Bile Formation and the Enterohepatic Circulation

Chapter 41

Bile Formation and the Enterohepatic Circulation Paul A. Dawson

41.1. INTRODUCTION Bile is a complex aqueous secretion that originates from hepatocytes and is modified distally by the biliary epithelium. As a basic “humor” in the body, the significance of bile had been recognized since antiquity. However, our understanding of bile was originally restricted to knowledge of its composition, and the mechanism of bile formation remained elusive until the mid-20th century with the advent of techniques to perfuse isolated livers and to study isolated hepatocytes.1 The concept that bile cycles between the liver and the gut, a “motus circularis bilis,” dates back more than 300 years to the work of Mauritius van Reverhorstand the elegant kinetic modeling by the Neapolitan mathematician, Giovanni Borelli.2 As the major biliary solute and driving force for bile flow, much attention has been focused on the mechanisms responsible for bile acid biosynthesis and enterohepatic cycling, and the relationship of those mechanisms to hepatic and gastrointestinal physiology.1,3

diversity in their chemical structures across the species, with modification to both the C19 steroid nucleus and the side chain. This large diversity is thought to be unique among classes of small molecule endobiotics, however, the evolutionary forces driving the variation remain poorly understood.8,9 In  vivo, bile acids exist primarily as sulfate conjugates of bile alcohols and as taurine (or glycine) aminoacyl-amidated conjugates of bile acids.10 The general structure of the steroid nucleus and side chain, position of hydroxyl groups, and hydrophobicity for the major mammalian bile acid species are shown in Fig. 41.1.

41.3.  MAJOR FUNCTIONS OF BILE AND BILE ACIDS

Bile acids are planar amphipathic molecules possessing a characteristic four-ring, 19-carbon perhydrocyclopentanophenanthrene nucleus and a multicarbon side chain. In all the vertebrates examined, cholesterol serves as the precursor for bile acid biosynthesis, whereby a waterinsoluble, hydrophobic membrane lipid is converted into water-soluble derivatives that can be excreted in bile.4 As a group, these molecules are termed “bile salts” or “bile acids,” and are included in the ST04 category (sterol lipids: Bile acids and derivatives) (http://www.lipidmaps.org) of the LIPID MAPS Lipid Classification System.5 Most bile acids can be assigned to three general structural classes according to the length of the side chain and functionality of the terminal polar group. The three classes are 27-carbon (C27) bile alcohols, C27 bile acids, and 24-carbon (C24) bile acids, with C24 bile acids being the predominant form in mammals.4,6 Bile acids are not known to be made by invertebrates.7 In vertebrates, bile acids show a ­remarkable

Bile formation and secretion is essential for life and fulfills a number of important functions in vertebrates.1 (1) Bile secretion is a major route for excretion of heavy metals, lipophilic endogenous compounds (endobiotics) such as bilirubin, cholesterol and steroids, and lipophilic exogenous compounds (xenobiotics) such as drugs, drug metabolites, and environmental toxins. (2) Bile is a critical digestive secretion and works in concert with saliva, gastric, and pancreatic secretions to facilitate the breakdown and assimilation of food.11 (3) Bile secretion plays a role in innate immunity and controlling intestinal microbes by serving as a conduit for the release of IgA antibodies.12 (4) Bile secretion is the vehicle for the excretion of bile acids, the major organic solutes in bile. As described in the next section, bile acids perform a variety of indispensable functions in the liver and gastrointestinal tract. Although best known for their ability to form micelles and facilitate absorption of lipids in the gut, the physiological functions of bile acids extend well beyond their role as simple detergents. The recognized functions ascribed to bile acids in the liver and gastrointestinal tract are summarized in Table 41.1.11,13 The major functions of bile acids include: (1) Inducing bile flow and hepatic secretion of biliary lipids (phospholipid

Physiology of the Gastrointestinal Tract. https://doi.org/10.1016/B978-0-12-809954-4.00041-4 © 2018 Elsevier Inc. All rights reserved.

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41.2.  STRUCTURE OF BILE ACIDS

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FIG. 41.1  Structure and hydrophobicity/hydrophilicity profile of bile acids. (A) Structure of the common bile acids. In humans, cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary bile acids synthesized by the hepatocyte. Primary bile acids in other species include muricholic acid (MCA) and ursodeoxycholic acid (UDCA). (B) Structure and hydrophobicity of bile acids. Hydroxyl groups that are oriented in the α-orientation are located below the steroid nucleus and are axial to the plane of the steroid nucleus. Hydroxyl groups that are in the β-orientation are located above the steroid nucleus and are equatorial to the plane of the steroid nucleus. The MCA escapes from this common rule as it contains a 6α-hydroxylated group that is equatorially positioned to the steroid nucleus plane. The equatorial location of hydroxyl groups confers polarity to the hydrophobic concave side of the steroid nucleus. Therefore, MCAs containing both 6α and 7β-oriented hydroxyl groups, and UDCA with its 7β-oriented hydroxyl group are more hydrophilic than other bile acids with the same number of hydroxyl groups axially positioned to the steroid nucleus. (Adapted with permission from Dawson PA. Bile acid metabolism. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier B.V.; 2015. p. 359.)

TABLE 41.1  Functions of Bile Acids in the Gastrointestinal Tract Tissue

Function

Whole body

• Elimination of cholesterol • Regulation of fat, glucose, and energy homeostasis by signaling through nuclear and G-protein-coupled receptors

Liver—hepatocyte

• Insertion of canalicular bile acid and phospholipid transporters • Induction of bile flow and biliary lipid secretion • Promotion of mitosis during hepatic regeneration • Regulation of gene expression via nuclear receptors (FXR, PXR, VDR)

Liver—endothelial cell

• Regulation of hepatic blood flow via activation of TGR5

Biliary tract— lumen

• Micellar solubilization of cholesterol • Micellar trapping of cholephilic xenobiotics • Antimicrobial actions • Calcium binding to prevent formation of calcium bilirubinate or salts of calcium phosphate, carbonate, or palmitate

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TABLE 41.1  Functions of Bile Acids in the Gastrointestinal Tract—cont’d Tissue

Function

Biliary tract— cholangiocytes

• Stimulation of bicarbonate secretion via CFTR and AE2

Gallbladder epithelium

• Modulation of cAMP-mediated secretion

Small intestine— lumen

• Micellar solubilization of dietary lipids, especially cholesterol, and fat-soluble vitamins

• Promotion of proliferation when bile duct is obstructed

• Promotion of mucin secretion

• Solubilization of lipophilic drugs and xenobiotics • Antimicrobial actions • Acceleration of protein hydrolysis by pancreatic proteases • Prevention of enteric hyperoxaluria

Small intestine— ileal enterocyte

• Regulation of gene expression via nuclear receptors (FXR, PXR, VDR) • Secretion of FGF19 to regulate hepatic bile acid synthesis • ASBT and CFTR-dependent induction of water secretion

Small intestine— other effects

• Secretion of antimicrobial factors by intestinal epithelium (FXR-mediated) • Activation of TGR5 on cholinergic neurons to inhibit intestinal contractility and delay small intestinal transit • Activation of TGR5 on enteroendocrine L-cells

Large intestine— colonic enterocyte

• Modulation of electrolyte absorption and secretion

Large intestine— other effects

• Alter intestinal motility

Gut microbiota

• Regulate microbial diversity and metabolism

• Activation of TGR5 on enteroendocrine L-cells

Adapted from Hofmann AF, Hagey LR. Cell Mol Life Sci 2008;65:2474.

and cholesterol). The active vectorial movement of bile acids from blood to the bile canaliculus creates an osmotic gradient, allowing water and small solutes to enter the biliary space by solvent drag. This is a major driving force for bile formation. (2) Digestion and absorption of dietary fats such as long-chain fatty acids, cholesterol, and fat-soluble vitamins. Bile acids form mix micelles with lipids and lipid digestion products to increase their aqueous solubility in the gut lumen, thereby enhancing their diffusion across the unstirred aqueous layer at the surface of the intestinal epithelium.14–16 Fat-soluble vitamins (A, D, E, K) are poorly absorbed from the intestinal lumen in the absence of bile acid micelles, and disturbances in the secretion or enterohepatic cycling of bile acids cause fat-soluble vitamin deficiency.17,18 Along with their major role in dietary lipid absorption, bile acids may facilitate intestinal absorption of dietary protein by promoting protein denaturation and accelerating hydrolysis by pancreatic proteases.19 (3) Bile acids play a complex role in maintaining cholesterol homeostasis. On one hand, bile acids increase cholesterol intake by promoting intestinal absorption of biliary and

dietary cholesterol. However, on the other hand, bile acids also promote cholesterol loss from the body. Bile acids are water-soluble end products of cholesterol catabolism and bile acid loss in the feces is quantitatively the second most important route for cholesterol elimination.20–22 Bile acids also promote hepatic secretion of cholesterol into bile by inducing bile flow and solubilizing biliary cholesterol, thereby enabling cholesterol to move from the liver to the intestinal lumen for elimination. (4) Bile acids contribute to the gut's antimicrobial defenses through direct bacteriostatic actions of bile acid-fatty acid mixed micelles and by signaling to induce expression of antimicrobial genes, thereby reducing small bowel bacterial translocation and intestinal inflammation.23–26 Bacterial overgrowth occurs in biliary fistula or bile duct-ligated animals, as well as in animals with experimental cirrhosis. In cirrhotic or cholestatic rats with bacterial overgrowth, feeding of conjugated bile acids or bile acid analogs ameliorates bacterial overgrowth, decreases bacterial translocation to intestinal lymph nodes, and decreases endotoxemia.23–26 (5) Bile acids regulate gut microbial diversity and vice versa under physiological and

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pathophysiological conditions.27–30 (6) Bile acids act to prevent the formation of calcium gallstones and calcium oxalate kidney stones.31–33 Conjugated bile acids, which are fully water soluble as calcium salts, prevent the formation of gallstone-enucleating precipitates of calcium bilirubinate or salts of calcium phosphate, calcium carbonate, or calcium palmitate by binding calcium in the biliary tract and gallbladder.33,34 In the small intestinal lumen, dietary oxalate is usually precipitated by calcium. However, in patients with severe ileal resection and in obese patients after bariatric surgery, excess amounts of dietary fatty acids and bile acids passing into the colon act as a sink for calcium, greatly lowering its intraluminal activity.35–37 As a result, oxalate remains in solution and is absorbed from the colon, leading to hyperoxaluria and an increased risk of renal stone formation. In the presence of an intact enterohepatic circulation, bile acids are present at sufficient luminal concentrations in the small intestine to facilitate fat absorption, thereby reducing steatorrhea, colonic fatty acid concentrations, and oxalate absorption.38 (7) Bile acids act as hormones to signal through nuclear and G-protein-coupled receptors in order to regulate the bile acid enterohepatic circulation, hepatic function, gut motility, and fat, glucose, and energy homeostasis.39–42

41.3.1  Bile Acids as Signaling Molecules Beyond their roles as simple detergents to facilitate dietary lipid absorption and cholesterol homeostasis, bile acids also function as signaling molecules. Bile acids activate specific nuclear receptors (farnesoid X receptor alpha, FXR; pregnane X receptor, PXR; vitamin D receptor, VDR), G-proteincoupled receptors (Takeda G-protein-coupled receptor, TGR5; muscarinic receptors; sphingosine-1-phosphate receptor 2, S1PR2), integrins (α5,β1-integrin), and cellsignaling pathways (protein kinase C, PKC; c-jun N-terminal kinase 1/2, JNK 1/2; serine/threonine protein kinase, AKT/ PKB; extracellular signal-regulated kinase, ERK 1/2; p38 mitogen-activated protein kinase, p38 MAPK; epidermal growth factor receptor, EGFR),41–43 with FXR and TGR5 being the best understood examples. Evidence of their signaling properties began to emerge in the 1980s and 1990s, when bile acids were shown to activate protein kinase C isoforms and exhibit cell growthmodulatory effects.44–46 However, the role of bile acids as hormones/signaling molecules was not firmly established until 1999, when bile acids were identified as ligands for the orphan nuclear receptor FXR (gene symbol: NR1H4).47–49 The FXR (FXRα) should not be confused with the related nuclear receptor, FXRβ. FXRβ (gene symbol: NR1H5) is a widely expressed nuclear receptor that is activated by lanosterol, but not by bile acids. Although expressed by many vertebrates and mammalian species including mice, rats, rabbits, and dogs, FXRβ (NR1H5) is a nonexpressed

p­ seudogene in humans and primates.50,51 For FXR, many of the major mammalian bile acids (both unconjugated as well as glycine or taurine conjugated) function as ligands, with the following rank order of potency: chenodeoxycholic acid > lithocholic acid ≈ deoxycholic acid > cholic acid. Notable exceptions to the list of bile acid FXR agonists are ursodeoxycholic acid and 6-hydroxylated bile acids species such as muricholic acid, which do not activate FXR or function as FXR antagonists.52,53 There are four major isoforms of FXR in mice and humans, which are generated through the use of alternative promoters and alternative splicing.54 Although all four FXR isoforms encode identical ligand binding domains, the abundance of the isoforms vary between tissues and there are differences in their relative transcriptional activity.55,56 As noted above, vertebrates exhibit a remarkable diversity in their bile acid chemical structures, and the FXR ligand binding domain appears to have coevolved with its bile acid ligand.57,58 The FXR is mainly expressed in the liver intestine, kidney, and adrenal. Consistent with its gastrointestinal expression, FXR plays important roles in the regulation of enterohepatic cycling of bile acids, feedback regulation of bile acid biosynthesis, and protection against bile acid-associated toxicity.59–62 In the liver, these functions include stimulating bile acid conjugation and export across the canalicular membrane into bile. In the small intestine, activation of FXR protects the enterocyte from bile acid overload by inducing expression of the ileal cytosolic ileal bile acid binding protein (IBABP; gene symbol: FABP6), the basolateral bile acid transporter subunits, organic solute transporter (OST) alpha (OSTα), and OST beta (OSTβ),63,64 and the endocrine polypeptide hormone fibroblast growth factor (FGF) 19 (mouse ortholog: FGF15), a central regulator of hepatic bile acid synthesis.65,66 With regard to general functions in the gastrointestinal tract, FXR induces expression of genes important for intestinal barrier function and antimicrobial defense,24 and has important antiproliferative and antiinflammatory properties.67–69 Bile acids also signal via the nuclear receptors PXR (gene symbol: NR1I2)70,71 and VDR (gene symbol: NR1I1).72,73 These receptors are activated by lithocholic acid, a hydrophobic and potentially cytotoxic secondary bile acid produced from chenodeoxycholic acid by intestinal anaerobic bacteria. With regard to ligand specificity, bile acids activate PXR with a rank order of potency: lithocholic acid > deoxycholic acid > cholic acid, and activate VDR with a rank order of potency: 3-oxo-lithocholic acid > deoxycholic acid > cholic acid. With regard to bile acid homeostasis, PXR or VDR primarily function to induce expression of enzymes involved in bile acid metabolism and detoxification,71,72,74 and likely play only a minor role in regulating bile acid biosynthesis.75,76 In 2002, two groups independently identified TGR5 (also called membrane-type bile acid receptor, M-BAR; G-protein-coupled bile acid receptor 1, GPBAR1; gene

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symbol: GPBAR1) as a Gαs-coupled bile acid receptor, which stimulates adenylate cyclase and increases intracellular cAMP levels.77,78 TGR5 is activated by conjugated and unconjugated bile acids, with the following rank order of potency: deoxycholic acid > lithocholic acid > chenodeoxycholic acid> cholic acid.78 Notably, there are bile acid ligand specificity differences between FXR and TGR5, which have been exploited to generate bile acid derivatives that selectively activate the individual receptors or serve as agonists for both.79–81 TGR5 is ubiquitously expressed, with highest levels of expression in gallbladder and moderate levels of expression in liver and intestine. In the liver, TGR5 is not expressed by hepatocytes. However TGR5 is expressed by Kupffer cells, where it is thought to play an immunomodulatory role, and by sinusoidal endothelial cells, where TGR5 functions to induce nitric oxide synthesis and regulate the hepatic microcirculation.82 With the growing appreciation of bile acids as signaling molecules, considerable study is being directed toward understanding the physiological functions of TGR5.83 For example, bile acid activation of TGR5 can regulate gallbladder filling, intestinal motility, and may have a role in bile acid-induced itch and the analgesia associated with cholestatic liver disease.84–87 There are also metabolic effects associated with TGR5 signaling in brown adipose, muscle, and macrophages.41,88–90

41.4.  BIOSYNTHESIS AND BIOTRANSFORMATION OF BILE ACIDS 41.4.1  Biosynthesis of Bile Acids Bile acids are synthesized from cholesterol in the pericentral hepatocytes of the hepatic acini.91 In this process, the hydrophobic substrate, cholesterol, is converted to a watersoluble, amphipathic product through a series of sterol ring hydroxylations and side chain oxidation steps. Bile acids synthesized by the hepatocyte are designated primary bile acids to distinguish them from the secondary bile acids that are formed by the reactions carried out by the host or gut microbiota, which include dehydroxylation, dehydrogenation (oxidation of a hydroxy group to an oxo group), oxidation, epimerization (changing an α-hydroxy group to a β-hydroxy group or vice versa), and esterification.3,92 Bile acid synthesis was originally thought to involve one major pathway, the “classical” or neutral pathway (cholesterol 7αhydroxylase pathway) that favors cholic acid biosynthesis.93 This paradigm was later modified by the discovery of a second pathway, the “alternative” or acidic pathway (oxysterol 7α-hydroxylase pathway) that favors the biosynthesis of chenodeoxycholic acid in humans and 6-hydroxylated bile acids such as muricholic acid and hyocholic acid in mice and rats.94,95 Details of the hepatocellular and biochemical mechanisms responsible for the metabolic channeling of cholesterol toward cholic acid versus chenodeoxycholic

acid/6-hydroxylated bile acids are still not clear, and the specificity is not absolute. The cholesterol 7α-hydroxylase pathway produces some chenodeoxycholic acid/6-hydroxylated bile acid whereas the alternative pathway can yield cholic acid.96–98 In the alternative pathway, the first step involves modification of the cholesterol side chain by C-24 (­sterol 24-hydroxylase; gene symbol: CYP46A1), C-25 (sterol 25hydroxylase; gene symbol: CH25H), or C-27 (sterol ­ 27-­hydroxylase; gene symbol: CYP27A1) sterol hydroxylases present in liver and extra-hepatic tissues such as brain. This reaction is then followed by an oxysterol 7αhydroxylation, which is mediated primarily by CYP7B1 in liver.39,94,99–102 Of these alternative hydroxylation pathways, the contribution of 27-hydroxycholesterol to bile acid synthesis is quantitatively most important.93,94 Although quantitatively a minor contributor to bile acid synthesis, the conversion of cholesterol to 24S-hydroxycholesterol functions as a major mechanism for cholesterol elimination from brain by facilitating sterol transfer across the bloodbrain barrier into the systemic circulation for excretion by the liver.103–105 The overall process of bile acid biosynthesis is complex, requiring the action of 16 enzymes that catalyze as many as 17 different reactions.102 In the classical pathway, the steroid nucleus is modified before the side chain, whereas in the alternative pathways, side chain modifications occur before or coincident with changes to the steroid nucleus. Cholesterol 7α-hydroxylase (gene symbol: CYP7A1) is the rate-limiting enzyme for bile acid synthesis via the classical pathway. However, the step catalyzed by the sterol 12αhydroxylase (gene symbol, CYP8B1) controls the amount of cholic acid synthesized and is an important determinant of the ratio of cholic acid to chenodeoxycholic acid and cholic acid to muricholic acid in human and mouse bile, respectively.106 In this capacity, CYP8B1 plays a critical role in modulating the composition and hydrophobicity of the bile acid pool. It should be noted that humans and mice have substantially different bile acid pool compositions. This reflects differences in bile acid conjugation (discussed below), synthesis of ursodeoxycholic acid as a primary bile acid in mice, and hydroxylation at the 6-position of the bile acid steroid nucleus in mice.107 In contrast, hydroxylation of bile acids at the 6-position is rare in humans, and detectable under only specialized circumstances, such as in fetal/early neonatal development or in certain cholestatic conditions.108–110 It has long been known that 6hydroxylation of bile acids alters their physicochemical and detergent properties, with mice having a more hydrophilic bile acid pool.111,112 With the recognition of bile acids as signaling molecules that act through nuclear and G-protein-coupled receptors, the human-rodent bile acid structural differences have gained additional importance. The 6-hydroxylation of bile acids dramatically alters their

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signaling properties, potentially limiting the human relevance of mouse models for the study of bile acid-related disease.113 The cytochrome P450 enzyme(s) (CYPs) responsible for 6-hydroxylation of bile acids were very recently identified.114 Correlative data had implicated members of the murine CYP3A family, particularly CYP3A11. However, analysis of knockout mice lacking CYP3A enzymes or all members of the CYP1A, CYP2C, CYP2D, or CYP3A families found that members of the murine CYP2C family, including at least CYP2C70, are required and directly mediate the first step in

6-hydroxylation of chenodeoxycholic acid to alpha-­ muricholic acid and ursodeoxycholic acid to beta-­muricholic acid.114,115 In contrast, the major human CYP2C enzyme, CYP2C9, was unable to oxidize bile acids, in agreement with the finding that 6-hydroxylated bile acid species are absent in humans under physiological conditions.114 The major bile acid biosynthetic pathways are summarized in Fig. 41.2. After their biosynthesis, bile acids are conjugated via a two-step process involving the generation of a bile acid-CoA by bile acid-CoA synthase and then amidation with taurine

FIG. 41.2  Bile acid synthesis pathways. Primary bile acids are synthesized by the hepatocyte. The major classical (neutral) pathway for bile acid synthesis begins with cholesterol 7α-hydroxylase (CYP7A1). Bile acid intermediates synthesized via this pathway are substrates for the sterol 12α-hydroxylase (CYP8B1), the rate-determining step in the production of cholic acid. In the minor alternative (acidic) pathway for bile acid synthesis, cholesterol is first hydroxylated on its side chain by sterol 27-hydroxylase (CYP27A1), sterol 25-hydroxylase, or sterol 24-hydroxylase (CYP46A1). Subsequent hydroxylation of the steroid nucleus is catalyzed by oxysterol 7α-hydroxylase (CYP7B1) or to a lesser extent by the distinct oxysterol 7α-hydroxylase, CYP39A1. The classical and alternative pathways converge at the enzymatic steps for the reduction and dehydrogenation of the steroid ring. The alternative pathway preferentially produces chenodeoxycholic acid. In mice, ursodeoxycholic acid is also synthesized as a primary bile acid in the liver. The cytochrome P450 (CYP2C70) then converts chenodeoxycholic acid and ursodeoxycholic acid to alpha-muricholic acid and beta-muricholic acid, respectively. After side chain oxidation and cleavage, bile acids are aminoacyl amidated to taurine or glycine. (Adapted with permission from Chiang JY. Bile acid metabolism and signaling. Compr Physiol 2013;3(3):1191–1212.)

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or glycine by bile acid-CoA: amino acid N-acyltransferase (BAAT).116 In most nonmammalian vertebrate species, bile acids are typically modified on their side by sulfation (for C27 bile alcohols) or conjugation with taurine or a taurine derivative (for C27 and C24 bile acids).4 In mammals, bile acids are primarily conjugated on their side chain to either taurine or glycine.4,6,117 Notably, the conjugation pattern varies considerably between different mammalian species, ranging from almost exclusively taurine in the rat, cat, mouse, sheep, and dog, to mostly glycine in the pig, hamster, guinea pig, and human, to exclusively glycine in the rabbit.4,6 The amino acid specificity for the conjugation of bile acids is controlled by the BAAT enzyme,118,119 and to a lesser degree, by the availability of the taurine precursor. However, the evolutionary forces driving the selection of a particular amino acid in different animal species are unclear. Taurine conjugated bile acids have a lower pKa than their respective glycine conjugates and are more likely to remain ionized and membrane impermeant. However, both the glycine and taurine amide linkages are more resistant to hydrolysis by the pancreatic carboxypeptidases, as compared to other amino acids. As such, both taurine and glycine-conjugated bile acids largely escape cleavage by host proteases in the intestinal lumen during the digestive process.120 Of the two major biosynthetic pathways, the classical (CYP7A1) pathway is quantitatively more important in rodents and humans.93 In mice, the classical pathway accounts for ~70% of the total bile acid synthesis in adults, and is the predominant pathway in neonates.121–123 In humans, the classical pathway accounts for more than 90% of bile acid synthesis, as evidenced by approximately 96% reduction in fecal bile acid excretion in an adult patient with an inherited CYP7A1 defect.96 In contrast to mice and adult humans,124 the alternative pathway is the predominant biosynthetic pathway in human neonates, as evidenced by low to undetectable CYP7A1 expression in newborns and the finding of severe cholestatic liver disease in infants with inherited oxysterol 7α-hydroxylase (CYP7B1) gene defects.125–127

41.4.2  Regulation of Bile Acid Biosynthesis Bile acid biosynthesis is regulated by bile acids, hormones, cytokines, growth factors, oxysterols, xenobiotics, and diurnal rhythm,39,102,128 reflecting the need to tightly control the body's bile acid load. It was recognized for many years that feedback inhibition of the rate-limiting enzyme CYP7A1 plays a major role in controlling bile acid biosynthesis.129,130 The major mechanism responsible for the regulation of CYP7A1 expression and bile acid synthesis have been elucidated over the past decade and are summarized in Fig. 41.3.

The major pathway for feedback regulation of bile acid synthesis involves FXR131–133 and gut-liver signaling via the endocrine polypeptide hormone FGF 19 (mouse ortholog: FGF15).65,66 In this pathway, bile acids activate FXR in ileal enterocytes to induce synthesis of FGF15/19. After its release by the enterocyte, FGF15/19 travels in the portal circulation to the hepatocyte where it signals via its cell surface receptor, a complex of the βKlotho protein and fibroblast growth factor receptor-4 (FGFR4), to repress CYP7A1 expression and bile acid synthesis.65,66,134 The dominant role of this pathway as the major physiological mechanism responsible for feedback repression of CYP7A1 expression is strongly supported by results obtained using knockout mouse models, including FGFR4, β-Klotho, FGF15, and tissue-specific FXR-null mice.62,65,135–137 Moreover, identification of this pathway helped to explain a series of puzzling experimental findings, which included the observation that intravenous infusion of bile acids into the bile-fistula rat was ineffective at downregulating hepatic bile acid synthesis as compared to intraduodenal infusion of bile acids138 and that bile acids were relatively weak inhibitors of bile acid synthesis when added directly to isolated hepatocytes in culture.139,140 This regulatory pathway appears to be conserved in humans and nonhuman primates, since circulating FGF19 levels inversely correlate with markers of hepatic bile acid biosynthesis, administration of inhibitory anti-FGFR4 antibodies stimulated bile acid synthesis in nonhuman primates, and administration of recombinant FGF19 to human subjects strongly repressed bile acid synthesis.66,141,142 After binding FGF15/19, FGFR4/β-Klotho signals through the docking protein fibroblast growth factor substrate 2 (FRS2α) and tyrosine-protein phosphatase nonreceptor type 11 (Shp2; gene symbol: PTPN11). Activation of Shp2 stimulates extracellular-signal-regulated kinase (ERK1/2) activity and blocks the activation of CYP7A1 gene expression by hepatic nuclear factor 4-alpha (HNF4α) and liver receptor homolog-1 (LRH1).143–147 Bile acids also regulate the expression of CYP8B1 by similar, but not identical pathways.62 The regulation of both CYP7A1 and CYP8B1 by bile acids and FGF15/19 appears to involve the orphan nuclear receptor, small heterodimer partner (SHP; gene symbol: NR0B2). For example: (1) SHP can antagonize LRH-1 or HNF4α-mediated activation of CYP7A1 and CYP8B1 expression, and (2) FGF15/19-mediated regulation of CYP7A1 and CYP8B1 is blunted in SHP null mice.62,65,137,148 Finally, it was very recently shown that FXR indirectly represses expression of other genes involved in bile acid biosynthesis (but not CYP7A1) by inducing expression of the transcriptional repressor v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), which interacts directly with the promoters of those genes.61 This complex network of receptors and

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FIG. 41.3  Mechanisms responsible for feedback negative regulation of hepatic bile acid synthesis. In the major physiological pathway, intestinal bile acids are taken up by the ASBT, and activate FXR to induce FGF15/19 expression in ileal enterocytes. The basolateral secretion of FGF15/19 protein may be facilitated by the endosomal membrane glycoprotein, Diet1. FGF15/19 is then carried in the portal circulation to the liver where it binds to its cell surface receptor, a complex of the receptor tyrosine kinase, FGFR4 and the associated protein β-Klotho. FGFR4/β-Klotho then signals through the docking protein FRS2α and the tyrosine phosphatase Shp2 to stimulate ERK1/2 phosphorylation and block activation of CYP7A1 gene expression by the nuclear factors HNF4α and LRH1. In a direct pathway that may be more significant under pathophysiological conditions, bile acids can activate FXR in hepatocytes to induce expression of SHP, an atypical orphan nuclear receptor. The SHP interacts with LRH-1 and HNF4α to block activation of CYP7A1. (Adapted with permission from Dawson PA. Bile acid metabolism. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier B.V.; 2015. p. 359.)

regulatory factors links the control of bile acid synthesis and bile acid composition to changes in ileal as well as hepatic bile acid levels. The receptors and protein factors that participate in the negative feedback regulation of bile acid synthesis are summarized in Table 41.2. With regard to the alternative bile acid biosynthetic pathway, bile acids can signal via FXR-MAFG to repress expression of important biosynthetic genes such as CYP27A1 and CYP7B1.61 However, the major mechanism for control of the alternative pathway appears to be posttranscriptional and involve the regulation of cholesterol delivery to the mitochondrial inner membrane, the site of cholesterol 27-hydroxylation.149,150 The process is thought to be similar to that for steroid hormone biosynthesis in adrenal gland, where steroidogenic acute regulatory protein D1 (StARD1)mediated transfer of cholesterol to the mitochondria inner

membrane is rate-limiting step.151 The liver expresses several different StAR-related lipid transfer (START) domaincontaining proteins in addition to StARD1,152 and StARD5 has been shown to directly bind primary bile acids.153 However, details of the molecular mechanisms underlying cholesterol trafficking, intramitochondrial cholesterol delivery, and their regulatory consequences for bile acid biosynthesis in hepatocytes still remains poorly understood.154

41.4.3  Biotransformation of Bile Acids During Enterohepatic Cycling Under physiological conditions, essentially all bile acids secreted into bile are conjugated. Conjugation with taurine or glycine increases the hydrophilicity of bile acids and the acidic strength of the side chain, in essence converting a

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TABLE 41.2  Regulation of Bile Acid Synthesis and Enterohepatic Cycling Protein (Gene)

Tissue

Description and Function in Bile Acid Metabolism

FXR (NR1H4)

Intestine, liver, kidney

Bile acid-activated nuclear receptor; regulation of bile acid synthesis, transport, and metabolism

SHP (NR0B2)

Liver, intestine

Nuclear receptor; negative feedback regulation of hepatic bile acid synthesis by antagonizing HNF4a, LRH1; regulation of bile acid transport and metabolism

HNF4a (NR2a1)

Liver, intestine

Nuclear receptor; positive regulator of CYP7A1 expression and hepatic bile acid synthesis

LRH1 (NR5A2)

Liver, intestine

Nuclear receptor; positive regulator of CYP7A1 expression and hepatic bile acid synthesis

PXR (NR1I2)

Liver, intestine

Bile acid and xenobiotic-activated nuclear receptor involved in detoxification of secondary bile acids

VDR (NR1I1)

Intestine

Vitamin D and bile acid-activated nuclear receptor; involved in detoxification of LCA

FGFR4 (FGFR4)

Ubiquitous

Membrane receptor; negative feedback regulation of CYP7A1 and hepatic bile acid synthesis

β-klotho (KLB)

Liver

Membrane co-receptor associated with FGFR4; confers liver specificity to FGFR4-FGF19 pathway; negative feedback regulation of Cyp7a1 and hepatic bile acid synthesis

FGF19 (FGF19)

Intestine

Protein growth factor; secreted by intestine in response to bile acids; regulates hepatic bile acid synthesis via FGFR4:b-klotho

TGR5

Ubiquitous

Bile acid-activated G-protein coupled receptor; regulates intestinal motility, metabolism

MafG

Ubiquitous

Transcription factor; negative regulation of hepatic bile acid synthesis and bile acid transport

FXR, farnesoid X-receptor; FGF19, fibroblast growth factor 19; FGFR4, fibroblast growth factor receptor 4; HNF4a, hepatocyte nuclear factor 4alpha; PXR, pregnane X-receptor; SHP, small heterodimer partner; VDR, vitamin D receptor.

weak acid (pKa > 5.0) to a strong acid (pKa ~3.9 for the glycine conjugate; pKa <2.0 for the taurine conjugate).155–157 As a result, conjugated bile acids are almost completely ionized under the pH conditions present in the lumen of the biliary tract and small intestine. The physiological consequence of conjugation is to decrease the passive diffusion of bile acids across cell membranes during their transit through the biliary tree and small intestine.158 Conjugated bile acids are also more soluble at acidic pH and more resistant to precipitation in the presence of high concentrations of calcium than unconjugated species.32 The net effect of conjugation is to maintain high intralumenal concentrations of bile acids to solubilize cholesterol and fat-soluble vitamins, and to facilitate lipid digestion and absorption down the length of the small intestine. The importance of bile acid conjugation is underscored by the finding that patients with inherited bile acid conjugation defects present with malabsorption of dietary triglyceride and fat-soluble vitamins.17,18,159,160 Most of the conjugated bile acids secreted into the small intestine are efficiently absorbed intact. However, a fraction of the bile acids undergoes deconjugation (cleavage of the amide bond linking the glycine or taurine to the bile acid side chain) and biotransformation by the gut microbiota.3,92 The bacterial modifications of primary bile acids are important for several reasons. First, these modifications increase the hydrophobicity and decrease the aqueous solubility of bile acids, resulting in a marked lowering of the monomeric concentration of bile acids in aqueous ­solution.156,161 This

in turn reduces the flux of bile acids across the ileal or colonic epithelium and increases bile acid loss in the feces. Second, the composition of the circulating pool of bile acids is influenced by the input of secondary bile acids from the ileum and colon. Notably, these secondary bile acids have detergent properties, signaling activities, and toxicities that are distinct from their primary bile acid precursors.13,162,163 Third, the bile acid composition affects the diversity and composition of the gut microbiome, which can have pleiotropic metabolic and physiologic effects.27 In the small intestine, unconjugated bile acids are passively or actively absorbed and returned to the liver. After hepatocellular uptake, the unconjugated bile acids are efficiently reconjugated to taurine or glycine and resecreted into bile, a process termed “bile acid repair” by Hofmann.10 In the colon, gut microbial deconjugation of bile acids may proceed to near completion prior to being excreted in the feces.164 Bile acid deconjugation (removal of the glycine of taurine N-acyl-amidation) by bacterial bile salt hydrolases is a “gateway reaction” and precedes subsequent biotransformation by the gut microbiota.165,166 In particular, bile acids must be deconjugated prior to the removal of the C-7 hydroxy group because the first step in that process is the formation of a coenzyme A derivative, which requires a weak acid such as the unconjugated bile acid side chain.167 As such, deconjugationresistant bile acid analogs such as cholylsarcosine are also resistant to dehydroxylation.168 Bacterial 7α-dehydroxylation converts cholic acid to deoxycholic acid, a dihydroxy bile

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acid with hydroxyl groups at the C-3 and C-12 positions, and chenodeoxycholic acid to lithocholic acid, a monohydroxy bile acid with a hydroxyl group at the C-3 position167 (see Fig. 41.4). In rats and mice, the 3,6,7-trihydroxy bile acids (α, β, ω-muricholic acid; hyocholic acid) are converted to the 3,6-dihydroxy bile acids, hyodeoxycholic acid (3α,6αdihydroxy-5β-cholanoic acid), and murideoxycholic acid (3α,6β-dihydroxy-5β-cholanoic acid).169 Besides undergoing 7-dehydroxylation during colonic transit, the hydroxy groups on the steroid nucleus may be modified by dehydrogenation, epimerization, or even elimination to form an unsaturated bile acid (with a double bond in the steroid nucleus). One of the more common bacterial modifications is epimerization of the 3α-hydroxy or 7α-hydroxy groups to their corresponding 3βor 7β-hydroxy forms.164,166,170 For example, the 7α-hydroxy group of chenodeoxycholic acid is epimerized to form the 3α,7β-dihydroxy bile acid, ursodeoxycholic acid, and lithocholic acid and deoxycholic acid are epimerized to their 3β-hydroxy-epimers, isolithocholic acid and isodeoxycholic acid.164,170 Note that in addition to these reactions, other gut microbiota-mediated modifications of bile acids have been detected such as fatty acyl esterification and polymerization. However, the quantitative significance of these reactions and the biochemical pathways responsible for their synthesis remain largely unexplored.3 A fraction of the 7-deoxy bile acids are absorbed from the colon and returned to the liver, where they are efficiently reconjugated with glycine or taurine and potentially 7αrehydroxylated. Hepatic bile acid 7α-rehydroxylation activity varies considerably between species and the enzyme(s) responsible has not been identified.171,172 The interspecies variation in hepatic bile acid 7α-rehydroxylation is reflected in the biliary deoxycholate concentration, which is low (from 0% to 10% of bile acids) in species that actively 7α-rehydroxylate deoxycholate such as rats, mice, guinea pigs, prairie dogs, and hamsters,173–176 and higher in species that cannot rehydroxylate deoxycholate, ranging from 15% to 30% in dogs and humans177,178 to greater than 90% in rabbits.179 Finally, in addition to reconjugation with taurine/ glycine, hepatocytes can epimerize iso(3β-hydroxy) bile acids to their 3α-hydroxy form, reduce the oxo groups on bile acids to hydroxyl groups, and modify bile acids by sulfation (sulfonation) or to a lesser extent, by glucuronidation.180–182 The secondary metabolism of bile acids in humans and mice is summarized in Fig. 41.4.

41.5.  ENTEROHEPATIC CIRCULATION OF BILE ACIDS As mentioned in the introduction, the concept of an enterohepatic circulation dates back more than 300  years.10 Anatomically, the gut-liver circulation can be subdivided into a portal and an extra-portal pathway. The extra-portal pathway consists primarily of the lymphatic drainage from

the intestine into the superior vena cava. Although this process is important for chylomicron particle-mediated transport of cholesterol, triglycerides, fat-soluble vitamins, and phospholipids, it plays little role in bile acid absorption. Bile acids undergo a portal enterohepatic circulation, whereby they are: (1) secreted into bile by the liver, (2) pass into the duodenum, (3) absorbed from the intestinal lumen at the distal ileum, (4) pass into the portal circulation, and (5) efficiently extracted by the liver for resecretion into bile. The enterohepatic circulation of bile acids is an extremely efficient process; less than 5% of the intestinal bile acids escape reabsorption and are eliminated in the feces.183 Thus, most of the bile acids secreted by the hepatocyte were previously secreted into the small intestine and returned to the liver in the portal circulation. During fasting, about half the bile acid pool is sequestered and concentrated approximately 10-fold in the gallbladder, resulting in lower levels of bile acids in the small intestine, portal vein, liver, and serum. However, basal rates of hepatic bile acid secretion are still present and there is continuous enterohepatic cycling of that portion of the bile acid pool that is not sequestered in the gallbladder.184,185 The gallbladder empties its contents in response to a meal and causes the release of cholecystokinin, and the newly secreted plus stored bile acids pass directly into the duodenum. In the digestive phase, the bile acid concentration in the small intestine is approximately 5–10 mM. During the inter-digestive phase, the sphincter of Oddi contracts and the gallbladder relaxes, causing a larger fraction of the secreted bile acids to enter the gallbladder for storage. Interestingly, bile acids have a direct role in promoting gallbladder filling by signaling directly via TGR5 or by stimulating ileal synthesis and release of FGF15/19, a polypeptide hormone that induces gallbladder relaxation.85,186 Thus, the enterohepatic cycling of bile acids increases during digestion and slows between meals and during overnight fasting. This rhythm is maintained even after cholecystectomy, where the fraction of the bile acids stored in proximal intestine is increased but bile acid metabolism and enterohepatic cycling is largely intact.187–189 A fraction (10%–50%, depending on the bile acid species) of the bile acids returning in the portal circulation escapes first pass hepatic extraction and spills into the systemic circulation. Bile acid binding to plasma proteins such as albumin reduces their glomerular filtration and minimizes their urinary excretion. In healthy humans, the kidney filters approximately 100 μmol of bile acids each day, but only 1–2 μmol of bile acid are excreted in urine because of a highly efficient tubular reabsorption.190 Even in patients with cholestatic liver disease, in whom plasma bile acid concentrations are greatly elevated, the 24-h urinary excretion of nonsulfated bile acids is significantly less than the quantity that undergoes glomerular filtration.191 Subsequent studies showed that bile acids in the glomerular filtrate are actively reabsorbed from the renal tubules by a

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FIG. 41.4  Biotransformation of bile acids by the gut microbiome. Gut bacterial metabolism includes deconjugation, 7-dehydroxylation, oxidation, and epimerization of bile acids. Major reactions are indicated in bold. Reversible reactions are indicated by the double arrows. Oxidation yields the respective oxo-bile acid species, which can be epimerized, converting the 7α-hydroxy group to 7β-hydroxyepimers (ursocholic acid, ursodeoxycholic acid), the 6β-hydroxy group to 6α-hydroxyepimers (hyodeoxycholic acid), and the 3α-hydroxy group to 3β-hydroxyepimers (isocholic acid, isodeoxycholic acid, isochenodeoxycholic acid, and isolithocholic acid). In human cecum, a significant fraction of the bile acids present are converted to their respective 3βepimers (iso-bile acids). In rats and mice, muricholic acid species (α, β, ω-muricholic acid) are converted to the 3,6-dihydroxy bile acids, murideoxycholic acid (3α,6β-dihydroxy-5β-cholanoic acid) and hyodeoxycholic acid (3α,6α-dihydroxy-5β-cholanoic acid). (Adapted with permission from Dawson PA. Bile acid metabolism. Biochemistry of lipids, lipoproteins and membranes. Amsterdam: Elsevier B.V.; 2015. p. 359.)

sodium-dependent mechanism.192,193 As in the ileum, the renal proximal tubule epithelium expresses the apical sodiumdependent bile acid transporter (ASBT; also called ileal bile acid transporter, IBAT; gene symbol: SLC10A2) as a salvage mechanism to conserve bile acids.194,195 In addition to expressing the ASBT on the apical surface, renal epithelial cells express OSTα-OSTβ196 on the basolateral membrane, thereby completing the route for bile acids to be taken up from the tubule lumen and exported into the systemic circulation.

41.6.  BILE SECRETION AND HEPATIC BILE ACID TRANSPORT 41.6.1  Overview of Bile Secretion The formation of canalicular bile is an osmotic process driven by active secretion of organic solutes into the canalicular lumen, followed by passive inflow of water, electrolytes, and other solutes.1,197 Canalicular bile flow is traditionally

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divided into two components: bile acid-dependent and bile acid-independent flow.198 Solutes such as conjugated bile acids that are actively pumped across the canalicular membrane generate bile flow and are termed primary solutes. Other primary solutes include conjugated bilirubin, glutathione, bicarbonate, and the glucuronide or sulfate conjugates of endobiotics and xenobiotics. Water, plasma electrolytes, calcium, glucose, amino acids, and other low-molecularweight solutes that flow passively into the canaliculus in response to the osmotic gradient are termed secondary solutes. The choleretic activity of each primary solute is defined as the volume of bile flow induced per amount of solute secreted. The apparent choleretic activity for different conjugated bile acid species ranges from 8 to 25 μL of bile flow induced per μmol of bile acid secreted.199 This activity is also influenced by the osmotic properties of mixed micelles in bile, as well as the permeability of paracellular junctions to other solutes that enter canalicular bile by solvent drag.200 In addition, certain unconjugated bile acids such as ursodeoxycholic acid or C23 side chain-shortened bile acid analogs such as nor-ursodeoxycholic acid can induce a bicarbonaterich “hypercholeresis,” which is defined as bile flow greater than can be explained simply by the bile acid osmotic effects.201,202 In that proposed mechanism, unconjugated bile acids in bile becomes pronated and are passively absorbed by the biliary epithelial cells (cholangiocytes). The unconjugated bile acid is then exported from the biliary epithelium into the venous drainage of the biliary tract, which empties into the portal vein or directly enters the liver, thereby returning the bile acids to hepatocytes for uptake and resecretion into bile.201,203 This “cholehepatic shunt pathway” carries a proton each time a molecule of bile acid is absorbed, thereby generating an additional bicarbonate ion from biliary carbon dioxide per cycle. The majority of bile flow is bile acid dependent in humans, whereas most of the bile flow in rodents is induced by the secretion of other anions.200,204 For example, the bile acid-dependent and bile acid-independent flow in rats has been estimated to be approximately 50 μL/kg-min and 70 μL/kg-min, respectively.200,204 Mouse models with a genetic defect in hepatic bile acid secretion exhibit relatively normal levels of bile flow.205 In contrast, a similar defect in humans is associated with significantly impaired bile flow (cholestasis).206 However, even in humans, secretion of other primary solutes by the hepatocyte and biliary epithelium contributes significantly to bile formation. Hepatobiliary excretion of reduced glutathione (GSH) and bicarbonate (HCO3 - ) constitute major components of the bile acid-independent fraction of bile flow.207,208 The ATP-dependent canalicular secretion of GSH via the multidrug resistance-associated protein-2 (MRP2; gene symbol: ABCC2) plays a particularly important role.209 Besides the ATP-dependent secretion of organic anions into bile, hepatic and biliary ATP-independent secretion

of bicarbonate via the HCO3 - /Cl− anion exchanger AE2 contributes to the bile acid-independent bile flow.210 The majority of this HCO3 - secretion is mediated by cholangiocytes lining the biliary tract211 in response to stimulation by a variety of hormones and neuropeptides such as secretin and vasoactive intestinal peptide.212 Biliary HCO3− secretion in humans far exceeds that of rodents and may be responsible for as much as 30% of total bile flow versus only 5%–10% in rodent models.200 In addition to secretion of HCO3 - , other ductular modifications to hepatic bile include the absorption of solutes such as glucose, amino acids, and bile acids, chloride secretion by CFTR and non-CFTR pathways, hydrolysis of GSH by γ-glutamylpeptidase, and movement of water through specific channels (aquaporins) and paracellularly.212,213

41.6.2  Overview of Hepatic Bile Acid Transport In the fasting state, bile acids are taken up predominantly by the periportal hepatocytes (the first hepatocytes of the liver acinus), whereas during feeding, more hepatocytes in the liver acinus participate in bile acid uptake.214,215 Conversely, perivenous (pericentral) hepatocytes are primarily responsible for bile acid synthesis.91 As a generalization, periportal hepatocytes absorb and secrete recirculating bile acids, whereas perivenous (pericentral) cells secrete predominantly newly synthesized bile acids. Hepatocellular uptake of bile acids occurs against an unfavorable electrochemical ion gradient and results in a 5–10-fold concentration gradient between the plasma filtrate present in the space of Disse and the hepatocyte cytosol.216 In lower vertebrates such as the little skate (Leucoraja erinacea), the uptake of bile acids (primarily the C27 bile alcohol sulfate: scymnol sulfate) is Na+ independent and mediated by a member of the organic anion transporting polypeptide (OATP) family.217,218 However, in higher vertebrates and all mammals studied to date, hepatocellular uptake of most conjugated bile acids under physiological conditions is Na+ dependent and mediated by the Na+-taurocholate cotransporting polypeptide (NTCP; gene symbol: SLC10A1).219 In contrast to conjugated bile acids, hepatocellular uptake of unconjugated bile acids is mediated by members of the OATP family with NTCP playing only a minor role.220,221 The transporters responsible for the enterohepatic circulation of bile acids have been identified and are shown in Fig. 41.5.3,222,223 After uptake, conjugated bile acids are shuttled across the hepatocyte to the canalicular membrane for secretion into bile; unconjugated bile acids follow a similar path after first undergoing N-acyl amidation (conjugation) to taurine or glycine.13 Although bile acid concentrations within the hepatocyte are presumed to be in the micromolar range, canalicular bile acid concentrations can be 1000-fold higher, necessitating active transport across the canalicu-

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FIG.  41.5  Enterohepatic circulation of bile acids showing the individual transport proteins in hepatocytes, ileocytes (ileal enterocytes), and renal proximal tubule cells. After their synthesis or reconjugation, taurine and glycine-conjugated bile acids (T/G-BA) are secreted into bile by the canalicular bile salt export pump (BSEP; gene symbol ABCB11). A fraction of the bile acids secreted into bile acids undergo cholehepatic shunting. In this pathway, conjugated bile acids are taken up by cholangiocytes via the apical sodium-dependent bile acid transporter (ASBT; gene symbol SLC10A2), and exported across the basolateral membrane via the heteromeric organic solute transporter OSTα-OSTβ (gene symbols: SLC51A-SLC51B), and possibly the multidrug resistance-associated protein-3 (MRP3; gene symbol ABCC3), for return to the hepatocyte. Ultimately, bile acids empty into the intestinal lumen. Conjugated bile acids are poorly absorbed in the proximal small intestine, but efficiently taken up by the ileal enterocytes via the ASBT. The bile acids bind to the ileal bile acid binding protein (IBABP; also called the ileal lipid binding protein, ILBP; gene symbol FABP6) in the cytosol, and are efficiently exported across the basolateral membrane into the portal circulation by OSTα-OSTβ. The multidrug resistance-associated protein-3 (MRP3; gene symbol ABCC3) is a minor contributor to basolateral export of native bile acids from the enterocyte, but may have a more significant role in export of any glucuronidated (U-BA) or sulfated (S-BA) bile acids that may be formed. Although most bile acids are absorbed in the small intestine, colonocytes express appreciable levels of MRP3 and OSTα-OSTβ. These carriers may serve to export unconjugated bile acids (BA) that were taken up by passive diffusion from the lumen of the colon. After their absorption from the intestine, bile acids travel back to the liver where they are cleared by the Na+-taurocholate cotransporting polypeptide (NTCP; gene symbol SLC10A1). Members of the organic anion transport protein family, OATP1B1 (gene symbol SLCO1B1) and OATP1B3 (gene symbol SLCO1B3) (Oatp1b2; gene symbol Slco1b2, in mice) also participate, and are particularly important for unconjugated bile acids. Under cholestatic conditions, unconjugated, conjugated, or modified (glucuronidated or sulfated) bile acids are effluxed across the basolateral (sinusoidal) membrane of the hepatocyte by OSTα-OSTβ, MRP3, or multidrug resistance-associated protein-4 (MRP4; gene symbol ABCC4) into the systemic circulation. Under normal physiological conditions, a fraction of the bile acid escapes first pass hepatic clearance enters the systemic circulation. The free bile acids are filtered by the renal glomerulus, efficiently reclaimed by the ASBT in the proximal tubules, and exported back into the systemic circulation, thereby minimizing their excretion in the urine. A fraction of the glucuronidated or sulfated bile acids can also be exported across the apical membrane by multidrug resistance-associated protein-2 (MRP2; gene symbol ABCC2). The tetrahydroxylated bile acids (H-BA) formed under pathophysiological conditions can be secreted into bile by the multidrug resistance protein (MDR1; gene symbol ABCB1; Mdr1a/b in mice). (Adapted with permission from Dawson PA, Hubbert ML, Rao A. Getting the mOST from OST: role of organic solute transporter, OSTalpha-OSTbeta, in bile acid and steroid metabolism. Biochim Biophys Acta 2010;1801(9):994–1004.)

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lar ­membrane via the ATP-dependent bile salt export pump (BSEP; gene symbol: ABCB11).224–226 A small amount of bile acid can be modified by the addition of sulfate or glucuronide, and is secreted into bile by other ABC transporters, primarily MRP2.227 Sulfation is particularly important for the monohydroxy bile acid, lithocholic acid. Under pathophysiological conditions, bile acids also undergo additional hydroxylation reactions in hepatocytes and these unusual tri- or tetrahydroxylated bile acid species are secreted into bile by MRP2228 and possibly by the breast cancer-related protein (BCRP; gene symbol: ABCG2)228 or P-glycoprotein (MDR1; gene symbol: ABCB1).229 Although sinusoidal membrane bile acid transport is overwhelming in the direction of uptake under normal physiological conditions, bile acids can also be effluxed from the interior of the hepatocyte into the space of Disse as a protective mechanism to reduce bile acid overload.230 This process may be analogous to the “hepatocyte-hopping” that was recently demonstrated for conjugated bilirubin and drugs.220 Under conditions when the bile acid load returning to periportal hepatocytes exceeds their canalicular bile acid export capacity, a fraction of the bile acids can be rerouted to sinusoidal blood by basolateral membrane efflux, and carried to more pericentral hepatocytes for reuptake and secretion into bile. This mechanism would dynamically recruit additional hepatocytes within the liver lobule for bile acid clearance and serve as an additional safeguard to protect the periportal hepatocytes from bile acid overload. The major transporters involved in hepatocyte sinusoidal bile acid efflux includes members of the MRP (ABCC) family of ATP-dependent transporters, MRP3231 and MRP4,232 and OSTα-OSTβ.233,234 MRP3 and MRP4 are expressed on the sinusoidal membrane of hepatocytes235,236 and have bile acid transport237 or glutathione-bile acid cotransport activity.238 The expression of these transporters is induced under cholestatic conditions to promote bile acid efflux,230,233,239 thus lowering the concentration in the hepatocyte and decreasing the likelihood of apoptosis or necrosis. The relative contribution of the different efflux transporters remains to be determined under different pathophysiological conditions. However, current results indicate especially significant roles for MRP4 and OSTα-OSTβ,232,234 whereas MRP3 may be more important for glucuronidated or sulfated bile acids.240 Overall, the increased expression of these transporters appears to be an important part of the adaptive response to conditions of bile acid overload, along with downregulation of the major hepatic bile acid uptake transporters, NTCP, and members of the OATP family.230

41.6.3  Bile Acid Uptake Across the Sinusoidal Membrane of the Hepatocyte The NTCP is the founding member of the SLC10 family of solute carrier proteins, which includes two bile acid ­transporters (SLC10A1/NTCP and SLC10A2/ASBT),

one steroid sulfate transporter (SLC10A6/SOAT), and four orphan carriers (SLC10A3, SLC10A4, SLC10A5, SLC10A7).241–243 The NTCP encodes a 349-amino acid (approximately 45 kDa) membrane glycoprotein that functions as an electrogenic sodium-solute cotransporter to mediate the uptake of all the major glycine/taurine-conjugated bile acids.244,245 Depending on the bile acid species and NTCP ortholog, unconjugated bile acids may be moderate or weak substrates,246 and sulfated bile acids appear to be only weakly transported.195 The NTCP accounts for most, if not all, hepatocellular sinusoidal membrane Na+dependent conjugated bile acid transport under physiological conditions in humans.247 The primary role of NTCP in the clearance of conjugated bile acids is strongly supported by recent genetic evidence in humans and mice.248–251 In 2015, a single pediatric patient with an isolated inherited NTCP deficiency and greatly elevated (25–100-fold) plasma levels of conjugated bile acids (hypercholanemia) was reported. In contrast to the inherited defects in bile acid synthesis, the patient exhibited no jaundice, itching (pruritus), fat malabsorption (steatorrhea), or obvious liver disease.248 Alternative transport mechanisms were unable to compensate for the loss of NTCP in this pediatric patient, but subsequent reports of additional NTCP-deficient adults with only mild hypercholanemia suggests that other transporters may contribute in a limited fashion to hepatocellular conjugated bile acid uptake.251 In contrast to humans, mice lacking NTCP maintain normal plasma levels of bile acids.250 This species difference is explained by efficient hepatic uptake of conjugated bile acids by mouse Oatp1a/ Oatp1b family members, including Oatp1a1.249 In addition to being the major hepatic bile acid uptake transporter, NTCP was recently identified as a cell surface receptor for binding of hepatitis B virus (HBV) and hepatitis delta virus (HDV).252 The HBV infection is initiated via lowaffinity binding of the HBV small surface protein to heparinsulfate proteoglycans, followed by high-affinity binding to a host receptor that allows for viral entry. The identity of the HBV entry receptor(s) had long remained a mystery, and a major breakthrough came in 2012 when Yan et  al. demonstrated that binding of HBV large surface protein pre-S1 domain myristoylated peptide (amino acids 2–48) to NTCP is essential for infection of human hepatocytes.252 This finding and subsequent analyses of NTCP-HBV interactions has led to rapid basic science and clinical advances in the field.253 In contrast to conjugated bile acids, the hepatic uptake of unconjugated bile acids is mediated primarily by an Na+independent mechanism.219,221,254 This Na+-independent bile acid transport involves several different OATPs, ­members of a gene family encoding 12 potential transmembrane domain proteins that share no sequence identity with the Na+-dependent bile acid transporters. The driving force for OATP-mediated organic anion uptake remains to be conclusively established, but may involve anion exchange

Bile Formation and the Enterohepatic Circulation Chapter | 41  945

or facilitative diffusion.255 The original Human Genome Organization (HUGO) Gene Nomenclature Committee designation for the supergene family of OATP solute carriers was SLC21A. However, confusion related to species differences led to the adoption of a species-independent classification and nomenclature system in 2004, designated OATP/ SLCO where OATP refers to the protein isoforms and SLCO (“SLC” = solute carrier, “O” = member of the OATP family) refers to the genes.256 The OATP/SLCO-type transporters include 11 human and 16 rat/mouse genes that fall within six subgroups.256,257 In humans, the liver expresses several different OATPs, including OATP1B1, OATP1B3, and OATP2B1. OATP1B1 (gene symbol: SLCO1B1; original protein name: OATP-C) and OATP1B3 (gene symbol: SLCO1B3; original protein name: OATP8) exhibit partially overlapping substrate specificities and account for the majority of hepatic Na+-independent bile acid clearance, as well as uptake of bilirubin glucuronides. OATP2B1 (gene symbol: SLCO2B1; original protein name OATP-B) does not transport bile acids, but functions in human liver along with OATP1B1 and OATP1B3 to transport organic anions and a variety of drugs or drug metabolites. Mice and rats encode only a single OATP1B ortholog (Oatp1b2; gene symbol: Slco1b2), and OATP1B1 and OATP1B3 arose in primates by duplication after divergence from rodents.256 Conversely, the human genome encodes only one member of the OATP1A family (OATP1A2), whereas the rodent genome includes multiple members (Oatp1a1, Oatp1a3, Oatp1a4, Oatp1a5, Oatp1a6) that arose from gene duplication and are clustered in the same chromosomal region (chromosome 6G2 in mouse). In mouse, several different bile acid-transporting Oatps are expressed on the hepatic sinusoidal membrane, including Oatp1a1, Oatp1a4, and Oatp1b2. Compelling genetic evidence comes from recent knockout mouse studies for a significant role of the Oatp1a/1b transporters, particularly Oatp1b2, in hepatic clearance of unconjugated bile acids,220,258 and these transporters participate in the clearance of conjugated bile acids when NTCP is inhibited.249 In humans, OATP1B1/ OATP1B3 plays a primary role in the hepatic clearance of unconjugated bile acids,259 and combined loss of the adjacent SLCO1B1 and SLCO1B3 genes on chromosome 12 causes Rotor Syndrome, a rare benign disorder characterized by elevated plasma levels of unconjugated bile acids and conjugated bilirubin.259

41.6.4  Canalicular Bile Acid Transport Since bile acids are potent detergents, their hepatocellular uptake and export must be carefully balanced to avoid intracellular accumulation and cytotoxicity. Regardless of their uptake mechanism, the returning as well as newly synthesized bile acids are shuttled to the canalicular membrane for secretion into bile by the ATP-dependent bile salt export pump (BSEP; gene symbol: ABCB11).225 BSEP's role as the

major canalicular bile acid efflux pump was confirmed with the identification of ABCB11 mutations in patients with progressive familial intrahepatic cholestasis (PFIC) type 2, a hepatic disorder characterized by biliary bile acid concentrations less than 1% of normal.206,260 When analyzed in transfected mammalian cells, BSEP transports conjugated as well as unconjugated bile acids,246 and in some species, such as humans but not rodents, BSEP also transports sulfated bile acids such as taurolithosulfocholate.261 However under physiological conditions, unconjugated bile acids are first conjugated to glycine or taurine prior to secretion, as evidenced by the low proportions of unconjugated bile acids typically found in bile (less than 5%). Although bile acids are the major physiological substrate, a variety of drugs such as cyclosporin, rifamycin, troglitazone, and glibenclamide also interact with BSEP as nonsubstrate inhibitors to impede bile acid export. This direct inhibition of BSEP is thought to be an important mechanism underlying druginduced hepatoxicity or cholestasis.225,262–264 The BSEP expression is induced when hepatocyte bile acid levels are elevated, such as following dietary challenge with bile acids,265 or under certain cholestatic conditions.266–268 This is due to a direct activation of the human and rodent BSEP gene expression by bile acids signaling via FXR.269–272 The induction of BSEP expression is not a universal property of all bile acid species and appears to correlate with FXR ligand specificity.48,273,274 In addition to transcriptional regulation, there is also posttranscriptional regulation of BSEP protein trafficking and localization to the canalicular membrane.225,275 This short-term regulation enables the hepatocyte to rapidly modulate bile acid secretion in response to bile acid load and to pathophysiological conditions such as cholestasis.276

41.6.5  Cholehepatic Shunt Pathway The cholehepatic shunt pathway was proposed to describe the cycle whereby unconjugated dihydroxy bile acids secreted into bile are passively absorbed by cholangiocytes lining the bile ducts, returned to the hepatocyte via the venous drainage, transported across the sinusoidal membrane, and resecreted into bile.201 Absorption of the protonated unconjugated bile acid molecule generates a bicarbonate anion, resulting in a bicarbonate-rich hypercholeresis (increased bile secretion).277 Cholehepatic shunting is important for the hypercholeresis induced by therapeutic doses of unconjugated C-24 dihydroxy bile acid ursodeoxycholic acid and C-23 bile acid analogs such as nor-ursodeoxycholic acid.201,278,279 At the doses administered, ursodeoxycholate exceeds the capacity for bile acid conjugation, whereas the side chain-shorted bile acid analog nor-ursodeoxycholic acid is resistant to conjugation with glycine or taurine, resulting in hepatic biliary secretion of the unconjugated bile acids at high concentrations.280,281 Because hepatic bile contains primarily conjugated bile acids, the contribution

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of cholehepatic shunting of unconjugated bile acids to bile flow was thought to be negligible under normal physiological conditions.201 However, the subsequent discovery that the biliary epithelium and gallbladder expresses the ASBT and OSTα-OSTβ provided a physiological mechanism for cholehepatic or cholecysthepatic shunting of ionized conjugated bile acids.282–286 In vivo data documenting cholehepatic or cholecysthepatic shunting for conjugated bile acids has been obtained using rat and mouse models,285,286 but the quantitative significance in humans or under different physiological or pathophysiological conditions still remains to be determined.287,288

41.7.  INTESTINAL ABSORPTION OF BILE ACIDS 41.7.1 Overview Bile acids are reclaimed through a combination of passive absorption in the proximal small intestine, active transport in the distal ileum, and passive absorption in the colon.3 In many species including humans, the bile acid pool includes glycine conjugates, unconjugated bile acids, and hydrophobic bile acid species.13 A fraction of the glycine conjugates and unconjugated bile acids are protonated at the acidic cell surface pH conditions of the intestinal mucosa and are absorbed by passive diffusion across the apical brush border membrane.289 Besides passive membrane diffusion, there is limited evidence of carrier-mediated transport of bile acids in the proximal small intestine of some species.290 In terminal ileum, the ASBT actively transports bile acids from the intestinal lumen across the apical brush border membrane.247,291 Bile acids are then shuttled to the basolateral membrane and effluxed into the portal circulation by OSTα-OSTβ.63 Several observations support the concept that terminal ileum is the major site of bile acid reabsorption in man and experimental animal models. These observations include the finding that there is little decrease in intraluminal bile acid concentration prior to the ileum292 and the appearance of bile acid malabsorption after ileal resection.38 Subsequent studies using in situ perfused intestinal segments to measure bile acid absorption289 demonstrated that ileal bile acid transport is a high capacity absorptive system sufficient to account for the biliary bile acid output. In mouse, bile acids are taurine-conjugated and hydrophilic,293 thereby limiting their nonionic diffusion. Inactivation of the ASBT in mice largely abolished intestinal bile acid absorption, suggesting that alternative uptake mechanisms are of only minor importance.294 Similarly in humans, inherited mutations in ASBT causes significant bile acid malabsorption.295 The general consensus from these studies was that ileal active transport is the major route for conjugated bile acid uptake, whereas passive or facilitative absorption down the length

of the small intestine is significant for unconjugated and some glycine-conjugated bile acids. However, the quantitative contribution of jejunal bile acid absorption in different species is still being debated.296 Little is known about intracellular transport of bile acids in the enterocyte. The ileal bile acid binding protein (IBABP, also called the ileal lipid binding protein, ILBP; gene symbol: FABP6) is a member of the fatty acid binding protein family and very abundant cytosolic protein in ileal enterocytes.297,298 The IBABP expression parallels that of the ASBT and is induced by bile acids acting through FXR.299 The IBABP preferentially binds bile acids, with a stoichiometry of two or three bile acids per molecular of IBABP and with highest affinity for taurine-conjugated bile acids versus glycine-conjugated and unconjugated species.300,301 It is likely that IBABP functions as an intracellular buffer to bind bile acids and facilitate their transcellular transport. In doing so, IBABP reduces bile acid-membrane interactions and protects the ileal enterocyte from bile acid cytotoxicity.302 Genetic evidence that IBABP plays a role in intestinal bile acid transport comes from studies of an IBABP-null mouse.303 In that model, apical to basolateral transport of taurocholate was reduced when analyzed using the inverted gut sac method of Wilson and Wiseman. In addition, bile acid metabolism was altered suggesting that IBABP may also be involved in enterocyte bile acid sensing.

41.7.2  Ileal Na+-Dependent Bile Acid Uptake Bile acids are actively transported across the ileal brush border membrane by the apical sodium-dependent bile acid transporter (ASBT).195,295 The ASBT transports all major species of bile acids, but favors trihydroxy over dihydroxy bile acids, and conjugated over unconjugated species.195 The intestinal absorption efficiency for an individual bile acid species depends on the affinity of ASBT and the length of time the bile acid is exposed to ASBT-expressing epithelium. As such, the substrate specificity and gradient of ASBT intestinal expression are important determinants of the bile acid pool composition,304 along with the hepatic expression of 12α-hydroxylase (CYP8B1) and the gut microbiota flora (levels of 7α-dehydroxlating bacteria). The properties of the ASBT satisfied all the functional criteria for ileal active bile acid uptake, including: (1) a strict sodium dependence for bile acid transport195; (2) specific transport of all the major species of bile acids with negligible uptake of nonbile acid solutes195,245; (3) specific intestinal expression in the terminal ileum; (4) similar ontogeny for ileal sodium-dependent taurocholate uptake and ASBT expression at fetal day 22 and postnatal day 17305,306; (5) targeted inactivation of the ASBT gene eliminates enterohepatic cycling of bile acids in mice294; (6) loss-offunction mutations in the human ASBT gene are associated

Bile Formation and the Enterohepatic Circulation Chapter | 41  947

with intestinal bile acid malabsorption.295 In addition to the ileal enterocyte, the ASBT is expressed in other tissues that serve to facilitate the enterohepatic circulation of bile acids, including the apical membrane of proximal renal convoluted tubule cells, large cholangiocytes, and gallbladder epithelial cells.247 Considering its central role in the enterohepatic circulation, inherited defects or dysfunctional regulation of the ASBT may play a role in the pathogenesis of a number of gastrointestinal disorders. For example, ASBT mutations were identified as a cause of Primary Bile Acid Malabsorption, a rare idiopathic disorder associated with chronic diarrhea beginning in early infancy, steatorrhea, interruption of the enterohepatic circulation of bile acids, and reduced plasma cholesterol levels.295 Other disorders associated with intestinal bile acid malabsorption that could potentially involve the ASBT in their pathogenesis or clinical phenotype include hypertriglyceridemia,307,308 idiopathic chronic diarrhea,309 chronic ileitis,310 gallstone disease,311,312 postcholecystectomy diarrhea,313 and irritable bowel syndrome.314

41.7.3  Ileal Basolateral Bile Acid Transport The unusual heteromeric transporter, OSTα-OSTβ was identified as the carrier responsible for bile acid export across the basolateral membrane of the ileal enterocyte, cholangiocytes, and renal proximal tubule cell.63 The breakthrough in this area came with the elegant expression cloning of an unusual transporter, OSTα-OSTβ, from the little skate (Raja erinacea)315 followed by cloning of the mammalian orthologs.316 In contrast to the previously identified bile acid carriers, co-expression of two distinct subunits: OSTα, a 340 amino acid polytopic membrane protein, and OSTβ, a 128 amino acid predicted type Ib membrane protein, was required for bile acid transport. Functional studies of the individual subunits to date indicate that co-expression and assembly of both subunits into a complex is required for their trafficking to the plasma membrane and solute transport activity.317–320 Soon after their cloning, OSTα-OSTβ was identified as a candidate ileal basolateral bile acid transporter using a transcriptional profiling approach.317 Support for a role of OSTα-OSTβ in basolateral bile acid transport includes: (1) intestinal expression of OSTα and OSTβ mRNA that generally follows that of the ASBT, with highest levels in ileum,196,317,321 (2) appropriate cellular localization on the lateral and basal membranes of ileal enterocyte,317 (3) expression of OSTα-OSTβ on the basolateral membrane of hepatocytes, cholangiocytes, and renal proximal tubule cells, other membranes exhibiting bile acid efflux,196 (4) efficient transport of the major bile acid species,196,317 (5) positive regulation of OSTα-OSTβ expression by bile acids acting via FXR,64,322 (6) targeted inactivation of the OSTα gene in mice resulted in impaired intestinal bile acid absorption and

altered bile acid metabolism,323–325 and (7) inherited mutations in the human OSTβ gene (SLC51B) cause congenital chronic diarrhea, a phenotype similar to that observed in patients with ASBT mutations.326

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