Apical Sodium-dependent Bile Acid Transporter Research Articles

OS019 - Novel anti-cholestatic treatment strategies by combining inhibition of the Apical sodium-dependent bile acid transporter with stimulation of urinary bile salt excretion or lowering bile salt synthesis

OS019 - Novel anti-cholestatic treatment strategies by combining inhibition of the Apical sodium-dependent bile acid transporter with stimulation of urinary bile salt excretion or lowering bile salt synthesis

Biomimetic and temporal-controlled nanocarriers with ileum transporter targeting for achieving oral administration of chemotherapeutic drugs

BackgroundOral chemotherapy is preferred for patients with cancer owing to its multiple advantages, including convenience, better patient compliance, and improved safety. Nevertheless, various physical barriers exist in this route that hamper the development of oral chemotherapeutic formulations, including destruction of drugs in the gastrointestinal tract (GIT), low permeability in enterocytes, and short residence time in the intestine. To overcome these limitations, it is necessary to design an efficient oral drug delivery system with high efficacy and improved safety.ResultsHerein, we designed novel glycocholic acid (GCA)-functionalized double layer nanoparticles (GCA-NPs), which can act via an endogenous pathway and in a temporally controlled manner in the intestine, to enhance the oral bioavailability of hydrophobic chemotherapeutic drugs such as paclitaxel (PTX). GCA-NPs were composed of quercetin (Qu)-modified liposomes (QL) coated with GCA-chitosan oligosaccharide conjugate (GCOS). The GCA-NPs thus prepared showed prolonged intestinal retention time and good GIT stability due to the presence of chitosan oligosaccharide (COS) and enhanced active transportation via intestinal apical sodium-dependent bile acid transporter (ASBT) due to the presence of GCA. GCA-NPs also efficiently inhibited intestinal P-gp induced by Qu. PTX-loaded GCA-NPs (PTX@GCA-NPs) had a particle size of 84 nm and an entrapment efficiency of 98% with good stability. As a result, the oral bioavailability of PTX was increased 19-fold compared to that of oral Taxol® at the same dose. Oral PTX@GCA-NPs displayed superior antitumor efficacy and better safety than Taxol® when administered intravenously.ConclusionsOur novel drug delivery system showed remarkable efficacy in overcoming multiple limitations and is a promising carrier for oral delivery of multiple drugs, which addresses several challenges in oral delivery in the clinical context.Graphical

Bile Acid Conjugation on Solid Nanoparticles Enhances ASBT‐Mediated Endocytosis and Chylomicron Pathway but Weakens the Transcytosis by Inducing Transport Flow in a Cellular Negative Feedback Loop

Bile acid‐modified nanoparticles provide a convenient strategy to improve oral bioavailability of poorly permeable drugs by exploiting specific interactions with bile acid transporters. However, the underlying mechanisms are unknown, especially considering the different absorption sites of free bile acids (ileum) and digested fat molecules from bile acid‐emulsified fat droplets (duodenum). Here, glycocholic acid (GCA)‐conjugated polystyrene nanoparticles (GCPNs) are synthesized and their transport in Caco‐2 cell models is studied. GCA conjugation enhances the uptake by interactions with apical sodium‐dependent bile acid transporter (ASBT). A new pathway correlated with both ASBT and chylomicron pathways is identified. Meanwhile, the higher uptake of GCPNs does not lead to higher transcytosis to the same degree compared with unmodified nanoparticles (CPNs). The pharmacological and genomics study confirm that GCA conjugation changes the endocytosis mechanisms and downregulates the cellular response to the transport at gene levels, which works as a negative feedback loop and explains the higher cellular retention of GCPNs. These findings offer a solid foundation in the bile acid‐based nanomedicine design, with utilizing advantages of the ASBT‐mediated uptake, as well as inspiration to take comprehensive consideration of the cellular response with more developed technologies.

Acrylamide induced glucose metabolism disorder in rats involves gut microbiota dysbiosis and changed bile acids metabolism

Acrylamide (AA) is a common food contaminant that causes glucose metabolism disorders (GMD). However, the underlying mechanism remains unclear. Female Sprague Dawley (SD) rats were treated with AA via gavage for 21 days, and the glucose and insulin levels, gut microbiota, intestinal barrier, and metabolism were analyzed. The results revealed that AA elevated serum glucose levels, reduced insulin levels and caused intestinal barrier injury. The 16S amplicon sequencing and non-targeted metabolomics showed that AA induced gut microbiota dysbiosis and bile acids (BAs) metabolism disorder. Specifically, AA decreased the abundance of Lactobacillus and Bacteroides in the cecal contents, and increased the cholic acid (CA) content in feces. Meanwhile, the expression of ileum apical sodium-dependent bile acid transporter (ASBT) responsible for CA reabsorption was suppressed. Further analysis indicated that BAs sensing nuclear receptor farnesoid X receptor (FXR) gene was activated and glucagon-like peptide-1 (GLP-1) which stimulates insulin secretion was downregulated. In addition, activation of FXR increased the expression of fibroblast growth factor 15 (FGF15), which resulted in the inhibition of hepatic BAs synthesis. Overall, this study demonstrated that AA-induced GMD is associated with the gut-microbiota-CA-FXR/GLP-1 axis. These findings add new knowledge to the AA-induced GMD and provide a basis for potential AA toxicity mitigation by manipulation of the gut microbiota.

Structural insights into the HBV receptor and bile acid transporter NTCP

Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.

L-Arabinose improves hypercholesterolemia via regulating bile acid metabolism in high-fat-high-sucrose diet-fed mice

BackgroundHypercholesterolemia is closely associated with an increased risk of cardiovascular diseases. l-Arabinose exhibited hypocholesterolemia properties, but underlying mechanisms have not been sufficiently investigated. This study aimed to elucidate the mechanisms of l-arabinose on hypocholesterolemia involving the enterohepatic circulation of bile acids.MethodsThirty six-week-old male mice were randomly divided into three groups: the control group and the high-fat-high-sucrose diet (HFHSD)-fed group were gavaged with distilled water, and the l-arabinose-treated group were fed HFHSD and received 400 mg/kg/day l-arabinose for 12 weeks. Serum and liver biochemical parameters, serum and fecal bile acid, cholesterol and bile acid metabolism-related gene and protein expressions in the liver and small intestine were analyzed.Resultsl-Arabinose supplementation significantly reduced body weight gain, lowered circulating low-density lipoprotein cholesterol (LDL-C) while increasing high-density lipoprotein cholesterol (HDL-C) levels, and efficiently alleviated hepatic inflammation and lipid accumulations in HFHSD-fed mice. l-Arabinose inhibited cholesterol synthesis via downregulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Additionally, l-arabinose might facilitate reverse cholesterol transport, evidenced by the increased mRNA expressions of low-density lipoprotein receptor (LDL-R) and scavenger receptor class B type 1 (SR-B1). Furthermore, l-arabinose modulated ileal reabsorption of bile acids mainly through downregulation of ileal bile acid-binding protein (I-BABP) and apical sodium-dependent bile acid transporter (ASBT), resulting in the promotion of hepatic synthesis of bile acids via upregulation of cholesterol-7α-hydroxylase (CYP7A1).Conclusionsl-Arabinose supplementation exhibits hypocholesterolemic effects in HFHSD-fed mice primarily due to regulation of bile acid metabolism-related pathways.

Exposure to the mycotoxin deoxynivalenol reduces the transport of conjugated bile acids by intestinal Caco-2 cells

Conjugated bile acids are synthesized in liver and subsequently secreted into the intestinal lumen from which they are actively reabsorbed and transported back to liver. The efficient enterohepatic circulation of conjugated bile acids is important to maintain homeostasis. The mycotoxin deoxynivalenol (DON) is a fungal secondary metabolite that contaminates cereal food. Upon human exposure, it can cause intestinal dysfunction. We explored the effects of DON exposure on the intestinal absorption of conjugated bile acids and the expression of bile acid transporters using an in vitro model based on Caco-2 cell layers grown in transwells. Our study shows that the transport rate of taurocholic acid (TCA) is decreased after 48-h pre-exposure of the Caco-2 cells to 2 µM DON, which is a realistic intestinal DON concentration. Exposure to DON downregulates expression of the genes coding for the apical sodium-dependent bile acid transporter (ASBT), the ileal bile acid-binding protein (IBABP) and the organic solute transporter α (OSTα), and it counteracts the agonist activity of Farnesoid X receptor (FXR) agonist GW4064 on these genes. In addition, the transport of ten taurine or glycine-conjugated bile acids in a physiological relevant mixture by the intestinal Caco-2 cell layers was decreased after pre-exposure of the cells to DON, pointing at a potential for DON-mediated accumulation of the conjugated bile acids at the intestinal luminal side. Together the results reveal that DON inhibits intestinal bile acid reabsorption by reducing the expression of bile acid transporters thereby affecting bile acid intestinal kinetics, leading to bile acid malabsorption in the intestine. Our study provides new insights into the hazards of DON exposure.

Effect of Enterohepatic Circulation on the Accumulation of Per- and Polyfluoroalkyl Substances: Evidence from Experimental and Computational Studies.

The pharmacokinetic characteristics of per- and polyfluoroalkyl substances (PFAS) affect their distribution and bioaccumulation in biological systems. The enterohepatic circulation leads to reabsorption of certain chemicals from bile back into blood and the liver and thus influences their elimination, yet its influence on PFAS bioaccumulation remains unclear. We explored the role of enterohepatic circulation in PFAS bioaccumulation by examining tissue distribution of various PFAS in wild fish and a rat model. Computational models were used to determine the reabsorbed fractions of PFAS by calculating binding affinities of PFAS for key transporter proteins of enterohepatic circulation. The results indicated that higher concentrations were observed in blood, the liver, and bile compared to other tissues for some PFAS in fish. Furthermore, exposure to a PFAS mixture on the rat model showed that the reabsorption phenomenon appeared during 8-12 h for most long-chain PFAS. Molecular docking calculations suggest that PFAS can bind to key transporter proteins via electrostatic and hydrophobic interactions. Further regression analysis adds support to the hypothesis that binding affinity of the apical sodium-dependent bile acid transporter is the most important variable to predict the human half-lives of PFAS. This study demonstrated the critical role of enterohepatic circulation in reabsorption, distribution, and accumulation of PFAS.

Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

Bile acid (BA) homeostasis is regulated by the extensive cross-talk between liver and intestine. Many bile-acid-activated signaling pathways have become attractive therapeutic targets for the treatment of metabolic disorders. In this study we investigated the regulatory mechanisms of BA in the intestine. We showed that the BA levels in the gallbladder and faeces were significantly increased, whereas serum BA levels decreased in systemic Krüppel-like factor 9 (Klf9) deficiency (Klf9-/-) mice. These phenotypes were also observed in the intestine-specific Klf9-deleted (Klf9vil-/-) mice. In contrast, BA levels in the gallbladder and faeces were reduced, whereas BA levels in the serum were increased in intestinal Klf9 transgenic (Klf9Rosa26+/+) mice. By using a combination of biochemical, molecular and functional assays, we revealed that Klf9 promoted the expression of apical sodium-dependent bile acid transporter (Asbt) in the terminal ileum to enhance BA absorption in the intestine. Reabsorbed BA affected liver BA synthetic enzymes by regulating Fgf15 expression. This study has identified a previously neglected transcriptional pathway that regulates BA homeostasis.

The Apical Sodium-Dependent Bile Acid Transporter in Cholangiocytes Is Not Required for the Generation of Bile Flow in Mice.

Bile flow is generated by the osmotic activity exerted by the secretion of bile acids and other solutes from hepatocytes into bile canaliculi. The bile duct system, lined by cholangiocytes, can subsequently alter the bile composition, but it has remained unclear whether bile acid transporters in cholangiocytes quantitatively contribute to the generation of bile flow in vivo. Classically, bile flow has been subclassified into a bile acid-dependent component, indicating the linear positive correlation between bile acid secretion and bile flow generation, and a bile acid-independent component, the Y-intercept of the bile acid secretion to bile flow correlation.

Redundant Functions of ERK1 and ERK2 Maintain Mouse Liver Homeostasis Through Down-Regulation of Bile Acid Synthesis.

Activation of extracellular signal–regulated kinase (ERK) 1/2 promotes hepatocyte proliferation in response to growth stimuli, but whether constitutive hepatocyte ERK1/2 signaling functions in liver physiology is unknown. To examine the role of ERK1/2 in hepatic homeostasis, the effects of a knockout of Erk1 and/or Erk2 in mouse liver were examined. The livers of mice with a global Erk1 knockout or a tamoxifen‐inducible, hepatocyte‐specific Erk2 knockout were normal. In contrast, Erk1/2 double‐knockout mice developed hepatomegaly and hepatitis by serum transaminases, histology, terminal deoxynucleotide transferase‐mediated deoxyuridine triphosphate nick end‐labeling, and assays of hepatic inflammation. Liver injury was associated with biochemical evidence of cholestasis with increased serum and hepatic bile acids and led to hepatic fibrosis and mortality. RNA sequencing and polymerase chain reaction analysis of double‐knockout mouse livers revealed that the rate‐limiting bile acid synthesis gene Cyp7a1 (cholesterol 7α‐hydroxylase) was up‐regulated in concert with decreased expression of the transcriptional repressor short heterodimer partner. Elevated bile acids were the mechanism of liver injury, as bile acid reduction by SC‐435, an inhibitor of the ileal apical sodium–dependent bile acid transporter, prevented liver injury. Conclusion: Constitutive ERK1 and ERK2 signaling has a redundant but critical physiological function in the down‐regulation of hepatic bile acid synthesis to maintain normal liver homeostasis.

Modulation of Bile Acid Metabolism to Improve Plasma Lipid and Lipoprotein Profiles.

New drugs targeting bile acid metabolism are currently being evaluated in clinical studies for their potential to treat cholestatic liver diseases, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Changes in bile acid metabolism, however, translate into an alteration of plasma cholesterol and triglyceride concentrations, which may also affect cardiovascular outcomes in such patients. This review attempts to gain insight into this matter and improve our understanding of the interactions between bile acid and lipid metabolism. Bile acid sequestrants (BAS), which bind bile acids in the intestine and promote their faecal excretion, have long been used in the clinic to reduce LDL cholesterol and, thereby, atherosclerotic cardiovascular disease (ASCVD) risk. However, BAS modestly but consistently increase plasma triglycerides, which is considered a causal risk factor for ASCVD. Like BAS, inhibitors of the apical sodium-dependent bile acid transporter (ASBTi’s) reduce intestinal bile acid absorption. ASBTi’s show effects that are quite similar to those obtained with BAS, which is anticipated when considering that accelerated faecal loss of bile acids is compensated by an increased hepatic synthesis of bile acids from cholesterol. Oppositely, treatment with farnesoid X receptor agonists, resulting in inhibition of bile acid synthesis, appears to be associated with increased LDL cholesterol. In conclusion, the increasing efforts to employ drugs that intervene in bile acid metabolism and signalling pathways for the treatment of metabolic diseases such as NAFLD warrants reinforcing interactions between the bile acid and lipid and lipoprotein research fields. This review may be considered as the first step in this process.

Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 β-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04μM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

Apical sodium-dependent bile acid transporter inhibition in children with Alagille syndrome

In The Lancet, Emmanuel Gonzales and colleagues 1 Gonzales E Hardikar W Stormon M et al. Efficacy and safety of maralixibat treatment in patients with Alagille syndrome and cholestatic pruritus (ICONIC): a randomised phase 2 study. Lancet. 2021; 398: 1581-1592 Google Scholar present the findings of the phase 2 placebo-controlled, randomised withdrawal period, open-label, extension ICONIC study of the apical sodium-dependent bile acid transporter (ASBT) inhibitor, maralixibat, in paediatric patients with Alagille syndrome. Alagille syndrome is a cholestatic liver disease that leads to pruritus, xanthomas, and impaired quality of life. Surgical interventions to interrupt the enterohepatic bile acid circulation or liver transplantation are often pursued 2 Wang KS Tiao G Bass LM et al. Analysis of surgical interruption of the enterohepatic circulation as a treatment for pediatric cholestasis. Hepatology. 2017; 65: 1645-1654 Google Scholar , 3 Kamath BM Ye W Goodrich NP et al. Outcomes of childhood cholestasis in Alagille syndrome: results of a multicenter observational study. Hepatol Commun. 2020; 4: 387-398 Google Scholar because there are no approved medical therapies. ASBT has a key role in the enterohepatic circulation of bile acids because it absorbs approximately 95% of bile acids from the terminal ileum. Because intrahepatic bile acids are thought to be the main driver behind hepatocellular injury in cholestatic diseases, pharmacological inhibition of ASBT has the potential to not only improve pruritus, but also disease trajectory. Studies in mouse models of cholestasis have shown that ASBT inhibition can decrease intrahepatic bile acid concentrations and improve fibrosis and disease progression. 4 Miethke AG Zhang W Simmons J et al. Pharmacological inhibition of apical sodium-dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice. Hepatology. 2016; 63: 512-523 Google Scholar , 5 Baghdasaryan A Fuchs CD Osterreicher CH et al. Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis. J Hepatol. 2016; 64: 674-681 Google Scholar Efficacy and safety of maralixibat treatment in patients with Alagille syndrome and cholestatic pruritus (ICONIC): a randomised phase 2 studyIn children with Alagille syndrome, maralixibat is, to our knowledge, the first agent to show durable and clinically meaningful improvements in cholestasis. Maralixibat might represent a new treatment paradigm for chronic cholestasis in Alagille syndrome. Full-Text PDF

Bile acid transporter-mediated oral absorption of insulin via hydrophobic ion-pairing approach

Despite many ongoing and innovative approaches, there are still formidable challenges in the clinical translation of oral peptide drugs into marketable products due to their low absorption and poor bioavailability. Herein, a novel nanocarrier platform was developed that employs a hydrophobic ion-pairing (HIP) of model peptide (insulin) and the anionic bile salt (sodium glycodeoxycholate, SGDC), and markedly improves intestinal absorption via the bile acid pathway. The developed HIP-nanocomplexes (C1 and C2) were optimized, characterized, and in vitro and in vivo evaluation were performed to assess oral efficacy of these system. The optimal molar ratios of C1 and C2-nanocomplexes were 30:1 and 6:1 (SGDC:insulin), respectively. Compared to the insulin solution, the C1 and C2 nanocomplexes significantly enhanced the permeation of insulin across the Caco-2 cell monolayers, with 6.36- and 4.05-fold increases in apparent permeability, respectively. Uptake mechanism studies were conducted using different endocytosis inhibitors and apical sodium-dependent bile acid transporter (ASBT)-transfected MDCK cells, which demonstrated the involvement of the energy-dependent ASBT-mediated active transport. Furthermore, the intrajejunal administration of C1 and C2 resulted in their pharmacological availabilities (PA) being 6.44% and 0.10%, respectively, indicating increased potential for C1, when compared to C2. Similarly, the PA and the relative bioavailability with intrajejunal administration of the C1 were 17.89-fold and 16.82-fold greater than those with intracolonic administration, respectively, confirming better jejunal absorption of C1. Overall, these findings indicate that the HIP-nanocomplexes could be a prominent platform for the effective delivery of peptides with improved intestinal absorption.

Modulatory Effect of Theaflavins on Apical Sodium-Dependent Bile Acid Transporter (ASBT) Activity.

Inhibiting apical sodium-dependent bile acid transporter (ASBT) has been identified as a potential strategy to reduce plasma cholesterol levels. Thus, in this study, we aimed to identify polyphenols that inhibited ASBT activity and to elucidate their mechanism. ASBT is responsible for most of the taurocholic acid (TC) uptake in Caco-2 cells. Of the 39 polyphenols examined, theaflavin (TF)-3-gallate (TF2A) and theaflavin-3'-gallate (TF2B) have been found to significantly reduce TC uptake in Caco-2 cells to 37.4 ± 2.8 and 33.8 ± 4.0%, respectively, of that in the untreated cells. The results from the TC uptake assay using N-acetylcysteine suggested that the inhibitory effect of TF2A and TF2B was attributed to the oxidization of their benzotropolone rings and their covalent bonding with ASBT's cysteine. TC uptake was reduced in the COS-7 cells expressing recombinant ASBT whose cysteine residues were mutated to alanine. Finally, the substrate concentration-dependent TC uptake assay showed that TFs competitively inhibited TC uptake.

Evaluation for Potential Drug-Drug Interaction of MT921 Using In Vitro Studies and Physiologically-Based Pharmacokinetic Models.

MT921 is a new injectable drug developed by Medytox Inc. to reduce submental fat. Cholic acid is the active pharmaceutical ingredient, a primary bile acid biosynthesized from cholesterol, endogenously produced by liver in humans and other mammals. Although individuals treated with MT921 could be administered with multiple medications, such as those for hypertension, diabetes, and hyperlipidemia, the pharmacokinetic drug–drug interaction (DDI) has not been investigated yet. Therefore, we studied in vitro against drug-metabolizing enzymes and transporters. Moreover, we predicted the potential DDI between MT921 and drugs for chronic diseases using physiologically-based pharmacokinetic (PBPK) modeling and simulation. The magnitude of DDI was found to be negligible in in vitro inhibition and induction of cytochrome P450s and UDP-glucuronosyltransferases. Organic anion transporting polypeptide (OATP)1B3, organic anion transporter (OAT)3, Na+-taurocholate cotransporting polypeptide (NTCP), and apical sodium-dependent bile acid transporter (ASBT) are mainly involved in MT921 transport. Based on the result of in vitro experiments, the PBPK model of MT921 was developed and evaluated by clinical data. Furthermore, the PBPK model of amlodipine was developed and evaluated. PBPK DDI simulation results indicated that the pharmacokinetics of MT921 was not affected by the perpetrator drugs. In conclusion, MT921 could be administered without a DDI risk based on in vitro study and related in silico simulation. Further clinical studies are needed to validate this finding.

Auxin transport mechanism of membrane transporter encoded by AEC gene of Bacillus licheniformis isolated from metagenome of Tapta Kund Hotspring of Uttrakhand, India

Members of group Bacillus are most widely occurring microbes in agricultural soil and they affect crop health in various ways. They directly stimulate plant growth either by augmenting nutrients availability, invigorating plants' defence mechanisms; repressing soil-borne phytopathogens or by producing growth-regulating hormones like auxins and cytokinins. It is a well known fact that indole-3- acetic acid (a type of auxin) is a vital biologically active phytohormone excreted by certain Bacillus species, but its molecular mechanism has not yet been described. In this study, the auxin efflux carrier gene is isolated from the metagenome of the Tapta Kund hot spring, Uttrakhand, India. In addition, auxin efflux carrier (AEC) transporter protein of Bacillus licheniformis is modeled and the 318 amino acid residues long protein was found homologous to the apical sodium-dependent bile acid transporter (ASBT) of Yersinia frederiksnii, with 10 transmembrane segments (TM1–10) split into different domains: a panel domain defined by TM1, 2, 6 and 7; and a core domain defined by TM3–5 and 8–10. Finally, the predicted Bacillus licheniformis AEC protein has also been phylogenetically evaluated and its detailed molecular transport mechanism was worked out using molecular dynamics simulation analysis. Conclusively, this study demonstrates the efflux mechanism of the substrate, Indole 3- acetic acid by AEC transporter protein.

Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium–dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na+‐taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na+‐taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium–dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.

Substrate Specificities and Inhibition Pattern of the Solute Carrier Family 10 Members NTCP, ASBT and SOAT.

Three carriers of the solute carrier family SLC10 have been functionally characterized so far. Na+/taurocholate cotransporting polypeptide NTCP is a hepatic bile acid transporter and the cellular entry receptor for the hepatitis B and D viruses. Its intestinal counterpart, apical sodium-dependent bile acid transporter ASBT, is responsible for the reabsorption of bile acids from the intestinal lumen. In addition, sodium-dependent organic anion transporter SOAT specifically transports sulfated steroid hormones, but not bile acids. All three carriers show high sequence homology, but significant differences in substrate recognition that makes a systematic structure-activity comparison attractive in order to define the protein domains involved in substrate binding and transport. By using stably transfected NTCP-, ASBT-, and SOAT-HEK293 cells, systematic comparative transport and inhibition experiments were performed with more than 20 bile acid and steroid substrates as well as different inhibitors. Taurolithocholic acid (TLC) was identified as the first common substrate of NTCP, ASBT and SOAT with K m values of 18.4, 5.9, and 19.3 µM, respectively. In contrast, lithocholic acid was the only bile acid that was not transported by any of these carriers. Troglitazone, BSP and erythrosine B were identified as pan-SLC10 inhibitors, whereas cyclosporine A, irbesartan, ginkgolic acid 17:1, and betulinic acid only inhibited NTCP and SOAT, but not ASBT. The HBV/HDV-derived myr-preS1 peptide showed equipotent inhibition of the NTCP-mediated substrate transport of taurocholic acid (TC), dehydroepiandrosterone sulfate (DHEAS), and TLC with IC50 values of 182 nM, 167 nM, and 316 nM, respectively. In contrast, TLC was more potent to inhibit myr-preS1 peptide binding to NTCP with IC50 of 4.3 µM compared to TC (IC50 = 70.4 µM) and DHEAS (IC50 = 52.0 µM). Based on the data of the present study, we propose several overlapping, but differently active binding sites for substrates and inhibitors in the carriers NTCP, ASBT, SOAT.