Intestinal FXR-mediated FGF15 production contributes to diurnal control of hepatic bile acid synthesis in mice

Abstract

Hepatic bile acid synthesis is subject to complex modes of transcriptional control, in which the bile acid-activated nuclear receptor farnesoid X receptor (FXR) in liver and intestine-derived, FXR-controlled fibroblast growth factor 15 (Fgf15) are involved. The Fgf15 pathway is assumed to contribute significantly to control of hepatic bile acid synthesis. However, scientific evidence supporting this assumption is primarily based on gene expression data. Using intestine-selective FXR knockout mice (iFXR-KO), we show that contribution of intestinal FXR-Fgf15 signalling in regulation of hepatic cholesterol 7α-hydroxylase (Cyp7A1) expression depends on time of the day with increased hepatic Cyp7A1 expression in iFXR-KO mice compared with controls exclusively during the dark phase. To assess the physiological relevance hereof, we determined effects of intestine-selective deletion of FXR on physiological parameters such as bile formation and kinetics of the enterohepatic circulation of bile acids. It appeared that intestinal FXR deficiency leads to a modest but significant increase in cholic acid pool size, without changes in fractional turnover rate. As a consequence, bile flow and biliary bile acid secretion rates were increased in iFXR-KO mice compared with controls. Feeding a bile acid-containing diet or treatment with a bile acid sequestrant similarly affected bile formation in iFXR-KO and control mice and induced similar changes in Cyp7A1 and Cyp8B1 expression patterns. In conclusion, this study is the first to demonstrate the physiological relevance of the contribution of the intestinal FXR-Fgf15 signalling pathway in control of hepatic bile acid synthesis. Fgf15 contributes to the regulation of hepatic bile acid synthesis in mice mainly during the dark phase. Expansion of the circulating bile acid pool as well as bile acid sequestration diminishes the contribution of intestinal FXR-Fgf15 signalling in control of hepatic bile acid synthesis and bile formation.

Figure 1

Relative gene expression pattern along the small Intestine. The small Intestine of male wild-type (WT) mice (open circles, n = 3) and male intestine-selective FXR knockout mice (solid circles, n = 3) was rinsed with PBS and six segments were excised; 0, 33, 66, 83, 90 and 100% (relative distance from stomach to distal ileum). Relative gene expression levels of Fxr and Fgf15 compared with 18S were analyzed using RT-PCR. Values are expressed as means ± s.d.

Figure 2

Liver morphology upon chow, 0.5% TCA- or 2% Colesevelam HCl-enriched diet feeding of WT and intestine-selective FXR knockout mice. Histological examination of liver morphology was assessed by hematoxylin/ eosin staining (see ‘Materials and Methods’ section). Liver morphology of WT and intestine-selective FXR knockout mice upon chow, 0.5% TCA- or 2% Colesevelam HCl-enriched diet feeding are represented at × 20 magnification. ‘^⋆’ Represents periportal hepatocytes; ^# represents pericentral hepatocytes.

Figure 3

Circadian expression profile of genes involved in the regulation of hepatic bile acid synthesis. Gene expression levels (relative to 18S expression) were measured at four time points (0700, 1300, 1900 and 0100 hours) in 6–7 female wild-type controls (open circles) or female intestine-selective FXR knockout mice (closed circles). Data are represented as mean ± s.d. Cyp7A1 and Cyp8B1 are key enzymes in bile acid synthesis, Shp is the second messenger of FXR and Dbp, Rev-erbα and E4bp4 are involved in maintaining the circadian rhythm of Cyp7A1 expression. ^‘#’ Represents a significant difference with P<0.05 between the two analyzed strains.

Figure 4

Circadian expression profile of intestinal genes. Gene expression levels (relative to 18S) were measured at four time points (0700, 1300, 1900 and 0100 hours) in ~4cm of terminal ileum proximal to the ileocecal sphincter either of female wild-type controls (open circles) or female intestine-selective FXR knockout mice (closed circles); N = 4. Data are represented as mean±s.d. Fgf15 is the growth factor whose expression is induced by active intestinal FXR; Ibabp, ileal bile acid-binding protein, is an intestinal FXR target gene. ^‘#’ Represents a significant difference with P<0.05 between the two analyzed strains.

Figure 5

Bile flow (a) and biliary output rates (b–d) in wild-type (WT) and intestine-selective FXR knockout mice fed either a chow, 0.5% TCA- or 2% Colesevelam HCl-enriched diet. After 0.5% TCA-enriched diet or 2% Colesevelam HCl-enriched diet feeding for three respectively 7 days, mice (N = 5) were subjected to bile cannulation. As a result bile flow and biliary output rates were obtained. Open bars: WT littermates, closed bars: intestine-selective FXR knockout mice. Values are expressed as means ± s.d. ^‘⋆’ Represents a statistical significance of P<0.05 between chow- and TCA-/Colesevelam HCl-fed mice. ^# is a statistical significance (P<0.05) between the two strain fed the same diet.

Figure 6

Effects of intestine-selective FXR deficiency on decay of [²H4]-cholate (a), pool size (b), fractional turnover rate (c) and cholate synthesis rate (d). Mice were subjected to bile cannulation, as described in experimental procedures section. (a–d) are derived from the [²H4]-cholate isotope enrichment measurements in plasma of wild-type (WT) and intestine-selective FXR knockout mice. N = 6 per strain: WT mice (open bars) and intestine-selective FXR knockout mice (closed bars). Data are represented as means±s.d. ^# significant difference (P<0.05) between the WT and intestine-selective FXR knockout (iFXR-KO) mice.

Figure 7

Relative gene expression of wild-type (WT) and intestine-selective FXR knockout mice fed either a chow, 0.5% TCA- or 2% Colesevelam HCl-enriched diet. Quantitative real-time PCR was performed on ~ 1.5 cm of terminal ileum proximal to the ileocecal sphincter as well as on liver tissue (expressed as relative expression compared with 18S). Total RNA of WT (open bars) and intestine-selective FXR knockout mice (closed bars) fed either a standard chow or a 0.5% TCA- or 2% Colesevelam HCl-enriched diet (samples taken between 0930 and 1300 hours). Data are represented as means ± s.d. ^‘⋆’ Represents a significant difference of P<0.05 within a strain when compared with the chow-fed condition. ^#P<0.05, WT vs intestine-selective FXR knockout mice.