroblast clocks correlate with donor chronotype. Given the complexity of circadian phenotype assessment in humans, the opportunity to study oscillator properties by using cultured primary cells has the potential to uncover molecular details difficult to assess directly in humans. Since altered properties of the circadian oscillator have been associated with many diseases including metabolic disorders and cancer, clock characteristics assessed in additional primary cell types using similar technologies might represent an important tool for exploring the connection between chronotype and disease, and for diagnostic purposes. Here, we review implications of this approach for gathering insights into human circadian rhythms and their function in health and disease.

Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis.

We previously reported an upregulation of the clock transcript BMAL1, correlating with TIMP1 expression in fresh-frozen samples from papillary thyroid carcinoma (PTC). Since frozen postoperative biopsy samples are difficult to obtain, we aimed to validate the application of high-precision NanoString analysis for formalin-fixed paraffin-embedded (FFPE) thyroid nodule samples and to screen for potential biomarkers associated with PTC. No significant differences were detected between fresh-frozen and FFPE samples. NanoString analysis of 51 transcripts in 17 PTC and 17 benign nodule samples obtained from different donors and in 24 pairs of benign and PTC nodules, obtained from the same donor (multinodular goiters), confirmed significant alterations in the levels of BMAL1, c-MET, c-KIT, TIMP1, and other transcripts. Moreover, we identified for the first time alterations in CHEK1 and BCL2 levels in PTC. A predictive score was established for each sample, based on the combined expression levels of BMAL1, CHEK1, c-MET, c-KIT and TIMP1. In combination with BRAF mutation analysis, this predictive score closely correlated with the clinicopathological characteristics of the analyzed thyroid nodules. Our study identified new thyroid transcripts with altered levels in PTC using the NanoString approach. A predictive score correlation coefficient might contribute to improve the preoperative diagnosis of thyroid nodules.

Thyroid circadian timing: roles in physiology and thyroid malignancies.

The circadian clock represents an anticipatory mechanism, well preserved in evolution. It has a critical impact on most aspects of the physiology of light-sensitive organisms. These rhythmic processes are governed by environmental cues (fluctuations in light intensity and temperature), an internal circadian timing system, and interactions between this timekeeping system and environmental signals. Endocrine body rhythms, including hypothalamic-pituitary-thyroid (HPT) axis rhythms, are tightly regulated by the circadian system. Although the circadian profiles of thyroid-releasing hormone (TRH), thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) in blood have been well described, relatively few studies have analyzed molecular mechanisms governing the circadian regulation of HPT axis function. In this review, we will discuss the latest findings in the area of complex regulation of thyroid gland function by the circadian oscillator. We will also highlight the molecular makeup of the human thyroid oscillator as well as the potential link between thyroid malignant transformation and alterations in the clockwork.

Circadian clock characteristics are altered in human thyroid malignant nodules.

CONTEXT: The circadian clock represents the body's molecular time-keeping system. Recent findings revealed strong changes of clock gene expression in various types of human cancers. OBJECTIVE: Due to emerging evidence on the connection between the circadian oscillator, cell cycle, and oncogenic transformation, we aimed to characterize the circadian clockwork in human benign and malignant thyroid nodules. DESIGN: Clock transcript levels were assessed by quantitative RT-PCR in thyroid tissues. To provide molecular characteristics of human thyroid clockwork, primary thyrocytes established from normal or nodular thyroid tissue biopsies were subjected to in vitro synchronization with subsequent clock gene expression analysis by circadian bioluminescence reporter assay and by quantitative RT-PCR. RESULTS: The expression levels of the Bmal1 were up-regulated in tissue samples of follicular thyroid carcinoma (FTC), and in papillary thyroid carcinoma (PTC), as compared with normal thyroid and benign nodules, whereas Cry2 was down-regulated in FTC and PTC. Human thyrocytes derived from normal thyroid tissue exhibited high-amplitude circadian oscillations of Bmal1-luciferase reporter expression and endogenous clock transcripts. Thyrocytes established from FTC and PTC exhibited clock transcript oscillations similar to those of normal thyroid tissue and benign nodules (except for Per2 altered in PTC), whereas cells derived from poorly differentiated thyroid carcinoma exhibited altered circadian oscillations. CONCLUSIONS: This is the first study demonstrating a molecular makeup of the human thyroid circadian clock. Characterization of the thyroid clock machinery alterations upon thyroid nodule malignant transformation contributes to understanding the connections between circadian clocks and oncogenic transformation. Moreover, it might help in improving the thyroid nodule preoperative diagnostics.

Biological rhythms and preeclampsia.

The impact of impaired circadian rhythm on health has been widely studied in shift workers and trans-meridian travelers. A part from its correlation with sleep and mood disorders, biological rhythm impairment is a recognized risk factor for cardiovascular diseases and breast cancer. Preeclampsia is a major public health issue, associated with a significant maternal and fetal morbidity and mortality worldwide. While the risks factors for this condition such as obesity, diabetes, pre-existing hypertension have been identified, the underlying mechanism of this multi-factorial disease is yet not fully understood. The disruption of the light/dark cycle in pregnancy has been associated with adverse outcomes. Slightly increased risk for "small for gestational age" babies, "low birth weight" babies, and preterm deliveries has been reported in shift working women. Whether altered circadian cycle represents a risk factor for preeclampsia or preeclampsia is itself linked with an abnormal circadian cycle is less clear. There are only few reports available, showing conflicting results. In this review, we will discuss recent observations concerning circadian pattern of blood pressure in normotensive and hypertensive pregnancies. We explore the hypothesis that circadian misalignments may represent a risk factor for preeclampsia. Unraveling potential link between circadian clock gene and preeclampsia could offer a novel approach to our understanding of this multi-system disease specific to pregnancy.

The circadian clock starts ticking at a developmentally early stage.

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.

Circadian gene expression in cultured cells.

In mammals, circadian oscillators not only exist in specialized neurons of the suprachiasmatic nucleus, but in almost all peripheral cell types. These oscillators are operative even in established fibroblast cell lines, such as Rat-1 cells or NIH3T3 cells, and in primary fibroblasts from mouse embryos or adult animals. This can be demonstrated by treating such cells for a short time period with high concentrations of serum or chemicals that activate a large number of known signaling pathways. The possibility of studying circadian rhythms in cultured cells should facilitate the biochemical and genetic dissection of the circadian clockwork and should promote the discovery of new clock components.