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1.
Gastroenterology ; 159(5): 1853-1865.e10, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32712104

RESUMEN

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4, also called FXR) is a ligand-activated transcription factor that, upon binding of bile acids, regulates the expression of genes involved in bile acid, fat, sugar, and amino acid metabolism. Transcript variants encode the FXR isoforms alpha 1, alpha 2, alpha 3, and alpha 4, which activate different genes that regulate metabolism. Little is known about the mechanisms by which the different isoforms regulate specific genes or how the expression of these genes affects the outcomes of patients given drugs that target FXR. METHODS: We determined genome-wide binding of FXR isoforms in mouse liver organoids that express individual FXR isoforms using chromatin immunoprecipitation, followed by sequencing analysis and DNA motif discovery. We validated regulatory DNA sequences by mobility shift assays and with luciferase reporters using mouse and human FXR isoforms. We analyzed mouse liver organoids and HepG2 cells that expressed the FXR isoforms using chromatin immunoprecipitation, quantitative polymerase chain reaction, and immunoblot assays. Organoids were analyzed for mitochondrial respiration, lipid droplet content, and triglyceride excretion. We used the FXR ligand obeticholic acid to induce FXR activity in organoids, cell lines, and mice. We collected data on the binding of FXR in mouse liver and the expression levels of FXR isoforms and gene targets in human liver tissue and primary human hepatocytes from the Gene Expression Omnibus. RESULTS: In mouse liver cells, 89% of sites that bound FXR were bound by only FXRα2 or FXRα4, via direct interactions with the DNA sequence motif ER-2. Via DNA binding, these isoforms regulated metabolic functions in liver cells, including carbon metabolism and lipogenesis. Incubation with obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis in organoids that expressed FXRα2 but not FXRα1. In human liver tissues, levels of FXRα2 varied significantly and correlated with expression of genes predicted to be regulated via an ER-2 motif. CONCLUSIONS: Most metabolic effects regulated by FXR in mouse and human liver cells are regulated by the FXRα2 isoform via specific binding to ER-2 motifs. The expression level of FXRα2 in liver might be used to predict responses of patients to treatment with FXR agonists.


Asunto(s)
Metabolismo Energético , Hepatocitos/metabolismo , Hígado/metabolismo , Motivos de Nucleótidos , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Sitios de Unión , Regulación de la Expresión Génica , Células Hep G2 , Humanos , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organoides/citología , Organoides/metabolismo , Unión Proteica , Isoformas de Proteínas , Receptores Citoplasmáticos y Nucleares/genética
2.
Gastroenterology ; 152(6): 1462-1476.e10, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28130067

RESUMEN

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. METHODS: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15NH4Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. RESULTS: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. CONCLUSIONS: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.


Asunto(s)
Amoníaco/metabolismo , Glutamina/biosíntesis , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Urea/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Proteínas en la Dieta/administración & dosificación , Expresión Génica , Hepatocitos , Hígado/enzimología , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteoma , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores
3.
Biochim Biophys Acta Mol Basis Dis ; 1863(3): 687-700, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27956139

RESUMEN

Nuclear receptors (NRs) are ligand-activated transcription factors regulating a large variety of processes involved in reproduction, development, and metabolism. NRs are ideal drug targets because they are activated by lipophilic ligands that easily pass cell membranes. Immortalized cell lines recapitulate NR biology poorly and generating primary cultures is laborious and requires a constant need for donor material. There is a clear need for development of novel preclinical model systems that better resemble human physiology. Uncertainty due to technical limitations early in drug development is often the cause of preclinical drugs not reaching the clinic. Here, we studied whether organoids, mini-organs derived from the respective mouse tissue's stem cells, can serve as a novel model system to study NR biology and targetability. We characterized mRNA expression profiles of the NR superfamily in mouse liver, ileum, and colon organoids. Tissue-specific expression patterns were largely maintained in the organoids, indicating their suitability for NR research. Metabolic NRs Fxrα, Lxrα, Lxrß, Pparα, and Pparγ induced expression of and binding to endogenous target genes. Transcriptome analyses of wildtype colon organoids stimulated with Rosiglitazone showed that lipid metabolism was the highest significant changed function, greatly mimicking the PPARs and Rosiglitazone function in vivo. Finally, using organoids we identify Trpm6, Slc26a3, Ang1, and Rnase4, as novel Fxr target genes. Our results demonstrate that organoids represent a framework to study NR biology that can be further expanded to human organoids to improve preclinical testing of novel drugs that target this pharmacologically important class of ligand activated transcription factors.


Asunto(s)
Colon/citología , Íleon/citología , Hígado/citología , Organoides/citología , Receptores Citoplasmáticos y Nucleares/genética , Células Madre/citología , Transcriptoma , Animales , Colon/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Íleon/metabolismo , Hígado/metabolismo , Ratones , Técnicas de Cultivo de Órganos/métodos , Organoides/metabolismo , ARN Mensajero/genética , Células Madre/metabolismo
4.
Biochim Biophys Acta ; 1812(8): 851-8, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21540105

RESUMEN

UNLABELLED: Hyperactivation of NF-κB is a key factor in the pathophysiology of inflammatory bowel disease (IBD). We previously showed that the bile salt nuclear Farnesoid X Receptor (FXR) counter-regulates intestinal inflammation, possibly via repression of NF-κB. Here, we examine whether mutual antagonism between NF-κB and FXR exists. FXR and its target genes IBABP and FGF15/19 expression were determined in HT29 colon carcinoma cells and ex vivo in intestinal specimens of wild type (WT) and Fxr-ko mice, treated with/without FXR ligands (GW4064/INT-747) and inflammatory stimuli (TNFα/IL-1ß). In addition, FXR activation was studied in vivo in WT and Fxr-ko mice with DSS-colitis. The involvement of NF-κB in decreasing FXR activity was investigated by reporter assays and Glutathione S-transferase pulldown assays. FXR target gene expression was highly reduced by inflammatory stimuli in all model systems, while FXR mRNA expression was unaffected. In line with these results, reporter assays showed reduced FXR transcriptional activity upon TNFα/IL-1ß stimulation. We show that this reduction in FXR activity is probably mediated by NF-κB, since overexpression of NF-κB subunits p50 and/or p65 also lead to inhibition of FXR activity. Finally, we report that p65 and p50 physically interact with FXR in vitro. CONCLUSIONS: Together, these results indicate that intestinal inflammation strongly reduces FXR activation, probably via NF-κB-dependent tethering of FXR. Therefore, FXR not only inhibits inflammation, but also is targeted by the inflammatory response itself. This could result in a vicious cycle where reduced FXR activity results in less repression of inflammation, contributing to development of chronic intestinal inflammation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Citocinas/fisiología , Mediadores de Inflamación/fisiología , Mucosa Intestinal/metabolismo , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Gut ; 60(4): 463-72, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21242261

RESUMEN

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterised by chronic intestinal inflammation, resulting from dysregulation of the mucosal immune system and compromised intestinal epithelial barrier function. The bile salt, nuclear farnesoid X receptor (FXR), was recently implicated in intestinal antibacterial defence and barrier function. The aim of this study was to investigate the therapeutic potential of FXR agonists in the treatment of intestinal inflammation in complementary in vivo and in vitro models. METHODS: Colitis was induced in wild-type (WT) and Fxr-null mice using dextran sodium sulfate, and in WT mice using trinitrobenzenesulfonic acid. Mice were treated with vehicle or the FXR agonist INT-747, and colitis symptoms were assessed daily. Epithelial permeability assays and cytokine expression analysis were conducted in mouse colon and enterocyte-like cells (Caco-2/HT29) treated with medium or INT-747. Inflammatory cytokine secretion was determined by ELISA in various human immune cell types. RESULTS: INT-747-treated WT mice are protected from DSS- and TNBS-induced colitis, as shown by significant reduction of body weight loss, epithelial permeability, rectal bleeding, colonic shortening, ulceration, inflammatory cell infiltration and goblet cell loss. Furthermore, Fxr activation in intestines of WT mice and differentiated enterocyte-like cells downregulates expression of key proinflammatory cytokines and preserves epithelial barrier function. INT-747 significantly decreases tumour necrosis factor α secretion in activated human peripheral blood mononuclear cells, purified CD14 monocytes and dendritic cells, as well as in lamina propria mononuclear cells from patients with IBD. CONCLUSIONS: FXR activation prevents chemically induced intestinal inflammation, with improvement of colitis symptoms, inhibition of epithelial permeability, and reduced goblet cell loss. Furthermore, FXR activation inhibits proinflammatory cytokine production in vivo in the mouse colonic mucosa, and ex vivo in different immune cell populations. The findings provide a rationale to explore FXR agonists as a novel therapeutic strategy for IBD.


Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Absorción Intestinal/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Células CACO-2 , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Colon/metabolismo , Citocinas/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Íleon/metabolismo , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/fisiopatología , Absorción Intestinal/fisiología , Ratones , Ratones Endogámicos C57BL , Receptores Citoplasmáticos y Nucleares/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ácido Trinitrobencenosulfónico , Factor de Necrosis Tumoral alfa/biosíntesis
6.
Hepatology ; 52(4): 1341-9, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20842631

RESUMEN

UNLABELLED: Pregnancy alters bile acid homeostasis and can unmask cholestatic disease in genetically predisposed but otherwise asymptomatic individuals. In this report, we show that normal pregnant mice have raised hepatic bile acid levels in the presence of procholestatic gene expression. The nuclear receptor farnesoid X receptor (FXR) regulates the transcription of the majority of these genes, and we show that both ablation and activation of Fxr prevent the accumulation of hepatic bile acids during pregnancy. These observations suggest that the function of Fxr may be perturbed during gestation. In subsequent in vitro experiments, serum from pregnant mice and humans was found to repress expression of the Fxr target gene, small heterodimer partner (Shp), in liver-derived Fao cells. Estradiol or estradiol metabolites may contribute to this effect because coincubation with the estrogen receptor (ER) antagonist fulvestrant (ICI 182780) abolished the repressive effects on Shp expression. Finally, we report that ERα interacts with FXR in an estradiol-dependent manner and represses its function in vitro. CONCLUSION: Ligand-activated ERα may inhibit FXR function during pregnancy and result in procholestatic gene expression and raised hepatic bile acid levels. We propose that this could cause intrahepatic cholestasis of pregnancy in genetically predisposed individuals.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Hígado/metabolismo , Preñez/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Estradiol/análogos & derivados , Estradiol/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Femenino , Fulvestrant , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Embarazo , Preñez/sangre , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis
7.
JHEP Rep ; 3(5): 100344, 2021 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-34604725

RESUMEN

BACKGROUND & AIMS: The interorgan crosstalk between the liver and the intestine has been the focus of intense research. Key in this crosstalk are bile acids, which are secreted from the liver into the intestine, interact with the microbiome, and upon absorption reach back to the liver. The bile acid-activated farnesoid X receptor (Fxr) is involved in the gut-to-liver axis. However, liver-to-gut communication and the roles of bile acids and Fxr remain elusive. Herein, we aim to get a better understanding of Fxr-mediated liver-to-gut communication, particularly in colon functioning. METHODS: Fxr floxed/floxed mice were crossed with cre-expressing mice to yield Fxr ablation in the intestine (Fxr-intKO), liver (Fxr-livKO), or total body (Fxr-totKO). The effects on colonic gene expression (RNA sequencing), the microbiome (16S sequencing), and mucus barrier function by ex vivo imaging were analysed. RESULTS: Despite relatively small changes in biliary bile acid concentration and composition, more genes were differentially expressed in the colons of Fxr-livKO mice than in those of Fxr-intKO and Fxr-totKO mice (3272, 731, and 1824, respectively). The colons of Fxr-livKO showed increased expression of antimicrobial genes, Toll-like receptors, inflammasome-related genes and genes belonging to the 'Mucin-type O-glycan biosynthesis' pathway. Fxr-livKO mice have a microbiome profile favourable for the protective capacity of the mucus barrier. The thickness of the inner sterile mucus layer was increased and colitis symptoms reduced in Fxr-livKO mice. CONCLUSIONS: Targeting of FXR is at the forefront in the battle against metabolic diseases. We show that ablation of Fxr in the liver greatly impacts colonic gene expression and increased the colonic mucus barrier. Increasing the mucus barrier is of utmost importance to battle intestinal diseases such as inflammatory bowel disease, and we show that this might be done by antagonising FXR in the liver. LAY SUMMARY: This study shows that the communication of the liver to the intestine is crucial for intestinal health. Bile acids are key players in this liver-to-gut communication, and when Fxr, the master regulator of bile acid homoeostasis, is ablated in the liver, colonic gene expression is largely affected, and the protective capacity of the mucus barrier is increased.

8.
Mol Metab ; 30: 221-229, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31767173

RESUMEN

OBJECTIVE: Coupling metabolic and reproductive pathways is essential for the survival of species. However, the functions of steroidogenic enzymes expressed in metabolic tissues are largely unknown. METHODS AND RESULTS: Here, we show that in the liver, the classical steroidogenic enzyme Cyp17a1 forms an essential nexus for glucose and ketone metabolism during feed-fast cycles. Both gain- and loss-of-function approaches are used to show that hepatic Cyp17a1 is induced by fasting, catalyzes the production of at least one hormone-ligand (DHEA) for the nuclear receptor PPARα, and is ultimately required for maintaining euglycemia and ketogenesis during nutrient deprivation. The feedback-loop that terminates Cyp17a1-PPARα activity, and re-establishes anabolic liver metabolism during re-feeding is mapped to postprandial bile acid-signaling, involving the receptors FXR, SHP and LRH-1. CONCLUSIONS: Together, these findings represent a novel paradigm of homeostatic control in which nutritional cues feed-forward on to metabolic pathways by influencing extragonadal steroidogenesis.


Asunto(s)
Hígado/metabolismo , PPAR alfa/metabolismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Glucosa/metabolismo , Células HEK293 , Hepatocitos/metabolismo , Homeostasis , Humanos , Cetonas/metabolismo , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidación-Reducción , Receptores Citoplasmáticos y Nucleares , Transducción de Señal , Esteroide 17-alfa-Hidroxilasa/fisiología
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