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Bile formation and secretion: An update

Published:February 19, 2021DOI:https://doi.org/10.1016/j.jhep.2021.02.011

      Summary

      Bile formation is a fundamental physiological process that is vital to the survival of all vertebrates. However, little was known about the mechanisms of this secretion until after World War II. Initial studies involved classic physiologic studies in animal models and humans, which progressed to include studies in isolated cells and membrane vesicles. The advent of molecular biology then led to the identification of specific transport systems that are the determinants of this secretion. Progress in this field was reviewed in the American Physiologic Society’s series on “Comprehensive Physiology” in 2013. Herein, we provide an in-depth update of progress since that time.

      Keywords

      Introduction

      The 1950s saw the beginning of the modern understanding of the mechanism of bile formation, starting with the observations of Brauer that bile production was energy dependent and did not rely on hydrostatic perfusion.
      • Brauer R.W.
      • Leong G.F.
      • Holloway R.J.
      Mechanics of bile secretion: effect of perfusion pressure and temperature on bile flow and secretion pressure.
      Subsequent studies by Sperber introduced the concept of osmotic filtration.
      • Sperber I.
      Secretion of organic anions in the formation of urine and bile.
      Bile salt dependent (canalicular bile flow) and independent (cholangiocyte secretion) fractions of bile were then characterised in animals and humans using radiolabelled inert solutes (erythritol or mannitol.
      • Wheeler H.O.
      • Mancusi-Ungaro P.L.
      Role of bile ducts during secretin choleresis in dogs.
      ,
      • Boyer J.L.
      • Klatskin G.
      Canalicular bile flow and bile secretory pressure: evidence for a non-bile salt dependent fraction in the isolated perfused rat liver.
      Immunochemical localisation of the Na+K+ ATPase on the basolateral sinusoidal membrane led to the concept that the hepatocyte is a polarised epithelial cell and made it possible to isolate apical (canalicular) and basolateral plasma membrane domains.
      • Blitzer B.L.
      • Boyer J.L.
      Cytochemical localization of Na+,K+-ATPase in the rat hepatocyte.
      This enabled the localisation of specific transporters to these domains using membrane vesicles including the milestone discovery of ATP-dependent bile salt transport at the canalicular membrane.
      • Adachi T.
      • Nakagawa H.
      • Chung I.
      • Hagiya Y.
      • Hoshijima K.
      • Noguchi N.
      • et al.
      Nrf2-dependent and -independent induction of ABC transporters ABCC1, ABCC2, and ABCG2 in HepG2 cells under oxidative stress.
      • Muller M.
      • Ishikawa T.
      • Berger U.
      • Klunemann C.
      • Lucka L.
      • Schreyer A.
      • et al.
      ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt.
      • Nishida T.
      • Gatmaitan Z.
      • Che M.X.
      • Arias I.M.
      Rat liver canalicular membrane vesicles containing an ATP-dependent bile acid transport system.
      • Stieger B.
      • O'Neill B.
      • Meier P.J.
      ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles.
      The development of isolated hepatocyte couplets and bile duct units enabled clarification of their cell-specific functions independent of the effects of blood flow.
      • Graf J.
      • Gautam A.
      • Boyer J.L.
      Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function.
      ,
      • Mennone A.
      • Alvaro D.
      • Cho W.
      • Boyer J.L.
      Isolation of small polarized bile duct units.
      By the 1990s, the molecular basis of these transport systems was defined. All of these developments were summarised in the American Physiologic Society’s series on “Comprehensive Physiology” in 2013, “Bile Formation and Secretion”. Since then, no in-depth update on this topic has been published. This review will focus on significant developments since that time. This will include new perspectives on the mechanisms of bile acid-dependent canalicular flow, new insights into the regulation of cholangiocyte secretion, and an update on the role of bile acids and other solute transporters in hepatocytes. The reader is referred to the previous review as needed for necessary background information.
      • Boyer J.L.
      Bile formation and secretion.

      Canalicular bile secretion

      According to classic concepts, canalicular bile formation is determined by a “mechano-osmotic” process, initiated at the hepatocyte as a consequence of concentrative trans canalicular membrane transport of bile acids, glutathione and other solutes via ATP-dependent transporters. These osmotic gradients result in the filtration of water primarily through aquaporins located in the apical and basolateral hepatocyte domains (Fig. 1A). A lobular gradient exists for bile acids which are transported predominately by periportal hepatocytes (so-called bile acid-dependent bile flow) to the relative exclusion of pericentral lobular cells where secretion is largely determined by bile acid-independent mechanisms (bile acid-independent bile flow). Slower flow in these pericentral zones may be the reason canalicular bile plugs tend to predominate there following cholestatic liver injury. The flow of bile is countercurrent to the direction of blood perfusing the liver and its movement from central regions of the lobule to the biliary ducts is facilitated by calcium-stimulated contractions in the pericanalicular actin-myosin microfilament complex, resulting in canalicular membrane contractions or “peristalsis”
      • Trampert D.C.
      • Nathanson M.H.
      Regulation of bile secretion by calcium signaling in health and disease.
      (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1Recent developments in our understanding of the mechanisms of hepatic transporter function.
      The classic theory of mechano-osmotic regulation of hepatic secretion now includes the role for apical and basolateral aquaporins (A), peristaltic pericanalicular actin contractions (B), regulation of paracellular fluid movement by tight junction proteins such as claudins (C), and the actions of apical and basolateral solute transporters (D). Protein-protein interactions with apical transporters such as BSEP (HAX-1, AP-2) (D) and MRP2 (ezrin) (D) control endocytosis and internalisation to and from the membrane, thus regulating bile acid secretion. In response to a bile acid load, the pericanalicular actin-myosin signalling complex can be sensed by the Hippo transcriptional co-activator YAP which can then translocate to the nucleus (E) and lead to increases in apical surface area. It is now known that NTCP can act as a receptor for HBV (F). Basolateral membrane transporters such as OATPs and OCT1 are now being recognised by the pharmaceutical industry for their action in transporting drugs, as well as endogenous anions and cations (G). OSTα-OSTβ plays a critical role in reducing the toxic levels of intracellular bile acids under cholestatic conditions (H). New disease-causing mutations have been described in many of these essential membrane transporters. AP-2, adaptor protein complex 2; AQP, aquaporin; BA, bile acid; BSEP, bile salt export pump; GSH, glutathione; HAX-1, HS-1-associated protein X-1; MDR3, multidrug resistance transporter 3; MRP2, multidrug resistance protein 2; NTCP, sodium dependent cotransporting polypeptide; OA-, organic anion; OATP, organic anion-transporting polypeptides; OC+, organic cation; OCT, organic cation transporter; OSTα-OSTβ, organic solute transporter α-β; YAP, yes-associated protein.
      The canalicular membrane is formed between adjacent hepatocytes and sealed by the junctional complex consisting of the zonula occludens. Claudins, 20-27 kD transmembrane proteins, form the major structural barrier in the tight junction – most clearly seen as a meshwork of strands in freeze fracture replicas. Claudins determine the permeability of the junction. Until recently it had been axiomatic that hydrostatic pressure played no role in the formation of bile. However, in claudin 2 knockout mice, bile flow is reduced by 50% despite normal excretion of bile acids, biliary lipids and bilirubin.
      • Matsumoto K.
      • Imasato M.
      • Yamazaki Y.
      • Tanaka H.
      • Watanabe M.
      • Eguchi H.
      • et al.
      Claudin 2 deficiency reduces bile flow and increases susceptibility to cholesterol gallstone disease in mice.
      Claudin 2 is expressed in tight junctions of the hepatocyte with its expression increasing down the lobular gradient
      • Rahner C.
      • Mitic L.L.
      • Anderson J.M.
      Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut.
      (Fig. 1C). Claudin 1, 2 and 3 are the dominant claudins in hepatocytes. Claudin 1 is also expressed in the tight junction and the lateral membrane of cholangiocytes while claudin 3 is expressed throughout the hepatocyte lobule as well as in cholangiocyte tight junctions. Claudin 2 is cation selective and is also permeable to water in several other tissues and cell lines. Lumens of both isolated hepatocyte couplets and bile duct units from claudin 2 null mice reduced their size more slowly in response to increases in osmotic gradients in their buffers, consistent with water movement via claudin 2. However, it remains unclear if paracellular water movement, and by extension hydrostatic pressure, plays a role in bile secretion in the human liver.
      • Boyer J.L.
      The hepatobiliary paracellular pathway: a paradigm revisited.
      Nevertheless, water movement into the canaliculus is likely to be influenced by changes in the expression of claudins as well as aquaporins, independently of the osmotic function of hepatic transporters.
      • Marinelli R.A.
      • Vore M.
      • Javitt N.B.
      Hepatic bile formation: canalicular osmolarity and paracellular and transcellular water flow.
      Canalicular bile secretion may be more than a mechano-osmotic process due to hepatic transporters, peristaltic actin contractions, and paracellular fluid movement.
      Using high-resolution confocal and scanning electron microscopy, Meyer et al.
      • Meyer K.
      • Ostrenko O.
      • Bourantas G.
      • Morales-Navarrete H.
      • Porat-Shliom N.
      • Segovia-Miranda F.
      • et al.
      A predictive 3D multi-scale model of biliary fluid dynamics in the liver lobule.
      developed a 3D model to simulate fluid dynamics in the bile canaliculus of the mouse hepatocyte. Their findings predict both bile velocity and pressure gradients within the liver lobule. Bile velocity accelerates exponentially from central to portal regions, rising about 3.5-fold and increasing to ~12 um/sec near the bile ducts. These studies also confirm early studies
      • Oshio C.
      • Phillips M.J.
      Contractility of bile canaliculi, implications of liver functions.
      ,
      • Watanabe N.
      • Tsukada N.
      • Smith C.
      • Phillips M.J.
      Motility of bile canaliculi in the living animal: implications for bile flow.
      that bile canalicular contractions (peristalsis) play an important role in bile movement, since paralysing actin-myosin with the Rho kinase inhibitor, fasudil, reduced bile velocity by ~50%. Peristalsis contributed 88% of the velocity in the central venous zone and up to 32% in the portal venous zone. Thus, both osmosis-dependent and peristalsis-dependent mechanisms contribute equally as determinants of canalicular bile flow. In contrast to the bile velocity central to portal gradient, their model predicted pressure gradients to be greatest in peri-central zones, decreasing about 30-fold from ~2,500 Pa (18.6 mmHg) in the central to portal regions and dropping rapidly near the bile ducts.
      Vartak and colleagues introduced further complexity to this subject by examining small molecular flux of bile acids and fluorescent organic anions using fluorescence loss after photoactivation (FLAP) and intravital arbitrary region image correlation spectroscopy (IVARICS) in mice.
      • Vartak N.
      • Guenther G.
      • Joly F.
      • Damle-Vartak A.
      • Wibbelt G.
      • Fickel J.
      • et al.
      Intravital dynamic and correlative imaging reveals diffusion-dominated canalicular and flow-augmented ductular bile flux.
      Their studies challenge the “mechano-osmotic” theory of canalicular bile flow and suggest that the bile canalicular network functions only as a reservoir or “standing water zone” into which organic solutes are transported with bile acids being transported predominately in pericentral zones of the lobule. Thereafter, bile acids diffuse bidirectionally in the canaliculi rather than moving with bulk flow until they reach the bile ducts where the addition of secretin-stimulated ions augments their directional flow, e.g. bile acids move in canaliculi by diffusion and by osmotic flow within the biliary duct system. However, it is not clear how canalicular peristalsis and species variation in cholangiocyte secretion influences these conclusions. In addition, technical concerns have been raised concerning the conclusions of this study (letter to editors in submission).
      It has long been known that bile acids influence the surface area of the bile canaliculus and stimulate hepatocyte cell polarity. Chronic infusions of bile acids (taurocholate but not dehydrocholate) increase phospholipid content in isolated plasma membrane preparations from rat livers, resulting in dilation, outpouching and side branching of bile canaliculi as observed by scanning electron microscopy.
      • Nemchausky B.A.
      • Layden T.J.
      • Boyer J.L.
      Effects of chronic choleretic infusions of bile acids on the membrane of the bile canaliculus.
      Fu and colleagues demonstrated that bile acids stimulated the formation of bile canaliculi through a cAMP-Epac-Mek-LKB1-AMPK pathway.
      • Fu D.
      • Wakabayashi Y.
      • Lippincott-Schwartz J.
      • Arias I.M.
      Bile acid stimulates hepatocyte polarization through a cAMP-Epac-MEK-LKB1-AMPK pathway.
      More recent studies in the mouse liver regeneration model indicate that liver resection expands the apical surface of hepatocytes and the actin phosphomyosin network
      • Meyer K.
      • Morales-Navarrete H.
      • Seifert S.
      • Wilsch-Braeuninger M.
      • Dahmen U.
      • Tanaka E.M.
      • et al.
      Bile canaliculi remodeling activates YAP via the actin cytoskeleton during liver regeneration.
      in response to an increase in bile acid load. This stimulates the translocation of the Hippo transcriptional co-activator Yes-associated protein (YAP) to the nucleus (Fig. 1E). YAP is a transcription factor that is enriched in the peri-apical region of the hepatocyte and is presumably retained there by elements of the tight junction zonula adherens. This pathway responds directly to changes in the peri-canalicular actin-myosin signalling complex. These findings indicate that the apical membrane of hepatocytes functions as a “self-regulatory” mechano-sensory system in response to bile acids.
      The hepatocyte apical membrane can act as a self-regulatory mechano-sensory system, responding to a bile acid load by increasing the canalicular surface area through various intracellular signalling pathways.
      It is well established that tauroursodeoxycholic acid (TUDCA) is taken up by the liver via the sodium dependent cotransporting polypeptide (NTCP); within the liver, TUDCA induces the insertion of the bile salt export pump (Bsep)
      • Kurz A.K.
      • Graf D.
      • Schmitt M.
      • Vom Dahl S.
      • Häussinger D.
      Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats.
      and the multidrug resistance protein 2 (Mrp2)
      • Beuers U.
      • Bilzer M.
      • Chittattu A.
      • Kullak-Ublick G.A.
      • Keppler D.
      • Paumgartner G.
      • et al.
      Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver.
      into the canalicular membrane, thereby stimulating bile acid secretion and bile flow. This process involves direct activation of the α5β1 integrin causing c-Src to activate the epidermal growth factor receptor, which then activates phosphorylation of the mitogen-activated protein kinases Erk-1/2 and p38MACK5, presumably accounting for TUDCA’s anti-cholestatic effects.
      • Gohlke H.
      • Schmitz B.
      • Sommerfeld A.
      • Reinehr R.
      • Häussinger D.
      α5 β1-integrins are sensors for tauroursodeoxycholic acid in hepatocytes.
      In contrast, norursodeoxycholic acid enters the liver by diffusion across the sinusoidal membrane, is relatively resistant to conjugation with taurine and glycine, has little effect on bile acid secretion, and is excreted into bile – by Mrp2 – where it stimulates a bicarbonate choleresis, thus accounting for the differential clinical effects of these bile acids.
      • Steinacher D.
      • Claudel T.
      • Trauner M.
      Therapeutic mechanisms of bile acids and nor-ursodeoxycholic acid in non-alcoholic fatty liver disease.
      Recent modelling demonstrates that TUDCA is a much stronger activator of the α5β1 integrin and that this functional selectivity with the α5β1 integrin may account for the differences in their choleretic properties.
      • Bonus M.
      • Sommerfeld A.
      • Qvartskhava N.
      • Görg B.
      • Ludwig B.S.
      • Kessler H.
      • et al.
      Evidence for functional selectivity in TUDC- and norUDCA-induced signal transduction via α(5)β(1) integrin towards choleresis.

      Cholangiocyte bile secretion

      Bile duct epithelial cells contribute a variable amount of volume to overall bile formation depending on the species, but may account for up to 40% of total daily secretion in humans. Both cyclic AMP and calcium-activated apical chloride channels promote bicarbonate secretion via the chloride/bicarbonate anion exchanger 2 (AE2). The type 3 isoform of the inositol 1,4,5-triphosphate receptor (ITPR3), is the most abundant calcium release channel in cholangiocytes and is located on the endoplasmic reticulum (ER) in the subapical region of the cell where it regulates bicarbonate and chloride secretion into bile. Loss of ITPR3 results in impaired cholangiocyte secretion in mouse models; this isoform is lost from cholangiocytes in human cholestatic diseases like primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).
      • Minagawa N.
      • Nagata J.
      • Shibao K.
      • Masyuk A.I.
      • Gomes D.A.
      • Rodrigues M.A.
      • et al.
      Cyclic AMP regulates bicarbonate secretion in cholangiocytes through release of ATP into bile.
      While secretin stimulates bile secretion in response to meals via receptors on the basolateral membrane that lead to increases in cellular cAMP and activation of cystic fibrosis transmembrane conductance regulator (CFTR), recent evidence suggests that calcium-mediated pathways may be more predominant. In addition to CFTR, cholangiocytes express an apical Ca2+-activated Cl channel which is encoded by the transmembrane member 16A gene, TMEM16A, also known as anoctamin-1 (ANO1). Studies in isolated cells and monolayers indicate that increases in [Ca2+]i increase Cl current density and transepithelial secretion to values 3- to 5x more, respectively, than cAMP.
      • Dutta A.K.
      • Woo K.
      • Doctor R.B.
      • Fitz J.G.
      • Feranchak A.P.
      Extracellular nucleotides stimulate Cl- currents in biliary epithelia through receptor-mediated IP3 and Ca2+ release.
      These findings indicate that TMEM16A, not CFTR, may represent the dominant Cl channel regulating anion efflux, and Ca2+-dependent signalling may represent the dominant pathway for modulating the anion permeability of the apical cholangiocyte membrane.
      Interestingly, in vitro studies in mouse, rat and human biliary epithelial cells
      • Li Q.
      • Dutta A.
      • Kresge C.
      • Bugde A.
      • Feranchak A.P.
      Bile acids stimulate cholangiocyte fluid secretion by activation of transmembrane member 16A Cl(-) channels.
      indicate that the bile acids ursodeoxycholic acid (UDCA) and TUDCA, but not taurocholic acid (TCA) or cholic acid, activate the TMEM16A chloride channel to stimulate cholangiocyte bile production; this mechanism depends on the uptake of UDCA and TUDCA by the apical sodium-dependent bile acid transporter (ASBT), their intracellular accumulation, ATP release and increased intracellular calcium. This connection between bile acid-dependent secretion and ductular bile formation (“hepatobiliary coupling”) provides a possible mechanism to explain the effect of these bile acids in cholestatic liver disease.
      Studies in normal human cholangiocytes and mouse cholangiocytes identified a small musculo-aponeurotic fibrosarcoma (Maf) protein that heterodimerises with the transcription factor NRF2, leading to translocation of NRF2 to the nucleus in response to oxidative stress.
      • Weerachayaphorn J.
      • Amaya M.J.
      • Spirli C.
      • Chansela P.
      • Mitchell-Richards K.A.
      • Ananthanarayanan M.
      • et al.
      Nuclear factor, erythroid 2-like 2 regulates expression of type 3 inositol 1,4,5-trisphosphate receptor and calcium signaling in cholangiocytes.
      NRF binds to Maf-recognition elements in the promoter of ITPR3 leading to loss of ITPR3 expression and diminished Ca2+-mediated bicarbonate secretion. Higher levels of NRF2 and lower expression levels of ITPR3 were also found in cholestatic animal models and human cholestatic disorders, suggesting that this may be a mechanism by which oxidative stress contributes to cholestatic liver injury, particularly in cholangiopathies.
      Cholangiocyte bile secretion is a complex process mediated by apical membrane proteins responding to changes in cAMP, lumenal ATP, and intracellular calcium levels.
      The ITPR3 is also regulated by micro RNAs (miRNAs) that play a role in regulating the epigenetic silencing of genes by targeting the 3’-untranslated region (UTR) of specific miRNAs. An analysis of the 3’-UTR of human InsP3R3 revealed 2 highly conserved binding sites for miR-506. Forskolin-stimulated fluid secretion was significantly reduced in bile duct units isolated from miR-506-transfected rats, suggesting that this miRNA is also a post-translational regulator of InsP3R3.
      • Ananthanarayanan M.
      • Banales J.M.
      • Guerra M.T.
      • Spirli C.
      • Munoz-Garrido P.
      • Mitchell-Richards K.
      • et al.
      Post-translational regulation of the type III inositol 1,4,5-trisphosphate receptor by miRNA-506.
      MicroR-506 was also found to be increased in the intrahepatic bile ducts and to negatively target AE2 in patients with PBC.
      • Banales J.M.
      • Sáez E.
      • Uriz M.
      • Sarvide S.
      • Urribarri A.D.
      • Splinter P.
      • et al.
      Up-regulation of microRNA 506 leads to decreased Cl-/HCO3- anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.
      miRNA125b and let7a are also involved in the stimulation of biliary cell proliferation by secretin.
      • Glaser S.
      • Meng F.
      • Han Y.
      • Onori P.
      • Chow B.K.
      • Francis H.
      • et al.
      Secretin stimulates biliary cell proliferation by regulating expression of microRNA 125b and microRNA let7a in mice.
      Current views on the regulation of bicarbonate secretion by cholangiocytes are summarised in Fig. 2, modified from.
      • Rodrigues M.A.
      • Gomes D.A.
      • Nathanson M.H.
      Calcium signaling in cholangiocytes: methods, mechanisms, and effects.
      In response to meals, secretin stimulates cyclic AMP in large cholangiocytes which activates CFTR chloride channels which then increase chloride in the bile duct lumen, thereby driving the Cl/HCO3 exchanger (AE2) leading to an increase in luminal bicarbonate. ATP is also released into the lumen via unexplained mechanisms and binds to P2Y receptors, stimulating intracellular ITPR3-mediated calcium release, which activates TMEM16A, adding to the chloride gradient that drives AE2. Hepatocytes may also contribute to this latter pathway by stimulating ATP release and, in the case of UDCA or TUDCA treatment, further increasing ITPR3-mediated calcium release as mentioned previously.
      Figure thumbnail gr2
      Fig. 2Regulation of bicarbonate secretion in cholangiocytes (from reference #
      • Rodrigues M.A.
      • Gomes D.A.
      • Nathanson M.H.
      Calcium signaling in cholangiocytes: methods, mechanisms, and effects.
      ).
      Separate signalling pathways that regulate secretion have been identified in large and small cholangiocytes. In large cholangiocytes, secretin receptors on the basolateral membrane link to formation of cAMP, which leads to the activation of CFTR, causing Cl efflux. This also stimulates exocytic release of ATP into the ductular lumen, through a mechanism that has not yet been identified. Luminal ATP then binds to apical P2Y receptors to stimulate intracellular Ca2+ release via ITPR3, which in turn activates Cl secretion through TMEM16A in the apical membrane. The resulting Cl gradient across the apical membrane drives the AE2 Cl/HCO3 exchanger, resulting in net HCO3 secretion. This pathway can also be activated directly by biliary ATP secreted from upstream hepatocytes. Alternatively, InsP3 formed from the stimulation of the M3 muscarinic receptor can stimulate secretion, although there is some evidence that this may act through Ca2+ released from ITPR1 and ITPR2 rather than ITPR3. Small cholangiocytes lack SRs and CFTRs, but have the same apical calcium signalling machinery to link to HCO3 secretion that is found in large cholangiocytes. Ach, acetyl choline; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; ITPR1/2/3, inositol 1,4,5-trisphosphate receptor isoform 1/2/3; M3R, M3 muscarinic acetylcholine receptor; P2YR, P2Y receptor; SR, secretin receptor; TMEM16A, transmembrane member 16A.

      Hepatic transporters

      Following the molecular identification of liver transporters, studies focused on how their expression was regulated both at the transcriptional and post-transcriptional level. The discovery of nuclear receptors (and their ligands) that activated gene expression were major advances, particularly the discovery that the farnesoid X receptor, originally an orphan receptor, was regulated by bile acids.
      • Karpen S.J.
      Bile acids go nuclear!.
      Many drugs, metabolites, and herbal compounds also exert their biologic properties as ligands of nuclear receptors. In addition, there is considerable coordination among different nuclear receptors in the regulation of gene expression that determines the hepatic clearance of bile acids, bilirubin, and xenobiotics. Post-transcriptional regulation also plays a major role, particularly in the insertion and removal of transporters from the plasma membrane during adaptation to changes in metabolic status and liver injury. Current drug discovery initiatives focus on these regulatory steps. While this subject is beyond the scope of the present review, the interested reader may find more details in these references
      • Boyer J.L.
      Nuclear receptor ligands: rational and effective therapy for chronic cholestatic liver disease?.
      ,
      • Halibasic E.C.T.
      Trauner M Bile acid transporteres and regulatory nuclear receptors in the liver and beyond.
      and should keep these underlying concepts in mind as information is updated for individual hepatic transporters.

      Sodium taurocholate cotransporting polypeptide (NTCP; SLC10A1)

      NTCP is the major liver-specific transporter for the uptake of conjugated bile acids from portal blood. More recently NTCP has also been identified as the receptor by which hepatitis B and the delta virus (HBV/HDV) enter the liver
      • Ni Y.
      • Lempp F.A.
      • Mehrle S.
      • Nkongolo S.
      • Kaufman C.
      • Fälth M.
      • et al.
      Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.
      (Fig. 1F). Myrcludex B, a synthetic peptide, has been developed that mimics the NTCP-binding domain of HBV. It blocks the ability of HBV and HDV to enter the liver and prevents infection in human-derived HepaRG cell lines.
      • Schulze A.
      • Schieck A.
      • Ni Y.
      • Mier W.
      • Urban S.
      Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction.
      Myrcludex B is currently in clinical trials for HBV/HDV where it transiently reduces viral levels and improves aminotransferase abnormalities.
      • Blank A.
      • Markert C.
      • Hohmann N.
      • Carls A.
      • Mikus G.
      • Lehr T.
      • et al.
      First-in-human application of the novel hepatitis B and hepatitis D virus entry inhibitor myrcludex B.
      Myrcludex B also increases serum levels of bile acids, but only in organic anion-transporting peptide (Oatp)- or Ntcp-deficient mice.
      • Slijepcevic D.
      • Roscam Abbing R.L.P.
      • Katafuchi T.
      • Blank A.
      • Donkers J.M.
      • van Hoppe S.
      • et al.
      Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.
      and partially protects mice from cholestatic injury in DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine)-fed, Atp8b1-G308V and bile duct ligated mice.
      • Slijepcevic D.
      • Roscam Abbing R.L.P.
      • Fuchs C.D.
      • Haazen L.C.M.
      • Beuers U.
      • Trauner M.
      • et al.
      Na(+) -taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice.
      These studies established that conjugated bile acids are transported by both Oatp and Ntcp in mice, in contrast to humans where NTCP is the dominant bile acid uptake system. Other studies indicate that Myrcludex B is cleared from plasma while elevated plasma levels of bile acids are prolonged, suggesting strong non-covalent binding to NTCP. Myrcludex can also transfer to newly synthesised NTCP peptides, thereby extending its blocking efficiency.
      • Donkers J.M.
      • Appelman M.D.
      • van de Graaf S.F.J.
      Mechanistic insights into the inhibition of NTCP by myrcludex B.
      Studies from Asian populations suggest that the NTCP rs2296651 variant (c.800C>T), is inversely correlated with susceptibility to HBV infection or HBV-related hepatocellular carcinoma (HCC). These individuals have a lower risk of HBV infection and are relatively protected from HBV infection and the development of HCC.
      • Binh M.T.
      • Hoan N.X.
      • Van Tong H.
      • Sy B.T.
      • Trung N.T.
      • Bock C.T.
      • et al.
      NTCP S267F variant associates with decreased susceptibility to HBV and HDV infection and decelerated progression of related liver diseases.
      ,
      • Wang P.
      • Mo R.
      • Lai R.
      • Xu Y.
      • Lu J.
      • Zhao G.
      • et al.
      Genetic variations of NTCP are associated with susceptibility to HBV infection and related hepatocellular carcinoma.
      Although still controversial, these findings suggest that polymorphisms that effect the expression of NTCP may influence geographic differences in susceptibility to the HBV virus and the development of HCC.
      Next generation sequencing has demonstrated that there are a number of rare SLC10A1 variants in the population. Transient transfection of a number of these variants into HEK293T cells has demonstrated a wide range of functional impairment in the uptake of TCA and rosuvastatin, with some variants reducing uptake to 0–13% of the wild-type transfect.
      • Russell L.E.
      • Zhou Y.
      • Lauschke V.M.
      • Kim R.B.
      In vitro functional characterization and in silico prediction of rare genetic variation in the bile acid and drug transporter, Na(+)-Taurocholate cotransporting polypeptide (NTCP, SLC10A1).
      These findings suggest that some rare variants may also affect bile acid homeostasis and/or hepatic drug uptake.
      Studies in humans with the NTCP rs2296651 variant (c.800C>T), demonstrate that sulfated bile acids are increased in the blood and urine of individuals as serum bile acid levels decline with time. Simultaneous studies in Slc10a1 mice showed marked increases in expression of sulfotransferases implicated in enhanced phase II (sulfation) detoxification of bile acids. These findings suggest that sulfation may be an important adaptive mechanism to decrease the toxic effects of bile acids and facilitate the clearance of bile acid overload both in mice and humans with Slc10a1/SLC10A1 deficiencies.
      • Mao F.
      • Liu T.
      • Hou X.
      • Zhao H.
      • He W.
      • Li C.
      • et al.
      Increased sulfation of bile acids in mice and human subjects with sodium taurocholate cotransporting polypeptide deficiency.
      NTCP deficiency in humans was first described in 2015 as a new inborn error of metabolism in an infant with mild growth retardation and hypotonia and delayed developmental milestones. Marked elevations in serum bile acids were detected with levels reaching 1,500 μM. Remarkably there was no evidence of cholestasis, pruritus or liver test abnormalities. Plasma levels of C4 (7α-hydroxy-4-cholesten-3-one) and fibroblast growth factor-19 (FGF19) were normal, indicating that bile acid synthesis and intestinal bile salt signalling were unaffected. Secondary bile acids were detected in the plasma, indicating that some enterohepatic circulation of bile salts was occurring. A single homozygous nonsynonymous mutation in the coding sequence resulted in an arginine to histidine substitution at position 252 that markedly reduced the uptake of TCA, a finding confirmed by the absence of the protein from the plasma membrane.
      • Vaz F.M.
      • Paulusma C.C.
      • Huidekoper H.
      • de Ru M.
      • Lim C.
      • Koster J.
      • et al.
      Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.
      Subsequently, a number of case reports of NTCP deficiency have been published, including 2 unrelated infants both with indirect hyperbilirubinaemia and hypercholanemia who were found to be compound heterozygotes of the variants c.800C>T and c.263T>C that presumably resulted in a conformational change in NTCP. The cause of the indirect hyperbilirubinaemia was speculated to result from bile acid competition with bilirubin for uptake by the OATPs.
      • Qiu J.W.
      • Deng M.
      • Cheng Y.
      • Atif R.M.
      • Lin W.X.
      • Guo L.
      • et al.
      Sodium taurocholate cotransporting polypeptide (NTCP) deficiency: identification of a novel SLC10A1 mutation in two unrelated infants presenting with neonatal indirect hyperbilirubinemia and remarkable hypercholanemia.
      Four additional patients with hypercholanaemia and NTCP deficiency (2 paediatric and 2 adults) that were compound heterozygotes for SLC10A1 variants c.800C>T aln c.595A>C were described in a study from China.
      • Li H.
      • Deng M.
      • Guo L.
      • Qiu J.W.
      • Lin G.Z.
      • Long X.L.
      • et al.
      Clinical and molecular characterization of four patients with NTCP deficiency from two unrelated families harboring the novel SLC10A1 variant c.595A>C (p.Ser199Arg).
      Total bile acid levels tended to decrease over time in the 2 paediatric cases, possibly related to age-dependent maturation of OATP1B1 and 1B3.
      • Mooij M.G.
      • Schwarz U.I.
      • de Koning B.A.
      • Leeder J.S.
      • Gaedigk R.
      • Samsom J.N.
      • et al.
      Ontogeny of human hepatic and intestinal transporter gene expression during childhood: age matters.
      NTCP has been identified as the receptor by which hepatitis B and the delta virus (HBV/HDV) enter the liver.
      Ntcp deficiency in mice leads to prolonged postprandial elevations in serum bile acids as originally described.
      • Slijepcevic D.
      • Kaufman C.
      • Wichers C.G.
      • Gilglioni E.H.
      • Lempp F.A.
      • Duijst S.
      • et al.
      Impaired uptake of conjugated bile acids and hepatitis b virus pres1-binding in na(+) -taurocholate cotransporting polypeptide knockout mice.
      Studies by Donkers et al. show that these mice are relatively resistant to weight gain compared to wild-type mice and do not develop hepatic steatosis or hyperlipidaemia. Their studies demonstrate that Ntcp knockout mice have a reduction in intestinal fat absorption and an increase in uncoupled respiration in brown adipose tissue that in combination leads to increased energy expenditure. They conclude that strategies that block NTCP-mediated bile acid uptake might be a novel approach for the treatment of obesity and associated fatty liver disease.
      • Donkers J.M.
      • Kooijman S.
      • Slijepcevic D.
      • Kunst R.F.
      • Roscam Abbing R.L.
      • Haazen L.
      • et al.
      NTCP deficiency in mice protects against obesity and hepatosteatosis.
      That this strategy might work in humans is suggested by earlier work that showed that in vitro treatment of primary human brown adipocytes with chenodeoxycholic acid (CDCA) or other specific TGR5 agonists increased mitochondrial uncoupling and type 2 iodothyronine deiodinase expression, an effect that was absent in human primary white adipocytes.
      • Broeders E.P.
      • Nascimento E.B.
      • Havekes B.
      • Brans B.
      • Roumans K.H.
      • Tailleux A.
      • et al.
      The bile acid chenodeoxycholic acid increases human Brown adipose tissue activity.

      Organic anion-transporting polypeptides (OATPs, SLCOs)

      OATPs are the primary transporters on the basolateral sinusoidal membrane of hepatocytes that determine the uptake and disposition of drugs and other xenobiotics (Fig. 1G). In addition, they mediate the transport of a wide variety of endogenous substrates and have led to an understanding of the molecular mechanisms of pharmacokinetic drug–drug interactions.
      • König J.
      • Müller F.
      • Fromm M.F.
      Transporters and drug-drug interactions: important determinants of drug disposition and effects.
      As such they are of fundamental interest to the pharmaceutical industry, as recently reviewed.
      • Schulte R.R.
      • Ho R.H.
      Organic anion transporting polypeptides: emerging roles in cancer pharmacology.
      Much of this information is beyond the scope of this current review. Herein, we will highlight a few recent developments.
      While more than 300 OATPs have been described in the SLCO superfamily,
      • Hagenbuch B.
      • Stieger B.
      The SLCO (former SLC21) superfamily of transporters.
      OATP1B1 and 1B3 are generally accepted as the major SLCOs that enable unconjugated bilirubin to be taken up by hepatocytes. However, the molecular mechanism remains to be determined and continues to be an area of controversy.
      • Čvorović J.
      • Passamonti S.
      Membrane transporters for bilirubin and its conjugates: a systematic review.
      Imaging studies of the liver and the delivery of anticancer agents also depend on these SLCOs.
      • Schulte R.R.
      • Ho R.H.
      Organic anion transporting polypeptides: emerging roles in cancer pharmacology.
      ,
      • Stieger B.
      • Hagenbuch B.
      Organic anion-transporting polypeptides.
      Several novel liver OATPs have recently been characterised. OATP1B3-1B7 is expressed on the smooth ER of both hepatocytes and enterocytes
      • Malagnino V.
      • Hussner J.
      • Seibert I.
      • Stolzenburg A.
      • Sager C.P.
      • Meyer Zu Schwabedissen H.E.
      LST-3TM12 is a member of the OATP1B family and a functional transporter.
      and is thought to be a splice variant of SLCO1B3, with the first part encoded by SLCO1B3 and the remainder by SLCO1B7. Both transcripts seem to be regulated by the same promoter and both are upregulated by the farnesoid X receptor (FXR), as is OATP1B3. Bile acids (TCA and lithocholate) are substrates for OATP1B3-1B7 and it is speculated that this transporter allows these bile acids to be exposed to bile acid-metabolising enzymes in the ER, such as the UDP-glucuronosyltransferases, thereby providing a protective effect from bile acid tissue toxicity.
      • Malagnino V.
      • Hussner J.
      • Issa A.
      • Midzic A.
      • Meyer Zu Schwabedissen H.E.
      OATP1B3-1B7, a novel organic anion transporting polypeptide, is modulated by FXR ligands and transports bile acids.
      Under normal conditions, OATP31A is expressed at low levels in human liver and its function is not known. However, Pan and colleagues discovered that, in cholestatic liver diseases, OATP31A is significantly upregulated on the sinusoidal membrane where it functions as an efflux transporter for bile acids. Its induction is mediated by bile acid upregulation of FGF19 which activates the transcription factor Sp1 and NF-kB binding to the OAPT3A1 promoter as part of the adaptive response to cholestasis.
      • Pan Q.
      • Zhang X.
      • Zhang L.
      • Cheng Y.
      • Zhao N.
      • Li F.
      • et al.
      Solute carrier organic anion transporter family member 3A1 is a bile acid efflux transporter in cholestasis.

      Organic solute transporter-alpha and beta (SLC51A and SLC51B)

      OSTα-OSTβ is a heteromeric organic solute transporter that was discovered ~20 years ago from expression cloning studies with RNA from the marine skate liver.
      • Wang W.
      • Seward D.J.
      • Li L.
      • Boyer J.L.
      • Ballatori N.
      Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.
      Widely expressed in a number of tissues, its physiologic function was not initially clear until it was identified on the basolateral membrane of the terminal ileum as the “missing link” in the enterohepatic circulation of bile acids.
      • Dawson P.
      • Hubbert M.
      • Haywood J.
      • Craddock A.L.
      • Zerangue N.
      • Christian W.V.
      • et al.
      The heteromeric organic solute transporter alpha-beta, Osta-Ostb, is an ileal basolateral bile acid transporter.
      A recent comprehensive review provides an extensive summary of its tissue expression in mammalian cells and cell culture systems.
      • Beaudoin J.J.
      • Brouwer K.L.R.
      • Malinen M.M.
      Novel insights into the organic solute transporter alpha/beta, OSTα/β: from the bench to the bedside.
      Nevertheless, the stoichiometry of the 2 subunits is still not known for any tissue. It remains unclear when and how the 2 subunits interact, although both are needed for translocation to the plasma membrane and for its function. Earlier studies
      • Soroka C.J.
      • Xu S.
      • Mennone A.
      • Lam P.
      • Boyer J.L.
      N-Glycosylation of the alpha subunit does not influence trafficking or functional activity of the human organic solute transporter alpha/beta.
      in HepG2 and COS7 cells suggested glycosylation was not necessary for the function of the transporter. However, immunoprecipitation of OSTβ did not co-precipitate the mature, complex glycosylated form of OSTα, suggesting that the primary interaction occurs earlier in the biosynthetic pathway and may be transient. Other studies
      • Xu S.
      • Soroka C.J.
      • Sun A.Q.
      • Backos D.S.
      • Mennone A.
      • Suchy F.J.
      • et al.
      A novel di-leucine motif at the N-terminus of human organic solute transporter beta is essential for protein association and membrane localization.
      demonstrated that a novel di-leucine motif at the N-terminus of human OSTβ is required for the interaction of the 2 subunits and for their membrane expression.
      Both subunits contain response elements in their promoters for FXR binding; they are thus upregulated by bile acids and in cholestatic liver diseases such as PBC, leading to the concept that this transporter is a major efflux pathway to protect hepatocytes from bile acid-induced liver injury
      • Boyer J.L.
      • Trauner M.
      • Mennone A.
      • Soroka C.J.
      • Cai S.Y.
      • Moustafa T.
      • et al.
      Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents.
      (Fig. 1H). Jackson and colleagues provide confirmatory evidence for this concept using sandwich cultured human hepatocytes exposed to 3 days of CDCA, following which OSTα-OSTβ mRNA and basolateral bile acid efflux were significantly increased in association with reduced intracellular concentrations of bile acids in hepatocytes.
      • Jackson J.P.F.K.
      • Brouwer K.R.
      Basolateral efflux transporters: a potentially important pathway for the prevention of cholestatic hepatotoxicity.
      OSTα-OSTβ is also elevated in patients with non-alcoholic steatohepatitis and primary biliary cholangitis, in whom serum bile acids are also increased.
      • Malinen M.M.
      • Ali I.
      • Bezençon J.
      • Beaudoin J.J.
      • Brouwer K.L.R.
      Organic solute transporter OSTα/β is overexpressed in nonalcoholic steatohepatitis and modulated by drugs associated with liver injury.
      The latter study also reported interactions with several drugs including troglitazone sulfate and macrocyclic antibiotics known to cause cholestasis. There have also been reports of other cholestatic drugs that inhibit OSTα-OSTβ, including clofazimine, ethinylestradiol and fidaxomin,
      • Malinen M.M.
      • Kauttonen A.
      • Beaudoin J.J.
      • Sjöstedt N.
      • Honkakoski P.
      • Brouwer K.L.R.
      Novel in vitro method reveals drugs that inhibit organic solute transporter alpha/beta (OSTα/β).
      suggesting that inhibition of OSTα-OSTβ may contribute to the mechanism of drug-induced cholestatic liver disease.
      Other regulatory mechanisms have also been identified including a novel retinoic acid response element (RARE) that responds to retinoic acid in human OSTβ but not OSTα. This RARE is also a constitutive androstane receptor binding site that functions synergistically with the FXR binding site.
      • Xu S.
      • Sun A.Q.
      • Suchy F.J.
      A novel RARα/CAR-mediated mechanism for regulation of human organic solute transporter-β gene expression.
      Hypoxia also stimulates the expression of both OST subunits in Huh7 cells and primary human hepatocytes and is regulated by the binding of HIF-1α (hypoxia-inducible factor-1-alpha) to hypoxia response elements, particularly in the promoter of OSTα.
      • Schaffner C.A.
      • Mwinyi J.
      • Gai Z.
      • Thasler W.E.
      • Eloranta J.J.
      • Kullak-Ublick G.A.
      The organic solute transporters alpha and beta are induced by hypoxia in human hepatocytes.
      Because NTCP and BSEP expression are both reduced by hypoxia,
      • Fouassier L.
      • Beaussier M.
      • Schiffer E.
      • Rey C.
      • Barbu V.
      • Mergey M.
      • et al.
      Hypoxia-induced changes in the expression of rat hepatobiliary transporter genes.
      this induction of OSTs may represent an adaptive response to help reduce intracellular bile acid levels.
      Finally, 2 separate reports have identified mutations in human OSTα-OSTβ. Sultan and colleagues report deficiencies in OSTβ in 2 brothers that resulted in chronic diarrhoea and steatorrhoea together with clinical features of cholestatic liver disease.
      • Sultan M.
      • Rao A.
      • Elpeleg O.
      • Vaz F.M.
      • Abu-Libdeh B.
      • Karpen S.J.
      • et al.
      Organic solute transporter-β (SLC51B) deficiency in two brothers with congenital diarrhea and features of cholestasis.
      Gao et al. identified a mutation in the alpha subunit in a child with unexplained liver transaminase elevations. Elevations in serum gamma-glutamyltransferase and increases in bile duct proliferation and fibrosis in these cases suggest injury to cholangiocytes in the absence of OSTα-OSTβ.
      • Gao E.
      • Cheema H.
      • Waheed N.
      • Mushtaq I.
      • Erden N.
      • Nelson-Williams C.
      • et al.
      Organic solute transporter alpha deficiency: a disorder with cholestasis, liver fibrosis, and congenital diarrhea.
      Ostα KO mice do not develop this phenotype, most likely because their bile acid pool is more hydrophilic than in humans.

      Recent studies of OSTα-OSTβ function

      Together with ASBT in the ileum, OSTs are key regulators that maintain bile acid pool homeostasis and defend against the accumulation of toxic levels of bile acids in tissues, largely via FXR/FGF19/FGFR4 signal transduction pathways. In Ostα-deficient mice, newborns exhibit significant increases in intestinal length and markedly decreased villous height, with goblet cells that accumulate on the apical surface of the ileal mucosa. In studies by Ferrebee and colleagues, a deletion of Asbt in these mice completely reversed these morphological abnormalities, supporting the concept that a major role of intestinal Ostα-Ostβ is to protect the ileum from bile acid toxicity.
      • Ferrebee C.B.
      • Li J.
      • Haywood J.
      • Pachura K.
      • Robinson B.S.
      • Hinrichs B.H.
      • et al.
      Organic solute transporter α-β protects ileal enterocytes from bile acid-induced injury.

      Organic cation transporter 1 (OCT1, SLC22A1)

      Human OCT1 is a polyspecific, bi-directional, facilitative diffusional 12 membrane spanning domain transporter on the sinusoidal membrane of hepatocytes that transfers small organic and neutral compounds (many of which are drugs) from the blood into the liver (Fig. 1G). The level of expression of OCT1 varies greatly and many single nucleotide variants have been described in the SLC22A1 gene that affects its function.
      • Hyrsova L.
      • Smutny T.
      • Trejtnar F.
      • Pavek P.
      Expression of organic cation transporter 1 (OCT1): unique patterns of indirect regulation by nuclear receptors and hepatospecific gene regulation.
      Metformin is a major substrate for OCT1 which is commonly used to treat type 2 diabetes mellitus. Genetic variants in OCT1 are thought to account for up to a third of patients failing to achieve glycaemic control.
      • Mofo Mato E.P.
      • Guewo-Fokeng M.
      • Essop M.F.
      • Owira P.M.O.
      Genetic polymorphisms of organic cation transporter 1 (OCT1) and responses to metformin therapy in individuals with type 2 diabetes: a systematic review.
      Thirty-four OCT1 polymorphisms have been identified in 10 different ethnic groups; these polymorphisms have been associated with both deleterious and positive therapeutic responses.
      • Seitz T.
      • Stalmann R.
      • Dalila N.
      • Chen J.
      • Pojar S.
      • Dos Santos Pereira J.N.
      • et al.
      Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1.
      Cancer chemotherapeutics, including sorafenib and anthracyclines such as daunorubicin,
      • Andreev E.
      • Brosseau N.
      • Carmona E.
      • Mes-Masson A.M.
      • Ramotar D.
      The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin.
      are also substrates for OCT1 and genetic variations may account for drug resistance in some cases. Significant downregulation of OCT1 expression has been described in several liver cancer tissues (including hepatocellular carcinoma, cholangiocarcinoma and hepatoblastoma) compared to adjacent normal tissue.
      • Li J.
      • Yang Z.
      • Tuo B.
      Role of OCT1 in hepatocellular carcinoma.
      OCT1 is also downregulated in cholestasis
      • Denk G.U.
      • Soroka C.J.
      • Mennone A.
      • Koepsell H.
      • Beuers U.
      • Boyer J.L.
      Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis.
      ,
      • Nies A.T.
      • Koepsell H.
      • Winter S.
      • Burk O.
      • Klein K.
      • Kerb R.
      • et al.
      Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver.
      While the physiologic function of OCT1 is not fully understood, certain endogenous substrates including choline, acetylcholine, agmatine, norepinephrine, and the monoamine neurotransmitters, dopamine and serotonin, have been described. More recently OCT1 has been shown to be a high-capacity transporter for thiamine, and Oct1-null mice developed energy dysfunction and reduced hepatic triglyceride levels, suggesting a role for OCT1 in metabolic liver disease.
      • Chen L.
      • Shu Y.
      • Liang X.
      • Chen E.C.
      • Yee S.W.
      • Zur A.A.
      • et al.
      OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin.
      Genome-wide association studies demonstrated that polymorphisms with reduced function were paradoxically associated with increases in cholesterol, LDL, and triglyceride levels and an increased risk of type 2 diabetes, possibly related to the effects on thiamine transport into the liver.
      • Liang X.
      • Yee S.W.
      • Chien H.C.
      • Chen E.C.
      • Luo Q.
      • Zou L.
      • et al.
      Organic cation transporter 1 (OCT1) modulates multiple cardiometabolic traits through effects on hepatic thiamine content.

      Bile salt export pump (ABCB11)

      BSEP – an apical canalicular domain ATP binding cassette (ABC) export pump – is the primary transporter of bile acids from the hepatocyte into bile and the rate limiting factor in the formation of bile salt-dependent bile flow. BSEP exists within lipid rich microdomains in canalicular membrane microvilii and in submembrane vesicles where it is recycled as part of the mechanism that regulates its expression and function. Post-transcriptional trafficking and apical recycling of BSEP is incompletely understood but requires protein-protein interactions, some of which have been described.
      • Soroka C.J.
      • Boyer J.L.
      Biosynthesis and trafficking of the bile salt export pump, BSEP: therapeutic implications of BSEP mutations.
      For example, both the AP-2 (adaptor protein complex 2) which binds to a tyrosine containing motif in the C-terminus of BSEP and HAX-1 (HS-1-associated protein X-1) which binds to the NBS domain of BSEP are involved in endocytic retrieval from the canalicular membrane (Fig. 1D). In contrast, the myosin II regulatory light chain influences trafficking of the transporter to the apical membrane. Przybylla and colleagues
      • Przybylla S.
      • Stindt J.
      • Kleinschrodt D.
      • Schulte Am Esch J.
      • Häussinger D.
      • Keitel V.
      • et al.
      Analysis of the bile salt export pump (ABCB11) interactome employing complementary approaches.
      applied 3 different techniques to identify 10 other BSEP-interacting proteins using co-immunoprecipitation, mass spectrometry and a membrane yeast two-hybrid assay (using a human DNA library), before verifying interactions using purified proteins. All 10 proteins except for radixin were integral or membrane-associated proteins in the early protein secretory pathway. This included bile-acyl-CoA synthetase, (BACS), the next to last ER-associated enzyme in the bile salt synthetic pathway. BACS was found to be localised in pseudocanalicular structures in HepaRG cells suggesting that BACS and BSEP interacted at or proximal to a canalicular site. BACS (SLC27A5) catalyses the first step in re-conjugation of bile acids to taurine or glycine.
      BSEP expression is tightly regulated by transactivation pathways, particularly by the nuclear receptor FXR (NR1H4) which is activated by bile acids. This important mechanism helps to prevent the toxic accumulation of bile acids in the liver. Two FXR genes exist, FXRα and FXRβ, although in humans FXRβ is a pseudogene. There are also 4 isoforms of FXRα with isoforms FXRα1 and 2 expressed in the liver, with FXRα2 being the more potent activator. Deng and his laboratory previously demonstrated that the relative expression of these isoforms was altered in the livers of patients with HCC where FXR-α1/FXR-α2 ratios were significantly increased, leading to a marked reduction in BSEP expression.
      • Chen Y.
      • Song X.
      • Valanejad L.
      • Vasilenko A.
      • More V.
      • Qiu X.
      • et al.
      Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma.
      Subsequent studies revealed that FXRα1 and FXRα2 regulate human BSEP through 2 distinct FXR response elements: IR1a and IR1b with the more potent regulator, FXRα2, transactivating human BSEP through IR1a, while FXRα1 weakly transactivated human BSEP through a newly identified IR1b. In contrast to IR1b, IR1a exhibited significant species-specific differences in expression, accounting for the species-specific differences in BSEP transactivation by FXRα1 and FXRα2.
      • Song X.
      • Chen Y.
      • Valanejad L.
      • Kaimal R.
      • Yan B.
      • Stoner M.
      • et al.
      Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor.
      Some mutations in BSEP result in primary familial intrahepatic cholestasis (PFIC) type 2. The Natural course and Prognosis of PFIC and Effect of biliary Diversion (NAPPED) consortium examined the effect of BSEP genotypes on outcome in a retrospective multicentre study of 264 patients with homozygous or compound heterozygous pathological ABCB11 mutations. This is the largest genetically defined cohort of patients with severe BSEP deficiency published to date. Cases were divided into 3 categories of severity based on predicted residual BSEP transport function. Overall survival correlated with genetic severity, with a median survival of the native liver of 20.4 years in category 1, 7.0 years in category 2, and only 3.5 years in the most severe category 3. The risk of HCC at age 15 and survival after surgical biliary diversion also correlated with genetic severity. Serum bile salt levels <102 μmol/L or at least a reduction of 75% predicted native liver survival of ≥ 15 years following biliary diversion. The study emphasises the importance of using genotypic data to guide therapy in paediatric and adult patients with this disease.
      • van Wessel D.B.E.
      • Thompson R.J.
      • Gonzales E.
      • Jankowska I.
      • Sokal E.
      • Grammatikopoulos T.
      • et al.
      Genotype correlates with the natural history of severe bile salt export pump deficiency.
      Overall survival of PFIC type 2 patients correlated with the genetic severity of the BSEP mutation, emphasizing the importance of using genotype data to guide therapeutic approaches.
      In another study
      • Ramos Pittol J.M.
      • Milona A.
      • Morris I.
      • Willemsen E.C.L.
      • van der Veen S.W.
      • Kalkhoven E.
      • et al.
      FXR isoforms control different metabolic functions in liver cells via binding to specific DNA motifs.
      different FXR isoforms were also found to control different metabolic functions in liver cells by binding to specific DNA motifs. Using organoids that expressed FXRα2 but not FXRα1, obeticholic acid increased mitochondrial pyruvate transport and reduced insulin-induced lipogenesis. These studies found that most metabolic effects regulated by FXR in mouse and human liver cells were regulated by the FXRα2 isoform via specific binding to ER-2 motifs. These studies further suggest that the liver expression levels of FXRα2 might predict responses to treatment with FXR agonists.
      Using the conjugated bile acid tracer [N-methyl-11C]cholylsarcosine, Kjaergaard et al. show that obeticholic acid, which is excreted into bile by BSEP, increased the hepatic transport of the tracer, and thus endogenous conjugated bile acids, from hepatocytes into the canaliculi in patients with PSC, thereby reducing the time hepatocytes are exposed to potentially cytotoxic bile acids.
      • Kjærgaard K.
      • Frisch K.
      • Sørensen M.
      • Munk O.L.
      • Hofmann A.F.
      • Horsager J.
      • et al.
      Obeticholic acid improves hepatic bile acid excretion in patients with primary biliary cholangitis.
      In a separate study in patients awaiting cholecystectomy, obeticholic acid treatment, which increases the biliary cholesterol saturation index, also enriched the bile with FGF19; this possibly led to relaxation, filling and delayed emptying of the gallbladder, thereby contributing to the formation of gallstones.
      • Al-Dury S.
      • Wahlström A.
      • Panzitt K.
      • Thorell A.
      • Ståhlman M.
      • Trauner M.
      • et al.
      Obeticholic acid may increase the risk of gallstone formation in susceptible patients.

      The multidrug resistance transporter 3 (MDR3, ABCB4)

      The function of MDR3 is to transport phosphatidycholine across the canalicular membrane into bile where it plays a major role in preventing bile acids from injuring the bile duct epithelium. Mutations in MDR3 result in 4 clinical disorders: PIFC3 in children, low phospholipid-associated cholelithiasis, intrahepatic cholestasis of pregnancy and a progressive cholangiopathy in adults including drug-induced liver injury.
      • Stättermayer A.F.
      • Halilbasic E.
      • Wrba F.
      • Ferenci P.
      • Trauner M.
      Variants in ABCB4 (MDR3) across the spectrum of cholestatic liver diseases in adults.
      ,
      • de Vries E.
      • Mazzetti M.
      • Takkenberg B.
      • Mostafavi N.
      • Bikker H.
      • Marzioni M.
      • et al.
      Carriers of ABCB4 gene variants show a mild clinical course, but impaired quality of life and limited risk for cholangiocarcinoma.
      Deficiencies in MDR3 are often misdiagnosed, particularly in children.
      • Schatz S.B.
      • Jüngst C.
      • Keitel-Anselmo V.
      • Kubitz R.
      • Becker C.
      • Gerner P.
      • et al.
      Phenotypic spectrum and diagnostic pitfalls of ABCB4 deficiency depending on age of onset.
      Sequencing of cholestasis-related genes often results in genetic variants of unknown clinical significance, creating a need to assess their functional phenotype. In one such attempt, Delaunay et al. expressed 12 ABCB4 variants in HepG2 and HEK293 cells. Three mutants were retained in the ER and could be rescued by cyclosporines. Five other mutants were expressed on the canalicular membrane but did not effectively excrete phosphatidylcholine. Two other mutants were not stable at the apical membrane. This approach enabled classification of these variants (nonsense or missense mutations, maturation defects, defective activity or stability or no detectable defect) and illustrates one approach for assessment of genotype-based treatments. In the future, high throughput screening might make personalised medicine a reality and enable selection of therapies based on a patient’s genetic defect.
      • Delaunay J.L.
      • Durand-Schneider A.M.
      • Dossier C.
      • Falguières T.
      • Gautherot J.
      • Davit-Spraul A.
      • et al.
      A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3.
      The rodent equivalent to MDR3 is Mdr2. Wang et al. produced double knockout Bsep and Mdr2 null mice to see if the highly hydrophilic bile acids that the Bsep mutant mice produce could protect against the liver injury normally observed in Mdr2-/- mice. Indeed, the bile acid composition of the double knockout mouse resembled the Bsep-/- mouse with significant increases in hydrophilic muricholic acids, tetrahydroxylated bile acids and reductions in the more hydrophobic cholic acid. Feeding tetrahydroxy bile acids to the Mdr2-/- mouse significantly reduced liver injury, suggesting a novel approach for the treatment of cholestasis.
      • Wang R.
      • Sheps J.A.
      • Liu L.
      • Han J.
      • Chen P.S.K.
      • Lamontagne J.
      • et al.
      Hydrophilic bile acids prevent liver damage caused by lack of biliary phospholipid in Mdr2(-/-) mice.

      The multidrug resistance protein 2 (MRP2, ABCC2)

      MRP2 is a canalicular membrane ABC transporter family member that functions to excrete conjugated organic anions, particularly bilirubin, bile acid glucuronides and drugs, into bile. Inhibition of MRP2 leads to jaundice. Chai and colleagues examined how MRP2 expression was regulated in patients with bile duct obstruction from gallstones. MRP2 protein but not mRNA was significantly reduced to 25% of normal controls, suggesting that post-transcriptional regulation was involved. Immunoprecipitation studies identified ezrin, but not radixin, in association with MRP2. Ezrin and radixin are members of the ERM family that tether proteins to actin filaments. Levels of phospho-ezrin Thr567 correlated with the amount of co-precipitated MRP2 in the cholestatic livers, whereas ezrin and radixin total protein levels were unchanged. Further findings suggested that activation of hepatic protein kinase C during cholestasis led to ezrin Thr567 phosphorylation resulting in MRP2 internalisation and ubiquitin ligase E3 GP78-dependent degradation in the proteasome (Fig. 1D). Dephosphorylation of ezrin allowed recycling of the ezrin-MRP2 complex to the canalicular membrane. These findings suggest that downregulation of MRP2 in patients with bile duct obstruction and possibly other causes of cholestasis could be involved in the development of jaundice.
      • Chai J.
      • He Y.
      • Cai S.Y.
      • Jiang Z.
      • Wang H.
      • Li Q.
      • et al.
      Elevated hepatic MRP3/ABCC3 expression in human obstructive cholestasis is mediated through TNFalpha and JNK/SAPK signaling pathway.

      Conclusions and future directions

      In this review, we have described some of the recent developments in the field of bile formation and secretion that have occurred since we last reviewed the subject in 2013.
      • Boyer J.L.
      Bile formation and secretion.
      Older concepts of bile salt dependent and independent secretion have given way to a much more complex understanding of the molecular mechanisms for both canalicular and cholangiocyte secretion. Much has also been learned about the role that genetic variants and mutations in human transporters play as a cause of clinical disease, particularly novel variants in NTCP that affect the entry of hepatitis B and mutations that result in hypercholanaemia, yet do not result in liver injury. What can we expect in the future? It is likely that deep sequencing of unexplained cases of cholestasis will continue to find additional genetic variants and mutations in transporters that influence the formation of bile, as recently described for BSEP and in separate case reports for human OSTα and OSTβ (Table 1). Drug therapies are increasingly likely to be based on knowledge of an individual’s genotype. These advances should bring “personalized medicine” closer to reality. Advances in imaging technologies should provide deeper insight into the determinants of canalicular bile flow and how drugs and other toxins result in cholestatic drug-induced liver injury. However, basic studies on these fundamental secretory processes, which determine both hepatic and cholangiocyte secretion, will continue to be required if new therapeutic strategies for cholestatic disorders are to be found. The possibility that inhibition of NTCP with a drug like Myrcludex might advance to the clinic not only for treatment of hepatitis B but also for cholestasis, is one recent example of how basic science is leading to potential therapeutic breakthroughs in this field.
      Table 1Disease-producing mutations in human hepatic transporters.
      TransporterMutationDisease/phenotypeReference
      NTCPp.S267FDecreased susceptibility to HBV and HBV-related HCC
      • Binh M.T.
      • Hoan N.X.
      • Van Tong H.
      • Sy B.T.
      • Trung N.T.
      • Bock C.T.
      • et al.
      NTCP S267F variant associates with decreased susceptibility to HBV and HDV infection and decelerated progression of related liver diseases.
      ,
      • Wang P.
      • Mo R.
      • Lai R.
      • Xu Y.
      • Lu J.
      • Zhao G.
      • et al.
      Genetic variations of NTCP are associated with susceptibility to HBV infection and related hepatocellular carcinoma.
      NTCPp.R252HConjugated hypercholanaemia
      • Vaz F.M.
      • Paulusma C.C.
      • Huidekoper H.
      • de Ru M.
      • Lim C.
      • Koster J.
      • et al.
      Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype.
      NTCPp.S267F, p.I88TIndirect hyperbilirubinemia, hypercholanaemia
      • Qiu J.W.
      • Deng M.
      • Cheng Y.
      • Atif R.M.
      • Lin W.X.
      • Guo L.
      • et al.
      Sodium taurocholate cotransporting polypeptide (NTCP) deficiency: identification of a novel SLC10A1 mutation in two unrelated infants presenting with neonatal indirect hyperbilirubinemia and remarkable hypercholanemia.
      NTCPp.S267F, p.S199RHypercholanaemia
      • Li H.
      • Deng M.
      • Guo L.
      • Qiu J.W.
      • Lin G.Z.
      • Long X.L.
      • et al.
      Clinical and molecular characterization of four patients with NTCP deficiency from two unrelated families harboring the novel SLC10A1 variant c.595A>C (p.Ser199Arg).
      OSTα/OSTβp.F27fsChronic diarrhoea and steatorrhoea, cholestasis
      • Sultan M.
      • Rao A.
      • Elpeleg O.
      • Vaz F.M.
      • Abu-Libdeh B.
      • Karpen S.J.
      • et al.
      Organic solute transporter-β (SLC51B) deficiency in two brothers with congenital diarrhea and features of cholestasis.
      OSTα/OSTβp.Q186Cholestasis, congenital diarrhoea
      • Gao E.
      • Cheema H.
      • Waheed N.
      • Mushtaq I.
      • Erden N.
      • Nelson-Williams C.
      • et al.
      Organic solute transporter alpha deficiency: a disorder with cholestasis, liver fibrosis, and congenital diarrhea.
      OCT1Multiple polymorphismsIncreased cholesterol, LDL and triglyceride levels
      • Seitz T.
      • Stalmann R.
      • Dalila N.
      • Chen J.
      • Pojar S.
      • Dos Santos Pereira J.N.
      • et al.
      Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1.
      BSEPp.E297G, p.D482GCholestasis
      • van Wessel D.B.E.
      • Thompson R.J.
      • Gonzales E.
      • Jankowska I.
      • Sokal E.
      • Grammatikopoulos T.
      • et al.
      Genotype correlates with the natural history of severe bile salt export pump deficiency.
      MDR3MultiplePFIC3, Cholelithiasis, ICP, progressive cholangiopathy
      • Stättermayer A.F.
      • Halilbasic E.
      • Wrba F.
      • Ferenci P.
      • Trauner M.
      Variants in ABCB4 (MDR3) across the spectrum of cholestatic liver diseases in adults.
      BSEP, bile salt export pump; HCC, hepatocellular carcinoma; ICP, intrahepatic cholangitis of pregnancy; MDR3, multidrug resistance transporter 3; NTCP, sodium dependent cotransporting polypeptide; OCT1, organic cation transporter 1; OSTα-OSTβ, organic solute transporter α-β; PFIC3, primary familial intrahepatic cholestasis type 3.

      Abbreviations

      AE2, Cl/HCO3 exchanger; ASBT, apical sodium-dependent bile acid transporter; BACS, bile-acyl-CoA synthetase; BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; CFTR, cystic fibrosis transembrane conductance regulator; FGF19, fibroblast growth factor 19; FXR, farnesoid X receptor; FXRE, FXR response elements; HCC, hepatocellular carcinoma; InsP3R3/ITPR3, type 3 isoform of the inositol 1,4,5-triphosphate receptor; MDR3, multidrug resistance transporter 3; MRP2, multidrug resistance protein 2; NRF2, nuclear factor, erythroid 2-Like 2; NTCP, sodium dependent cotransporting polypeptide; OATP, organic anion-transporting polypeptides; OCT, organic cation transporter; OSTα-OSTβ, organic solute transporter α-β; PBC, primary biliary cholangitis; PFIC, primary familial intrahepatic cholestasis; PSC, primary sclerosing cholangitis; RARE, retinoic acid response element; TCA, taurocholic acid; TGR5, Takeda G protein-coupled receptor 5; TMEM16A, transmembrane member 16A; TUDCA, tauroursodeoxycholic acid; SLCO, solute carrier organic anion; UDCA, ursodeoxycholic acid; UTR, untranslated region; YAP, Yes-associated protein.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors’ contributions

      Both authors contributed to the writing and editing of this review. Dr. Soroka designed Fig. 1.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is the supplementary data to this article:

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