Regulation of the mouse organic solute transporter alpha-beta, Ostalpha-Ostbeta, by bile acids

The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter alpha-beta (Ostalpha-Ostbeta) expression were investigated. Expression of Ostalpha-Ostbeta mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ostalpha and Ostbeta promoters and reporter constructs containing Ostalpha and Ostbeta 5'-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ostalpha and Ostbeta promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ostalpha and Ostbeta promoters by bile acids was mediated through LRH-1 elements. Ostalpha and Ostbeta promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ostalpha and Ostbeta mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ostalpha and Ostbeta intestinal protein expressions correlated with mRNA expression. The mouse Ostalpha and Ostbeta promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ostalpha-Ostbeta expression to the bile acid flux.     

Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT

Apical sodium-dependent bile acid transporter (ASBT) is responsible for the absorption of bile acids from the intestine. A decrease in ASBT function and expression has been implicated in diarrhea associated with intestinal inflammation. Whether infection with pathogenic microorganisms such as the enteropathogenic Escherichia coli (EPEC) affect ASBT activity is not known. EPEC is a food-borne enteric pathogen that translocates bacterial effector molecules via type three secretion system (TTSS) into host cells and is a major cause of infantile diarrhea. We investigated the effects of EPEC infection on ileal ASBT function utilizing human intestinal Caco2 cells and HEK-293 cells stably transfected with ASBT-V5 fusion protein (2BT cells). ASBT activity was significantly inhibited following 60 min infection with EPEC but not with nonpathogenic E. coli. Mutations in bacterial escN, espA, espB, and espD, the genes encoding for the elements of bacterial TTSS, ablated EPEC inhibitory effect on ASBT function. Furthermore, mutation in the bacterial BFP gene encoding for bundle-forming pili abrogated the inhibition of ASBT by EPEC, indicating the essential role for bacterial aggregation and the early attachment. The inhibition by EPEC was associated with a significant decrease in the V(max) of the transporter and a reduction in the level of ASBT on the plasma membrane. The inhibition of ASBT by EPEC was blocked in the presence of protein tyrosine phosphatase inhibitors. Our studies provide novel evidence for the alterations in the activity of ASBT by EPEC infection and suggest a possible effect for EPEC in influencing intestinal bile acid homeostasis.     

Characterization, cDNA cloning, and functional expression of mouse ileal sodium-dependent bile acid transporter

Mouse ileal sodium dependent bile acid transporter (ISBT) was characterized using isolated enterocytes. Only enterocytes from the most distal portion showed Na+-dependent [3H]taurocholate uptake. Northern blot analysis using a probe against mouse ISBT revealed the expression of mouse ISBT mRNA to be restricted to the distal ileum. The Km and Vmax for Na+-dependent [3H]taurocholate transport into isolated ileocytes were calculated as 27 microM and 360 pmol/mg protein/min, respectively. Uptake of [3H]taurocholate was inhibited by N-ethylmaleimide. We have cloned ISBT cDNA from mouse ileum. The cDNA included the entire open reading frame coding 348 amino acid protein with seven hydrophobic segments and two N-glycosylation sites. COS-7 cells transfected with the expression vector containing this cDNA expressed Na+-dependent [3H]taurocholate uptake activity with a Km of 34 microM.     

Targeted deletion of the ileal bile acid transporter eliminates enterohepatic cycling of bile acids in mice

The ileal apical sodium bile acid cotransporter participates in the enterohepatic circulation of bile acids. In patients with primary bile acid malabsorption, mutations in the ileal bile acid transporter gene (Slc10a2) lead to congenital diarrhea, steatorrhea, and reduced plasma cholesterol levels. To elucidate the quantitative role of Slc10a2 in intestinal bile acid absorption, the Slc10a2 gene was disrupted by homologous recombination in mice. Animals heterozygous (Slc10a2+/-) and homozygous (Slc10a2-/-) for this mutation were physically indistinguishable from wild type mice. In the Slc10a2-/- mice, fecal bile acid excretion was elevated 10- to 20-fold and was not further increased by feeding a bile acid binding resin. Despite increased bile acid synthesis, the bile acid pool size was decreased by 80% and selectively enriched in cholic acid in the Slc10a2-/- mice. On a low fat diet, the Slc10a2-/- mice did not have steatorrhea. Fecal neutral sterol excretion was increased only 3-fold, and intestinal cholesterol absorption was reduced only 20%, indicating that the smaller cholic acid-enriched bile acid pool was sufficient to facilitate intestinal lipid absorption. Liver cholesteryl ester content was reduced by 50% in Slc10a2-/- mice, and unexpectedly plasma high density lipoprotein cholesterol levels were slightly elevated. These data indicate that Slc10a2 is essential for efficient intestinal absorption of bile acids and that alternative absorptive mechanisms are unable to compensate for loss of Slc10a2 function.     

Apical sodium bile acid transporter and ileal lipid binding protein in gallstone carriers

Although a cholesterol supersaturation of gallbladder bile has been identified as the underlying pathophysiologic defect, the molecular pathomechanism of gallstone formation in humans remains poorly understood. A deficiency of the apical sodium bile acid transporter (ASBT) and ileal lipid binding protein (ILBP) in the small intestine may result in bile acid loss into the colon and might promote gallstone formation by reducing the bile acid pool and increasing the amount of hydrophobic bile salts. To test this hypothesis, protein levels and mRNA expression of ASBT and ILBP were assessed in ileal mucosa biopsies of female gallstone carriers and controls. Neither ASBT nor ILBP levels differed significantly between gallstone carriers and controls. However, when study participants were subgrouped by body weight, ASBT and ILBP protein were 48% and 67% lower in normal weight gallstone carriers than in controls (P < 0.05); similar differences were found for mRNA expression levels. The loss of bile transporters in female normal weight gallstone carriers was coupled with a reduction of protein levels of hepatic nuclear factor 1alpha and farnesoid X receptor. In conclusion, in normal weight female gallstone carriers, the decreased expression of ileal bile acid transporters may form a molecular basis for gallstone formation.     

Diminished gene expression of ileal apical sodium bile acid transporter explains impaired absorption of bile acid in patients with hypertriglyceridemia

Patients with type IV hyperlipoproteinemia, particularly those with familial hypertriglyceridemia (FHT), have impaired absorption of bile acid, a defect that may contribute to the hypertriglyceridemia ( J. Lipid Res. 1995. 36: 96;-107). To determine whether this absorption defect is a result of abnormal expression of the ileal apical sodium bile acid transporter (ASBT) gene, we biopsied the terminal ileum at colonoscopy in 28 subjects, 13 with hypertriglyceridemia and 15 control subjects. Of the 13 hypertriglyceridemic subjects, 10 had lipid profiles compatible with FHT (elevated very low density lipoprotein [VLDL] triglycerides with normal LDL cholesterol). ASBT mRNA levels were measured in these biopsies by RNase protection assay, using glyceraldehyde dehydrogenase mRNA as a reference. ASBT protein was quantitated by Western blotting with an antibody to the carboxy-terminal 20 amino acids of the protein. The mean +/- SEM ASBT mRNA level in the control group was 205.7 +/- 19.9 (arbitrary units) compared with 138. 7 +/- 19.1 for all 13 hypertriglyceridemics (P = 0.03) and 141.7 +/- 20.8 in the 10 FHT patients (P = 0.05). Commensurate with these mRNA levels, the mean ASBT protein level in the control group was 126.2 +/- 22.6 versus 58.8 +/- 13.8 in hypertriglyceridemics (P = 0.02) and 61.8 +/- 15.2 in the FHT patients (P = 0.05).We conclude that impaired absorption of bile acid in type IV hypertriglyceridemia results from diminished expression of the ASBT gene in terminal ileum.     

A 14-amino acid sequence with a beta-turn structure is required for apical membrane sorting of the rat ileal bile acid transporter

The rat ileal sodium-dependent bile acid transporter (Asbt) is a polytopic membrane glycoprotein, which is specifically expressed on the apical domain of the ileal brush-border membrane. In the present study, an essential 14-amino acid (aa 335-348) sorting signal was defined on the cytoplasmic tail of Asbt with two potential phosphorylation sites motifs for casein kinase II ((335)SFQE) and protein kinase C (PKC) ((339)TNK). Two-dimension NMR spectra analysis demonstrated that a tetramer, (340)NKGF, which overlaps with the potential PKC site within the 14-mer signal sequence, adopts a type I beta-turn conformation. Replacement of the potential phosphorylation residue Ser(335) and Thr(339) with alanine or deletion of either the 4 ((335)SFQE) or 10 aa (338-348, containing (339)TNKGF) from the C terminus of Asbt resulted in a significantly decreased initial bile acid transport activity and increased the basolateral distribution of the mutants by 2-3-fold compared with that of wild type Asbt. Deletion of the entire last 14 amino acids (335-348) from the C terminus of Asbt abolished the apical expression of the truncated Asbt. Moreover, replacement of the cytoplasmic tail of the liver basolateral membrane protein, Na(+)/taurocholate cotransporting polypeptide, with the 14-mer peptide tail of Asbt redirected the chimera to the apical domain. In contrast, a chimera consisting of the 14-mer peptide of Asbt fused with green fluorescent protein was expressed in an intracellular transport vesicle-like distribution in transfected Madin-Darby canine kidney and COS 7 cells. This suggests that the apical localization of the 14-mer peptide requires a membrane anchor to support proper targeting. The results from biological reagent treatment and low temperature shift (20 degrees C) suggests that Asbt follows a transport vesicle-mediated apical sorting pathway that is brefeldin A-sensitive and insensitive to protein glycosylation, monensin treatment, and low temperature shift.     

Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.     

Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents

Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.     

The structure of the human 4F2hc-LAT1 heteromeric amino acid transporter

<正>Amino acids are biologically important molecules that serve as energy source, building blocks of proteins, precursors for metabolites and signaling compounds in all living cells.Transport of proteinogenic amino acids and related substances across biological membranes is mediated by membrane embedded proteins:the amino acid transporters. These membrane proteins belong to different solute carrier (SLC)families (http://slc.bioparadigms.org) and transfer amino acids in and out between compartments inside cells, between different cells and between organs. Amino acid transporters have diverse important physiological functions and their absence, overexpression and malfunction can lead to human diseases.     

The nuclear receptor for bile acids, FXR, transactivates human organic solute transporter-alpha and -beta genes

Bile acids are synthesized from cholesterol in the liver and are excreted into bile via the hepatocyte canalicular bile salt export pump. After their passage into the intestine, bile acids are reabsorbed in the ileum by sodium-dependent uptake across the apical membrane of enterocytes. At the basolateral domain of ileal enterocytes, bile acids are extruded into portal blood by the heterodimeric organic solute transporter OSTalpha/OSTbeta. Although the transport function of OSTalpha/OSTbeta has been characterized, little is known about the regulation of its expression. We show here that human OSTalpha/OSTbeta expression is induced by bile acids through ligand-dependent transactivation of both OST genes by the nuclear bile acid receptor/farnesoid X receptor (FXR). FXR agonists induced endogenous mRNA levels of OSTalpha and OSTbeta in cultured cells, an effect that was not discernible upon inhibition of FXR expression by small interfering RNAs. Furthermore, OST mRNAs were induced in human ileal biopsies exposed to the bile acid chenodeoxycholic acid. Reporter constructs containing OSTalpha or OSTbeta promoters were transactivated by FXR in the presence of its ligand. Two functional FXR binding motifs were identified in the OSTalpha gene and one in the OSTbeta gene. Targeted mutation of these elements led to reduced inducibility of both OST promoters by FXR. In conclusion, the genes encoding the human OSTalpha/OSTbeta complex are induced by bile acids and FXR. By coordinated control of OSTalpha/OSTbeta expression, bile acids may adjust the rate of their own efflux from enterocytes in response to changes in intracellular bile acid levels.     

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter,OSTalpha-OSTbeta

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.     

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.     

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MbetaCD. The inhibition in ASBT activity by MbetaCD was blocked in the cells treated with MbetaCD-cholesterol complexes. Kinetic analysis revealed that MbetaCD treatment decreased the V(max) of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis.     

SC-435, an ileal apical sodium-codependent bile acid transporter inhibitor alters mRNA levels and enzyme activities of selected genes involved in hepatic cholesterol and lipoprotein metabolism in guinea pigs

We have demonstrated that SC-435, an apical sodium codependent bile acid transporter (ASBT) inhibitor, lowers plasma low-density lipoprotein cholesterol (LDL-C) concentrations in guinea pigs. The purpose of this study was to further examine the hypocholesterolemic effects of SC-435, by measuring the activity and RNA expression of regulatory enzymes of hepatic cholesterol and lipoprotein metabolism. In addition, the use of a combination (COMBO) therapy with simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, was also tested. Male Hartley guinea pigs were randomly allocated to one of three diets (n=10 per group), for 12 weeks. The control diet contained no ASBT inhibitor or simvastatin. The monotherapy diet (ASBTi) contained 0.1% of SC-435. The COMBO therapy consisted of a lower dose of SC-435 (0.03%) and 0.05% simvastatin. Cholesterol ester transfer protein (CETP) and HMG-CoA reductase mRNA abundance were determined using RT-PCR techniques. Hepatic HMG-CoA reductase and cholesterol 7-hydroxylase (CYP7) activities were measured by radioisotopic methods. Compared to the control group, CETP activity was 34% and 56% lower with ASBTi and COMBO, respectively. Similarly, CETP mRNA expression was reduced by 36% and 73% in ASBTi and COMBO groups, respectively. Cholesterol 7-hydroxylase and HMG-CoA reductase activities were increased 2-fold with ASBTi and COMBO treatments, respectively. Likewise, HMG-CoA reductase mRNA expression was increased 33% with ASBTi treatment. These results suggest that both SC-435 monotherapy and combination therapy lower LDL cholesterol concentrations by altering both hepatic cholesterol homeostasis and the intravascular processing of lipoproteins in guinea pigs.