A High-Throughput Physiological Screen Reveals a Conserved Catecholamine-Dependent Axis Regulating Systemic Metabolic Flexibility

A High-Throughput Physiological Screen Reveals a Conserved Catecholamine-Dependent Axis Regulating Systemic Metabolic Flexibility | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Letter A High-Throughput Physiological Screen Reveals a Conserved Catecholamine-Dependent Axis Regulating Systemic Metabolic Flexibility Antonio Vidal-Puig, Guillaume Bidault, Samuel Virtue, Martin Dale, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8028437/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Metabolic flexibility, the capacity to switch between carbohydrate and lipid fuels, is fundamental to metabolic health, yet its genetic regulation has not been systematically explored in vivo . To address this gap, we developed a high-throughput physiological screen in mice using the respiratory exchange ratio range (ΔRER) as quantitative index of metabolic flexibility. Screening over 800 knockout lines identified Cyb561, a vesicular cytochrome required for catecholamine biosynthesis, as an unexpected regulator of systemic substrate utilisation. Mice lacking Cyb561 exhibited impaired lipolysis, blunted brown adipose thermogenesis, and delayed lipid clearance, while maintaining preserved glucose tolerance. Under high-fat feeding or at thermoneutrality (28°C), Cyb561 deficiency exacerbated insulin resistance, revealing metabolic vulnerability. Remarkably, despite impaired lipolysis and BAT dysfunction, Cyb561 knockout mice did not develop increased adiposity. Mechanistically, we demonstrate that catecholamines transcriptionally couple lipid uptake and release in white adipose tissue, thereby sustaining lipid turnover and maintaining systemic metabolic flexibility. Together, these findings establish catecholamine tone as a key determinant of whole-body fuel switching and demonstrate that high-throughput ΔRER phenotyping offers a powerful and sensitive approach for large-scale analysis of the genetic determinants of metabolic flexibility. Biological sciences/Physiology/Metabolism/Fat metabolism Biological sciences/Physiology/Metabolism/Homeostasis Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity Biological sciences/Biological techniques/High-throughput screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Main Metabolic flexibility, the ability to adaptively switch between lipid and carbohydrate oxidation, is a hallmark of metabolic health and resilience to nutritional stress. Fuel preference dynamically shifts with physiological state: carbohydrates are primarily oxidised during feeding and acute exercise, whereas lipids dominate during fasting, sleep, and endurance activity 1 . Impaired fuel switching, or metabolic inflexibility, is closely linked to insulin resistance, obesity, and cardiovascular diseases 2 – 4 . Despite its well-established pathophysiological significance, assessing metabolic flexibility in humans remains labour-intensive, invasive, and costly, limiting large-scale genetic discovery. To overcome these limitations, rodents provide a robust model for assessing metabolic flexibility. Indirect calorimetry enables continuous measurement of the respiratory exchange ratio (RER), a well-established proxy for substrate utilisation, and mice display marked diurnal oscillations in fuel preference. We quantified metabolic flexibility using ΔRER, defined as the difference between the highest and lowest 10% of RER values 5 , providing an integrated index of fuel-switching capacity. Across multiple diets, fasting states, and experimental sites, ΔRER values proved highly reproducible and minimally affected by short-term variations in body weight or food intake (Supplementary Results; Extended Data Figs. 1 and 2 ), establishing this parameter as a reliable and scalable metric for large-scale screening. Metabolic screen identifies Akt2 and Cyb561 as regulators of fuel flexibility. To uncover novel regulators of metabolic flexibility, we analysed indirect calorimetry data from over 800 knockout mouse lines generated through the International Mouse Phenotyping Consortium (IMPC) metabolic pipeline at the Wellcome Trust Sanger Institute (WTSI) 6 , 7 (Fig. 1 a). Two lines were classified as hits according to the WTSI/IMPC criteria 7 for altered ΔRER: Akt2 and Cyb561. Akt2 encodes a central mediator of anabolic insulin signalling 8 , 9 and its loss impairs insulin responsiveness and oxidative capacity, both hallmarks of metabolic inflexibility. The identification of Akt2 validates the screening approach and demonstrates its reproducibility across sites, as indicated by the consistent compression of RER observed at both WTSI and UCAM (Extended Data Fig. 2 ). In contrast, Cyb561 emerged as a previously unrecognised regulator of systemic metabolic flexibility. Cyb561 encodes an ascorbate-dependent electron transporter required for catecholamine biosynthesis 10 , 11 . Consistent with the WTSI results (Fig. 1 b), Cyb561 knockout mice phenotyped at UCAM exhibited reduced ΔRER (Fig. 1 c,d). Notably, the screen identified genes acting in opposing metabolic pathways, anabolic (Akt2) and catabolic (Cyb561), whose loss similarly reduced ΔRER, underscoring the bidirectional regulation of systemic metabolic flexibility. Catecholamine deficiency disrupts lipid handling and predisposes to insulin resistance. Consistent with impaired catecholamine signalling, Cyb561-KO mice exhibited reduced fasting lipid oxidation, reflected by elevated daytime RER values (Fig. 1 c), likely due to defective adipose tissue lipolysis 12 . In line with this, circulating triacylglycerol, non-esterified fatty acids, and glycerol were all decreased in Cyb561-KO mice (Fig. 2 a–c). Despite normal fed glycaemia and insulin levels (Extended Data Fig. 2 a,b), these mice developed fasting hypoglycaemia while maintaining normal glucose and pyruvate tolerance (Fig. 2 d–h; Extended Data Fig. 3 e,f), indicating preserved glucose disposal and hepatic gluconeogenic capacity. Collectively, these findings suggest a compensatory shift toward glucose utilisation during fasting, recapitulating the adipose-driven metabolic inflexibility previously described in PPARγ2-deficient mice 5 . Acute nutritional challenges unmasked subjacent metabolic defects in Cyb561-KO mice. Upon refeeding, knockout mice displayed exaggerated insulin responses without overt hyperglycaemia (Fig. 2 i,j; Extended Data Fig. 3 g–j), and lipid tolerance tests revealed markedly delayed triglyceride clearance (Fig. 2 k,l; Extended Data Fig. 3 k). We next examined the response of male Cyb561-KO mice to chronic nutrient excess induced by two months of high-fat diet (HFD) feeding. Under HFD, Cyb561-deficient mice consistently exhibited lower fasting glucose and preserved glucose tolerance (Extended Data Fig. 4 a–c), yet maintained normoglycaemia at the expense of elevated fed-state insulin levels (Extended Data Fig. 4 d,e), indicative of systemic insulin resistance confirmed by insulin tolerance tests (Fig. 2 m,n). As in chow-fed conditions, Cyb561-KO mice under HFD showed exacerbated abnormalities in the fasting–refeeding response (Fig. 2 o–r). Circulating triglycerides, NEFA, and glycerol remained reduced, reinforcing the presence of defective lipolysis during HFD (Extended Data Fig. 4 f–h). Together, these findings demonstrate that catecholamine deficiency impairs adaptive fuel utilisation, rendering mice metabolically vulnerable under nutritional stress.. Impaired substrate switching persists when BAT thermogenesis is suppressed Catecholamines are key activators of BAT thermogenesis 13 . In Cyb561-KO mice, BAT noradrenaline (NA) levels were undetectable (Fig. 3 a). Histological analysis revealed fewer but enlarged lipid droplets per cell (Fig. 3 b), accompanied by altered expression of genes regulating lipid uptake and lipolysis, indicating increased storage and reduced mobilisation (Extended Data Fig. 5a,b). Thermogenic gene expression was diminished under both chow and high-fat diet conditions, and Ucp1 protein content per BAT pad was markedly reduced (Extended Data Fig. 5c,d). Functionally, maximal thermogenic capacity induced by NA was strongly blunted in Cyb561-KO mice (Fig. 3 d,e), and acute cold exposure elicited a weaker induction of thermogenic genes (Extended Data Fig. 5e). Together, these findings confirm that loss of catecholamine tone compromises BAT responsiveness. BAT serves as a major sink for circulating lipids 14 and its dysfunction could account for the metabolic inflexibility observed in Cyb561-KO mice. To test this, we housed wild-type and Cyb561-KO female mice at 28°C, a condition that suppresses BAT activity and approximates human thermal physiology. As at room temperature, Cyb561-deficient mice displayed reduced lipolytic activity during the light phase (Fig. 3 f–h), fasting hypoglycaemia (Fig. 3 i), and preserved glucose tolerance and fasting–refeeding responses (Extended Data Fig. 6a–g). However, unlike at room temperature, fed glucose levels were significantly elevated in knockout mice (Fig. 3 g), accompanied by a trend toward higher insulin levels (Extended Data Fig. 6h), revealing a glucose-handling defect apparent only when thermogenic demand was reduced. Importantly, even under these warm, low-sympathetic conditions, Cyb561-KO mice continued to exhibit slower triglyceride clearance than wild-type controls (Fig. 3 k,l), indicating defective lipid uptake independent of BAT thermogenesis Together, these findings demonstrate that catecholamine deficiency disrupts systemic metabolic flexibility across both glucose and lipid pathways, even when BAT thermogenesis is suppressed, establishing a BAT-independent role for catecholamines in coordinating whole-body fuel switching. Catecholamines coordinate lipid turnover without altering adiposity Despite the combined impairment of white adipose tissue (WAT) lipolysis and BAT thermogenesis, Cyb561-KO mice maintained normal body weight and adiposity across diets and housing temperatures (Fig. 4 a,b and Extended Data Fig. 7a–d). To directly assess lipid turnover in WAT, we placed wild-type (WT) mice on a fat-free diet and tracked the loss of linoleic acid (C18:2ω6), an essential fatty acid that must be obtained from the diet. Turnover was faster in subcutaneous (scWAT) than gonadal WAT (gWAT), with estimated losses of ~ 0.66% per day and ~ 0.42% per day, respectively (Fig. 4 c). Extrapolated to whole-body fat mass, this corresponds to ~ 100 mg per day of lipid flux in a mouse with 4 g of fat. Given the lack of increased adiposity in Cyb561–KO mice, these data indicate that diminished mobilisation was offset by proportionally decreased lipid uptake. In Cyb561-deficient mice, the absence of increased adiposity despite reduced lipolytic activity implies that diminished lipid mobilisation was offset by proportionally reduced lipid uptake. Without this compensatory mechanism, Cyb561-KO mice would be expected to accumulate ~ 5 g additional fat over two months. Together, these findings indicate that catecholamines coordinate lipid flux in WAT, maintaining a balance between lipid release and uptake rather than controlling net fat storage. Gene expression analyses revealed a coordinated suppression of lipid release and uptake pathways in Cyb561-KO WAT. In scWAT, both lipolytic genes ( Atgl, Hsl ) and genes mediating lipid uptake ( Lpl, Fatp1, Cd36 ) were downregulated (Fig. 4 d,e), indicating that catecholamines co-regulate lipid input and output pathways. To reinforce that catecholamines dynamically modulate this coupling, we exposed mice to cold, a potent activator of adipose sympathetic outflow. Cold exposure significantly increased the expression of both lipid uptake and release genes in WAT and tended to strengthen their correlation (Extended Data Fig. 7e,f), further supporting a catecholamine-dependent regulatory mechanism. This coupling extended beyond our model. Across both mice and humans, expression of lipid uptake genes positively correlated with lipolytic gene expression in all tested depots (Fig. 4 f–i), suggesting an evolutionarily conserved mechanism aligning adipose lipid flux. Notably, correlations between lipid-release and re-esterification genes remained stable over time (84 days) in humans (Fig. 4 i), underscoring the robustness of this transcriptional coordination. Together, these findings define a conserved transcriptional programme in WAT whereby catecholamines coordinate lipid uptake and release, maintaining lipid flux and systemic energy balance during metabolic stress or impaired thermogenesis. Discussion Although metabolic flexibility is a defining feature of metabolic health, its physiological regulation remains poorly understood. Here, we overcome key limitations in assessing this trait at scale in vivo , establishing a framework for systematic discovery of its genetic determinants. By integrating large-scale physiological phenotyping with molecular analyses, we identify Cyb561, emerging alongside Akt2, and catecholamine signalling as critical regulators of systemic metabolic flexibility. The striking phenotypic parallels between Cyb561 and Akt2 knockout mice, despite their roles in ostensibly opposing hormonal pathways, suggest that catecholamines and insulin converge over longer timescales to coordinate lipid flux and maintain systemic metabolic flexibility. Mechanistically, Cyb561 deletion caused profound BAT dysfunction and markedly reduced circulating non-esterified fatty acids, glycerol, and triglycerides, consistent with defective WAT lipolysis. Yet, despite impaired lipid mobilisation, Cyb561-KO mice did not accumulate excess adiposity under any dietary or thermal condition. Instead, we show that catecholamines synchronise lipid mobilisation and uptake in WAT, thereby sustaining lipid turnover and maintaining substrate flexibility across energetic states. Gene expression analyses in mice and humans reveal tight, evolutionarily conserved co-regulation of lipid uptake and lipolytic pathways across depots and over time, enabling adipose tissue to act as a dynamic metabolic buffer. Loss of catecholamine tone disrupts this coordination, diminishing lipid flux, impairing fuel switching, and predisposing to insulin resistance under metabolic stress. The persistence of defective lipid handling at thermoneutrality highlights a BAT-independent role for catecholamines, with WAT emerging as a key effector. Notably, Cyb561-KO mice developed fed-state hyperglycaemia and hyperinsulinaemia when housed at 28°C, a defect masked at room temperature. This may reflect a redistribution of glucose toward thermogenic processes under mild cold stress, consistent with evidence that skeletal muscle non-shivering thermogenesis primarily relies on glucose oxidation, whereas BAT thermogenesis depends largely on lipids 14 , 15 . These observations carry translational relevance, as humans typically reside under thermoneutral conditions and depend more on WAT and skeletal muscle than on BAT for adaptive thermogenesis. While our study establishes a central role for catecholamines in regulating lipid uptake and metabolic adaptability, several questions remain. Adrenergic signals also influence LPL activity in heart, skeletal muscle, and BAT, thereby modulating triglyceride clearance and systemic fuel partitioning 14 , 16 – 18 . Consequently, the phenotype of Cyb561-KO mice may in part reflect impaired lipid disposal in these peripheral tissues. Catecholamines additionally suppress insulin secretion 19 , 20 . However, given the pronounced adipose lipid defects and the absence of fasting hyperinsulinaemia, the elevated fed insulin levels observed in Cyb561-deficient mice most likely stem from peripheral insulin resistance. Taken together, while contributions from multiple organs cannot be excluded, dysfunction of WAT appears to be the predominant driver of metabolic inflexibility in this model. While our study establishes a key role for catecholamines in lipid uptake and metabolic adaptability, several questions remain. Adrenergic signals also modulate LPL activity in heart, skeletal muscle, and BAT, affecting triglyceride clearance and fuel partitioning 14 , 16 – 18 . Consequently, the phenotype of Cyb561-KO mice could partly reflect impaired lipid disposal in these tissues. Catecholamines also suppress insulin secretion 19 , 20 . However, given the pronounced adipose lipid defects and absence of fasting hyperinsulinaemia, the elevated fed insulin levels in Cyb561-deficient mice most likely stem from peripheral insulin resistance. Overall, while contributions from multiple organs cannot be excluded, dysfunction of WAT appears to be the predominant driver of metabolic inflexibility in this model. Although the phenotype is most plausibly due to loss of catecholamine biosynthesis, CYB561 may have catecholamine-independent roles in ascorbate recycling, iron metabolism, and redox homeostasis 21 – 23 , which could, in turn, influence energy metabolism. Nonetheless, the near-complete depletion of noradrenaline in BAT, together with the well-established role of catecholamines in lipid mobilisation and thermogenesis, strongly supports adrenergic deficiency as the primary driver of the observed phenotype. Future studies should clarify central versus peripheral contributions and the molecular mechanisms linking adrenergic tone to adipocyte lipid uptake. Parallel transcriptomic effects in human adipose tissue suggest potential translational relevance. This association may be clinically relevant given the associations of β-blocker therapy and age-related sympathetic decline with reduced metabolic flexibility and increased diabetes risk 24 , 25 . In summary, our high-throughput ΔRER-based screen identifies catecholamine signalling as a central regulator of whole-body fuel switching. By coupling adrenergic tone to lipid turnover and insulin sensitivity, we uncover a conserved axis that maintains systemic metabolic flexibility and highlight a pathway that could be therapeutically targeted to enhance metabolic resilience in obesity and diabetes. Methods Animal models Cyb561 tm1a(EUCOMM)Wtsi knockout (KO) mice and AKT2 tm1a(EUCOMM)Wtsi KO mice were obtained from the Wellcome Trust Sanger Institute as part of the IMPC. AKT2 tm1.1mbb KO mice were obtained from Jackson Laboratory and phenotyped at the University of Cambridge on a C57BL/6J background, while all other lines were maintained on a C57BL/6N background. Homozygous KO and wild-type littermates were generated by mating heterozygous animals. Additional wild-type C57BL/6J mice were purchased from Charles River UK. Ages and sexes of animals are indicated in the figure legends. Mice were housed in specific pathogen-free facilities under 12-hour light/12-hour dark cycles at 21°C unless otherwise stated. All procedures were approved by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB) under the UK Animals (Scientific Procedures) Act 1986 (Amendment Regulations 2012). Diets Animals were fed standard breeder’s chow (Safe Diets DS-105), a high-fat diet containing 45% of kcal from fat (Research Diets D12451, Brogaarden, Denmark), or a fat-free diet (SDS 820416, based on AIN-93M with soybean oil replaced by cornstarch). The Sanger MGP used a Western diet (SDS Western RD 829100, UK). All diets were provided ad libitum. Human Subjects Human studies included two cohorts. In cohort 1, subcutaneous adipose tissue biopsies were obtained from 19 individuals undergoing bariatric bypass surgery, from which adipose tissue macrophages were isolated. Clinical characteristics of these participants have been published previously 26 . All participants provided written informed consent. Cohort 2 consisted of 60 obese individuals aged 20–55 years with BMI 30–40 kg/m², who had no type 2 diabetes, no history of childhood obesity, and no prior bariatric surgery. Participants were enrolled in a 12-week outpatient study evaluating weight loss interventions 27 and were randomised to one of three interventions: calorie restriction (− 600 kcal/day), calorie restriction plus moderate exercise (~ 10% of daily energy expenditure), or calorie restriction plus the serotonin-norepinephrine reuptake inhibitor sibutramine. For this study, 26 biopsies from 13 participants were analysed. Participants were stratified into “losers” (≥ 5 kg weight loss by day 84) and “stable” (± 2 kg weight change). Ethical approval for the human studies was obtained from the GlaxoSmithKline protocol review panel, the Cambridge Local Research Ethics Committee (08/H0308/10), and the Wellcome Trust Clinical Research Facility Scientific Advisory Board. Metabolic tests Oral glucose tolerance tests were performed after overnight fasting from 16:00 to 09:00. Mice were acclimatised to the procedure room for 1 hour before receiving a fixed oral glucose dose of 2 g/kg, based on the average weight of the control group. Blood glucose measurements were taken at 0, 10, 20, 30, 60, 90, and 120 minutes. Insulin tolerance tests were conducted following a 6-hour fast from 08:00 to 14:00. Mice were administered 1 U/kg Actrapid insulin intraperitoneally at a dose normalised to the average weight of the control group. Lipid tolerance tests were performed after overnight fasting. Mice were gavaged with 200 µl olive oil, and blood samples were collected at 0, 1, 2, 4, and 6 hours for triglyceride analysis. For hepatic gluconeogenesis assessment, mice were fasted overnight and injected intraperitoneally with sodium pyruvate (2 g/kg). Blood glucose was measured 0, 10, 20, 30, 60, 90, and 120 minutes. Fast refed were performed after overnight fasting from 16:00 to 09:00. Mice were acclimatised to the procedure room for 1 hour. After a basal blood sample, food was provided ad libitum and blood samples taken 2- and 6-hours post-refeeding. Data were analysed by measuring the incremental area under the curve (oGTT, fast-refed, LTT) or the area over the curve (ITT) 28 . Norepinephrine-Stimulated Energy Expenditure Mice were anaesthetised with pentobarbital (60 mg/kg) and placed in a temperature-controlled calorimetry chamber (28°C, 2.4 L volume, Creative Scientific UK). Baseline oxygen consumption and carbon dioxide production were measured every 4 minutes until stable, after which mice received 1 µg/kg norepinephrine and 30 mg/kg pentobarbital subcutaneously. Energy expenditure was monitored for a further 28 minutes and calculated using the modified Weir equation: EE (J/min) = 15.818 × VO₂ (ml/min) + 5.176 × VCO₂ (ml/min). Altered temperature housing Acute cold exposure experiments involved exposure to 5°C in a temperature- and humidity-controlled cabinet after prior acclimation to 21°C. One mouse was culled every 30 minutes over 3.5 hours to enable regression analysis of transcriptional responses. During chronic cold exposure, mice were housed at 24°C or at 5°C in a temperature- and humidity-controlled cabinet for 4 weeks. To reduce BAT activity, animals were housed at 28°C in a temperature- and humidity-controlled cabinet for six weeks. Indirect Calorimetry and Metabolic Flexibility Indirect calorimetry was performed using custom-built chambers (Creative Scientific UK) for up to 48 hours. Gas concentrations were recorded every 11 minutes at a flow rate of 400 ml/min. Energy expenditure was calculated using the modified Weir equation. Metabolic flexibility was defined as the amplitude of the respiratory exchange ratio (RER), calculated as the difference between the mean of the highest 10% and the mean of the lowest 10% of RER values 5 . Serum Biochemistry Serum triglycerides and glycerol were measured on a Dimension RXL analyser (Siemens Healthcare), and insulin was quantified using electrochemiluminescence immunoassay (MesoScale Discovery). Noradrenaline levels in brown adipose tissue were measured using HPLC with electrochemical detection 29 . RNA Extraction and qPCR RNA was extracted using STAT-60 (AMS Biotech) and reverse transcribed into cDNA using the Promega Reverse Transcriptase System. qPCR was performed using TaqMan or SYBR Green chemistry on an ABI QuantStudio 5 (Applied Biosystems). Gene expression was normalised to the geometric mean of housekeeping genes: 18s , 36b4 , and Tbp (mouse) or 18S, CYCA , and GUSB (human). Western Blotting Western blotting was performed using RIPA-extracted protein quantified by Bio-Rad DC assay. Proteins (10 µg/lane) were denatured at 95°C for 5 minutes, separated on 4–12% gradient SDS-PAGE gels (Novex NuPage), transferred using iBlot 2, and probed with antibodies including Ucp1 (ab10983, Abcam) and β-tubulin (ab6046, Abcam). Histology Brown adipose tissue was fixed in formalin, paraffin-embedded, sectioned at 4 µm, and stained with H&E. Imaging was performed using an Axioscan Z1 microscope (20× objective). Lipid droplet quantification was performed using Halo software (Indica Labs). GC-MS Fatty Acid Analysis Lipids were extracted using the Folch method, esterified using BF₃-methanol, and analysed by GC-MS. Methyl esters were quantified based on integrated peak areas against reference standards (Thames Restek, UK) as previously described 30 . Statistical analysis Statistical analyses were performed using GraphPad Prism, SPSS (v26/27), or JASP. Outliers were identified using ROUT testing, and any removed outliers are noted in the Source Data files. Statistical significance was defined as p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Greenhouse–Geisser correction was applied for repeated-measures two-way ANOVA. For comparisons using Student’s t-test, an F-test was first applied to assess equality of variance; when variances were significantly different, Welch’s t-test was used instead. Specific tests used for each dataset are detailed in the figure legends, and the test results are presented in the Source Data files. Declarations Competing interests The authors declare no competing interests. Acknowledgments This work was supported by the British Heart Foundation [grant number RG/F/23/110110] and the Medical Research Council [grant number MC_UU_00039]. We thank the disease model core (DMC) for their excellent technical assistance with animal phenotyping, which is supported by the Medical Research Council [grant number MC_UU_00039]; and the Wellcome Trust [grant number 226800/Z/22/Z]. We thank the Core Biochemical Assay Laboratory (CBAL) which is supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312); and the Wellcome Trust [grant number 226800/Z/22/Z]. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a Creative Commons Attribution (CC-BY) licence to any Author Accepted Manuscript version arising from this submission. Data availability All data supporting this study are available in the accompanying Source Data files. No custom code was used. Additional information is available from the corresponding author upon reasonable request. References Goodpaster BH, Sparks LM (2017) Metabolic Flexibility in Health and Disease. 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Vidal-Puig","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYFCCA4wPPlSAaAY2hgcI4QR8WpgNZ5yBakFSh08LA5s0bxspWvgbzxhI8M7bJsd3/vCzBwkMdvK6DcwPPzC2peHUInHgjIGB5LbbxpI30swNEhiSDbcdYDOWYGzLweOVYwkJhttuJ264wcMmkQAMjG0HGMwYGNsqcOqQB2o5kDjndv2G82fAWuy3HWD/hleLwYHDBxsONtxOMDiQA9aSuO0AD8gW3A4zPHD4MGPDsduGM2+kmUkkGCQnbzvMUyyRcA639+VuHGz//afmtjwoxCQ+VNjZbjvevvHDh7Jk3N6XOIDiTiBmZiAQkfwN+GRHwSgYBaNgFAABAGlWXfkeFiQXAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4220-9577","institution":"University of Cambridge","correspondingAuthor":true,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Vidal-Puig","suffix":""},{"id":547060563,"identity":"d828856b-489c-4ad7-b65c-c8bc2cd087a8","order_by":1,"name":"Guillaume Bidault","email":"","orcid":"","institution":"Cambridge University","correspondingAuthor":false,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Bidault","suffix":""},{"id":547060564,"identity":"017b491a-fad1-45ee-b399-aa2fed27dd69","order_by":2,"name":"Samuel Virtue","email":"","orcid":"https://orcid.org/0000-0002-9545-5432","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Virtue","suffix":""},{"id":547060565,"identity":"0b3d3f31-651a-4e6c-815a-a24a289e324f","order_by":3,"name":"Martin Dale","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Dale","suffix":""},{"id":547060566,"identity":"36444a62-ed28-4ca2-aeff-a9ada9cf5085","order_by":4,"name":"Agnes Lukasic","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Agnes","middleName":"","lastName":"Lukasic","suffix":""},{"id":547060567,"identity":"e7770d83-5aed-4c2d-9d5a-3704c735f946","order_by":5,"name":"Vivian Peirce","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Vivian","middleName":"","lastName":"Peirce","suffix":""},{"id":547060568,"identity":"ae88ece9-cc14-4f93-af2c-7e4384bbe78b","order_by":6,"name":"Lee Roberts","email":"","orcid":"https://orcid.org/0000-0002-1455-5248","institution":"University of Leeds","correspondingAuthor":false,"prefix":"","firstName":"Lee","middleName":"","lastName":"Roberts","suffix":""},{"id":547060569,"identity":"47323ef1-a4df-4c07-873b-b9fbb460ea53","order_by":7,"name":"Gavin Jarvis","email":"","orcid":"","institution":"University of Sunderland","correspondingAuthor":false,"prefix":"","firstName":"Gavin","middleName":"","lastName":"Jarvis","suffix":""},{"id":547060570,"identity":"db110caa-50f3-4ba7-a925-e2747a70a3c5","order_by":8,"name":"Jeffrey Dalley","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"","lastName":"Dalley","suffix":""},{"id":547060571,"identity":"3d376c4b-0a45-4d37-952b-0210808b701b","order_by":9,"name":"Julian Griffin","email":"","orcid":"https://orcid.org/0000-0003-1336-7744","institution":"University of Aberdeen","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Griffin","suffix":""},{"id":547060572,"identity":"392cdd29-eb25-4e0a-bf58-f9de457b2bfe","order_by":10,"name":"Barbara Weijer","email":"","orcid":"","institution":"Ziekenhuisgroep Twente","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Weijer","suffix":""},{"id":547060573,"identity":"f3563707-61c9-40bb-9faf-cc4539a2d8ef","order_by":11,"name":"Mireille Serlie","email":"","orcid":"","institution":"Amsterdam UMC Department of Endocrinology and Metabolism","correspondingAuthor":false,"prefix":"","firstName":"Mireille","middleName":"","lastName":"Serlie","suffix":""},{"id":547060574,"identity":"a8c51912-0814-4c62-a5e1-37a36431e898","order_by":12,"name":"Antonella Napolitano","email":"","orcid":"","institution":"Glaxo Smith Kline","correspondingAuthor":false,"prefix":"","firstName":"Antonella","middleName":"","lastName":"Napolitano","suffix":""},{"id":547060575,"identity":"c5069b7a-0a11-4d73-981c-4a5ac23f0b42","order_by":13,"name":"Michele Vacca","email":"","orcid":"https://orcid.org/0000-0002-1973-224X","institution":"University of Bari","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Vacca","suffix":""},{"id":547060576,"identity":"fa4ad1f9-cb2d-4896-92d5-6af50a3302c2","order_by":14,"name":"Davide Chiarugi","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Davide","middleName":"","lastName":"Chiarugi","suffix":""}],"badges":[],"createdAt":"2025-11-04 11:57:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8028437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8028437/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97234175,"identity":"099a5a89-fb60-4616-91ba-ff6f9ffc77a4","added_by":"auto","created_at":"2025-12-02 10:05:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":215635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic flexibility screening identifies Akt2 and Cyb561 as regulators of fuel utilisation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Schematic of the WTSI MGP pipeline used to screen for regulators of metabolic flexibility.\u003c/p\u003e\n\u003cp\u003eb, ΔRER values in Cyb561 knockout (KO) and wild-type (WT) mice at the Wellcome Trust Sanger Institute (WTSI) (males: WT n=24, KO n=9).\u003c/p\u003e\n\u003cp\u003ec, Representative RER traces showing reduced flexibility in Cyb561-deficient male mice at the University of Cambridge (UCAM) (n=10).\u003c/p\u003e\n\u003cp\u003ed, ΔRER values in Cyb561-KO and WT mice at the UCAM (males: n=10; females: WT n=8, KO n=6) measured by indirect calorimetry at 4 months. ΔRER was calculated over 24 h to match WTSI analysis.\u003c/p\u003e\n\u003cp\u003eData are mean ± SEM and were analysed by two-way ANOVA. G, genotype; S, sex.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/6d97bc04307e15add425f16f.png"},{"id":97234177,"identity":"529558d1-5350-4461-9eee-d3a8526c0489","added_by":"auto","created_at":"2025-12-02 10:05:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatecholamine deficiency impairs lipid metabolism and induces insulin resistance during high-fat feeding.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-c, Serum triacylglycerol (TAG), non-esterified fatty acids (NEFA), and glycerol (males: WT n=10, KO n=8; females: WT n=8, KO n=6 [TAG, NEFA]; WT n=8, KO n=5 [glycerol]).\u003c/p\u003e\n\u003cp\u003ed, Fasted blood glucose in WT and Cyb561-KO mice (males: WT n=10, KO n=9; females: WT n=17, KO n=14).\u003c/p\u003e\n\u003cp\u003ee, f, Oral glucose tolerance test (oGTT) in chow-fed mice (males: WT n=10, KO n=7; females: WT n=8, KO n=6).\u003c/p\u003e\n\u003cp\u003eg, h, Plasma insulin during oGTT (males: WT n=7, KO n=5; females: WT n=5, KO n=4).\u003c/p\u003e\n\u003cp\u003ei, j, Plasma insulin during fast–refed challenge (males: WT n=6, KO n=5; females: WT n=5, KO n=4).\u003c/p\u003e\n\u003cp\u003ek, l, Serum TAG during lipid tolerance test (LTT) in WT and Cyb561-KO mice (males: n=9; females: WT n=9, KO n=7).\u003c/p\u003e\n\u003cp\u003em, n, Blood glucose during insulin tolerance test (ITT) in high-fat diet (HFD)–fed males (WT n=10, KO n=8).\u003c/p\u003e\n\u003cp\u003eo-r, Plasma insulin and glucose during fast–refed challenge in HFD-fed males (n=10).\u003c/p\u003e\n\u003cp\u003eData are mean ± SEM and were analysed by two-way ANOVA (a–m, o, q) or Student’s t-test (n, p, r). G, genotype; S, sex; T, time. All mice were phenotyped at 4–5 months.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/90cb1344bcfc6e7abfd5c3ad.png"},{"id":97234176,"identity":"5fd51455-9482-423a-b96a-2b9646ad2fc5","added_by":"auto","created_at":"2025-12-02 10:05:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":260601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatecholamines regulate metabolic flexibility independently of brown adipose tissue activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, Noradrenaline (NA) levels in brown adipose tissue (BAT) from WT and Cyb561-KO mice (males: WT n=10, KO n=9; females: WT n=7, KO n=6).\u003c/p\u003e\n\u003cp\u003eb, Representative BAT images of chow-fed male mice: tissue cross-section (left), H\u0026amp;E staining (10×, middle), and lipid droplet/nuclear classification (right).\u003c/p\u003e\n\u003cp\u003ec, Lipid droplet number (left) per cell and lipid droplet size (right) in BAT of chow-fed male mice (WT n=10, KO n=9).\u003c/p\u003e\n\u003cp\u003ed, Thermogenic gene expression in BAT of chow-fed (n=10) and HFD-fed (WT=10, KO=8) male mice.\u003c/p\u003e\n\u003cp\u003ee, Energy expenditure response to NA in male mice (WT n=6, KO n=5).\u003c/p\u003e\n\u003cp\u003ef-h, Serum triacylglycerol (TAG), non-esterified fatty acids (NEFA), and glycerol in females housed at 28 °C (WT n=11, KO n=9).\u003c/p\u003e\n\u003cp\u003ei, j, Fasted and fed blood glucose in females housed at 28 °C (WT n=11, KO n=10; fed: WT n=10, KO n=8).\u003c/p\u003e\n\u003cp\u003ek, l, Serum TAG during LTT in chow-fed females housed at 28 °C (WT n=11, KO n=9).\u003c/p\u003e\n\u003cp\u003eData are mean ± SEM and were analysed by two-way ANOVA (a, d, e, k) or Student’s t-test (c, f-j and l). G, genotype; D, diet; S, sex; T, time. All mice were phenotyped at 4–5 months.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/85b3ad3b015ec96195d949e2.png"},{"id":97251060,"identity":"ca1dd03a-6646-410c-9e84-ed5b78971c7b","added_by":"auto","created_at":"2025-12-02 13:15:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":193063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatecholamines couple lipid uptake and release in white adipose tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea, b, Body and organ weights of WT and Cyb561-KO mice on chow diet (males: WT n=10, KO n=9; females: WT n=8, KO n=6).\u003c/p\u003e\n\u003cp\u003ec, Gonadal (gWAT) and subcutaneous (scWAT) white adipose tissue lipid turnover, measured by the rate of disappearance of C18:2n6 on no-fat diet in male C57BL/6J mice. Each dot represents an individual male mouse.\u003c/p\u003e\n\u003cp\u003ed, e, Expression of lipolytic and lipid uptake/esterification genes in scWAT of chow- and HFD-fed male mice (n=10 per group, except KO HFD n=8).\u003c/p\u003e\n\u003cp\u003ef, Correlation between \u003cem\u003eAtgl\u003c/em\u003e and \u003cem\u003eFatp1\u003c/em\u003e expression in murine gWAT (n=34, C57BL/6J, various diets).\u003c/p\u003e\n\u003cp\u003eg, Gene expression correlation matrix (r², p-values) for scWAT from 19 human subjects.\u003c/p\u003e\n\u003cp\u003eh, Correlations of \u003cem\u003eATGL\u003c/em\u003e with \u003cem\u003eDGAT2\u003c/em\u003e in human subcutaneous (scWAT), mesenteric (mWAT), and omental (oWAT) adipose tissue (n=19).\u003c/p\u003e\n\u003cp\u003ei, Correlations of \u003cem\u003eATGL\u003c/em\u003e with \u003cem\u003eDGAT1\u003c/em\u003e in human WAT over time (n=13).\u003c/p\u003e\n\u003cp\u003eData are mean ± SEM and were analysed by two-way ANOVA (a, b, d, e) or linear regression (c, f–i). G, genotype; D, diet. All mice were phenotyped at 4–5 months.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/566cf27805d239cf20a1dab2.png"},{"id":97252593,"identity":"d573795f-f524-43bd-a47b-c17736b2cc9b","added_by":"auto","created_at":"2025-12-02 13:22:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1573664,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/2fea3cbf-41d1-417d-9475-027378fe7e01.pdf"},{"id":97234180,"identity":"468d466a-d631-491c-ac02-c0494ebf1f5d","added_by":"auto","created_at":"2025-12-02 10:05:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1752523,"visible":true,"origin":"","legend":"","description":"","filename":"Extendeddata.docx","url":"https://assets-eu.researchsquare.com/files/rs-8028437/v1/6597a3af87450564647ec768.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A High-Throughput Physiological Screen Reveals a Conserved Catecholamine-Dependent Axis Regulating Systemic Metabolic Flexibility","fulltext":[{"header":"Main","content":"\u003cp\u003eMetabolic flexibility, the ability to adaptively switch between lipid and carbohydrate oxidation, is a hallmark of metabolic health and resilience to nutritional stress. Fuel preference dynamically shifts with physiological state: carbohydrates are primarily oxidised during feeding and acute exercise, whereas lipids dominate during fasting, sleep, and endurance activity\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Impaired fuel switching, or metabolic inflexibility, is closely linked to insulin resistance, obesity, and cardiovascular diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Despite its well-established pathophysiological significance, assessing metabolic flexibility in humans remains labour-intensive, invasive, and costly, limiting large-scale genetic discovery.\u003c/p\u003e\u003cp\u003eTo overcome these limitations, rodents provide a robust model for assessing metabolic flexibility. Indirect calorimetry enables continuous measurement of the respiratory exchange ratio (RER), a well-established proxy for substrate utilisation, and mice display marked diurnal oscillations in fuel preference. We quantified metabolic flexibility using ΔRER, defined as the difference between the highest and lowest 10% of RER values\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, providing an integrated index of fuel-switching capacity. Across multiple diets, fasting states, and experimental sites, ΔRER values proved highly reproducible and minimally affected by short-term variations in body weight or food intake (Supplementary Results; Extended Data Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), establishing this parameter as a reliable and scalable metric for large-scale screening.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolic screen identifies Akt2 and Cyb561 as regulators of fuel flexibility.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo uncover novel regulators of metabolic flexibility, we analysed indirect calorimetry data from over 800 knockout mouse lines generated through the International Mouse Phenotyping Consortium (IMPC) metabolic pipeline at the Wellcome Trust Sanger Institute (WTSI)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Two lines were classified as hits according to the WTSI/IMPC criteria\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e for altered ΔRER: Akt2 and Cyb561.\u003c/p\u003e\u003cp\u003eAkt2 encodes a central mediator of anabolic insulin signalling\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and its loss impairs insulin responsiveness and oxidative capacity, both hallmarks of metabolic inflexibility. The identification of Akt2 validates the screening approach and demonstrates its reproducibility across sites, as indicated by the consistent compression of RER observed at both WTSI and UCAM (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn contrast, Cyb561 emerged as a previously unrecognised regulator of systemic metabolic flexibility. Cyb561 encodes an ascorbate-dependent electron transporter required for catecholamine biosynthesis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Consistent with the WTSI results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), Cyb561 knockout mice phenotyped at UCAM exhibited reduced ΔRER (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec,d). Notably, the screen identified genes acting in opposing metabolic pathways, anabolic (Akt2) and catabolic (Cyb561), whose loss similarly reduced ΔRER, underscoring the bidirectional regulation of systemic metabolic flexibility.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCatecholamine deficiency disrupts lipid handling and predisposes to insulin resistance.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsistent with impaired catecholamine signalling, Cyb561-KO mice exhibited reduced fasting lipid oxidation, reflected by elevated daytime RER values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), likely due to defective adipose tissue lipolysis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In line with this, circulating triacylglycerol, non-esterified fatty acids, and glycerol were all decreased in Cyb561-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). Despite normal fed glycaemia and insulin levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b), these mice developed fasting hypoglycaemia while maintaining normal glucose and pyruvate tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u0026ndash;h; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee,f), indicating preserved glucose disposal and hepatic gluconeogenic capacity. Collectively, these findings suggest a compensatory shift toward glucose utilisation during fasting, recapitulating the adipose-driven metabolic inflexibility previously described in PPARγ2-deficient mice \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAcute nutritional challenges unmasked subjacent metabolic defects in Cyb561-KO mice. Upon refeeding, knockout mice displayed exaggerated insulin responses without overt hyperglycaemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei,j; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;j), and lipid tolerance tests revealed markedly delayed triglyceride clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek,l; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek).\u003c/p\u003e\u003cp\u003eWe next examined the response of male Cyb561-KO mice to chronic nutrient excess induced by two months of high-fat diet (HFD) feeding. Under HFD, Cyb561-deficient mice consistently exhibited lower fasting glucose and preserved glucose tolerance (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c), yet maintained normoglycaemia at the expense of elevated fed-state insulin levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,e), indicative of systemic insulin resistance confirmed by insulin tolerance tests (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em,n). As in chow-fed conditions, Cyb561-KO mice under HFD showed exacerbated abnormalities in the fasting\u0026ndash;refeeding response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo\u0026ndash;r). Circulating triglycerides, NEFA, and glycerol remained reduced, reinforcing the presence of defective lipolysis during HFD (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u0026ndash;h).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTogether, these findings demonstrate that catecholamine deficiency impairs adaptive fuel utilisation, rendering mice metabolically vulnerable under nutritional stress..\u003c/p\u003e\n\u003ch3\u003eImpaired substrate switching persists when BAT thermogenesis is suppressed\u003c/h3\u003e\n\u003cp\u003eCatecholamines are key activators of BAT thermogenesis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In Cyb561-KO mice, BAT noradrenaline (NA) levels were undetectable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Histological analysis revealed fewer but enlarged lipid droplets per cell (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), accompanied by altered expression of genes regulating lipid uptake and lipolysis, indicating increased storage and reduced mobilisation (Extended Data Fig.\u0026nbsp;5a,b). Thermogenic gene expression was diminished under both chow and high-fat diet conditions, and Ucp1 protein content per BAT pad was markedly reduced (Extended Data Fig.\u0026nbsp;5c,d). Functionally, maximal thermogenic capacity induced by NA was strongly blunted in Cyb561-KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e), and acute cold exposure elicited a weaker induction of thermogenic genes (Extended Data Fig.\u0026nbsp;5e). Together, these findings confirm that loss of catecholamine tone compromises BAT responsiveness.\u003c/p\u003e\u003cp\u003eBAT serves as a major sink for circulating lipids\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and its dysfunction could account for the metabolic inflexibility observed in Cyb561-KO mice. To test this, we housed wild-type and Cyb561-KO female mice at 28\u0026deg;C, a condition that suppresses BAT activity and approximates human thermal physiology. As at room temperature, Cyb561-deficient mice displayed reduced lipolytic activity during the light phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u0026ndash;h), fasting hypoglycaemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), and preserved glucose tolerance and fasting\u0026ndash;refeeding responses (Extended Data Fig.\u0026nbsp;6a\u0026ndash;g). However, unlike at room temperature, fed glucose levels were significantly elevated in knockout mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg), accompanied by a trend toward higher insulin levels (Extended Data Fig.\u0026nbsp;6h), revealing a glucose-handling defect apparent only when thermogenic demand was reduced. Importantly, even under these warm, low-sympathetic conditions, Cyb561-KO mice continued to exhibit slower triglyceride clearance than wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek,l), indicating defective lipid uptake independent of BAT thermogenesis\u003c/p\u003e\u003cp\u003eTogether, these findings demonstrate that catecholamine deficiency disrupts systemic metabolic flexibility across both glucose and lipid pathways, even when BAT thermogenesis is suppressed, establishing a BAT-independent role for catecholamines in coordinating whole-body fuel switching.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCatecholamines coordinate lipid turnover without altering adiposity\u003c/h2\u003e\u003cp\u003eDespite the combined impairment of white adipose tissue (WAT) lipolysis and BAT thermogenesis, Cyb561-KO mice maintained normal body weight and adiposity across diets and housing temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b and Extended Data Fig.\u0026nbsp;7a\u0026ndash;d).\u003c/p\u003e\u003cp\u003eTo directly assess lipid turnover in WAT, we placed wild-type (WT) mice on a fat-free diet and tracked the loss of linoleic acid (C18:2ω6), an essential fatty acid that must be obtained from the diet. Turnover was faster in subcutaneous (scWAT) than gonadal WAT (gWAT), with estimated losses of ~\u0026thinsp;0.66% per day and ~\u0026thinsp;0.42% per day, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Extrapolated to whole-body fat mass, this corresponds to ~\u0026thinsp;100 mg per day of lipid flux in a mouse with 4 g of fat. Given the lack of increased adiposity in Cyb561\u0026ndash;KO mice, these data indicate that diminished mobilisation was offset by proportionally decreased lipid uptake.\u003c/p\u003e\u003cp\u003eIn Cyb561-deficient mice, the absence of increased adiposity despite reduced lipolytic activity implies that diminished lipid mobilisation was offset by proportionally reduced lipid uptake. Without this compensatory mechanism, Cyb561-KO mice would be expected to accumulate\u0026thinsp;~\u0026thinsp;5 g additional fat over two months. Together, these findings indicate that catecholamines coordinate lipid flux in WAT, maintaining a balance between lipid release and uptake rather than controlling net fat storage.\u003c/p\u003e\u003cp\u003eGene expression analyses revealed a coordinated suppression of lipid release and uptake pathways in Cyb561-KO WAT. In scWAT, both lipolytic genes (\u003cem\u003eAtgl, Hsl\u003c/em\u003e) and genes mediating lipid uptake (\u003cem\u003eLpl, Fatp1, Cd36\u003c/em\u003e) were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,e), indicating that catecholamines co-regulate lipid input and output pathways. To reinforce that catecholamines dynamically modulate this coupling, we exposed mice to cold, a potent activator of adipose sympathetic outflow. Cold exposure significantly increased the expression of both lipid uptake and release genes in WAT and tended to strengthen their correlation (Extended Data Fig.\u0026nbsp;7e,f), further supporting a catecholamine-dependent regulatory mechanism.\u003c/p\u003e\u003cp\u003eThis coupling extended beyond our model. Across both mice and humans, expression of lipid uptake genes positively correlated with lipolytic gene expression in all tested depots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u0026ndash;i), suggesting an evolutionarily conserved mechanism aligning adipose lipid flux. Notably, correlations between lipid-release and re-esterification genes remained stable over time (84 days) in humans (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), underscoring the robustness of this transcriptional coordination.\u003c/p\u003e\u003cp\u003eTogether, these findings define a conserved transcriptional programme in WAT whereby catecholamines coordinate lipid uptake and release, maintaining lipid flux and systemic energy balance during metabolic stress or impaired thermogenesis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough metabolic flexibility is a defining feature of metabolic health, its physiological regulation remains poorly understood. Here, we overcome key limitations in assessing this trait at scale \u003cem\u003ein vivo\u003c/em\u003e, establishing a framework for systematic discovery of its genetic determinants. By integrating large-scale physiological phenotyping with molecular analyses, we identify Cyb561, emerging alongside Akt2, and catecholamine signalling as critical regulators of systemic metabolic flexibility. The striking phenotypic parallels between Cyb561 and Akt2 knockout mice, despite their roles in ostensibly opposing hormonal pathways, suggest that catecholamines and insulin converge over longer timescales to coordinate lipid flux and maintain systemic metabolic flexibility.\u003c/p\u003e\u003cp\u003eMechanistically, Cyb561 deletion caused profound BAT dysfunction and markedly reduced circulating non-esterified fatty acids, glycerol, and triglycerides, consistent with defective WAT lipolysis. Yet, despite impaired lipid mobilisation, Cyb561-KO mice did not accumulate excess adiposity under any dietary or thermal condition. Instead, we show that catecholamines synchronise lipid mobilisation and uptake in WAT, thereby sustaining lipid turnover and maintaining substrate flexibility across energetic states. Gene expression analyses in mice and humans reveal tight, evolutionarily conserved co-regulation of lipid uptake and lipolytic pathways across depots and over time, enabling adipose tissue to act as a dynamic metabolic buffer. Loss of catecholamine tone disrupts this coordination, diminishing lipid flux, impairing fuel switching, and predisposing to insulin resistance under metabolic stress.\u003c/p\u003e\u003cp\u003eThe persistence of defective lipid handling at thermoneutrality highlights a BAT-independent role for catecholamines, with WAT emerging as a key effector. Notably, Cyb561-KO mice developed fed-state hyperglycaemia and hyperinsulinaemia when housed at 28\u0026deg;C, a defect masked at room temperature. This may reflect a redistribution of glucose toward thermogenic processes under mild cold stress, consistent with evidence that skeletal muscle non-shivering thermogenesis primarily relies on glucose oxidation, whereas BAT thermogenesis depends largely on lipids\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These observations carry translational relevance, as humans typically reside under thermoneutral conditions and depend more on WAT and skeletal muscle than on BAT for adaptive thermogenesis.\u003c/p\u003e\u003cp\u003eWhile our study establishes a central role for catecholamines in regulating lipid uptake and metabolic adaptability, several questions remain. Adrenergic signals also influence LPL activity in heart, skeletal muscle, and BAT, thereby modulating triglyceride clearance and systemic fuel partitioning\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Consequently, the phenotype of Cyb561-KO mice may in part reflect impaired lipid disposal in these peripheral tissues. Catecholamines additionally suppress insulin secretion\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, given the pronounced adipose lipid defects and the absence of fasting hyperinsulinaemia, the elevated fed insulin levels observed in Cyb561-deficient mice most likely stem from peripheral insulin resistance. Taken together, while contributions from multiple organs cannot be excluded, dysfunction of WAT appears to be the predominant driver of metabolic inflexibility in this model.\u003c/p\u003e\u003cp\u003eWhile our study establishes a key role for catecholamines in lipid uptake and metabolic adaptability, several questions remain. Adrenergic signals also modulate LPL activity in heart, skeletal muscle, and BAT, affecting triglyceride clearance and fuel partitioning\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Consequently, the phenotype of Cyb561-KO mice could partly reflect impaired lipid disposal in these tissues. Catecholamines also suppress insulin secretion\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, given the pronounced adipose lipid defects and absence of fasting hyperinsulinaemia, the elevated fed insulin levels in Cyb561-deficient mice most likely stem from peripheral insulin resistance. Overall, while contributions from multiple organs cannot be excluded, dysfunction of WAT appears to be the predominant driver of metabolic inflexibility in this model.\u003c/p\u003e\u003cp\u003eAlthough the phenotype is most plausibly due to loss of catecholamine biosynthesis, CYB561 may have catecholamine-independent roles in ascorbate recycling, iron metabolism, and redox homeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, which could, in turn, influence energy metabolism. Nonetheless, the near-complete depletion of noradrenaline in BAT, together with the well-established role of catecholamines in lipid mobilisation and thermogenesis, strongly supports adrenergic deficiency as the primary driver of the observed phenotype.\u003c/p\u003e\u003cp\u003eFuture studies should clarify central versus peripheral contributions and the molecular mechanisms linking adrenergic tone to adipocyte lipid uptake. Parallel transcriptomic effects in human adipose tissue suggest potential translational relevance. This association may be clinically relevant given the associations of β-blocker therapy and age-related sympathetic decline with reduced metabolic flexibility and increased diabetes risk\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn summary, our high-throughput ΔRER-based screen identifies catecholamine signalling as a central regulator of whole-body fuel switching. By coupling adrenergic tone to lipid turnover and insulin sensitivity, we uncover a conserved axis that maintains systemic metabolic flexibility and highlight a pathway that could be therapeutically targeted to enhance metabolic resilience in obesity and diabetes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal models\u003c/strong\u003e\u003cp\u003eCyb561\u003csup\u003etm1a(EUCOMM)Wtsi\u003c/sup\u003e knockout (KO) mice and AKT2\u003csup\u003etm1a(EUCOMM)Wtsi\u003c/sup\u003e KO mice were obtained from the Wellcome Trust Sanger Institute as part of the IMPC. AKT2\u003csup\u003etm1.1mbb\u003c/sup\u003e KO mice were obtained from Jackson Laboratory and phenotyped at the University of Cambridge on a C57BL/6J background, while all other lines were maintained on a C57BL/6N background. Homozygous KO and wild-type littermates were generated by mating heterozygous animals. Additional wild-type C57BL/6J mice were purchased from Charles River UK. Ages and sexes of animals are indicated in the figure legends. Mice were housed in specific pathogen-free facilities under 12-hour light/12-hour dark cycles at 21\u0026deg;C unless otherwise stated. All procedures were approved by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB) under the UK Animals (Scientific Procedures) Act 1986 (Amendment Regulations 2012).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDiets\u003c/strong\u003e\u003cp\u003eAnimals were fed standard breeder\u0026rsquo;s chow (Safe Diets DS-105), a high-fat diet containing 45% of kcal from fat (Research Diets D12451, Brogaarden, Denmark), or a fat-free diet (SDS 820416, based on AIN-93M with soybean oil replaced by cornstarch). The Sanger MGP used a Western diet (SDS Western RD 829100, UK). All diets were provided ad libitum.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHuman Subjects\u003c/strong\u003e\u003cp\u003eHuman studies included two cohorts. In cohort 1, subcutaneous adipose tissue biopsies were obtained from 19 individuals undergoing bariatric bypass surgery, from which adipose tissue macrophages were isolated. Clinical characteristics of these participants have been published previously \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. All participants provided written informed consent.\u003c/p\u003e\u003cp\u003eCohort 2 consisted of 60 obese individuals aged 20\u0026ndash;55 years with BMI 30\u0026ndash;40 kg/m\u0026sup2;, who had no type 2 diabetes, no history of childhood obesity, and no prior bariatric surgery. Participants were enrolled in a 12-week outpatient study evaluating weight loss interventions \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and were randomised to one of three interventions: calorie restriction (\u0026minus;\u0026thinsp;600 kcal/day), calorie restriction plus moderate exercise (~\u0026thinsp;10% of daily energy expenditure), or calorie restriction plus the serotonin-norepinephrine reuptake inhibitor sibutramine. For this study, 26 biopsies from 13 participants were analysed. Participants were stratified into \u0026ldquo;losers\u0026rdquo; (\u0026ge;\u0026thinsp;5 kg weight loss by day 84) and \u0026ldquo;stable\u0026rdquo; (\u0026plusmn;\u0026thinsp;2 kg weight change). Ethical approval for the human studies was obtained from the GlaxoSmithKline protocol review panel, the Cambridge Local Research Ethics Committee (08/H0308/10), and the Wellcome Trust Clinical Research Facility Scientific Advisory Board.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMetabolic tests\u003c/strong\u003e\u003cp\u003eOral glucose tolerance tests were performed after overnight fasting from 16:00 to 09:00. Mice were acclimatised to the procedure room for 1 hour before receiving a fixed oral glucose dose of 2 g/kg, based on the average weight of the control group. Blood glucose measurements were taken at 0, 10, 20, 30, 60, 90, and 120 minutes.\u003c/p\u003e\u003cp\u003eInsulin tolerance tests were conducted following a 6-hour fast from 08:00 to 14:00. Mice were administered 1 U/kg Actrapid insulin intraperitoneally at a dose normalised to the average weight of the control group.\u003c/p\u003e\u003cp\u003eLipid tolerance tests were performed after overnight fasting. Mice were gavaged with 200 \u0026micro;l olive oil, and blood samples were collected at 0, 1, 2, 4, and 6 hours for triglyceride analysis.\u003c/p\u003e\u003cp\u003eFor hepatic gluconeogenesis assessment, mice were fasted overnight and injected intraperitoneally with sodium pyruvate (2 g/kg). Blood glucose was measured 0, 10, 20, 30, 60, 90, and 120 minutes.\u003c/p\u003e\u003cp\u003eFast refed were performed after overnight fasting from 16:00 to 09:00. Mice were acclimatised to the procedure room for 1 hour. After a basal blood sample, food was provided ad libitum and blood samples taken 2- and 6-hours post-refeeding.\u003c/p\u003e\u003cp\u003eData were analysed by measuring the incremental area under the curve (oGTT, fast-refed, LTT) or the area over the curve (ITT)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNorepinephrine-Stimulated Energy Expenditure\u003c/strong\u003e\u003cp\u003eMice were anaesthetised with pentobarbital (60 mg/kg) and placed in a temperature-controlled calorimetry chamber (28\u0026deg;C, 2.4 L volume, Creative Scientific UK). Baseline oxygen consumption and carbon dioxide production were measured every 4 minutes until stable, after which mice received 1 \u0026micro;g/kg norepinephrine and 30 mg/kg pentobarbital subcutaneously. Energy expenditure was monitored for a further 28 minutes and calculated using the modified Weir equation: EE (J/min)\u0026thinsp;=\u0026thinsp;15.818 \u0026times; VO₂ (ml/min)\u0026thinsp;+\u0026thinsp;5.176 \u0026times; VCO₂ (ml/min).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAltered temperature housing\u003c/strong\u003e\u003cp\u003eAcute cold exposure experiments involved exposure to 5\u0026deg;C in a temperature- and humidity-controlled cabinet after prior acclimation to 21\u0026deg;C. One mouse was culled every 30 minutes over 3.5 hours to enable regression analysis of transcriptional responses. During chronic cold exposure, mice were housed at 24\u0026deg;C or at 5\u0026deg;C in a temperature- and humidity-controlled cabinet for 4 weeks.\u003c/p\u003e\u003cp\u003eTo reduce BAT activity, animals were housed at 28\u0026deg;C in a temperature- and humidity-controlled cabinet for six weeks.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eIndirect Calorimetry and Metabolic Flexibility\u003c/strong\u003e\u003cp\u003eIndirect calorimetry was performed using custom-built chambers (Creative Scientific UK) for up to 48 hours. Gas concentrations were recorded every 11 minutes at a flow rate of 400 ml/min. Energy expenditure was calculated using the modified Weir equation. Metabolic flexibility was defined as the amplitude of the respiratory exchange ratio (RER), calculated as the difference between the mean of the highest 10% and the mean of the lowest 10% of RER values\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSerum Biochemistry\u003c/strong\u003e\u003cp\u003eSerum triglycerides and glycerol were measured on a Dimension RXL analyser (Siemens Healthcare), and insulin was quantified using electrochemiluminescence immunoassay (MesoScale Discovery). Noradrenaline levels in brown adipose tissue were measured using HPLC with electrochemical detection\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA Extraction and qPCR\u003c/strong\u003e\u003cp\u003eRNA was extracted using STAT-60 (AMS Biotech) and reverse transcribed into cDNA using the Promega Reverse Transcriptase System. qPCR was performed using TaqMan or SYBR Green chemistry on an ABI QuantStudio 5 (Applied Biosystems). Gene expression was normalised to the geometric mean of housekeeping genes: \u003cem\u003e18s\u003c/em\u003e, \u003cem\u003e36b4\u003c/em\u003e, and \u003cem\u003eTbp\u003c/em\u003e (mouse) or \u003cem\u003e18S, CYCA\u003c/em\u003e, and \u003cem\u003eGUSB\u003c/em\u003e (human).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003cp\u003eWestern blotting was performed using RIPA-extracted protein quantified by Bio-Rad DC assay. Proteins (10 \u0026micro;g/lane) were denatured at 95\u0026deg;C for 5 minutes, separated on 4\u0026ndash;12% gradient SDS-PAGE gels (Novex NuPage), transferred using iBlot 2, and probed with antibodies including Ucp1 (ab10983, Abcam) and β-tubulin (ab6046, Abcam).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003cp\u003eBrown adipose tissue was fixed in formalin, paraffin-embedded, sectioned at 4 \u0026micro;m, and stained with H\u0026amp;E. Imaging was performed using an Axioscan Z1 microscope (20\u0026times; objective). Lipid droplet quantification was performed using Halo software (Indica Labs).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eGC-MS Fatty Acid Analysis\u003c/strong\u003e\u003cp\u003eLipids were extracted using the Folch method, esterified using BF₃-methanol, and analysed by GC-MS. Methyl esters were quantified based on integrated peak areas against reference standards (Thames Restek, UK) as previously described\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism, SPSS (v26/27), or JASP. Outliers were identified using ROUT testing, and any removed outliers are noted in the Source Data files. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Greenhouse\u0026ndash;Geisser correction was applied for repeated-measures two-way ANOVA. For comparisons using Student\u0026rsquo;s t-test, an F-test was first applied to assess equality of variance; when variances were significantly different, Welch\u0026rsquo;s t-test was used instead. Specific tests used for each dataset are detailed in the figure legends, and the test results are presented in the Source Data files.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was supported by the British Heart Foundation [grant number RG/F/23/110110] and the Medical Research Council [grant number MC_UU_00039]. We thank the disease model core (DMC) for their excellent technical assistance with animal phenotyping, which is supported by the Medical Research Council [grant number MC_UU_00039]; and the Wellcome Trust [grant number 226800/Z/22/Z]. We thank the Core Biochemical Assay Laboratory (CBAL) which is supported by the NIHR Cambridge Biomedical Research Centre (NIHR203312); and the Wellcome Trust [grant number 226800/Z/22/Z]. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a Creative Commons Attribution (CC-BY) licence to any Author Accepted Manuscript version arising from this submission.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll data supporting this study are available in the accompanying Source Data files. No custom code was used. Additional information is available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoodpaster BH, Sparks LM (2017) Metabolic Flexibility in Health and Disease. 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Nat Metab 3:1150\u0026ndash;1162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42255-021-00440-5\u003c/span\u003e\u003cspan address=\"10.1038/s42255-021-00440-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8028437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8028437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetabolic flexibility, the capacity to switch between carbohydrate and lipid fuels, is fundamental to metabolic health, yet its genetic regulation has not been systematically explored \u003cem\u003ein vivo\u003c/em\u003e. To address this gap, we developed a high-throughput physiological screen in mice using the respiratory exchange ratio range (ΔRER) as quantitative index of metabolic flexibility. Screening over 800 knockout lines identified Cyb561, a vesicular cytochrome required for catecholamine biosynthesis, as an unexpected regulator of systemic substrate utilisation.\u003c/p\u003e\u003cp\u003eMice lacking Cyb561 exhibited impaired lipolysis, blunted brown adipose thermogenesis, and delayed lipid clearance, while maintaining preserved glucose tolerance. Under high-fat feeding or at thermoneutrality (28\u0026deg;C), Cyb561 deficiency exacerbated insulin resistance, revealing metabolic vulnerability. Remarkably, despite impaired lipolysis and BAT dysfunction, Cyb561 knockout mice did not develop increased adiposity. Mechanistically, we demonstrate that catecholamines transcriptionally couple lipid uptake and release in white adipose tissue, thereby sustaining lipid turnover and maintaining systemic metabolic flexibility.\u003c/p\u003e\u003cp\u003eTogether, these findings establish catecholamine tone as a key determinant of whole-body fuel switching and demonstrate that high-throughput ΔRER phenotyping offers a powerful and sensitive approach for large-scale analysis of the genetic determinants of metabolic flexibility.\u003c/p\u003e","manuscriptTitle":"A High-Throughput Physiological Screen Reveals a Conserved Catecholamine-Dependent Axis Regulating Systemic Metabolic Flexibility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 10:05:43","doi":"10.21203/rs.3.rs-8028437/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fbef3621-4707-48cc-af83-8cce7a80ac22","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58201572,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"},{"id":58201573,"name":"Biological sciences/Physiology/Metabolism/Homeostasis"},{"id":58201574,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Obesity"},{"id":58201575,"name":"Biological sciences/Biological techniques/High-throughput screening"}],"tags":[],"updatedAt":"2025-12-02T10:05:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 10:05:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8028437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8028437","identity":"rs-8028437","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}