Structure of the bile acid transporter and HBV receptor NTCP.

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually1,2. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes3. However, the molecular basis for the virus-transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs.

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.

Structural basis of sodium-dependent bile salt uptake into the liver.

The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+-taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP 'gated-pore' transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.

SLC10A2 deficiency-induced congenital chronic bile acid diarrhea and stunting.

BACKGROUND: Diarrhea is a common occurrence in children below the age of 5 years. In chronic cases, it induces malnutrition that severely stunts growth. Bile acid diarrhea (BAD), caused by malabsorption of bile acid (BA), is a rare form of chronic diarrhea seldom observed in pediatric patients. Here, we present a clinical report on a novel case of chronic BAD, with severe stunting in an infant, induced by a homozygous mutation of SLC10A2. METHODS: We performed DNA extraction, whole-exome sequencing analysis, and mutation analysis of SLC10A2 to obtain genetic data on the patient. We subsequently analyzed the patient's clinical and genetic data. RESULTS: The patient's clinical manifestations were chronic diarrhea with increased BAs in the feces and extreme stunting, which was diagnosed as BAD. A homozygous mutation of SLC10A2 at the c.313T>C (rs201206937) site was detected. CONCLUSION: Our report reveals the youngest case illustrating the characteristics of BAD induced by genetic variant at 313T>C, and the second case entailing a clear association between a SLC10A2 genetic mutation and the onset of BAD. Our findings expand the mutant spectrum of the SLC10A2 gene and contribute to the refinement of the genotype-phenotype mapping of severe stunting induced by pediatric BAD. Moreover, they highlight the value of molecular genetic screening for diagnosing BAD in young patients.

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.

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 to ASBT 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.

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.

A clinically relevant polymorphism in the Na+/taurocholate cotransporting polypeptide (NTCP) occurs at a rheostat position.

Conventionally, most amino acid substitutions at "important" protein positions are expected to abolish function. However, in several soluble-globular proteins, we identified a class of nonconserved positions for which various substitutions produced progressive functional changes; we consider these evolutionary "rheostats". Here, we report a strong rheostat position in the integral membrane protein, Na+/taurocholate (TCA) cotransporting polypeptide, at the site of a pharmacologically relevant polymorphism (S267F). Functional studies were performed for all 20 substitutions (S267X) with three substrates (TCA, estrone-3-sulfate, and rosuvastatin). The S267X set showed strong rheostatic effects on overall transport, and individual substitutions showed varied effects on transport kinetics (Km and Vmax) and substrate specificity. To assess protein stability, we measured surface expression and used the Rosetta software (https://www.rosettacommons.org) suite to model structure and stability changes of S267X. Although buried near the substrate-binding site, S267X substitutions were easily accommodated in the Na+/TCA cotransporting polypeptide structure model. Across the modest range of changes, calculated stabilities correlated with surface-expression differences, but neither parameter correlated with altered transport. Thus, substitutions at rheostat position 267 had wide-ranging effects on the phenotype of this integral membrane protein. We further propose that polymorphic positions in other proteins might be locations of rheostat positions.

Innovative HBV Animal Models Based on the Entry Receptor NTCP.

Hepatitis B is a major global health problem, with an estimated 257 million chronically infected patients and almost 1 million deaths per year. The causative agent is hepatitis B virus (HBV), a small, enveloped, partially double-stranded DNA virus. HBV has a strict species specificity, naturally infecting only humans and chimpanzees. Sodium taurocholate co-transporting polypeptide (NTCP), a bile acid transporter expressed on hepatocytes, has been shown to be one of the key factors in HBV infection, playing a crucial role in the HBV entry process in vitro and in vivo. Variations in the amino acid sequence of NTCP can inhibit HBV infection and, therefore, contributes, in part, to the species barrier. This discovery has revolutionized the search for novel animal models of HBV. Indeed, it was recently shown that variations in the amino acid sequence of NTCP represent the sole species barrier for HBV infection in macaques. Here, we review what is known about HBV entry through the NTCP receptor and highlight how this knowledge has been harnessed to build new animal models for the study of HBV pathogenesis and curative therapies.

The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling.

SLC10A7 represents an orphan member of the Solute Carrier Family SLC10. Recently, mutations in the human SLC10A7 gene were associated with skeletal dysplasia, amelogenesis imperfecta, and decreased bone mineral density. However, the exact molecular function of SLC10A7 and the mechanisms underlying these pathologies are still unknown. For this reason, the role of SLC10A7 on intracellular calcium signaling was investigated. SLC10A7 protein expression was negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. Whereas SLC10A7 knockout HAP1 cells showed significantly increased calcium influx after thapsigargin, ionomycin and ATP/carbachol treatment, SLC10A7 overexpression reduced this calcium influx. Intracellular Ca2+ levels were higher in the SLC10A7 knockout cells and lower in the SLC10A7-overexpressing cells. The SLC10A7 protein co-localized with STIM1, Orai1, and SERCA2. Most of the previously described human SLC10A7 mutations had no effect on the calcium influx and thus were confirmed to be functionally inactive. In the present study, SLC10A7 was established as a novel negative regulator of intracellular calcium signaling that most likely acts via STIM1, Orai1 and/or SERCA2 inhibition. Based on this, SLC10A7 is suggested to be named as negative regulator of intracellular calcium signaling (in short: RCAS).

Bile acid transporter mediated STC/Soluplus self-assembled hybrid nanoparticles for enhancing the oral drug bioavailability.

The nano-particulate system for oral delivery faces a big challenge across the gastrointestinal bio-barriers. The aim was to explore the potential applications of bile acid transporter mediated the self-assembled hybrid nanoparticles (SHNPs) of sodium taurocholate (STC) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (Soluplus) for augmenting the oral delivery of poorly water-soluble drugs. Felodipine (FLDP) was chosen as a model drug. The self-assembly of STC with Soluplus to load FLDP and the microstructure of the SHNPs were confirmed using molecular simulation, STC determination by high performance liquid chromatography (HPLC) and transmission electron microscope. Results showed that STC was integrated with Soluplus on the surface of nanoparticles by hydrophobic interactions. The permeability of FLDP loaded STC/Soluplus SHNPs was STC dependent in the ileum, which was inhibited by the higher concentrations of STC and the inhibitor of apical sodium-dependent bile acid transporter (ASBT). STC/Soluplus (1:9) SHNPs significantly improved the drug loading of FLDP, achieved the highest permeability of FLDP and realized 1.6-fold of the area under the curve (AUC) of Soluplus self-assembled nanoparticles (SNPs). A water-quenching fluorescent probe P4 was loaded into the STC/Soluplus SHNPs, which verified that the SHNPs were transferred intactly across the ileum. In conclusion, STC/Soluplus SHNPs via ASBT are a potential strategy for enhancing the oral bioavailability of poorly water-soluble drugs.

Clinical and molecular characterization of four patients with NTCP deficiency from two unrelated families harboring the novel SLC10A1 variant c.595A>C (p.Ser199Arg).

Sodium taurocholate cotransporting polypeptide (NTCP), a carrier protein encoded by solute carrier family 10 member 1 (SLC10A1), is expressed in the basolateral membrane of hepatocytes, where it is responsible for the uptake of bile acids from plasma into hepatocytes. The first patient with NTCP deficiency was described in 2015. A limited number of such patients have been reported in the literature and their genotypic and phenotypic features require further investigation. The current study investigated 4 patients with NTCP deficiency from two unrelated families. The patients were subjected to SLC10A1 genetic analysis and it was revealed that all patients were compound heterozygous for the c.800C>T (p.Ser267Phe) and c.595A>C (p.Ser199Arg) SLC10A1 variants. To the best of the authors' knowledge, the latter variant had not been previously reported. Further analysis in 50 healthy individuals did not identify carriers. The c.595A>C (p.Ser199Arg) variant exhibited co­segregation with hypercholanemia and exhibited a relatively conserved amino acid when compared with homologous peptides. Moreover, SWISS­MODEL prediction revealed that the mutation affected the conformation of the NTCP molecule. The 4 patients demonstrated varying degrees of hypercholanemia while a downward trend in the plasma levels of total bile acids (TBA) in 2 pediatric patients and occasionally normal TBA level in an adult case were observed. The results indicated an autosomal recessive trait for NTCP deficiency, supported the primary role of NTCP in the uptake of bile acids from plasma and suggested that hepatic uptake of bile acids may occur by means other than NTCP uptake. Moreover, the novel missense variant c.595A>C(p.Ser199Arg) enriched the SLC10A1 mutation spectrum and may serve as a new genetic marker for the molecular diagnosis and genetic counseling of NTCP deficiency.

Site-Directed Alkylation Detected by In-Gel Fluorescence (SDAF) to Determine the Topology Map and Probe the Solvent Accessibility of Membrane Proteins.

The topology of helix-bundle membrane proteins provides low-resolution structural information with regard to the number and orientation of membrane-spanning helices, as well as the sidedness of intra/extra-cellular domains. In the past decades, several strategies have been developed to experimentally determine the topology of membrane proteins. However, generally, these methods are labour-intensive, time-consuming and difficult to implement for quantitative analysis. Here, we report a novel approach, site-directed alkylation detected by in-gel fluorescence (SDAF), which monitors the fluorescent band shift caused by alkylation of the EGFP-fused target membrane protein bearing one single introduced cysteine. In-gel fluorescence provides a unique readout of target membrane proteins with EGFP fusion from non-purified samples, revealing a distinct 5 kDa shift on SDS-PAGE gel due to conjugation with mPEG-MAL-5K. Using the structurally characterised bile acid transporter ASBTNM as an example, we demonstrate that SDAF generates a topology map consistent with the crystal structure. The efficiency of mPEG-MAL-5K modification at each introduced cysteine can easily be quantified and analysed, providing a useful tool for probing the solvent accessibility at a specific position of the target membrane protein.

Homo- and heterodimerization is a common feature of the solute carrier family SLC10 members.

The solute carrier family SLC10 consists of seven members, including the bile acid transporters Na+/taurocholate co-transporting polypeptide (NTCP) and apical sodium-dependent bile acid transporter (ASBT), the steroid sulfate transporter SOAT as well as four orphan carriers (SLC10A3, SLC10A4, SLC10A5 and SLC10A7). Previously, homodimerization of NTCP, ASBT and SOAT was described and there is increasing evidence that carrier oligomerization is an important regulatory factor for protein sorting and transport function. In the present study, homo- and heterodimerization were systematically analyzed among all SLC10 carriers (except for SLC10A3) using the yeast-two-hybrid membrane protein system. Strong homodimerization occurred for NTCP/NTCP, ASBT/ASBT and SLC10A7/SLC10A7. Heterodimerization was observed for most of the SLC10 carrier combinations. Heterodimerization of NTCP was additionally investigated by co-localization of NTCP-GFP and NTCP-mScarlet with respective SLC10 carrier constructs. NTCP co-localized with SLC10A4, SLC10A5, SOAT and SLC10A7. This co-localization was most pronounced for SLC10A4 and was additionally confirmed by co-immunoprecipitation. Interestingly, SLC10 carrier co-expression decreased the taurocholate transport function of NTCP for most of the analyzed constructs, indicating that SLC10 carrier heterodimerization is of functional relevance. In conclusion, homo- and heterodimerization is a common feature of the SLC10 carriers. The relevance of this finding for regulation and transport function of the SLC10 carriers in vivo needs further investigation.

Use of Indocyanine Green (ICG), a Medical Near Infrared Dye, for Enhanced Fluorescent Imaging-Comparison of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) and Sodium-Taurocholate Cotransporting Polypeptide (NTCP) Reporter Genes.

Molecular and cellular imaging in living organisms have ushered in an era of comprehensive understanding of intracellular and intercellular events. Currently, more efforts have been focused on the infrared fluorescent dyes that facilitate deeper tissue visualization. Both sodium taurocholate cotransporting polypeptide (NTCP) and organic-anion-transporting polypeptide 1B3 (OATP1B3) are capable of carrying indocyanine green (ICG) into the cytoplasm. We compared the feasibility of NTCP and OATP1B3 as reporter genes in combination with ICG. NTCP and OATP1B3 were transduced into HT-29 cells. Genetically modified HT-29 cells were inoculated into nude mice. ICG was administered in vitro and in vivo and the signals were observed under confocal microscopy, flow cytometry, multimode microplate reader, and an in vivo imaging system. Both NTCP- and OATP1B3-expressing cells and xenografts had higher ICG intensities. The OATP1B3-expressing xenograft has a higher ICG uptake than the NTCP-expressing xenograft. NTCP or OATP1B3 combined with ICG could serve as a noninvasive imaging modality for molecular and cellular imaging. OATP1B3 outperforms NTCP in terms of in vivo imaging.

Structural basis for functional interactions in dimers of SLC26 transporters.

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.

Bioinspired Artificial Nanodecoys for Hepatitis†B Virus.

A facile route is presented for fabricating a new class of nanomimics that overexpress hepatitis B virus (HBV) receptor by a natural biosynthetic procedure against HBV infection. A nine-transmembrane HBV-specific receptor, human sodium taurocholate co-transporting polypeptide (hNTCP), was engineered to naturally immobilize it onto the cellular surface and subsequently trigger the budding of hNTCP-anchoring membrane vesicles (hNTCP-MVs) that favor the HBV virion. hNTCP-MVs could rapidly block HBV infection in cell models. Furthermore, hNTCP-MVs treatment could effectively prevent viral infection, spreading, and replication in a human-liver-chimeric mouse model of HBV infection. Our findings demonstrate the receptor-mediated antiviral effect of hNTCP-MVs to trick HBV and offer novel opportunities for further development of antiviral strategies in nanomedicine.

Rare genetic variants in the sodium-dependent organic anion transporter SOAT (SLC10A6): Effects on transport function and membrane expression.

Sulfo-conjugated steroid hormones, such as dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate or estrone-3-sulfate are abundant in the body, but are biologically inactive at classical androgen and estrogen steroid receptors. However, after carrier-mediated import and de-conjugation by the steroid sulfatase, these compounds participate in the overall steroid regulation of reproductive organs. The sodium-dependent organic anion transporter SOAT, coded by the SLC10A6 gene, is specific for the transport of steroid sulfates and is highly expressed in testicular germ cells, including pachytene spermatocytes, secondary spermatocytes, and round spermatids. Therefore, SOAT is supposed to be involved in the regulation of spermatogenesis and male fertility. In the present study, the SLC10A6 gene was analyzed for rare genetic variants, which might affect transport function or membrane expression of SOAT. Among the 31 SOAT variants analyzed, L44P, Q75R, P107L, G109S, S112F, N113K, S133F, G241D, G263E, G294R, and Y308N showed no transport activity for DHEAS at all. In the case of P107L, G241D, G263E, and Y308N, this was most likely due to significantly reduced expression in the plasma membrane. Other variants are located directly at (Q75R, S112F, N113K) or close to (G109S, S133F, and G263E) the supposed SOAT Na+ binding sites and thus could disable the sodium-coupled transport cycle. If these loss-of-function SOAT variants are more frequent in men with impaired spermatogenesis or infertility needs further investigation.

Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers.

The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self-associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co-expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.

Functional nanoparticles exploit the bile acid pathway to overcome multiple barriers of the intestinal epithelium for oral insulin delivery.

Oral absorption of protein/peptide-loaded nanoparticles is often limited by multiple barriers of the intestinal epithelium. In addition to mucus translocation and apical endocytosis, highly efficient transepithelial absorption of nanoparticles requires successful intracellular trafficking, especially to avoid lysosomal degradation, and basolateral release. Here, the functional material, deoxycholic acid-conjugated chitosan, is synthesized and loaded with the model protein drug insulin into deoxycholic acid-modified nanoparticles (DNPs). The DNPs designed in this study are demonstrated to overcome multiple barriers of the intestinal epithelium by exploiting the bile acid pathway. In Caco-2 cell monolayers, DNPs are internalized via apical sodium-dependent bile acid transporter (ASBT)-mediated endocytosis. Interestingly, insulin degradation in the epithelium is significantly prevented due to endolysosomal escape of DNPs. Additionally, DNPs can interact with a cytosolic ileal bile acid-binding protein that facilitates the intracellular trafficking and basolateral release of insulin. In rats, intravital two-photon microscopy also reveals that the transport of DNPs into the intestinal villi is mediated by ASBT. Further pharmacokinetic studies disclose an oral bioavailability of 15.9% in type I diabetic rats after loading freeze-dried DNPs into enteric-coated capsules. Thus, deoxycholic acid-modified chitosan nanoparticles can overcome multiple barriers of the intestinal epithelium for oral delivery of insulin.