ABSTRACT
In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment1. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes1. Here we use a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. Our findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis.
Subject(s)
Chloride Channels/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Eating/physiology , Intestines/physiology , Zinc/metabolism , Animals , Drosophila melanogaster/genetics , Enterocytes/metabolism , Female , Food Preferences , Homeostasis , Insect Vectors , Insulin/metabolism , Ion Channel Gating , Larva/genetics , Larva/growth & development , Larva/metabolism , Lysosomes/metabolism , Male , Oocytes/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , XenopusABSTRACT
Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.
Subject(s)
Cell Differentiation , Cell Proliferation , Epithelial Cells/physiology , Intestinal Mucosa/physiology , Proto-Oncogene Proteins c-ret/metabolism , Animals , Drosophila , Gene Expression Regulation , Humans , Mice , Wnt Signaling PathwayABSTRACT
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.
Subject(s)
Energy Metabolism , Hepatocytes/metabolism , Liver/metabolism , Nucleotide Motifs , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Binding Sites , Gene Expression Regulation , Hep G2 Cells , Humans , Liver/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Organoids/cytology , Organoids/metabolism , Protein Binding , Protein Isoforms , Receptors, Cytoplasmic and Nuclear/geneticsABSTRACT
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.
Subject(s)
Ammonia/metabolism , Glutamine/biosynthesis , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Urea/metabolism , Animals , Bile Acids and Salts/metabolism , Chenodeoxycholic Acid/analogs & derivatives , Chenodeoxycholic Acid/pharmacology , Dietary Proteins/administration & dosage , Gene Expression , Hepatocytes , Liver/enzymology , Male , Metabolome , Mice , Mice, Inbred C57BL , Mice, Knockout , Proteome , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitorsABSTRACT
Inflammatory Bowel Disease (IBD) is a multifactorial disorder involving dysregulation of the immune response and bacterial translocation through the intestinal mucosal barrier. Previously, we have shown that activation of the bile acid sensor Farnesoid X Receptor (FXR), which belongs to the family of nuclear receptors, improves experimental intestinal inflammation, decreasing expression of pro-inflammatory cytokines and protecting the intestinal barrier. Here, we aimed to investigate the immunological mechanisms that ameliorate colitis when FXR is activated. We analyzed by FACS immune cell populations in mesenteric lymph nodes (MLN) and in the spleen to understand whether FXR activation alters the systemic immune response. We show that FXR activation by obeticholic acid (OCA) has systemic anti-inflammatory effects that include increased levels of plasma IL-10, inhibition of both DSS-colitis associated decrease in splenic dendritic cells (DCs) and increase in Tregs. Impact of OCA on DC relative abundance was seen in spleen but not MLN, possibly related to the increased FXR expression in splenic DCs compared to MLN DCs. Moreover, FXR activation modulates the chemotactic environment in the colonic site of inflammation, as Madcam1 expression is decreased, while Ccl25 is upregulated. Together, our data suggest that OCA treatment elicits an anti-inflammatory immune status including retention of DCs in the spleen, which is associated with decreased colonic inflammation. Pharmacological FXR activation is therefore an attractive new drug target for treatment of IBD.
Subject(s)
Colitis/chemically induced , Colitis/immunology , Dendritic Cells/immunology , Dextran Sulfate , Receptors, Cytoplasmic and Nuclear/immunology , Spleen/immunology , Animals , Chemotaxis , Colitis/pathology , Colon/cytology , Colon/immunology , Colon/pathology , Dendritic Cells/pathology , Interleukin-10/immunology , Male , Mice, Inbred C57BL , Spleen/cytology , Spleen/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologyABSTRACT
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.
Subject(s)
Colon/cytology , Ileum/cytology , Liver/cytology , Organoids/cytology , Receptors, Cytoplasmic and Nuclear/genetics , Stem Cells/cytology , Transcriptome , Animals , Colon/metabolism , Gene Expression , Gene Expression Regulation , Ileum/metabolism , Liver/metabolism , Mice , Organ Culture Techniques/methods , Organoids/metabolism , RNA, Messenger/genetics , Stem Cells/metabolismABSTRACT
BACKGROUND & AIMS: The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. METHODS: hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. RESULTS: Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTß), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. CONCLUSIONS: Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS.
Subject(s)
Chenodeoxycholic Acid/analogs & derivatives , DNA/genetics , Gene Expression Regulation , Liver/pathology , Non-alcoholic Fatty Liver Disease/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Animals , Chenodeoxycholic Acid/pharmacology , Disease Models, Animal , Gene Expression Profiling , Humans , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Microarray Analysis , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/metabolism , Polymerase Chain Reaction , Promoter Regions, Genetic , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/biosynthesis , Transcriptional ActivationABSTRACT
The farnesoid X receptor (FXR) is a ligand-activated transcription factor belonging to the nuclear receptor (NR) superfamily. FXR plays an important role in positively regulating genes (transactivation) involved in bile acid homeostasis, fat and glucose metabolism. Recently, it has become clear that an additional important role for FXR consists of downregulating genes involved in inflammation. Because of this broad spectrum of regulated genes, therapeutically targeting FXR with full agonists will likely result in adverse side effects, in line with what is described for other NRs. It may therefore be necessary to develop selective FXR modulators. However, the molecular mechanisms that distinguish between FXR-mediated transactivation and transrepression are currently unknown. For other NRs, post-translational modifications such as SUMOylation and phosphorylation have been reported to be unique to either transactivation or transrepression. Here, we review current knowledge on post-translational regulation of FXR with respect to transactivation and transrepression. Ultimately, increased understanding of the different mechanisms of transactivation and transrepression of nuclear receptors will aid in the development of NR drugs with fewer side effects.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Bile Acids and Salts/metabolism , Inflammation/drug therapy , RNA-Binding Proteins/metabolism , Humans , Protein Processing, Post-TranslationalABSTRACT
Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.
Subject(s)
DAX-1 Orphan Nuclear Receptor , Estrogen Receptor alpha , Hypothalamus , Kisspeptins , Animals , Female , Mice , Arcuate Nucleus of Hypothalamus/metabolism , Estradiol/metabolism , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Hypothalamus/metabolism , Kisspeptins/genetics , Kisspeptins/metabolism , DAX-1 Orphan Nuclear Receptor/genetics , DAX-1 Orphan Nuclear Receptor/metabolismABSTRACT
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.
Subject(s)
Bile Acids and Salts/metabolism , Liver/metabolism , Pregnancy, Animal/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Animals , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/metabolism , Female , Fulvestrant , Gene Expression Profiling , Humans , Mice , Mice, Inbred C57BL , Pregnancy , Pregnancy, Animal/blood , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesisABSTRACT
Rodents undergo gestational hepatomegaly to meet the increased metabolic demands on the maternal liver during pregnancy. This is an important physiological process, but the mechanisms and signals driving pregnancy-induced liver growth are not known. Here, we show that liver growth during pregnancy precedes maternal body weight gain, is proportional to fetal number, and is a result of hepatocyte hypertrophy associated with cell-cycle progression, polyploidy, and altered expression of cell-cycle regulators p53, Cyclin-D1, and p27. Because circulating reproductive hormones and bile acids are raised in normal pregnant women and can cause liver growth in rodents, these compounds are candidates for the signal driving gestational liver enlargement in rodents. Administration of pregnancy levels of reproductive hormones was not sufficient to cause liver growth, but mouse pregnancy was associated with increased serum bile acid levels. It is known that the bile acid sensor Fxr is required for normal recovery from partial hepatectomy, and we demonstrate that Fxr(-/-) mice undergo gestational liver growth by adaptive hepatocyte hyperplasia. This is the first identification of any component that is required to maintain the normal mechanisms of gestational hepatomegaly and also implicates Fxr in a physiologically normal process that involves control of the hepatocyte cell cycle. Understanding pregnancy-induced hepatocyte hypertrophy in mice could suggest mechanisms for safely increasing functional liver capacity in women during increased metabolic demand.
Subject(s)
Liver Regeneration/physiology , Liver/physiology , Pregnancy, Animal/physiology , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Cholates/metabolism , Cholates/pharmacology , Constitutive Androstane Receptor , Cyclin D1/genetics , Cyclin D1/metabolism , Energy Metabolism/physiology , Female , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/physiology , Liver/cytology , Liver/growth & development , Mice , Mice, Mutant Strains , Ovariectomy , Polyploidy , Pregnancy , S Phase/drug effects , S Phase/physiologyABSTRACT
The intestinal-derived secondary bile acid (BA) lithocholic acid (LCA) is hepatotoxic and is implicated in the pathogenesis of cholestatic diseases. LCA is an endogenous ligand of the xenobiotic nuclear receptor pregnane X receptor (PXR), but there is currently no consensus on the respective roles of hepatic and intestinal PXR in mediating protection against LCA in vivo. Under the conditions reported here, we show that mice lacking Pxr are resistant to LCA-mediated hepatotoxicity. This unexpected phenotype is found in association with enhanced urinary BA excretion and elevated basal expression of drug metabolism enzymes and the hepatic sulfate donor synthesis enzyme Papss2 in Pxr(-/-) mice. By subsequently comparing molecular responses to dietary and intraperitoneal administration of LCA, we made two other significant observations: 1) LCA feeding induces intestinal, but not hepatic, drug-metabolizing enzymes in a largely Pxr-independent manner; and 2) in contrast to LCA feeding, bypassing first-pass gut transit by intraperitoneal administration of LCA did induce hepatic detoxification machinery and in a Pxr-dependent manner. These data reconcile important discrepancies in the reported molecular responses to this BA and suggest that Pxr plays only a limited role in mediating responses to gut-derived LCA. Furthermore, the route of administration must be considered in the future planning and interpretation of experiments designed to assess hepatic responses to BAs, orally administered pharmaceuticals, and dietary toxins.
Subject(s)
Inactivation, Metabolic/physiology , Intestinal Mucosa/metabolism , Lithocholic Acid/pharmacokinetics , Liver/metabolism , Receptors, Steroid/metabolism , Administration, Oral , Alanine Transaminase/blood , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Bile Acids and Salts/blood , Bile Acids and Salts/metabolism , Bile Acids and Salts/urine , Body Weight/drug effects , Chemical and Drug Induced Liver Injury/blood , Chemical and Drug Induced Liver Injury/pathology , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P450 Family 2 , Enzyme Induction/drug effects , Female , Gene Expression/drug effects , Injections, Intraperitoneal , Intestines/drug effects , Lithocholic Acid/administration & dosage , Lithocholic Acid/pharmacology , Liver/drug effects , Liver/pathology , Male , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Multienzyme Complexes/genetics , Pregnane X Receptor , Steroid Hydroxylases/genetics , Sulfate Adenylyltransferase/genetics , Sulfotransferases/geneticsABSTRACT
The nuclear hormone receptor Dax1 functions during development as a testes-determining gene. However, the phenotype of male mice lacking Dax1 is strain-dependent due to the background-specific abundance of male-determining Sry gene-transcripts. We hypothesised that inter-individual variation in Sry mRNA-abundance would result in a spectrum of phenotypes even within-strain. We found that while all XY C57BL/6J mice lacking Dax1 presented as phenotypic females, there was a marked inter-individual variability in measures of fertility. Indeed, we report rare occasions where sex-reversed mice had measures of fertility comparable to those in control females. On two occasions, these sex-reversed XY mice were able to give birth to live offspring following mating to stud-males. As such, this work documents within-strain variability in phenotypes of XY mice lacking Dax1, and reports for the first time a complete sex-reversal capable of achieving live birth in these mice.
Subject(s)
DAX-1 Orphan Nuclear Receptor/genetics , Disorders of Sex Development/genetics , Sex Determination Processes/physiology , Sex-Determining Region Y Protein/genetics , Testis/physiology , Animals , Biological Variation, Individual , Female , Fertility , Genetic Background , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Parturition , PhenotypeABSTRACT
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.
Subject(s)
Liver/metabolism , PPAR alpha/metabolism , Steroid 17-alpha-Hydroxylase/metabolism , Animals , Bile Acids and Salts/metabolism , Glucose/metabolism , HEK293 Cells , Hepatocytes/metabolism , Homeostasis , Humans , Ketones/metabolism , Lipogenesis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxidation-Reduction , Receptors, Cytoplasmic and Nuclear , Signal Transduction , Steroid 17-alpha-Hydroxylase/physiologyABSTRACT
Fibroblast growth factor 19 (FGF19) is a gut-derived peptide hormone that is produced following activation of Farnesoid X Receptor (FXR). FGF19 is secreted and signals to the liver, where it contributes to the homeostasis of bile acid (BA), lipid and carbohydrate metabolism. FGF19 is a promising therapeutic target for the metabolic syndrome and cholestatic diseases, but enthusiasm for its use has been tempered by FGF19-mediated induction of proliferation and hepatocellular carcinoma. To inform future rational design of FGF19-variants, we have conducted temporal quantitative proteomic and gene expression analyses to identify FGF19-targets related to metabolism and proliferation. Mice were fasted for 16 hours, and injected with human FGF19 (1 mg/kg body weight) or vehicle. Liver protein extracts (containing "light" lysine) were mixed 1:1 with a spike-in protein extract from 13C6-lysine metabolically labelled mouse liver (containing "heavy" lysine) and analysed by LC-MS/MS. Our analyses provide a resource of FGF19 target proteins in the liver. 189 proteins were upregulated (≥ 1.5 folds) and 73 proteins were downregulated (≤ -1.5 folds) by FGF19. FGF19 treatment decreased the expression of proteins involved in fatty acid (FA) synthesis, i.e., Fabp5, Scd1, and Acsl3 and increased the expression of Acox1, involved in FA oxidation. As expected, FGF19 increased the expression of proteins known to drive proliferation (i.e., Tgfbi, Vcam1, Anxa2 and Hdlbp). Importantly, many of the FGF19 targets (i.e., Pdk4, Apoa4, Fas and Stat3) have a dual function in both metabolism and cell proliferation. Therefore, our findings challenge the development of FGF19-variants that fully uncouple metabolic benefit from mitogenic potential.
Subject(s)
Energy Metabolism , Fibroblast Growth Factors/pharmacology , Liver/metabolism , Proteome , Proteomics , Animals , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Energy Metabolism/drug effects , Energy Metabolism/genetics , Gene Expression , Humans , Liver/drug effects , Male , Mice , Proteomics/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant ProteinsABSTRACT
The Farnesoid X receptor (FXR) regulates bile salt, glucose and cholesterol homeostasis by binding to DNA response elements, thereby activating gene expression (direct transactivation). FXR also inhibits the immune response via tethering to NF-κB (tethering transrepression). FXR activation therefore has therapeutic potential for liver and intestinal inflammatory diseases. We aim to identify and develop gene-selective FXR modulators, which repress inflammation, but do not interfere with its metabolic capacity. In a high-throughput reporter-based screen, mometasone furoate (MF) was identified as a compound that reduced NF-κB reporter activity in an FXR-dependent manner. MF reduced mRNA expression of pro-inflammatory cytokines, and induction of direct FXR target genes in HepG2-GFP-FXR cells and intestinal organoids was minor. Computational studies disclosed three putative binding modes of the compound within the ligand binding domain of the receptor. Interestingly, mutation of W469A residue within the FXR ligand binding domain abrogated the decrease in NF-κB activity. Finally, we show that MF-bound FXR inhibits NF-κB subunit p65 recruitment to the DNA of pro-inflammatory genes CXCL2 and IL8. Although MF is not suitable as selective anti-inflammatory FXR ligand due to nanomolar affinity for the glucocorticoid receptor, we show that separation between metabolic and anti-inflammatory functions of FXR can be achieved.
Subject(s)
Gene Expression Regulation , Inflammation/genetics , Inflammation/metabolism , Mometasone Furoate/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Gene Expression , Gene Expression Regulation/drug effects , Genes, Reporter , Hep G2 Cells , Humans , Ligands , Mice , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Mometasone Furoate/chemistry , Mometasone Furoate/pharmacology , NF-kappa B/metabolism , Promoter Regions, Genetic , Protein Binding , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND AND AIMS: Intrahepatic cholestasis of pregnancy (ICP) is characterized by liver impairment, pruritus, and elevated maternal serum bile acids. It can cause premature delivery and intrauterine death. Bile acid synthesis, metabolism, and transport are regulated by the bile acid sensor FXR, and we hypothesized that genetic variation in FXR confers susceptibility to ICP. METHODS: The coding regions and intron/exon boundaries of FXR were sequenced in 92 British ICP cases of mixed ethnicity. Subsequently, a case-control study of allele frequencies of these variants in 2 independent cohorts of Caucasian ICP patients and controls was performed. Variants were cloned into an FXR expression plasmid and tested in functional assays. RESULTS: We identified 4 novel heterozygous FXR variants (-1g>t, M1V, W80R, M173T) in ICP. W80R was not present in Caucasians and M1V was detected uniquely in 1 British case. M173T and -1g>t occur both in Caucasian cases and controls, and we found a significant association of M173T with ICP (OR, 3.2; 95% confidence interval, 1.1-11.2; P = .02) when the allele frequencies of both Caucasian cohorts were analyzed together. We demonstrate functional defects in either translation efficiency or activity for 3 of the 4 variants (-1g>t, M1V, M173T). CONCLUSIONS: This is the first report of functional variants in FXR. We propose that these variants may predispose to ICP, and because FXR has a central role in regulating bile and lipid homeostasis they may be associated with other cholestatic and dyslipidemic disorders.