Bile Formation and the Enterohepatic Circulation




Abstract


Hepatic bile formation serves a number of important functions such as critical roles in the excretion of lipid-soluble xenobiotics and endobiotics, intestinal lipid digestion and absorption, and cholesterol homeostasis. The production of bile is dependent on the hepatic synthesis, transport, and canalicular secretion of bile acids, the predominant organic anions in bile. The process also requires an intact enterohepatic circulation since the majority of hepatic bile acids have undergone intestinal reabsorption. Hepatic and gastrointestinal physiology can be profoundly affected by disturbances in bile acid biosynthesis, biliary secretion, and enterohepatic cycling. This chapter will review the physiology of bile formation and the enterohepatic circulation of bile acids, with particular emphasis on the transporters and signaling pathways involved.




Keywords

Bile acids, Transporters, Nuclear receptors, Liver, Intestine

 





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. 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. 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.





Structure of 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 water- insoluble, hydrophobic membrane lipid is converted into water-soluble derivatives that can be excreted in bile. 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) ( www.lipidmaps.org ) of the LIPID MAPS Lipid Classification System. 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. Bile acids are not known to be made by invertebrates. In vertebrates, bile acids show a remarkable 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. In vivo, bile acids exist primarily as sulfate conjugates of bile alcohols and as taurine (or glycine) aminoacyl-amidated conjugates of bile acids. 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 .




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.)





Major Functions of Bile and Bile Acids


Bile formation and secretion is essential for life and fulfills a number of important functions in vertebrates. (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. (3) Bile secretion plays a role in innate immunity and controlling intestinal microbes by serving as a conduit for the release of IgA antibodies. (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 .



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

Biliary tract—cholangiocytes


  • Stimulation of bicarbonate secretion via CFTR and AE2



  • Promotion of proliferation when bile duct is obstructed

Gallbladder epithelium


  • Modulation of cAMP-mediated secretion



  • Promotion of mucin secretion

Small intestine—lumen


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



  • 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



  • Activation of TGR5 on enteroendocrine L-cells

Gut microbiota


  • Regulate microbial diversity and metabolism


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


The major functions of bile acids include: (1) Inducing bile flow and hepatic secretion of biliary lipids (phospholipid 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. 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. 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. (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. 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. 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. (5) Bile acids regulate gut microbial diversity and vice versa under physiological and pathophysiological conditions. (6) Bile acids act to prevent the formation of calcium gallstones and calcium oxalate kidney stones. 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. 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. 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. (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.



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-protein-coupled receptors (Takeda G-protein-coupled receptor, TGR5; muscarinic receptors; sphingosine-1-phosphate receptor 2, S1PR2), integrins (α5,β1-integrin), and cell- signaling 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), 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 growth-modulatory effects. 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 ). 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 pseudogene in humans and primates. 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. There are four major isoforms of FXR in mice and humans, which are generated through the use of alternative promoters and alternative splicing. 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. 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. 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. 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), and the endocrine polypeptide hormone fibroblast growth factor (FGF) 19 (mouse ortholog: FGF15), a central regulator of hepatic bile acid synthesis. With regard to general functions in the gastrointestinal tract, FXR induces expression of genes important for intestinal barrier function and antimicrobial defense, and has important antiproliferative and antiinflammatory properties.


Bile acids also signal via the nuclear receptors PXR (gene symbol: NR1I2 ) and VDR (gene symbol: NR1I1 ). 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, and likely play only a minor role in regulating bile acid biosynthesis.


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 symbol: GPBAR1 ) as a G αs -coupled bile acid receptor, which stimulates adenylate cyclase and increases intracellular cAMP levels. TGR5 is activated by conjugated and unconjugated bile acids, with the following rank order of potency: deoxycholic acid > lithocholic acid > chenodeoxycholic acid > cholic acid. 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. 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. With the growing appreciation of bile acids as signaling molecules, considerable study is being directed toward understanding the physiological functions of TGR5. 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. There are also metabolic effects associated with TGR5 signaling in brown adipose, muscle, and macrophages.





Biosynthesis and Biotransformation of Bile Acids



Biosynthesis of Bile Acids


Bile acids are synthesized from cholesterol in the pericentral hepatocytes of the hepatic acini. In this process, the hydrophobic substrate, cholesterol, is converted to a water-soluble, 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. Bile acid synthesis was originally thought to involve one major pathway, the “classical” or neutral pathway (cholesterol 7α-hydroxylase pathway) that favors cholic acid biosynthesis. 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. 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. 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 25- hydroxylase; 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 7α-hydroxylation, which is mediated primarily by CYP7B1 in liver. Of these alternative hydroxylation pathways, the contribution of 27-hydroxycholesterol to bile acid synthesis is quantitatively most important. 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 blood-brain barrier into the systemic circulation for excretion by the liver.


The overall process of bile acid biosynthesis is complex, requiring the action of 16 enzymes that catalyze as many as 17 different reactions. 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 12α-hydroxylase (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. 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. 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. It has long been known that 6- hydroxylation of bile acids alters their physicochemical and detergent properties, with mice having a more hydrophilic bile acid pool. 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 signaling properties, potentially limiting the human relevance of mouse models for the study of bile acid-related disease. The cytochrome P450 enzyme(s) (CYPs) responsible for 6-hydroxylation of bile acids were very recently identified. 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. 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. The major bile acid biosynthetic pathways are summarized in Fig. 41.2 .




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.)


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 or glycine by bile acid-CoA: amino acid N-acyltransferase (BAAT). 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). In mammals, bile acids are primarily conjugated on their side chain to either taurine or glycine. 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. The amino acid specificity for the conjugation of bile acids is controlled by the BAAT enzyme, 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 p K a 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.


Of the two major biosynthetic pathways, the classical (CYP7A1) pathway is quantitatively more important in rodents and humans. In mice, the classical pathway accounts for ~ 70% of the total bile acid synthesis in adults, and is the predominant pathway in neonates. 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. In contrast to mice and adult humans, 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.



Regulation of Bile Acid Biosynthesis


Bile acid biosynthesis is regulated by bile acids, hormones, cytokines, growth factors, oxysterols, xenobiotics, and diurnal rhythm, 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. 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 .




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.)


The major pathway for feedback regulation of bile acid synthesis involves FXR and gut-liver signaling via the endocrine polypeptide hormone FGF 19 (mouse ortholog: FGF15). 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 β-Klotho protein and fibroblast growth factor receptor-4 (FGFR4), to repress CYP7A1 expression and bile acid synthesis. 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. 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 acids and that bile acids were relatively weak inhibitors of bile acid synthesis when added directly to isolated hepatocytes in culture. 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. 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). Bile acids also regulate the expression of CYP8B1 by similar, but not identical pathways. 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. 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. This complex network of receptors and 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 .



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.


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. 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. 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. The liver expresses several different StAR-related lipid transfer (START) domain-containing proteins in addition to StARD1, and StARD5 has been shown to directly bind primary bile acids. 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.



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 weak acid (p K a > 5.0) to a strong acid (p K a ~ 3.9 for the glycine conjugate; p K a < 2.0 for the taurine conjugate). 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. 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. 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.


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. 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. 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. Third, the bile acid composition affects the diversity and composition of the gut microbiome, which can have pleiotropic metabolic and physiologic effects. 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. In the colon, gut microbial deconjugation of bile acids may proceed to near completion prior to being excreted in the feces.


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. 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. As such, deconjugation-resistant bile acid analogs such as cholylsarcosine are also resistant to dehydroxylation. Bacterial 7α-dehydroxylation converts cholic acid to deoxycholic acid, a dihydroxy bile 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 position (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,6α-dihydroxy-5β-cholanoic acid), and murideoxycholic acid (3α,6β-dihydroxy-5β-cholanoic acid). 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 3β- or 7β-hydroxy forms. 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. 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.




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 3β-epimers (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.)


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 7α-rehydroxylated. Hepatic bile acid 7α-rehydroxylation activity varies considerably between species and the enzyme(s) responsible has not been identified. 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, and higher in species that cannot rehydroxylate deoxycholate, ranging from 15% to 30% in dogs and humans to greater than 90% in rabbits. 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. The secondary metabolism of bile acids in humans and mice is summarized in Fig. 41.4 .





Enterohepatic Circulation of Bile Acids


As mentioned in the introduction, the concept of an enterohepatic circulation dates back more than 300 years. 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. 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. 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. 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.


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. 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. Subsequent studies showed that bile acids in the glomerular filtrate are actively reabsorbed from the renal tubules by a sodium-dependent mechanism. As in the ileum, the renal proximal tubule epithelium expresses the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter, IBAT; gene symbol: SLC10A2 ) as a salvage mechanism to conserve bile acids. In addition to expressing the ASBT on the apical surface, renal epithelial cells express OSTα-OSTβ 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.





Bile Secretion and Hepatic Bile Acid Transport



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. Canalicular bile flow is traditionally divided into two components: bile acid-dependent and bile acid-independent flow. 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-molecular-weight 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. 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. 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 bicarbonate-rich “hypercholeresis,” which is defined as bile flow greater than can be explained simply by the bile acid osmotic effects. 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. 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. 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. Mouse models with a genetic defect in hepatic bile acid secretion exhibit relatively normal levels of bile flow. In contrast, a similar defect in humans is associated with significantly impaired bile flow (cholestasis). 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 ( <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='HCO3−’>HCO3HCO3−
HCO 3 −
) constitute major components of the bile acid-independent fraction of bile flow. The ATP-dependent canalicular secretion of GSH via the multidrug resistance-associated protein-2 (MRP2; gene symbol: ABCC2 ) plays a particularly important role.


Besides the ATP-dependent secretion of organic anions into bile, hepatic and biliary ATP-independent secretion of bicarbonate via the <SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='HCO3−’>HCO3HCO3−
HCO 3 −
/Cl anion exchanger AE2 contributes to the bile acid-independent bile flow. The majority of this <SPAN role=presentation tabIndex=0 id=MathJax-Element-3-Frame class=MathJax style="POSITION: relative" data-mathml='HCO3−’>HCO3HCO3−
HCO 3 −
secretion is mediated by cholangiocytes lining the biliary tract in response to stimulation by a variety of hormones and neuropeptides such as secretin and vasoactive intestinal peptide. Biliary HCO 3 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. In addition to secretion of <SPAN role=presentation tabIndex=0 id=MathJax-Element-4-Frame class=MathJax style="POSITION: relative" data-mathml='HCO3−’>HCO3HCO3−
HCO 3 −
, 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.



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. Conversely, perivenous (pericentral) hepatocytes are primarily responsible for bile acid synthesis. 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. 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. 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 ). 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. The transporters responsible for the enterohepatic circulation of bile acids have been identified and are shown in Fig. 41.5 .


Apr 21, 2019 | Posted by in ABDOMINAL MEDICINE | Comments Off on Bile Formation and the Enterohepatic Circulation
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