Role of FXR in the Adaptive Response to Bile Acids during Pregnancy

The bile acid receptor FXR maintains bile acid homeostasis through the dynamic regulation of transporters, detoxification and biosynthesis enzymes. Intrahepatic cholestasis of pregnancy (ICP) is caused by disturbance in bile acid homeostasis in predisposed individuals. The first genetic variants in FXR, identified in an ICP population, are investigated in this thesis using in vitro approaches and a functional effect is demonstrated. Reproductive hormones are implicated in the aetiology of ICP but experiments in mice suggest that estrogen and progesterone alone are unlikely to be the cause of gestational cholestasis. Pregnancy increases the metabolic demand on the maternal liver and, in mice, causes maternal hepatomegaly that is associated with pro-cholestatic gene expression and raised serum and hepatic bile acids. Gestational hepatomegaly is characterised in detail for the first time and Fxr is shown to be required to maintain the normal mechanisms driving this process. Furthermore, pro-cholestatic gene expression and the fact that hepatic bile acids do not accumulate in pregnant Fxr-/- or cholate-fed mice suggest that for unknown reasons, gestation could be a period of reduced Fxr function. Conclusions: Pregnancy causes perturbed bile acid homeostasis in mice. This may be a result of Fxr playing a complex role in mediating, or responding to, the metabolic demands of gestation. Furthermore, FXR protects individuals from developing pregnancy-specific cholestatic disease

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

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.Peer reviewe

Role of FXR in the adaptive response to bile acids during pregnancy

The bile acid receptor FXR maintains bile acid homeostasis through the dynamic regulation of transporters, detoxification and biosynthesis enzymes. Intrahepatic cholestasis of pregnancy (ICP) is caused by disturbance in bile acid homeostasis in predisposed individuals. The first genetic variants in FXR, identified in an ICP population, are investigated in this thesis using in vitro approaches and a functional effect is demonstrated. Reproductive hormones are implicated in the aetiology of ICP but experiments in mice suggest that estrogen and progesterone alone are unlikely to be the cause of gestational cholestasis. Pregnancy increases the metabolic demand on the maternal liver and, in mice, causes maternal hepatomegaly that is associated with pro-cholestatic gene expression and raised serum and hepatic bile acids. Gestational hepatomegaly is characterised in detail for the first time and Fxr is shown to be required to maintain the normal mechanisms driving this process. Furthermore, pro-cholestatic gene expression and the fact that hepatic bile acids do not accumulate in pregnant Fxr-/- or cholate-fed mice suggest that for unknown reasons, gestation could be a period of reduced Fxr function. Conclusions: Pregnancy causes perturbed bile acid homeostasis in mice. This may be a result of Fxr playing a complex role in mediating, or responding to, the metabolic demands of gestation. Furthermore, FXR protects individuals from developing pregnancy-specific cholestatic disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Quantitative liver proteomics identifies FGF19 targets that couple metabolism and proliferation

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

Splenic dendritic cell involvement in FXR-mediated amelioration of DSS colitis

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

FGF19 modulates expression of proteins involved in metabolism and cell survival.

<p>(A) IPA of pathways enriched in mice treated with FGF19 for 12h compared to vehicle control. For the analysis, proteins with fold change ≥1.3 FGF19 over vehicle were included. Pathways related to physiology or disease that were significantly enriched (p-value < 0.01) are ranked in function of their activation z-score and grouped into functional classes. (B) Venn Diagram representation of proteins changed upon FGF19 treatment that are involved in metabolism and cell survival/cancer, inferred from the metabolic (#) and cell survival (*) pathways depicted in panel A. Fold change upon FGF19 treatment for proteins classified in metabolism, cell survival/cancer pathways or both is shown.</p

FGF19-mediated regulation of liver protein expression resolved by quantitative proteomics.

<p>(A) Schematic representation of the experimental outline to determine the hepatic proteomic profile of mice treated with FGF19 or Veh for 12 h. (n = 3) (B) Frequency plot of proteins identified in vehicle-treated Wt mice based on their total log2 heavy/light normalized ratio. The plot is representative of mean Wt untreated condition to show the basal efficiency of the heavy spike-in added to the light samples. Percentage of proteins with a log2 heavy/light normalized ratio included in interval (-1,+1) is shown. (C) Protein ranking based on changes of the log2 light/heavy normalized ratio induced by FGF19 when comparing FGF19 treatment for 12h to vehicle control. Number of proteins, of which expression was decreased (≤ -1.5 fold), unchanged or increased (≥1.5 fold) are indicated. (C) Volcano plot depicting the protein changes induced by FGF19 after 12h treatment. Plots are accompanied by tables listing the significant upregulated or downregulated proteins with fold change >1.5 (n = 3; p<0.05).</p

FGF19 elicits expression changes in target genes of tumorigenic regulators.

<p>(A) Ingenuity upstream regulator analysis applied to protein changes observed upon FGF19 treatment for 12h. Prediction of upstream regulators is based on the overlap between the dataset proteins and the genes that are regulated by a transcription factor/hormone/compound, based on the knowledge included in Ingenuity database (overlap p-value <0.01). Green bars, upstream regulators with positive activation z-score; grey bars, upstream regulators with negative activation z-score.</p

Intestinal Detoxification Limits the Activation of Hepatic Pregnane X Receptor by Lithocholic AcidS⃞

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

FGF19 stimulation affects mRNA expression of genes involved in metabolism and cell survival.

<p>(A) Schematic representation of the experimental outline to determine gene expression changes occurring upon FGF19 treatment for 0, 15 min, 1h, 2h, 4h and 12h. (B) Hepatic expression of genes involved in metabolism (Cyp7a1, Acox1, Acsl3), proliferation (Egfr, c-Fos, Hdlbp, Anxa2) or both (Stat3, Apoa4, Apoe, Fas, Gtpbp4) was determined by Real Time qPCR. (n = 5–6). Data are normalized to Gapdh expression and expressed as mean ± SEM.</p