The conjugation-resistant bile acid norUDCA cures liver fibrosis but impairs systemic energy metabolism

The conjugation-resistant bile acid norUDCA cures liver fibrosis but impairs systemic energy metabolism | 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 Article The conjugation-resistant bile acid norUDCA cures liver fibrosis but impairs systemic energy metabolism Joerg Heeren, Ioannis Evangelakos, Esther Verkade, Folkert Kuipers, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8280795/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bile acids (BAs) play an important role in systemic metabolic improvements following bariatric surgery. In this study, we found that orally administered norursodeoxycholic acid (norUDCA), a conjugation-resistant C23 derivative of naturally occurring UDCA, accumulated in peripheral organs including heart and brown adipose tissue (BAT). Moreover, norUDCA decreased systemic levels of endogenous conjugated BAs, while increasing unconjugated BAs. Notably, in addition to beneficial effects in a cholestatic liver disease model, norUDCA also lowered plasma glucose and fat mass in mice, suggesting that this BA derivative could be repurposed for treating obesity-associated cardiometabolic diseases. Metabolic energy expenditure studies, however, revealed that norUDCA-treated mice developed intolerance to cold stress, a phenotype exacerbated in mice lacking adipose ATGL-dependent lipolysis. Transcriptomic and metabolic analyses demonstrated tissue remodeling in heart and BAT that involved pronounced changes in energy substrate utilization, including enhanced cardiac glucose uptake. Importantly, co-administration of a low-carb diet prevented cold stress-induced metabolic deficits. Mechanistic studies in human engineered heart tissue indicated that norUDCA impaired mitochondrial respiration and thereby compromised contractile function. In conclusion, these data suggest that conjugation- resistant BA derivatives like norUDCA impair myocardial and BAT energetics by altering glucose, lipid, and energy metabolism, particularly during catabolic cold stress conditions. Biological sciences/Physiology/Metabolism/Fat metabolism Health sciences/Gastroenterology/Gastrointestinal system/Liver/Hepatic portal vein Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic syndrome Biological sciences/Chemical biology/Metabolic pathways Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Main Bile acids (BAs) are cholesterol derivatives that are synthesized in hepatocytes by consecutive steps of enzymatic reactions finalized by conjugation to taurine or glycine. The unconjugated (UBAs) and conjugated (CBAs) species present in the circulating BA pool vary considerably in their biochemical and biophysical properties, which has important physiological and pathological consequences 1 . For example, patients suffering from metabolic dysfunction-associated steatohepatitis, alcoholic liver cirrhosis, or primary biliary cholangitis (PBC) frequently exhibit elevated total BAs, particularly CBAs 2 . These pathological conditions are further characterized by higher CBA/UBA ratios 3 , reflecting underlying alterations in the gut microbiome and its capacity for BA-transformation, including the generation of secondary BAs 4 . Supporting a potentially detrimental role of CBAs, a recent study demonstrated that the inhibition of BA conjugation through deletion of the hepatic bile acid–CoA: amino acid N-acyltransferase (BAAT) enhanced tumor-specific T cell responses and reduced tumor growth in liver 5 . In line, conjugation of the gut microbiome-derived secondary BAs 3-oxoLCA and isoalloLCA reduced their immunomodulatory effects on T helper and regulatory T cells 6 , indicating that conjugation also of secondary BAs influences adaptive immune responses. Similarly, enriching the BA-pool with hydrophilic BAs, such as ursodeoxycholic acid (UDCA), an approved standard therapy for the treatment of PBC 7 , boosts T cell function and consequently abolishes liver injury and concomitant tumors growth in various mouse models 5 , 8 – 10 . On the other hand, BA de-conjugation by bacterial bile salt hydrolases (BSHs) in the gut generates UBAs that are less efficient than their conjugated counterparts in the emulsification of dietary lipids and may lead to dysregulated lipid absorption and weight loss 11 , 12 . Growing evidence suggest that the balance between BA-conjugation and microbial de-conjugation is an important determinant for systemic metabolism, such as during Roux-en-Y gastric bypass (RYGB) surgery. This weight loss procedure causes increased BA levels, predominantly of glycine-conjugated BAs in the postprandial phase 13 , which are associated with altered rates of glucose and lipid oxidation 14 . Another example that a higher CBA to UBA ratio regulates systemic energy metabolism is based on the observation that under conditions of BAT activation by cold exposure, adaptive changes in endogenous BA metabolism were associated with efficient heat production as wells as improved lipid and lipoprotein metabolism in mice 15 , 16 . In another mouse study, focusing on gut microbiome changes in response to cold exposure, the beneficial effects on adiposity and adaptive thermogenesis also involved enhanced CBA production and a higher CBA to UBA ratio 17 . Notably, metabolomics analysis of human plasma samples following BAT activation by the β3-adrenoreceptor agonist mirabegron revealed that BA metabolism was the most affected pathway 18 , 19 . C23 BAs with a shortened side chain, such as norchenodeoxycholic acid (norCDCA), norcholic acid (norCA) and norUDCA, have been developed to improve therapeutic properties of naturally occurring BAs 20 . These nor-BAs are inefficiently amidated, i.e. they are highly resistant to taurine or glycine conjugation as well as microbial biotransformation. Accordingly, they are secreted in an unconjugated form into bile, which induces hypercholeresis accompanied by increased bicarbonate output 21 , thereby alleviating toxic effects particularly of hydrophobic BAs 20 . This is in particular the case for norUDCA, a derivative that combines hydrophilicity with conjugation resistance, and has been successfully used for therapeutic intervention in preclinical models and patients with cholestatic as well as fibrotic liver diseases 22 – 25 . Moreover, norUDCA has been shown to modulate regulatory networks influencing immune-metabolism of T cells, attenuating T cell-driven inflammatory diseases in gut and liver 26 , 27 . Importantly, unconjugated norUDCA can reach plasma concentration that are approximately 10-fold higher as compared to other orally administered endogenous BAs including UDCA 26 . However, the consequences of this systemic spill-over for peripheral tissues such as muscle, heart, brown or white fat depots remain elusive. Given its high bioavailability in the systemic circulation, here we investigated in mice whether norUDCA could serve as a potential and suitable treatment to enhance energy expenditure and thermogenesis. We found that the accumulation of norUDCA in peripheral organs was associated with reduced adiposity and lower blood glucose. Unexpectedly, however, norUDCA treatment resulted in decreased energy expenditure and core body temperature in response to energy-demanding cold exposure. Metabolic tracer and mechanistic studies indicate a critical role of norUDCA for fuel uptake, mitochondrial respiration and whole-body energy metabolism, particularly during adaptive thermogenesis induced by cold exposure. Moreover, our data suggest that elevated systemic levels of norUDCA together with the concomitant increase in endogenous unconjugated BAs cause alterations in glucose and lipid homeostasis that contribute to metabolic inflexibility in cardiac and adipose tissues, impairing their capacity to effectively utilize energy substrates for metabolic processes. Results NorUDCA treatment ameliorates cholestatic liver disease but confers intolerance to cold exposure To investigate the impact of conjugation-resistance of BAs for systemic energy metabolism, we first compared UDCA versus norUDCA treatment in wild type and Cyp2c70 −/− mice. The latter is a preclinical model with a more hydrophobic bile acid pool that results in spontaneous cholestatic liver disease, which is based on the deficient conversion of CDCA into the hydrophilic muricholic acids 28 , 29 . In the experimental setup, wild type and Cyp2c70 −/− mice were fed a regular chow (control) or diets supplemented with 0.5% of either UDCA or norUDCA for a period of one week. Both BAs equally improved the inflammatory and fibrotic liver phenotype in Cyp2c70 −/− mice (Extended Data Fig. 1 a-c), which is in line with a previous study using UDCA 8 . In caecum, norUDCA levels accumulated to a concentration that was 5-fold higher than that of UDCA (Extended Data Fig. 1 d). Notably, norUDCA but not UDCA significantly reduced body weight (Fig. 1 a), which was mainly explained by reduced fat mass (Extended data Fig. 2 a) and lower weights of adipose tissue depots (Fig. 1 b). Treatment with norUDCA led to larger gall bladders (Fig. 1 b, Extended Data Fig. 2 b), which was probably a result of hypercholeresis 23 , 30 . The weights of other organs including liver (Fig. 1 c) and heart (Fig. 1 b) were unaffected. As the reduced fat mass suggested enhanced energy expenditure and heat production in BAT and muscle, we determined the effects of norUDCA on adaptive thermogenic responses. To this end, wild type mice fed a normal chow (control) or chow supplemented with norUDCA were housed at thermoneutral conditions (30 o C) or exposed to cold (6 o C). Unexpectedly, the norUDCA-treated mice housed at 6°C were completely cold-intolerant and 80% died within 8 hours of cold exposure (Fig. 1 d). Furthermore, indirect calorimetry revealed that mice on the norUDCA-supplemented diet exhibited progressively lower respiration rates and body temperatures even when the ambient temperatures were gradually reduced from 30 o C to only 16 o C (Fig. 1 e-h, Extended Data Fig. 2 c-f). These data indicate that norUDCA treatment reduces whole body energy expenditure, thermogenesis and critically impacts survival during cold acclimation. NorUDCA accumulates in the systemic circulation and peripheral tissues together with endogenous unconjugated BAs To determine whether norUDCA exerts its effects on energy expenditure in peripheral organs directly via the circulation, we measured CBA und UBA species in wild type mice fed a normal chow or a norUDCA-supplemented diet. Dietary exposure resulted in decreased fecal CBAs and increased total fecal bile acid content, the latter resulting from the exogenous norUDCA (Fig. 2 a). Despite the high fecal excretion, concentrations of norUDCA in systemic plasma reached values of approximately 200 µM (Fig. 2 b). Remarkably, concentrations of endogenous UBAs were also increased in plasma with total levels of ~ 20µM. Thus, UBA levels by far exceed those of CBAs with ~ 2 µM, indicating a pronounced spillover of UBAs into the systemic circulation (Fig. 2 b). To determine the exposure of peripheral organs to norUDCA, we orally administered ³H-norUDCA and compared its organ uptake at four hours with that of simultaneously administered ¹⁴C-cholic acid (¹⁴C-CA) in mice fed a chow or a norUDCA-supplemented chow diet. Irrespective of diet composition, almost 90% of radiolabeled CA was detected in the intestine and only minute amounts were found in the liver (Fig. 2 c). In contrast, only ~ 30% of 3 H-norUDCA ended up in the intestine and a similar amount was found in the liver (Fig. 2 c, d), which argues for efficient cholehepatic shunting between the bile duct epithelium and liver of norUDCA as compared to CA. Remarkably, a substantial amount of 3 H-norUDCA compared to 14 C-CA was detected in peripheral tissues such as heart and BAT, an effect that was even more pronounced in norUDCA-preconditioned mice (Fig. 2 e-f, Extended Data Fig. 3 a-d). Our data demonstrate considerable exposure of key metabolic tissues to circulating norUDCA and other UBAs. NorUDCA induces tissue remodeling and alters energy substrate utilization in heart and BAT Metabolic functions of BAT and heart are critical for physiological responses to cold stress by increasing heat production and blood flow, respectively 31 . To understand the effects of norUDCA in an unbiased manner, bulk RNA sequencing of heart (Fig. 3 a-c) and BAT (Fig. 3 d-f) from mice on regular chow or on norUDCA-supplemented chow was performed. Volcano plot analysis indicated a high number of differentially expressed genes (DEG) between the groups both in heart (Fig. 3 a, Supplementary Table 1) and BAT (Fig. 3 d, Supplementary Table 2). In hearts, top upregulated genes upon norUDCA feeding were related to ventricular remodeling such as Atf3 and Myh7 , as well as those mediating mitochondrial biogenesis like Ppargc1a (Fig. 3 a). Furthermore, Slc2a1 and Slc2a4 encoding the glucose transporters GLUT1 and GLUT4, respectively, were upregulated whereas Pdk4 , known to inhibit pyruvate dehydrogenase, was reduced. The changes in expression of a number of metabolic genes were confirmed by qPCR in an independent study (Extended Data Fig. 4 a). Gene ontology (GO) analysis of DEG data revealed that a few metabolic pathways were downregulated (Fig. 3 b). On the other hand, pathways related to glucose metabolism, e.g., pyruvate metabolism, glycolysis and gluconeogenesis, as well as tissue remodeling pathways including proteasome, ferroptosis and mitophagy were upregulated in the hearts (Fig. 3 c). In BAT, the expression of key thermogenic markers including Ucp1 , Dio2 and Adrb3 was downregulated (Fig. 3 d). Consistently, pathways related to core thermogenic functions such as oxidative phosphorylation, fatty acid metabolism and thermogenesis were suppressed in the norUDCA-treated group (Fig. 3 e). In line, qPCR showed altered expression of lipogenic genes such as Fasn and Scd1 (Extended Data Fig. 4 b). Similar to the findings in heart, pathways related to tissue remodeling were upregulated in BAT of norUDCA-treated mice (Fig. 3 f). The functional relevance of the alterations in metabolic pathways were confirmed by radioactive tracer studies showing impaired fatty acid (Fig. 3 g) but unaltered glucose uptake (Fig. 3 h) by BAT. Despite compromised lipid uptake, a BAT-specific increase in proteins promoting intravascular processing of triglyceride-rich lipoproteins (GPIHBP1, LPL) and fatty acid uptake (CD36) was observed (Extended Data Fig. 4 c-f), suggesting an induction of compensatory yet futile mechanisms to replenish energy stores in thermogenic adipose tissues of norUDCA-treated mice. In the heart, norUDCA caused a several-fold increase in the uptake of glucose (Fig. 3 h), while the uptake of fatty acids (Fig. 3 g) was preserved. This observation, in accordance with the increased expression of genes regulating glucose transport and metabolism (Fig. 3 a-c), point toward an increased reliance of the heart on glucose as energy source and impaired fatty acid utilization in BAT in response to norUDCA supplementation. NorUDCA shifts cardiac energy metabolism towards glucose utilization Given the relevance of alterations in glucose metabolism in the context of heart failure 32 , the specificity of norUDCA on cardiac glucose uptake compared to other bile acids was determined. For this purpose, 3 H-deoxyglucose was injected into wild type mice which were fed a chow diet supplemented with norUDCA, UDCA, CDCA, CA, the secondary bile acid deoxycholic acid (DCA), the semisynthetic FXR agonist obeticholic acid (OCA), or the synthetic de-conjugation resistant bile acid cholylsarcosine. Among all the bile acid species tested, only norUDCA significantly increased 3 H-deoxyglucose uptake in the heart (Fig. 4 a). Supplementation with norUDCA but also with CA and DCA resulted in higher glucose uptake into white adipose tissue depots, while no significant changes were detected in other organs investigated (Fig. 4 a). The effect of norUDCA on cardiac glucose disposal was also observed in the FVB mouse strain, hyperlipidemic Apoa5 -deficient FVB and Cyp2c70 −/− mice (Extended Data Fig. 4 g-h). We next determined whether the microbiome is involved in mediating the effect of norUDCA on shifting energy substrate utilization. For this purpose, we depleted the gut bacteria by antibiotic treatment (Abx), known to decrease the turnover of bile acids and to elevate plasma levels of CBAs 33 . Of note, the heart of norUDCA-treated mice show a tendency to even higher 3 H-deoxyglucose uptake after Abx treatment (Fig. 4 b), indicating that microbial processing is not essential for the observed norUDCA effect on glucose metabolism. Of note, already 6 hours after oral administration of norUDCA the heart internalized larger amounts of 3 H-deoxyglucose (Fig. 4 c), indicating an acute effect that is independent of processes induced by chronic administration, e.g. loss in fat mass. Next, we performed metabolic flux studies to determine how norUDCA and the associated higher glucose uptake impacts intracellular glucose metabolism. To achieve this goal, control and norUDCA-fed mice received an intravenous bolus of 13 C-labeleled glucose and enrichment of 13 C in glucose metabolites (schematic diagram in Fig. 4 d) was assessed by mass spectrometry in heart (Fig. 4 e) and BAT (Fig. 4 f). In line with higher glucose uptake, norUDCA treatment caused a markedly increase in glucose metabolism as indicated by higher enrichments of 13 C in intermediates of glycolysis including lactate and citrate cycle in heart (Fig. 4 e) but not in BAT (Fig. 4 f). Overall, cardiac tissue of mice treated with norUDCA exhibits a profound increase in glucose uptake and utilization, a phenotype often related to heart failure and cardiac hypertrophy 32 . Lipolysis and ketone bodies compensate norUDCA-induced defects in energy homeostasis and thermogenesis Normal cardiac function relies on the continuous supply of fatty acids as energy substrate 32 . These free acids delivered to the heart are derived from either adipose tissue lipolysis and/or hydrolysis of triglyceride-rich lipoproteins by lipoprotein lipase 34 . To further unravel disturbances in energy substrate utilization, plasma levels of glucose, non-esterified fatty acids (NEFAs) and ketone bodies were quantified in mice fed with norUDCA for 1 day, 3 days and 7 days, respectively (Fig. 5 a-c). Glucose levels were strongly reduced at day 1 and remained at lower levels on the following days, which coincides with increased uptake by heart (Fig. 5 d) but not by BAT (Fig. 5 e). In contrast, NEFAs and ketone bodies were increased in plasma of norUDCA-treated mice (Fig. 5 b-c), which was accompanied by a marked increase in the uptake of the ketone body β-hydroxybutyrate in BAT and to a lesser extent by the heart (Fig. 5 f-g). The higher levels of ketone bodies are probably triggered by an increased flow of fatty acids from white adipose tissues to liver, as supported by increased lipolysis in adipose tissue explants of norUDCA-treated mice (Fig. 5 h). In line, higher expression of adipose tissue triglyceride lipase (ATGL) encoded by Pnpla2 was found in WAT of norUDCA-treated mice (Fig. 5 i). To directly study the effect of ATGL, we performed indirect calorimetry studies in whole body Pnpla2 deficient mice with transgenic cardiac ATGL overexpression, which prevents lipid accumulation and heart dysfunction described for the whole body knockout mice 35 . Of note, these mice lacking ATGL in adipose tissues displayed reduced energy expenditure even at 22°C after norUDCA treatment (Fig. 5 j-k, Extended Data Fig. 5 a-d). However, under low carb dietary conditions favoring ketone body production, energy expenditure was not compromised by norUDCA supplementation even when the temperature was gradually decreased to 16°C (Fig. 5 l-n). Overall, these data indicate that adipose ATGL-mediated lipolysis and subsequent hepatic ketone body production are critical in maintaining energy homeostasis in norUDCA-treated mice. Moreover, dietary approaches allow to overcome disturbed energy substrate utilization in norUDCA fed animals, thereby preventing cold stress-induced metabolic deficits. NorUDCA impairs mitochondrial respiration and disturbs contractile function of human engineered heart tissue The profound increase in cardiac glucose uptake and lactate production suggested a negative effect of norUDCA on mitochondrial function. Surprisingly, the overall architecture of cardiomyocytes and mitochondrial structure visualized by electron microscopy were not conspicuously altered by norUDCA treatment (Fig. 6 a-b). Similarly, no morphological changes were observed in thermogenic adipocytes and mitochondria of BAT (Extended Data Fig. 6 a-b). In addition, the levels of selected OXPHOS complex proteins were comparably abundant in heart mitochondria isolated from control and norUDCA-treated mice (Fig. 6 c-d). However, respirometry demonstrated a massive decline in oxidation of energy substrates in heart mitochondria acutely treated with 100 µM norUDCA (Fig. 6 e), a concentration similar to that detected in the systemic circulation of norUDCA-supplemented mice (Fig. 2 b). Of note, in engineered heart tissues generated from human induced pluripotent stem cells (see model in Fig. 6 f), incubation with norUDCA impaired both relaxation time and force generation gradually during incubation, which was not observed using equal concentrations of unconjugated CDCA (Fig. 6 g-j). Taken together, these data indicate that norUDCA internalized by cardiomyocytes impairs contractile function by inhibiting mitochondrial respiration. Discussion Despite recent progress in understanding the structural diversity and the specific functions of individual BAs, their divergence in the capacity to regulate hepatic and systemic metabolic adaptations remains underexplored. Given the known role of endogenous BAs in stimulating energy expenditure via the G-protein coupled receptor TGR5 in skeletal muscle and BAT both in mice and humans 36 – 39 , a major goal of this study was to investigate the potential beneficial effects of conjugation-resistant norUDCA, a C23-norderivative of UDCA, on systemic energy metabolism. Next to its known anti-fibrotic effects in the hepatobiliary system, its higher systemic concentration compared to naturally occurring bile acids makes norUDCA an attractive candidate to treat obesity-associated comorbidities such as dyslipidemia, diabetes and cardiovascular disease 1 , 40 . In the current study, we confirmed and complement previous reports that modifying the BA-pool towards higher hydrophilicity with UDCA or norUDCA completely prevents liver inflammation and fibrosis in Cyp2c70 −/− mice, a mouse model with characteristics of human cholestasis 28 . In metabolic turnover studies, we found a marked enrichment of norUDCA in peripheral organs that was associated with changes in energy substrate utilization in heart and BAT. Against our expectation that norUDCA would be pro-thermogenic, however, we observed that norUDCA caused a drastic drop in whole body energy expenditure and thermogenesis under catabolic conditions triggered by cold stress. It was found that norUDCA negatively affected heart and BAT function and thus compromised the survival of animals. The primary reasons for the strong effect can either relate to the high systemic concentration of unconjugated norUDCA, or be the result of increased endogenous UBA levels in plasma, causing a tremendous shift in the CBA to UBA ratio that might have contributed to the effects on heart and BAT described in the current study. Higher UBA levels could be the result of impaired hepatic conjugation that has been reported to be mediated by C23 nor-bile acid derivatives 41 , or result from competition of excessive norUDCA with other UBAs during hepatic extraction from the portal blood 42 . Besides, high UBAs may be a consequence of the activation of microbial bile salt hydrolase (BSH) through norUDCA 43 , 44 , which is mirrored by decreased CBAs in feces found in our study. In any event, it is very likely that the high ratio of UBA to CBA in plasma provokes the negative effects on energy expenditure and heat production. Notably, uncoupled respiration for heat production in BAT is mediated by elevated CBAs rather than UBAs 45 . Moreover, beneficial effects of cold on adiposity and adaptive thermogenesis is associated with a higher CBA to UBA ratio 16 , 17 . Similarly, the metabolic effects of BAs in Roux-en-Y gastric bypass bariatric surgery are related to high levels CBAs and a concomitant decrease in UBA levels 13 . Notably, the phenotype of mice treated with norUDCA shares similarities with that observed after biliopancreatic diversion. The shortened route of enterohepatic circulation in this weight loss intervention characterized by inefficient lipid absorption results in significantly elevated UBAs 46 . In the future, it would be interesting to study preclinical models and patients undergoing this surgical intervention with regard to cardiac and BAT fuel oxidation and energy expenditure. Remarkably, the metabolic deficits observed in our study were highly specific for norUDCA, since adverse effects were not observed for the closely related UDCA or other BAs. The uptake, as shown by tracer studies using radiolabeled norUDCA, and in consequence the accumulation of norUDCA within extrahepatic organs could be mediated by the organic anion transporting polypeptide 1 (OATP1), a bile acid transporter that is expressed in multiple tissues. OATP1 has been demonstrated to efficiently transport unconjugated nor bile acids but not other UBAs such as CDCA 47 . An excess of BAs is known to impair cardiac function 48 , 49 , impact energy metabolism and BAT thermogenesis 45 , 50 , as well as mitochondrial function 51 , 52 . These effects were observed in both mice and humans with implications for overall energy metabolism and heart function 53 – 55 . The relationship between “cardiotoxic” BAs and cardiac dysfunction is only witnessed in the context of liver disorders 56 , 57 . Particularly, hydrophobic BAs with high cellular cytotoxicity mediate such pathological effects in vitro , whereas hydrophilic BAs, such as UDCA or muricholic acids even counteracted the effect of lipophilic BAs 58 . NorUDCA that is even more hydrophilic than UDCA (critical micellar concentration is 17 mM for norUDCA versus 7 mM for UDCA; 59 ) has not been reported to cause adverse cellular effects, making it highly unlikely to act as a typical hydrophobic cytotoxic BA. It is well established that a dysbalance between glucose and fatty acid oxidation is detrimental for cardiac function 32 . In the current study norUDCA caused hypoglycemia as well as tissue remodeling and inflammation in heart and BAT, which we linked to impaired mitochondrial respiration of palmitic acid and increased flux of cardiac glucose. Still, these metabolic changes/adaptions seem not to compromise the vital functions of animals at room temperature, but deteriorates during cold exposure, causing decompensation of systemic energy homeostasis and death of the animals. Together, our data indicate that disturbed energy substrate utilization underlies the adverse effects of norUDCA, a notion that is supported by our observation that feeding a low-carb ketogenic diet prevented norUDCA-induced cold intolerance. Ketone bodies are easy to oxidize fuels for peripheral tissues that are produced in the liver from fatty acids released under catabolic conditions by ATGL-mediated lipolysis in the adipose tissues 60 . Notably, cardiac glucose uptake increased > 10-fold during acute cold exposure in mice with reduced adipose tissue lipolysis 61 and was associated with pathological remodeling of the heart during chronic cold stress. The relevance of this pathway is highlighted by our observation that mice lacking ATGL in adipose tissues succumb to norUDCA supplementation already at room temperature. In wild type mice housed at room temperature, the higher levels of non-esterified fatty acids and ketone bodies can compensate energy disturbances induced by norUDCA supplementation. Most likely, enhanced lipolysis is stimulated via activation of the Gs-coupled bile acid receptor TGR5 62 . Although norUDCA is a rather poor ligand for TGR5 63,64 , it is present in very high concentrations, and furthermore, also other UBAs such as CDCA and DCA are highly elevated. Careful consideration must be given when discussing the potential clinical relevance of our findings, as there are species differences between mice and humans regarding their cardiac metabolism and their significant differences in BA metabolism. For instance, it remains unclear whether treatment in humans results in norUDCA levels that are similar to those in preclinical models after norUDCA supplementation. In contrast to rodents, norUDCA in humans is efficiently glucuronidated 59 and subsequently excreted via the urine, which may limit its plasma accumulation. Nevertheless, norUDCA can reach significant levels in the systemic circulation in humans, as suggested by a pharmacokinetic study in six healthy male volunteers. These probands were treated with a single oral dose of 1500 mg radiolabelled norUDCA and a peak concentration reached a level 65 is similar to those observed in the current mouse study. Thus, norUDCA could impact systemic energy metabolism in humans, in particular under stress conditions. Future studies are mandatory to determine the effects on glucose disposal and energy expenditure in a clinical setting, taking into account that norUDCA treatment only in combination with catabolic stress may alter cardiac performance. Methods Animal models All animal experiments were approved by the Behörde für Gesundheit und Verbraucherschutz Hamburg (Germany) or by the Austrian Federal Ministry of Education, Science and Research (Austria). All animals were housed with a 12-h light-dark cycle in humidity and temperature-controlled conditions and permitted ad libitum consumption of water and a standard mouse diet. Cyp2c70 −/− , ApoA5 −/− and Atgl-MHC mice and the respective control littermates were bred and housed in the animal facility of the UKE or Graz. Mice were randomized based on body weight and fed for 7 days a chow diet (Altromin 1324) or a chow diet supplemented with norUDCA, UDCA, CDCA, DCA, CA, OCA (0.05%) or cholylsacosin at 0.5% (w/w) with ad libitum access to food and water. For antibiotic treatment, neomycin (Sigma, #N6386), bacitracin (Sigma, #11702) and streptomycin (Sigma, #S6501) were diluted in the drinking water, all at a concentration of 1g/l. Organ harvests and metabolic turnover studies were performed after a 4 h fasting period, and the mice were anesthetized with a lethal dose of ketamine and xylazine. Cardiac blood was drawn with syringes containing 0.5 M EDTA. Animals were perfused with PBS containing 10 U/ml heparin, and then the organs were harvested and immediately stored at − 80°C for further analysis. Body composition analysis was performed by echoMRI (Zinsser Analytic). Indirect calorimetry Indirect calorimetry was performed using either TSE Phenomaster system (TSE systems) or the PROMETHION systems (Sable Systems) in a temperature- and humidity-controlled chamber. During the experiments, all mice were housed in single cages under a 12 h light: 12 h dark cycle and had ad libitum access to food and water. The animals were fed regular chow with/without 0.5% norUDCA or a low-carb diet (ssniff, E15660) with/without 0.5% norUDCA. Gene expression Tissues were lysed in TRIzol (Ambion, Life Technologies) using a TissueLyser (Qiagen). Nucleic acids were extracted with chloroform, and total RNA was isolated using the RNA purification kit NucleoSpin®RNA II (Macherey & Nagel). RNA concentration was determined with NanoDrop, and 400 ng of RNA was used for reverse transcription into cDNA by using the III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed on a QuantStudio™ 5 Real-Time PCR System using the following TaqMan® on-demand primer sets (Invitrogen): Acaca : Mm01304285_m1, Acly : Mm00652520_m1, Ccl2 : Mm00441242_m1, Ccl5 : Mm01302428_m1, Cd68 : Mm03047343_m1, Col1a1 : Mm00801666_g1, Cpt1 : Mm00550438_m1, Cxcl10 : Mm00445235_m1, Fasn : Mm00662319_m1, Il1b :Mm00434228_m1, Lipe : Mm00495359_m1, Lpl : Mm00434764_m1, Mmp12 : Mm00500554_m1, Mmp13 : Mm00439491_m1), Pdk4 : Mm00443325_m1, Pfkfb4 : Mm00557176_m1, Pnpla2 : Mm00503040_m1, Ppargc1a : Mm00447183_m1, Scd1 : Mm00772290_m1, Slc2a1 (encoding Glut1): Mm00441480_m1, Slc2a4 (encoding Glut4): Mm01245502_m1, Tbp : Mm00446973_m1, Timp1 : Mm00441818_m1, Tnfa : Mm00443258_m1). mRNA levels were normalized to the level of the housekeeping gene TATA-box binding protein ( Tbp ) mRNA, and the results were displayed as relative gene expression normalized to the experimental control group, following calculations using the 2-ΔΔCt method. RNA sequencing For transcriptomics, RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized utilizing random hexamer primers, followed by the second strand cDNA synthesis. Libraries prepared using Novogene NGS RNA Library Prep Set (PT042) was sequenced on Illumina NovaSeq 6000 platform S4 flow cell. RNA-seq data are aligned using Hisat2 v2.0.5. Reads are assigned to transcripts using featureCounts. Differential expression analysis of the data was performed using edgeR. The P values were adjusted using the Benjamini & Hochberg method. Local version of the gene set enrichment analysis tool http://www.broadinstitute.org/gsea/index.jsp was used, GO, KEGG (Kyoto Encyclopedia of genes and Genomens) and Reactome were used for GSEA independently. Plasma parameters Plasma was isolated by centrifugation of EDTA-spiked blood for 10 min at 10.000 rpm at 4°C in a bench top centrifuge. Glucose was determined in tail blood using Accu-Check Aviva test strips (ROCHE). Non esterified fatty acids (NEFAs) were determined photometrically using the NEFA-HR (2) Assay (FUJIFILM). Ketone bodies were determined using the Autokit Total Ketone Bodies Assay (FUJIFILM). Glycerol was determined calorimetrically using the Free Glycerol Reagent (SIGMA, #F6428) and the Glycerol Standard Solution (SIGMA, #G7793) as reference. Respirometry Oxygen consumption rate was analyzed in heart mitochondria by high-resolution respirometry using O2k-technology chambers (Oroboros Instruments). Mitochondria were isolated following the “Laboratory protocol: isolation of rat heart mitochondria” 66 from whole mice hearts. Mice were euthanized by cervical dislocation and whole hearts were isolated using sterile surgical equipment. Hearts were places in ice cold isolation buffer until further processing. Heart tissue was later minced by sterile scissors and further homogenized in pre-cooled Teflon potter in buffer containing protease. Homogenates were left digesting on ice under constant shaking. After digestion with protease, samples were centrifuged to remove the non-homogenized pieces of tissue and supernatant containing mitochondria was further centrifuged until solid pellets of mitochondria were observed. Mitochondrial pellets were re-suspended in the ice-cold isolation buffer. Oxygen consumption was determined in the mitochondria that were pre-incubated without/with 100 µM norUDCA for 30 min in pre-calibrated 2 ml chambers with continuous stirring. Then, sequential addition of palmitoyl-CoA (0.04 mM), ADP (2 mM), malate (0.1 mM and 2 mM), pyruvate (5 mM), glutamate (10 mM), cytochrome C (0.01 mM), oligomycin (5 nM) and finally antimycin A (2.5 µM) to determine substrate specificity and membrane integrity of the mitochondria throughout the experiment. Western blotting For SDS–PAGE, perfused organs were harvested and homogenized with a TissueLyzer (Qiagen) in 10x excess of (v/w) RIPA buffer (50 mM Tris–HCl pH 7.4, 5 mM EDTA, 150 mM sodium chloride, 1 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium ortho-vanadate, 1% (NP-40) supplemented with cOmplete mini protease inhibitor cocktail tablets (Roche), and phosphatase inhibitor cocktail (Bimake.com). After centrifugation at 16.000 g for 10 min, the clear soluble middle layer of the lysate was taken, and protein concentration was assessed using the method of Lowry. Then 20 µg of total protein was denatured at 55°C for 10 min in a NuPAGE reducing sample buffer (Invitrogen) and separated on 10% SDS–polyacrylamide Tris–glycine gels. Proteins were transferred to nitrocellulose membranes in a wet blotting system. Equal loading was confirmed by Ponceau S (Serva) staining. Subsequently, the membranes were washed twice in TBS-T (20 mM Tris, 150 mM sodium chloride, 0.1% (v/v) Tween 20) and blocked for 1 h in 5% milk powder (Sigma) in TBS-T at room temperature. Primary antibodies were incubated (5% BSA in TBS-T) overnight at 4°C, and secondary antibodies were diluted in 5% milk powder in TBS-T. Detection was performed with enhanced chemiluminescence using an Amersham Imager 600 (GE Healthcare). The following primary antibodies were used: rabbit polyclonal anti-CD36 (1:1000, Novus biologicals, NB400-144), goat polyclonal anti-LPL (1:1000, kind gift from Andre Bensadoun, Cornell University), rat monoclonal anti-GPIHBP1 (1:1000, kind gift from Stephen G. Young, UCLA), mouse monoclonal total OXPHOS WB cocktail (1:500, abcam, ab110413), rabbit monoclonal anti-gamma-Tubulin (1:1000, abcam, ab179503), rabbit monoclonal anti-AKT (1:1000, cell signaling, #9272). The following secondary antibodies (in a dilution of 1:5.000) were used: HRP goat anti-rabbit (Jackson ImmunoResearch Labs, #111-035-144), HRP goat anti-mouse (Jackson ImmunoResearch Labs, #115-035-003), HRP donkey anti-rat (Jackson ImmunoResearch Labs, #712-035-150), and HRP mouse anti-goat (Jackson ImmunoResearch Labs, #205-035-108). Bile acid measurement Bile acids were quantified by HPLC coupled to electrospray ionization tandem mass spectrometry as described 67 . Briefly, plasma or cecal samples were prepared by a methanol liquid-liquid extraction. Quantitative measurement of bile acids was performed using a LC-ESI-QqQ system run multiple reaction monitoring (MRM) mode. HPLC analysis was performed using NEXERA X2 LC-30AD HPLC PUMP (Shimadzu, Tokyo, Japan) equipped with a Kinetex C18 column (100 Å, 150 mm × 2.1 mm i.d., Phenomenex, Torrance, CA, USA). For HPLC a mobile phase A consisting of water and a mobile phase B consisting of acetonitrile methanol (3/1 v/v) both enriched with 0.1% formic acid and 20 mM ammonium acetate was used. The column was coupled to QqQ: Q trap 5500 System (SCIEX, Darmstadt, Germany). Peaks were identified and quantified by comparing retention times, as well as MRM transitions and peak areas, respectively, to particular corresponding standard chromatograms. Metabolomic enrichment analysis For flux analysis, mice treated with/without norUDCA received a bolus i.p. injection of uniformly labeled 13 C-glucose (1g/kg body weight). Mice were sacrified 30 min after injection and organs were harvested for metabolomics. Each x mg tissue sample was mixed with five times the µl amount of ice-cold extraction solvent acetonitrile (ACN) with H 2 O (1:1) and homogenized using a TissueLyser II (30 Hz, 10 min; Retsch Qiagen). After centrifugation (4°C, 2 min, 14000 rpm), 250 µl supernatant were mixed with 250 µl ACN: H 2 O for a second extraction step. After vortexing for 1 min and centrifugation (4°C, 10 min, 14000 rpm), the supernatant was transferred to a new tube and evaporated to dryness (SpeedVac, Eppendorf). Extracted metabolites of heart and BAT were analyzed by LC-MS/MS using an adapted method described by Buescher et al., 2010 68 . Prior to measurement, each sample was re-suspended in 100 µl H 2 O and 10 µl were injected onto an Agilent 1290 II infinity UPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) coupled on-line with a QTRAP® 6500 + mass spectrometer (Sciex, Framingham, USA). Chromatographic separation was achieved with a XSelect HSS T3 XP column (2.1 x 150 mm, 2.5 µm, 100 Å; Waters, Milford, MA, USA) connected to an XP VanGuard® cartridge (HSS T3, 2.1 x 5 mm, 2.5 µM; Waters, Milford, MA, USA). Mobile phase A and mobile phase B were 10 mM tributylamine, 10 mM acetic acid, 5% methanol and 2% 2-propanol (pH 7.1) in water and 100% 2-propanol, respectively. Metabolites were eluted with the following non-linear gradient: 0-15.5 min 0.4 mL/min, 15.5–16.5 min 0.4 − 0.15 mL/min, 16.5–23 min 0.15 mL/min, 23–27 min 0.15–0.4 mL/min, 27–33 min 0.4 mL/min. The autosampler was kept at 5°C and the column oven was set to 40°C. For identification and quantitation, a scheduled multiple reaction monitoring (MRM) method in negative mode electrospray ionization was used with specific transitions for every metabolite and isotopologue. Data acquisition was performed using the Analyst® software (v. 1.7.0) and peak integration was done in SciexOS® Software (v. 3.0.0., Sciex). All isotopologue measurement values were corrected for 1.1% of 13 C-natural abundance 69 . Metabolic turnover studies To quantify the uptake of radiolabeled bile acids, mice received an oral gavage enriched with 3 H-norUDCA (74 kBq per mouse) and 14 C-CA (37 kBq per mouse). Four hours after gavage, mice were anaesthetized and organs were harvested. To quantify the uptake of energy substrates, mice were i.v. injected with 14 C-DOG (7.4 kBq per mouse), 3 H-DOG (14.8 kBq per mouse), albumin-bound 14 C-oleic acid (7.4 kBq per mouse) or 14 C-hydroxybutyrate (7.4 kBq per mouse). Mice were anaesthetized 15 min after injection, blood was collected by cardiac puncture, perfused with PBS via the left heart ventricle and organs were harvested. In both setups, organs were dissolved in Solvable (Perkin Elmer) for scintillation counting using a Perkin Elmer Tricarb Scintillation Counter. Histology and immunohistochemistry Immunohistochemical stainings were performed on paraffin-embedded tissues using standard procedures. Briefly, liver tissues were fixed in 3.7% formaldehyde in PBS solution and later embedded in paraffin. Stainings were performed using 4 µm sections cut on a Leica microtome and mounted on Histobond slides (Marienfeld-Superior). The following primary antibodies were used in 3% BSA (Sigma): rat monoclonal anti-LY6C (1:200, abcam, ab15627), rabbit monoclonal anti-CK19 (1:200, abcam, ab52625). Horseradish peroxidase (HRP) coupled donkey-anti-rat (Jackson Immunoresearch, #712-036-153) and horseradish peroxidase (HRP) goat anti-rabbit (Jackson ImmunoResearch Labs, #111-035-144) were used as secondary antibodies. After secondary antibody incubation, sections were washed with PBS 3-times for 10 min. Staining was performed using an abcam DAB kit following the manufacturer’s instructions. After DAB-staining, the slides were rinsed with PBS to stop the chromogenic reaction and counterstained with hematoxilin for 2 min. Slides were incubated under running tap water for 10 min to achieve bluing of the hematoxilin. Afterwards, the slides were dehydrated and mounted using Eukitt. Images were taken using a NikonA1 Ti microscope equipped with a DS-Fi-U3 brightfield camera. Ex vivo lipolysis assay White adipose tissue pieces (~ 25-35mg) were incubated in 500 µl Dulbecco's Modified Eagle Medium (Gibco, #11965092) supplemented with 2% fatty acid-free bovine serum albumin (Sigma, #A8806). After 30 min, basal lipolysis was determined by measuring the released free fatty acids and glycerol in the media, using the NEFA-HR (2) Assay (FUJIFILM) and the Free Glycerol Reagent (SIGMA, #F6428), respectively. Protein content of the individual adipose tissue explant pieces was measured with the Lowry method for normalization. Engineered heart tissues An established control line of human induced pluripotent stem cells (hiPSC, hiPSCreg code: UKEi001-A, ERC001 XX) was used to differentiate cardiomyocytes as recently described 70 . Briefly, master/working cell bank hiPSCs aliquots 71 were expanded in FTDA media on Geltrex-coated cell culture vessels 72 . Embryoid bodies were generated in spinner flasks. Ventricular cardiomyocytes were differentiated in suspension/EB format by growth factor/small molecule cocktails into mesodermal progenitor cells and subsequently into cardiomyocytes. Collagenase-dissociated hiPSC-CM were either cryopreserved or used directly for the generation of engineered heart tissues (EHT). Differentiation efficiency was determined by FACS analysis for troponin T. Fibrin-based strip-format EHTs were generated with 1.0 x 106 hiPSC-CM per construct 70 . EHTs were cultivated for approximately 21 days in EHT medium (10% horse serum, 1% penicillin-streptomycin, 33 µg/ml aprotinin, 10 µg/ml insulin, 200 µM tranexamic acid), in 24 well plates. Cell culture media was changed on Mondays, Wednesday and Fridays. Functional assessment was performed by video-optical recording of spontaneous EHT contraction and calculation of force based on deflection during contraction 73 . EHT were equilibrated in measurement medium (DMEM, horse serum 2%) overnight. After baseline contractility recording EHTs were incubated in the presence of vehicle (0.9% NaCl) or bile acids. Recording of contractility was performed at 2h, 24h, 48 h, and 72 h of incubation. Electron microscopy For electron microscopy mice were sacrificed with a lethal dose of ketamin/xylazine injection anesthesia and perfused with PBS. Organs were cut and directly transferred into fixative (4% PFA, 1% GA in PBS) and stored at 4°C. Then, tissues were dissected with a razor blade and rinsed three times in 0.1 M sodium cacodylate buffer (pH 7.2–7.4) and osmicated using 1% osmium tetroxide in cacodylate buffer. Following osmication, the samples were dehydrated using ascending ethyl alcohol concentration steps, followed by two rinses in propylene oxide. Infiltration of the embedding medium was performed by immersing the pieces in a 1:1 mixture of propylene oxide and Epon and finally in neat Epon and hardened at 60°C. Semithin sections (0.5 µm) were prepared for light microscopy mounted on glass slides and stained for 1 min with 1% Toluidine blue. Ultrathin sections (60 nm) were cut and mounted on copper grids. Sections were stained using uranyl acetate and lead citrate. Thin sections were examined and photographed using an EM902 (Zeiss) electron microscope. Statistical methods Data are expressed as mean ± S.E.M. Comparisons of two groups were examined using Students T-Test. Comparison of three or more groups were analyzed using ANOVA. GraphPad Prism and Microsoft Excel were used for all statistical analyses. The statistical parameters can be found in the figure legends. P < 0.05 was considered to be statistically significant. Declarations Conflict of interest The authors declare no competing interests. Author contributions Conception: I.E., J.H., T.M. and F.K. Experimental design: G.E., J.H. and T.M. Investigation and Methodology: I.E., E.V., J.K.R., M.V., M.H., A.W., S.G., M.M.F., K.G., D.S., U.R.-K., A.Z., R.B., R.F., J.F.d.B. and A.H. Formal analysis: I.E., E.V., J.K.R., M.V., M.H., A.W., S.G., M.M.F., K.G., D.S., U.R.-K., A.H., T.M., and J.H. Drafting the manuscript: I.E., J.H., L.S., T.M. and F.K. Critical review and discussion: M.V.B., L.S., C.S., A.H. and F.K. Funding acquisition: JH, CS, AW and LS. All authors read and approved the paper. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to JH, CS, AW and LS (SFB-Transregio 333, project-ID: 450149205) and by the Austrian Science Fund FWF, DK-MCD W1226 to AZ and MV. References Perino A, Demagny H, Velazquez-Villegas L, Schoonjans K (2021) Molecular Physiology of Bile Acid Signaling in Health, Disease, and Aging. Physiol Rev 101:683–731 Sang C et al (2021) Bile Acid Profiles Are Distinct among Patients with Different Etiologies of Chronic Liver Disease. J Proteome Res 20:2340–2351 Zhang YL, Li ZJ, Gou HZ, Song XJ, Zhang L (2022) The gut microbiota-bile acid axis: A potential therapeutic target for liver fibrosis. Front Cell Infect Microbiol 12:945368 Jia B, Park D, Hahn Y, Jeon CO (2020) Metagenomic analysis of the human microbiome reveals the association between the abundance of gut bile salt hydrolases and host health. Gut Microbes 11:1300–1313 Varanasi SK et al (2025) Bile acid synthesis impedes tumor-specific T cell responses during liver cancer. Science 387:192–201 Hang S et al (2019) Bile acid metabolites control T(H)17 and T(reg) cell differentiation. Nature 576:143–148 Trampert DC, Kunst RF, van de Graaf (2024) S.F.J. Targeting bile salt homeostasis in biliary diseases. Curr Opin Gastroenterol 40:62–69 de Boer JF et al (2021) Cholangiopathy and Biliary Fibrosis in Cyp2c70-Deficient Mice Are Fully Reversed by Ursodeoxycholic Acid. Cell Mol Gastroenterol Hepatol 11:1045–1069 Shen Y et al (2022) Ursodeoxycholic acid reduces antitumor immunosuppression by inducing CHIP-mediated TGF-beta degradation. Nat Commun 13:3419 Yoshimoto S et al (2013) Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499:97–101 Joyce SA et al (2014) Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut. Proc Natl Acad Sci U S A 111:7421–7426 Riba A et al (2020) Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment. Sci Transl Med 12 Werling M et al (2013) Enhanced fasting and post-prandial plasma bile acid responses after Roux-en-Y gastric bypass surgery. Scand J Gastroenterol 48:1257–1264 Simonen M et al (2012) Conjugated bile acids associate with altered rates of glucose and lipid oxidation after Roux-en-Y gastric bypass. Obes Surg 22:1473–1480 Evangelakos I et al (2021) Oxysterol 7-alpha Hydroxylase (CYP7B1) Attenuates Metabolic-Associated Fatty Liver Disease in Mice at Thermoneutrality. Cells 10 Worthmann A et al (2017) Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis. Nat Med 23:839–849 Zietak M et al (2016) Altered Microbiota Contributes to Reduced Diet-Induced Obesity upon Cold Exposure. Cell Metab 23:1216–1223 Baskin AS et al (2018) Regulation of Human Adipose Tissue Activation, Gallbladder Size, and Bile Acid Metabolism by a beta3-Adrenergic Receptor Agonist. Diabetes 67:2113–2125 O'Mara AE et al (2020) Chronic mirabegron treatment increases human brown fat, HDL cholesterol, and insulin sensitivity. J Clin Invest 130:2209–2219 Beuers U et al (2025) The history and future of bile acid therapies. J Hepatol 83:1172–1188 Yeh HZ et al (1997) Effect of side chain length on biotransformation, hepatic transport, and choleretic properties of chenodeoxycholyl homologues in the rodent: studies with dinorchenodeoxycholic acid, norchenodeoxycholic acid, and chenodeoxycholic acid. Hepatology 26:374–385 Fickert P et al (2017) norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis. J Hepatol 67:549–558 Fickert P et al (2013) Differential effects of norUDCA and UDCA in obstructive cholestasis in mice. J Hepatol 58:1201–1208 Halilbasic E et al (2009) Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice. Hepatology 49:1972–1981 Moustafa T et al (2012) Alterations in lipid metabolism mediate inflammation, fibrosis, and proliferation in a mouse model of chronic cholestatic liver injury. Gastroenterology 142:140–151e112 Zhu C et al (2021) 24-Norursodeoxycholic acid reshapes immunometabolism in CD8(+) T cells and alleviates hepatic inflammation. J Hepatol 75:1164–1176 Zhu C et al (2025) 24-Nor-ursodeoxycholic acid improves intestinal inflammation by targeting T(H)17 pathogenicity and transdifferentiation. Gut 74:1079–1093 de Boer JF et al (2020) A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice. J Lipid Res 61:291–305 Takahashi S et al (2016) Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans. J Lipid Res 57:2130–2137 Gurantz D et al (1991) Hypercholeresis induced by unconjugated bile acid infusion correlates with recovery in bile of unconjugated bile acids. Hepatology 13:540–550 Morrison SF, Nakamura K (2019) Central Mechanisms for Thermoregulation. Annu Rev Physiol 81:285–308 Glatz JFC et al (2020) Re-balancing cellular energy substrate metabolism to mend the failing heart. Biochim Biophys Acta Mol Basis Dis 1866:165579 Out C et al (2015) Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4. J Hepatol 63:697–704 Da Dalt L, Cabodevilla AG, Goldberg IJ, Norata GD (2023) Cardiac lipid metabolism, mitochondrial function, and heart failure. Cardiovasc Res 119:1905–1914 Schoiswohl G et al (2010) Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids. J Lipid Res 51:490–499 Watanabe M et al (2006) Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 439:484–489 Teodoro JS et al (2014) Enhancement of brown fat thermogenesis using chenodeoxycholic acid in mice. Int J Obes (Lond) 38:1027–1034 Broeders EP et al (2015) The Bile Acid Chenodeoxycholic Acid Increases Human Brown Adipose Tissue Activity. Cell Metab 22:418–426 Velazquez-Villegas LA et al (2018) TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue. Nat Commun 9:245 Evangelakos I, Heeren J, Verkade E, Kuipers F (2021) Role of bile acids in inflammatory liver diseases. Semin Immunopathol 43:577–590 Czuba B, Vessey DA (1982) The effect of bile acid structure on the activity of bile acid-CoA:glycine/taurine-N-acetyltransferase. J Biol Chem 257:8761–8765 Dawson PA, Lan T, Rao A (2009) Bile acid transporters. J Lipid Res 50:2340–2357 Grander C et al (2023) 24-Norursodeoxycholic acid ameliorates experimental alcohol-related liver disease and activates hepatic PPARgamma. JHEP Rep 5:100872 Bourgin M et al (2021) Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health. Microorganisms 9 Donkers JM et al (2019) NTCP deficiency in mice protects against obesity and hepatosteatosis. JCI Insight 5 Ferrannini E et al (2015) Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion. Diabetes 64:3377–3385 Hata S et al (2003) Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake. Am J Physiol Gastrointest Liver Physiol 285:G829–839 Desai MS et al (2011) Cardiac structural and functional alterations in infants and children with biliary atresia, listed for liver transplantation. Gastroenterology 141(1272):1264–1272 Desai MS et al (2010) Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis. Hepatology 51:2097–2107 Thomas C et al (2009) TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab 10:167–177 Rose AJ et al (2011) Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor. Cell Metab 14:123–130 Zhou W, Lew B, Choi H, Kim K, Anakk S (2024) Chenodeoxycholic Acid-Loaded Nanoparticles Are Sufficient to Decrease Adipocyte Size by Inducing Mitochondrial Function. Nano Lett 24:1642–1649 Joubert P (1978) An in vivo investigation of the negative chronotropic effect of cholic acid in the rat. Clin Exp Pharmacol Physiol 5:1–8 Rainer PP et al (2013) Bile acids induce arrhythmias in human atrial myocardium–implications for altered serum bile acid composition in patients with atrial fibrillation. Heart 99:1685–1692 Tong DH (2023) Bile acid binds heart failure and inflammation. Nat Metab 5:1260–1261 Vasavan T et al (2018) Heart and bile acids - Clinical consequences of altered bile acid metabolism. Biochim Biophys Acta Mol Basis Dis 1864:1345–1355 Desai MS et al (2017) Bile acid excess induces cardiomyopathy and metabolic dysfunctions in the heart. Hepatology 65:189–201 Zavecz JH, Battarbee HD (2010) The role of lipophilic bile acids in the development of cirrhotic cardiomyopathy. Cardiovasc Toxicol 10:117–129 Hofmann AF et al (2005) Novel biotransformation and physiological properties of norursodeoxycholic acid in humans. Hepatology 42:1391–1398 Grabner GF, Xie H, Schweiger M, Zechner R (2021) Lipolysis: cellular mechanisms for lipid mobilization from fat stores. Nat Metab 3:1445–1465 Choi Y et al (2021) Adipose Lipolysis Regulates Cardiac Glucose Uptake and Function in Mice under Cold Stress. Int J Mol Sci 22 Perino A, Schoonjans K (2022) Metabolic Messengers: bile acids. Nat Metab 4:416–423 Sato H et al (2008) Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies. J Med Chem 51:1831–1841 Marchiano S et al (2022) Beneficial effects of UDCA and norUDCA in a rodent model of steatosis are linked to modulation of GPBAR1/FXR signaling. Biochim Biophys Acta Mol Cell Biol Lipids 1867:159218 Halibasic ET, Fickert M, Proels P, Hauswald M, Greinwald K, Tewes R (2020) Glucuronidation directs systemically available norursodeoxcholic acid (norUDCA) to the kidney. J Hepatol 73:S468 Sumbalova Z, Krumschnabel. G (2016) Lаbоrаtоry prоtосоl: isоlаtiоn оf rаt hеаrt mitосhоndriа. Mitосhоndriаl Physiоlоgy Nеtwоrk 15:1–2 John C et al (2014) A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids. J Chromatogr A 1371:184–195 Buescher JM, Moco S, Sauer U, Zamboni N (2010) Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82:4403–4412 Heinrich P et al (2018) Correcting for natural isotope abundance and tracer impurity in MS-, MS/MS- and high-resolution-multiple-tracer-data from stable isotope labeling experiments with IsoCorrectoR. Sci Rep 8:17910 Breckwoldt K et al (2017) Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc 12:1177–1197 Shibamiya A et al (2020) Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research. Curr Protoc Stem Cell Biol 55:e127 Frank S, Zhang M, Scholer HR, Greber B (2012) Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions. PLoS ONE 7:e41958 Mannhardt I et al (2016) Human Engineered Heart Tissue: Analysis of Contractile Force. Stem Cell Rep 7:29–42 Additional Declarations There is NO Competing Interest. Supplementary Files 251128SupplementalFiguresEvangelakosetal2025.pdf Extended data Fig. 1. NorUDCA and UDCA alleviate liver inflammation and fibrosis in Cyp2c70 -/- mice. Wild type and Cyp2c70 -/- mice were fed a regular chow, or chow diet supplemented with either 0.5% UDCA or 0.5% norUDCA for 7 days under standard housing conditions ( n=4-5 ). a, Representative images of LY6C and CK19 stainings of liver sections. b-d, Hepatic expression of fibrosis marker genes (b), inflammation marker genes (c), and cecal bile acid concentration (d). Error bars are shown as SEM. Statistical analysis was performed two-way ANOVA (same letter denotes groups that are not significantly different from each other, with p < 0.05). Extended data Fig. 2. NorUDCA reduces fat mass and increases gall bladder size. Mice were fed a regular chow (control), or chow diet supplemented with norUDCA for 7 days with housing at room temperature. a, Change of fat and lean mass determined by echoMRI ( n=7 ). b, Representative image of gallbladder size of chow (control) and norUDCA-treated mouse under standard housing conditions. c-f, Quantification of indirect calorimetry and body temperature measurements from the experiments shown in Fig. 1 separately calculated for the light and dark phases. CO 2 production (c), O 2 consumption (d), respiratory exchange rate (e), body core temperature (f). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with Student’s T-Test. *p<0.05, **p<0.01,***p<0.005. Extended data Fig. 3. norUDCA accumulates in metabolically active organs. a-d Wild type mice were fed a regular chow (control), or chow diet supplemented with 0.5% norUDCA before oral administration of 3 H-norUDCA and 14 C-CA ( n=8 ). Four hours later, percentage of applied tracers was determined in muscle (a), inguinal white adipose tissue (iWAT) (b), gonadal white adipose tissue (gWAT) (c), and plasma (d). Statistical analysis was performed with two-way ANOVA (a-d). *p<0.05, **p<0.01,***p<0.005. Extended data Fig. 4. norUDCA shifts cardiac energy metabolism towards glucose utilization. a, Mice were fed a regular chow (control), or chow diet supplemented with norUDCA at 0.5% for 7 days with housing at room temperature. a-b, Expression of metabolic genes in heart (a) and in BAT (b). c-f, Western blot analysis and quantification of lipoprotein-processing proteins from samples of heart (c, e) and BAT (d, f). g-h, Organ-specific uptake of intravenously administered 3 H-deoxyglucose in FVB wild type vs ApoA5 -/- mice on the FVB background (g), and in wild type controls vs Cyp2c70 -/- mice (h). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed either with Student’s T-Test (a-f) or two-way ANOVA (g-h). = p<0.05, = p<0.01, = p<0.005. Extended data Fig. 5. ATGL compensates for norUDCA-induced defects in energy homeostasis and thermogenesis at room temperature. a-d, Indirect calorimetry of ATGL-MHC mice fed with chow followed by norUDCA-enriched diet for the indicated time period at room temperature. CO 2 production (a), O 2 consumption (b), quantification of indirect calorimetry measurements separately calculated for the light and dark phases for CO 2 production (c) and O 2 consumption (d). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with Student’s T-Test. *= p<0.05. Extended data Fig. 6. Electron microscopy of BAT from mice and model depicting the effects of norUDCA on metabolically active organs at room temperature and at cold. a-b, Mice were fed a regular chow (control), or chow diet supplemented with norUDCA for 7 days with housing at room temperature. Representative electron microscopy pictures of BAT from control (a) and norUDCA-treated mice (b). Schematic model of norUDCA effects at room temperature and under cold stimulus (c). SupplementaryTable1.xlsx Table 1 SupplementaryTable2.xlsx Table 2 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":68587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNorUDCA reduces adipose tissue weights and compromises the thermogenic response to cold exposure. a-c\u003c/strong\u003e, Wild type mice were fed a regular chow (control), or chow diet supplemented with either 0.5% UDCA or 0.5% norUDCA. Body weight change (\u003cstrong\u003ea\u003c/strong\u003e), organ weights (\u003cstrong\u003eb\u003c/strong\u003e) and liver weights (\u003cstrong\u003ec\u003c/strong\u003e) after 7 day treatment under standard housing conditions (\u003cem\u003en=6\u003c/em\u003e). \u003cstrong\u003ed\u003c/strong\u003e, Survival of mice throughout 8 hour acclimation to the indicated environmental temperature after feeding a chow diet (control) or a chow diet supplemented with 0.5% norUDCA for one week at standard housing temperature (\u003cem\u003en=8\u003c/em\u003e). \u003cstrong\u003ee-h\u003c/strong\u003e, Indirect calorimetry and body temperature measurements were performed under feeding a chow (control) or norUDCA diet (\u003cem\u003en=6\u003c/em\u003e). The dietary regimen was started at time point zero and the ambient temperature was gradually reduced as indicated (blue line). CO\u003csub\u003e2 \u003c/sub\u003eproduction (\u003cstrong\u003ee\u003c/strong\u003e), O\u003csub\u003e2 \u003c/sub\u003econsumption (\u003cstrong\u003ef\u003c/strong\u003e), respiratory exchange rate (\u003cstrong\u003eg\u003c/strong\u003e), body core temperature (\u003cstrong\u003eh\u003c/strong\u003e) were continuously recorded. Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with one-way ANOVA. *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/1a3f9391f994a01c4e1b9e09.png"},{"id":97873675,"identity":"f39844db-58fb-4516-8afe-6fb6ff9ab617","added_by":"auto","created_at":"2025-12-10 10:45:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003enorUDCA effects on bile acid pool and enrichment in metabolically active organs.\u003c/strong\u003e \u003cstrong\u003ea-b\u003c/strong\u003e, Wild type mice were fed a regular chow (control), or chow diet supplemented with 0.5% norUDCA. Bile acid concentrations in feces (\u003cstrong\u003ea\u003c/strong\u003e) and systemic blood plasma (\u003cstrong\u003eb\u003c/strong\u003e) after 7 day treatment under standard housing conditions (\u003cem\u003en=7\u003c/em\u003e) \u003cstrong\u003ec-f\u003c/strong\u003e, Wild type mice were fed a regular chow (control), or chow diet supplemented with 0.5% norUDCA before oral administration of \u003csup\u003e33\u003c/sup\u003eH-norUDCA and \u003csup\u003e14\u003c/sup\u003eC-CA (\u003cem\u003en=8\u003c/em\u003e). Four hours later, percentage of applied tracers was determined in intestine (\u003cstrong\u003ec\u003c/strong\u003e), liver (\u003cstrong\u003ed\u003c/strong\u003e), heart (\u003cstrong\u003ee\u003c/strong\u003e) and BAT (\u003cstrong\u003ef\u003c/strong\u003e). Statistical analysis was performed with Student’s T-Test (\u003cstrong\u003ea-b\u003c/strong\u003e) or with two-way ANOVA (\u003cstrong\u003ec-f\u003c/strong\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/1684650fb1f7f534f9d70e7d.png"},{"id":97873696,"identity":"2b50d5f0-310c-4e60-a30c-757170f445f9","added_by":"auto","created_at":"2025-12-10 10:45:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003enorUDCA drives transcriptional remodelling and alters energy source utilization in heart and brown adipose tissue. a-h, \u003c/strong\u003eWild type mice were fed a regular chow (control), or chow diet supplemented with 0.5% norUDCA for 7 days with housing at room temperature. Bulk RNAseq (\u003cem\u003en=4\u003c/em\u003e) of heart (\u003cstrong\u003ea-c\u003c/strong\u003e) and BAT\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ed-f\u003c/strong\u003e) were performed to identify differentially expressed genes (\u003cstrong\u003ea, d\u003c/strong\u003e), and to characterize downregulated (\u003cstrong\u003eb, e\u003c/strong\u003e) and upregulated (\u003cstrong\u003ec, f\u003c/strong\u003e) pathways determined by gene ontology analysis. \u003cstrong\u003eg-h\u003c/strong\u003e, Organ-specific uptake of intravenously administered albumin-bound \u003csup\u003e14\u003c/sup\u003eC-oleic acid (\u003cem\u003en=4\u003c/em\u003e) (\u003cstrong\u003eg\u003c/strong\u003e) and \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose (\u003cem\u003en=5-8\u003c/em\u003e) (\u003cstrong\u003eh\u003c/strong\u003e) was determined 15 min after injection. Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with Student’s T-Test. *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/419b5543c9cc5c393506f990.png"},{"id":97900726,"identity":"5e43c5f6-3ee0-4650-bf4c-ff304124dfdc","added_by":"auto","created_at":"2025-12-10 15:45:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003enorUDCA shifts cardiac energy metabolism towards glucose utilization. a, \u003c/strong\u003eMice were fed a regular chow (control), or chow diet supplemented with various bile acids at 0.5% (except OCA, 0.05%) for 7 days with housing at room temperature. Organ-specific uptake of intravenously administered \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose (\u003cem\u003en=5-6\u003c/em\u003e). \u003cstrong\u003eb,\u003c/strong\u003e Mice were treated without or with an antibiotic cocktail to deplete gut bacteria and fed a regular chow (control) or a chow diet supplemented with norUDCA for 7 days at room temperature. Organ-specific uptake of \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose was determined 15 min after intravenous administration (\u003cem\u003en=6\u003c/em\u003e). \u003cstrong\u003ec\u003c/strong\u003e, Saline or norUDCA were administered by oral gavage. Organ-specific uptake of \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose was determined 15 min after intravenous administration 8 hours after gavage. \u003cstrong\u003ed-f\u003c/strong\u003e, Mice were fed a regular chow (control), or chow diet supplemented with norUDCA at 0.5% for 7 days with housing at room temperature. Subsequently, \u003csup\u003e13\u003c/sup\u003eC-(U) glucose was intravenously injected and 30 min later organs for metabolite enrichment analysis (\u003cem\u003en=4-5\u003c/em\u003e). Schematic model showing \u003cem\u003ein vivo\u003c/em\u003e stable isotope labelling of glycolysis and citric acid cycle (\u003cstrong\u003ed\u003c/strong\u003e) that were quantified in heart (\u003cstrong\u003ee\u003c/strong\u003e) and BAT (\u003cstrong\u003ef\u003c/strong\u003e) to determine \u003csup\u003e13\u003c/sup\u003eC enrichment in metabolites. Error bars are shown as SEM. Statistical analysis was performed either with one-way ANOVA (\u003cstrong\u003ea\u003c/strong\u003e), two-way ANOVA (\u003cstrong\u003eb\u003c/strong\u003e) or Student’s T-Test (\u003cstrong\u003ec, e, f\u003c/strong\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005, ****p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/1f18bd2417a7741f661f59f3.png"},{"id":97873698,"identity":"23a4b448-1207-4540-a1fd-22c9e1581a75","added_by":"auto","created_at":"2025-12-10 10:45:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79505,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipolysis and ketone bodies compensate norUDCA-induced defects in energy homeostasis and thermogenesis. a-g, \u003c/strong\u003eMice were fed a regular chow (control), or chow diet supplemented with norUDCA for 1 day, 3 days or 7 days with housing at room temperature. \u003cstrong\u003ea-c\u003c/strong\u003e, Circulating levels of glucose (\u003cstrong\u003ea\u003c/strong\u003e), non esterified fatty acids (NEFA) (\u003cstrong\u003eb\u003c/strong\u003e), and ketone bodies (\u003cstrong\u003ec\u003c/strong\u003e) were determined (\u003cem\u003en=7\u003c/em\u003e). \u003cstrong\u003ed-g\u003c/strong\u003e, Uptake of \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose (\u003cstrong\u003ed, e\u003c/strong\u003e) and \u003csup\u003e14\u003c/sup\u003eC-hydroxybutyrate (\u003cstrong\u003ef, g\u003c/strong\u003e) by heart (\u003cstrong\u003ed, f\u003c/strong\u003e) and by BAT (\u003cstrong\u003ee, g\u003c/strong\u003e) was determined 15 min after intravenous administration (\u003cem\u003en=6\u003c/em\u003e). \u003cstrong\u003eh\u003c/strong\u003e, Release of NEFA and glycerol from white adipose tissue explants isolated from mice fed a chow (control) or chow diet supplemented with norUDCA (\u003cem\u003en=3\u003c/em\u003e). \u003cstrong\u003ei\u003c/strong\u003e, Expression of lipogenic and lipolytic genes in white adipose tissues isolated from control and norUDCA-fed mice (\u003cem\u003en=7\u003c/em\u003e). \u003cstrong\u003ej-k\u003c/strong\u003e, Indirect calorimetry was performed at room temperature in lipolysis-deficient ATGL-MHC and control mice fed a chow for 3 days followed by feeding a norUDCA-containing chow diet for 5 days (\u003cem\u003en=2-3\u003c/em\u003e). Consumption of O\u003csub\u003e2 \u003c/sub\u003e(\u003cstrong\u003ej\u003c/strong\u003e) and production of CO\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003ek\u003c/strong\u003e) are shown. \u003cstrong\u003el-n, \u003c/strong\u003eIndirect calorimetry\u003cstrong\u003e \u003c/strong\u003ewas performed in wild type mice that were fed a low-carb diet or low-carb diet supplemented with norUDCA. Production of CO\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003el\u003c/strong\u003e), consumption of O\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003em\u003c/strong\u003e) and respiratory exchange ratio (\u003cstrong\u003en\u003c/strong\u003e) were recorded at various housing temperatures as indicated by the red line (\u003cem\u003en=3\u003c/em\u003e). Error bars are shown as SEM. Statistical analysis was performed with one-way ANOVA (\u003cstrong\u003ea-g\u003c/strong\u003e), or Student’s T-Test (\u003cstrong\u003eh-i\u003c/strong\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01,*** p\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/1506696868e2dd25132680a1.png"},{"id":97900611,"identity":"0aa44fd9-0136-4d3c-8bca-d1166630431a","added_by":"auto","created_at":"2025-12-10 15:45:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":898208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003enorUDCA deteriorates mitochondrial respiration and causes functional defects in human engineered heart tissues. a-e\u003c/strong\u003e, Mice were fed a regular chow (control), or chow diet supplemented with norUDCA for 7 days with housing at room temperature. Representative electron microscopy pictures of heart from control (\u003cstrong\u003ea\u003c/strong\u003e) and norUDCA-treated mice (\u003cstrong\u003eb\u003c/strong\u003e).Western blot analysis of OXPHOS protein complexes from cardiac tissues (\u003cstrong\u003ec, d\u003c/strong\u003e). High resolution respirometry of isolated heart mitochondria (\u003cstrong\u003ee\u003c/strong\u003e) (\u003cem\u003en=6\u003c/em\u003e). \u003cstrong\u003ef-j\u003c/strong\u003e, As depicted in the schematic model (\u003cstrong\u003ef\u003c/strong\u003e), engineered heart tissues (EHT) were generated from human induced pluripotent stem cells (hiPSCs). \u003cstrong\u003eg-j\u003c/strong\u003e, EHT were incubated with CDCA (\u003cstrong\u003eg, h\u003c/strong\u003e) or norUDCA (\u003cstrong\u003ei, j\u003c/strong\u003e), and relaxation time (\u003cstrong\u003eg, i\u003c/strong\u003e) and force (\u003cstrong\u003eh, j\u003c/strong\u003e) were measured at indicated time points (\u003cem\u003en=6-7\u003c/em\u003e). Error bars are shown as SEM. Statistical analysis was performed either with Student’s T-Test (\u003cstrong\u003ed, e\u003c/strong\u003e) or one-way ANOVA (\u003cstrong\u003eg-j\u003c/strong\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/7e741fc8c07f993753b2dd3d.png"},{"id":99319813,"identity":"379370f3-913d-4a37-8d79-e47af32f2650","added_by":"auto","created_at":"2025-12-31 16:37:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2732229,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/93cc8bb4-b536-4e6d-8e90-68d63a89a588.pdf"},{"id":97873702,"identity":"eb948c0b-1395-4c1b-a384-a16094a9435c","added_by":"auto","created_at":"2025-12-10 10:45:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1560842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtended data Fig. 1. NorUDCA and UDCA alleviate liver inflammation and fibrosis in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Cyp2c70\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice. \u003c/strong\u003eWild type and \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice were fed a regular chow, or chow diet supplemented with either 0.5% UDCA or 0.5% norUDCA for 7 days under standard housing conditions (\u003cem\u003en=4-5\u003c/em\u003e). \u003cstrong\u003ea\u003c/strong\u003e, Representative images of LY6C and CK19 stainings of liver sections. \u003cstrong\u003eb-d\u003c/strong\u003e, Hepatic expression of fibrosis marker genes (\u003cstrong\u003eb\u003c/strong\u003e), inflammation marker genes (\u003cstrong\u003ec\u003c/strong\u003e), and cecal bile acid concentration (\u003cstrong\u003ed\u003c/strong\u003e). Error bars are shown as SEM. Statistical analysis was performed two-way ANOVA (same letter denotes groups that are not significantly different from each other, with p \u0026lt; 0.05). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data Fig. 2. NorUDCA reduces fat mass and increases gall bladder size. \u003c/strong\u003eMice were fed a regular chow (control), or chow diet supplemented with norUDCA for 7 days with housing at room temperature. \u003cstrong\u003ea\u003c/strong\u003e, Change of fat and lean mass determined by echoMRI (\u003cem\u003en=7\u003c/em\u003e). \u003cstrong\u003eb,\u003c/strong\u003e Representative image of gallbladder size of chow (control) and norUDCA-treated mouse under standard housing conditions. \u003cstrong\u003ec-f\u003c/strong\u003e, Quantification of indirect calorimetry and body temperature measurements from the experiments shown in Fig. 1 separately calculated for the light and dark phases. CO\u003csub\u003e2 \u003c/sub\u003eproduction (\u003cstrong\u003ec\u003c/strong\u003e), O\u003csub\u003e2 \u003c/sub\u003econsumption (\u003cstrong\u003ed\u003c/strong\u003e), respiratory exchange rate (\u003cstrong\u003ee\u003c/strong\u003e), body core temperature (\u003cstrong\u003ef\u003c/strong\u003e). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with Student’s T-Test. *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data Fig. 3. norUDCA accumulates in metabolically active organs.\u003c/strong\u003e \u003cstrong\u003ea-d\u003c/strong\u003e Wild type mice were fed a regular chow (control), or chow diet supplemented with 0.5% norUDCA before oral administration of \u003csup\u003e3\u003c/sup\u003eH-norUDCA and \u003csup\u003e14\u003c/sup\u003eC-CA (\u003cem\u003en=8\u003c/em\u003e). Four hours later, percentage of applied tracers was determined in muscle (\u003cstrong\u003ea\u003c/strong\u003e), inguinal white adipose tissue (iWAT) (\u003cstrong\u003eb\u003c/strong\u003e), gonadal white adipose tissue (gWAT) (\u003cstrong\u003ec\u003c/strong\u003e), and plasma (\u003cstrong\u003ed\u003c/strong\u003e). Statistical analysis was performed with two-way ANOVA (\u003cstrong\u003ea-d\u003c/strong\u003e). *p\u0026lt;0.05, **p\u0026lt;0.01,***p\u0026lt;0.005.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data Fig. 4. norUDCA shifts cardiac energy metabolism towards glucose utilization. a, \u003c/strong\u003eMice were fed a regular chow (control), or chow diet supplemented with norUDCA at 0.5% for 7 days with housing at room temperature. \u003cstrong\u003ea-b\u003c/strong\u003e, Expression of metabolic genes in heart (\u003cstrong\u003ea\u003c/strong\u003e) and in BAT (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec-f\u003c/strong\u003e, Western blot analysis and quantification of lipoprotein-processing proteins from samples of heart (\u003cstrong\u003ec, e\u003c/strong\u003e) and BAT (\u003cstrong\u003ed, f\u003c/strong\u003e). \u003cstrong\u003eg-h\u003c/strong\u003e, Organ-specific uptake of intravenously administered \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose in FVB wild type vs \u003cem\u003eApoA5\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice on the FVB background (\u003cstrong\u003eg\u003c/strong\u003e), and in wild type controls vs \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/- \u003c/em\u003e\u003c/sup\u003emice (\u003cstrong\u003eh\u003c/strong\u003e). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed either with Student’s T-Test (\u003cstrong\u003ea-f\u003c/strong\u003e) or two-way ANOVA (\u003cstrong\u003eg-h\u003c/strong\u003e). *= p\u0026lt;0.05, **= p\u0026lt;0.01,***= p\u0026lt;0.005.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data Fig. 5. ATGL compensates for norUDCA-induced defects in energy homeostasis and thermogenesis at room temperature. a-d, \u003c/strong\u003eIndirect calorimetry of ATGL-MHC mice fed with chow followed by norUDCA-enriched diet for the indicated time period at room temperature. CO\u003csub\u003e2 \u003c/sub\u003eproduction (a), O\u003csub\u003e2 \u003c/sub\u003econsumption (b), quantification of indirect calorimetry measurements separately calculated for the light and dark phases for CO\u003csub\u003e2 \u003c/sub\u003eproduction (\u003cstrong\u003ec\u003c/strong\u003e) and O\u003csub\u003e2 \u003c/sub\u003econsumption (\u003cstrong\u003ed\u003c/strong\u003e). Error bars indicate standard error of the mean (SEM). Statistical analysis was performed with Student’s T-Test. *= p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtended data Fig. 6. Electron microscopy of BAT from mice and model depicting the effects of norUDCA on metabolically active organs at room temperature and at cold. a-b\u003c/strong\u003e, Mice were fed a regular chow (control), or chow diet supplemented with norUDCA for 7 days with housing at room temperature. Representative electron microscopy pictures of BAT from control (\u003cstrong\u003ea\u003c/strong\u003e) and norUDCA-treated mice (\u003cstrong\u003eb\u003c/strong\u003e). Schematic model of norUDCA effects at room temperature and under cold stimulus (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"251128SupplementalFiguresEvangelakosetal2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/22ba10980d8c7e2bc60a8931.pdf"},{"id":97900654,"identity":"da5af4da-6d7a-4156-b624-781f91d9b54c","added_by":"auto","created_at":"2025-12-10 15:45:42","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2059461,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/a0ff5ee6babc22c697edca14.xlsx"},{"id":97900263,"identity":"4d49b411-5b30-4dc7-ba3d-08110ebca7a3","added_by":"auto","created_at":"2025-12-10 15:45:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1502665,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8280795/v1/348bae270881eb8f5f74d456.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The conjugation-resistant bile acid norUDCA cures liver fibrosis but impairs systemic energy metabolism","fulltext":[{"header":"Main","content":"\u003cp\u003eBile acids (BAs) are cholesterol derivatives that are synthesized in hepatocytes by consecutive steps of enzymatic reactions finalized by conjugation to taurine or glycine. The unconjugated (UBAs) and conjugated (CBAs) species present in the circulating BA pool vary considerably in their biochemical and biophysical properties, which has important physiological and pathological consequences \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. For example, patients suffering from metabolic dysfunction-associated steatohepatitis, alcoholic liver cirrhosis, or primary biliary cholangitis (PBC) frequently exhibit elevated total BAs, particularly CBAs \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These pathological conditions are further characterized by higher CBA/UBA ratios \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, reflecting underlying alterations in the gut microbiome and its capacity for BA-transformation, including the generation of secondary BAs \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Supporting a potentially detrimental role of CBAs, a recent study demonstrated that the inhibition of BA conjugation through deletion of the hepatic bile acid\u0026ndash;CoA: amino acid N-acyltransferase (BAAT) enhanced tumor-specific T cell responses and reduced tumor growth in liver \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In line, conjugation of the gut microbiome-derived secondary BAs 3-oxoLCA and isoalloLCA reduced their immunomodulatory effects on T helper and regulatory T cells \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, indicating that conjugation also of secondary BAs influences adaptive immune responses. Similarly, enriching the BA-pool with hydrophilic BAs, such as ursodeoxycholic acid (UDCA), an approved standard therapy for the treatment of PBC \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, boosts T cell function and consequently abolishes liver injury and concomitant tumors growth in various mouse models \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. On the other hand, BA de-conjugation by bacterial bile salt hydrolases (BSHs) in the gut generates UBAs that are less efficient than their conjugated counterparts in the emulsification of dietary lipids and may lead to dysregulated lipid absorption and weight loss \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Growing evidence suggest that the balance between BA-conjugation and microbial de-conjugation is an important determinant for systemic metabolism, such as during Roux-en-Y gastric bypass (RYGB) surgery. This weight loss procedure causes increased BA levels, predominantly of glycine-conjugated BAs in the postprandial phase \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which are associated with altered rates of glucose and lipid oxidation \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Another example that a higher CBA to UBA ratio regulates systemic energy metabolism is based on the observation that under conditions of BAT activation by cold exposure, adaptive changes in endogenous BA metabolism were associated with efficient heat production as wells as improved lipid and lipoprotein metabolism in mice \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In another mouse study, focusing on gut microbiome changes in response to cold exposure, the beneficial effects on adiposity and adaptive thermogenesis also involved enhanced CBA production and a higher CBA to UBA ratio \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Notably, metabolomics analysis of human plasma samples following BAT activation by the β3-adrenoreceptor agonist mirabegron revealed that BA metabolism was the most affected pathway \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eC23 BAs with a shortened side chain, such as norchenodeoxycholic acid (norCDCA), norcholic acid (norCA) and norUDCA, have been developed to improve therapeutic properties of naturally occurring BAs \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These nor-BAs are inefficiently amidated, i.e. they are highly resistant to taurine or glycine conjugation as well as microbial biotransformation. Accordingly, they are secreted in an unconjugated form into bile, which induces hypercholeresis accompanied by increased bicarbonate output\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, thereby alleviating toxic effects particularly of hydrophobic BAs \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This is in particular the case for norUDCA, a derivative that combines hydrophilicity with conjugation resistance, and has been successfully used for therapeutic intervention in preclinical models and patients with cholestatic as well as fibrotic liver diseases \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, norUDCA has been shown to modulate regulatory networks influencing immune-metabolism of T cells, attenuating T cell-driven inflammatory diseases in gut and liver \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Importantly, unconjugated norUDCA can reach plasma concentration that are approximately 10-fold higher as compared to other orally administered endogenous BAs including UDCA \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the consequences of this systemic spill-over for peripheral tissues such as muscle, heart, brown or white fat depots remain elusive.\u003c/p\u003e\u003cp\u003eGiven its high bioavailability in the systemic circulation, here we investigated in mice whether norUDCA could serve as a potential and suitable treatment to enhance energy expenditure and thermogenesis. We found that the accumulation of norUDCA in peripheral organs was associated with reduced adiposity and lower blood glucose. Unexpectedly, however, norUDCA treatment resulted in decreased energy expenditure and core body temperature in response to energy-demanding cold exposure. Metabolic tracer and mechanistic studies indicate a critical role of norUDCA for fuel uptake, mitochondrial respiration and whole-body energy metabolism, particularly during adaptive thermogenesis induced by cold exposure. Moreover, our data suggest that elevated systemic levels of norUDCA together with the concomitant increase in endogenous unconjugated BAs cause alterations in glucose and lipid homeostasis that contribute to metabolic inflexibility in cardiac and adipose tissues, impairing their capacity to effectively utilize energy substrates for metabolic processes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eNorUDCA treatment ameliorates cholestatic liver disease but confers intolerance to cold exposure\u003c/h2\u003e\u003cp\u003eTo investigate the impact of conjugation-resistance of BAs for systemic energy metabolism, we first compared UDCA versus norUDCA treatment in wild type and \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice. The latter is a preclinical model with a more hydrophobic bile acid pool that results in spontaneous cholestatic liver disease, which is based on the deficient conversion of CDCA into the hydrophilic muricholic acids \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In the experimental setup, wild type and \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were fed a regular chow (control) or diets supplemented with 0.5% of either UDCA or norUDCA for a period of one week. Both BAs equally improved the inflammatory and fibrotic liver phenotype in \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c), which is in line with a previous study using UDCA \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In caecum, norUDCA levels accumulated to a concentration that was 5-fold higher than that of UDCA (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Notably, norUDCA but not UDCA significantly reduced body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), which was mainly explained by reduced fat mass (Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and lower weights of adipose tissue depots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Treatment with norUDCA led to larger gall bladders (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), which was probably a result of hypercholeresis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The weights of other organs including liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) were unaffected. As the reduced fat mass suggested enhanced energy expenditure and heat production in BAT and muscle, we determined the effects of norUDCA on adaptive thermogenic responses. To this end, wild type mice fed a normal chow (control) or chow supplemented with norUDCA were housed at thermoneutral conditions (30\u003csup\u003eo\u003c/sup\u003eC) or exposed to cold (6\u003csup\u003eo\u003c/sup\u003eC). Unexpectedly, the norUDCA-treated mice housed at 6\u0026deg;C were completely cold-intolerant and 80% died within 8 hours of cold exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Furthermore, indirect calorimetry revealed that mice on the norUDCA-supplemented diet exhibited progressively lower respiration rates and body temperatures even when the ambient temperatures were gradually reduced from 30\u003csup\u003eo\u003c/sup\u003eC to only 16\u003csup\u003eo\u003c/sup\u003eC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-h, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-f). These data indicate that norUDCA treatment reduces whole body energy expenditure, thermogenesis and critically impacts survival during cold acclimation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eNorUDCA accumulates in the systemic circulation and peripheral tissues together with endogenous unconjugated BAs\u003c/h3\u003e\n\u003cp\u003eTo determine whether norUDCA exerts its effects on energy expenditure in peripheral organs directly \u003cem\u003evia\u003c/em\u003e the circulation, we measured CBA und UBA species in wild type mice fed a normal chow or a norUDCA-supplemented diet. Dietary exposure resulted in decreased fecal CBAs and increased total fecal bile acid content, the latter resulting from the exogenous norUDCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Despite the high fecal excretion, concentrations of norUDCA in systemic plasma reached values of approximately 200 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Remarkably, concentrations of endogenous UBAs were also increased in plasma with total levels of ~\u0026thinsp;20\u0026micro;M. Thus, UBA levels by far exceed those of CBAs with ~\u0026thinsp;2 \u0026micro;M, indicating a pronounced spillover of UBAs into the systemic circulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). To determine the exposure of peripheral organs to norUDCA, we orally administered \u0026sup3;H-norUDCA and compared its organ uptake at four hours with that of simultaneously administered \u0026sup1;⁴C-cholic acid (\u0026sup1;⁴C-CA) in mice fed a chow or a norUDCA-supplemented chow diet. Irrespective of diet composition, almost 90% of radiolabeled CA was detected in the intestine and only minute amounts were found in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In contrast, only\u0026thinsp;~\u0026thinsp;30% of \u003csup\u003e3\u003c/sup\u003eH-norUDCA ended up in the intestine and a similar amount was found in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d), which argues for efficient cholehepatic shunting between the bile duct epithelium and liver of norUDCA as compared to CA. Remarkably, a substantial amount of \u003csup\u003e3\u003c/sup\u003eH-norUDCA compared to \u003csup\u003e14\u003c/sup\u003eC-CA was detected in peripheral tissues such as heart and BAT, an effect that was even more pronounced in norUDCA-preconditioned mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d). Our data demonstrate considerable exposure of key metabolic tissues to circulating norUDCA and other UBAs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eNorUDCA induces tissue remodeling and alters energy substrate utilization in heart and BAT\u003c/h3\u003e\n\u003cp\u003eMetabolic functions of BAT and heart are critical for physiological responses to cold stress by increasing heat production and blood flow, respectively \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To understand the effects of norUDCA in an unbiased manner, bulk RNA sequencing of heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c) and BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-f) from mice on regular chow or on norUDCA-supplemented chow was performed. Volcano plot analysis indicated a high number of differentially expressed genes (DEG) between the groups both in heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, Supplementary Table\u0026nbsp;1) and BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Supplementary Table\u0026nbsp;2). In hearts, top upregulated genes upon norUDCA feeding were related to ventricular remodeling such as \u003cem\u003eAtf3\u003c/em\u003e and \u003cem\u003eMyh7\u003c/em\u003e, as well as those mediating mitochondrial biogenesis like \u003cem\u003ePpargc1a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, \u003cem\u003eSlc2a1\u003c/em\u003e and \u003cem\u003eSlc2a4\u003c/em\u003e encoding the glucose transporters GLUT1 and GLUT4, respectively, were upregulated whereas \u003cem\u003ePdk4\u003c/em\u003e, known to inhibit pyruvate dehydrogenase, was reduced. The changes in expression of a number of metabolic genes were confirmed by qPCR in an independent study (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Gene ontology (GO) analysis of DEG data revealed that a few metabolic pathways were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). On the other hand, pathways related to glucose metabolism, e.g., pyruvate metabolism, glycolysis and gluconeogenesis, as well as tissue remodeling pathways including proteasome, ferroptosis and mitophagy were upregulated in the hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In BAT, the expression of key thermogenic markers including \u003cem\u003eUcp1\u003c/em\u003e, \u003cem\u003eDio2\u003c/em\u003e and \u003cem\u003eAdrb3\u003c/em\u003e was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Consistently, pathways related to core thermogenic functions such as oxidative phosphorylation, fatty acid metabolism and thermogenesis were suppressed in the norUDCA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). In line, qPCR showed altered expression of lipogenic genes such as \u003cem\u003eFasn\u003c/em\u003e and \u003cem\u003eScd1\u003c/em\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Similar to the findings in heart, pathways related to tissue remodeling were upregulated in BAT of norUDCA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The functional relevance of the alterations in metabolic pathways were confirmed by radioactive tracer studies showing impaired fatty acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) but unaltered glucose uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh) by BAT. Despite compromised lipid uptake, a BAT-specific increase in proteins promoting intravascular processing of triglyceride-rich lipoproteins (GPIHBP1, LPL) and fatty acid uptake (CD36) was observed (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-f), suggesting an induction of compensatory yet futile mechanisms to replenish energy stores in thermogenic adipose tissues of norUDCA-treated mice. In the heart, norUDCA caused a several-fold increase in the uptake of glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh), while the uptake of fatty acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) was preserved. This observation, in accordance with the increased expression of genes regulating glucose transport and metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c), point toward an increased reliance of the heart on glucose as energy source and impaired fatty acid utilization in BAT in response to norUDCA supplementation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eNorUDCA shifts cardiac energy metabolism towards glucose utilization\u003c/h3\u003e\n\u003cp\u003eGiven the relevance of alterations in glucose metabolism in the context of heart failure \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, the specificity of norUDCA on cardiac glucose uptake compared to other bile acids was determined. For this purpose, \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose was injected into wild type mice which were fed a chow diet supplemented with norUDCA, UDCA, CDCA, CA, the secondary bile acid deoxycholic acid (DCA), the semisynthetic FXR agonist obeticholic acid (OCA), or the synthetic de-conjugation resistant bile acid cholylsarcosine. Among all the bile acid species tested, only norUDCA significantly increased \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003eH-deoxyglucose uptake in the heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Supplementation with norUDCA but also with CA and DCA resulted in higher glucose uptake into white adipose tissue depots, while no significant changes were detected in other organs investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The effect of norUDCA on cardiac glucose disposal was also observed in the FVB mouse strain, hyperlipidemic \u003cem\u003eApoa5\u003c/em\u003e-deficient FVB and \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-h). We next determined whether the microbiome is involved in mediating the effect of norUDCA on shifting energy substrate utilization. For this purpose, we depleted the gut bacteria by antibiotic treatment (Abx), known to decrease the turnover of bile acids and to elevate plasma levels of CBAs \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Of note, the heart of norUDCA-treated mice show a tendency to even higher \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003eH-deoxyglucose uptake after Abx treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating that microbial processing is not essential for the observed norUDCA effect on glucose metabolism. Of note, already 6 hours after oral administration of norUDCA the heart internalized larger amounts of \u003csup\u003e3\u003c/sup\u003eH-deoxyglucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), indicating an acute effect that is independent of processes induced by chronic administration, e.g. loss in fat mass. Next, we performed metabolic flux studies to determine how norUDCA and the associated higher glucose uptake impacts intracellular glucose metabolism. To achieve this goal, control and norUDCA-fed mice received an intravenous bolus of \u003csup\u003e13\u003c/sup\u003eC-labeleled glucose and enrichment of \u003csup\u003e13\u003c/sup\u003eC in glucose metabolites (schematic diagram in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) was assessed by mass spectrometry in heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) and BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). In line with higher glucose uptake, norUDCA treatment caused a markedly increase in glucose metabolism as indicated by higher enrichments of \u003csup\u003e13\u003c/sup\u003eC in intermediates of glycolysis including lactate and citrate cycle in heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) but not in BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Overall, cardiac tissue of mice treated with norUDCA exhibits a profound increase in glucose uptake and utilization, a phenotype often related to heart failure and cardiac hypertrophy \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eLipolysis and ketone bodies compensate norUDCA-induced defects in energy homeostasis and thermogenesis\u003c/h3\u003e\n\u003cp\u003eNormal cardiac function relies on the continuous supply of fatty acids as energy substrate \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. These free acids delivered to the heart are derived from either adipose tissue lipolysis and/or hydrolysis of triglyceride-rich lipoproteins by lipoprotein lipase \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To further unravel disturbances in energy substrate utilization, plasma levels of glucose, non-esterified fatty acids (NEFAs) and ketone bodies were quantified in mice fed with norUDCA for 1 day, 3 days and 7 days, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). Glucose levels were strongly reduced at day 1 and remained at lower levels on the following days, which coincides with increased uptake by heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) but not by BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). In contrast, NEFAs and ketone bodies were increased in plasma of norUDCA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-c), which was accompanied by a marked increase in the uptake of the ketone body β-hydroxybutyrate in BAT and to a lesser extent by the heart (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g). The higher levels of ketone bodies are probably triggered by an increased flow of fatty acids from white adipose tissues to liver, as supported by increased lipolysis in adipose tissue explants of norUDCA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). In line, higher expression of adipose tissue triglyceride lipase (ATGL) encoded by \u003cem\u003ePnpla2\u003c/em\u003e was found in WAT of norUDCA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei). To directly study the effect of ATGL, we performed indirect calorimetry studies in whole body \u003cem\u003ePnpla2\u003c/em\u003e deficient mice with transgenic cardiac ATGL overexpression, which prevents lipid accumulation and heart dysfunction described for the whole body knockout mice \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Of note, these mice lacking ATGL in adipose tissues displayed reduced energy expenditure even at 22\u0026deg;C after norUDCA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej-k, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-d). However, under low carb dietary conditions favoring ketone body production, energy expenditure was not compromised by norUDCA supplementation even when the temperature was gradually decreased to 16\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003el-n). Overall, these data indicate that adipose ATGL-mediated lipolysis and subsequent hepatic ketone body production are critical in maintaining energy homeostasis in norUDCA-treated mice. Moreover, dietary approaches allow to overcome disturbed energy substrate utilization in norUDCA fed animals, thereby preventing cold stress-induced metabolic deficits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eNorUDCA impairs mitochondrial respiration and disturbs contractile function of human engineered heart tissue\u003c/h2\u003e\u003cp\u003eThe profound increase in cardiac glucose uptake and lactate production suggested a negative effect of norUDCA on mitochondrial function. Surprisingly, the overall architecture of cardiomyocytes and mitochondrial structure visualized by electron microscopy were not conspicuously altered by norUDCA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Similarly, no morphological changes were observed in thermogenic adipocytes and mitochondria of BAT (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). In addition, the levels of selected OXPHOS complex proteins were comparably abundant in heart mitochondria isolated from control and norUDCA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-d). However, respirometry demonstrated a massive decline in oxidation of energy substrates in heart mitochondria acutely treated with 100 \u0026micro;M norUDCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), a concentration similar to that detected in the systemic circulation of norUDCA-supplemented mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Of note, in engineered heart tissues generated from human induced pluripotent stem cells (see model in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), incubation with norUDCA impaired both relaxation time and force generation gradually during incubation, which was not observed using equal concentrations of unconjugated CDCA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-j). Taken together, these data indicate that norUDCA internalized by cardiomyocytes impairs contractile function by inhibiting mitochondrial respiration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite recent progress in understanding the structural diversity and the specific functions of individual BAs, their divergence in the capacity to regulate hepatic and systemic metabolic adaptations remains underexplored. Given the known role of endogenous BAs in stimulating energy expenditure via the G-protein coupled receptor TGR5 in skeletal muscle and BAT both in mice and humans \u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, a major goal of this study was to investigate the potential beneficial effects of conjugation-resistant norUDCA, a C23-norderivative of UDCA, on systemic energy metabolism. Next to its known anti-fibrotic effects in the hepatobiliary system, its higher systemic concentration compared to naturally occurring bile acids makes norUDCA an attractive candidate to treat obesity-associated comorbidities such as dyslipidemia, diabetes and cardiovascular disease \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In the current study, we confirmed and complement previous reports that modifying the BA-pool towards higher hydrophilicity with UDCA or norUDCA completely prevents liver inflammation and fibrosis in \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, a mouse model with characteristics of human cholestasis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In metabolic turnover studies, we found a marked enrichment of norUDCA in peripheral organs that was associated with changes in energy substrate utilization in heart and BAT. Against our expectation that norUDCA would be pro-thermogenic, however, we observed that norUDCA caused a drastic drop in whole body energy expenditure and thermogenesis under catabolic conditions triggered by cold stress. It was found that norUDCA negatively affected heart and BAT function and thus compromised the survival of animals. The primary reasons for the strong effect can either relate to the high systemic concentration of unconjugated norUDCA, or be the result of increased endogenous UBA levels in plasma, causing a tremendous shift in the CBA to UBA ratio that might have contributed to the effects on heart and BAT described in the current study. Higher UBA levels could be the result of impaired hepatic conjugation that has been reported to be mediated by C23 nor-bile acid derivatives \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, or result from competition of excessive norUDCA with other UBAs during hepatic extraction from the portal blood \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Besides, high UBAs may be a consequence of the activation of microbial bile salt hydrolase (BSH) through norUDCA \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which is mirrored by decreased CBAs in feces found in our study. In any event, it is very likely that the high ratio of UBA to CBA in plasma provokes the negative effects on energy expenditure and heat production.\u003c/p\u003e\u003cp\u003eNotably, uncoupled respiration for heat production in BAT is mediated by elevated CBAs rather than UBAs \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Moreover, beneficial effects of cold on adiposity and adaptive thermogenesis is associated with a higher CBA to UBA ratio \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Similarly, the metabolic effects of BAs in Roux-en-Y gastric bypass bariatric surgery are related to high levels CBAs and a concomitant decrease in UBA levels \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Notably, the phenotype of mice treated with norUDCA shares similarities with that observed after biliopancreatic diversion. The shortened route of enterohepatic circulation in this weight loss intervention characterized by inefficient lipid absorption results in significantly elevated UBAs \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In the future, it would be interesting to study preclinical models and patients undergoing this surgical intervention with regard to cardiac and BAT fuel oxidation and energy expenditure.\u003c/p\u003e\u003cp\u003eRemarkably, the metabolic deficits observed in our study were highly specific for norUDCA, since adverse effects were not observed for the closely related UDCA or other BAs. The uptake, as shown by tracer studies using radiolabeled norUDCA, and in consequence the accumulation of norUDCA within extrahepatic organs could be mediated by the organic anion transporting polypeptide 1 (OATP1), a bile acid transporter that is expressed in multiple tissues. OATP1 has been demonstrated to efficiently transport unconjugated nor bile acids but not other UBAs such as CDCA \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAn excess of BAs is known to impair cardiac function \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, impact energy metabolism and BAT thermogenesis \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, as well as mitochondrial function \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These effects were observed in both mice and humans with implications for overall energy metabolism and heart function \u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The relationship between \u0026ldquo;cardiotoxic\u0026rdquo; BAs and cardiac dysfunction is only witnessed in the context of liver disorders \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Particularly, hydrophobic BAs with high cellular cytotoxicity mediate such pathological effects \u003cem\u003ein vitro\u003c/em\u003e, whereas hydrophilic BAs, such as UDCA or muricholic acids even counteracted the effect of lipophilic BAs \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. NorUDCA that is even more hydrophilic than UDCA (critical micellar concentration is 17 mM for norUDCA versus 7 mM for UDCA; \u003csup\u003e59\u003c/sup\u003e) has not been reported to cause adverse cellular effects, making it highly unlikely to act as a typical hydrophobic cytotoxic BA. It is well established that a dysbalance between glucose and fatty acid oxidation is detrimental for cardiac function \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In the current study norUDCA caused hypoglycemia as well as tissue remodeling and inflammation in heart and BAT, which we linked to impaired mitochondrial respiration of palmitic acid and increased flux of cardiac glucose. Still, these metabolic changes/adaptions seem not to compromise the vital functions of animals at room temperature, but deteriorates during cold exposure, causing decompensation of systemic energy homeostasis and death of the animals.\u003c/p\u003e\u003cp\u003eTogether, our data indicate that disturbed energy substrate utilization underlies the adverse effects of norUDCA, a notion that is supported by our observation that feeding a low-carb ketogenic diet prevented norUDCA-induced cold intolerance. Ketone bodies are easy to oxidize fuels for peripheral tissues that are produced in the liver from fatty acids released under catabolic conditions by ATGL-mediated lipolysis in the adipose tissues \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Notably, cardiac glucose uptake increased\u0026thinsp;\u0026gt;\u0026thinsp;10-fold during acute cold exposure in mice with reduced adipose tissue lipolysis \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and was associated with pathological remodeling of the heart during chronic cold stress. The relevance of this pathway is highlighted by our observation that mice lacking ATGL in adipose tissues succumb to norUDCA supplementation already at room temperature. In wild type mice housed at room temperature, the higher levels of non-esterified fatty acids and ketone bodies can compensate energy disturbances induced by norUDCA supplementation. Most likely, enhanced lipolysis is stimulated via activation of the Gs-coupled bile acid receptor TGR5 \u003csup\u003e62\u003c/sup\u003e. Although norUDCA is a rather poor ligand for TGR5 \u003csup\u003e63,64\u003c/sup\u003e, it is present in very high concentrations, and furthermore, also other UBAs such as CDCA and DCA are highly elevated.\u003c/p\u003e\u003cp\u003eCareful consideration must be given when discussing the potential clinical relevance of our findings, as there are species differences between mice and humans regarding their cardiac metabolism and their significant differences in BA metabolism. For instance, it remains unclear whether treatment in humans results in norUDCA levels that are similar to those in preclinical models after norUDCA supplementation. In contrast to rodents, norUDCA in humans is efficiently glucuronidated \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and subsequently excreted via the urine, which may limit its plasma accumulation. Nevertheless, norUDCA can reach significant levels in the systemic circulation in humans, as suggested by a pharmacokinetic study in six healthy male volunteers. These probands were treated with a single oral dose of 1500 mg radiolabelled norUDCA and a peak concentration reached a level \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e is similar to those observed in the current mouse study. Thus, norUDCA could impact systemic energy metabolism in humans, in particular under stress conditions. Future studies are mandatory to determine the effects on glucose disposal and energy expenditure in a clinical setting, taking into account that norUDCA treatment only in combination with catabolic stress may alter cardiac performance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnimal models\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the Beh\u0026ouml;rde f\u0026uuml;r Gesundheit und Verbraucherschutz Hamburg (Germany) or by the Austrian Federal Ministry of Education, Science and Research (Austria). All animals were housed with a 12-h light-dark cycle in humidity and temperature-controlled conditions and permitted ad libitum consumption of water and a standard mouse diet. \u003cem\u003eCyp2c70\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eApoA5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and Atgl-MHC mice and the respective control littermates were bred and housed in the animal facility of the UKE or Graz. Mice were randomized based on body weight and fed for 7 days a chow diet (Altromin 1324) or a chow diet supplemented with norUDCA, UDCA, CDCA, DCA, CA, OCA (0.05%) or cholylsacosin at 0.5% (w/w) with ad libitum access to food and water. For antibiotic treatment, neomycin (Sigma, #N6386), bacitracin (Sigma, #11702) and streptomycin (Sigma, #S6501) were diluted in the drinking water, all at a concentration of 1g/l. Organ harvests and metabolic turnover studies were performed after a 4 h fasting period, and the mice were anesthetized with a lethal dose of ketamine and xylazine. Cardiac blood was drawn with syringes containing 0.5 M EDTA. Animals were perfused with PBS containing 10 U/ml heparin, and then the organs were harvested and immediately stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further analysis. Body composition analysis was performed by echoMRI (Zinsser Analytic).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIndirect calorimetry\u003c/h2\u003e\u003cp\u003eIndirect calorimetry was performed using either TSE Phenomaster system (TSE systems) or the PROMETHION systems (Sable Systems) in a temperature- and humidity-controlled chamber. During the experiments, all mice were housed in single cages under a 12 h light: 12 h dark cycle and had ad libitum access to food and water. The animals were fed regular chow with/without 0.5% norUDCA or a low-carb diet (ssniff, E15660) with/without 0.5% norUDCA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGene expression\u003c/h2\u003e\u003cp\u003eTissues were lysed in TRIzol (Ambion, Life Technologies) using a TissueLyser (Qiagen). Nucleic acids were extracted with chloroform, and total RNA was isolated using the RNA purification kit NucleoSpin\u0026reg;RNA II (Macherey \u0026amp; Nagel). RNA concentration was determined with NanoDrop, and 400 ng of RNA was used for reverse transcription into cDNA by using the III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed on a QuantStudio\u0026trade; 5 Real-Time PCR System using the following TaqMan\u0026reg; on-demand primer sets (Invitrogen): \u003cem\u003eAcaca\u003c/em\u003e: Mm01304285_m1, \u003cem\u003eAcly\u003c/em\u003e: Mm00652520_m1, \u003cem\u003eCcl2\u003c/em\u003e: Mm00441242_m1, \u003cem\u003eCcl5\u003c/em\u003e: Mm01302428_m1, \u003cem\u003eCd68\u003c/em\u003e: Mm03047343_m1, \u003cem\u003eCol1a1\u003c/em\u003e: Mm00801666_g1, \u003cem\u003eCpt1\u003c/em\u003e: Mm00550438_m1, \u003cem\u003eCxcl10\u003c/em\u003e: Mm00445235_m1, \u003cem\u003eFasn\u003c/em\u003e: Mm00662319_m1, \u003cem\u003eIl1b\u003c/em\u003e:Mm00434228_m1, \u003cem\u003eLipe\u003c/em\u003e: Mm00495359_m1, \u003cem\u003eLpl\u003c/em\u003e: Mm00434764_m1, \u003cem\u003eMmp12\u003c/em\u003e: Mm00500554_m1, \u003cem\u003eMmp13\u003c/em\u003e: Mm00439491_m1), \u003cem\u003ePdk4\u003c/em\u003e: Mm00443325_m1, \u003cem\u003ePfkfb4\u003c/em\u003e: Mm00557176_m1, \u003cem\u003ePnpla2\u003c/em\u003e: Mm00503040_m1, \u003cem\u003ePpargc1a\u003c/em\u003e: Mm00447183_m1, \u003cem\u003eScd1\u003c/em\u003e: Mm00772290_m1, \u003cem\u003eSlc2a1\u003c/em\u003e (encoding Glut1): Mm00441480_m1, \u003cem\u003eSlc2a4\u003c/em\u003e (encoding Glut4): Mm01245502_m1, \u003cem\u003eTbp\u003c/em\u003e: Mm00446973_m1, \u003cem\u003eTimp1\u003c/em\u003e: Mm00441818_m1, \u003cem\u003eTnfa\u003c/em\u003e: Mm00443258_m1). mRNA levels were normalized to the level of the housekeeping gene TATA-box binding protein (\u003cem\u003eTbp\u003c/em\u003e) mRNA, and the results were displayed as relative gene expression normalized to the experimental control group, following calculations using the 2-ΔΔCt method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing\u003c/h2\u003e\u003cp\u003eFor transcriptomics, RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized utilizing random hexamer primers, followed by the second strand cDNA synthesis. Libraries prepared using Novogene NGS RNA Library Prep Set (PT042) was sequenced on Illumina NovaSeq 6000 platform S4 flow cell. RNA-seq data are aligned using Hisat2 v2.0.5. Reads are assigned to transcripts using featureCounts. Differential expression analysis of the data was performed using edgeR. The P values were adjusted using the Benjamini \u0026amp; Hochberg method. Local version of the gene set enrichment analysis tool \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.broadinstitute.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"http://www.broadinstitute.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e was used, GO, KEGG (Kyoto Encyclopedia of genes and Genomens) and Reactome were used for GSEA independently.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePlasma parameters\u003c/h2\u003e\u003cp\u003ePlasma was isolated by centrifugation of EDTA-spiked blood for 10 min at 10.000 rpm at 4\u0026deg;C in a bench top centrifuge. Glucose was determined in tail blood using Accu-Check Aviva test strips (ROCHE). Non esterified fatty acids (NEFAs) were determined photometrically using the NEFA-HR (2) Assay (FUJIFILM). Ketone bodies were determined using the Autokit Total Ketone Bodies Assay (FUJIFILM). Glycerol was determined calorimetrically using the Free Glycerol Reagent (SIGMA, #F6428) and the Glycerol Standard Solution (SIGMA, #G7793) as reference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRespirometry\u003c/h2\u003e\u003cp\u003eOxygen consumption rate was analyzed in heart mitochondria by high-resolution respirometry using O2k-technology chambers (Oroboros Instruments). Mitochondria were isolated following the \u0026ldquo;Laboratory protocol: isolation of rat heart mitochondria\u0026rdquo; \u003csup\u003e66\u003c/sup\u003e from whole mice hearts. Mice were euthanized by cervical dislocation and whole hearts were isolated using sterile surgical equipment. Hearts were places in ice cold isolation buffer until further processing. Heart tissue was later minced by sterile scissors and further homogenized in pre-cooled Teflon potter in buffer containing protease. Homogenates were left digesting on ice under constant shaking. After digestion with protease, samples were centrifuged to remove the non-homogenized pieces of tissue and supernatant containing mitochondria was further centrifuged until solid pellets of mitochondria were observed. Mitochondrial pellets were re-suspended in the ice-cold isolation buffer. Oxygen consumption was determined in the mitochondria that were pre-incubated without/with 100 \u0026micro;M norUDCA for 30 min in pre-calibrated 2 ml chambers with continuous stirring. Then, sequential addition of palmitoyl-CoA (0.04 mM), ADP (2 mM), malate (0.1 mM and 2 mM), pyruvate (5 mM), glutamate (10 mM), cytochrome C (0.01 mM), oligomycin (5 nM) and finally antimycin A (2.5 \u0026micro;M) to determine substrate specificity and membrane integrity of the mitochondria throughout the experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting\u003c/h2\u003e\u003cp\u003eFor SDS\u0026ndash;PAGE, perfused organs were harvested and homogenized with a TissueLyzer (Qiagen) in 10x excess of (v/w) RIPA buffer (50 mM Tris\u0026ndash;HCl pH 7.4, 5 mM EDTA, 150 mM sodium chloride, 1 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium ortho-vanadate, 1% (NP-40) supplemented with cOmplete mini protease inhibitor cocktail tablets (Roche), and phosphatase inhibitor cocktail (Bimake.com). After centrifugation at 16.000 g for 10 min, the clear soluble middle layer of the lysate was taken, and protein concentration was assessed using the method of Lowry. Then 20 \u0026micro;g of total protein was denatured at 55\u0026deg;C for 10 min in a NuPAGE reducing sample buffer (Invitrogen) and separated on 10% SDS\u0026ndash;polyacrylamide Tris\u0026ndash;glycine gels. Proteins were transferred to nitrocellulose membranes in a wet blotting system. Equal loading was confirmed by Ponceau S (Serva) staining. Subsequently, the membranes were washed twice in TBS-T (20 mM Tris, 150 mM sodium chloride, 0.1% (v/v) Tween 20) and blocked for 1 h in 5% milk powder (Sigma) in TBS-T at room temperature. Primary antibodies were incubated (5% BSA in TBS-T) overnight at 4\u0026deg;C, and secondary antibodies were diluted in 5% milk powder in TBS-T. Detection was performed with enhanced chemiluminescence using an Amersham Imager 600 (GE Healthcare). The following primary antibodies were used: rabbit polyclonal anti-CD36 (1:1000, Novus biologicals, NB400-144), goat polyclonal anti-LPL (1:1000, kind gift from Andre Bensadoun, Cornell University), rat monoclonal anti-GPIHBP1 (1:1000, kind gift from Stephen G. Young, UCLA), mouse monoclonal total OXPHOS WB cocktail (1:500, abcam, ab110413), rabbit monoclonal anti-gamma-Tubulin (1:1000, abcam, ab179503), rabbit monoclonal anti-AKT (1:1000, cell signaling, #9272). The following secondary antibodies (in a dilution of 1:5.000) were used: HRP goat anti-rabbit (Jackson ImmunoResearch Labs, #111-035-144), HRP goat anti-mouse (Jackson ImmunoResearch Labs, #115-035-003), HRP donkey anti-rat (Jackson ImmunoResearch Labs, #712-035-150), and HRP mouse anti-goat (Jackson ImmunoResearch Labs, #205-035-108).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eBile acid measurement\u003c/h2\u003e\u003cp\u003eBile acids were quantified by HPLC coupled to electrospray ionization tandem mass spectrometry as described \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Briefly, plasma or cecal samples were prepared by a methanol liquid-liquid extraction. Quantitative measurement of bile acids was performed using a LC-ESI-QqQ system run multiple reaction monitoring (MRM) mode. HPLC analysis was performed using NEXERA X2 LC-30AD HPLC PUMP (Shimadzu, Tokyo, Japan) equipped with a Kinetex C18 column (100 \u0026Aring;, 150 mm \u0026times; 2.1 mm i.d., Phenomenex, Torrance, CA, USA). For HPLC a mobile phase A consisting of water and a mobile phase B consisting of acetonitrile methanol (3/1 v/v) both enriched with 0.1% formic acid and 20 mM ammonium acetate was used. The column was coupled to QqQ: Q trap 5500 System (SCIEX, Darmstadt, Germany). Peaks were identified and quantified by comparing retention times, as well as MRM transitions and peak areas, respectively, to particular corresponding standard chromatograms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomic enrichment analysis\u003c/h2\u003e\u003cp\u003eFor flux analysis, mice treated with/without norUDCA received a bolus i.p. injection of uniformly labeled \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-glucose (1g/kg body weight). Mice were sacrified 30 min after injection and organs were harvested for metabolomics. Each x mg tissue sample was mixed with five times the \u0026micro;l amount of ice-cold extraction solvent acetonitrile (ACN) with H\u003csub\u003e2\u003c/sub\u003eO (1:1) and homogenized using a TissueLyser II (30 Hz, 10 min; Retsch Qiagen). After centrifugation (4\u0026deg;C, 2 min, 14000 rpm), 250 \u0026micro;l supernatant were mixed with 250 \u0026micro;l ACN: H\u003csub\u003e2\u003c/sub\u003eO for a second extraction step. After vortexing for 1 min and centrifugation (4\u0026deg;C, 10 min, 14000 rpm), the supernatant was transferred to a new tube and evaporated to dryness (SpeedVac, Eppendorf). Extracted metabolites of heart and BAT were analyzed by LC-MS/MS using an adapted method described by Buescher et al., 2010 \u003csup\u003e68\u003c/sup\u003e. Prior to measurement, each sample was re-suspended in 100 \u0026micro;l H\u003csub\u003e2\u003c/sub\u003eO and 10 \u0026micro;l were injected onto an Agilent 1290 II infinity UPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) coupled on-line with a QTRAP\u0026reg; 6500\u0026thinsp;+\u0026thinsp;mass spectrometer (Sciex, Framingham, USA). Chromatographic separation was achieved with a XSelect HSS T3 XP column (2.1 x 150 mm, 2.5 \u0026micro;m, 100 \u0026Aring;; Waters, Milford, MA, USA) connected to an XP VanGuard\u0026reg; cartridge (HSS T3, 2.1 x 5 mm, 2.5 \u0026micro;M; Waters, Milford, MA, USA). Mobile phase A and mobile phase B were 10 mM tributylamine, 10 mM acetic acid, 5% methanol and 2% 2-propanol (pH 7.1) in water and 100% 2-propanol, respectively. Metabolites were eluted with the following non-linear gradient: 0-15.5 min 0.4 mL/min, 15.5\u0026ndash;16.5 min 0.4\u0026thinsp;\u0026minus;\u0026thinsp;0.15 mL/min, 16.5\u0026ndash;23 min 0.15 mL/min, 23\u0026ndash;27 min 0.15\u0026ndash;0.4 mL/min, 27\u0026ndash;33 min 0.4 mL/min. The autosampler was kept at 5\u0026deg;C and the column oven was set to 40\u0026deg;C. For identification and quantitation, a scheduled multiple reaction monitoring (MRM) method in negative mode electrospray ionization was used with specific transitions for every metabolite and isotopologue. Data acquisition was performed using the Analyst\u0026reg; software (v. 1.7.0) and peak integration was done in SciexOS\u0026reg; Software (v. 3.0.0., Sciex). All isotopologue measurement values were corrected for 1.1% of \u003csup\u003e13\u003c/sup\u003eC-natural abundance \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eMetabolic turnover studies\u003c/h2\u003e\u003cp\u003eTo quantify the uptake of radiolabeled bile acids, mice received an oral gavage enriched with \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003eH-norUDCA (74 kBq per mouse) and \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC-CA (37 kBq per mouse). Four hours after gavage, mice were anaesthetized and organs were harvested. To quantify the uptake of energy substrates, mice were i.v. injected with \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC-DOG (7.4 kBq per mouse), \u003csup\u003e3\u003c/sup\u003eH-DOG (14.8 kBq per mouse), albumin-bound \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003eC-oleic acid (7.4 kBq per mouse) or \u003csup\u003e14\u003c/sup\u003eC-hydroxybutyrate (7.4 kBq per mouse). Mice were anaesthetized 15 min after injection, blood was collected by cardiac puncture, perfused with PBS via the left heart ventricle and organs were harvested. In both setups, organs were dissolved in Solvable (Perkin Elmer) for scintillation counting using a Perkin Elmer Tricarb Scintillation Counter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eHistology and immunohistochemistry\u003c/h2\u003e\u003cp\u003eImmunohistochemical stainings were performed on paraffin-embedded tissues using standard procedures. Briefly, liver tissues were fixed in 3.7% formaldehyde in PBS solution and later embedded in paraffin. Stainings were performed using 4 \u0026micro;m sections cut on a Leica microtome and mounted on Histobond slides (Marienfeld-Superior). The following primary antibodies were used in 3% BSA (Sigma): rat monoclonal anti-LY6C (1:200, abcam, ab15627), rabbit monoclonal anti-CK19 (1:200, abcam, ab52625). Horseradish peroxidase (HRP) coupled donkey-anti-rat (Jackson Immunoresearch, #712-036-153) and horseradish peroxidase (HRP) goat anti-rabbit (Jackson ImmunoResearch Labs, #111-035-144) were used as secondary antibodies. After secondary antibody incubation, sections were washed with PBS 3-times for 10 min. Staining was performed using an abcam DAB kit following the manufacturer\u0026rsquo;s instructions. After DAB-staining, the slides were rinsed with PBS to stop the chromogenic reaction and counterstained with hematoxilin for 2 min. Slides were incubated under running tap water for 10 min to achieve bluing of the hematoxilin. Afterwards, the slides were dehydrated and mounted using Eukitt. Images were taken using a NikonA1 Ti microscope equipped with a DS-Fi-U3 brightfield camera.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003elipolysis assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWhite adipose tissue pieces (~\u0026thinsp;25-35mg) were incubated in 500 \u0026micro;l Dulbecco's Modified Eagle Medium (Gibco, #11965092) supplemented with 2% fatty acid-free bovine serum albumin (Sigma, #A8806). After 30 min, basal lipolysis was determined by measuring the released free fatty acids and glycerol in the media, using the NEFA-HR (2) Assay (FUJIFILM) and the Free Glycerol Reagent (SIGMA, #F6428), respectively. Protein content of the individual adipose tissue explant pieces was measured with the Lowry method for normalization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eEngineered heart tissues\u003c/h2\u003e\u003cp\u003eAn established control line of human induced pluripotent stem cells (hiPSC, hiPSCreg code: UKEi001-A, ERC001 XX) was used to differentiate cardiomyocytes as recently described \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Briefly, master/working cell bank hiPSCs aliquots \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e were expanded in FTDA media on Geltrex-coated cell culture vessels \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Embryoid bodies were generated in spinner flasks. Ventricular cardiomyocytes were differentiated in suspension/EB format by growth factor/small molecule cocktails into mesodermal progenitor cells and subsequently into cardiomyocytes. Collagenase-dissociated hiPSC-CM were either cryopreserved or used directly for the generation of engineered heart tissues (EHT). Differentiation efficiency was determined by FACS analysis for troponin T. Fibrin-based strip-format EHTs were generated with 1.0 x 106 hiPSC-CM per construct \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. EHTs were cultivated for approximately 21 days in EHT medium (10% horse serum, 1% penicillin-streptomycin, 33 \u0026micro;g/ml aprotinin, 10 \u0026micro;g/ml insulin, 200 \u0026micro;M tranexamic acid), in 24 well plates. Cell culture media was changed on Mondays, Wednesday and Fridays. Functional assessment was performed by video-optical recording of spontaneous EHT contraction and calculation of force based on deflection during contraction \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. EHT were equilibrated in measurement medium (DMEM, horse serum 2%) overnight. After baseline contractility recording EHTs were incubated in the presence of vehicle (0.9% NaCl) or bile acids. Recording of contractility was performed at 2h, 24h, 48 h, and 72 h of incubation.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eElectron microscopy\u003c/h2\u003e\u003cp\u003eFor electron microscopy mice were sacrificed with a lethal dose of ketamin/xylazine injection anesthesia and perfused with PBS. Organs were cut and directly transferred into fixative (4% PFA, 1% GA in PBS) and stored at 4\u0026deg;C. Then, tissues were dissected with a razor blade and rinsed three times in 0.1 M sodium cacodylate buffer (pH 7.2\u0026ndash;7.4) and osmicated using 1% osmium tetroxide in cacodylate buffer. Following osmication, the samples were dehydrated using ascending ethyl alcohol concentration steps, followed by two rinses in propylene oxide. Infiltration of the embedding medium was performed by immersing the pieces in a 1:1 mixture of propylene oxide and Epon and finally in neat Epon and hardened at 60\u0026deg;C. Semithin sections (0.5 \u0026micro;m) were prepared for light microscopy mounted on glass slides and stained for 1 min with 1% Toluidine blue. Ultrathin sections (60 nm) were cut and mounted on copper grids. Sections were stained using uranyl acetate and lead citrate. Thin sections were examined and photographed using an EM902 (Zeiss) electron microscope.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eStatistical methods\u003c/h2\u003e\u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Comparisons of two groups were examined using Students T-Test. Comparison of three or more groups were analyzed using ANOVA. GraphPad Prism and Microsoft Excel were used for all statistical analyses. The statistical parameters can be found in the figure legends. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eConception: I.E., J.H., T.M. and F.K. Experimental design: G.E., J.H. and T.M. Investigation and Methodology: I.E., E.V., J.K.R., M.V., M.H., A.W., S.G., M.M.F., K.G., D.S., U.R.-K., A.Z., R.B., R.F., J.F.d.B. and A.H. Formal analysis: I.E., E.V., J.K.R., M.V., M.H., A.W., S.G., M.M.F., K.G., D.S., U.R.-K., A.H., T.M., and J.H. Drafting the manuscript: I.E., J.H., L.S., T.M. and F.K. Critical review and discussion: M.V.B., L.S., C.S., A.H. and F.K. Funding acquisition: JH, CS, AW and LS. All authors read and approved the paper.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to JH, CS, AW and LS (SFB-Transregio 333, project-ID: 450149205) and by the Austrian Science Fund FWF, DK-MCD W1226 to AZ and MV.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePerino A, Demagny H, Velazquez-Villegas L, Schoonjans K (2021) Molecular Physiology of Bile Acid Signaling in Health, Disease, and Aging. Physiol Rev 101:683\u0026ndash;731\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSang C et al (2021) Bile Acid Profiles Are Distinct among Patients with Different Etiologies of Chronic Liver Disease. J Proteome Res 20:2340\u0026ndash;2351\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang YL, Li ZJ, Gou HZ, Song XJ, Zhang L (2022) The gut microbiota-bile acid axis: A potential therapeutic target for liver fibrosis. Front Cell Infect Microbiol 12:945368\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia B, Park D, Hahn Y, Jeon CO (2020) Metagenomic analysis of the human microbiome reveals the association between the abundance of gut bile salt hydrolases and host health. Gut Microbes 11:1300\u0026ndash;1313\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVaranasi SK et al (2025) Bile acid synthesis impedes tumor-specific T cell responses during liver cancer. Science 387:192\u0026ndash;201\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHang S et al (2019) Bile acid metabolites control T(H)17 and T(reg) cell differentiation. Nature 576:143\u0026ndash;148\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrampert DC, Kunst RF, van de Graaf (2024) S.F.J. Targeting bile salt homeostasis in biliary diseases. Curr Opin Gastroenterol 40:62\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Boer JF et al (2021) Cholangiopathy and Biliary Fibrosis in Cyp2c70-Deficient Mice Are Fully Reversed by Ursodeoxycholic Acid. Cell Mol Gastroenterol Hepatol 11:1045\u0026ndash;1069\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen Y et al (2022) Ursodeoxycholic acid reduces antitumor immunosuppression by inducing CHIP-mediated TGF-beta degradation. Nat Commun 13:3419\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoshimoto S et al (2013) Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499:97\u0026ndash;101\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoyce SA et al (2014) Regulation of host weight gain and lipid metabolism by bacterial bile acid modification in the gut. Proc Natl Acad Sci U S A 111:7421\u0026ndash;7426\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiba A et al (2020) Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment. Sci Transl Med 12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWerling M et al (2013) Enhanced fasting and post-prandial plasma bile acid responses after Roux-en-Y gastric bypass surgery. Scand J Gastroenterol 48:1257\u0026ndash;1264\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSimonen M et al (2012) Conjugated bile acids associate with altered rates of glucose and lipid oxidation after Roux-en-Y gastric bypass. Obes Surg 22:1473\u0026ndash;1480\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEvangelakos I et al (2021) Oxysterol 7-alpha Hydroxylase (CYP7B1) Attenuates Metabolic-Associated Fatty Liver Disease in Mice at Thermoneutrality. \u003cem\u003eCells\u003c/em\u003e 10\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWorthmann A et al (2017) Cold-induced conversion of cholesterol to bile acids in mice shapes the gut microbiome and promotes adaptive thermogenesis. Nat Med 23:839\u0026ndash;849\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZietak M et al (2016) Altered Microbiota Contributes to Reduced Diet-Induced Obesity upon Cold Exposure. Cell Metab 23:1216\u0026ndash;1223\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaskin AS et al (2018) Regulation of Human Adipose Tissue Activation, Gallbladder Size, and Bile Acid Metabolism by a beta3-Adrenergic Receptor Agonist. Diabetes 67:2113\u0026ndash;2125\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eO'Mara AE et al (2020) Chronic mirabegron treatment increases human brown fat, HDL cholesterol, and insulin sensitivity. J Clin Invest 130:2209\u0026ndash;2219\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeuers U et al (2025) The history and future of bile acid therapies. J Hepatol 83:1172\u0026ndash;1188\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYeh HZ et al (1997) Effect of side chain length on biotransformation, hepatic transport, and choleretic properties of chenodeoxycholyl homologues in the rodent: studies with dinorchenodeoxycholic acid, norchenodeoxycholic acid, and chenodeoxycholic acid. Hepatology 26:374\u0026ndash;385\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFickert P et al (2017) norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis. J Hepatol 67:549\u0026ndash;558\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFickert P et al (2013) Differential effects of norUDCA and UDCA in obstructive cholestasis in mice. J Hepatol 58:1201\u0026ndash;1208\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHalilbasic E et al (2009) Side chain structure determines unique physiologic and therapeutic properties of norursodeoxycholic acid in Mdr2-/- mice. Hepatology 49:1972\u0026ndash;1981\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoustafa T et al (2012) Alterations in lipid metabolism mediate inflammation, fibrosis, and proliferation in a mouse model of chronic cholestatic liver injury. Gastroenterology 142:140\u0026ndash;151e112\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu C et al (2021) 24-Norursodeoxycholic acid reshapes immunometabolism in CD8(+) T cells and alleviates hepatic inflammation. J Hepatol 75:1164\u0026ndash;1176\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu C et al (2025) 24-Nor-ursodeoxycholic acid improves intestinal inflammation by targeting T(H)17 pathogenicity and transdifferentiation. Gut 74:1079\u0026ndash;1093\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Boer JF et al (2020) A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice. J Lipid Res 61:291\u0026ndash;305\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTakahashi S et al (2016) Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans. J Lipid Res 57:2130\u0026ndash;2137\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGurantz D et al (1991) Hypercholeresis induced by unconjugated bile acid infusion correlates with recovery in bile of unconjugated bile acids. Hepatology 13:540\u0026ndash;550\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorrison SF, Nakamura K (2019) Central Mechanisms for Thermoregulation. Annu Rev Physiol 81:285\u0026ndash;308\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGlatz JFC et al (2020) Re-balancing cellular energy substrate metabolism to mend the failing heart. Biochim Biophys Acta Mol Basis Dis 1866:165579\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOut C et al (2015) Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4. J Hepatol 63:697\u0026ndash;704\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDa Dalt L, Cabodevilla AG, Goldberg IJ, Norata GD (2023) Cardiac lipid metabolism, mitochondrial function, and heart failure. Cardiovasc Res 119:1905\u0026ndash;1914\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchoiswohl G et al (2010) Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids. J Lipid Res 51:490\u0026ndash;499\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe M et al (2006) Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation. Nature 439:484\u0026ndash;489\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeodoro JS et al (2014) Enhancement of brown fat thermogenesis using chenodeoxycholic acid in mice. Int J Obes (Lond) 38:1027\u0026ndash;1034\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroeders EP et al (2015) The Bile Acid Chenodeoxycholic Acid Increases Human Brown Adipose Tissue Activity. Cell Metab 22:418\u0026ndash;426\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVelazquez-Villegas LA et al (2018) TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue. Nat Commun 9:245\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEvangelakos I, Heeren J, Verkade E, Kuipers F (2021) Role of bile acids in inflammatory liver diseases. Semin Immunopathol 43:577\u0026ndash;590\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCzuba B, Vessey DA (1982) The effect of bile acid structure on the activity of bile acid-CoA:glycine/taurine-N-acetyltransferase. J Biol Chem 257:8761\u0026ndash;8765\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDawson PA, Lan T, Rao A (2009) Bile acid transporters. J Lipid Res 50:2340\u0026ndash;2357\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrander C et al (2023) 24-Norursodeoxycholic acid ameliorates experimental alcohol-related liver disease and activates hepatic PPARgamma. JHEP Rep 5:100872\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBourgin M et al (2021) Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health. Microorganisms 9\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDonkers JM et al (2019) NTCP deficiency in mice protects against obesity and hepatosteatosis. JCI Insight 5\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerrannini E et al (2015) Increased Bile Acid Synthesis and Deconjugation After Biliopancreatic Diversion. Diabetes 64:3377\u0026ndash;3385\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHata S et al (2003) Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake. Am J Physiol Gastrointest Liver Physiol 285:G829\u0026ndash;839\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesai MS et al (2011) Cardiac structural and functional alterations in infants and children with biliary atresia, listed for liver transplantation. Gastroenterology 141(1272):1264\u0026ndash;1272\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesai MS et al (2010) Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis. Hepatology 51:2097\u0026ndash;2107\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThomas C et al (2009) TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metab 10:167\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRose AJ et al (2011) Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor. Cell Metab 14:123\u0026ndash;130\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou W, Lew B, Choi H, Kim K, Anakk S (2024) Chenodeoxycholic Acid-Loaded Nanoparticles Are Sufficient to Decrease Adipocyte Size by Inducing Mitochondrial Function. Nano Lett 24:1642\u0026ndash;1649\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoubert P (1978) An in vivo investigation of the negative chronotropic effect of cholic acid in the rat. Clin Exp Pharmacol Physiol 5:1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRainer PP et al (2013) Bile acids induce arrhythmias in human atrial myocardium\u0026ndash;implications for altered serum bile acid composition in patients with atrial fibrillation. Heart 99:1685\u0026ndash;1692\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTong DH (2023) Bile acid binds heart failure and inflammation. Nat Metab 5:1260\u0026ndash;1261\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasavan T et al (2018) Heart and bile acids - Clinical consequences of altered bile acid metabolism. Biochim Biophys Acta Mol Basis Dis 1864:1345\u0026ndash;1355\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDesai MS et al (2017) Bile acid excess induces cardiomyopathy and metabolic dysfunctions in the heart. Hepatology 65:189\u0026ndash;201\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZavecz JH, Battarbee HD (2010) The role of lipophilic bile acids in the development of cirrhotic cardiomyopathy. Cardiovasc Toxicol 10:117\u0026ndash;129\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHofmann AF et al (2005) Novel biotransformation and physiological properties of norursodeoxycholic acid in humans. Hepatology 42:1391\u0026ndash;1398\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGrabner GF, Xie H, Schweiger M, Zechner R (2021) Lipolysis: cellular mechanisms for lipid mobilization from fat stores. Nat Metab 3:1445\u0026ndash;1465\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChoi Y et al (2021) Adipose Lipolysis Regulates Cardiac Glucose Uptake and Function in Mice under Cold Stress. Int J Mol Sci 22\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePerino A, Schoonjans K (2022) Metabolic Messengers: bile acids. Nat Metab 4:416\u0026ndash;423\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSato H et al (2008) Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure-activity relationships, and molecular modeling studies. J Med Chem 51:1831\u0026ndash;1841\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarchiano S et al (2022) Beneficial effects of UDCA and norUDCA in a rodent model of steatosis are linked to modulation of GPBAR1/FXR signaling. Biochim Biophys Acta Mol Cell Biol Lipids 1867:159218\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHalibasic ET, Fickert M, Proels P, Hauswald M, Greinwald K, Tewes R (2020) Glucuronidation directs systemically available norursodeoxcholic acid (norUDCA) to the kidney. J Hepatol 73:S468\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSumbalova Z, Krumschnabel. G (2016) Lаbоrаtоry prоtосоl: isоlаtiоn оf rаt hеаrt mitосhоndriа. Mitосhоndriаl Physiоlоgy Nеtwоrk 15:1\u0026ndash;2\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJohn C et al (2014) A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids. J Chromatogr A 1371:184\u0026ndash;195\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBuescher JM, Moco S, Sauer U, Zamboni N (2010) Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82:4403\u0026ndash;4412\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeinrich P et al (2018) Correcting for natural isotope abundance and tracer impurity in MS-, MS/MS- and high-resolution-multiple-tracer-data from stable isotope labeling experiments with IsoCorrectoR. Sci Rep 8:17910\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBreckwoldt K et al (2017) Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc 12:1177\u0026ndash;1197\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShibamiya A et al (2020) Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research. Curr Protoc Stem Cell Biol 55:e127\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrank S, Zhang M, Scholer HR, Greber B (2012) Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions. PLoS ONE 7:e41958\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMannhardt I et al (2016) Human Engineered Heart Tissue: Analysis of Contractile Force. Stem Cell Rep 7:29\u0026ndash;42\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8280795/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8280795/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBile acids (BAs) play an important role in systemic metabolic improvements following bariatric surgery. In this study, we found that orally administered norursodeoxycholic acid (norUDCA), a conjugation-resistant C23 derivative of naturally occurring UDCA, accumulated in peripheral organs including heart and brown adipose tissue (BAT). Moreover, norUDCA decreased systemic levels of endogenous conjugated BAs, while increasing unconjugated BAs. Notably, in addition to beneficial effects in a cholestatic liver disease model, norUDCA also lowered plasma glucose and fat mass in mice, suggesting that this BA derivative could be repurposed for treating obesity-associated cardiometabolic diseases. Metabolic energy expenditure studies, however, revealed that norUDCA-treated mice developed intolerance to cold stress, a phenotype exacerbated in mice lacking adipose ATGL-dependent lipolysis. Transcriptomic and metabolic analyses demonstrated tissue remodeling in heart and BAT that involved pronounced changes in energy substrate utilization, including enhanced cardiac glucose uptake. Importantly, co-administration of a low-carb diet prevented cold stress-induced metabolic deficits. Mechanistic studies in human engineered heart tissue indicated that norUDCA impaired mitochondrial respiration and thereby compromised contractile function. In conclusion, these data suggest that conjugation- resistant BA derivatives like norUDCA impair myocardial and BAT energetics by altering glucose, lipid, and energy metabolism, particularly during catabolic cold stress conditions.\u003c/p\u003e","manuscriptTitle":"The conjugation-resistant bile acid norUDCA cures liver fibrosis but impairs systemic energy metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 10:45:40","doi":"10.21203/rs.3.rs-8280795/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bb0e6a97-827e-4614-8125-357a2c6b38d8","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59331620,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"},{"id":59331621,"name":"Health sciences/Gastroenterology/Gastrointestinal system/Liver/Hepatic portal vein"},{"id":59331622,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic syndrome"},{"id":59331623,"name":"Biological sciences/Chemical biology/Metabolic pathways"}],"tags":[],"updatedAt":"2025-12-30T13:25:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 10:45:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8280795","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8280795","identity":"rs-8280795","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}