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Targeted Disruption of the Nuclear Receptor FXR/BAR Impairs Bile Acid and Lipid Homeostasis
Abstract
Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.
... CYP7A1 is a rate limiting enzyme that catalyzes bile acid synthesis [30], but CYP7A1 was not changed in the Pex16 Alb-Cre mice [30]. As an intracellular bile acid sensor, the nuclear receptor farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis [31]. FXR was almost undetectable in the Pex16 Alb-Cre mice ( Figure 6C), implying that the elevation of serum bile acids is associated with the impaired regulation in FXR on ...
... CYP7A1 is a rate limiting enzyme that catalyzes bile acid synthesis [30], but CYP7A1 was not changed in the Pex16 Alb-Cre mice [30]. As an intracellular bile acid sensor, the nuclear receptor farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis [31]. FXR was almost undetectable in the Pex16 Alb-Cre mice ( Figure 6C), implying that the elevation of serum bile acids is associated with the impaired regulation in FXR on bile acid homeostasis. ...
Obesity results in hepatic fat accumulation, i.e., steatosis. In addition to fat overload, impaired fatty acid β-oxidation also promotes steatosis. Fatty acid β-oxidation takes place in the mitochondria and peroxisomes. Usually, very long-chain and branched-chain fatty acids are the first to be oxidized in peroxisomes, and the resultant short chain fatty acids are further oxidized in the mitochondria. Peroxisome biogenesis is regulated by peroxin 16 (PEX16). In liver-specific PEX16 knockout (Pex16Alb-Cre) mice, hepatocyte peroxisomes were absent, but hepatocytes proliferated, and liver mass was enlarged. These results suggest that normal liver peroxisomes restrain hepatocyte proliferation and liver sizes. After high-fat diet (HFD) feeding, body weights were increased in PEX16 floxed (Pex16fl/fl) mice and adipose-specific PEX16 knockout (Pex16AdipoQ-Cre) mice, but not in the Pex16Alb-Cre mice, suggesting that the development of obesity is regulated by liver PEX16 but not by adipose PEX16. HFD increased liver mass in the Pex16fl/fl mice but somehow reduced the already enlarged liver mass in the Pex16Alb-Cre mice. The basal levels of serum triglyceride, free fatty acids, and cholesterol were decreased, whereas serum bile acids were increased in the Pex16Alb-Cre mice, and HFD-induced steatosis was not observed in the Pex16Alb-Cre mice. These results suggest that normal liver peroxisomes contribute to the development of liver steatosis and obesity.
... Because of active absorption, immune and epithelial cells in the ileum are exposed to high, potentially toxic BA concentrations 9,10 . Ileal enterocytes manage BA toxicity via the nuclear receptor (NR), FXR/Nr1h4, which upon activation by intracellular BAs promotes expression of several adaptive genes, including Fapb6 (to increase BA-binding capacity), OSTα/β (SLC51A/SLC51B; to increase basolateral membrane efflux capacity), and Fgf15 (FGF19 in humans; to restrict hepatic BA synthesis) [11][12][13][14][15][16][17][18] . In parallel, immune cells in the ileal lamina propria leverage an orthogonal NR, CAR/Nr1i3, to activate a 'hepatocyte-like' transcriptional response upon BA exposure that limits oxidative stress and inflammation 19,20 . ...
... The copyright holder for this preprint this version posted https://doi.org/10.1101/2024.02. 16.580658 doi: bioRxiv preprint biotransformation, producing major secondary BAs deoxycholic acid (DCA, from CA) and lithocholic acid (LCA, from CDCA), and utilizing enzymes encoded by the bile acid-induced (bai) operon, a common genetic trait of several commensal genera (e.g., Clostridia, Bacillus) 26 . ...
Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ∼0.3 μmoles/g. Asbt-mediated transport accounts for ∼80% of this pool and amplifies size, whereas passive absorption explains the remaining ∼20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ∼5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.
... CYP7A1 is a rate limiting enzyme to catalyze bile acid synthesis [30], but CYP7A1 was not changed in the Pex16 Alb-Cre mice [30]. As an intracellular bile acid sensor, the nuclear receptor farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis [31]. FXR was almost undetectable in the Pex16 Alb-Cre mice ( Figure 6C), implying that the elevation of serum bile acids is associated with the impaired regulation of FXR on bile acid homeostasis. ...
Obesity results in hepatic fat accumulation, i.e., steatosis. Besides the fat overload, impaired fatty acid β-oxidation also promotes steatosis. Fatty acid β-oxidation takes place in mitochondria and peroxisomes. Usually very long chain and branched-chain fatty acids are first oxidized in peroxisomes, and the resultant short chain fatty acids are further oxidized in mitochondria. Peroxisome biogenesis is regulated by peroxin 16 (PEX16). In liver-specific PEX16 knockout (Pex16Alb-Cre) mice, hepatocyte peroxisomes were absent, but hepatocytes proliferated, and liver mass was enlarged. These results suggest that normal liver peroxisomes restrain hepatocyte proliferation and liver sizes. After high fat diet (HFD) feeding, body weights were increased in PEX16 floxed (Pex16fl/fl) mice and adipose-specific PEX16 knockout (Pex16AdipoQ-Cre) mice but not in the Pex16Alb-Cre mice, suggesting that the development of obesity is regulated by liver PEX16 but not by adipose PEX16. However, it was previously reported that HFD-induced obesity in the Pex16AdipoQ-Cre mice was more severe than in the wild type mice, which was not observed until 27 weeks of HFD feeding. Thus, liver initiates the development of obesity, which is enhanced by abnormal adipocytes. HFD increased liver mass in the Pex16fl/fl mice but somehow reduced the already enlarged liver mass in the Pex16Alb-Cre mice. Basal levels of serum triglyceride, free fatty acids and cholesterol were decreased whereas serum bile acids were increased in the Pex16Alb-Cre mice, and HFD-induced steatosis was not observed in the Pex16Alb-Cre mice. These results suggest that normal liver peroxisomes contribute to the development of liver steatosis and obesity.
... Loss of Fxr in the MHOs abolished the gene expression changes induced by GW4064, but not those induced by FGF19 (Fig. 5B-E), indicating that FGF19 can act via FXR-independent pathways to regulate bile acid metabolism. Additionally, consistent with previous findings in vivo (Sinal et al., 2000), the Fxr −/− MHOs, but not the Fxr −/− fetal-like HOs, showed lipid accumulation and elevated expression of genes involved in lipid synthesis (Fig. 5F,G). These findings show that the MHOs we have generated in vitro could complement or even replace the in vivo mouse models for genetic and pharmacological assays in bile acid metabolism studies. ...
Hepatocyte organoids (HOs) generated in vitro recently are powerful tools for liver regeneration. However, the reported HOs were mostly fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studying metabolic regulations and therapeutic testing for liver disorders. We report development of novel culture conditions that contains optimized levels of Triiodothyronine (T3) with the removal of growth factors, enabled successful generation of mature hepatocyte organoids (MHOs) with metabolic functions characteristic of adult livers of both mouse and human origins. We showed that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic and non-alcoholic fatty liver diseases as well hepatocyte proliferation, injury, and cell fate changes. Notably, the MHOs derived from human fetal hepatoblasts also showed improved hepatitis B virus (HBV) infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially a robust research tools for therapeutic development as well.
... ICC confirmed its location to the nucleus of HLCs while in protocol 1 only unspecific background staining was detected ( Figure 2B). FXR is a nuclear hormone receptor, involved in liver metabolism (Repa and Mangelsdorf, 2000;Sinal et al., 2000). Expression of FXR in iPSC-derived HLCs was recently found in vitro to be associated with increased maturity as observed in PHHs (Nell et al., 2022). ...
The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases, their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients, enabling the study of disease-associated mutations and, when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately, the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study, we optimized our previously published protocol by fine-tuning the initial cell number, exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple, cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB, HNF4α, and CYP3A4) and protein (ALB) expression, as well as significantly elevated inducible CYP3A4 activity.
... In fact, our previous study revealed a significant increase in gene expression of Fgf15 in the distal ileum of sCSDS mice on day 13 after an sCSDS period (days 1-10) [11]. Feedback inhibition of Cyp7a1 transcription by FXR is one of the most important mechanisms in bile acid homeostasis [21]. Furthermore, FXR negatively regulates Slc10a2/ASBT, the main bile acid transport system in ileal enterocytes [22]. ...
The gut microbiota plays a crucial role in both the pathogenesis and alleviation of host depression by modulating the brain-gut axis. We have developed a murine model of human depression called the subchronic and mild social defeat stress (sCSDS) model, which impacts not only behavior but also the host gut microbiota and gut metabolites, including bile acids. In this study, we utilized liquid chromatography/mass spectrometry (LC/MS) to explore the effects of sCSDS on the mouse fecal bile acid profile. sCSDS mice exhibited significantly elevated levels of deoxycholic acid (DCA) and lithocholic acid (LCA) in fecal extracts, leading to a notable increase in total bile acids and 7α-dehydroxylated secondary bile acids. Consequently, a noteworthy negative correlation was identified between the abundances of DCA and LCA and the social interaction score, an indicator of susceptibility in stressed mice. Furthermore, analysis of the colonic microbiome unveiled a negative correlation between the abundance of CDCA and Turicibacter. Additionally, DCA and LCA exhibited positive correlations with Oscillospiraceae and Lachnospiraceae but negative correlations with the Eubacterium coprostanoligenes group. These findings suggest that sCSDS impacts the bidirectional interaction between the gut microbiota and bile acids and is associated with reduced social interaction, a behavioral indicator of susceptibility in stressed mice.
... All experimental procedures followed the Guide for the Care and Use of Experimental Animals and were approved by the Animal Care and Use Committee of West China Hospital, Sichuan University (20231127008). Male 6-week-old Fxr-null mice (C57BL/6 J background) were cared for as previously described [52]. Male 6-week-old C57BL/6 mice (Gempharmatech Co., Ltd., Nanjing, Jiangsu, China) weighing 20-25 g were maintained under a standard 12 h light/12 h dark cycle environment with free access to water and rodent chow (Double lion experimental animal feed technology Co., Ltd., Suzhou, China). ...
Aspirin is a non-steroidal, anti-inflammatory drug often used long term. However, long-term or large doses will cause gastrointestinal adverse reactions. To explore the mechanism of intestinal damage, we used non-targeted metabolomics; farnesoid X receptor (FXR) knockout mice, which were compared with wild-type mice; FXR agonists obeticholic acid (OCA) and chenodeoxycholic acid (CDCA); and endothelin-producing inhibitor estradiol to explore the mechanisms of acute and chronic intestinal injuries induced by aspirin from the perspective of molecular biology. Changes were found in the bile acids taurocholate acid (TCA) and tauro-β-muricholic acid (T-β-MCA) in the duodenum, and we detected a significant inhibition of FXR target genes. After additional administration of the FXR agonists OCA and CDCA, duodenal villus damage and inflammation were effectively improved. The results in the FXR knockout mice and wild-type mice showed that the overexpression of endothelin 1 (ET-1) was independent of FXR regulation after aspirin exposure, whereas CDCA was able to restore the activation of ET-1, which was induced by aspirin in wild-type mice in an FXR-dependent manner. The inhibition of ET-1 production could also effectively protect against small bowel damage. Therefore, the study revealed the key roles of the FXR and ET-1 pathways in acute and chronic aspirin-induced intestinal injuries, as well as strategies on alleviating aspirin-induced gastrointestinal injury by activating FXR and inhibiting ET-1 overexpression.
... Progressive familial intrahepatic cholestasis (PFIC) is a rare group of genetic disorders that affect the liver's ability to excrete bile constituents, resulting in impaired bile flow, subsequent intrahepatic cholestasis, and progressive liver damage and failure (1,2). Farnesoid X receptor (FXR), encoded by the NR1H4 gene, is a nuclear receptor (NR) responsive to bile acids (BA) and a key regulator of BA metabolism, playing a pivotal role in maintaining BA homeostasis by controlling BA synthesis, transport, and detoxification (3,4). ...
Nuclear receptor farnesoid X receptor (FXR) acts as a key regulator of bile acid pool homeostasis and metabolism. Within the enterohepatic circulation, reabsorbed bile acids act as agonists on FXR, which transcriptionally controls the synthesis and transport of bile acids. Binding occurs in the ligand binding domain (LBD), favoring a conformational change to the active state in which helix 12 interacts with the LBD to form an interaction surface for nuclear co-activators. The homozygous missense variant T296I, identified in a PFIC5 patient, is located close to the critical helix 12 interaction. Here, we identified reduced transcriptional activity of the variant protein on the downstream targets BSEP and SHP in vitro and within the patient's liver. Analysis of the structural dynamics of the conformational change from an inactive to an active state of the FXR LBD with molecular dynamics simulations revealed that while the wildtype protein frequently transitions into the active state, this movement and the necessary perfect placement of helix 12 was significantly impeded in the T296I mutated protein. To our knowledge, this is the first study to describe the conformational change from an inactive to an active state of the FXR LBD. This might be useful for new therapeutic approaches targeting the activation of FXR. Overall, combining in vivo data with in vitro and in silico experiments, we suggest a molecular mechanism underlying the PFIC phenotype of a patient with an FXR missense variant.
... Furthermore, the activation of hepatic FXR directly impacts the upregulation of the Abcb11 gene, which is responsible for encoding the BSEP (Sun, 2021). Sinal et al. demonstrated that fortifying the diet with 1 % cholic acid increased mortality in Fxr knock-out mice by approximately 30 % on day seven; Serum bile acid levels in fxr KO mice were 23-fold higher than in wild-type mice, which may be attributed to the impaired secretion of bile acids, resulting in the accumulation of bile acids within hepatocytes (Sinal et al., 2000). Another study demonstrated animals show FXR− /− mice exhibit rapid supersaturation of bile with cholesterol, cholesterol crystal precipitation in the gallbladder, increased hydrophobicity of bile salts, and gallbladder inflammation (Moschetta et al., 2004). ...
Cholestasis is a hepatobiliary condition that manifests as acute or chronic and results from disruptions in the bile flow, formation, or secretion processes. The Farnesoid X receptor (FXR) is a vital target for the therapy of cholestasis since it regulates BA homeostasis. Despite the discovery of multiple active FXR agonists, there are still no effective treatments for cholestasis. Papaverine is identified as an FXR agonist.This study investigates papaverine's efficacy and probable mechanism in protecting against alpha naphthylisothiocyanate (ANIT) induced cholestasis. Thirty male albino rats were divided into three groups, each with ten rats. Group I (control) rats were administered 1 mL/kg corn oil 48 h before sacrifice; group II rats were orally administered 100 mg/kg ANIT. Group III received a 200 mg/kg dosage of papaverine over seven consecutive days. A single dose of ANIT at a concentration of 100 mg/kg was orally administered on the fifth day; group II and III animals were euthanized 48 h after inducing cholestasis, and serum concentrations of liver function tests and total bile acid (TBA) were measured. Besides measuring the inflammatory mediator's tumor necrosis factor-alpha (TNF-α) and interleukin 1 (IL-1β), antioxidant markers such as superoxide dismutase (SOD) and glutathione (GSH) were also assessed. The findings indicated the enhancement in the liver function test and total bile acids, as well as in liver histology; papaverine significantly lowered TNF-α and IL-1β while SOD and GSH significantly increased. Additionally, papaverine upregulates Fxr gene expression, bile salt export pump (Besp), small heterodimer partner (shp), hepatocyte nuclear factor 1α (Hnfα), nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase (Ho-1), NAD(P)H quinone oxidoreductase 1 (Nqo1). Furthermore, papaverine increased protein expressions of Sirtuin1.
(SIRT 1), FXR, HO-1, and BSEP levels in the rats' livers. The protective effects of papaverine may be attributed to the activation of FXR signaling pathways. These findings revealed that papaverine protects against ANIT-induced Cholestasis.
... The eFGF19-mediated gut-liver signaling axis, which involves enteric FXR engagement and hepatic FGFR4-KLB machinery, is vital as "cholestat" for maintaining a normal negative feedback control mechanism on BA content. These pathways are key to tightly regulating BA homeostasis and preventing BA overshoot, as demonstrated in all Fgf15-, Fgfr4-, Klb-, or Fxr-deficient mouse models [1,17,52,53]. Furthermore, circulating FGF19 levels are increased in patients with PBC and primary sclerosing cholangitis (PSC), along with hepatic BA levels. ...
Human fibroblast growth factor 19 (FGF19, or FGF15 in rodents) plays a central role in controlling bile acid (BA) synthesis through a negative feedback mechanism. This process involves a postprandial crosstalk between the BA‐activated ileal farnesoid X receptor and the hepatic Klotho beta (KLB) coreceptor complexed with fibrobalst growth factor receptor 4 (FGFR4) kinase. Additionally, FGF19 regulates glucose, lipid, and energy metabolism by coordinating responses from functional KLB and FGFR1‐3 receptor complexes on the periphery. Pharmacologically, native FGF19 or its analogs decrease elevated BA levels, fat content, and collateral tissue damage. This makes them effective in treating both cholestatic diseases such as primary biliary or sclerosing cholangitis (PBC or PSC) and metabolic abnormalities such as nonalcoholic steatohepatitis (NASH). However, chronic administration of FGF19 drives oncogenesis in mice by activating the FGFR4‐dependent mitogenic or hepatic regenerative pathway, which could be a concern in humans. Agents that block FGF19 or FGFR4 signaling have shown great potency in preventing FGF19‐responsive hepatocellular carcinoma (HCC) development in animal models. Recent phase 1/2 clinical trials have demonstrated promising results for several FGF19‐based agents in selectively treating patients with PBC, PSC, NASH, or HCC. This review aims to provide an update on the clinical development of both analogs and antagonists targeting the FGF19–FGFR4 signaling pathway for patients with cholestatic, metabolic, and cancer diseases. We will also analyze potential safety and mechanistic concerns that should guide future research and advanced trials.
... In mice, an impaired synergistic relationship within the gut microbiota can lead to gut dysbiosis and reduce the activation of important receptors such as membrane G proteincoupled receptor TGR5 and nuclear bile acid receptor FXR. Such impairment can lead to a decrease in secondary bile acids synthesis, followed by the retention of bile salt, bacterial overgrowth, and liver disease that may potentially progress to liver failure [57,58]. One example would be hepatic encephalopathy, a typical disease model of a dysregulated gutliver-brain axis. ...
This review presents a new perspective on the exacerbation of autism spectrum disorder (ASD) by per- and polyfluoroalkyl substances (PFAS) through the gut–liver–brain axis. We have summarized evidence reported on the involvement of the gut microbiome and liver inflammation that led to the onset and exacerbation of ASD symptoms. As PFAS are toxicants that particularly target liver, this review has comprehensively explored the possible interaction between PFAS and acetaminophen, another liver toxicant, as the chemicals of interest for future toxicology research. Our hypothesis is that, at acute dosages, acetaminophen has the ability to aggravate the impaired conditions of the PFAS-exposed liver, which would further exacerbate neurological symptoms such as lack of social communication and interest, and repetitive behaviors using mechanisms related to the gut–liver–brain axis. This review discusses their potential interactions in terms of the gut–liver–brain axis and signaling pathways that may contribute to neurological diseases.
... Bile acids, cholesterol, triglycerides, and proatherogenic serum lipoproteins are all increased in Fxr-deficient mice. 52 The major bile acid cholate is produced by the sterol 12-hydroxylase CYP8B1. 53 Involved in the production of oxysterol and the oxidation of the sterol side-chain, CYP27A1 functions as a sterol 27-/26-hydroxylase. ...
... In the liver, BAs, such as CDCA, DCA, and CA, activate FXR and further induce the protein expression of the small heterodimer partner (SHP), thereby suppressing the protein expression of SREBP-1c and leading to a decrease in hepatic TG, plasma TG, and plasma lipoproteins [12,[27][28][29]. Consistent with previous reports, our results showed that the protein expression levels of SHP and FXR were increased in the liver in the CP group compared to the CON group, while the protein expression levels of ApoA1 and SREBP1c were decreased in the CP group. ...
Coprophagy prevention (CP) affects the growth performance, hepatic lipid synthesis, and gut microbiota in rabbits. Supplementation with Clostridium butyricum (C. butyricum, Strain number: CCTCC M 2019962) has been found to improve growth performance in rabbits. However, it remains unknown whether C. butyricum can ameliorate the effects of CP on hepatic lipid synthesis and the underlying mechanisms are yet to be elucidated. Therefore, this study aimed to investigate the impact of CP on hepatic lipid synthesis and the underlying mechanism based on the gut–liver axis. The findings revealed that supplementation with C. butyricum could reverse CP-related growth performance, lipid accumulation, bile acid synthesis, and inflammation. Furthermore, C. butyricum exerted protective effects on the gut by preserving intestinal barrier integrity and modulating gut microbiota composition; these factors may represent potential mechanisms through which C. butyricum improves CP-related outcomes. Specifically, C. butyricum reshaped the microbiota by increasing butyric acid levels, thereby maintaining secondary bile acid (deoxycholic acid, chenodeoxycholic acid) balance and attenuating the inhibitory effects of the FXR/SHP pathway on lipid synthesis (SREBP1c/ApoA1). Moreover, the activation of butyrate/GPR43pathway by C. butyricum reduced damage to the intestinal barrier (ZO-1/Occludin/Claudin1) and restored the gut immune microenvironment in CP rabbits. In summary, supplementation with C. butyricum can alleviate the adverse effects of CP on growth performance and hepatic lipid synthesis by modulating the gut–liver axis.
... Overexpression of RORα reduces diet-induced hepatic lipid accumulation, and its inverse agonist SR1001 regulates intestinal excretion of cholesterol by upregulating ABCG5/G8 [82,83] NR1F3 RORγ Sterols Knockout of RORγ may reduce bile acid synthesis by decreasing levels of Cyp8b1, Cyp7b1, and Cyp27a1 [84] NR1H4 FXRα Bile acids FXRα can promote CYP7A1 expression through FGF19 and SHP/LRH-1 pathway, competitively inhibit LXRα to promote CETP transcription and reduce liver cholesterol uptake and accumulation [85][86][87] NR1H3 LXRα Oxysterols LXRα activates ABCG5/8 to promote the excretion of cholesterol in bile. LXRα can promote cholesterol effection in gallbladder epithelium by inducing the expression of ABCA1. ...
As the most prevalent chronic liver disease globally, NAFLD encompasses a pathological process that ranges from simple steatosis to NASH, fibrosis, cirrhosis, and HCC, closely associated with numerous extrahepatic diseases. While the initial etiology was believed to be hepatocyte injury caused by lipid toxicity from accumulated triglycerides, recent studies suggest that an imbalance of cholesterol homeostasis is of greater significance. The role of nuclear receptors in regulating liver cholesterol homeostasis has been demonstrated to be crucial. This review summarizes the roles and regulatory mechanisms of nuclear receptors in the 3 main aspects of cholesterol production, excretion, and storage in the liver, as well as their cross talk in reverse cholesterol transport. It is hoped that this review will offer new insights and theoretical foundations for the study of the pathogenesis and progression of NAFLD and provide new research directions for extrahepatic diseases associated with NAFLD.
... Furthermore, they can regulate glucose and lipid metabolism in the liver [17]. An increasing amount of experimental and clinical evidence indicates that BAs hold exceptional potential as a therapeutic approach for fatty liver disease, hypercholesterolemia, and metabolic diseases [18][19][20]. BA receptors are drawing attention as potential therapeutic targets for liver illnesses because BAs perform their numerous biological actions by attaching to their receptors [21]. More research has revealed that modifications to the gut microbiota affect the host's BA profiles, most notably in the way taurine-conjugated BAs interact with the intestinal farnesol receptor (FXR) [22]. ...
Berberine (BBR) is a natural alkaloid with multiple biotical effects that has potential as a treatment for fatty liver hemorrhagic syndrome (FLHS). However, the mechanism underlying the protective effect of BBR against FLHS remains unclear. The present study aimed to investigate the effect of BBR on FLHS induced by a high-energy, low-protein (HELP) diet and explore the involvement of the gut microbiota and bile acid metabolism in the protective effects. A total of 90 healthy 140-day-old Hy-line laying hens were randomly divided into three groups, including a control group (fed a basic diet), a HELP group (fed a HELP diet), and a HELP+BBR group (high-energy, high-protein diet supplemented with BBR instead of maize). Our results show that BBR supplementation alleviated liver injury and hepatic steatosis in laying hens. Moreover, BBR supplementation could significantly regulate the gut’s microbial composition, increasing the abundance of Actinobacteria and Romboutsia. In addition, the BBR supplement altered the profile of bile acid. Furthermore, the gut microbiota participates in bile acid metabolism, especially taurochenodeoxycholic acid and α-muricholic acid. BBR supplementation could regulate the expression of genes and proteins related to glucose metabolism, lipid synthesis (FAS, SREBP-1c), and bile acid synthesis (FXR, CYP27a1). Collectively, our findings demonstrate that BBR might be a potential feed additive for preventing FLHS by regulating the gut microbiota and bile acid metabolism.
... FXR, which is highly expressed in the liver and gastrointestinal tract, can be conjugated by bile acids, the highest affinity of which is chenodeoxycholic acid (CDCA). 13 Modulation of inflammatory responses in myeloid immune cells by FXR agonists attenuates multiple sclerosis and experimental autoimmune encephalomyelitis in mice. 14,15 The traditional view holds that biliary diseases cause bile reflux, and that bile acids, the main constituents of bile, play an important role in the pathogenesis of AP. 16 However, growing research on the properties of bile acids as signaling molecules in recent years has led to a new understanding of the pathophysiological mechanisms underlying AP. ...
Bile acids are altered and associated with prognosis in patients with acute pancreatitis (AP). Here, we conduct targeted metabolomic analyses to detect bile acids changes in patients during the acute (n = 326) and the recovery (n = 133) phases of AP, as well as in healthy controls (n = 60). Chenodeoxycholic acid (CDCA) decreases in the acute phase, increases in the recovery phase, and is associated with pancreatic necrosis. CDCA and its derivative obeticholic acid exhibit a protective effect against acinar cell injury in vitro and pancreatic necrosis in murine models, and RNA sequencing reveals that the oxidative phosphorylation pathway is mainly involved. Moreover, we find that overexpression of farnesoid X receptor (FXR, CDCA receptor) inhibits pancreatic necrosis, and interfering expression of FXR exhibits an opposite phenotype in mice. Our results possibly suggest that targeting CDCA is a potential strategy for the treatment of acinar cell necrosis in AP, but further verification is needed.
... 18 In FXR-/-mice, higher levels of triglycerides and cholesterol were observed and supplementation with a selective agonist for FXR reversed hyperlipemia. 29 FXR agonists might decrease lipid synthesis in the liver and lipid absorption in the intestine, which could be a latent therapy for nonalcoholic fatty liver disease. 30 The process of autophagy was proven to be an essential component in lipid metabolism, especially under conditions of nutrient deprivation and/or starvation. ...
Bile acids play a crucial role in promoting intestinal nutrient absorption and biliary cholesterol excretion, thereby protecting the liver from cholesterol accumulation and bile acid toxicity. Additionally, bile acids can bind to specific nuclear and membrane receptors to regulate energy expenditure and specific functions of particular tissues. Vascular calcification refers to the pathological process of calcium-phosphate deposition in blood vessel walls, which serves as an independent predictor for cardiovascular adverse events. In addition to aging, this pathological change is associated with aging-related diseases such as atherosclerosis, hypertension, chronic kidney disease, diabetes mellitus, and osteoporosis. Emerging evidence suggests a close association between the bile acid network and these aforementioned vascular calcification-associated conditions. Several bile acids have been proven to participate in calcium-phosphate metabolism, affecting the transdifferentiation of vascular smooth muscle cells and thus influencing vascular calcification. Targeting the bile acid network shows potential for ameliorating these diseases and their concomitant vascular calcification by regulating pathways such as energy metabolism, inflammatory response, oxidative stress, and cell differentiation. Here, we present a summary of the metabolism and functions of the bile acid network and aim to provide insights into the current research on the profound connections between the bile acid network and these vascular calcification-associated diseases, as well as the therapeutic potential.
... 93 Nuclear bile acid receptor farnesoid X receptor deficient mice showed increased hepatic cholesterol and triglyceride content by suppressing NF-κB and regulating gluconeogenesis via phosphoenolpyruvate carboxykinase. 94 In addition, changes to the microbiome also change to metabolism of food substrates, such as increased production of certain short-chain fatty acids (SCFAs) and depletion of choline. SCFAs, including acetic, propionic, and butyric acid, further affect energy metabolism, immunity, and adipose tissue expansion. ...
The present systematic review and meta-analysis aimed to summarize the associations between gut microbiota composition and non-alcoholic fatty liver disease. To compare the differences between individuals with or without NAFLD, the standardized mean difference and 95% confidence interval were computed for each α-diversity index and relative abundance of gut microbes. The β-diversity indices were summarized in a qualitative manner. A total of 54 studies with 8894 participants were included. Overall, patients with NAFLD had moderate reduction in α-diversity indices including Shannon (SMD = À0.36, 95% CI = [À0.53, À0.19], p < 0.001) and Chao 1 (SMD = À0.42, 95% CI = [À0.68, À0.17], p = 0.001), but no significant differences were found for Simpson, observed species, phylogenetic diversity , richness, abundance-based coverage estimator, and evenness (p ranged from 0.081 to 0.953). Over 75% of the included studies reported significant differences in β-diversity. Although there was substantial interstudy heterogeneity, especially for analyses at the phylum, class, and family levels, the majority of the included studies showed alterations in the depletion of anti-inflammatory microbes (i.e., Ruminococcaceae and Coprococcus) and the enrichment of proinflammatory microbes (i.e., Fusobacterium and Escherichia) in patients with NAFLD. Perturbations in gut microbiota were associated with NAFLD, commonly reflected by a reduction in beneficial species and an increase in the pathogenic species.
Bile acids, once considered mere dietary surfactants, now emerge as critical modulators of macronutrient (lipid, carbohydrate, protein) metabolism and the systemic pro-inflammatory/anti-inflammatory balance. Bile acid metabolism and signaling pathways play a crucial role in protecting against, or if aberrant, inducing cardiometabolic, inflammatory, and neoplastic conditions, strongly influencing health and disease. No curative treatment exists for any bile acid influenced disease, while the most promising and well-developed bile acid therapeutic was recently rejected by the FDA. Here, we provide a bottom-up approach on bile acids, mechanistically explaining their biochemistry, physiology, and pharmacology at canonical and non-canonical receptors. Using this mechanistic model of bile acids, we explain how abnormal bile acid physiology drives disease pathogenesis, emphasizing how ceramide synthesis may serve as a unifying pathogenic feature for cardiometabolic diseases. We provide an in-depth summary on pre-existing bile acid receptor modulators, explain their shortcomings, and propose solutions for how they may be remedied. Lastly, we rationalize novel targets for further translational drug discovery and provide future perspectives. Rather than dismissing bile acid therapeutics due to recent setbacks, we believe that there is immense clinical potential and a high likelihood for the future success of bile acid therapeutics.
High temperature and humidity in the environment are known to be associated with discomfort and disease, yet the underlying mechanisms remain unclear. We observed a decrease in plasma glucagon-like peptide-1 levels in response to high-temperature and humidity conditions. Through 16S rRNA gene sequencing, alterations in the gut microbiota composition were identified following exposure to high temperature and humidity conditions. Notably, changes in the gut microbiota have been implicated in bile acid synthesis. Further analysis revealed a decrease in lithocholic acid levels in high-temperature and humidity conditions. Subsequent in vitro experiments demonstrated that lithocholic acid increases glucagon-like peptide-1 secretion in NCI-H716 cells. Proteomic analysis indicated upregulation of farnesoid X receptor expression in the ileum. In vitro experiments revealed that the combination of lithocholic acid with farnesoid X receptor inhibitors resulted in a significant increase in GLP-1 levels compared to lithocholic acid alone. In this study, we elucidate the mechanism by which reduced lithocholic acid suppresses glucagon-like peptide 1 via farnesoid X receptor activation under high-temperature and humidity condition.
Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as nonalcoholic fatty liver disease, is becoming a significant contributor to chronic liver disease globally, surpassing other etiologies, such as viral hepatitis. Prevention and early treatment strategies to curb its growing prevalence are urgently required. Recent evidence suggests that targeting the gut microbiota may help treat and alleviate disease progression in patients with MAFLD. This review aims to explore the complex relationship between MAFLD and the gut microbiota in relation to disease pathogenesis. Additionally, it delves into the therapeutic strategies targeting the gut microbiota, such as diet, exercise, antibiotics, probiotics, synbiotics, glucagon-like peptide-1 receptor agonists, and fecal microbiota transplantation, and discusses novel biomarkers, such as microbiota-derived testing and liquid biopsy, for their diagnostic and staging potential. Overall, the review emphasizes the urgent need for preventive and therapeutic strategies to address the devastating consequences of MAFLD at both individual and societal levels and recognizes that further exploration of the gut microbiota may open avenues for managing MAFLD effectively in the future.
Innate lymphoid cells (ILCs) can promote host defense, chronic inflammation, or tissue protection and are regulated by cytokines and neuropeptides. However, their regulation by diet and microbiota-derived signals remains unclear. We show that an inulin fiber diet promotes Tph1-expressing inflammatory ILC2s (ILC2INFLAM) in the colon, which produce IL-5 but not tissue-protective amphiregulin (AREG), resulting in the accumulation of eosinophils. This exacerbates inflammation in a murine model of intestinal damage and inflammation in an ILC2- and eosinophil-dependent manner. Mechanistically, the inulin fiber diet elevated microbiota-derived bile acids, including cholic acid (CA) that induced expression of ILC2-activating IL-33. In IBD patients, bile acids, their receptor farnesoid X receptor (FXR), IL-33, and eosinophils were all upregulated compared with controls, implicating this diet–microbiota–ILC2 axis in human IBD pathogenesis. Together, these data reveal that dietary fiber–induced changes in microbial metabolites operate as a rheostat that governs protective versus pathologic ILC2 responses with relevance to precision nutrition for inflammatory diseases.
Nonalcoholic fatty liver disease (NAFLD) is a global health concern with a high prevalence and increasing economic burden, but official medicine remains unavailable. Farnesoid X receptor (FXR), a nuclear receptor member, is one of the most promising drug targets for NAFLD therapy that plays a crucial role in modulating bile acid, glucose, and lipid homeostasis, as well as inhibits hepatic inflammation and fibrosis. However, the rejection of the FXR agonist, obecholic acid, by the Food and Drug Administration for treating hepatic fibrosis raises a question about the functions of FXR in NAFLD progression and the therapeutic strategy to be used. Natural products, such as FXR modulators, have become the focus of attention for NAFLD therapy with fewer adverse reactions. The anti-NAFLD mechanisms seem to act as FXR agonists and antagonists or are involved in the FXR signaling pathway activation, indicating a promising target of FXR therapeutic prospects using natural products. This review discusses the effective mechanisms of FXR in NAFLD alleviation, and summarizes currently available natural products such as silymarin, glycyrrhizin, cycloastragenol, berberine, and gypenosides, for targeting FXR, which can facilitate development of naturally targeted drug by medicinal specialists for effective treatment of NAFLD.
Although the medicinal properties of colchicine (COL) have been widely known for centuries, its toxicity has been the subject of controversy. The narrow therapeutic window causes COL to induce gastrointestinal adverse effects even when taken at recommended doses, mainly manifested as nausea, vomiting, and diarrhea. However, the mechanism of COL-induced gastrointestinal toxic reactions remains obscure. In the present study, the mice were dosed with COL (2.5 mg/kg b.w./day) for a week to explore the effect of COL on bile acid metabolism and the mechanism of COL-induced diarrhea. The results showed that COL treatment affected liver biochemistry in mice, resulting in a significant down-regulation of the mRNA expression levels of bile acid biosynthesis regulators Cyp7a1, Cyp8b1, Cyp7b1, and Cyp27a1 in liver tissues. The mRNA expression levels of bile acid transporters Ntcp, Oatp1, Mrp2, Ibabp, Mrp3, Osta, and Ostb in liver and ileum tissues were also significantly down-regulated. In addition, COL treatment significantly inhibited the mRNA expression levels of Fxr and its downstream target genes Shp, Lrh1, and Fgf15 in liver and ileum tissues, affecting the feedback regulation of bile acid biosynthesis. More importantly, the inhibition of COL on bile acid transporters in ileal and hepatic tissues affected bile acid recycling in the ileum as well as their reuptake in the liver, leading to a significantly increased accumulation of bile acids in the colon, which may be an important cause of diarrhea. In conclusion, our study revealed that COL treatment affected bile acid biosynthesis and enterohepatic circulation, thereby disrupting bile acid metabolic homeostasis in mice.
Obesity, a chronic condition marked by the excessive accumulation of adipose tissue, not only affects individual well-being but also significantly inflates healthcare costs. The physiological excess of fat manifests as triglyceride (TG) deposition within adipose tissue, with white adipose tissue (WAT) expansion via adipocyte hyperplasia being a key adipogenesis mechanism. As efforts intensify to address this global health crisis, understanding the complex interplay of contributing factors becomes critical for effective public health interventions and improved patient outcomes. In this context, gut microbiota-derived metabolites play an important role in orchestrating obesity modulation. Microbial lipopolysaccharides (LPS), secondary bile acids (BA), short-chain fatty acids (SCFAs), and trimethylamine (TMA) are the main intestinal metabolites in dyslipidemic states. Emerging evidence highlights the microbiota’s substantial role in influencing host metabolism and subsequent health outcomes, presenting new avenues for therapeutic strategies, including polyphenol-based manipulations of these microbial populations. Among various agents, caffeine emerges as a potent modulator of metabolic pathways, exhibiting anti-inflammatory, antioxidant, and obesity-mitigating properties. Notably, caffeine’s anti-adipogenic potential, attributed to the downregulation of key adipogenesis regulators, has been established. Recent findings further indicate that caffeine’s influence on obesity may be mediated through alterations in the gut microbiota and its metabolic byproducts. Therefore, the present review summarizes the anti-adipogenic effect of caffeine in modulating obesity through the intestinal microbiota and its metabolites.
Bile salts have an established role in the emulsification and intestinal absorption of dietary lipids, and their homeostasis is tightly controlled by various transporters and regulators in the enterohepatic circulation. Notably, emerging evidence points toward bile salts as major modulators of cardiometabolic disease (CMD), an umbrella disease of disorders affecting the heart and blood vessels that is caused by systemic metabolic diseases such as Type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), the latter encompassing also metabolic dysfunction-associated steatohepatitis (MASH). The underlying mechanisms of protective effects of bile salts are their hormonal properties, enabling them to exert versatile metabolic effects by activating various bile salt-responsive signaling receptors with the nuclear farnesoid X receptor (FXR) and the Takeda G-protein-coupled receptor 5 (TGR5) as most extensively investigated. Activation of FXR and TGR5 is involved in the regulation of glucose, lipid and energy metabolism, and inflammation. Bile salt-based therapies directly targeting FXR and TGR5 signaling have been evaluated for their therapeutic potential in CMD. More recently, therapeutics targeting bile salt transporters thereby modulating bile salt localization, dynamics, and signaling, have been developed and evaluated in CMD. Here, we discuss the current knowledge on the contribution of bile salt signaling in the pathogenesis of CMD and the potential of bile salt-based therapies for the treatment of CMD.
Lipid droplets (LDs) are organelles that store excess lipids and provide fatty acids for energy production during starvation. LDs are also essential for cellular maintenance, but excessive accumulation of LDs triggers various cancers in addition to metabolic diseases such as diabetes. In this study, we aimed to develop a strategy to identify new genes that reduces accumulation of LDs in cancer cells using an RNA interference (RNAi) screening system employing artificial sequence‐enriched shRNA libraries. Monitoring LDs by fluorescent activated cell sorting, the subsequently collected cumulative LDs cells, and shRNA sequence analysis identified a clone that potentially functioned to accumulate LDs. The clone showed no identical sequence to human Refseq. It showed very similar sequence to seven genes by allowing three mismatches. Among these genes, we identified the mediator complex subunit 6 ( MED6 ) gene as a target of this shRNA. Silencing of MED6 led to an increase in LD accumulation and expression of the marker genes, PLIN2 and DGAT1 , in fatty cells. MED6 is a member of the mediator complex that regulates RNA polymerase II transcription through transcription factor II. Some mediator complexes play important roles in both normal and pathophysiological transcription processes. These results suggest that MED6 transcriptionally regulates the genes involved in lipid metabolism and suppresses LD accumulation.
Background
Imbalances in bile acid (BA) synthesis and metabolism are involved in the onset of diabetes and depression in humans and rodents. However, the role of BAs and the farnesoid X receptor (FXR)/fibroblast growth factor (FGF) 15 signaling pathway in the development of diabetes and depression is still largely unknown. Therefore, we investigated the potential molecular mechanisms of BAs that may be associated with glucolipid metabolism disorders in diabetic mice subjected to chronic stress.
Methods
The type 2 diabetes mellitus (T2DM) mouse model was induced by feeding mice a high-fat diet and administering an intraperitoneal injection of streptozotocin (STZ). The chronic unpredictable mild stress (CUMS) procedure was performed by introducing a series of mild stressors. Forty mice were randomly divided into the regular chow feeding group and the high-fat diet feeding group. After two weeks of feeding, the mice were randomly divided into four groups: the Control group, CUMS group, T2DM group, and T2DM+CUMS group. The T2DM group and T2DM+CUMS group received an intraperitoneal injection of STZ to induce the T2DM model. The CUMS and T2DM+CUMS groups were exposed to CUMS to induce depressive-like phenotypes. Blood and tissue samples were obtained for pertinent analysis and detection.
Results
Compared with the T2DM mice, T2DM+CUMS mice had higher blood glucose and lipid levels, insulin resistance, inflammation of the liver and pancreas, impaired liver function, and increased total bile acids. These changes were accompanied by attenuated FXR signaling. Chronic stress was found to attenuate FXR expression and its downstream target, FGF15, in the ileum when compared with the T2DM group.
Conclusion
FXR may play a role in the diabetic disorder of glucolipid metabolism when aggravated by chronic stress. FXR and its downstream target, FGF15, may be therapeutic targets for treating comorbid T2DM and depression.
Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-specific liver disease. It is characterized by pruritus, abnormal liver function and elevated total bile acid (TBA) levels, increasing the risk of maternal and fetal adverse outcomes. Its etiology remains poorly elucidated. Over the years, various omics techniques, including metabolomics, microbiome, genomics, etc., have emerged with the advancement of bioinformatics, providing a new direction for exploring the pathogenesis, diagnosis and treatment of ICP. In this review, we first summarize the role of bile acids and related components in the pathogenesis of ICP and then further illustrate the results of omics studies.
Background and Aims
We have previously shown in a model of hepatic ischaemia/reperfusion injury that the farnesoid X receptor (FXR) agonist obeticholic acid (OCA) restores reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK), an inverse modulator of metalloproteases (MMPs) and inhibitor of the sheddases ADAM10 and ADAM17 involved in inflammation and fibrogenesis. Here, the effects of FXR agonists OCA and INT‐787 on hepatic levels of RECK, MMPs, ADAM10 and ADAM17 were compared in a diet‐induced ob/ob mouse model of non‐alcoholic steatohepatitis (NASH).
Methods
Lep ob/ob NASH mice fed a high‐fat diet (HFD) or control diet (CD) for 9 weeks (wks) were treated with OCA or INT‐787 0.05% dosed via HFD admixture (30 mg/kg/day) or HFD for further 12 wks. Serum alanine transaminase (ALT) and inflammatory cytokines, liver RECK, MMP‐2 and MMP‐9 activity as well as ADAM10, ADAM17, collagen deposition (Sirius red), hepatic stellate cell activation (α‐SMA) and pCK ⁺ reactive biliary cells were quantified.
Results
Only INT‐787 significantly reduced serum ALT, IL‐1β and TGF‐β. A downregulation of RECK expression and protein levels observed in HFD groups (at 9 and 21 wks) was counteracted by both OCA and INT‐787. HFD induced a significant increase in liver MMP‐2 and MMP‐9; OCA administration reduced both MMP‐2 and MMP‐9 while INT‐787 markedly reduced MMP‐2 expression. OCA and INT‐787 reduced both ADAM10 and ADAM17 expression and number of pCK ⁺ cells. INT‐787 was superior to OCA in decreasing collagen deposition and α‐SMA levels.
Conclusion
INT‐787 is superior to OCA in controlling specific cell types and clinically relevant anti‐inflammatory and antifibrotic molecular mechanisms in NASH.
Aging and cancer are increasingly becoming big challenges for public health worldwide due to increased human life expectancy. Meanwhile, aging is one of the major risk factors for cancer. In December 2019, the first International Conference on Aging and Cancer was held in Haikou, Hainan province (island), China, preluding the establishment of the International Center for Aging and Cancer (ICAC) at Hainan, an institute dedicated to the research at the intersection of aging and cancer. Since then, the ICAC has hosted the annual conference each December in Hainan. The 2022 ICAC conference, with the theme of “promoting longevity with less cancer,” invited 17 internationally renowned scientists to share their new research and insights. Topics included DNA methylation in rejuvenation, development, and cellular senescence; lifespan regulation and longevity manipulation; metabolism and aging; cellular senescence and diseases; and novel therapeutics for cancer and antiaging/anticancer drug discovery. The forum highlighted the interconnectedness of aging and senescence with cancer evolution and risk. Although there is hope for preventing diseases like cancer by modulating systems that also control lifespan, attention has to be paid to the conflicting needs and competing demands in human biology.
Bile acids (BAs) facilitate the absorption of dietary lipids and vitamins and have also been identified as signaling molecules involved in regulating their own metabolism, glucose and lipid metabolism, as well as immunity. Disturbances in BA homeostasis are associated with various enterohepatic and metabolic diseases, such as cholestasis, nonalcoholic steatohepatitis, inflammatory bowel disease, and obesity. As a key regulator, the nuclear orphan receptor farnesoid X receptor (FXR, NR1H4) precisely regulates BA homeostasis by transcriptional regulation of genes involved in BA synthesis, metabolism, and enterohepatic circulation. FXR is widely regarded as the most potential therapeutic target. Obeticholic acid is the only FXR agonist approved to treat patients with primary biliary cholangitis, but its non-specific activation of systemic FXR also causes high-frequency side effects. In recent years, developing tissue-specific FXR-targeting drugs has become a research highlight. This article provides a comprehensive overview of the role of tissue-specific intestine/liver FXR in regulating genes involved in BA homeostasis and briefly discusses tissue-specific FXR as a therapeutic target for treating diseases. These findings provide the basis for the development of tissue-specific FXR modulators for the treatment of enterohepatic and metabolic diseases associated with BA dysfunction
Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 ( Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are 2-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (6 male, 9 female), and NETosis severity correlates with clinical markers of kidney disease. This suggests potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.
High vs. low aerobic capacity significantly impacts the risk for metabolic diseases. Rats selectively bred for high or low intrinsic aerobic capacity differently modify hepatic bile acid metabolism in response to high-fat diets (HFD). Here we tested if a bile acid sequestrant would alter hepatic and whole-body metabolism differently in rats with high and low aerobic capacity fed a 1-week HFD. Male rats (8 months of age) that were artificially selected to be high (HCR) and low-capacity runners (LCR) with divergent intrinsic aerobic capacities were transitioned from a low-fat diet (LFD, 10% fat) to a HFD (45% fat) with or without a bile acid sequestrant (BA-Seq, 2% Cholestyramine Resin) for 7 days while maintained in an indirect calorimetry system. HFD+BA-Seq increased fecal excretion of lipids and bile acids and prevented weight and fat mass gain in both strains. Interestingly, HCR rats had increased adaptability to enhance fecal bile acid and lipid loss, resulting in more significant energy loss than their LCR counterpart. In addition, BA-Seq induced a greater expression of hepatic CYP7A1 gene expression, the rate-limiting enzyme of bile acid synthesis in HCR rats both on HFD and HFD+BA-Seq diets. HCR displayed a more significant reduction of RQ in response to HFD than LCR, but HFD+BA-Seq lowered RQ in both groups compared to HFD alone, demonstrating a pronounced impact on metabolic flexibility. In conclusion, BA-Seq provides uniform metabolic benefits for metabolic flexibility and adiposity, but rats with higher aerobic capacity display adaptability for hepatic bile acid metabolism.
Intestinal bile acid-binding protein (I-BABP) iis a cytosolic protein that binds bile acids (BAs) with a high affinity. In the small intestine, its expression is restricted to the ileum where it is involved in the enterohepatic circulation of BAs, Using the human enterocyte-like Caco-2 cell line, we have recently shown that BAs increased I-BABP gene expression. To determine whether this regulation occurs in vivo, the effect of BA depletion or supplementation was studied in mice, A dramatic drop in I-BABP mRNA levels was observed in mice treated with the BA-binding resin cholestyramine, whereas an increase was found in animals fed with taurocholic acid. BAs are physiological ligands for the nuclear farnesoid X receptor (FXR), Both FXR and I-BABP are co-expressed along the small intestine and in Caco-2 cells. To determine the role of FXR in the regulation of I-BABP expression, the promoter of the human I-BABP gene was cloned. In Caco-2 cells, cotransfection of FXR and RXR alpha is required to obtain the full transactivation of the I-BABP promoter by BAs, Deletion and mutation analyses demonstrate that the FXR/RXR alpha heterodimer activates transcription through an inverted repeat bile acid responsive element located in position -160/-148 of the human I-BABP promoter. In conclusion, we show that FXR is a physiological BA sensor that is likely to play an essential role in BA homeostasis through the regulation of genes involved in their enterohepatic circulation.
Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.
Oestrogens are involved in the growth, development and homeostasis of a number of tissues1. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER)2. Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery3,4. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17-oestradiol, and the selective antagonist raloxifene5, at resolutions of 3.1 and 2.6 Å, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.
Recent in vitro studies have provided evidence that hepatic lipase (HL) facilitates the selective uptake of HDL cholesteryl esters (CE), but the in vivo physiological relevance of this process has not been demonstrated. To evaluate the role that HL plays in facilitating the selective uptake of HDL-CE in vivo, we studied the metabolism of [³H]CEt, ¹²⁵I-labeled apolipoprotein (apo) A-I, and ¹³¹I-labeled apoA-II-labeled HDL in HL-deficient mice. Kinetic analysis revealed similar catabolism of ¹²⁵I-labeled apoA-I (as well as ¹³¹I-labeled apoA-II) in C57BL controls and HL deficient mice, with fractional catabolic rates (FCR) of 2.17 ± 0.15 and 2.16 ± 0.11 d⁻¹ (2.59 ± 0.14 and 2.67 ± 0.13 d⁻¹, respectively). In contrast, despite similar hepatic scavenger receptor BI expression, HL-deficient mice had delayed clearance of [³H]CEt compared to controls (FCR = 3.66 ± 0.29 and 4.41 ± 0.18 d⁻¹, P < 0.05). The hepatic accumulation of [³H]CEt in HL-deficient mice (62.3 ± 2.1% of total) was significantly less than in controls (72.7 ± 3.0%), while the [³H]CEt remaining in the plasma compartment increased (20.7 ± 1.8% and 12.6 ± 0.5%) (P < 0.05, all).
In summary, HL deficiency does not alter the catabolism of apoA-I and apoA-II but decreases the hepatic uptake and the plasma clearance of HDL-CE. These data establish for the first time an important role for HL in facilitating the selective uptake of HDL-CE in vivo.
—Lambert, G., M. J. A. Amar, P. Martin, J. Fruchart-Najib, B. Föger, R. D. Shamburek, H. B. Brewer, Jr., and S. Santamarina-Fojo. Hepatic lipase deficiency decreases the selective uptake of HDL-cholesteryl esters in vivo. J. Lipid Res. 2000. 41: 667–672.
Retinoic acid and thyroid hormone receptors can act alternatively as ligand-independent repressors or ligand-dependent activators, based on an exchange of N-CoR or SMRT-containing corepressor complexes for coactivator complexes in response to ligands. We provide evidence that the molecular basis of N-CoR recruitment is similar to that of coactivator recruitment, involving cooperative binding of two helical interaction motifs within the N-CoR carboxyl terminus to both subunits of a RAR-RXR heterodimer. The N-CoR and SMRT nuclear receptor interaction motifs exhibit a consensus sequence of LXX I/H I XXX I/L, representing an extended helix compared to the coactivator LXXLL helix, which is able to interact with specific residues in the same receptor pocket required for coactivator binding. We propose a model in which discrimination of the different lengths of the coactivator and corepressor interaction helices by the nuclear receptor AF2 motif provides the molecular basis for the exchange of coactivators for corepressors, with ligand-dependent formation of the charge clamp that stabilizes LXXLL binding sterically inhibiting interaction of the extended corepressor helix.
Canalicular secretion of bile salts is a vital function of the vertebrate liver, yet the molecular identity of the involved ATP-dependent carrier protein has not been elucidated. We cloned the full-length cDNA of the sister of P-glycoprotein (spgp; Mr approximately 160,000) of rat liver and demonstrated that it functions as an ATP-dependent bile salt transporter in cRNA injected Xenopus laevis oocytes and in vesicles isolated from transfected Sf9 cells. The latter demonstrated a 5-fold stimulation of ATP-dependent taurocholate transport as compared with controls. This spgp-mediated taurocholate transport was stimulated solely by ATP, was inhibited by vanadate, and exhibited saturability with increasing concentrations of taurocholate (Km approximately 5 microM). Furthermore, spgp-mediated transport rates of various bile salts followed the same order of magnitude as ATP-dependent transport in canalicular rat liver plasma membrane vesicles, i.e. taurochenodeoxycholate > tauroursodeoxycholate = taurocholate > glycocholate = cholate. Tissue distribution assessed by Northern blotting revealed predominant, if not exclusive, expression of spgp in the liver, where it was further localized to the canalicular microvilli and to subcanalicular vesicles of the hepatocytes by in situ immunofluorescence and immunogold labeling studies. These results indicate that the sister of P-glycoprotein is the major canalicular bile salt export pump of mammalian liver.
Recently we reported that a P-450c27/25 cDNA probe hybridizes to two RNA species of about 1.9 and 2.3-2.4 kilobase pairs (kb) in some rat tissues. To understand the molecular relationship between the two mRNAs, we have isolated and characterized a cDNA for the larger, previously uncharacterized 2.3-kb mRNA species. The 2.3-kb cDNA is identical to the previously reported 1.9-kb P-450c27/25 cDNA excepting a 400-nucleotide-long 5' extension. The terminal 291 nucleotides of this extension exhibit 100% complementarity with the 5'-translated region of the mRNA belonging to a family of growth hormone-inducible serine protease inhibitors (SPI). Northern blot analysis, using strand-specific probes, and S1 nuclease protection revealed the presence of the 2.3-kb mRNA exhibiting the sequence characteristics of the larger cDNA. These results were further confirmed by polymerase chain reaction amplification of reverse transcribed RNA. Expression of the 2.3-kb cDNA in COS cells resulted in the correct mitochondrial targeting of a 52-kDa protein exhibiting the properties of P-450c27/25. Furthermore, both the 1.9- and 2.3-kb mRNAs appear to direct the synthesis of a similarly sized 55-kDa precursor protein in a reticulocyte lysate system. Restriction mapping, polymerase chain reaction amplification and partial sequencing of a 25-kb genomic DNA clone suggest the proximal location of the SPI and the P-450c27/25 protein coding regions in the rat genome on either side of a common overlap region. The results also show that the P-450c27/25 mRNAs are regulated by growth hormone in parallel to the SPI mRNAs. These results collectively suggest that a growth hormone-inducible SPI family mRNA and the P-450c27/25 mRNA are encoded by two closely linked, possibly overlapping genes.
The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7 alpha-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7 alpha-hydroxylase. DNA sequence analysis of the cDNA revealed a 7 alpha-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7 alpha-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7 alpha-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7 alpha-hydroxylase mRNA levels. The enzymatic activity of 7 alpha-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7 alpha-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis.
The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.
To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the alpha isoform of mouse PPAR (mPPAR alpha) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR alpha protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional-activation of target genes were not observed. These results clearly demonstrate that mPPAR alpha is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR alpha-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.
The hepatic transport systems mediating bile acid uptake and excretion undergo independent, stage-specific expression during development in the rat. In this study, the mechanisms underlying ontogenic regulation of both the Na(+)-dependent basolateral bile acid transporter and canalicular bile acid transporter/ecto-ATPase were examined. Steady state mRNA levels for the basolateral transporter were less than 20% of adult values prior to birth, increased to 35% on the first postnatal day, and reached adult levels by 1 week of age. This was paralleled by transcription rates, which were low prior to birth, reached 47% by day 1, and were maximal by 1 week of age. Steady state mRNA levels for ecto-ATPase were 12% of adult values prior to birth and showed a 2-fold increase by the first day of life. Thereafter, there was a gradual increase in mRNA for this transporter, with adult levels being reached at 4 weeks of age. Transcription rates paralleled this increment, although adult levels were reached earlier. Surprisingly, for both transporters, the full complement of protein was present well before adult levels of mRNA were reached. The basolateral protein was expressed at 82% of adult levels on the first day of life but was of lower apparent molecular mass (39 kDa), a difference that persisted until 4 weeks of age. N-Glycanase digestion suggested that this difference could be fully accounted for by N-linked glycosylation. The ecto-ATPase protein was present at 33% of adult levels prior to birth, 77% by 1 day, and 84% of adult levels by 1 week of age. Unlike the basolateral transporter, the apparent molecular weight of this protein did not change during development. In summary, the ontogeny of bile acid transporters on the plasma membrane of the hepatocyte is complex and appears to be regulated at transcriptional, translational, and post-translational levels.
The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units. Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold. The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain. Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors.
Normal, chimeric-transgenic, and transgenic mice have been used to study the axial patterns of ileal lipid-binding protein gene (Ilbp) expression during and after completion of gut morphogenesis. Ilbp is initially activated in enterocytes in bidirectional wave that expands proximally in the ileum and distally to the colon during late gestation and the first postnatal week. This activation occurs at the same time that a wave of cytodifferentiation of the gut endoderm is completing its unidirectional journey from duodenum to colon. The subsequent contraction of Ilbp's expression domain, followed by its reexpansion from the distal to proximal ileum, coincides with a critical period in gut morphogenesis (postnatal days 7-28) when its proliferative units (crypts) form, establish their final stem cell hierarchy, and then multiply through fission. The wave of reactivation is characterized by changing patterns of Ilbp expression: (a) at the proximal most boundary of the wave, villi contain a mixed population of scattered ileal lipid-binding protein (ILBP)-positive and ILBP-negative enterocytes derived from the same monoclonal crypt; (b) somewhat more distally, villi contain vertical coherent stripes of wholly ILBP-positive enterocytes derived from monoclonal crypts and adjacent, wholly ILBP-negative stripes of enterocytes emanating from other monoclonal crypts; and (c) more distally, all the enterocytes on a villus support Ilbp expression. Functional mapping studies of Ilbp's promoter in transgenic mice indicate that nucleotides -145 to +48 contain cis-acting elements sufficient to produce an appropriately directed distal-to-proximal wave of Ilbp activation in the ileum, to maintain an appropriate axial distribution of monophenotypic wholly reporter-positive villi in the distal portion of the ileum, as well as striped and speckled villi in the proximal portion of its expression domain, and to correctly support reporter production in villus-associated ileal enterocytes. Nucleotides -417 to -146 of Ilbp contain a "temporal" suppressor that delays initial ileal activation of the gene until the second postnatal week. Nucleotides -913 to -418 contain a temporal suppressor that further delays initial activation of the gene until the third to fourth postnatal week, a spatial suppressor that prohibits gene expression in the proximal quarter of the ileum and in the proximal colon, and a cell lineage suppressor that prohibits expression in goblet cells during the first two postnatal weeks.
Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.
The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.
We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.
Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D.(1993) Am. J. Physiol. 264, G528-G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a Km of 26.3 microM and a Vmax of 117 pmol/mg protein/min. The Km value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodium-dependent transport of taurocholate and cholate.
Thyroid hormone receptors bind DNA with highest affinity as heterodimers with retinoid X receptors, and such heterodimers generally are thought to be the biological mediators of thyroid hormone action. An alternative splice product of the thyroid hormone receptor alpha gene, thyroid hormone receptor variant alpha2, does not bind thyroid hormone and functions as a weak dominant negative inhibitor of thyroid hormone action. Thyroid hormone receptor variant alpha2 is missing one-half of the ninth heptad, a region of the bona fide receptor thought to be important for heterodimerization with retinoid X receptors. The role of the ninth heptad in heterodimerization has been evaluated further. Thyroid hormone receptor variant alpha2-retinoid X receptor heterodimers form on a subset of direct repeat response elements but not on palindromic or inverted palindromic elements. Restoration of the missing ninth heptad sequence is critical for restoring heterodimerization on the palindromic DNA, but either the ninth heptad amino acids or a stretch of alanines is equally able to restore heterodimerization on the inverted palindrome. Thus, the role of the ninth heptad in heterodimerization differs on direct repeat, palindromic, and inverted palindromic response elements, suggesting that the protein-protein interactions differ on each of these elements. The dominant negative activity of thyroid hormone receptor variant alpha2 requires DNA binding, but the relatively weak nature of the dominant negative activity is only partially explained by the weak DNA binding.
Accumulation of cholesterol causes both repression of genes controlling cholesterol biosynthesis and cellular uptake and induction of cholesterol 7alpha-hydroxylase, which leads to the removal of cholesterol by increased metabolism to bile acids. Here, we report that LXRalpha and LXRbeta, two orphan members of the nuclear receptor superfamily, are activated by 24(S), 25-epoxycholesterol and 24(S)-hydroxycholesterol at physiologic concentrations. In addition, we have identified an LXR response element in the promoter region of the rat cholesterol 7alpha-hydroxylase gene. Our data provide evidence for a new hormonal signaling pathway that activates transcription in response to oxysterols and suggest that LXRs play a critical role in the regulation of cholesterol homeostasis.
A subset of patients with high plasma HDL concentrations have enhanced rather than reduced atherosclerosis. We have developed a new transgenic mouse model overexpressing human lecithin-cholesteryl acyltransferase (LCAT) that has elevated HDL and increased diet-induced atherosclerosis. LCAT transgenic mouse HDLs are abnormal in both composition and function. Liver uptake of [3H]cholesteryl ether incorporated in transgenic mouse HDL was reduced by 41% compared with control HDL, indicating ineffective transport of HDL-cholesterol to the liver and impaired reverse cholesterol transport. Analysis of this LCAT-transgenic mouse model provides in vivo evidence for dysfunctional HDL as a potential mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels.
The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units. Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold. The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain. Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors.
Normal, chimeric-transgenic, and transgenic mice have been used to study the axial patterns of ileal lipid-binding protein gene (Ilbp) expression during and after completion of gut morphogenesis. Ilbp is initially activated in enterocytes in bidirectional wave that expands proximally in the ileum and distally to the colon during late gestation and the first postnatal week. This activation occurs at the same time that a wave of cytodifferentiation of the gut endoderm is completing its unidirectional journey from duodenum to colon. The subsequent contraction of Ilbp's expression domain, followed by its reexpansion from the distal to proximal ileum, coincides with a critical period in gut morphogenesis (postnatal days 7-28) when its proliferative units (crypts) form, establish their final stem cell hierarchy, and then multiply through fission. The wave of reactivation is characterized by changing patterns of Ilbp expression: (a) at the proximal most boundary of the wave, villi contain a mixed population of scattered ileal lipid-binding protein (ILBP)-positive and ILBP-negative enterocytes derived from the same monoclonal crypt; (b) somewhat more distally, villi contain vertical coherent stripes of wholly ILBP-positive enterocytes derived from monoclonal crypts and adjacent, wholly ILBP-negative stripes of enterocytes emanating from other monoclonal crypts; and (c) more distally, all the enterocytes on a villus support Ilbp expression. Functional mapping studies of Ilbp's promoter in transgenic mice indicate that nucleotides -145 to +48 contain cis-acting elements sufficient to produce an appropriately directed distal-to-proximal wave of Ilbp activation in the ileum, to maintain an appropriate axial distribution of monophenotypic wholly reporter-positive villi in the distal portion of the ileum, as well as striped and speckled villi in the proximal portion of its expression domain, and to correctly support reporter production in villus-associated ileal enterocytes. Nucleotides -417 to -146 of Ilbp contain a "temporal" suppressor that delays initial ileal activation of the gene until the second postnatal week. Nucleotides -913 to -418 contain a temporal suppressor that further delays initial activation of the gene until the third to fourth postnatal week, a spatial suppressor that prohibits gene expression in the proximal quarter of the ileum and in the proximal colon, and a cell lineage suppressor that prohibits expression in goblet cells during the first two postnatal weeks.
We have cloned a murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane-transport proteins from mouse liver. The cloned cDNA insert of 2783 bp with an open reading frame of 2011 bp codes for a 12-transmembrane 670-amino-acid protein with highest amino acid identity with the: rat Oatp1. When expressed in Xenopus laevis oocytes, the mouse Oatp exhibited the same substrate specificity as the rat Oatp1. Besides the common Oatp substrates bromosulphophthalein, taurocholate, oestrone 3-sulphate and ouabain, the new mouse Oatp also mediates transport of the Oatp1-specific magnetic-resonance-imaging agent gadoxetate. The Oatp2-specific cardiac glycoside digoxin, however, is not transported. Kinetic analyses performed for taurocholate and oestrone 3-sulphate revealed apparent K-m values of 12 mu M and 5 mu M respectively. Northern-blot analysis demonstrated a predominant expression in the liver with an additional moderate expression in the kidney. Taken together, the amino acid identity, the functional characteristics and the tissue distribution suggest that we have isolated the murine orthologue of the rat Oatp1, and consequently the identified protein will be called Oatp1. Using fluorescence in Situ hybridization, the murine Oatp1 gene was mapped to chromosome XA3-A5.
The nuclear receptor PPARγ/RXRα heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARγ and RXRα LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARγ agonist rosiglitazone or GI262570, and coactivator peptides. The PPARγ/RXRα heterodimer is asymmetric, with each LBD deviated ∼10° from the C2 symmetry, allowing the PPARγ AF-2 helix to interact with helices 7 and 10 of RXRα. The heterodimer interface is composed of conserved motifs in PPARγ and RXRα that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARγ/RXRα heterodimer by 9cRA.
We demonstrate that mice lacking the oxysterol receptor, LXRα, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXRα (−/−) mice fail to induce transcription of the gene encoding cholesterol 7α-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXRα (−/−) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXRα as the major sensor of dietary cholesterol.
In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5′-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the −840/+23, −329/+23, and −195/+23 mCAT constructs. A −76/+23 construct showed no regulation by bile acids. When a DNA fragment (−110/−86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. ‘Super’-electrophoretic mobility shift assays (EMSA) indicated that both HNF1α and C/EBP bind to the −110 to −86 bp DNA fragment. Recombinant rat HNF1α and C/EBPα competitively bound to this DNA fragment. ‘Super’-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1α, but not C/EBP, binding to the −110/−86 bp DNA fragment. A four base pair substitution mutation (−103 to −99) in this sequence eliminated TCA and TDCA regulation of the (−840/+23) construct. The substitution mutation also eliminated (>95%) HNF1α, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between −110 and −86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1α binding to the CYP27 promoter.
LXRα and -β are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile
acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring
oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many
of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly
to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series
of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation
of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also
be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction
of an oxygen on the sterol B-ring results in a ligand with LXRα-subtype selectivity. These results support the hypothesis
that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach
can be used to design potent LXR ligands for pharmacologic use.
The liver X receptors (LXRs) are a family of transcription factors that were first identified as orphan members of the nuclear receptor superfamily. The identification of a specific class of oxidized derivatives of cholesterol as ligands for the LXRs has been crucial to helping understand the function of these receptors in vivo and first suggested their role in the regulation of lipid metabolism. Confirmation of this role has come from the recent analysis of LXR-deficient mice, which has demonstrated the essential function of one of these receptors in the liver as a major sensor of dietary cholesterol.
Oxysterols are important regulatory molecules of diverse biological processes such as cholesterol homeostasis, bile acid synthesis and apoptosis. Recent findings led to the suggestion that some of these functions are mediated by the nuclear receptors LXRα and LXRβ owing to their potential to bind a group of naturally occurring oxysterols as their ligands. In this report, we compare the genomic structure and the promoter regions of the two mouse LXR genes. In addition, we show evidence for the presence of a processed, but truncated LXRβ pseudogene in the mouse genome. RACE-PCR on mouse liver cDNA demonstrates the presence of more than one defined transcription initiation site for both genes. The LXRα and LXRβ promoter regions are GC-rich and contain a number of putative Sp1 binding sites but lack obvious TATA and CAAT boxes. A database search revealed several sequence motifs in the LXR promoter regions that resemble known transcription factor binding sites. Most striking is the identification of one potential NFκB and seven potential Ets-protein binding sites in the LXRβ promoter, suggesting an important role for this receptor in the haematopoietic/immune system.
Hepatic bile salt secretion and bile formation are essential functions of the mammalian liver, and the rate-limiting step of hepatocellular secretion of bile salts is canalicular secretion. Recently, the rat sister-of-p-glycoprotein/bilesaltexportpump (spgp/BSEP) was demonstrated to encode for the rat ATP-dependent canalicular bile salt export protein, and mutations of human BSEP were identified as the cause of PFIC 2. Since mouse models are vital for studies in hepatocellular transport and metabolism, cloning and characterization of the murine gene are essential. In this study, we have cloned a full-length, functional cDNA for the mBsep. The deduced amino acid sequence encodes for a 1321-amino-acid protein and is 94% similar to rat and 89% similar to human bsep. Western immunoblotting using an antibody directed against a carboxy-terminal peptide of mbsep protein reveals a 160 kDa protein, which is highly enriched in mouse canalicular membranes. Transfection of mBSEP into Sf-9 insect cells or mammalian Balb-3T3 cells confers functional transport of the bile salt taurocholate. The mBsep mRNA is expressed in murine liver, but not in other tissues. Hepatic mBsep levels appear highly regulated, being markedly diminished in both LPS and estrogen models of cholestasis. These data are important for further murine studies of hepatocellular transport physiology and metabolism.
Molecular cloning and transcriptional activation studies have revealed a new protein similar to the steroid hormone receptors and which responds specifically to vitamin A metabolites. This protein is substantially different in primary structure and ligand specificity from the products of the previously described retinoic acid receptor gene family. By indicating the existence of an additional pathway through which retinoic acid may exert its effects, these data lead to a re-evaluation of retinoid physiology.
A full-length cDNA for the human liver mitochondrial cytochrome P-450 CYP27 was cloned from a human hepatoma HepG2 cDNA library and then subcloned into the mammalian expression vector pSG5. When CYP27 cDNA was transfected into COS-1 transformed monkey kidney cells along with adrenodoxin cDNA, transfected cells revealed a 10- to 20-fold higher vitamin D3-25-hydroxylase activity than nontransfected cells. Transfected cells were capable of 25-hydroxylation of vitamin D3, 1 alpha-hydroxyvitamin D3 and 1 alpha-hydroxydihydrotachysterol3. In each case they also showed the ability to 26(27)-hydroxylate the cholesterol-like (D3) side chain. The relative rates of 25- and 26(27)-hydroxylation of 1 alpha-hydroxyvitamin D3 approximately mimicked the ratio of products observed in HepG2 cells. Vitamin D2 and 1 alpha-hydroxyvitamin D2, both with the ergosterol-like side chain, were 24- and 26(27)-hydroxylated by CYP27. The rate of side-chain 24-, 25-, or 26(27)-hydroxylation was greater for 1 alpha-hydroxylated vitamin D analogs than for their nonhydroxylated counterparts. We conclude that CYP27 is capable of 24-, 25-, and 26(27)-hydroxylation of vitamin D analogs and that the nature of products is partially dictated by the side chain of the substrate. This work has revealed that the cytochrome P-450 CYP27 may be important in the metabolism of vitamin D analogs used as drugs.
We have used a yeast genetic system to isolate cDNAs encoding proteins that specifically interact with the ligand-binding domain of human retinoid X receptor-alpha (RXR alpha). A number encoded portions of two known RXR heterodimer partners, the retinoic acid receptor (RAR) and the peroxisome proliferator activated receptor. Of four additional RXR-interacting proteins (RIPs) selected for further study two, RIP14 and RIP15, are previously unidentified orphan members of the nuclear receptor superfamily. Two others, RIP110 and RIP13, do not show significant similarities to previously reported proteins. RIP110 interacts with LexA-RXR only in yeast cells grown in the presence of the RXR ligand 9-cis-RA, while the interaction of the four receptor superfamily members and RIP13 is unaffected by the presence or absence of 9-cis-RA. RIP110 and RIP13 also interact in yeast with several other members of the receptor superfamily, but RIP14 and RIP15 interact only with RXR. Analysis of larger cDNA clones demonstrates that there are at least two isoforms of RIP14 that differ in the N-terminal (A and B) and hinge (D) domains. Northern blot analysis indicates that RIP14 is expressed specifically in liver and kidney, while RIP15 is expressed in every tissue tested. Both RIP14 and 15 bind as heterodimers with RXR to the RA response element (RARE) from the promoter of the RAR beta 2 isoform (the beta RARE), and RIP14 and RXR heterodimers also bind the ecdysone response element from the Drosophila heat shock protein 27 promoter. Both heterodimers also bind to several synthetic RAREs and other elements. In cotransfections, neither RIP14 nor RIP15 trans-activates a reporter containing multiple copies of the beta RARE under any of a variety of conditions, suggesting that their activities are dependent on the binding of as yet unidentified specific ligands or on activation by other processes.
Nuclear hormone receptors comprise a superfamily of ligand-modulated transcription factors that mediate the transcriptional activities of steroids, retinoids, and thyroid hormones. A growing number of related proteins have been identified that possess the structural features of hormone receptors, but that lack known ligands. Known as orphan receptors, these proteins represent targets for novel signaling molecules. We have isolated a mammalian orphan receptor that forms a heterodimeric complex with the retinoid X receptor. A screen of candidate ligands identified farnesol and related metabolites as effective activators of this complex. Farnesol metabolites are generated intracellularly and are required for the synthesis of cholesterol, bile acids, steroids, retinoids, and farnesylated proteins. Intermediary metabolites have been recognized as transcriptional regulators in bacteria and yeast. Our results now suggest that metabolite-controlled intracellular signaling systems are utilized by higher organisms.
Three overlapping cholesterol 7 alpha-hydroxylasecDNAs were cloned from total mouse liver RNA by a combination of the polymerase chain reaction and mouse liver cDNA library screening. One of the cDNA clones was used to screen a mouse genomic library; three genomic clones were isolated and one of them was fully characterized. The mouse cholesterol 7 alpha-hydroxylase gene spans approximately 10 kb. The gene contains six exons including a 1509-bp open reading frame encoding 503 amino acid residues. The predicted amino acid sequence of mouse cholesterol 7 alpha-hydroxylase showed 93 and 82% identity to the rat and human enzymes, respectively. The transcription initiation site is mapped to 64 bases 5' of the translation initiation codon. Several transcription factor binding sequences are identified in the 5' flanking region of the mouse cholesterol 7 alpha-hydroxylase gene.
Pig liver mitochondria were found to catalyze 27-, 25- and 24-hydroxylation of cholesterol at relative rates of about 1:0.2:0.04. An apparently homogeneous preparation of pig liver mitochondrial cytochrome P-450-27 was found to catalyze the same three hydroxylations at about the same relative rates when reconstituted with adrenodoxin and adrenodoxin reductase. The 24-hydroxycholesterol formed was shown to consist of one of the two possible stereoisomers. When using specifically deuterium-labeled substrates a significant isotope effect was observed in the case of 24-hydroxylation (KH/KD > 10), but not 25-hydroxylation (KH/KD = 1.1), or 27-hydroxylation (KH/KD = 1.1). The difference between the 24-hydroxylation and the other two hydroxylations may be due to different interactions between cholesterol and the same enzyme, with a resulting difference with respect to the rate-limiting step in the reaction. The physiological significance of the mitochondrial 24-hydroxylation is discussed.
Triglyceride, free cholesterol, and total cholesterol were quantified in lipid extracts of liver and thoracic aorta from nonhuman primates using commercially available enzymatic reagents. Lipids were solubilized in water by the addition of Triton X-100. Results of the enzymatic assays compared favorably with chemical assays of lipids separated by thin-layer chromatography. In addition to saving time, the present method has the advantage of measuring each lipid class from a single sample preparation. Furthermore, the procedure has been adapted for use with microtiter plates that conserve both sample and reagent.
hct-1 (hippocampal transcript) was detected in a differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in rat liver and kidney, though at much lower levels; expression was
barely detectable in testis, ovary, and adrenal. In liver, unlike brain, expression was sexually dimorphic; hepatic expression
was greatly reduced in female rats. In mouse, brain expression was widespread, with the highest levels being detected in corpus
callosum; only low levels were detected in liver. Sequence analysis of rat and mouse hct-1 cDNAs revealed extensive homologies with cytochrome P450s (CYPs), a diverse family of heme-binding monooxygenases that metabolize
a range of substrates including steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol 7α-hydroxylase (CYP7) and contains a postulated steroidogenic
domain present in other steroid-metabolizing CYPs but clearly represents a type of CYP not previously reported. Genomic Southern
analysis suggests that a single gene corresponding to hct-1 is present in mouse, rat, and human. hct-1 is unusual in that, unlike all other CYPs described, the primary site of expression is in the brain. Similarity to CYP7 and
other steroid-metabolizing CYPs may argue that hct-1 (CYP7B) plays a role in steroid metabolism in brain, notable because of the documented ability of brain-derived steroids
(neurosteroids) to modulate cognitive function in vivo.
A direct comparison of jejunal and ileal absorption rates of bile acids has not been reported. The aim of this study was to compare the relative transport rates of different bile acids in the jejunum and ileum.
Jejunal and ileal rabbit intestinal segments were separately perfused with bile acid solutions, and dose-response curves were obtained for taurocholate, ursodeoxycholate, chenodeoxycholate, deoxycholate, and their glycoconjugates. Membrane fluidity and bile acid transport were assessed in brush border membrane vesicles.
Taurocholate showed active transport in the ileum and no transport in the jejunum. Unconjugated bile acids showed passive diffusion in the two tracts, whereas glycoconjugated bile acids showed both components of transport in the ileum and passive diffusion in the jejunum (lower in the latter). A higher membrane fluidity and lower cholesterol-to-phospholipid ratio were found in the jejunum. Ursodeoxycholate reduced bile acid uptake into membrane vesicles from both ileum and jejunum.
Active transport is limited to the ileum. Passive diffusion is higher through a less fluid membrane with a higher cholesterol-to-phospholipid ratio in the ileum than in the jejunum. Ursodeoxycholate inhibition may be at the level of a facilitated, sodium-independent diffusion in the jejunum.
SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain
found in other family members but lacks the conserved DNA binding domain. In the yeast two-hybrid system, SHP interacted with
several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor,
and the orphan receptor MB67. SHP also interacted directly with these receptors in vitro. In mammalian cells, SHP specifically
inhibited transactivation by the superfamily members with which it interacted. These results suggest that SHP functions as
a negative regulator of receptor-dependent signaling pathways.
Retinoids are crucial regulators of a wide variety of processes in both developing and adult animals. These effects are thought to be mediated by the retinoic acid (RA) receptors and the retinoid X receptors (RXRs). We have identified an additional retinoid-activated receptor that is neither a retinoic acid receptors nor an RXR. RXR-interacting protein 14 (RIP14), a recently described orphan member of the nuclear receptor superfamily, can be activated by either all-trans-RA (tRA) or the synthetic retinoid TTNPB [[E]-4-[2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid].RIP14 binds to DNA as a heterodimer with RXR. In the presence of either tRA or TTNPB, the addition of 9-cis-RA or the RXR-specific agonist LG1069 [4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tertrahydro-2-naphthyl)ethenyl]benzoic acid] results in additional activation. Mutations of the ligand-dependent transcriptional activation functions indicate that TTNPB activates the RIP14 component of the RIP14-RXR heterodimer, that 9-cis-RA and LG1069 activate RXR, and that tRA activates via both RIP14 and RXR. Despite the very effective activation of RIP14 by tRA or TTNPB, relatively high concentrations of these compounds are required, and no evidence for direct binding of either compound was obtained using several approaches. These results suggest that RIP14 is the receptor for an as-yet-unidentified retinoid metabolite.