Sodium-dependent Bile Acid Transporter Research Articles

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.

Substrate binding in the bile acid transporter ASBTYf from Yersinia frederiksenii.

Apical sodium-dependent bile acid transporter (ASBT) retrieves bile acids from the small intestine and plays a pivotal role in enterohepatic circulation. Currently, high-resolution structures are available for two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii), from which an elevator-style alternating-access mechanism has been proposed for substrate transport. A key concept in this model is that the substrate binds to the central cavity of the transporter so that the elevator-like motion can expose the bound substrate alternatingly to either side of the membrane during a transport cycle. However, no structure of an ASBT has been solved with a substrate bound in its central cavity, so how a substrate binds toASBT remains to be defined. In this study, molecular docking, structure determination and functional analysis were combined to define and validate the details of substrate binding in ASBTYf. The findings provide coherent evidence to provide a clearer picture of how the substrate binds in the central cavity of ASBTYf that fits the alternating-access model.

Inhibitors of the Apical Sodium-Dependent Bile Acid Transporter (ASBT) as Promising Drugs

Inhibitors of the Apical Sodium-Dependent Bile Acid Transporter (ASBT) as Promising Drugs

An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter.

Apical sodium-dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator-style alternating-access model, which was deduced mainly by comparing high-resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward-facing structure (PDB entry 4n7x) was obtained with an Na+-binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+- or substrate-binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild-type ASBTYf. These data support the hypothesis that the PDB entry 4n7x-like structure represents a functional outward-facing conformation of ASBTYf in its transport cycle.

Lactobacillus delbrueckii Interfere With Bile Acid Enterohepatic Circulation to Regulate Cholesterol Metabolism of Growing-Finishing Pigs via Its Bile Salt Hydrolase Activity.

Microbiota-targeted therapies for hypercholesterolemia get more and more attention and are recognized as an effective strategy for preventing and treating cardiovascular disease. The experiment was conducted to investigate the cholesterol-lowering mechanism of Lactobacillus delbrueckii in a pig model. Twelve barrows (38.70 ± 5.33 kg) were randomly allocated to two groups and fed corn–soybean meal diets with either 0% (Con) or 0.1% Lactobacillus delbrueckii (Con + LD) for 28 days. L. delbrueckii–fed pigs had lower serum contents of total cholesterol (TC), total bile acids (TBAs), and triglyceride, but higher fecal TC and TBA excretion. L. delbrueckii treatment increased ileal Lactobacillus abundance and bile acid (BA) deconjugation and affected serum and hepatic BA composition. Dietary L. delbrueckii downregulated the gene expression of ileal apical sodium-dependent bile acid transporter (ASBT) and ileal bile acid binding protein (IBABP), and hepatic farnesoid X receptor (FXR), fibroblast growth factor (FGF19), and small heterodimer partner (SHP), but upregulated hepatic high-density lipoprotein receptor (HDLR), low-density lipoprotein receptor (LDLR), sterol regulatory element binding protein-2 (SREBP-2), and cholesterol-7α hydroxylase (CYP7A1) expression. Our results provided in vivo evidence that L. delbrueckii promote ileal BA deconjugation with subsequent fecal TC and TBA extraction by modifying ileal microbiota composition and induce hepatic BA neosynthesis via regulating gut–liver FXR–FGF19 axis.

Effect of Zanthoxylum alkylamides on lipid metabolism and its mechanism in rats fed with a high-fat diet.

This research aimed at exploring the effect of Zanthoxylum alkylamides on lipid metabolism and its potential mechanisms using high-fat diet rat model. Treatment with Zanthoxylum alkylamides for 6weeks, food efficiency and atherogenic index of the low, medium, and high doses of Zanthoxylum alkylamides-treated groups were significantly reduced. Meanwhile, the histopathological structure of the livers showed that hepatic steatosis in the groups treated with Zanthoxylum alkylamides was reduced, particularly the HD group. Moreover, the related genes were studied, such as, liver X receptor (LXR), cholesterol 7 alpha-hydroxylase (CYP7A1), hepatic 3-hydroxyl-2-methylglutaryl CoA (HMG-CoA) reductase, sterol regulatory element-binding protein 2 (SREBP-2), ileal bile acid-binding protein (IBABP), sodium-dependent bile acid transporter (ASBT), and transient receptor potential vanilloid subtype1 (TRPV1). These results demonstrated that Zanthoxylum alkylamides could ameliorate abnormal lipid metabolism in rats fed with a high-fat diet. The underlying mechanism may be the downregulation of the expression levels of cholesterol synthesis and bile acid reabsorption-related genes, reduction of endogenous cholesterol synthesis, and increase in bile acid and neutral sterol excretion. PRACTICAL APPLICATIONS: High-energy diet is a potential risk of lipid metabolic disorder. Many studies have shown that hyperlipidemia can lead to atherosclerosis and even hemangioma, cerebral thrombosis, coronary heart disease, and other diseases, which seriously threaten human health. Therefore, seeking an effective and safe way to prevent the obesity-related disease is necessary. This research found that Zanthoxylum alkylamide could ameliorate abnormal lipid metabolism in rats fed with a high-fat diet. The underlying mechanism may be the downregulation of the expression levels of cholesterol synthesis and ileal absorption of bile acid genes, reduction of endogenous cholesterol synthesis, and increase in bile acid and neutral sterol excretion. Therefore, Zanthoxylum alkylamide has the potential for preventing or alleviating high-energy intake-related obesity.

A Novel Fluorescence-Based Method to Evaluate Ileal Apical Sodium-Dependent Bile Acid Transporter ASBT.

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122μM and Vmax 1.49 pmol/oocyte/30min for peak 1, 30.7μM and 1.34 pmol/oocyte/30min for peak 2, and 40.6μM and 2.36 pmol/oocyte/30min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na+ free condition. Furthermore, in Caco-2cells, tauro-nor-THCA-24-DBD uptake was also Na+-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.

S-acylation status of bile acid transporter hASBT regulates its function, metabolic stability, membrane expression, and phosphorylation state

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (Jmax) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.

The Beneficial Effects of Apical Sodium-Dependent Bile Acid Transporter Inactivation Depend on Dietary Fat Composition.

ScopeThe apical sodium‐dependent bile acid transporter (ASBT, SLC10A2) is important in the enterohepatic cycling of bile acids and thereby in the intestinal absorption of lipids. ASBT inhibition has been shown to improve aspects of the metabolic syndrome, but the underlying mechanisms have remained unclear. Here, the effect of ASBT inhibition on the uptake of specific fatty acids and its consequences for diet‐induced obesity and non‐alcoholic fatty liver disease (NAFLD) are investigated.MethodsIntestinal fat absorption is determined in mice receiving an ASBT inhibitor and in Asbt−/− mice. Metabolic disease development is determined in Asbt−/− mice receiving a low‐fat control diet (LFD) or high‐fat diet (HFD) rich in saturated fatty acids (SFAs) or PUFAs.ResultsBoth ASBT inhibition and Asbt gene inactivation reduce total fat absorption, particularly of SFAs. Asbt gene inactivation lowers bodyweight gain, improves insulin sensitivity, and decreases the NAFLD activity score upon feeding a HFD rich in SFAs, but not in PUFAs.ConclusionsThe beneficial metabolic effects of ASBT inactivation on diet‐induced obesity depend on decreased intestinal absorption of SFAs, and thus on the dietary fatty acid composition. These findings highlight the importance of dietary fatty acid composition in the therapeutic effects of ASBT inhibition.

Serum Cholesterol-Lowering Activity of β-Sitosterol Laurate Is Attributed to the Reduction of Both Cholesterol Absorption and Bile Acids Reabsorption in Hamsters.

The research was performed to delineate how β-sitosterol laurate (β-SLE) consumption influenced serum and hepatic lipids. The results showed that 220 mg/5 mL oil/kg body weight of β-SLE robustly reduced serum total triglyceride and cholesterol levels and the epididymal adipocyte size, and efficiently protected hepatic polyunsaturated fatty acids against lipid peroxidation through superoxide dismutase and glutathione transferase activity enhancement and malondialdehyde level reduction. Based on the changes of fecal cholesterol contents, fecal and hepatic bile acid (BAs) levels, and related protein expression, it was concluded that the mechanisms for lowering serum cholesterol by β-SLE involved (i) the enhanced excretion of fecal cholesterol via down-regulation of intestinal Niemann-Pick C1-like 1 protein; (ii) the increased conversion from cholesterol to primary BAs via up-regulation of cholesterol-7α-hydroxylase and sterol 27-hydroxylase, which was induced by the reduced BAs reabsorption through up-regulating ileal apical sodium-dependent bile acid transporter and ileal bile acid-binding protein.

Bile acid transporter-mediated oral drug delivery

Bile acids are synthesized in the liver, stored in the gallbladder, and secreted into the duodenum at meals. Apical sodium-dependent bile acid transporter (ASBT), an ileal Na+-dependent transporter, plays the leading role of bile acid absorption into enterocytes, where bile acids are delivered to basolateral side by ileal bile acid binding protein (IBABP) and then released by organic solute transporter OSTα/β. The absorbed bile acids are delivered to the liver via portal vein. In this process called “enterohepatic recycling”, only 5% of the bile acid pool (~3 g in human) is excreted in feces, indicating the large recycling capacity and high transport efficacy of ASBT-mediated absorption. Therefore, bile acid transporter-mediated oral drug delivery has been regarded as a feasible and potential strategy to improve the oral bioavailability. This review introduces the key factors in enterohepatic recycling, especially the mechanism of bile acid uptake by ASBT, and the development of bile acid-based oral drug delivery for ASBT-targeting, including bile acid-based prodrugs, bile acid/drug electrostatic complexation and bile acid-containing nanocarriers. Furthermore, the specific transport pathways of bile acid in enterocytes are described and the recent finding of lymphatic delivery of bile acid-containing nanocarriers is discussed.

The benefit of elobixibat in chronic constipation is associated with faecal deoxycholic acid but not effects of altered microbiota

Elobixibat, a novel inhibitor of apical sodium-dependent bile acid transporter for treating chronic constipation, increases colonic bile acid concentrations, stimulating bowel function. However, it is not clear which bile acids are altered, or whether altered gut microbiota are associated with functional effects that may alter bowel function. To investigate the effects of elobixibat on changes in the faecal concentrations of total and individual bile acids and in faecal microbiota. This was a prospective, single-centre study. After baseline period, patients received 10mg daily of elobixibat for 2weeks. We evaluated the effects on bowel function, changes in faecal bile acid concentrations and composition of gut bacteria, before and after elobixibat administration. In the 30 patients analysed, the frequency of pre- and post-treatment bowel movements per fortnight was 7 and 10 (P<0.001), respectively. The pre-treatment faecal bile acid concentration increased significantly from 10.9 to 15.0µg/g stool post-treatment (P=0.030), with a significant increase in faecal deoxycholic acid (pre-treatment 3.94µg/g stool to post-treatment 5.02µg/g stool, P=0.036) and in glycine-conjugated deoxycholic and chenodeoxycholic acids. Shannon index was significantly decreased, but there were no significant changes at the genus and phylum levels. Short term treatment with elobixibat increased the concentrations of total bile acids and deoxycholic acid and decreased the diversity of faecal microbiota. The biological effects of elobixibat are associated with its effects on secretory bile acids, rather than the structural changes of an altered faecal microbiota.

From the Editor's Desk…

From the Editor's Desk…

Synthetic G protein-coupled bile acid receptor agonists and bile acids act via basolateral receptors in ileal and colonic mucosa.

The G protein-coupled bile acid (BA) receptor, GPBA (previously named TGR5), mediates BA gastrointestinal (GI) activities. Our aim was to elucidate the mucosal and motility responses to selective GPBA agonists compared with conjugated BA (eg, taurodeoxycholate, TDCA) in mouse and human colon. Ion transport responses to GPBA agonists or BAs were measured in mucosal preparations with intact submucous innervation, from C57Bl/6, PYY-/-, or GPBA-/- mice and compared with GPBA signaling in human colon. We also investigated the mechanisms underlying GPBA agonism in mucosae and on natural fecal pellet propulsion. GPBA agonist Merck V stimulated basolateral responses involving peptide YY (PYY), cholinergic, and 5-HT mechanisms in colonic mucosa. The PYY-mediated GPBA signal was glucose-sensitive. Luminal TDCA crossed the epithelial lining via the apical sodium-dependent BA transporter (ASBT) and its inhibitor, GSK2330672 significantly reduced luminal, but not basolateral TDCA activity. Merck V also slowed natural fecal pellet progression in wild-type and PYY-/- colons but not in GPBA-/- colon, while TDCA increased motility in wild-type colon. The antimotile GPBA effect was reversed by blockade of glucagon-like peptide 1 (GLP-1) receptors or nitric oxide synthase, indicating involvement of GLP-1 and nitric oxide. We conclude that several different targets within the lamina propria express GPBA, including L cells (that release PYY and GLP-1), enterochromaffin cells and neurons (that release 5-HT), and other enteric neurons. Furthermore, luminal-conjugated BAs require transport across the epithelium via ASBT in order to activate basolateral GPBA.

IBD and Bile Acid Absorption: Focus on Pre-clinical and Clinical Observations.

Inflammatory bowel disease (IBD) causes chronic inflammation affecting the GI tract. It is classified as consisting of Crohn’s Disease (CD) and Ulcerative Colitis (UC). Bile Acid absorption is altered in both pre-clinical models of Inflammatory Bowel Disease (IB) and in human IBD. The bile acid transporter apical sodium dependent bile acid transporter (ASBT) showed decreased expression in rats with TNBS colitis. Decreased ASBT expression has also been described in murine, canine and rabbit models of intestinal inflammation. Human IBD studies have shown that an inflamed ileum can interrupt enterohepatic recirculation of bile acid, which could be due to inflammatory cytokine induced repression of the ASBT promoter. There are different hypotheses as to why ASBT is downregulated during CD. In addition, one study has demonstrated the beneficial effect of a glucocorticoid on ASBT expression, when treating IBD. Our aim in this paper was to systematically review various aspects of bile acid malabsorption in animal models of intestinal inflammation, as well as in IBD.

1662-P: Lower Expression of Bile Acid Transporters and Fibroblast Growth Factor 19 in Mucosa Biopsies from the Ileocecal Region in Persons with Type 2 Diabetes Compared with Healthy Controls

Background and Aims: Bile acids stimulate the secretion of gluco-regulatory and appetite-regulating gut hormones (including glucagon-like peptide 1 and fibroblast factor 19 (FGF19)) and increase energy expenditure. Changes in postprandial concentrations of circulating bile acids (primarily determined by the apical sodium-dependent bile acid transporter (ASBT) and organic solute transporter α/β (OSTα/β) in the terminal ileum) have been linked to type 2 diabetic pathophysiology. Methods: In a cross-sectional study, 12 persons with type 2 diabetes (T2D) (mean[range] age 51[34-63] years, BMI 26.8[23.7-31.5] kg/m2, HbA1c 48[36-85] mmol/mol) and 12 gender, age and BMI-matched nondiabetic controls (CTRLs) (age 50[41-67] years, BMI 27.1[20.3-30.8] kg/m2, HbA1c 34[29-43] mmol/mol) underwent upper and lower double-balloon enteroscopy with retrieval of mucosal biopsies from every 30 cm along the entire intestinal tract. The biopsies were subjected to full mRNA sequencing and here, the expression of ASBT, OSTα/β and FGF19 is reported. Results: For both groups, robust expression (reads per kilobase million (RPKM) ≥1) of ASBT and OSTα/β was evident in the duodenum and this increased along the small intestine, with highest expression of ASBT in the ileocecal region and highest expression of OSTα/β in terminal ileum. Compared to healthy controls, persons with T2D exhibited significantly lower ASBT and OSTα/β expression in the ileocecal region (P&amp;lt;0.05). Compared to persons with T2D in whom expression of FGF19 was below 1 RPKM in all locations, the control group had robust and significantly greater expression of FGF19 in the terminal ileum (P&amp;lt;0.01). Conclusions: T2D is associated with reduced expression of ASBT, OSTα/β and FGF19 in the terminal ileum and the proximal colon. This may point to a link between dysfunctional enterohepatic circulation of bile acids and type 2 diabetic pathophysiology. Disclosure H.H. Nerild: Employee; Spouse/Partner; Novo Nordisk A/S. A. Brønden: None. A. El Haddouchi: None. T. Jorsal: None. D.P. Sonne: None. J. Jelsing: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. K. Rigbolt: Employee; Self; Gubra. T. Vilsbøll: Advisory Panel; Self; AstraZeneca, Mundipharma International, Novo Nordisk A/S, Sun Pharmaceutical Industries Ltd. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Medscape, Merck Sharp &amp; Dohme Corp., Sanofi. F.K. Knop: Advisory Panel; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Mundipharma International, Novo Nordisk A/S, Sanofi. Consultant; Self; Carmot Therapeutics, Inc., Eli Lilly and Company, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Gubra, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Speaker’s Bureau; Self; AstraZeneca, Lupin Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Norgine B.V., Novo Nordisk A/S.

Changes in the gut microbiota caused by ASBTi suppress intrahepatic steatosis and inflammation in a mouse model of non-alcoholic fatty liver disease (NAFLD)

Abstract Although the estimated prevalence of NAFLD is approximately 25%, an effective treatment has not been established. Recently, gut microbiota has been shown to affect the pathogenesis of NAFLD. In this study, the protective effect of Apical Sodium-dependent Bile acid Transporter inhibitor (ASBTi) against NAFLD was investigated in mice fed a high-fat diet for 12 weeks (HFD mice) via changing their gut microbiota. ASBTi mice were mice treated with ASBTi (2.5 μmol/kg/day) during HFD feeding. For fecal microbiota transplantation (FMT), stools were collected from HFD mice and ASBTi mice. Fecal materials obtained from HFD mice and ASBTi mice were transplanted to normal mice, fed HFD for 12 weeks, and designated as HFD-FMT mice and ASBTi-FMT mice, respectively. Microbiota in these groups of mice were analyzed by 16S rRNA gene sequencing. The degrees of hepatic steatosis and inflammation in these mice were determined pathologically. The shannon index of the microbiota from ASBTi mice (3.31±0.19) and ASBTi-FMT mice (3.29±0.18) was significantly high as compared to that from HFD mice (2.90±0.19) and HFD-FMT mice (2.89±0.16). Steatosis and inflammation in the liver of HFD mice and HFD-FMT mice were shown to be pathologically severe and NAFLD Activity Score (NAS) was 7.67±0.58 in HFD mice and 5.67±2.08 in HFD-FMT mice. However, those in the liver of ASBTi mice and ASBTi-FMT mice were improved and NAS was reduced in ASBTi mice (1.67±0.58) and in ASBTi-FMT mice (2.67±0.58). These results suggest that changes in the gut microbiota caused by ASBTi suppress intrahepatic steatosis and inflammation in NAFLD mice. Elobixibat can be expected to prevent fatty liver.

Mechanism of Bile Acid Reabsorption in the Biliopancreatic Limb After Duodenal-Jejunal Bypass in Rats.

Bile acids (BAs) are important in the metabolic effects of bariatric surgery. Most BAs are reabsorbed in the ileum and recycled back to the liver. We have reported that this enterohepatic circulation was shortened by duodenal-jejunal bypass (DJB), and the biliopancreatic (BP)-limb plays an important role in reabsorption of BAs. However, the mechanism of BA reabsorption in BP-limb remains uncertain. We aimed to investigate the mechanisms of BA reabsorption after DJB, especially focusing on carrier-mediated transport of BAs and the impact of the presence or absence of lipids on BA reabsorption. Otsuka-Long-Evans-Tokushima fatty rats or Sprague-Dawley rats were assigned to a control group and DJB group. BA levels in the divided small intestine were quantified with liquid chromatography-mass spectrometry. Labeled BA was injected and perfused with BA transporter inhibitors or mixture of lipids in the isolated BP-limb, and bile was sampled and analyzed. Conjugated BA levels in the BP-limb were significantly higher than that of the control group. BA absorption tended to decrease by the apical sodium-dependent BA transporter inhibitor and was significantly decreased by the organic anion-transporting peptide (OATP) inhibitor. BA absorption tended to increase in the absence of lipid solutions compared with that in the presence of lipid solutions. We attributed the increased BA reabsorption in the BP-limb to lack of food in the BP-limb, which contains concentrated BAs and no lipids. OATP played an important role in BA reabsorption in the BP-limb. Therefore, BAs would be reabsorbed in different manners after DJB.

Functional Characterization of Position 271 in NTCP, a Predicted Rheostat Location

BackgroundHuman Na+/taurocholate cotransporting polypeptide (NTCP) is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. It is important for the enterohepatic circulation of bile acids, such as taurocholate (TCA), but it also transports other compounds including estrone‐3‐sulfate (E3S) and rosuvastatin. We previously identified a rheostat position at a naturally occurring polymorphism, S267F. That is, when other amino acids were substituted into this position, progressive changes in function were observed.AimThe purpose of the current study is to determine whether position N271, a predicted rheostat, is indeed another rheostat position in NTCP.MethodsThe crystal structures of two bacterial NTCP homologs were used to model the structure of human NTCP in the inward‐open and outward‐open conformations. Mutations were then introduced into the structural models and the energetic effect on protein stability was evaluated using the Rosetta software. Based on these calculations we predicted that N271 is a rheostat. Mutants for position N271 were generated using site‐directed mutagenesis and the resulting proteins were transiently expressed in HEK293 cells. The functions of the mutants were characterized by measuring uptake of radiolabeled model substrates: [3H]‐taurocholate, [3H]‐estrone‐3‐sulfate, and [3H]‐rosuvastatin. Plasma membrane expression levels were determined by surface biotinylation assays.ResultsInitial transport studies using nanomolar concentrations of TCA, E3S, and rosuvastatin indicated a variety of intermediate functional outcomes for the mutants. Furthermore, this alteration in function was substrate dependent, which is consistent with our findings for S267. Some mutants, such as N271A, transport substrates similar to wild‐type NTCP, while others, like N271K, show variation depending on the substrate. N271P consistently transports all substrates minimally. In addition, there is no correlation between the initial uptake results and the Rosetta energies.Summary and ConclusionsInitial transport studies demonstrate that position N271 is a rheostat position, as predicted by the alteration in Rosetta energy scores. However, there was no correlation between the calculated energy scores and the functional results. This suggests that functional outcomes of specific amino acid replacements at position N271 may arise from more complex conformational changes than were captured by the two modeled states. Additional experiments should help to determine to what extent the observed change in function is due to variations in the substrate affinity, the turnover number or the transport capacity of the mutants.Support or Funding InformationP30 GM118247, GM077336, and W. M. Keck Foundation

S-acylation modulates the function of the apical sodium-dependent bile acid transporter in human cells

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several posttranslational modifications. One reversible posttranslational modification is S-acylation, involving the covalent attachment of fatty acids to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. Using the acyl resin-assisted capture method, we found that the majority of ASBT (∼80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 μm for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 μm for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 μm for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 μm for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated fatty acids to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated.