Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies

Abstract

Background & aims: Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs).

Methods: Bile secretion and BA homeostasis were investigated in Cftr(tm1Unc) (Cftr-/-) and CftrΔF508 (ΔF508) mice.

Results: Cftr-/- and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr-/- mice were enlarged and had defects in emptying, based on (99m)technetium-mebrofenin scintigraphy or post-prandial variations in gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr-/- mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide-a myorelaxant factor for the gallbladder. The BA pool was larger in Cftr-/- than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr-/- mice were much lower than normal. Expression of genes that are induced by BAs, including fibroblast growth factor-15 and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr-/- mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr-/- mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr-/- mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr-/- mice.

Conclusions: Cftr-/- and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver.

Figure 1

Gallbladder emptying defect in cftr^−/− mice. Overnight-fed cftr^−/− mice and cftr^+/+ control littermates were subjected to (A) laparotomy and macroscopic examination of the abdominal cavity, showing enlarged gallbladders in cftr^−/− mice in comparison with cftr^+/+controls (right and left panels, respectively, arrows). (B and C) Scintigraphic analyses of gallbladder motor function. (B) Typical images recorded at the indicated times after intravenous injection of ^99mTc-mebrofenin. (C) Representative time-activity curves generated over regions of interest.

Figure 2

Mechanisms of gallbladder emptying defect in cftr^−/− mice. cftr^−/− mice and cftr^+/+ control littermates were subjected to the analysis of (A and B) gallbladder motor function assessed by ^99mTc-mebrofenin scintigraphy, (A) in basal fed conditions and (B) 1 week later, in the same animals, after the subcutaneous injection of CCK-8. In each condition, the time-course of gallbladder radioactivity (expressed as a percentage of total injected activity) is shown in individual animals (left panels), and the corresponding gallbladder ejection fractions (right panels) were calculated at 45 and 120 minutes (means ± standard error of the mean [ SEM]). (C) Gallbladder bile volumes after overnight fasting or feeding (means ± SEM of 15 animals). (D) pH (means ± SEM) of the duodenum (n = 15), of hepatic bile (n = 4), and of gallbladder bile (n = 15). (E) Expression of the Vip and Cck genes, analyzed by quantitative reverse-transcription polymerase chain reaction, in the duodenum. The mRNA levels are shown relative to the mean value in cftr^+/+ mice (means ± SEM of 6 animals).

Figure 3

BA composition and pool size in cftr^−/−mice. BA analyses were performed in cftr^−/− mice and cftr^+/+ control litter-mates, by HPLC-tandem mass spectrometry, to determine (A) the proportions of primary BAs (cholic acid, muricholic acid, chenodeoxycholic acid, and hyocholic acid), secondary BAs (deoxycholic acid, hyodeoxycholic acid, and lithocholic acid), and ursodeoxycholic acid (UDCA), including their conjugates, in liver tissue (n = 5), gallbladder bile (n = 15), and portal blood (n = 15). Histograms show means (standard error of the mean [ SEM] and total concentrations are shown in Table 1). (B) BA pool size and fecal output (means ± SEM of 8 animals).

Figure 4

Changes in gene expression underlying BA homeostasis in cftr^−/−mice. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (A) in the terminal ileum, (B) in the liver, and (C) in the gallbladder of cftr^−/− mice and cftr^+/+ control littermates. The mRNA levels are shown relative to the mean value in cftr^+/+ mice (means ± standard error of the mean of 6 animals). (D) Protein levels were analyzed by immunoblot in membrane fractions prepared from pooled samples of terminal ileum (n = 2) or gallbladder (n = 10), and were quantified by densitometry. Two bands for ASBT represent different glycosylated forms, as confirmed by peptide: N-glycosidase F digestion (not shown).

Figure 5

Visualization of the cholecystohepatic shunt and effect of cholecystectomy on BA homeostasis in cftr^−/− mice. (A) Cholyl-(Ne-NBD)-lysine was injected into the gallbladder of cftr^−/− mice, after cystic duct ligation. Twenty minutes later, the accumulation of cholyl-(Ne-NBD)-lysine was visible within bile canaliculi, predominantly in the periportal area (lower panel, representative of 5 animals); basal fluorescence in the liver from a noninjected animal is shown in comparison (upperpanel). (B-E)cftr^−/− mice with intact gallbladders or that had undergone cholecystectomy 1 month earlier were subjected to reverse-transcription polymerase chain reaction analyses of (B) ileal gene expression, (C) hepatic gene expression, and to HPLC-tandem mass spectrometry analyses. The HPLC-tandem mass spectrometry analyses were performed for (D) BA pool size and (E) individual BAs in portal blood to determine total BA concentrations and the proportion of primary BAs (cholic acid, muricholic acid, chenodeoxycholic acid, and hyocholic acid), secondary BAs (deoxycholic acid, hyodeoxycholic acid, and lithocholic acid), and ursodeoxycholic acid [ UDCA], including their conjugates. The mRNA levels are shown relative to the mean value in cftr^+/+ mice (means ± standard error of the mean of 6 animals). *P < .05 vs cftr^+/+ (as shown in Figure 4A and B).

Figure 6

Proposed model of BA recycling in CF. As compared with (A) normal conditions, (B) the duodenum in CF is abnormally acidic and overproduces the relaxing factor VIP, which is also locally produced, together with FGF15, by the gallbladder. Gallbladder emptying is impaired, impeding BA influx into the intestine. BA transporters (closed circles) are down-regulated in the ileum and increased in the gallbladder, providing a cholecystohepatic shunt for BAs. This pathway maintains the level of BA transport back to the liver, and results in a lower proportion of secondary BAs.