University of Groningen. The enterohepatic circulation of bile salts in health and disease Hulzebos, Christian Victor

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1 University of Groningen The enterohepatic circulation of bile salts in health and disease Hulzebos, Christian Victor IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2004 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Hulzebos, C. V. (2004). The enterohepatic circulation of bile salts in health and disease: a kinetic approach Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter 1 General introduction and scope of the thesis 9

3 Chapter 1 Introduction The production of bile is an important function of the liver. Bile salts, which are the major organic constituents of bile, are essential for a number of physiologically important functions of bile 1. Bile salts are biological detergents and solubilize biliary lipids (phospholipids, cholesterol) into mixed micelles. In addition, hepatobiliary excretion of bile salts provides the main driving force for generation of bile flow 1;4. Hepatic bile formation is crucial for the hepatobiliary secretion of many insoluble or protein-bound substances (e.g., bilirubin, heavy metals) and drug metabolites, which can subsequently be eliminated in the feces. In the intestine, bile salts facilitate intestinal absorption of dietary fats, including fat-soluble vitamins (A,D,E,K) 1 : in the absence of intestinal bile salts, e.g., during cholestasis, a significant percentage of dietary lipids is lost into the feces 5-7. Intestinal bile salts also influence release of gastrointestinal peptides, e.g., cholecystokinine Bile salts are efficiently conserved in the enterohepatic circulation, implying that after hepatobilary secretion in the intestine, the majority of bile salts is reabsorbed, return to the liver and subsequently rescreted into bile. Bile salts are essential in cholesterol homeostasis. Only a small fraction (~10%) of cholesterol is excreted in unmetabolized form and even less is converted into steroid hormones. Conversion of cholesterol into bile salts and their subsequent fecal excretion provides the major route for elimination of excess cholesterol (~90%). Recent studies indicate that bile salts also modulate hepatic triglyceride metabolism and the clearance of triglyceride-rich lipoproteins from the circulation Disturbances in bile salt metabolism have been found to underlie diseases as diverse as cholestasis and familial hypertriglyceridemia in humans. Conversely, patients with liver disease may present with abnormalities in bile salt metabolism, including a reduced bile salt synthesis, altered bile salt composition and bile salt pool size 14. Bile salt kinetic parameters, i.e., pool size (the amount of bile salts in the body), fractional turnover rate (the portion of the pool that is newly synthesized per day), and synthesis rate may represent markers that reflect hepatocellular function and serve as a sensitive diagnostic tool to detect liver disease even before the onset of other symptoms. Measurement of bile salt kinetics can thus provide information on liver graft function after liver transplantation as well as on effects of various drugs suspected to interfere with bile formation. In previous studies, such measurements required invasive procedures including monitoring of bile flow 15 and radioactive isotope techniques 16. Isotope dilution techniques have been accepted as the preferred method to study bile salt metabolism in vivo and have contributed significantly to the present knowledge of bile salt (patho)physiology in humans Without the need to interrupt the enterohepatic circulation, this method allows simultaneous determination of kinetic parameters. The conventional stable isotope approaches required a number of relatively large plasma samples which precludes its use in children or in commonly used laboratory animals. Introduction of novel derivatisation modalities and analytical procedures, described in this thesis, requires only minute amounts of plasma and thus opens the possibility to study bile salt kinetics in children as well as in small animal models. The newly 10

4 The enterohepatic circulation of bile salts developed stable isotope technique uses [ 2 H 4 ]-cholate as labeled bile salt. Cholate is a major primary bile salt species and comprises 30 to 50% (humans) or 50 to 80% (rodents) of the total bile salt pool. Therefore, cholate pool size, fractional turnover rate (FTR) and synthesis rate are kinetic parameters that allow description of whole body bile salt kinetics. Application of this novel procedure allows to study the effects of diseases and drugs, as well as the physiological importance of newly identified proteins on the kinetics of the enterohepatic circulation of bile salts in humans and (genetically modified) laboratory animals. Research described in this thesis addresses various aspects of bile salt metabolism in animal models and in children after liver transplantation. Animal studies were conducted in various genetically-modified mouse models, with special reference to hepatic (farnesoid X receptor, multidrug resistance 2 P-glycoprotein) and intestinal proteins involved in enterohepatic circulation of bile salts. Moreover, we determined effects of established and potentially novel drugs on bile salt and lipid metabolism in rodents (cyclosporin A, FXR ligand) and in children after liver transplantation (cyclosporin A). In the following paragraphs a condensed overview of current knowledge relevant for this research is provided. Bile formation Primary bile is formed by the hepatocytes or liver parenchymal cells. Hepatocytes are clustered in acines within so-called hepatic lobules (Figure 1). These lobules are functionally divided in a periportal zone (around the vena portae), an intermediate zone, and a pericentral zone (around the central vein). Bile is secreted into a complex system of bile canaliculi which coalesce into progressively larger ducts, finally reaching the common hepatic bile duct. Together with the cystic bile duct, the hepatic bile duct forms the common bile duct which delivers the bile into the duodenum. Approximately 600 to 1200 ml of bile is produced per day in adult humans 25. Hepatic bile formation involves canalicular and ductular processes. Canalicular secretion of bile salts generates the bile salt-dependent bile flow (BSDF) whereas canalicular and ductular secretion processes provide the driving force for the bile salt-independent flow (BSIF) 1;26. Primary hepatic bile can be manipulated during its passage through the ductular system; the formation of secondary bile. Hepatobiliary secretion of bile acids into the canaliculi induces the flow of plasma water and solutes across apical membranes of hepatocytes and epithelial cells lining the biliary tree, i.e., the cholangiocytes, and their tight junctions until iso-osmolality is restored. Because bile salts are concentrated up to 1000-fold in bile, active transport by hepatocytes occurs against a steep concentration gradient. This active transport mechanism represents the major driving force for BSDF and ultimately for hepatic bile formation. The BSIF is driven by the canalicular secretion of glutathione disulfide and inorganic electrolytes and ductular secretion of inorganic electrolytes, inducing movement of water across apical membrane of cholangiocytes, putatively mediated by aquaporins 27;28. Some of these transport proteins, involved in water and ion transport, are located in vesicles and redistribute to the apical membrane of cholangiocytes in response to choleretic stimuli

5 Chapter 1 In the common hepatic bile duct, bile contains water (82 w/v %), bile salts (12 w/v %), lecithin and other phospholipids (4 w/v %) and unesterified cholesterol (0.7 w/v %). Quantitatively minor constituents are conjugated bilirubin (responsible for the green-yellowish colour), electrolytes (with a plasmalike composition, but lower in chloride and bicarbonate), proteins (e.g., IgA, hormones), and depending on on their use, drugs and their metabolic products. Figure 1. Hepatic lobules are hexagonal in shape and histologically centered around a terminal hepatic venule (central vein), an intermediate zone, and a periportal zone (around the vena portae). The portal tracts are positioned at the angles of the hexagon and consists of an hepatic artery, a portal vein and a bile duct. The hepatic acinus concept is more functionally defined. It is centred on a portal tract and is located between 2 or more terminal hepatic venules. The acinus is divided into three zones (1,2 and 3) based on the metabolic function. Blood flows from the portal tracts through the sinusoids to the venules. Zone 1 is closest to portal tract and receives the most oxygenated blood while zone 3 is pericentral localized, receiving the least amount of oxygenated blood. In contrast to blood flow, bile flow is directed from zone 3 to 1, and collected into the bile duct in the portal tract (reprinted with permission from Elsevier: Wheater s Functional Histology. A Text and Colour Atlas p275). 12

6 The enterohepatic circulation of bile salts Biosynthesis of bile salts Bile salts are synthesized from cholesterol in the liver 1. In the biosynthesis of bile salts, the side chain of cholesterol is truncated and hydroxylgroups are added to the nucleus by the actions of a series of enzymes. This cascade of enzymatic catabolism of cholesterol to bile salts, which occurs only in the liver, can be divided into two pathways (Figure 2) 2. In the neutral or classical pathway, modifications of the steroid nucleus precede those of the side chain. In this pathway, cholesterol 7α-hydroxylase (CYP7A1), which is exclusively expressed in the liver, catalyzes the conversion of cholesterol into 7α-hydroxycholesterol. This is generally considered the rate-controlling step in bile salt biosynthesis and subject to negative feedback of enterohepatic cycling of bile salts 3. In the acidic or alternative pathway, sterol-27-hydroxylase (CYP27A1), which is ubiquitously expressed, catalyzes oxidation of the side chain of cholesterol to 27-hydroxycholesterol prior to changes in the steroid nucleus. Finally, both pathways merge and C27-3ß-steroid dehydrogenase converts the 7α-hydroxycholesterol formed via both pathways into cholate (a 3αOH,7αOH,12αOH-trihydroxy bile salt) or the less hydrophylic species chenodeoxycholate (a 3αOH,7αOH-dihydroxy bile salt). Pericentral hepatocytes are probably the major site of de novo bile acid synthesis 1 ; CYP7A1 is expressed at a higher level in the hepatocytes surrounding the central vein compared to hepatocytes in the periportal zone 29. In healthy humans, the classical pathway contributes for ~80% to total bile salt synthesis, whereas in rodents the contribution of the alternative pathway may be larger (~45%) 30;31. Yet, proteins of the classic pathway also quantitatively regulate bile salt synthesis in mice since disruption of the cholesterol 7α-hydroxylase gene (Cyp7a1) in mice results in reduced bile salt synthesis, whereas disruption of Cyp7b1, encoding oxysterol 7α-hydroxylase, a key enzyme essential in the alternative pathway only minimally affects bile salt synthesis 32;33. The primary bile salts thus synthesized, cholate and chenodeoxycholate, contain hydrophobic and hydrophylic domains which render these molecules amphipathic in nature. After synthesis, more than 99 % of primary bile acids is conjugated with either the amino acid taurine (predominantly in rodents) or glycine (predominantly in humans), at the terminal (C24) carboxyl group 34. Hydrophylicity of bile salts is hereby increased, which promotes aqueous solubility at intestinal ph 1. The majority of biliary bile salts, associated into mixed micelles (composed of bile salts, cholesterol, and phosphatidylcholine), are stored in the gallbladder. Upon gallbladder contraction, bile is released into the small intestine where bile salts act as detergents to solubilize dietary fats and lipid-soluble vitamins. In the intestine, particularly in the colon, conjugated cholate and chenodeoxycholate may undergo deconjugation and are subjected to dehydroxylation by bacterial flora, resulting in so-called secondary bile acids, deoxycholate and lithocholate, respectively, and a tertiary species, ursodeoxycholate The majority of bile salts is reabsorbed in the ileum, transported into the portal blood and returns to the liver to complete an efficient enterohepatic circulation (Figure 3) 38. De novo bile salt biosynthesis compensates for fecal bile salt loss to maintain pool size under steady state conditions. Bile salt synthesis has a minor contribution 13

7 Chapter 1 to total biliary bile salt secretion: bile salt synthesis rate is ~0.02 mmol/h, whereas bile salt secretion after a meal may be as high as ~5 mmol/h 1. Bile formation is mainly dependent on the feedback inhibition of the enterohepatic circulation of bile salts 1. The bile salt pool (approximately g in size in human adults) recirculates 10 to 20 times per day in the enterohepatic circulation 25. The frequency of bile salt cycling is partly depending on the amount of dietary fat in the intestine; the bile salt pool may circulate several times during and after a fatty meal. Figure 2. The cascade of enzymatic catabolism of cholesterol to bile salts can be divided into two pathways 2. In the neutral or classical pathway, modifications of the steroid nucleus precede those of the side chain. In this pathway, cholesterol 7α-hydroxylase (CYP7A1) catalyzes the conversion of cholesterol into 7α-hydroxycholesterol which is considered the rate-limiting step in bile salt synthesis 3. In the acidic or alternative pathway, sterol-27-hydroxylase (CYP27A1) catalyzes oxidation of the side chain of cholesterol to 27 hydroxycholesterol before changes in the steroid nucleus. Finally, both pathways merge and cholate or the less hydrophylic species chenodeoxycholate is formed. 14

8 The enterohepatic circulation of bile salts Figure 3. Bile salt transporters in hepatocytes, cholangiocytes and enterocytes. Ntcp is the major uptake protein, which mediates the sodium-dependent uptake of conjugated bile salts at the sinusoidal, i.e., basolateral, plasma membrane of hepatocytes. Oatps are multispecific organic substrate carriers, which mediate hepatocellular uptake of conjugated and mainly unconjugated primary or secondary bile salts, but also of other substrates (e.g., bilirubin, steroids, or drugs) in exchange with efflux of an intracellular compound. Canalicular secretion of bile salts is mediated by the canalicular bile salt export pump, Bsep, whereas sulfated or glucuronidated bile salts are transported by the multidrug resistant protein2, Mrp2. Biliary excretion of phospholipids is mediated by Mdr2 (MDR3 in humans). Asbt facilitates uptake of conjugated bile salt species in cholangiocytes and enterocytes. Bile salt efflux from the basolateral membrane of cholangiocytes may involve a truncated isoform of Asbt (t-asbt) and/ or the multidrug-resistance protein 3 (Mrp3) or the organic anion transporting polypeptide OATP-A. According to cholehepatic shunt concept (small circle), cholangiocytes absorb (un)conjugated bile salts. Subsequently, these bile salts return to the liver via the periductular capillary plexus to be resecreted into the biliary tree. The majority of conjugated bile salts are retained in the small intestine and require active sodium-dependent reabsorption. Absorption of conjugated (trihydroxy) bile salts, which are the least able to diffuse across the apical membrane of the enterocyt in the terminal ileum is, to a large extent, mediated by Asbt. In the cytosol of enterocytes, bile salts interact with Ibabp. Basolateral efflux of bile salts from enterocytes may be mediated by t-asbt, Mrp3 and Ostα/β and forms the last step before the (un)conjugated bile acids enter the mesenteric venous blood to the portal system and return to the liver, which completes the enterohepatic circulation (large circle). 15

9 Chapter 1 Bile salt transport in the liver Hepatobiliary transport involves uptake of bile salts across the sinusoidal or basolateral membrane, intracellular bile salt transport through a variety of mechanisms, and then active canalicular (re)secretion of bile salts into the canalicular space between hepatocytes. Sinusoidal bile salt uptake After their appearance in the portal circulation the majority of bile salts is, depending on their hydrophobicity and charge, efficiently taken up by one of several transport proteins encoded by the solute carrier gene family (Slc) 39. The Na + -taurocholate cotransporting polypeptide (Ntcp; Slc10a1) is an unidirectional uptake system, which mediates the sodium-dependent uptake of conjugated bile salts at the sinusoidal plasma membrane of hepatocytes (Figure 3) 40. Coupled transport with sodium and potassium (Na + /K + -exchange) provides the energy required for bile salts to traverse across the lipid bilayer of the hepatocyte. The fact that Ntcp is homogeneously distributed along the liver acinus, while bile salt transport is predominantly localized to the periportal zone even at high bile salt concentrations, indicates an excess transport capacity that is able to accommodate large variations in the amount of bile salts presented to the liver. At higher bile salt fluxes, more hepatocytes along the acinus will be effectively involved in bile salt transport. In parallel with Ntcp, sodium-dependent hepatocellular uptake of bile salts may be mediated by microsomal epoxide hydrolase, although its contribution to overall bile salt uptake is still unclear 41;42. Organic anion transporting polypeptides (Oatp; Slc21a) function like antiporters or exchange proteins (Figure 3). Oatps are multispecific organic substrate carriers, which mediate hepatocellular uptake of conjugated and mainly unconjugated primary or secondary bile salts, but also of other substrates (e.g., bilirubin, steroids, or drugs) in exchange for an intracellular compound. To date, three hepatic Oatps have been identified in humans (OATPB, OATP-C, which is similar to Oatp2 in rodents, and OATP8) and three in rodents (Oatp1, Oatp2, Oatp4) 43;44. In addition to sodium-dependent and sodium-independent bile salt uptake systems, deconjugated hydrophobic bile acids may be able to traverse the hepatocellular bilayer by simple passive diffusion. Intracellular bile salt transport The exact mechanism of intracellular bile salt transport in the liver is not known. Intracellular bile salt transport from uptake to biliary secretion occurs very rapidly. For example, within 10 minutes after injection of a radioactively labelled bile salt into an isolated rat liver, intrahepatic radioactivity has diminished 39. It has been hypothesized that at least two mechanisms exist: transport in association with bile salt-binding proteins and vesicullar transport. The bulk of bile salts are bound to cytosolic proteins (including 3α-hydroxysteroid dehydrogenase (3α-HSD), glutathione S-transferase, liver fatty acid-binding proteins (L-FABP), and dihydrodiol 16

10 The enterohepatic circulation of bile salts hydrogenase) and traverse the cell mainly by diffusion. Inhibition of 3α-HSD results in redistribution of bile salts out of the cytosol in vitro and a delay in biliary bile salt excretion in vivo 45. Vesicle-mediated transport has been implicated to be involved in intracellular trafficking, in particular of secondary or tertiary bile salt species. Yet, vesicular transport contributes to overall intracellular transport only under circumstances of strongly increased hepatic bile salt fluxes 46. Canalicular bile salt secretion Canalicular secretion of bile salts is mediated by the canalicular bile salt export pump (Bsep, Abcb11), an adenosine triphosphate (ATP)-dependent bile salt transporter encoded by the Abcb11 gene on human chromosome 2q24-31 (Figure 3) 47. Abcb11 belongs to the ATP-binding cassette (ABC) transporter superfamily, which is composed of highly conserved proteins sharing ATP-binding domains. About 50 ABC transporters have been identified to date, which are classified in 7 groups (A-G) based on their structure and homology. ABC transporters hydrolyze ATP and translocate a variety of molecules across lipid membranes by active transport or by conducting the transport via stimulation of other translocation mechanisms 26. ABC transporters are present in subcellular compartments. Delivery to the canalicular membrane follows physiological stimuli, e.g., the need to secrete bile salts during and after a fatty meal 48. Bsep is responsible for active canalicular secretion of mainly conjugated bile acids into bile. In humans, mutations in the gene encoding Bsep are associated with low biliary bile salt concentrations and intrahepatic cholestasis, also known as progressive familial intrahepatic cholestasis (PFIC-2). This disease is further characterized by extreme pruritus, growth failure, and progression to livercirrhosis in the first decade of life 49. In contrast, Bsep knockout mice (Bsep (-/-) ) show a reduced biliary bile salt secretion rate (~70%) but a relatively mild cholestasis. The presence of an alternative bile salt transport system is anticipated, because biliary secretion of muricholate (a rodent-specific bile salt species) and tetrahydroxylated bile salts still occured and the bile salt output and bile flow in Bsep (-/-) mice fed a bile saltenriched diet significantly increased 50;51. Another canalicular protein involved in hepatobiliary bile salt efflux is the canalicular multispecific organic anion transporter (cmoat), also known as the multidrugresistant protein2 (Mrp2, Abcc2), which is a member of the multidrug resistance protein family (Figure 3) 52. Apart from its role in dianionic bile salt transport, Mrp2 functions as an efflux protein for organic anions, including glucuronide or sulfate conjugated compounds, like bilirubin diglucuronide and sulphated bile salts. Mutations or deletions in the MRP2 gene result in conjugated hyperbilirubinemia, which is known as the Dubin-Johnson syndrome 53. Bile salt transport in cholangiocytes After hepatobiliary secretion, hepatic bile is exposed to epithelial cells, i.e., cholangiocytes. These cells form the lining of the biliary tree which, apart from its anatomic pipeline function, is functionally involved in biliary transport and bile formation 54. In addition to electrolytes and water, cholangiocytes are capable to 17

11 Chapter 1 take up bile salts from the primary bile. Cholangiocytes have been suggested to influence the BSDF via their role in the so-called cholehepatic shunt pathway (Figure 3). The cholehepatic shunt concept was originally proposed to explain the hypercholeresis observed after administration of certain (unconjugated) bile salt species 55. According to this concept, cholangiocytes absorb by passive diffusion unconjugated bile salts after their protonation. Subsequently, these bile salts return to the liver via the periductular capillary plexus to be resecreted into the biliary tree, thereby promoting additional movement of water. Bile salt transport proteins have recently been identified at the apical and basolateral membranes of cholangiocytes, which suggests that, in addition to unconjugated bile salts, active reabsorption of conjugated bile salts from bile can occur 56. The apical sodiumdependent bile salt transporter (Asbt, Slc10a2) is, in addition to the terminal ileum, present at the apical membrane of cholangiocytes (Figure 3). Asbt facilitates uptake of conjugated bile salt species 57;58. Bile salt efflux from the basolateral membrane of cholangiocytes may involve a truncated isoform of Asbt (t-asbt) and/ or the multidrug-resistance protein 3 (Mrp3, Abcc3), which has been identified as a conjugate export pump, or the organic anion transporting polypeptide OATP-A (Figure 3) Especially during conditions associated with bile duct proliferation, such as extrahepatic cholestasis, cholehepatic circulation of bile salts could protect the liver from further bile salt toxicity via feedback repression of de novo bile salts synthesis. For ursodeoxycholate (UDCA), used as a therapeutic agent in various cholestatic liver diseases, indications for cholehepatic shunting were reported 63. Bile salt transport in the intestine Intestinal processes of the enterohepatic circulation include uptake of bile salts across the apical membrane of the enterocyte, intracellular bile salt transport, and basolateral secretion into the capilaries of the venous portal system. Apical bile salt uptake The vast majority of bile salts is efficiently reabsorbed from the small intestine through a combination of active sodium-dependent absorption in the distal ileum, sodium-independent absorption, and passive diffusion in the proximal small intestine 39. Whereas unconjugated bile salts are absorbed in significant portions by passive diffusion in the jejunum and proximal ileum, the majority of conjugated bile salts is retained in the lumen of the small intestine and hence require active sodium-dependent reabsorption 25. Absorption of conjugated (trihydroxy) bile salts, which are the least able to diffuse across the apical membrane of the enterocyt is, to a large extent, mediated by the apical sodium-dependent bile salt transporter (Asbt, Slc10a1) localized in the apical domain of the enterocyte of the distal ileum (Figure 3) 64. The gene encoding rat Asbt, Slc10a2, shares 63% homology with its transporter family member Ntcp (Slc10a). Asbt activity in the distal ileum ensures almost complete recovery of bile salts not absorbed in the former part of the small intestine, thereby minimalizing the occurence of secretory diarrhea caused by an excess of colonic bile salts. Moreover, beyond the distal 18

12 The enterohepatic circulation of bile salts ileum, bile salts are not required for micelle formation and fat absorption. The physiological importance of Asbt for the enterohepatic circulation is underlined by several observations. Mice in which the Asbt gene is disrupted show an increased fecal loss and synthesis rate of bile salts and a decreased bile salt pool size 65. In humans, mutations in the ASBT gene have been reported in patients with so-called primary bile salt malabsorption (PBAM) 66. In this disease, a dysfunctional ASBT protein is associated with congenital diarrhea, steatorrhea, and growth retardation. Nonfunctional Asbt results in interruption of the enterohepatic circulation of bile salts leading to an increased turnover and a diminished pool size of bile salts in these patients. Abnormal bile salt absorption associated with reduced expression levels of ASBT in patients with type IV hypertriglyceridemia also supports the important role of Asbt in bile salt reabsorption 67. The identification of the organic anion transport protein 3 (Oatp3), which is 80% identical to Oatp1, as a sodium-independent bile salt uptake system in the brush border membrane of jejunal enterocytes of the small intestine in rats suggests a significant role of this transporter for overall intestinal bile salt absorption. In rats, jejunal absorption of taurine conjugated cholate accounts upto 50% of total bile salt absorption 68. At present, it remains to be established whether functional expression of bile salt transporting-oatps also occurs in humans, although intestinal expression of OATP-A has been reported and evidence for jejunal absorption of conjugated bile salts exist, suggesting a more proximal absorption of conjugated bile salts 68. Intracellular bile salt transport In the cytosol of enterocytes bile salts may interact with cytosolic proteins, including 3α-HSD. In enterocytes of the ileum, a specific cytosolic protein is expressed. This cytosolic protein is also known as the ileal lipid-binding protein or ileal bile acid-binding protein, respectively (Ilbp or Ibabp) 69. Ibabp is a member of the so-called liver fatty acid binding protein (L-FABP) family, which comprises a large number of proteins assumed to assist in the intracellular transport of fatty acids and other hydrophobic compounds. Although Ibabp has been proposed as an important mediator of transcellular bile salt movement through the enterocytes, functional studies to establish the physiological role of this protein have not been reported sofar. Basolateral bile salt excretion Basolateral excretion is the last step before the (un)conjugated bile acids enter the mesenteric venous blood to the portal system. The precise mechanism has still to be elucidated. Yet, several candidate proteins exist for the basolateral efflux of bile salts from enterocytes. One of them is the multidrug-resistant related protein3 (Mrp3) which transports organic anions, including bile salts. Mrp3 is expressed in enterocytes of the small intestine (Figure 3) 60. A splice variant of Asbt, truncated Asbt (t-asbt), has also been documented to be present in the basolateral membrane of enterocytes (Figure 3). Because this protein has the capacity to exchange anions and because from earlier experiments it was 19

13 Chapter 1 concluded that a bile salt anion exchange mechanism is present in the basolateral membrane, t-asbt is putatively involved in basolateral bile salt export in the small intestine 59. Recently, the so-called organic solute transporter alpha and beta (Ost-α/β) complex has been proposed as a new tranporter that may facilitate basolateral bile salt transport 70. Regulation of the enterohepatic circulation It has been known for many years that bile salts repress their own biosynthesis and regulate bile flow. Furthermore, regulatory mechanisms exist controlling bile salt uptake and clearance systems to prevent intracellular accumulation of bile salts, which is harmful to the cells. Thus, virtual all steps in the enterohepatic ciculation are subjected to regulatory mechanisms. Regulation of proteins involved in bile salt synthesis and transport occurs to some extent by posttranscriptional mechanisms but mainly at the level of gene transcription (Figure 4). Interaction of bile salts with so-called nuclear receptors, ligand-activated transcription factors of a highly conserved gene family that are selectively expressed in enterohepatic tissues, plays a major role in transcriptional regulation of genes critically involved in various processes of bile salt and lipid homeostasis. The nuclear receptors and their target genes that have been identified to date have been extensively reviewed elsewhere 2;71. In short, nuclear receptors function as intracellular sensors to protect cells from excess of potentially harmful compounds, including bile salts. For the scope of this thesis, the role of FXR in coordinating the expression of genes involved in bile salt homeostasis is adressed in more detail. FXR (NR1H4) belongs to the NR1 family of nuclear receptors and is highly expressed in liver, intestine, kidney and adrenal gland. Bile salts can bind and activate the nuclear receptor FXR. The most effective activator of FXR is CDCA. After FXR heterodimerizes with the retinoid receptor (RXR), the bile salt-activated FXR-RXR complex effectively regulates the transcription of several genes considered crucial in bile salt synthesis and transport 72. Regulation of bile salt synthesis in the liver Synthesis of bile salts is mainly regulated at the transcriptional level, particular of the Cyp7a1 gene 2. Co-ordinately regulated feedback and feedforward mechanisms result in bile salt-mediated down- or upregulation, respectively, of transcription of Cyp7a1. This feedback regulation is, in part, mediated by bile salt-activated FXR. Transcriptional repression of Cyp7a1 is achieved indirectly via a regulatory cascade involving other liver-specific factors, including the small heterodimer partner (SHP; NROB2) 72. Bile salt-activated FXR-RXR heterodimers induce expression of SHP, which then inactivates Cyp7a1 expression by binding to liver receptor homologue-1 (LRH-1), also called fetal transcription factor (FTF) (NR5A2) and hepatocyte nuclear factor factor (HNF) 4α, both competence factors for Cyp7a1 expression 72. Sterol 12α-hydroxylase (Cyp8b1), the enzyme that controls the ratio in which the primary bile salt species cholate and chenodeoxycholate are being formed, is also negatively controlled by bile salts in a FXR-dependent manner 73. Thus, increased intracellular bile salt levels are able to repress their own synthesis 20

14 The enterohepatic circulation of bile salts Figure 4. The general structure and function of nuclear hormone receptors. The upper pannel represents the functional domain stucture of a nuclear receptor. It contains from the N towards the C-terminus, activation function domain1 (AF1), DNA binding domain (DBD), hinge region (D), ligand binding domain (LBD), and activation function domain 2 (AF2). The primary functions of the DBD and LBD are to recognize specific DNA sequences and ligands, respectively. The lower pannel shows FXR-mediated regulation of bile salt metabolism in the hepatocyte. Bile salts ( ) are endogenous ligands for FXR. Bile salt-activated FXR forms heterodimers with RXR (bound by retinoids, i.e., 9-cis retinoic acid ( )). FXR indirectly affects gene transcription by induction of SHP, which mediates transcriptional repression of Cyp7a1 and Cyp8b1 (bile salt synthesis) and Ntcp (bile salt uptake), as well as upregulation of Bsep (bile salt efflux). to prevent further intracellular accumulation. The role of FXR in coordinating the expression of genes involved in bile salt synthesis has been established in FXR knockout mice and by the use of FXR agonists 74;75. It has been demonstrated that, apart from FXR/ SHP-dependent regulatory pathways, FXR/ SHP-independent pathways exist in which the Kupffer cells (macrophages) in the liver are putatively involved 76;77. It has been shown that bile salts can interact with Kupffer cells 21

15 Chapter 1 before transport into hepatocytes. Interaction between bile salts and Kupffer cells induces the expression of inflammatory cytokines, which also repress Cyp7a1 expression 77. Recently, functional involvement of peroxisome proliferator-activated receptors, i.e., PPARα in the suppression of bile salt synthesis was demonstrated in vitro and in vivo 78. Upon PPAR activation by fibrates (hypolipidemic drugs) in cultured rat hepatocytes and in rodents, activities of cholesterol 7α-hydroxylase and sterol 27-hydroxylase activities were suppressed, paralleled by a similar reduction of the respective mrnas and reduced fecal bile salt loss 78. Conversion of cholesterol to bile salts can be promoted by activation of the liver X receptor (LXR), a nuclear receptor that can bind oxysterols, metabolites of cholesterol. Oxysterols are synthesized when an excess of cholesterol accumulates in the liver. After binding of oxysterols to LXR, Cyp7a1 gene expression increases in rodents, but this mode of regulation appears to be absent in humans, probably due to the lack an LXR-responsive element in the promotor of CYP7A1 79;80. Regulation of hepatobiliary transport. The major hepatocytic uptake system for bile salts, Ntcp (Slc 10a1), is also regulated by a concerted action of FXR and SHP 81. In addition this, cytokines appear to be involved in transcriptional regulation of Ntcp. In vitro, in WIF-B rat hepatoma hybrid cells, Ntcp messenger RNA (mrna) expression was significantly reduced after exposure to cytokines 76. Macrophages and their ability to secrete cytokines may be essential in mediating the endotoxin-induced decrease of hepatocellular bile salt uptake. In addition to this transcriptional regulation by bile salt-activated FXR and cytokines, Ntcp activity appears to be regulated at a posttranscriptional level: translocation of Ntcp from a preformed intracellular pool to the basolateral membrane may represent an important mechanism for rapid regulation of basolateral bile salt uptake 39. High bile salt fluxes induced by a bile salt-enriched diet or partial hepatectomy result in reduction of Ntcp mrna and/or protein expression 82. In general, expression of Ntcp is only marginally affected by large variations in transhepatic bile salt fluxes, suggesting that this transporter is abundantly expressed under normal physiological conditions. The liver is thus prepared for adequate handling a large variation in amounts of bile salts, e.g., during postprandial periods. Oatp is also regulated at transcriptional and posttranscriptional levels. Although not consistently, down-regulation of Oatp1 mrna and protein levels upon bile salt feeding has been reported, especially in the periportal region where bile salt concentrations are supposedly highest 83. Oatp1 is also down-regulated after bile duct ligation and after partial hepatectomy. Together, these results might indicate that reduction of Oatp expression contributes to protection of the liver against intracellular bile salt accumulation and the associated bile salt toxicity. Activation of the bile salt efflux pump Bsep by bile salts may represent an adaptive mechanism to prevent excess of intracellular bile salts. Bile salts induce Bsep expression in an FXR-mediated manner 84. Adaptation of hepatocellular bile salt transport capacity induced by high bile salt fluxes is also achieved by canalicular insertion of Bsep, indicating posttranscriptional regulatory mechanisms. 22

16 The enterohepatic circulation of bile salts However, high intracellular bile salt concentrations in vivo as induced by bile duct ligation cause a decrease in Bsep expression 85 : in this situation other regulatory factors apparently overrule bile salt regulation 82. Regulation of bile salt transport in the intestine Apart from regulating their own synthesis and hepatobiliary transport, bile salts appear also to be able to regulate their own intestinal reabsorption. From ontogenetic studies it has become clear that the developmental pattern of Asbt and Ibabp, two proteins considered to be involved in intestinal bile salt reabsorption, are closely interrelated 86. Increased postnatal expression of both Asbt and Ibabp coincides with functional bile salt transport. Recent studies indicate that mouse Asbt, but not rat Asbt, is subjected to negative feedback regulation mediated by LRH-1 activation 87. This explains, at least in part, the reported contradictory effects of manipulating the bile salt pool on the expression of Asbt 88. In contrast, intestinal expression of Ibabp, considered to be involved in intracellular trafficking of bile salts is strongly induced by bile salt-activated FXR in mice and in rats 69;72;88;89. Bile salt kinetics: characterizing the dynamics of the enterohepatic circulation Enterohepatic cycling of bile salts involves hepatic uptake and synthesis, hepatobiliary transport, and intestinal reabsorption of bile salts. These processes are essential for bile salt and lipid metabolism, as evidenced by the various phenotypic characteristics and disease states caused by dysfunction of proteins involved in this cascade. Knowledge of key proteins involved in bile salt metabolism has staggeringly increased in the past couple of years and, together with improved molecular tools, detailed analysis ot their molecular regulation and function has become possible. Yet, to fully appreciate the physiological importance of the various proteins, in vivo techniques that quantify actual metabolic fluxes are required. Isotope dilution techniques have been used to measure bile salt kinetics in vivo, which allows to calculate bile salt synthesis, pool size and turnover in order to characterize the dynamics of the enterohepatic circulation. To delineate potential clinical implications of these parameters an overview of current knowledge of the individual parameters on bile salt kinetics is presented in the following paragraph. Bile salt kinetic parameters: pool size, fractional turnover rate and synthesis rate The size and composition of the bile salt pool are critical for adequate bile formation and lipid absorption. The crucial role of bile salt pool size in lipid absorption is evident from studies performed in preterm infants. In general, premature neonates have a smaller bile salt pool as compared to full-term neonates or infants. The small bile salt pool in preterm neonates is associated with low intraluminal bile salt concentrations, frequently below their critical 23

17 Chapter 1 micellar concentration, which coincides with inefficient absorption of fat 20. Limited synthesis of bile salts and incomplete intestinal recovery of bile salts due to immaturity of intestinal proteins may underlie the small bile salt pool size. Early in life the bile salt pool increases, leading to increased hepatobiliary bile salt secretion rates and a more efficient intestinal fat absorption 20;90. Thus, the bile salt pool size seems to develop in response to maturational changes in the machinery of proteins involved in enterohepatic cycling of bile salts. Yet, at comparable bile salt pool sizes, fat absorption in neonates fed human milk is more efficient than in neonates fed a cow s milk formula, implying that other factors than bile salts are also important for fat absorption. For example, human milk contains a lipase (bile salt-stimulated lipase) that is considered to have an important role in fat absorption 91. In adult humans, age, race, and body size showed no statistically significant relationship with bile salt pool size, in constrast to gender 92. Limited data are available with respect to effects of dietary factors on bile salt pool size. In neonates, a more pronounced increase in bile salt pool size was observed in infants fed human milk compared to formula-fed infants suggesting that dietary factors affect pool size independent of maturation 20. In adult humans, no consistent effects on cholate pool size were found while fed solid diets or fatrich liquid diets containing either corn oil or coconut oil, 93 whereas bile salt pool sizes increased after institution of a fat- and protein-restricted diet 94. Recently, effects of both low- and high-fat feeding on bile salt kinetics have been described. Although total pool size of primary bile salts remained unchanged, differential effects on bile salt composition and individual pool sizes were observed 95. In humans, the rate of gallbladder emptying and short bowel transit time are determinants of bile salt pool size Incomplete gallbladder emptying or a delay in intestinal transit time coincide with a reduced cycling frequency and an enlargement of the bile salt pool size. In contrast, when intestinal transit time of bile salts increases, bile salt pool size decreases. Thus, an inverse relationship exists between the cycling frequency and the bile salt pool size. Variations in bile salt pool size are compensated for by an altered cycling frequency, so that hepatobilary secretion rate and probably intestinal bile salt delivery are maintained 99. The influence of intestinal bile salt delivery on bile salt pool size has been studied in an experimental primate model. It appeared that short term regulation of the bile salt pool can be accomplished via interfering with the bile salt cycling frequency and fecal losses; an acute increase in intestinal bile salts via infusion in the small bowel increased bile salt cycling frequency which resulted in increased fecal losses and maintenance of bile salt pool size. In contrast, in fasted animals with diminished intestinal bile salt input, reduced bile salt cycling and fecal losses help to preserve bile salt pool size 100. It has been shown that, apart from fat absorption, the bile salt pool size and its composition are important determinants of hepatobiliary secretion rates of cholesterol and phospholipids, factors that affect the lithogenicity of bile Chenodeoxycholate pool size has been found to be negatively correlated with biliary cholesterol saturation, suggesting a role in the etiology of cholesterol gallstone formation 93. Ursodeoxycholate (UDCA), a hydrophilic bile salt, is an effective litholytic drug that promotes the dissolution of cholesterol gallstones, 24

18 The enterohepatic circulation of bile salts by decreasing biliary cholesterol saturation 104;105. Moreover, UDCA modifies the bile salt pool composition by decreasing levels of endogenous, hydrophobic bile acids while increasing the proportion of the non-toxic hydrophilic UDCA, and has a choleretic effect 102;106;107. The bile salt pool size can also be manipulated by other drugs. For example, enlargement of the bile salt pool has been observed in neonates, whose mothers were treated with dexamethasone or phenobarbital, probably caused by drug-induced upregulation or increased activity of hepatic and/ or intestinal proteins involved in bile salt metabolism. The bile salt pool size is influenced by various diseases as diverse as inborn errors of bile salt synthesis, liver and intestinal diseases, endocrine disorders and hyperlipidemias 108. Some of these disorders may directly affect key enzymes in bile salt synthesis (e.g., cerebrotendinous xanthomatosis), whereas others result in changed expression of transport proteins involved in bile salt metabolism (e.g., diabetes mellitus) or alterations in the enterohepatic cycling time (e.g., celiac sprue) 109. Measurement of fecal bile salt excretion, or, in kinetic terms, bile salt turnover, provides information on the portion of the bile salt pool that is newly synthesized per day. The fractional turnover rate of bile salts is found to be inversely correlated with bile salt pool size, indicating that with a large bile salt pool, the portion that is lost via the feces is smaller 20. Measurements of turnover rates have provided useful knowledge of sterol balances in, for example, patients with various types of hyperlipoproteinemia or in patiens with cholelithiasis Bile salt synthesis which, under steady state conditions, maintains bile salt pool size can be demonstrated as early as after 15 weeks of gestation 114. In human neonates, bile salt synthesis rates are relatively high when compared to adults 23. Bile salt synthesis rates increase in the first year of life, coinciding with an increased activity of cholesterol 7α-hydroxylase (CYP7A1) and maturation of ileal absorption, resulting in enlargement of the bile salt pool. In humans, a large variation in the daily conversion of cholesterol to bile salts exists, for reasons unrevealed to date 115. The significant contribution of bile salt synthesis to cholesterol homeostasis is illustrated by progressive accumulation of cholesterol and accelerated atherosclerosis in inborn errors of bile salt synthesis 116. A relationship between bile salt and lipid metabolism has emerged from various other studies, targeting bile salt synthesis as a therapeutic option to lower serum cholesterol levels Outline and scope of the thesis Bile salts are of crucial importance in mammalian physiology, i.e., as generators of bile flow, as promotors of biliary lipid excretion, as lipid solubilizers and as end products of cholesterol catabolism. The recent identification of bile salts as signaling molecules, affecting expression of genes in control of bile salt, lipid, and glucose metabolism through nuclear receptors, has revolutionized traditional concepts and has promoted research on bile salt metabolism tremendously. A fascinating aspect of bile salt metabolism is their enterohepatic cycling, which involves coordinated action of transporter proteins in liver and intestine. Many of these proteins have been identified to date by modern cloning techniques. 25

19 Chapter 1 Development of methodology to fully appreciate the role of these proteins in whole body bile salt metabolism in vivo has been a major effort in many laboratories. For many decades, isotope dilution methods have been succesfully applied in adults and provided insight into in vivo bile salt kinetic parameters: the bile salt pool size, the fractional turnover rate, and the bile salt synthesis rate. Yet, a serious limitation of the conventional approaches was the requirement to collect a series of relatively large blood samples, which precluded its use in children or commonly used (small) experimental animal models. In chapter 2 an adapted stable isotope dilution technique using [ 2 H 4 ]-cholate with novel derivatisation modalities and analytical procedures is described that overcomes this limitation, as only extremely small blood samples are required. The availability of this novel method to measure bile salt kinetics in vivo together with the construction of genetically-modified, i.e., knockout or transgenic mouse models, in parallel with the development of nuclear receptor ligands, has stimulated research on kinetics of bile salts in the enterohepatic circulation. Scope of the research described in this thesis was to unravel the role of hepatic and intestinal proteins on the enterohepatic circulation of bile salts in vivo. In addition, effects of established and potentially novel drugs on bile salt and lipid metabolism were determined. Effects of cyclosporin A (CsA), a commonly used immunosuppressant in transplantation medicine and in auto-immunological disorders, on bile salt metabolism were studied. In chapter 3 we characterized the effects of CsA on the enterohepatic circulation of cholate with respect to kinetic parameters and in relation to the expression of relevant transporters in liver and intestine in rats. CsA appeared to enhance efficacy of intestinal cholate reabsorption through increased Asbt protein expression in the distal ileum, which contributed to maintenance of cholate pool size. Thus, in addition to hepatic regulation of bile salt homeostasis, it was demonstrated that regulation of bile salt pool size occured also at the intestinal level. Studies described in chapter 4 aimed to elucidate whether CsA treatment affects bile salt metabolism in pediatric patients after liver transplantation, and whether such effects would be related to CsA-associated hyperlipidemia, a well known side effect of CsA. CsA inhibited bile salt synthesis and increased plasma concentrations of cholesterol and triglycerides in pediatric liver transplant patients. Suppression of bile salt synthesis by long-term CsA treatment may contribute to hyperlipidemia and thus to increased risk for cardiovascular disease. Nuclear receptors control a wide variety of genes important for metabolism of bile salts as well as of lipids. One of these proteins is the farnesoid X receptor (FXR; NR1H4), which acts as an intracellular bile salt sensor, effectively coordinating the expression of genes involved in bile salt and lipid metabolism. Although the role of FXR as an important transcription factor has been firmly established, predominantly in in vitro studies, the physiological role of FXR in controlling the enterohepatic circulation of bile salts has not been addressed sofar. The role of FXR in control of in vivo bile formation and the kinetics of the enterohepatic circulation of bile salts has been determined by quantifying bile salt kinetics using the newly developed stable isotope dilution method. The impact of FXR-deficiency on bile formation and bile salt kinetics in FXR-deficient (Fxr (-/-) ) 26