Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein

The bile salt-activated farnesoid X receptor (FXR; NR1H4) controls expression of several genes considered crucial in maintenance of bile salt homeostasis. We evaluated the physiological consequences of FXR deficiency on bile formation and on the kinetics of the enterohepatic circulation of cholate, the major bile salt species in mice. The pool size, fractional turnover rate, synthesis rate, and intestinal absorption of cholate were determined by stable isotope dilution and were related to expression of relevant transporters in the livers and intestines of FXR-deficient (Fxr-/-) mice. Fxr-/- mice showed only mildly elevated plasma bile salt concentrations associated with a 2.4-fold higher biliary bile salt output, whereas hepatic mRNA levels of the bile salt export pump were decreased. Cholate pool size and total bile salt pool size were increased by 67 and 39%, respectively, in Fxr-/- mice compared with wild-type mice. The cholate synthesis rate was increased by 85% in Fxr-/- mice, coinciding with a 2.5-fold increase in cholesterol 7alpha-hydroxylase (Cyp7a1) and unchanged sterol 12alpha-hydroxylase (Cyp8b1) expression in the liver. Despite a complete absence of ileal bile acid-binding protein mRNA and protein, the fractional turnover rate and cycling time of the cholate pool were not affected. The calculated amount of cholate reabsorbed from the intestine per day was approximately 2-fold higher in Fxr-/- mice than in wild-type mice. Thus, the absence of FXR in mice is associated with defective feedback inhibition of hepatic cholate synthesis, which leads to enlargement of the circulating cholate pool with an unaltered fractional turnover rate. The absence of ileal bile acid-binding protein does not negatively interfere with the enterohepatic circulation of cholate in mice.     

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.     

Effects of cholecystectomy upon bile salt kinetics in the guinea pig

The effects of cholecystectomy upon bile salt kinetics were studied in normal guinea pigs. After cholecystectomy, bile salt pool size decreased, fractional daily turnover rate increased, and the rate of bile salt synthesis was unchanged. These data indicate that an increased frequency of bile salt enterohepatic cycling is sufficient to produce alterations in bile salt kinetics. Abnormalities of bile salt synthesis need not be present in order for a reduction in pool size to occur.     

Bile-salt-dependent and independent choleresis induced by bucolome in the rat.

Choleresis induced by bucolome (BC) (1-cyclohexyl-5-n-butyl-2,4,6-trioxoperhydropyrimidine) was studied in male Wistar rats. [14C]Erythritol and mannitol clearance studies indicated this choleresis to be of canalicular origin. In 1-h continuous bile collection studies, immediately after the interruption of enterohepatic circulation (acute interruption), both bile flow and bile salt excretion rates were significantly increased in rats administered BC. However, the bile salt excretion rate fell rather rapidly in BC-administered rats, while the bile flow rate was fairly constant during this 1-h period. Thus, unlike the situation in control rats, bile flow rate was not significantly correlated with the bile salt excretion rate in BC-administered rats. In rats that had an external bile fistula open for 16-20 h (chronic interruption of enterohepatic circulation) the bile flow rate was also significantly increased by BC administration, while the bile salt excretion rate was not changed after BC administration. It is suggested that BC induced bile-salt-independent choleresis in both experimental rat groups (acute and chronic interruption of enterohepatic circulation). In addition, BC appeared to increase the bile-salt-dependent fraction of bile in rats with acute interruption of enterohepatic circulation, possibly by mobilizing the bile salt pooled in the intestinal content and (or) intestinal wall.     

Biliary response to food in chickens with intact or interrupted enterohepatic circulation

The biliary secretion in response to food has been studied in chicken. In this species, a well defined biliary postprandial response with a clear vesicular component can be estimated. The bile salt independent fraction increases after feeding. Our results show that gallbladder bile has a lower osmotic power. The biliary response to food when the enterohepatic circulation is blocked, diminishes. The relationship between bile salt independent fraction values, whether the vesicular duct is tied or not, remains constant when the enterohepatic circulation is interrupted.     

Estimation of bile acid pool sizes from their spillover into systemic blood

We have examined the possibility of assessing primary bile acid pool sizes from the spillover of the bile acids into systemic blood after intestinal exposure to the total endogenous bile acid pool; the studies were carried out in 16 healthy subjects. Bile acid spillover was calculated as the integrated area under the curve of bile acid conjugates in serum of each primary bile acid class in response to a well-defined sustained cholecystokinin-induced stimulus of the enterohepatic circulation for 55 min causing complete gallbladder emptying. Serum levels of each species of primary bile acid conjugates were measured by two specific and sensitive radioimmunoassays, one for conjugated cholate and one for conjugated chenodeoxycholate. Primary bile acid pool sizes determined with [24-14C]cholic acid and [24-14C]chenodeoxycholic acid according to Lindstedt (1957. Acta Physiol. Scand. 40:1-9) served as reference. Bile acid conjugates of both species reached a peak 70 min after the start of the cholecystokinin infusion, probably reflecting simultaneous intestinal absorption of both primary bile acids in this model. Highly significant linear correlations were found between the integrated areas under the curve and primary bile acid pool sizes, which were closer for chenodeoxycholate (n = 16, r = 0.81, P less than 0.001) than for cholate (n = 16, r = 0.74, P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.     

Increased 25-hydroxyvitamin D levels in portal blood following cholecystokinin injection in the dog

To establish whether an enterohepatic circulation of the metabolites of vitamin D exists, polyethylene catheters were cannulated into the portal vein of dogs. The dogs were then starved for 24 h and injected with cholecystokinin (CCK) to induce gall bladder contraction. At various time intervals thereafter blood samples were collected from the portal and the saphena veins, and sera prepared and analyzed for the metabolites of vitamin D. The serum levels of 25-hydroxyvitamin D [25(OH)D] were found to be significantly higher in the portal blood when compared with levels in peripheral blood following CCK injection. Since portal blood collects nutrients absorbed from the gut and as the dogs were starved for 24 h prior to blood collection, the only source of the increased concentrations of 25(OH)D in portal blood is likely to be bile. These findings support the notion that an enterohepatic circulation of 25(OH)D does exist under normal physiological conditions.     

The human liver-specific homolog of very long-chain acyl-CoA synthetase is cholate:CoA ligase

Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acyl-CoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both non-isotopic and radioactive assays. Other long- and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis.     

Variation in Hepatic Bile Composition Following Cholecystectomy in Patients with Previous Gallstones

The composition of fasting hepatic bile was analyzed in 63 samples from 8 patients following cholecystectomy to determine if bile was lithogenic in patients with previous cholesterol gallstones after removal of the gallbladder. Bile specimens were obtained from t-tubes over a 7-20 day study period following re-establishment of the enterohepatic circulation. Bile composition varied on a day to day basis in each patient. 18 of 63 samples were lithogenic according to criteria of Admirand and Small while 35 of 63 samples were lithogenic according to criteria of Hegardt and Dam. Variations in the composition of hepatic bile appeared related to changes in the excretion rate of bile acids. These studies demonstrate that hepatic bile may be lithogenic after cholecystectomy and indicate that factors other than sequestration of the bile acid pool in the gallbladder influence the enterohepatic circulation of bile acids and the lithogenicity of bile.     

Bile acid pool changes and regulation of cholate synthesis in experimental diabetes

The effect of alloxan-diabetes and insulin treatment in bile acid pool size and composition, bile acid secretion and cholic acid synthesis was investigated in the rat. The size of the cholate pool was significantly increased 4 days after diabetes induction. It reached a constant size three times that of control animals after 2 weeks of diabetes. Changes in bile acid pool size and secretion were directly dependent of the insulin deficiency state since they were reversed by insulin treatment and were not influenced by the caloric intake of the animal nor the pharmacologic effect of alloxan. Biliary cholate secretion was also 3-fold increased in diabetic rats and it accounted for more than 80% of the total bile acids compared to 60% in the control group. The calculated daily rate of cholate synthesis was increased in diabetic rats and the circadian rhythm of cholate synthesis was abolished in this condition. Therefore, it was shown that the negative feedback mechanism that regulates bile acid snythesis was deleted in diabetes. This mechanism was partially restored after 2 weeks of insulin treatment. These studies demonstrated that bile acid metabolism was profoundly changed in alloxan-diabetic rats and suggested that insulin may play an important role in the regulation of bile acid snythesis and intestinal absorption.     

Pathogenesis of human cholesterol cholelithiasis.

The pathogenesis of cholesterol cholelithiasis in humans has been studied by means of three techniques. The cholesterol-solubilizing capacity of bile may be determined by estimation of the relative composition of the three major lipid constituents of bile. Consistent reduction in the cholesterol-carrying capacity of gallbladder bile of persons with gallstones when compared with normal subjects has not been shown. Normal subjects frequently have supersaturated bile. Secretion rates of biliary lipids have been estimated by two methods; with the method that appears to be more physiologic no change in lipid secretion rates was found in gallstone patients. Bile acid pool size has been measured by isotope dilution techniques; it is reduced in patients with gallstones. It is not clear whether this reduction is important in the pathogenesis of cholesterol cholelithiasis, for the bile acid secretion rate is normal because of an increased rate of cycling of the pool through the enterohepatic circulation. The role of the gallbladder in the genesis of cholesterol cholelithiasis may be more important than has been realized.     

Effects of sphincterectomy on bile acid enterohepatic circulation in dogs

Relative rates of bile enterohepatic circulation (EHC) and bile acid pool distribution were compared in intact and sphincterectomized dogs with portacaval shunt. There was no significant difference in the rates of EHC or in the bile acid pool distribution in the groups of animals. Feeding and cholecystokinin administration caused similar increases in bile acid EHC rates in sphincterectomized and intact animals. It was concluded that the sphincter of Oddi has little or no effect on these aspects of bile acid metabolism in dogs.     

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)

The apical Na(+)-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported (3)H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.     

Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate

The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.     

Control of Bile Salt Synthesis

THE regulation of bile salt synthesis in man is still poorly understood. The human liver synthesizes a trihydroxy and a dihydroxy bile acid, cholic and chenodeoxycholic acid, each conjugated with glycine or taurine. These, together with deoxycholic acid, a dihydroxy bile acid which results from the 7-α dehydroxylation of cholic acid by intestinal bacteria, constitute approximately 98% of the bile salt pool which circulates enterohepatically during digestion of a meal¹. Lindstedt found that the composition of the bile salt pool varies little from day to day in any one individual² and this has also been our experience on analysis of 48 bile samples collected on different days from 16 volunteers. However, the evidence that bile salt synthesis is regulated by a negative feedback mechanism is scanty³ apart from the increased synthesis that follows interruption of the enterohepatic circulation^4–6.     

Effects of acute administration of taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.     

Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat

Synthesis of bile salts is regulated through negative feedback inhibition by bile salts returning to the liver. Individual bile salts have not been distinguished with regard to inhibitory potential. We assessed inhibition of bile salt synthesis by either cholate or its taurine conjugate in bile fistula rats. After allowing synthesis to maximize, baseline synthesis was determined by measuring bile salt output in four consecutive 6-hr periods. Next, sodium cholate (+[(14)C]cholate) or taurocholate (+[(14)C]taurocholate) was infused into the jugular vein for 36 hr and bile was collected in 6-hr aliquots. Hepatic flux of exogenous bile salt was determined by measuring output of radioactivity in bile divided by specific activity of the infusate. Synthesis was determined during the last four 6-hr periods of infusion by subtracting exogenous bile salt secretion from the total bile salt output. Thirteen studies using cholate and 13 using taurocholate were performed. Hepatic flux of infused bile salt varied from 1 to 12 micro mol/100 g per rat per hr. Percent suppression of synthesis varied directly with hepatic flux of exogenous bile salt for both cholate and taurocholate in a linear fashion (r = 0.66, P < 0.01 and r = 0.87, P < 0.0005, respectively). Slope of the taurocholate line was 7.82 (% suppression/ micro mol per 100 g per hr), while slope of the cholate line was 3.66 (P < 0.05), indicating that taurocholate was approximately twice as potent as cholate in suppression of synthesis. At fluxes of 10-12 micro mol/100 g per hr, taurocholate suppressed synthesis 84 +/- 8 (SEM) % while cholate suppressed synthesis only 42 +/- 12% (P < 0.02). The x-intercept of the taurocholate line was 0.65 ( micro mol/100 g per hr), while that of the cholate line was -1.01 (NS) suggesting that the threshold for initial suppression of synthesis did not differ for these two bile salts. We conclude that taurocholate is a more effective inhibitor of hepatic bile salt synthesis than cholate, and that intestinal deconjugation of bile salts may play a role in the regulation of synthesis.-Pries, J. M., A. Gustafson, D. Wiegand, and W. C. Duane. Taurocholate is more potent than cholate in suppression of bile salt synthesis in the rat.     

Ursodeoxycholate modulates bile flow and bile salt pool independently from the cystic fibrosis transmembrane regulator (Cftr) in mice

Cystic fibrosis liver disease (CFLD) is treated with ursodeoxycholate (UDCA). Our aim was to evaluate, in cystic fibrosis transmembrane regulator knockout (Cftr(-/-)) mice and wild-type controls, whether the supposed therapeutic action of UDCA is mediated via choleretic activity or effects on bile salt metabolism. Cftr(-/-) mice and controls, under general anesthesia, were intravenously infused with tauroursodeoxycholate (TUDCA) in increasing dosage or were fed either standard or UDCA-enriched chow (0.5% wt/wt) for 3 wk. Bile flow and bile composition were characterized. In chow-fed mice, we analyzed bile salt synthesis and pool size of cholate (CA). In both Cftr(-/-) and controls intravenous TUDCA stimulated bile flow by ～250% and dietary UDCA by ～500%, compared with untreated animals (P < 0.05). In non-UDCA-treated Cftr(-/-) mice, the proportion of CA in bile was higher compared with that in controls (61 ± 4 vs. 46 ± 4%; P < 0.05), accompanied by an increased CA synthesis [16 ± 1 vs. 10 ± 2 μmol·h(-1)·100 g body wt (BW)(-1); P < 0.05] and CA pool size (28 ± 3 vs. 19 ± 1 μmol/100 g BW; P < 0.05). In both Cftr(-/-) and controls, UDCA treatment drastically reduced the proportion of CA in bile below 5% and diminished CA synthesis (2.3 ± 0.3 vs. 2.2 ± 0.4 μmol·day(-1)·100 g BW(-1); nonsignificant) and CA pool size (3.6 ± 0.6 vs. 1.5 ± 0.3 μmol/100 g BW; P < 0.05). Acute TUDCA infusion and chronic UDCA treatment both stimulate bile flow in cystic fibrosis conditions independently from Cftr function. Chronic UDCA treatment reduces the hydrophobicity of the bile salt pool in Cftr(-/-) mice. These results support a potential beneficial effect of UDCA on bile flow and bile salt metabolism in cystic fibrosis conditions.