Ileal Apical Sodium-dependent Bile Acid Research Articles

Enterobacteria Modulate Intestinal Bile Acid Transport and Homeostasis through Apical Sodium-Dependent Bile Acid Transporter (SLC10A2) Expression

In our study, ampicillin (AMP)-mediated decrease of enterobacteria caused increases in hepatic bile acid concentration through (at least in part) elevation of bile acid synthesis in C57BL/6N mice. We investigated the involvement of enterobacteria on intestinal bile acid absorption in AMP-treated mice in the present study. Fecal enterobacterial levels and fecal bile acid excretion rates were markedly decreased in mice treated with AMP (100 mg/kg) for 3 days, whereas bile acid concentrations in portal blood were significantly increased compared with those in mice treated with a vehicle. Ileal apical sodium-dependent bile acid transporter (SLC10A2) mRNA levels and ileal SLC10A2 protein levels in brush-border membranes were significantly increased compared with those in mice treated with the vehicle. In AMP-treated mice, total bile acid levels were increased, whereas levels of enterobacteria-biotransformed bile acid, taurodeoxycholic acid, and cholic acid were decreased in intestinal lumen. These phenomena were also observed in farnesoid X receptor-null mice treated with AMP for 3 days. Discontinuation of AMP administration after 3 days (vehicle administration for 4 days) increased levels of fecal enterobacteria, fecal bile acid excretion, and taurodeoxycholic acid and cholic acid in the intestinal lumen, whereas the discontinuation decreased ileal SLC10A2 expression and bile acid concentrations in the portal blood. Coadministration of taurodeoxycholic acid or cholic acid decreased ileal SLC10A2 expression in mice treated with AMP. These results suggest that enterobacteria-mediated bile acid biotransformation modulates intestinal bile acid transport and homeostasis through down-regulation of ileal SLC10A2 expression.

Green tea catechin EGCG inhibits ileal apical sodium bile acid transporter ASBT

Green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) is responsible for reabsorption of bile acids. The present studies were, therefore, designed to investigate the modulation of ASBT function and membrane expression by green tea catechins in human embryonic kidney HEK-293 cells stably transfected with ASBT-V5 fusion protein and intestinal Caco-2 monolayers. Our data showed that ASBT activity was significantly decreased by (-)-epigallocatechin-3-gallate (EGCG) but not other green tea catechins. Inhibition of PKC, phosphatidylinositol 3-kinase, and MAPK-dependent pathways failed to block the reduction in ASBT activity by EGCG. Kinetics studies showed a significant decrease in the V(max) of the transporter, whereas total ASBT content on the plasma membrane was unaltered by EGCG. Concomitant with the decrease in ASBT function, EGCG significantly reduced ASBT pool in the detergent-insoluble fraction, while increasing its presence in the detergent-soluble fraction of plasma membrane. Furthermore, EGCG decreased the association of ASBT with floating lipid raft fractions of cellular membrane on Optiprep density gradient. In conclusion, our data demonstrate a novel role of lipid rafts in the modulation of ASBT function by the dietary component EGCG, which may underlie the hypocholesterolemic effects of green tea.

A Variant of the SLC10A2 Gene Encoding the Apical Sodium-Dependent Bile Acid Transporter Is a Risk Factor for Gallstone Disease

BackgroundCholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated. As one of these mechanisms, a decreased expression of the ileal apical sodium-dependent bile acid transporter gene SLC10A2 in gallstone carriers was described previously. In this study the SLC10A2 gene was investigated to identify novel genetic variants and their association with gallstone formation.Methodology/Principal FindingsStudy subjects were selected with the presence or absence of gallstones confirmed by ultrasound and medical history. Genomic DNA was obtained from blood leukocytes. Sequence analysis was performed of all six exonic and flanking regions as well as of 2,400 base pairs of the SLC10A2 promoter in a cohort of gallstone carriers and control subjects from Stuttgart, Germany. Genotype frequencies of newly identified genetic variants (n = 6) and known single nucleotide polymorphisms (n = 24) were established using MALDI-TOF mass spectrometry. Six new genetic variants were found within the SLC10A2 gene. Although none of the variants was linked to gallstone disease in the Stuttgart cohort overall, the minor allele of SNP rs9514089 was more prevalent in male non-obese gallstone carriers (p = 0.06680, OR = 11.00). In a separate population from Aachen, Germany, the occurrence of rs9514089 was two-fold higher in gallstone patients (22%) than in corresponding controls (11%) (p = 0.00995, OR = 2.19). In the pooled Aachen/Stuttgart cohort rs9514089 was highly significantly linked to cholelithiasis (p = 0.00767, OR = 2.04). A more frequent occurrence was observed for male gallstone carriers (22%) compared to controls (9%) (p = 0.01017, OR = 2.99), for the total normal weight group (p = 0.00754, OR = 2.90), and for male non-obese gallstone patients (p = 0.01410, OR = 6.85). Moreover, for the minor allele of rs9514089 an association with low plasma cholesterol levels was found especially in gallstone carriers (p = 0.05).Conclusions/SignificanceWe have identified SLC10A2 as a novel susceptibility gene for cholelithiasis in humans. Comprehensive statistical analysis provides strong evidence that rs9514089 is a genetic determinant especially in male non-obese gallstone carriers. The minor allele of rs9514089 is related to differences in plasma cholesterol levels among the subjects.

W1651 Ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT) Is Upregulated in Rat Model of Diabetes Mellitus

W1651 Ileal Apical Sodium-Dependent Bile Acid Transporter (ASBT) Is Upregulated in Rat Model of Diabetes Mellitus

Reduced ileal expression of OSTα-OSTβ in non-obese gallstone disease

Cholelithiasis is a multifactorial process, and several mechanisms have been postulated. A decreased expression of the ileal apical sodium-dependent bile acid transporter (ASBT) and of the cytosolic ileal lipid binding protein (ILBP) was recently described in female non-obese patients. The role of the recently identified organic solute transporters alpha and beta (OSTalpha, OSTbeta) in gallstone pathogenesis remains unclear. Therefore, we performed analysis of OSTalpha-OSTbeta in gallstone patients according to body weight. Ileal mucosal biopsies were collected during routine colonoscopy from female gallstone carriers (n = 19) and controls (n = 34). OSTalpha-OSTbeta mRNA expression was measured using the LightCycler sequence detection system; protein was analyzed by immunohistochemistry and Western blot. The mRNA expression of OSTalpha-OSTbeta was significantly reduced (OSTalpha: 3.3-fold, P = 0.006; OSTbeta: 2.6-fold, P = 0.03) in normal-weight but not overweight gallstone carriers compared with controls. OSTalpha-OSTbeta protein levels also showed a reduction by 40-67%. The expression of OSTalpha-OSTbeta correlated positively with ASBT (r = 0.65, 0.58, respectively), ILBP (r = 0.77, 0.67), and the farnesoid X receptor (r = 0.58, 0.50). Fibroblast growth factor-19 showed a 2.8-fold reduction (P = 0.06), and liver receptor homolog-1 showed a 2-fold reduction (P = 0.04) in non-obese patients. In conclusion, an impaired function of all three ileal bile acid transporters may lead to low ileal bile acid reabsorption and an altered bile acid pool composition and therefore may contribute to the formation of gallstones in non-obese patients.

Identification of the Transporters Involved in the Hepatobiliary Transport and Intestinal Efflux of Methyl 1-(3,4-Dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) Glucuronide, a Pharmacologically Active Metabolite of S-8921

The glucuronide conjugate of methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921; S-8921G) is a 6000-fold more potent inhibitor of an ileal apical sodium-dependent bile acid transporter (SLC10A2) than S-8921 and is responsible for the hypocholesterolemic effect of S-8921 in rats. Because S-8921G is formed in the intestine and liver, the present study investigated the transporters involved in the secretion of S-8921G that govern its exposure to the target site and thereby play an important role in its pharmacological action. Organic anion transporting polypeptide (OATP) 1B1- and OATP1B3-expressing cells exhibited saturable accumulation of S-8921G with K(m) values (micromolar) of 1.9. The uptake of [14C]S-8921G by human cryopreserved hepatocytes was saturable and sodium-independent. Comparison of protein expression between the cDNA transfectants and hepatocytes suggests that the contribution of OATP1B1, OATP1B3, and Na+-taurocholate cotransporting polypeptide to the hepatic uptake of S-8921G is 63, 35, and 2.6%, respectively. The basal-to-apical transport of S-8921G was enhanced in Madin-Darby canine kidney cells expressing both OATP1B1 and multidrug resistance-associated protein (MRP) 2. In Mrp2-deficient mutant rats [Eisai hyperbilirubinemic rats (EHBR)], the biliary excretion clearance based on the plasma concentration was 20% of the normal value, whereas the pharmacokinetic parameters did not show any significant change in Bcrp-/- mice. Furthermore, the secretion clearance of S-8921G to the mucosal side was also significantly lower in everted jejunum sacs from EHBR (9.18 and 20.8 microl/min/g tissue). These results suggest that MRP2 is responsible for the secretion of S-8921G to the intestinal lumen and bile and that OATP1B1 and OATP1B3 account for the hepatic uptake. These transporters deliver S-8921G to the target site of its pharmacological action.

Effects of bile acids on expression of the human apical sodium dependent bile acid transporter gene

Using a luciferase reporter assay in both LMH cells and Caco2 cells we found that certain bile acids including unconjugated deoxycholic and others transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) at concentrations ranging from 20 to 300 μM. Confirming this effect, addition of deoxycholic acid to fresh human ileal biopsies caused an approximate 40% increase in endogenous ASBT mRNA production. Promoter deletion analysis indicated the effect of bile acids was mediated by a response element located in the downstream half of the 5′-UTR, a region known to contain a retinoic acid (RXR/RAR) response element and an activated protein-1 (AP-1) response element. Site-directed mutagenesis of the RAR/RXR response element actually enhanced response to deoxycholic acid. Site-directed mutagenesis of the downstream AP-1 response element reduced activation by deoxycholic acid while deletion of this response element completely eliminated this response. The epidermal growth factor (EGF) receptor inhibitor, AG1478, completely eliminated the response to bile acid while the mitogen-activated protein extracellular signal-regulated kinase cascade (MEK) inhibitor, U0126, partially inhibited the response to bile acid. These studies demonstrate that certain bile acids stimulate ASBT gene expression acting on the down-stream AP-1 response element via the EGF receptor and MEK cascade.

Transactivation of the human apical sodium-dependent bile acid transporter gene by human serum

Using a luciferase reporter assay we found that human serum transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) promoter three to fourfold. Confirming this effect, addition of human serum to both Caco-2 cells and fresh human ileal biopsies caused an approximate 2.0-fold increase in endogenous ASBT mRNA production. Alteration of non-esterified fatty acid (NEFA) content and cortisol content did not affect the transactivation potential of serum. Site-directed mutagenesis of response elements for corticosteroid, peroxisome proliferation-activated α (PPARα), hepatocyte nuclear factor 1α (HNF1α), and retinoic acid (RAR/RXR) did not affect transactivation potential of serum. Three putative serum response elements (SRE) were identified on the promoter, but all were determined inactive using site-directed mutagenesis and electrophoretic mobility shift assay. Promoter deletion analysis demonstrated that >80% of the response to serum was located within the last 273 bp of the 5′-UTR, an area containing one of two activate protein 1 (AP-1) response elements. Site-directed mutagenesis of this downstream AP-1 response element reduced the effect of serum on the promoter by about 50% while full deletion of the response element completely eliminated the effect of serum. These studies demonstrate that one or more constituents of human stimulate ASBT gene expression largely via the down-stream AP-1 response element.

Cloning and Functional Characterization of Human Sodium-dependent Organic Anion Transporter (SLC10A6)

We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K(m) values of 28.7, 12.0, and 11.3 microm, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K(i) value of 0.24 mum. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(omega-sulfooxyethyl)pyrene, bromosulfophthalein, 2- and 4-sulfooxymethylpyrene, and alpha-naphthylsulfate. Among these inhibitors, 2- and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K(i) values of 4.3 and 5.5 microm, respectively, and they were also transported by SOAT-HEK293 cells.

Glucuronidation Converting Methyl 1-(3,4-Dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) to a Potent Apical Sodium-Dependent Bile Acid Transporter Inhibitor, Resulting in a Hypocholesterolemic Action

Methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) is a novel inhibitor of the ileal apical sodium-dependent bile acid transporter (ASBT/SLC10A2) developed for the treatment of hypercholesterolemia. The present study investigated the hypocholesterolemic action of S-8921 glucuronide (S-8921G) in rats. The plasma concentration of S-8921G was higher than that of S-8921 after single oral administration of S-8921 in normal rats, and S-8921G was excreted into the bile (13% dose). Oral administration of either S-8921 or S-8921G reduced the serum total cholesterol, particularly nonhigh-density lipoprotein cholesterol, in hypercholesterolemic normal rats. In Gunn rats devoid of UDP glucuronosyltransferase-1A activity, S-8921G was undetectable both in the plasma and bile specimens, and only S-8921G administration significantly reduced the serum nonhigh-density lipoprotein cholesterol. An in vitro inhibition study showed that glucuronidation converts S-8921 to a 6000-fold more potent inhibitor of human ASBT (K(i) = 18 nM versus 109 microM). S-8921G was detected both in the portal plasma and loop when S-8921 was administered into the loop of the rat jejunum, although the cumulative amount of S-8921G recovered in the bile was 5-fold greater than that in the loop. The uptake of S-8921G by freshly prepared rat hepatocytes was saturable, and sodium-dependent and -independent systems were involved. Organic anions, such as bromosulfophthalein, estrone 3-sulfate, and taurocholic acid, inhibited the uptake. These results suggest that UDP glucuronosyltransferase-1 isoforms play a critical role in the hypocholesterolemic action of S-8921 by converting S-8921 to a more potent ASBT inhibitor, and organic anion transporter(s) are also involved in its pharmacological action through the biliary excretion of S-8921G.

Bile Salt Excretory Pump

Bile acids are the major determinant and driving force for the generation of bile flow. Bile acid transport across the canalicular membrane is primarily an ATP-dependent process. The predominant transporter is the bile salt excretory pump (BSEP, ABCB11), a member of the adenosine triphosphate-binding cassette (ABC) family of transporters. Regulatory mechanisms that can coordinate the genes encoding bile acid transport proteins are critically important to avoid hepatocyte damage from intracellar accumulation of bile acids. Bile salts are natural ligands for several nuclear hormone receptors expressed in liver and intestine. Nuclear receptors are transcription factors that bind specific ligands such as bile acids and regulate gene expression according to the metabolic requirements of the cell. In cloning of the BSEP gene, we found a binding site in the promoter for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with the 9-cis retinoid receptor (RXR alpha), and when bound by bile acids and retinoic acid, the complex effectively activates the transcription of BSEP. There is a growing body of evidence for the activation of nuclear hormone receptors through the remodeling of chromatin by histone modification involving acetylation, in concert with methylation of H3 and H4 histones. We have recently demonstrated a role for the coactivator-associated arginine methyltransferase 1 (CARM1), as a coactivator of the FXR/RXR receptor and regulator of FXR responsive genes such as BSEP. Chromatin immunoprecipitation showed that the bile acid-dependent activation of the human BSEP is associated with a simultaneous increase of FXR and CARM1 occupation of the BSEP promoter. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP. Our work on the role of nuclear receptors in regulation of bile acid homeostasis has led to an increased understanding of the pathogenesis of the disorder, progressive familial intrahepatic cholestasis, type 1 (PFIC1) or Byler disease. The gene mutated in PFIC1 is called FIC1 and codes for a type IV P-type ATPase whose function is unknown. Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with PFIC1. Ileal FXR and short heterodimer partner (an inhibitory nuclear receptor) messenger RNA levels were reduced in the same 3 patients. In studies of cells after antisense-mediated knock-down of endogenous FIC1, the activity of the ileal apical bile acid transporter promoter was enhanced, whereas the activities of the human FXR and BSEP promoters were reduced. Nuclear but not cytoplasmic localization of FXR is markedly decreased in FIC1-negative cells, indicating that FIC1 is necessary for posttranslational modifications necessary for the nuclear translocation of FXR. This defect leads to enhanced ileal bile salt uptake and impaired canalicular bile salt secretion by BSEP. In PFIC1, an increased load of bile acids is retained in the liver leading to cholestasis and progressive liver injury.

Identification of Functionally Relevant Residues of the Rat Ileal Apical Sodium-dependent Bile Acid Cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).

Effects of low‐ and high‐ isoflavone soy protein‐based diets on cholesterol metabolism and enterohepatic bile salt recirculation in mice

Mice consuming a high-isoflavone containing soy protein diet (HIS) for six weeks had dramatic changes in hepatic and intestinal mRNA expression of genes involved in the cholesterol catabolism and bile acid synthesis pathway as compared to mice consuming a low-isoflavone containing soy protein diet (LIS). Diets were atherogenic, containing 35% of calories from fat and 1.25% cholesterol. In particular, hepatic mRNA levels of cholesterol 7-alpha hydroxylase (cyp7a1), the rate-limiting enzyme for conversion of cholesterol to bile acid, were increased approximately 9-fold in HIS-fed mice. The mRNA levels of other genes in the biliary pathway were moderately increased in HIS- vs. LIS-fed mice, including oxysterol 7-alpha hydroxylase (cyp7b1), sterol 12-alpha hydroxylase (cyp8b1) and cholesterol 27 hydroxylase (cyp27a1). Concurrently, the mRNA levels of the ileal apical sodium-dependent bile acid transporter (asbt or slc10a2) were reduced in both liver and intestinal samples (approximately 55% reduction in both). The Asbt transporter is responsible for the reabsorption of bile salts from the intestine back into the bloodstream for hepatic uptake. Mice consuming the HIS diet also had reduced serum total bile acid levels as compared to LIS-consuming mice. These data provide support at the molecular level for the hypothesis explaining the hypocholesterolemic effect of soy intake through increased conversion of cholesterol to bile salts as well as disruption of enterohepatic recirculation of bile salts. Support: The Solae Company and NIHAT000826

Cholesterol modulates human intestinal sodium-dependent bile acid transporter

Bile acids are efficiently absorbed from the intestinal lumen via the ileal apical sodium-dependent bile acid transporter (ASBT). ASBT function is essential for maintenance of cholesterol homeostasis in the body. The molecular mechanisms of the direct effect of cholesterol on human ASBT function and expression are not entirely understood. The present studies were undertaken to establish a suitable in vitro experimental model to study human ASBT function and its regulation by cholesterol. Luminal membrane bile acid transport was evaluated by the measurement of sodium-dependent 3H-labeled taurocholic acid (3H-TC) uptake in human intestinal Caco-2 cell monolayers. The relative abundance of human ASBT (hASBT) mRNA was determined by real-time PCR. Transient transfection and luciferase assay techniques were employed to assess hASBT promoter activity. Caco-2 cell line was found to represent a suitable model to study hASBT function and regulation. 25-Hydroxycholesterol (25-HCH; 2.5 microg/ml for 24 h) significantly inhibited Na(+)-dependent 3H-TC uptake in Caco-2 cells. This inhibition was associated with a 50% decrease in the V(max) of the transporter with no significant changes in the apparent K(m). The inhibition in hASBT activity was associated with reduction in both the level of hASBT mRNA and its promoter activity. Our data show the inhibition of hASBT function and expression by 25-HCH in Caco-2 cells. These data provide novel evidence for the direct regulation of human ASBT function by cholesterol and suggest that this phenomenon may play a central role in cholesterol homeostasis.

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

Association of the 16-kDa Subunit c of Vacuolar Proton Pump with the Ileal Na+-dependent Bile Acid Transporter: PROTEIN-PROTEIN INTERACTION AND INTRACELLULAR TRAFFICKING

The rat ileal apical sodium-dependent bile acid transporter (Asbt) transports conjugated bile acids in a Na+-dependent fashion and localizes specifically to the apical surface of ileal enterocytes. The mechanisms that target organic anion transporters to different domains of the ileal enterocyte plasma membrane have not been well defined. Previous studies (Sung, A.-Q., Arresa, M. A., Zeng, L., Swaby, I'K., Zhou, M. M., and Suchy, F. J. (2001) J. Biol. Chem. 276, 6825-6833) from our laboratory demonstrated that rat Asbt follows an apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift. Furthermore, a 14-mer signal sequence that adopts a beta-turn conformation is required for apical localization of rat Asbt. In this study, a vacuolar proton pump subunit (VPP-c, the 16-kDa subunit c of vacuolar H+-ATPase) has been identified as an interacting partner of Asbt by a bacterial two-hybrid screen. A direct protein-protein interaction between Asbt and VPP-c was confirmed in an in vitro pull-down assay and in an in vivo mammalian two-hybrid analysis. Indirect immunofluorescence confocal microscopy demonstrated that the Asbt and VPP-c colocalized in transfected COS-7 and MDCK cells. Moreover, bafilomycin A1 (a specific inhibitor of VPP) interrupted the colocalization of Asbt and VPP-c. A taurocholate influx assay and membrane biotinylation analysis showed that treatment with bafilomycin A1 resulted in a significant decrease in bile acid transport activity and the apical membrane localization of Asbt in transfected cells. Thus, these results suggest that the apical membrane localization of Asbt is mediated in part by the vacuolar proton pump associated apical sorting machinery.

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

The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler's disease (progressive familial intrahepatic cholestasis type 1) are unknown. 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. 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. 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.

Inhibition of the apical sodium-dependent bile acid transporter reduces LDL cholesterol and apoB by enhanced plasma clearance of LDL apoB.

Cloning of the ileal apical sodium-dependent bile acid transporter (ASBT) has identified a new pharmacological target for the modulation of plasma lipoproteins. The objective of this study was to determine whether a novel, specific, minimally absorbed ASBT inhibitor (SC-435) decreases LDL cholesterol through the alteration of plasma apoB kinetics. Miniature pigs were treated for 21 days with 10 mg/kg/day of SC-435 or placebo. SC-435 decreased plasma cholesterol by 9% and LDL cholesterol by 20% with no effect on other lipids. Autologous (131)I-VLDL, (125)I-LDL, and [(3)H]-leucine were injected simultaneously to determine apoB kinetics. LDL apoB concentrations decreased significantly by 10% resulting entirely from an increase in LDL-apoB fractional catabolic rate. SC-435 had no effect on either total LDL apoB production or VLDL apoB converted to LDL. SC-435 increased VLDL apoB production by 22%; however, the concentration was unchanged as a result of increased VLDL apoB direct removal. SC-435 increased hepatic mRNA and enzymatic activity for both cholesterol 7alpha-hydroxylase and HMG-CoA reductase. Hepatic LDL receptor mRNA increased significantly, whereas apoB expression was unaffected. A low dose of the ASBT inhibitor, SC-435, significantly reduces plasma LDL cholesterol through enhanced LDL receptor-mediated LDL apoB clearance, secondary to increased expression of cholesterol 7alpha-hydroxylase.

Ontogenetic Development and Spatial Distribution of the Ileal Apical Sodium-Dependent Bile Acid Transporter and the Ileal Lipid-Binding Protein in ApoE Knockout and C57BL/6 Mice

Background: Although apoE-/- mice are characterized by hypercholesterolemia, the bile acid enterohepatic circulation, which plays a crucial role in cholesterol homeostasis, has not been examined in these mice. The differences between apoE-/- and C57BL/6 mice in expression of the ileal ASBT and ILBP and in intestinal bile acid absorption were studied. Methods: The intestinal tissues of the fetal, neonatal and post-weaning mice were processed for immunohistochemistry. Body retention and fecal excretion of 75SeHCAT were measured. The bile acid pool size and its composition were analysed by HPLC. Results: In apoE-/- and C57BL/6 mice, the bile acid pool size was 75 ± 13 and 78 ± 13 μmol/ 100 g body weight, respectively, while the ratio of cholic acid/ β -muricholic acid was 1.8 ± 0.3 and 1.4 ± 0.3 ( P < 0.05), respectively. The daily body retention of 75SeHCAT was 48% ± 1.8% in C57 black mice and 58.4% ± 2.7% in apoE-/- mice ( P < 0.05). In both mouse strains, ASBT expression in the small intestine was found in the near-term fetal and post-weaning mice, while ILBP expression was found in all postnatal mice. In the post-weaning mice, ILBP expression was limited to the distal 25%-30% of the small intestine, while ASBT expression was limited to the distal 18%. Conclusions : The bile acid enterohepatic circulation in apoE-/- mice probably does not differ greatly from that in C57BL/6 mice.

Increased expression of ileal apical sodium-dependent bile acid transporter in postpartum rats.

The expression and activity of the apical ileal sodium-dependent bile acid transporter (asbt) was examined in the small intestine of control, pregnant, and lactating postpartum rats 2, 12, and 21 days after delivery. Western blot analysis of brush border membrane vesicles (BBMV) prepared from different regions of the small intestine demonstrated that expression of asbt was maximal in the most distal segments for all experimental groups, was not substantially affected in pregnant and 2-day postpartum rats, and was significantly increased in 12- and 21-day postpartum rats. Analysis of mRNA suggested that asbt protein was regulated at the posttranscriptional level in postpartum rats. Increased expression of asbt protein postpartum was maximal (approximately 2-fold) in the proximal region of the ileum, consistent with a 60% increase in taurocholate (TC) transport in BBMV from the proximal ileum in 14- to 21-day postpartum rats relative to control rats. Absorption of TC, determined from the intact proximal ileum using an intestinal loop model, demonstrated a 30% increase in TC uptake per unit weight of tissue in 14- to 21-day postpartum rats relative to control rats. Together with the marked increase in intestinal mass observed at peak lactation, these data indicate a significant increase in asbt-mediated reclamation of bile acids in the intestine of lactating rats.