Conditional Gata4 deletion in mice induces bile acid absorption in the proximal small intestine

Abstract

Background and aims: The transcription factor GATA4 is expressed throughout most of the small intestine except distal ileum, and restricts expression of the apical sodium-dependent bile acid transporter (ASBT), the rate-limiting intestinal bile acid transporter, to distal ileum. The hypothesis was tested that reduction of GATA4 activity in mouse small intestine results in an induction of bile acid transport in proximal small intestine sufficient to restore bile acid absorption and homeostasis after ileocaecal resection (ICR).

Methods: Bile acid homeostasis was characterised in non-surgical, sham or ICR mice using two recombinant Gata4 models in which Asbt expression is induced to different levels.

Results: Reduction of intestinal GATA4 activity resulted in an induction of ASBT expression, bile acid absorption and expression of bile acid-responsive genes in proximal small intestine, and a reduction of luminal bile acids in distal small intestine. While faecal bile acid excretion and bile acid pool size remained unchanged, the bile acid pool became more hydrophilic due to a relative increase in tauro-beta-muricholate absorption. Furthermore, proximal induction of Asbt in both Gata4 mutant models corrected ICR-associated bile acid malabsorption, reversing the decrease in bile acid pool size and increase in faecal bile acid excretion and hepatic cholesterol 7alpha-hydroxylase expression.

Conclusions: Reduction of intestinal GATA4 activity induces bile acid absorption in proximal small intestine without inducing major changes in bile acid homeostasis. This induction is sufficient to correct bile acid malabsorption caused by ICR in mice.

Figure 1

Intestinal Gata4 deletion results in an induction of apical sodium-dependent bile acid transporter (ASBT) expression in proximal small intestine. (a) Schematic representation of intestinal sampling. The grey bars numbered 1–5 indicate the ~1.0 cm segments used for RNA isolation. The white bars with Roman numerals I –IV indicate the ~7 cm segments used for the isolation of brush border membrane vesicles. (b) Real-time reverse transcriptase–PCR reveals a proximal induction of Asbt mRNA in G4ap mice. *p<0.05, **p<0.01, as compared with Wt-controls, n=4 in each group. Values are presented relative to the mean value of the Wt-control segment 5 samples. (c) Western analysis shows a proximal induction of ASBT protein in G4ap mice. β-Actin was used as a loading control.

Figure 2

Intestinal Gata4 deletion results in an induction of taurocholate (TC) uptake in proximal small intestine, and a depletion of luminal bile acids in distal small intestine. Ex vivo measurements of TC transport in everted gut sacs show (a) a significant increase in mucosal-to-serosal transport of radioactively labelled TC in proximal intestine of G4ap mice as compared with Wt-controls, and (b) a corresponding increase in tissue-associated TC. *p<0.05, **p<0.01, ***p<0.001, as compared with Wt-controls, n=5 in each group. In vivo segmental analysis reveals that the amount of bile acid in luminal contents (c) and tissue (d) is reduced in distal small intestine of G4ap mice as compared with Wt-controls. *p<0.05, **p<0.01, as compared with Wt-controls, n=6 for G4ap mice, n=5 for Wt-control mice.

Figure 3

Intestinal Gata4 deletion results in an increase in proximal and a decrease in distal expression of intestinal genes regulated by bile acids. Real-time reverse transcriptase–PCR analyses of RNA from intestinal segments along the length of small intestine show a general increase in ileal lipid-binding protein (Ilbp), organic solute transporter (Ost)α and β, and fibroblast growth factor 15 (Fgf15) mRNA abundances in proximal segments, and a decrease in distal segments of G4ap mice as compared with Wt-controls. *p<0.05, **p<0.01, ***p<0.001, as compared with Wt-controls, n=4 in each group. Values are presented relative to the mean value of the Wt-control segment 5 samples.

Figure 4

Intestinal Gata4 recombination results in an enrichment of tauro-β-muricholate (TBMC) and reduction of taurocholate (TC) in the bile acid pool. (a) Real-time reverse transcriptase–PCR of apical sodium-dependent bile acid transporter (Asbt) mRNA in Wt-control jejunum (segment 3) and ileum (segment 5), G4Δex2 jejunum and G4ap jejunum shows a 22% and 69% transformation to wild-type ileal levels in jejunum of G4Δex2 and G4ap mice, respectively. n=6–10 in each group. (b) Bile acid (BA) pool size, as determined by the total bile acid content in liver, gall bladder and small intestine, is similar among Wt-control, G4Δex2 and G4ap mice, but demonstrates an increase in TBMC, and decrease in TC and other bile acids in G4Δex2 and G4ap mice as compared with Wt-controls. (c) The TBMC/TC ratio of the bile acid pool reveals a 2.2-fold increase in G4Δex2 mice and a 7.2-fold (p<0.001) increase in G4ap mice as compared with Wt-controls. (d) The hydrophobicity index of the bile acid pool reveals a 1.7-fold (p<0.001) decrease in G4Δex2 mice and a 2.2-fold (p<0.001) decrease in G4ap mice as compared with Wt-controls. (n=4–7 in each group).

Figure 5

Tauro-β-muricholate (TBMC) enrichment of the bile acid pool after intestinal Gata4 deletion is due to an increase in TBMC uptake. (a) Real-time reverse transcriptase–PCR shows that the mRNAs for hepatic bile acid biosynthetic enzymes, Cyp7a1, Cyp8b1, Cyp27 and Cyp7b1, are similar between Wt-control and G4ap mice (n=5 in each group). Values are presented relative to the mean value of Wt-control liver samples. (b) Segmental analysis of the bile acid composition of luminal contents reveals that the TMBC/taurocholate (TC) ratio as a percentage of segment I increases distally in Wt-controls, but decreases distally in the G4ap mice. *p<0.05 as compared with Wt-controls, n=3–6 in each group.

Figure 6

Intestinal Gata4 recombination restores bile acid absorption after ileocaecal resection (ICR). (a) Real-time reverse transcriptase–PCR on RNA obtained from jejunum shows an induction of the apical sodium-dependent bile acid transporter (Asbt) mRNA in sham-operated and ICR G4Δex2 and G4ap mice. Values are presented relative to the mean value of the sham-operated G4ap samples. *p<0.05 as compared with sham-operated mice, n=3–7 in each group. (b) Faecal bile acid content is increased in Wt-control ICR mice as compared with their sham-operated counterparts, but approached or returned to normal levels in G4Δex2 and G4ap mice that underwent ICR. **p<0.01 as compared with sham-operated mice, n=3–7 in each group.

Figure 7

Intestinal Gata4 recombination prevents a loss of bile acids in the body pool after ICR. Bile acid (BA) pool size, as determined by the total bile acid content in liver, gall bladder and small intestine, is lower in Wt-control mice that underwent ileocaecal resection (ICR) as compared with sham-operated mice. Bile acid pool size in G4Δex2 and G4ap mice that underwent ICR remains similar to that of their sham-operated counterparts. *p<0.05, as compared with sham-operated mice, n=3–7 in each group.

Figure 8

Intestinal Gata4 recombination eliminates the need for compensatory upregulation in bile acid synthesis after ileocaecal resection (ICR). Real-time reverse transcriptase–PCR shows an increase in Cyp7a1 and a decrease in Shp mRNA abundance in liver of Wt-control mice that underwent ICR as compared with sham-operated mice, whereas no major differences were found between sham-operated and ICR G4Δex2 and G4ap mice. *p<0.05, **p<0.01, as compared with sham-operated mice, n=3–7 in each group. Values are presented relative to the mean value of the sham-operated Wt-control liver samples.