Absence of dysfunctional ileal sodium-bile acid cotransporter gene mutations in patients with adult-onset idiopathic bile acid malabsorption

BACKGROUND: A congenital form of idiopathic intestinal bile acid malabsorption (IBAM) has been associated with dysfunctional mutations in the ileal apical sodium-dependent bile acid transporter (ASBT). The aim of this study was to determine whether mutations in the ASBT gene (SLC10A2) predispose to the development of adult-onset idiopathic bile acid malabsorption and chronic watery diarrhea. METHODS: Genomic DNA was obtained from 13 adult IBAM patients previously diagnosed on the basis of clinical data, response to cholestyramine, and abnormal 75Se-homocholic acid taurine (SeHCAT) test values. The ASBT gene was screened for the presence of mutations or polymorphisms by single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing. RESULTS: ASBT gene polymorphisms were detected in 5 of the 13 adult IBAM patients. Four patients were heterozygous for a common polymorphism in exon 3, leading to an alanine to serine substitution at codon 171 (A171S). An additional subject was heterozygous for a polymorphism in exon 1 that causes a valine to isoleucine substitution at codon 98 (V981). These functional polymorphisms were also found in unaffected subjects and do not appear to affect ASBT function. CONCLUSIONS: Adult-onset IBAM is not directly related to dysfunctional mutations in the coding region or intron/exon junctions of the SLC10A2 gene. In the absence of apparent ileal disease or intestinal motility defects, inappropriate down-regulation of the ileal bile acid transporter or defects in ileocyte transfer of bile acids into the portal circulation could explain this form of adult IBAM.     

Absence of Dysfunctional Ileal Sodium?Bile Acid Cotransporter Gene Mutations in Patients with Adult-Onset Idiopathic Bile Acid Malabsorption

Background: A congenital form of idiopathic intestinal bile acid malabsorption (IBAM) has been associated with dysfunctional mutations in the ileal apical sodium-dependent bile acid transporter (ASBT). The aim of this study was to determine whether mutations in the ASBT gene (SLC10A2) predispose to the development of adult-onset idiopathic bile acid malabsorption and chronic watery diarrhea. Methods: Genomic DNA was obtained from 13 adult IBAM patients previously diagnosed on the basis of clinical data, response to cholestyramine, and abnormal ⁷⁵Se-homocholic acid taurine (SeHCAT) test values. The ASBT gene was screened for the presence of mutations or polymorphisms by single-stranded conformation polymorphism analysis (SSCP) and DNA sequencing. Results: ASBT gene polymorphisms were detected in 5 of the 13 adult IBAM patients. Four patients were heterozygous for a common polymorphism in exon 3, leading to an alanine to serine substitution at codon 171 (A171S). An additional subject was heterozygous for a polymorphism in exon 1 that causes a valine to isoleucine substitution at codon 98 (V98I). These functional polymorphisms were also found in unaffected subjects and do not appear to affect ASBT function. Conclusions: Adult-onset IBAM is not directly related to dysfunctional mutations in the coding region or intron/exon junctions of the SLC10A2 gene. In the absence of apparent ileal disease or intestinal motility defects, inappropriate down-regulation of the ileal bile acid transporter or defects in ileocyte transfer of bile acids into the portal circulation could explain this form of adult IBAM.     

Inhibition of ileal bile acid transport lowers plasma cholesterol levels by inactivating hepatic farnesoid X receptor and stimulating cholesterol 7 alpha-hydroxylase

We investigated the effect of SC-435, a competitive inhibitor of ileal apical sodium-dependent bile acid cotransporter (ASBT) on ileal bile acid absorption and the hepatic nuclear receptor FXR (farnesoid X receptor), which regulates cholesterol 7 alpha-hydroxylase (CYP7A1) activity and mRNA levels. Eighteen New Zealand White (NZW) rabbits were divided into 2 groups: controls (n = 10) and fed SC-435 125 mg/kg/d for 1 week (n = 8). In rabbits treated with SC-435, fecal bile acid outputs increased by more than 8 times, reflecting substantial bile acid malabsorption. Plasma cholesterol levels decreased 26%, while bile acid pool sizes and biliary bile acid outputs did not change after treatment. CYP7A1 activity increased 64% and mRNA rose by 4 times after treatment. The expression of FXR target genes in the liver, short heterodimer partner (SHP) and bile salt export pump (BSEP), decreased 11.6 and 2.6 times, respectively, after treatment, which indicates inactivation of hepatic FXR. However, the mRNA levels of ileal bile acid binding protein (IBABP) did not change significantly, while ileal ASBT mRNA expression increased by 2.4 times after treatment. Rabbits treated with SC-435 developed ileal bile acid malabsorption, which decreased the return of bile acids (FXR ligands) to the liver to inactivate hepatic FXR, which upregulated CYP7A1 and lowered plasma cholesterol levels. Although fecal bile acid malabsorption was substantial, increased bile acid production from hepatic cholesterol kept biliary bile acid outputs intact. Thus, a new balance was reached in the liver, where increased bile acid synthesis compensated for diminished ileal bile acid absorption to maintain the circulating enterohepatic bile acid pool.     

Bile acid-induced negative feedback regulation of the human ileal bile acid transporter

Ileal expression of the apical sodium-dependent bile acid transporter (ASBT) in the rat is unaffected by bile salts, yet in the mouse it is under negative-feedback regulation. The bile acid responsiveness of human ASBT is unknown. The human ASBT promoter linked to a luciferase reporter was studied in Caco-2 cells treated with chenodeoxycholic acid (CDCA) and transfected with expression plasmids for farnesoid X-receptor (FXR), short heterodimer partner (SHP), and retinoic acid receptor/retinoid X receptor (RAR/RXR). CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT messenger RNA levels and a 78% reduction in human ASBT promoter activity. A dominant negative FXR abrogated the response to CDCA. Site-directed mutagenesis of an RAR/RXR cis element in the human ASBT promoter reduced its activity by 50% and eliminated the bile acid response. Retinoic acid activated the human ASBT promoter fourfold. SHP repressed the activity of the ASBT promoter and reduced activation by retinoic acid. Antisense mediated knock-down of SHP in Caco-2 cells partially offset the bile acid mediated repression of ASBT promoter activity. In conclusion, the human ASBT is positively regulated by retinoic acid. Bile acids induce a negative feedback regulation of human ASBT via an FXR-mediated, SHP-dependent effect upon RAR/RXR activation of ASBT.     

Human ileal bile acid transporter gene ASBT (SLC10A2) is transactivated by the glucocorticoid receptor

BACKGROUND: Patients with Crohn's disease suffer from intestinal bile acid malabsorption. Intestinal bile acid absorption is mediated by the apical sodium dependent bile acid transporter ASBT/IBAT (SLC10A2). In rats, ASBT is induced by glucocorticoids. AIMS: To study whether human ASBT is activated by glucocorticoids and to elucidate the mechanism of regulation. PATIENTS AND METHODS: ASBT expression in ileal biopsies from patients with Crohn's disease and from healthy subjects was quantified by western blot. ASBT promoter function was studied in luciferase assays and by electrophoretic mobility shift assay. RESULTS: In 16 patients with Crohn's disease, ASBT expression was reduced to 69 (7.5)% compared with healthy controls (mean (SEM); p = 0.01). In 10 healthy male volunteers, ASBT protein expression was increased 1.34 (0.11)-fold (mean (SEM); p<0.05) after 21 days' intake of budesonide (9 mg/day) whereas expression of the peptide transporter 1 was unaffected. Reporter constructs of the human ASBT promoter were activated 15-20-fold by coexpression of the glucocorticoid receptor (GR) and exposure to the GR ligands dexamethasone or budesonide. Two glucocorticoid response elements in the ASBT promoter, arranged as inverted hexanucleotide repeats (IR3 elements), conferred inducibility by GR and dexamethasone in a heterologous promoter context and were shown to bind GR in mobility shift assays. CONCLUSIONS: Human ASBT is induced by glucocorticoids in vitro and in vivo. Induction of ASBT by glucocorticoids could be beneficial in patients with Crohn's disease who exhibit reduced ASBT expression. This study identifies ASBT as a novel target of glucocorticoid controlled gene regulation in the human intestine.     

c-Fos is a critical mediator of inflammatory-mediated repression of the apical sodium-dependent bile acid transporter

BACKGROUND & AIMS: Ileal bile acid malabsorption is present in Crohn's ileitis. The molecular mechanisms of regulation of the apical sodium-dependent bile acid transporter (ASBT) by inflammatory cytokines in vitro and in vivo are investigated. METHODS: Transient transfection studies of the human, mouse, and rat ASBT promoters and Northern analyses were performed in cells treated with the inflammatory cytokines and/or various activator protein-1 constructs. Rat ASBT promoter transgenic, wild-type, and c-fos-null mice were treated with indomethacin to assess the response to acute inflammation of the ileal mucosa. RESULTS: In Caco-2 cells, ASBT messenger RNA expression was reduced 65% after interleukin-1beta treatment, while c-fos and c-jun were up-regulated 2-fold. Human ASBT promoter activity was enhanced by c-jun and repressed by a dominant negative c-jun, c-fos, or a dominant negative c-fos. Meanwhile, c-fos antisense treatment activated the human ASBT promoter 5-fold and not only abrogated interleukin-1beta-mediated repression but led to a paradoxical increase in ASBT promoter activity. Indomethacin-induced acute ileitis led to repression of ASBT in wild-type mice and in the transgenic rat ASBT promoter reporter, while paradoxical activation of ASBT was seen in c-fos-null mice. Indomethacin-induced ileal injury was greater in the c-fos-null mice compared with the wild-type littermates. CONCLUSIONS: Human, rat, and mouse ASBT is inhibited by inflammatory cytokines via direct interactions of c-fos with the ASBT promoter.     

Primary bile acid diarrhoea without an ileal carrier defect: quantification of active bile acid transport across the ileal brush border membrane.

Unexplained bile acid malabsorption associated with diarrhoea that responds to cholestyramine was first described in 1973 but convincing evidence of the proposed mechanism--a defective active ileal bile acid transport--has never been substantiated. Active bile acid transport was quantified in vitro using brush border membrane vesicles prepared from terminal ileal biopsy specimens from 10 patients who fulfilled the criteria of idiopathic bile acid diarrhoea. They were recruited from 181 patients with bile acid malabsorption of various causes. Transport was quantified as in vitro Na+ dependent bile acid transport (INBAT), expressed as pmol taurocholate/mg brush border membrane protein/15 seconds, and in vitro Na+ dependent bile acid local transport capacity (INBALTC), expressed as pmol taurocholate/g ileal biopsy tissue/15 seconds. The lowest INBAT and INBALTC values in the 10 patients with idiopathic bile acid diarrhoea were well above the 10th centile values of a control group of 132 patients. Both INBAT (mean (range) 88 (30-136)) and INBALTC (158 (85-268)) values were significantly higher in the 10 patients than in the control group (INBAT: mean (range) 63 (1-244), INBALTC: mean (range) 98 (1-408)). Quantification of active ileal bile acid transport in these 10 patients with idiopathic bile acid malabsorption suggests that a genetic (carrier) defect is rare in adults.     

Accelerated regional bowel transit and overweight shown in idiopathic bile acid malabsorption

OBJECTIVE: Overweight has recently been shown to accelerate small bowel transit. The role of gut transit and body weight in idiopathic bile acid malabsorption (IBAM) is unclear. We have prospectively studied gastrointestinal transit and body mass index (BMI) in patients with IBAM. METHODS: One hundred and ten patients with chronic diarrhea were prospectively included for transit measurements. All patients underwent a gastroscopy and colonoscopy, 75SeHCAT test for detection of bile acid malabsorption and calculation of BMI. Forty-three patients (15 men) had IBAM. A newly developed radiological procedure was used to measure gastrointestinal transit during one visit. The results were compared to results obtained in 83 healthy subjects. RESULTS: Colonic transit in women with IBAM was 0.8 (0.3-1.5) days versus 1.5 (1.0-3.7) days in healthy women (median and percentile 10 and 90; p < 0.0001). In men with IBAM it was 0.8 (0.1-1.0) days; in healthy men it was 1.3 (0.8-1.9) days, p < 0.0001. Segmental colonic transit was accelerated only in the distal colon in men and women with IBAM compared with healthy subjects. Small bowel transit time in women with IBAM was 1.9 (1.1-3.0) h versus 3.3 (1.5-6.3) h in healthy women, p= 0.0002. In men with IBAM it was 2.1 (1.2-3.2) h and 2.5 (1.4-4.3) h in healthy men (p= 0.04). BMI in patients with IBAM was 27.3 (20.4-33.8) kg/m2 and in healthy subjects it was 23.8 (20.5-26.2) kg/m2, p < 0.0001. CONCLUSION: Accelerated small bowel and distal colonic transit as well as overweight are probably involved in the pathophysiology of IBAM.     

Functional characterization of genetic variants in the apical sodium-dependent bile acid transporter (ASBT; SLC10A2)

Background and Aim: The major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium‐dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity. Methods: Single nucleotide polymorphisms in SLC10A2 from genomic DNA of ethnically‐defined healthy individuals were identified using a polymerase chain reaction (PCR)‐based temperature gradient capillary electrophoresis (TGCE) system. A heterologous gene expression system was used to assess transport activity of SLC10A2 nonsynonymous variants and missense mutations. Total and cell surface protein expression of wild‐type and variant ASBT was assessed by Western blot analysis and immunofluorescence confocal microscopy. Expression of ASBT mRNA and protein was also measured in human intestinal samples. Results: The studies revealed two nonsynonymous SNPs, 292G>A and 431G>A, with partially impaired in vitro taurocholate transport. A novel variant, 790A>G, was also shown to exhibit near complete loss of taurocholate transport, similar to the previously identified ASBT missense mutations. Examination of ASBT protein expression revealed no significant differences in expression or trafficking to the cell surface among variants versus wild‐type ASBT. Analysis of ASBT mRNA and protein expression in human intestinal samples revealed modest intersubject variability. Conclusions: Genome sequencing and in vitro studies reveal the presence of multiple functionally relevant variants in SLC10A2 that may influence bile acid homeostasis and physiology.     

FXR, a bile acid receptor and biological sensor

Bile acid synthesis is a major pathway for cholesterol disposal and thus represents a potential therapeutic target pathway for the treatment of hypercholesterolemia. Recently, the nuclear farnesoid X receptor (FXR) was identified as a bile acid receptor and biological sensor for the regulation of bile acid biosynthesis. FXR was shown to regulate cholesterol metabolism in two ways: (1) chenodeoxycholic acid (CDCA), a primary bile acid, binds directly to and activates FXR, which then mediates the feedback suppression by bile acids of cholesterol 7 alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis from cholesterol; and (2) FXR participates in the activation of intestinal bile acid binding protein (IBABP), which is involved in the enterohepatic circulation of bile acids. Thus FXR constitutes a potential therapeutic target that can be modulated to enhance the removal of cholesterol from the body.     

Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity

BACKGROUND & AIMS: The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler's disease (progressive familial intrahepatic cholestasis type 1) are unknown. METHODS: Interactions among the apical sodium-dependent bile acid transporter, the farnesoid X receptor (FXR), and familial intrahepatic cholestasis-1 were studied in the ileum of children with progressive familial intrahepatic cholestasis type 1 and in Caco-2 cells. RESULTS: Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with progressive familial intrahepatic cholestasis type 1. Paradoxically, ileal lipid-binding protein mRNA expression was repressed, suggesting a central defect in bile acid response. Ileal FXR and short heterodimer partner mRNA levels were reduced in the same 3 patients. In Caco-2 cells, antisense-mediated knock-down of endogenous familial intrahepatic cholestasis-1 led to up-regulation of apical sodium-dependent bile acid transporter and down-regulation of FXR, ileal lipid-binding protein, and short heterodimer partner mRNA. In familial intrahepatic cholestasis-1-negative Caco-2 cells, the activity of the human apical sodium-dependent bile acid transporter promoter was enhanced, whereas the human FXR and bile salt excretory pump promoters' activities were reduced. Overexpression of short heterodimer partner but not of the FXR abrogated the effect of familial intrahepatic cholestasis-1 antisense oligonucleotides. FXR cis-element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from familial intrahepatic cholestasis-1-negative Caco-2 cells. CONCLUSIONS: Loss of familial intrahepatic cholestasis-1 leads to diminished nuclear translocation of the FXR, with the subsequent potential for pathologic alterations in intestinal and hepatic bile acid transporter expression. Marked hypercholanemia and cholestasis are predicted to develop, presumably because of both enhanced ileal uptake of bile salts via up-regulation of the apical sodium-dependent bile acid transporter and diminished canalicular secretion of bile salts secondary to down-regulation of the bile salt excretory pump.     

Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor

Obstruction of bile flow results in bacterial proliferation and mucosal injury in the small intestine that can lead to the translocation of bacteria across the epithelial barrier and systemic infection. These adverse effects of biliary obstruction can be inhibited by administration of bile acids. Here we show that the farnesoid X receptor (FXR), a nuclear receptor for bile acids, induces genes involved in enteroprotection and inhibits bacterial overgrowth and mucosal injury in ileum caused by bile duct ligation. Mice lacking FXR have increased ileal levels of bacteria and a compromised epithelial barrier. These findings reveal a central role for FXR in protecting the distal small intestine from bacterial invasion and suggest that FXR agonists may prevent epithelial deterioration and bacterial translocation in patients with impaired bile flow.     

Prognosis of adult-onset idiopathic bile acid malabsorption.

BACKGROUND: From 1986 to 1993, 150 patients were investigated with the 75Se-homocholic acid taurine (SeHCAT) test as a late step in the investigation of chronic diarrhoea. On basis of low SeHCAT values and response to cholestyramine treatment, 33 patients were initially classified as having idiopathic bile acid malabsorption (IBAM). The aim was to describe the long-term clinical course of the disease and to assess the reliability of the SeHCAT test in diagnosing IBAM. METHODS: The methods included 1) clinical follow-up with patient interview combined with information from medical records and 2) repeated SeHCAT test. RESULTS: The diagnosis of IBAM had to be revised in three cases (inflammatory bowel disease in two patients, Clostridium difficile infection in one). Six patients were lost to follow-up and a further four patients were excluded from re-examination either because of old age (>80 years) or bowel resection, leaving 20 patients for re-examination, of which 16 completed both clinical follow-up and a new SeHCAT test. The median duration of symptoms before initial SeHCAT test was 2.5 (1-30) years. In 13 of 16 patients symptoms persisted, and SeHCAT values remained low and almost identical to the initial value after a median observation time of 88 (51-113) months. Despite initial response to treatment with cholestyramine, six patients had to discontinue treatment because of adverse effects or other compliance problems. In three patients the SeHCAT value showed a considerable increase, and bowel function had correspondingly normalized in these cases. CONCLUSION: The study confirms the reliability of the SeHCAT test in diagnosing IBAM. Despite adult onset of symptoms, only a few patients improve after several years' observation. Treatment with cholestyramine is generally effective but not always tolerated.     

Bile acid malabsorption and bile acid diarrhea in intestinal resection

Bile acid fecal excretion and dihydroxy bile acid concentration in the fecal water of patients with large (N=6) and small (N=8) ileal resection, colectomy (N=5), and healthy controls (N=10) have been studied in order to evaluate the degree of bile acid malabsorption and the occurrence of bile acid diarrhea in intestinal resections of different extent. Bile acid malabsorption was severe in large ileal resections, mild in small ones, and slight in colectomy. The fecal pH seems to be a limiting factor in the occurrence of a bile acid diarrhea, playing a critical role in determining the dihydroxy bile acid solubility in the fecal water. These results seem to suggest that the bile acids may induce water secretion in the colon not only in small but also in large ileal resections.     

Biliary lipid composition in idiopathic bile acid malabsorption

Background—Chronic diarrhoea is the clinicalhallmark of patients presenting with idiopathic bile acidmalabsorption. Its pathogenesis is unknown; colonic water secretion canbe induced by dihydroxy bile acids, but it is not known whetherenrichment of the bile acid pool with these bile acids occurs in suchpatients. Furthermore, bile acid malabsorption is known to affectbiliary lipid composition, but no information is available for theidiopathic type.
Aims—To verify: (a) whetherdiarrhoea in patients with idiopathic bile acid malabsorption isassociated with enrichment of the bile acid pool with dihydroxy bileacids; and (b) whether supersaturation with cholesterol ofduodenal bile occurs in such patients as a result of chronic bile acid depletion.
Patients—Thirteen patients with idiopathic bileacid malabsorption diagnosed according to abnormal ⁷⁵SeHCATtest and absence of other organic diseases, and 23 control subjects.
Methods—Bile rich duodenal fluid was collectedduring intravenous ceruletide infusion in the fasting state. Biliarylipids were analysed by enzymatic assays and bile acids by highperformance liquid chromatography.
Results—Patients with idiopathic bile acidmalabsorption had a cholesterol saturation index similar to controls.Bile acid composition showed only a decrease in percentage cholic acid(29(2)% versus 36 (2)%; p<0.05); the dihydroxy:trihydroxy bile acid ratio was similar to controls.
Conclusions—Patients with idiopathic bile acidmalabsorption do not have an increased risk of forming cholesterolgallstones. The mechanism of diarrhoea does not seem to depend on anenrichment of the bile acid pool with dihydroxy bile acids.

Keywords:primary bile acid malabsorption; bile acids; diarrhoea; ⁷⁵SeHCAT; biliary lipids; cholesterol saturationindex