Transcriptomic analysis of human primary breast cancer identifies fatty acid oxidation as a target for metformin.

BACKGROUND: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. METHODS: Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. RESULTS: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. CONCLUSIONS: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations. CLINICAL TRIAL REGISTRATION: NCT01266486.

Challenging metabolic tissues with fructose: tissue-specific and sex-specific responses.

Excessive consumption of free sugars (which typically includes a composite of glucose and fructose) is associated with an increased risk of developing chronic metabolic diseases including obesity, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes and cardiovascular disease. Determining the utilisation, storage and fate of dietary sugars in metabolically relevant tissues is fundamental to understanding their contribution to metabolic disease risk. To date, the study of fructose metabolism has primarily focused on the liver, where it has been implicated in impaired insulin sensitivity, increased fat accumulation and dyslipidaemia. Yet we still have only a limited understanding of the mechanisms by which consumption of fructose, as part of a mixed meal, may alter hepatic fatty acid synthesis and partitioning. Moreover, surprisingly little is known about the metabolism of fructose within other organs, specifically subcutaneous adipose tissue, which is the largest metabolically active organ in the human body and is consistently exposed to nutrient fluxes. This review summarises what is known about fructose metabolism in the liver and adipose tissue and examines evidence for tissue-specific and sex-specific responses to fructose.

Bone morphogenetic protein 2 is a depot-specific regulator of human adipogenesis.

BACKGROUND: Bone morphogenetic proteins (BMPs) regulate adipogenesis but it is not clear whether they influence regional adipose tissue (AT) development in humans. OBJECTIVE: To characterise BMP2 expression, BMP2-SMAD1/5/8 signalling, and BMP2's potential effect on proliferation and adipogenesis in human subcutaneous abdominal and gluteal AT and its constituent preadipocytes. METHODS: BMP2 expression was measured in whole AT and immortalised preadipocytes via qPCR and Western blot; secreted/circulating BMP2 was measured by ELISA. The effect of BMP2 on preadipocyte proliferation was evaluated using a fluorescent assay. BMP2's effect on adipogenesis in immortalised preadipocytes was determined via qPCR of adipogenic markers and cellular triacylglycerol (TAG) accumulation. BMP2-SMAD1/5/8 signalling was assessed in immortalised preadipocytes via Western blot and qPCR of ID1 expression. RESULTS: BMP2 was expressed and released by abdominal and gluteal AT and preadipocytes. Exogenous BMP2 dose dependently promoted adipogenesis in abdominal preadipocytes only; 50 ng/ml BMP2 increased PPARG2 expression (10-fold compared to vehicle, p < 0.001) and TAG accumulation (3-fold compared to vehicle; p < 0.001). BMP2 stimulated SMAD1/5/8 phosphorylation and ID1 expression in abdominal and gluteal preadipocytes but this was blocked by 500 nM K02288, a type 1 BMP receptor inhibitor (p < 0.001). Co-administration of 500 nM K02288 also inhibited the pro-adipogenic effect of 50 ng/ml BMP2 in abdominal cells; >90% inhibition of TAG accumulation (p < 0.001) and ~50% inhibition of PPARG2 expression (p < 0.001). The endogenous iron regulator erythroferrone reduced BMP2-SMAD1/5/8 signalling by ~30% specifically in subcutaneous abdominal preadipocytes (p < 0.01), suggesting it plays a role in restricting the expansion of the body's largest AT depot during energy deficiency. Additionally, a waist-hip ratio-increasing common polymorphism near BMP2 is an eQTL associated with ~15% lower BMP2 expression in abdominal and gluteal AT (p < 0.05) as well as altered adipocyte size in male abdominal AT (p < 0.05). CONCLUSIONS: These data implicate BMP2-SMAD1/5/8 signalling in depot-specific preadipocyte development and abdominal AT expansion in humans.

Correction to: Bone morphogenetic protein 2 is a depot-specific regulator of human adipogenesis.

We erroneously published the original Article with an incorrect Copyright line. This has been updated in the XML, PDF and HTML versions of this Article.

Role of developmental transcription factors in white, brown and beige adipose tissues.

In this review we discuss the role of developmental transcription factors in adipose tissue biology with a focus on how these developmental genes may contribute to regional variation in adipose tissue distribution and function. Regional, depot-specific, differences in lipid handling and signalling (lipolysis, lipid storage and adipokine/lipokine signalling) are important determinants of metabolic health. At a cellular level, preadipocytes removed from their original depot and cultured in vitro retain depot-specific functional properties, implying that these are intrinsic to the cells and not a function of their environment in situ. High throughput screening has identified a number of developmental transcription factors involved in embryological development, including members of the Homeobox and T-Box gene families, that are strongly differentially expressed between regional white adipose tissue depots and also between brown and white adipose tissue. However, the significance of depot-specific developmental signatures remains unclear. Developmental transcription factors determine body patterning during embryogenesis. The divergent developmental origins of regional adipose tissue depots may explain their differing functional characteristics. There is evidence from human genetics that developmental genes determine adipose tissue distribution: in GWAS studies a number of developmental genes have been identified as being correlated with anthropometric measures of adiposity and fat distribution. Additionally, compelling functional studies have recently implicated developmental genes in both white adipogenesis and the so-called 'browning' of white adipose tissue. Understanding the genetic and developmental pathways in adipose tissue may help uncover novel ways to intervene with the function of adipose tissue in order to promote health.

The value of neck adipose tissue as a predictor for metabolic risk in health and type 2 diabetes.

Upper-body adiposity is adversely associated with metabolic health whereas the opposite is observed for the lower-body. The neck is a unique upper-body fat depot in adult humans, housing thermogenic brown adipose tissue (BAT), which is increasingly recognised to influence whole-body metabolic health. Loss of BAT, concurrent with replacement by white adipose tissue (WAT), may contribute to metabolic disease, and specific accumulation of neck fat is seen in certain conditions accompanied by adverse metabolic consequences. Yet, few studies have investigated the relationships between neck fat mass (NFM) and cardiometabolic risk, and the influence of sex and metabolic status. Typically, neck circumference (NC) is used as a proxy for neck fat, without considering other determinants of NC, including variability in neck lean mass. In this study we develop and validate novel methods to quantify NFM using dual x-ray absorptiometry (DEXA) imaging, and subsequently investigate the associations of NFM with metabolic biomarkers across approximately 7000 subjects from the Oxford BioBank. NFM correlated with systemic insulin resistance (Homeostatic Model Assessment for Insulin Resistance; HOMA-IR), low-grade inflammation (plasma high-sensitivity C-Reactive Protein; hsCRP), and metabolic markers of adipose tissue function (plasma triglycerides and non-esterified fatty acids; NEFA). NFM was higher in men than women, higher in type 2 diabetes mellitus compared with non-diabetes, after adjustment for total body fat, and also associated with overall cardiovascular disease risk (calculated QRISK3 score). This study describes the development of methods for accurate determination of NFM at scale and suggests a specific relationship between NFM and adverse metabolic health.

An optimised protocol for the investigation of insulin signalling in a human cell culture model of adipogenesis.

While there is no standardized protocol for the differentiation of human adipocytes in culture, common themes exist in the use of supra-physiological glucose and hormone concentrations, and an absence of exogenous fatty acids. These factors can have detrimental effects on some aspects of adipogenesis and adipocyte function. Here, we present methods for modifying the adipogenic differentiation protocol to overcome impaired glucose uptake and insulin signalling in human adipose-derived stem cell lines derived from the stromal vascular fraction of abdominal and gluteal subcutaneous adipose tissue. By reducing the length of exposure to adipogenic hormones, in combination with a physiological glucose concentration (5 mM), and the provision of exogenous fatty acids (reflecting typical dietary fatty acids), we were able to restore early insulin signalling events and glucose uptake, which were impaired by extended use of hormones and a high glucose concentration, respectively. Furthermore, the addition of exogenous fatty acids greatly increased the storage of triglycerides and removed the artificial demand to synthesize all fatty acids by de novo lipogenesis. Thus, modifying the adipogenic cocktail can enhance functional aspects of human adipocytes in vitro and is an important variable to consider prior to in vitro investigations into adipocyte biology.

Metformin maintains intrahepatic triglyceride content through increased hepatic de novo lipogenesis.

OBJECTIVE: Metformin is a first-line pharmacotherapy in the treatment of type 2 diabetes, a condition closely associated with non-alcoholic fatty liver disease (NAFLD). Although metformin promotes weight loss and improves insulin sensitivity, its effect on intrahepatic triglyceride (IHTG) remains unclear. We investigated the effect of metformin on IHTG, hepatic de novo lipogenesis (DNL), and fatty acid (FA) oxidation in vivo in humans. DESIGN AND METHODS: Metabolic investigations, using stable-isotope tracers, were performed in ten insulin-resistant, overweight/obese human participants with NAFLD who were treatment naïve before and after 12 weeks of metformin treatment. The effect of metformin on markers of s.c. adipose tissue FA metabolism and function, along with the plasma metabolome, was investigated. RESULTS: Twelve weeks of treatment with metformin resulted in a significant reduction in body weight and improved insulin sensitivity, but IHTG content and FA oxidation remained unchanged. Metformin treatment was associated with a significant decrease in VLDL-triglyceride (TG) concentrations and a significant increase in the relative contribution of DNL-derived FAs to VLDL-TG. There were subtle and relatively few changes in s.c. adipose tissue FA metabolism and the plasma metabolome with metformin treatment. CONCLUSIONS: We demonstrate the mechanisms of action of metformin whereby it improves insulin sensitivity and promotes weight loss, without improvement in IHTG; these observations are partly explained through increased hepatic DNL and a lack of change in FA oxidation.

The effects of endogenously- and exogenously-induced hyperketonemia on exercise performance and adaptation.

Elevating blood ketones may enhance exercise capacity and modulate adaptations to exercise training; however, these effects may depend on whether hyperketonemia is induced endogenously through dietary carbohydrate restriction, or exogenously through ketone supplementation. To determine this, we compared the effects of endogenously- and exogenously-induced hyperketonemia on exercise capacity and adaptation. Trained endurance athletes undertook 6 days of laboratory based cycling ("race") whilst following either: a carbohydrate-rich control diet (n = 7; CHO); a carbohydrate-rich diet + ketone drink four-times daily (n = 7; Ex Ket); or a ketogenic diet (n = 7; End Ket). Exercise capacity was measured daily, and adaptations in exercise metabolism, exercise physiology and postprandial insulin sensitivity (via an oral glucose tolerance test) were measured before and after dietary interventions. Urinary ß-hydroxybutyrate increased by ⁓150-fold and ⁓650-fold versus CHO with Ex Ket and End Ket, respectively. Exercise capacity was increased versus pre-intervention by ~5% on race day 1 with CHO (p < 0.05), by 6%-8% on days 1, 4, and 6 (all p < 0.05) with Ex Ket and decreased by 48%-57% on all race days (all p > 0.05) with End Ket. There was an ⁓3-fold increase in fat oxidation from pre- to post-intervention (p < 0.05) with End Ket and increased perceived exercise exertion (p < 0.05). No changes in exercise substrate metabolism occurred with Ex Ket, but participants had blunted postprandial insulin sensitivity (p < 0.05). Dietary carbohydrate restriction and ketone supplementation both induce hyperketonemia; however, these are distinct physiological conditions with contrasting effects on exercise capacity and adaptation to exercise training.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

De novo lipogenesis in the differentiating human adipocyte can provide all fatty acids necessary for maturation.

The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used (13)C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation.

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation.

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing, microarrays and can be utilized in functional in vitro/in vivo studies.

Reversibility of metabolic and morphological changes associated with chronic exposure of pancreatic islet beta-cells to fatty acids.

Pancreatic beta-cells metabolise both lipid and glucose nutrients but chronic exposure (>24 h) to elevated fatty acid (FA) concentrations results in deleterious metabolic and morphological changes. The aims of this study were to assess the adaptive morphological, metabolic and secretory responses of islet beta-cells to exposure and removal of FA. Isolated mouse islets and INS-1 beta-cells were exposed to oleate or palmitate (0.5 mM) or a 1:1 mixture of both FA for 48 h prior to a 24 h period without FA. Subsequent changes in lipid storage and composition (triglycerides, TG and phospholipids, PL), gene expression, beta-cell morphology and glucose-stimulated insulin secretion (GSIS) were determined. Intracellular TG content increased during exposure to FA and was lower in cells subsequently incubated in FA-free media (P < 0.05); TG storage was visible as oil red O positive droplets (oleate) by light microscopy or 'splits' (palmitate) by electron microscopy. Significant desaturation of beta-cell FA occurred after exposure to oleate and palmitate. After incubation in FA-free media, there was differential handling of specific FA in TG, resulting in a profile that tended to revert to that of control cells. FA treatment resulted in elevated lipolysis of intracellular TG, increased FA oxidation and reduced GSIS. After incubation in FA-free media, oxidation remained elevated but inhibition of FA oxidation with etomoxir (10 microM) had no effect on the improvement in GSIS. The beta-cell demonstrates metabolic flexibility as an adaptive response to ambient concentrations of FA.

Report of a member-led meeting: how stable isotope techniques can enhance human nutrition research.

A Nutrition Society member-led meeting was held on 9 January 2020 at The University of Surrey, UK. Sixty people registered for the event, and all were invited to participate, either through chairing a session, presenting a '3 min lightning talk' or by presenting a poster. The meeting consisted of an introduction to the topic by Dr Barbara Fielding, with presentations from eight invited speakers. There were also eight lightning talks and a poster session. The meeting aimed to highlight recent research that has used stable isotope tracer techniques to understand human metabolism. Such studies have irrefutably shaped our current understanding of metabolism and yet remain a mystery to many. The meeting aimed to de-mystify their use in nutrition research.

Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) begins with steatosis, where a mixed macrovesicular pattern of large and small lipid droplets (LDs) develops. Since in vitro models recapitulating this are limited, the aims of this study were to develop mixed macrovesicular steatosis in immortalized hepatocytes and investigate effects on intracellular metabolism by altering nutritional substrates. METHODS: Huh7 cells were cultured in 11 mM glucose and 2% human serum (HS) for 7 days before additional sugars and fatty acids (FAs), either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS), or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS), were added for 7 days. FA metabolism, lipid droplet characteristics, and transcriptomic signatures were investigated. RESULTS: Between the LFLS and LFHS conditions, there were few notable differences. In the HFHS condition, intracellular triacylglycerol (TAG) was increased and the LD pattern and distribution was similar to that found in primary steatotic hepatocytes. HFHS-treated cells had lower levels of de novo-derived FAs and secreted larger, TAG-rich lipoprotein particles. RNA sequencing and gene set enrichment analysis showed changes in several pathways including those involved in metabolism and cell cycle. CONCLUSIONS: Repeated doses of HFHS treatment resulted in a cellular model of NAFLD with a mixed macrovesicular LD pattern and metabolic dysfunction. Since these nutrients have been implicated in the development of NAFLD in humans, the model provides a good physiological basis for studying NAFLD development or regression in vitro.

RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro.

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity.

Measuring Human Lipid Metabolism Using Deuterium Labeling: In Vivo and In Vitro Protocols.

Stable isotopes are powerful tools for tracing the metabolic fate of molecules in the human body. In this chapter, we focus on the use of deuterium (2H), a stable isotope of hydrogen, in the study of human lipid metabolism within the liver in vivo in humans and in vitro using hepatocyte cellular models. The measurement of de novo lipogenesis (DNL) will be focussed on, as the synthesis of fatty acids, specifically palmitate, has been gathering momentum as being implicated in cellular dysfunction, which may be involved in the development of non-alcoholic fatty liver disease (NAFLD). Therefore, this chapter focusses specifically on the use of 2H2O (heavy water) to measure hepatic DNL.

Regional fat depot masses are influenced by protein-coding gene variants.

Waist-to-hip ratio (WHR) is a prominent cardiometabolic risk factor that increases cardio-metabolic disease risk independently of BMI and for which multiple genetic loci have been identified. However, WHR is a relatively crude proxy for fat distribution and it does not capture all variation in fat distribution. We here present a study of the role of coding genetic variants on fat mass in 6 distinct regions of the body, based on dual-energy X-ray absorptiometry imaging on more than 17k participants. We find that the missense variant CCDC92S70C, previously associated with WHR, is associated specifically increased leg fat mass and reduced visceral but not subcutaneous central fat. The minor allele-carrying transcript of CCDC92 is constitutively more highly expressed in adipose tissue samples. In addition, we identify two coding variants in SPATA20 and UQCC1 that are associated with arm fat mass. SPATA20K422R is a low-frequency variant with a large effect on arm fat only, and UQCC1R51Q is a common variant reaching significance for arm but showing similar trends in other subcutaneous fat depots. Our findings support the notion that different fat compartments are regulated by distinct genetic factors.

MicroRNA-196a links human body fat distribution to adipose tissue extracellular matrix composition.

BACKGROUND: Abdominal fat mass is associated with metabolic risk whilst gluteal femoral fat is paradoxically protective. MicroRNAs are known to be necessary for adipose tissue formation and function but their role in regulating human fat distribution remains largely unexplored. METHODS: An initial microarray screen of abdominal subcutaneous and gluteal adipose tissue, with validatory qPCR, identified microRNA-196a as being strongly differentially expressed between gluteal and abdominal subcutaneous adipose tissue. FINDINGS: We found that rs11614913, a SNP within pre-miR-196a-2 at the HOXC locus, is an eQTL for miR-196a expression in abdominal subcutaneous adipose tissue (ASAT). Observations in large cohorts showed that rs11614913 increased waist-to-hip ratio, which was driven specifically by an expansion in ASAT. In further experiments, rs11614913 was associated with adipocyte size. Functional studies and transcriptomic profiling of miR-196a knock-down pre-adipocytes revealed a role for miR-196a in regulating pre-adipocyte proliferation and extracellular matrix pathways. INTERPRETATION: These data identify a role for miR-196a in regulating human body fat distribution. FUND: This work was supported by the Medical Research Council and Novo Nordisk UK Research Foundation (G1001959) and Swedish Research Council. We acknowledge the OBB-NIHR Oxford Biomedical Research Centre and the British Heart Foundation (BHF) (RG/17/1/32663). Work performed at the MRC Epidemiology Unit was funded by the United Kingdom's Medical Research Council through grants MC_UU_12015/1, MC_PC_13046, MC_PC_13048 and MR/L00002/1.

The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication.

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.