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Background & Aims Serum microRNA (miRNA) levels are known to change in non-alcoholic fatty liver disease (NAFLD) and may serve as useful biomarkers. This study aimed to profile miRNAs comprehensively at all NAFLD stages. Methods We profiled 2,083 serum miRNAs in a discovery cohort (183 cases with NAFLD representing the complete NAFLD spectrum and 10 population controls). miRNA libraries generated by HTG EdgeSeq were sequenced by Illumina NextSeq. Selected serum miRNAs were profiled in 372 additional cases with NAFLD and 15 population controls by quantitative reverse transcriptase PCR. Results Levels of 275 miRNAs differed between cases and population controls. Fewer differences were seen within individual NAFLD stages, but miR-193a-5p consistently showed increased levels in all comparisons. Relative to NAFL/non-alcoholic steatohepatitis (NASH) with mild fibrosis (stage 0/1), 3 miRNAs (miR-193a-5p, miR-378d, and miR378d) were increased in cases with NASH and clinically significant fibrosis (stages 2–4), 7 (miR193a-5p, miR-378d, miR-378e, miR-320b, miR-320c, miR-320d, and miR-320e) increased in cases with NAFLD activity score (NAS) 5–8 compared with lower NAS, and 3 (miR-193a-5p, miR-378d, and miR-378e) increased but 1 (miR-19b-3p) decreased in steatosis, activity, and fibrosis (SAF) activity score 2–4 compared with lower SAF activity. The significant findings for miR-193a-5p were replicated in the additional cohort with NAFLD. Studies in Hep G2 cells showed that following palmitic acid treatment, miR-193a-5p expression decreased significantly. Gene targets for miR-193a-5p were investigated in liver RNAseq data for a case subgroup (n = 80); liver GPX8 levels correlated positively with serum miR-193a-5p. Conclusions Serum miR-193a-5p levels correlate strongly with NAFLD activity grade and fibrosis stage. MiR-193a-5p may have a role in the hepatic response to oxidative stress and is a potential clinically tractable circulating biomarker for progressive NAFLD. Lay summary MicroRNAs (miRNAs) are small pieces of nucleic acid that may turn expression of genes on or off. These molecules can be detected in the blood circulation, and their levels in blood may change in liver disease including non-alcoholic fatty liver disease (NAFLD). To see if we could detect specific miRNA associated with advanced stages of NAFLD, we carried out miRNA sequencing in a group of 183 patients with NAFLD of varying severity together with 10 population controls. We found that a number of miRNAs showed changes, mainly increases, in serum levels but that 1 particular miRNA miR-193a-5p consistently increased. We confirmed this increase in a second group of cases with NAFLD. Measuring this miRNA in a blood sample may be a useful way to determine whether a patient has advanced NAFLD without an invasive liver biopsy., Graphical abstract, Highlights • Serum miRNA was sequenced in 183 NAFLD cases of varying severity and 10 population controls. • Plasma levels of miR-193a-5p were significantly increased in patients with advanced fibrosis, high NAS scores, or high SAF scores. • Other miRNAs including miR378d and miR378e were also significantly increased in certain comparisons. • The findings for miR-193a-5p were replicated in a cohort of 372 additional NAFLD cases.

The Farnesoid X receptor (FXR) is a nuclear receptor which is activated by bile acids. Bile acids function in solubilization of dietary fats and vitamins in the intestine. In addition, bile acids have been increasingly recognized to act as signaling molecules involved in energy metabolism pathways, amongst others via activating FXR. Upon activation by bile acids, FXR controls the expression of many genes involved in bile acid, lipid, glucose and amino acid metabolism. An inability to properly use and store energy substrates may predispose to metabolic disorders, such as obesity, diabetes, cholestasis and non-alcoholic fatty liver disease. These diseases arise through a complex interplay between genetics, environment and nutrition. Due to its function in metabolism, FXR is an attractive treatment target for these disorders. The regulation of FXR expression and activity occurs both at the transcriptional and at the post-transcriptional level. It has been shown that FXR can be phosphorylated, SUMOylated and acetylated, amongst other modifications, and that these modifications have functional consequences for DNA and ligand binding, heterodimerization and subcellular localization of FXR. In addition, these post-translational modifications may selectively increase or decrease transcription of certain target genes. In this review, we provide an overview of the posttranslational modifications of FXR and discuss their potential involvement in cholestatic and metabolic disorders.

Necrotizing enterocolitis (NEC) is an often lethal, inflammatory disease of the preterm intestine. The underdeveloped immune system plays an important role; however, the initial trigger for NEC development is likely a damaged intestinal epithelial layer. We hypothesize that due to incomplete maturation of different epithelial cell lineages, nutrients and bacteria are able to damage the epithelial cells and cause the (immature) inflammatory response, food intolerance and malabsorption seen in NEC. Intestinal organoid research has shown that maturation of intestinal epithelial cell lineages is orchestrated by two key signaling pathways: Wnt and Notch. In NEC, these pathways are dysregulated by hyperactivation of Toll-like-receptor-4. Breastfeeding decreases the risk of developing NEC compared to formula milk. Here, we review the intricate link between breast milk components, Wnt and Notch signaling and intestinal epithelial maturation. We argue that (nutritional) interventions regulating these pathways may decrease the risk of NEC development in preterm infants.

Metabolic nuclear receptors are ligand-activated transcription factors which control a wide range of metabolic processes and signaling pathways in response to nutrients and xenobiotics. Targeting these NRs is at the forefront of our endeavours to generate novel treatment options for diabetes, metabolic syndrome and fatty liver disease. Numerous splice variants have been described for these metabolic receptors. Structural changes, as a result of alternative splicing, lead to functional differences among NR isoforms, resulting in the regulation of different metabolic pathways by these NR splice variants. In this review, we describe known splice variants of FXR, LXRs, PXR, RXR, LRH-1, CAR and PPARs. We discuss their structure and functions, and elaborate on the regulation of splice variant abundance by nutritional signals. We conclude that NR splice variants pose an intriguing new layer of complexity in metabolic signaling, which needs to be taken into account in the development of treatment strategies for metabolic diseases.

Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample’s metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data. Electronic supplementary material The online version of this article (10.1007/s11306-020-01717-8) contains supplementary material, which is available to authorized users.

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.

The farnesoid X receptor (FXR) belongs to the nuclear receptor family and is activated by bile acids. Multiple, chemically rather diverse, FXR agonists have been developed and several of these compounds are currently tested in clinical trials for NAFLD and cholestasis. Here, we investigated possible FXR-agonism or antagonism of existing FDA/EMA-approved drugs. By using our recently developed FRET-sensor, containing the ligand binding domain of FXR (FXR-LBD), 1280 FDA-approved drugs were screened for their ability to activate FXR in living cells using flow cytometry. Fifteen compounds induced the sensor for more than twenty percent above background. Real-time confocal microscopy confirmed that avermectin B1a, gliquidone, nicardipine, bepridil and triclosan activated the FRET sensor within two minutes. These compounds, including fluticasone, increased mRNA expression of FXR target genes OSTα and OSTβ in Huh7 cells, and in most cases also of MRP2, SHP and FGF19. Finally, avermectin B1a, gliquidone, nicardipine and bepridil significantly increased IBABP promoter activity in a luciferase reporter assay in a dose-dependent manner. In conclusion, six FDA/EMA-approved drugs currently used in the clinical practice exhibit moderate agonistic FXR activity. This may on the one hand explain (undesired) side-effects, but on the other hand may form an opportunity for polypharmacology.

Nuclear receptors (NRs) are ligand-inducible transcription factors that play critical roles in metazoan development, reproduction, and physiology and therefore are implicated in a broad range of pathologies. The transcriptional activity of NRs critically depends on their interaction(s) with transcriptional coregulator proteins, including coactivators and corepressors. Short leucine-rich peptide motifs in these proteins (LxxLL in coactivators and LxxxIxxxL in corepressors) are essential and sufficient for NR binding. With 350 different coregulator proteins identified to date and with many coregulators containing multiple interaction motifs, an enormous combinatorial potential is present for selective NR-mediated gene regulation. However, NR-coregulator interactions have often been determined experimentally on a one-to-one basis across diverse experimental conditions. In addition, NR-coregulator interactions are difficult to predict because the molecular determinants that govern specificity are not well established. Therefore, many biologically and clinically relevant NR-coregulator interactions may remain to be discovered. Here, we present a comprehensive overview of 3696 NR-coregulator interactions by systematically characterizing the binding of 24 nuclear receptors with 154 coregulator peptides. We identified unique ligand-dependent NR-coregulator interaction profiles for each NR, confirming many well-established NR-coregulator interactions. Hierarchical clustering based on the NR-coregulator interaction profiles largely recapitulates the classification of NR subfamilies based on the primary amino acid sequences of the ligand-binding domains, indicating that amino acid sequence is an important, although not the only, molecular determinant in directing and fine-tuning NR-coregulator interactions. This NR-coregulator peptide interactome provides an open data resource for future biological and clinical discovery as well as NR-based drug design.

Bile acids are amphipathic molecules that were previously known to serve as fat solubilizers in the intestine in postprandial conditions. In the last two decades, bile acids have been recognized as signaling molecules regulating energy metabolism pathways via, amongst others, the farnesoid X receptor (FXR). Upon bile acid activation, FXR controls expression of genes involved in bile acid, lipid, glucose and amino acid metabolism. In addition, FXR activation has been shown to limit the inflammatory response. The central role of FXR in various aspects of metabolism and inflammation makes FXR an attractive drug target for several diseases, such as obesity, metabolic syndrome, non-alcoholic steatohepatitis, cholestasis and chronic inflammatory diseases of the liver and intestine. However, most of the currently available compounds impact on all discovered FXR-mediated functions and may have, on top of beneficial effects, undesired biological actions depending on the disease. Therefore, research efforts are increasingly focused on the development of selective FXR modulators, i.e. selective bile acid receptor modulators (SBARMs), aimed at limiting the potential side-effects of conventional full FXR agonists upon chronic treatment. Here, we review the rationale for the design of SBARMs comprising dissociation between metabolic and inflammatory signaling, gene-selective and tissue-specific targeting. We discuss the potential structural mechanisms underlying the binding properties of dissociating ligands of FXR in light of ongoing efforts on the generation of dissociated ligands for otxher nuclear receptors, as well as their pharmacological and therapeutic potential.

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 super family 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 Fxra, Lxr alpha, Lxr beta, Ppar alpha, and Ppary 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, Angl, 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. (C) 2016 Elsevier B.V. All rights reserved.

Guggulsterone is a promiscuous ligand for endocrine and metabolic lipid receptors traditionally used to treat a number of diseases including diabesity, hyperlipidemia, atherosclerosis, and osteoarthritis. Although relatively weak, its activity at the farnesoid X receptor (FXR) is particularly intriguing as guggulsterone acts as an antagonist with a peculiar ability of gene selective modulation. We report here a chemical biology study with the aim to further characterize the biological action of guggulsterone at the FXR and to obtain further insights into the functional role played by noncanonical FXR binding pockets S2 and S3. Our results suggest that the FXR accessory pockets might act as potential targets for small molecules able to modulate the metabolic activation of the receptor without affecting the anti-inflammatory activity thus revealing a new approach for disclosing selective FXR modulators that might bypass potential side-effects from chronic treatments.

The nuclear receptor Farnesoid X Receptor (FXR) is activated by bile acids and controls multiple metabolic processes, including bile acid, lipid, carbohydrate, amino acid and energy metabolism. Vitamin A is needed for proper metabolic and immune control and requires bile acids for efficient intestinal absorption and storage in the liver. Here, we analyzed whether FXR regulates vitamin A metabolism. Compared to control animals, FXR-null mice showed strongly reduced (>90%) hepatic levels of retinol and retinyl palmitate and a significant reduction in lecithin retinol acyltransferase (LRAT), the enzyme responsible for hepatic vitamin A storage. Hepatic reintroduction of FXR in FXR-null mice induced vitamin A storage in the liver. Hepatic vitamin A levels were normal in intestine-specific FXR-null mice. Obeticholic acid (OCA, 3 weeks) treatment rapidly reduced (>60%) hepatic retinyl palmitate levels in mice, concurrent with strongly increased retinol levels (>5-fold). Similar, but milder effects were observed in cholic acid (12 weeks)-treated mice. OCA did not change hepatic LRAT protein levels, but strongly reduced all enzymes involved in hepatic retinyl ester hydrolysis, involving mostly post-transcriptional mechanisms. In conclusion, vitamin A metabolism in the mouse liver heavily depends on the FXR and FXR-targeted therapies may be prone to cause vitamin A-related pathologies.

The amount of dietary protein is associated with intestinal disease in different vertebrate species. In humans, this is exemplified by the association between high-protein intake and fermentation metabolite concentrations in patients with inflammatory bowel disease. In production animals, dietary protein intake is associated with postweaning diarrhea in piglets and with the occurrence of wet litter in poultry. The underlying mechanisms by which dietary protein contributes to intestinal problems remain largely unknown. Fermentation of undigested protein in the hindgut results in formation of fermentation products including short-chain fatty acids, branched-chain fatty acids, ammonia, phenolic and indolic compounds, biogenic amines, hydrogen sulfide, and nitric oxide. Here, we review the mechanisms by which these metabolites may cause intestinal disease. Studies addressing how different metabolites induce epithelial damage rely mainly on cell culture studies and occasionally on mice or rat models. Often, contrasting results were reported. The direct relevance of such studies for human, pig, and poultry gut health is therefore questionable and does not suffice for the development of interventions to improve gut health. We discuss a roadmap to improve our understanding of gut metabolites and microbial species associated with intestinal health in humans and production animals and to determine whether these metabolite/bacterial networks cause epithelial damage. The outcomes of these studies will dictate proof-of-principle studies to eliminate specific metabolites and or bacterial strains and will provide the basis for interventions aiming to improve gut health.

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.

Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5-4 h) to 5-100 μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH. publisher: Elsevier articletitle: Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: Role of glycine conjugates journaltitle: Toxicology in Vitro articlelink: http://dx.doi.org/10.1016/j.tiv.2013.10.020 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved. ispartof: Toxicology in Vitro vol:28 issue:2 pages:218-230 ispartof: location:England status: published

Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in Forster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na+-taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter αβ (OSTαβ) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. Conclusion: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time. (HEPATOLOGY 2013)

Item does not contain fulltext 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 24h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7days. RESULTS: Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTalpha) and SLC51B (OSTbeta), 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.

Article first published online 10 August 2016., Background: Smoking has a negative impact on Crohn's disease (CD), but the mechanisms underlying this association are unclear. We compared the gut microbiota composition of smoking with nonsmoking patients with CD using a metagenomic approach. Methods: Stool samples and clinical data were collected from current smokers and nonsmokers with CD from France and the Netherlands, matched for country, gender, age, disease activity, and body mass index. Fecal DNA was sequenced on an Illumina HiSeq 2500. On average, 40 million paired-end reads were generated per sample. Gene richness and the Shannon index were computed to assess microbial diversity. Wilcoxon's signed-rank tests for paired samples were performed to detect differences between the 2 groups. Results: In total, 21 smoking and 21 nonsmoking patients with CD were included. Compared with nonsmoking patients, gut microbial gene richness (P = 0.01), genus diversity (P < 0.01), and species diversity (P = 0.01) were decreased in smoking patients. This was accompanied by a reduced relative abundance of the genera Collinsella (P = 0.02), Enterorhabdus (P = 0.02), and Gordonibacter (P = 0.02) in smokers. No statistically significant differences at the species level were observed, although smokers had lower proportions of Faecalibacterium prausnitzii (P = 0.10). Conclusions: Gut microbial diversity is reduced in smokers with CD compared with nonsmokers with CD. The microbial profile differs between these groups at the genus level. Future studies should evaluate whether intestinal microbes mediate the adverse effects of smoking in CD.

The gut epithelium is covered by mucus consisting of mucin polymers connected via disulfide bonds. The mucus layer limits exposure of epithelial cells to toxins and bacteria. A recent study has shown that sulfide, produced by certain bacteria, reduces disulfide bonds in the mucus network. The resulting breaks in the mucus barrier allow exposure of the epithelium to bacteria and toxins, causing inflammation. In this opinion article we argue that this mechanism may be involved in the etiology and/or severity of inflammatory bowel disease (IBD) because IBD is associated with decreased mucus barrier function, altered microbial species, and increased sulfide concentrations. Increasing the mucus integrity by reducing sulfide concentrations in the intestine may be a novel therapeutic option for IBD.

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 a 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. RI van Mil, Saskia/C-3751-2009

The Farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). BAs are amphipathic molecules that serve as fat solubilizers in the intestine under postprandial conditions. In the post-absorptive state, BAs bind FXR in the hepatocytes, which in turn provides feedback signals on BA synthesis and transport and regulates lipid, glucose and amino acid metabolism. Therefore, FXR acts as a homeostat of all three classes of nutrients, fats, sugars and proteins. Here we re-analyze the function of FXR in the perspective of nutritional metabolism, and discuss the role of FXR in liver energy homeostasis in postprandial, post-absorptive and fasting/starvation states. FXR, by regulating nutritional metabolism, represses autophagy in conditions of nutrient abundance, and controls the metabolic needs of proliferative cells. In addition, FXR regulates inflammation via direct effects and via its impact on nutrient metabolism. These functions indicate that FXR is an attractive therapeutic target for liver diseases.

Milona A, Owen BM, van Mil S, Dormann D, Mataki C, Boudjelal M, Cairns W, Schoonjans K, Milligan S, Parker M, White R, Williamson C. The normal mechanisms of pregnancy-induced liver growth are not maintained in mice lacking the bile acid sensor Fxr. Am J Physiol Gastrointest Liver Physiol 298: G151-G158, 2010. First published October 8, 2009; doi:10.1152/ajpgi.00336.2009.-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.

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.

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.

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.

Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.

FIC1 disease collectively refers to a group of autosomal-recessive familial liver disorders characterized by intrahepatic cholestasis due to mutations in the ATP8B1 gene (initially named FIC1). Classically, FIC1 disease comprises two different disorders: progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis (BRIC). However, we now view these two disorders as two ends of a continuum. Current therapeutic strategies for FIC1 disease, both medical and surgical, may relieve symptoms, but are presently insufficiently evaluated. ATP8B1 encodes a protein belonging to a recently defined subfamily of P-type ATPases. The biochemical and cellular functions of its product, FIC1, and the mechanisms by which its absence or dysfunction leads to cholestasis are currently elusive. Further studies to elucidate FIC1's function will be essential to unravel the pathogenesis of FIC1 disease. Such studies will also have a general impact on our understanding of the molecular mechanisms of bile formation and may therefore improve clinical management of both hereditary and acquired forms of cholestasis.

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.

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.

Progressive familial intrahepatic cholestasis (PFIC) type 1, 2 and 3 are due to mutations in ATP8B1, ABCB11 and ABCB4, respectively. Each of these genes encodes a hepatocanalicular transporter, which is essential for the proper formation of bile. Mutations in ABCB4 can result in progressive cholestatic disease, while mutations in ATP8B1 and ABCB11 can result both in episodic cholestasis, referred to as benign recurrent intrahepatic cholestasis (BRIC) type 1 and 2, as well as in progressive cholestatic disease. This suggests a clinical continuum and these diseases are therefore preferably referred to as ATP8B1 deficiency and ABCB11 deficiency. Similarly PFIC type 3 is designated as ABCB4 deficiency. Heterozygous mutations in each of these transporters can also be associated with intrahepatic cholestasis of pregnancy. This review summarizes the pathophysiology, clinical features and current as well as future therapeutic options for progressive familial- and benign recurrent intrahepatic cholestasis as well as intrahepatic cholestasis of pregnancy.

The nuclear receptor Farnesoid X Receptor (FXR) critically regulates nascent bile formation and bile acid enterohepatic circulation. Bile acids and FXR play a pivotal role in regulating hepatic inflammation and regeneration as well as in regulating extent of inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. Recent evidence suggests, that the bile acid-FXR interaction is involved in the pathophysiology of a wide range of diseases of the liver, biliary and gastrointestinal tract, such as cholestatic and inflammatory liver diseases and hepatocellular carcinoma, inflammatory bowel disease and inflammation-associated cancer of the colon and esophagus. In this review we discuss current knowledge of the role the bile acid-FXR interaction has in (patho)physiology of the liver, biliary and gastrointestinal tract, and proposed underlying mechanisms, based on in vitro data and experimental animal models. Given the availability of highly potent synthetic FXR agonists, we focus particularly on potential relevance for human disease.

Background & 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.

Mutations in ATP8B1 are associated with FIC1 disease, an autosomal recessive disorder in which intrahepatic cholestasis is the predominant manifestation. ATP8B1 encodes FIC1, which is expressed in several tissues, most prominently in the intestine, pancreas, and stomach and, to a much lesser extent, in the liver. In this study, Fic1 localization and expression during postnatal development was examined in healthy mice. Immunoblot and RT-PCR analysis indicated Fic1 is expressed abundantly in regions of the adult gastrointestinal tract of humans and mice. Immunohistochemistry revealed that Fic1 was localized to the apical membranes of enterocytes, pancreatic acinar cells, gastric pit epithelial cells, and hepatocytes and cholangiocytes. Subsequent analysis of early postnatal expression revealed that Fic1 expression in the small intestine was limited or absent at the age of 7 and 14 d and increased significantly with maturation. In contrast, pancreatic, hepatic, and gastric Fic1 expression was not diminished during the first 3 wk of postnatal development. In conclusion, these data show that Fic1 is expressed in a tissue-specific and developmentally regulated fashion at the apical membranes of epithelial cells. We speculate that the developing bile salt pool in the maturing intestine accounts for the increase in Fic1 protein expression in this tissue.

Background & Aims: Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are hereditary liver disorders; PFIC is characterized by severe progressive liver disease whereas BRIC patients have intermittent attacks of cholestasis without permanent liver damage. Mutations in ATP8B1 are present in PFIC type 1 and in a subset of BRIC patients. We hypothesized that a genetically distinct form of BRIC is associated with mutations in ABCB11 . This gene encodes the bile salt export pump (BSEP) and is mutated in PFIC type 2. Methods: Patients from 20 families were included; all had a normal ATP8B1 sequence. Sequencing of all 27 coding exons including the splice junctions of ABCB11 revealed 8 distinct mutations in 11 patients from 8 different families: one homozygous missense mutation (E297G) previously described in PFIC2 patients, 6 novel missense mutations, and one putative splice site mutation. Results: In 12 families, no mutations in ATB8B1 or ABCB11 were detected. Pancreatitis is a known extrahepatic symptom in BRIC caused by ATP8B1 mutations, but was not present in BRIC patients with mutations in ABCB11 . In contrast, cholelithiasis was observed in 7 of 11 BRIC patients with mutations in ABCB11 , but has not been described in ATP8B1 -affected BRIC patients. Conclusions: Mutations in ABCB11 are associated with BRIC, and consistent with the genetic classification of PFIC into 2 subtypes, we propose that this disorder be named BRIC type 2.

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1-3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened.

Background/Aims : Progressive familial intrahepatic cholestasis characterized by normal serum gamma-glutamyltransferase activity can be due to mutations in familial intrahepatic cholestasis type 1 ( FIC1 ) ( ATP8B1 ), a gene expressed in several organs. In some cases, it is associated with extrahepatic features. We searched for FIC1 mutations and analyzed the outcome of extrahepatic features after liver transplantation in two children with this form of progressive familial intrahepatic cholestasis associated with chronic unexplained diarrhea and short stature. Methods : FIC1 sequence was determined after polymerase chain reaction (PCR) of genomic lymphocyte DNA and/or reverse transcription-PCR of liver or lymphocyte RNA. Results : A homozygous amino acid change deletion was found in one child. The second child harboured compound heterozygous missense and nonsense mutations. In both children, despite successful liver transplantation, evolution (follow-up: 9.5–11 years) was characterized by exacerbation of diarrhea and no catch-up of stature growth, and appearance of liver steatosis. Conclusions : Progressive familial intrahepatic cholestasis characterized by normal serum gamma-glutamyltransferase activity and extrahepatic features corresponds to progressive familial intrahepatic cholestasis type 1. Extrahepatic symptomatology is not corrected or may be aggravated by liver transplantation, impairing life quality.

Background/Aims : FIC1 (familial intrahepatic cholestasis 1) is affected in two clinically distinct forms of hereditary cholestasis, namely progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis. Here we examined the subcellular localization of this protein within the liver. Methods : Antibodies raised against different epitopes of human FIC1 were used for immunoblot analysis and immunohistochemical detection of FIC1. Results : Immunoblot analysis of intestine and liver tissue extracts from human, rat and mouse origin indicated that the antibodies raised against FIC1 specifically detected FIC1 as a 140-kDa protein. In the liver homogenate of a PFIC1 patient, FIC1 could not be detected. Analysis of isolated rat liver membrane vesicles indicated that this protein is predominantly present in the canalicular membrane fraction. Immunohistochemical detection of the protein in liver sections confirmed that FIC1 was present in the canalicular membrane, whereas no staining was observed in the PFIC1 patients liver. Double label immunofluorescence of murine liver revealed that FIC1 colocalized with cytokeratin 7 in cholangiocytes. Conclusions : The localization of FIC1 in the canalicular membrane and cholangiocytes suggests that it may directly or indirectly play a role in bile formation since mutations in FIC1 are associated with severe symptoms of cholestasis.

Background The bile acid-activated nuclear receptor Farnesoid X Receptor (FXR) is critical in maintaining intestinal barrier integrity and preventing bacterial overgrowth. Patients with Crohn's colitis (CC) exhibit reduced ileal FXR target gene expression. FXR agonists have been shown to ameliorate inflammation in murine colitis models. We here explore the feasibility of pharmacological FXR activation in CC. Methods Nine patients with quiescent CC and 12 disease controls were treated with the FXR ligand chenodeoxycholic acid (CDCA; 15 mg/kg/day) for 8 days. Ileal FXR activation was assessed in the fasting state during 6 hrs after the first CDCA dose and on day 8, by quantification of serum levels of fibroblast growth factor (FGF) 19. Since FGF19 induces gallbladder (GB) refilling in murine models, we also determined concurrent GB volumes by ultrasound. On day 8 ileal and cecal biopsies were obtained and FXR target gene expression was determined. Results At baseline, FGF19 levels were not different between CC and disease controls. After the first CDCA dose, there were progressive increases of FGF19 levels and GB volumes during the next 6 hours in CC patients and disease controls (FGF19: 576 resp. 537% of basal; GB volumes: 190 resp. 178% of basal) without differences between both groups, and a further increase at day 8. In comparison with a separate untreated control group, CDCA affected FXR target gene expression in both CC and disease controls, without differences between both groups. Conclusions Pharmacological activation of FXR is feasible in patients with CC. These data provide a rationale to explore the anti-inflammatory properties of pharmacological activation of FXR in these patients. Trial Registration TrialRegister.nl NTR2009

A Functional Variant of the Farnesoid X Receptor (FXR) Predisposes to Ileocolonic Localization of Crohn's Disease Rian M. Nijmeijer, Raffaella Maria Gadaleta, Karel J. Van Erpecum, Adriaan A. van Bodegraven, J. Bart A. Crusius, Gerard Dijkstra, Daniel W. Hommes, Dirk J. De Jong, Cyriel Ponsioen, Hein W. Verspaget, Rinse K. Weersma, Christien J. van der Woude, Marguerite E. Schipper, Cisca Wijmenga, Saskia W. van Mil, Bas Oldenburg

In 17 patients GC samples were reevaluated for histological characteristics, 14 were MSIH and 3 failed in MSI testing, however IHC stainings for the affected MMR protein were negative in all these patients. Six (35%), 4 (23%) and 7 (41%) GC samples showed intestinal type, diffuse type and mixed type characteristics respectively. In 13 GC samples, the tissue surrounding GC could be evaluated. Intestinal metaplasia was diagnosed in 5 (38%) samples and atrophic gastritis in 2 (15%) samples. Pre-malignant gastric lesions were evenly distributed throughout intestinal, diffuse and mixed type gastric cancer samples (p=0.40). Conclusions: This study demonstrates that GC develops along similar histopathologic pathways as sporadic gastric cancer in LS. Pre-malignant lesions in tissue surrounding GC were evident in >50% of samples. These data support upper GI surveillance endoscopy in LS subjects.

The bile salt nuclear receptor Farnesoid X Receptor (FXR) was recently implicated in intestinal antibacterial defense and barrier function. We aimed to study its role in pathogenesis of inflammatory bowel diseasE. colitis was induced in Wild Type (WT) and FXR knock-out (ko) mice (n=8 to 10 for each group) by Dextran Sodium Sulphate (DSS: 2.5% in drinking water, 10 days) with or without synthetic FXR ligand 6-Ethyl Chenodeoxycholic Acid (6ECDCA: 5 mg/kg/day, 13 days, starting 3 days before DSS). Colitis symptoms were checked daily and intestinal permeability (FITC-dextran assay), bile salt composition (HPLC), histology and colonic inflammatory gene expression (Q-PCR) determined. mRNA expression of FXR and target genes was determined in IBD patient biopsies. Underlying mechanisms were explored in complementary In Vitro experiments. 6ECDCA-treated WT but not FXR ko mice were protected from DSS-induced colitis, as shown by highly significant reduction of body weight loss, rectal bleeding, colonic shortening, normalization of intestinal permeability, 49% reduction in blinded histological score and 55% reduction in goblet cell loss. Only in 6ECDCA-treated WT mice, mRNA levels of pro-inflammatory genes (IL-1β, IL-6, IL10 and MCP-1) were strongly down-regulated while antibacterial defense gene iNOS was upregulated. 6ECDCA was enriched in bile of both WT and FXR ko mice (9.8% and 5.3% of total bile salts, respectively). However intestinal expression of FXR target genes FGF15 and SHP was increased 4.5and 19-fold with 6ECDCA treatment only in WT. In patients with quiescent Crohn colitis (n=17), mRNA expression of FXR and SHP was significantly altered compared to patients with ulcerative colitis (n=16) or healthy controls (n=17). In differentiated CaCo2 cells grown on trans-well plates, FXR activation by GW4064 prevented DSS-dependent loss of integrity of the monolayer. In differentiated HT29 cells, TNFαinduced 20-fold increase of IL-1β expression was abolished by GW4064-dependent FXR activation. In reporter assays, the FXR agonist GW4064 prevented TNFα-induced NF-κB activity in HEK293 cells transfected with WT FXR, but no effect was achieved with FXR mutant W469A (defective in Ligand Binding Domain), indicating FXR-mediated inhibition of NF-κB signalling. In conclusion, FXR activation protects against experimental murine colitis, supposedly by preserving the intestinal barrier and inhibitingNF-κB activity. Currently available potent synthetic FXR agonists may offer new therapeutic strategies for inflammatory bowel disease.

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., Highlights • The classical steroidogenic enzyme, Cyp17a1, is upregulated in liver during fasting. • CYP17a1 produces a hormone-ligand for the nuclear receptor PPARα and affects glucose and lipid handling in the liver. • Hepatic Cyp17a1 is essential for maintaining glycaemia and ketones during fasting. • Bile acids, via a nuclear receptor cascade, repress hepatic Cyp17a1 as part of the re-feeding response.

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-1β). 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-1β 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.