Gut microbiota inhibit Asbt-dependent intestinal bile acid reabsorption via Gata4

Abstract

Background & aims: Regulation of bile acid homeostasis in mammals is a complex process regulated via extensive cross-talk between liver, intestine and intestinal microbiota. Here we studied the effects of gut microbiota on bile acid homeostasis in mice.

Methods: Bile acid homeostasis was assessed in four mouse models. Germfree mice, conventionally-raised mice, Asbt-KO mice and intestinal-specific Gata4-iKO mice were treated with antibiotics (bacitracin, neomycin and vancomycin; 100 mg/kg) for five days and subsequently compared with untreated mice.

Results: Attenuation of the bacterial flora by antibiotics strongly reduced fecal excretion and synthesis of bile acids, but increased the expression of the bile acid synthesis enzyme CYP7A1. Similar effects were seen in germfree mice. Intestinal bile acid absorption was increased and accompanied by increases in plasma bile acid levels, biliary bile acid secretion and enterohepatic cycling of bile acids. In the absence of microbiota, the expression of the intestinal bile salt transporter Asbt was strongly increased in the ileum and was also expressed in more proximal parts of the small intestine. Most of the effects of antibiotic treatment on bile acid homeostasis could be prevented by genetic inactivation of either Asbt or the transcription factor Gata4.

Conclusions: Attenuation of gut microbiota alters Gata4-controlled expression of Asbt, increasing absorption and decreasing synthesis of bile acids. Our data support the concept that under physiological conditions microbiota stimulate Gata4, which suppresses Asbt expression, limiting the expression of this transporter to the terminal ileum. Our studies expand current knowledge on the bacterial control of bile acid homeostasis.

Keywords: Antibiotic treatment; Asbt; Bile acid reabsorption; Bile acid synthesis; Cyp7a1; Enterohepatic circulation; Fgf15; Gata4; Germfree; Gut microbiota; Intestinal bacteria.

Fig. 1. Bile acid homeostasis in germfree mice.

(A) Total BA concentration in feces (μmol/g feces) of germfree and conventional mice. (B) Bile flow (μl/min/100 g bodyweight) measured by bile cannulation and (C) secretion of biliary BAs (nmol/min/100 g bodyweight) in germfree and conventional mice. Median ± range; n = 6–9/group; *p <0.05.

Fig. 2. Antibiotic treatment alters bile acid composition, recirculation and gene expression in the enterohepatic circulation.

(A) Fecal excretion of BAs in antibiotic-treated and control mice (μmol/24 h/100 g bodyweight). (B) Cholic acid turnover rate (μmol/24 h), (C) total plasma BA levels (μM), (D) bile flow (μl/min/100 g bodyweight) and (E) secretion of biliary BAs (nmol/min/100 g bodyweight) in antibiotic-treated and control mice. (F) Hepatic mRNA expression in antibiotic-treated and control mice. (G) Asbt mRNA expression in different segments of the small intestine. (H) Representative western blot of ileal Asbt from three control and three antibiotic-treated mice. (I) Ileal mRNA expression of BA-responsive genes. (J) mRNA expression of BA-responsive genes in the jejunum. Median ± range; n = 8/group; *p <0.05.

Fig. 3. Bile acid homeostasis in Asbt-KO mice treated with antibiotics.

(A) Fecal BA excretion (μmol/24 h/100 g bodyweight) in wildtype and Asbt-KO mice upon antibiotic treatment. (B) Secretion of biliary BAs (nmol/min/100 g bodyweight). (C) Bile flow (μl/min/100 g bodyweight). (D) Total plasma BAs (μM) in antibiotic-treated and control wildtype and Asbt-KO mice. (E) Hepatic mRNA expression of Cyp7a1, Cyp8b1 and Hmgcr. (F) Biliary cholesterol secretion (nmol/min/100 g bodyweight) in Asbt-KO and wildtype mice. (G) Ileal Asbt protein expression. (H) Ileal mRNA expression of BA-responsive genes. (I) Ileal FGF15 protein levels. (J) mRNA expression of Fgf15 and Shp in jejunum of wildtype and Asbt-KO mice. Median ± range; n = 8/group; *p <0.05 WT vs. WT + AB, ^‡p <0.05 WT vs. Asbt-KO, ^#p <0.05 Asbt-KO vs. Asbt-KO + AB.

Fig. 4. Bile acid homeostasis in Gata4-iKO mice upon antibiotic treatment.

(A) Relative mRNA expression of Asbt in different segments of the small intestine from wildtype and Gata4-iKO mice. (B) Fecal excretion of BAs (μmol/24 h/100 g bodyweight). (C) Bile flow (μl/min/100 g bodyweight). (D) Biliary BA secretion (nmol/min/100 g bodyweight) in wildtype and Gata4-iKO mice. (E) Total plasma BA levels (μM). Median ± range; n = 6–7/group; *p <0.05 WT vs. WT + AB; ^‡p <0.05 WT vs. Gata4-iKO; ^#p <0.05 Gata4-iKO vs. Gata4-iKO + AB.