UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Hepatic bile salt uptake
The role of NTCP revisited
Slijepčević, D.
Publication date
2019
Document Version
Final published version
License
Other
Link to publication
Citation for published version (APA):
Slijepčević, D. (2019). Hepatic bile salt uptake: The role of NTCP revisited.
General rights
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), other than for strictly personal, individual use, unless the work is under an open
content license (like Creative Commons).
Disclaimer/Complaints regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please
let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material
inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter
to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You
will be contacted as soon as possible.
-The role of NTCP
revisited-Davor Slijepcevic
paranifmen
Riemer T. de Vries
Financial support for printing of this thesis was kindly provided by
Academisch Medisch Centrum (AMC) Amsterdam,
Nederlandse Vereniging voor Hepatologie (NVH),
Dr. Falk Pharma Benelux B.V., Intercept Pharma, ChipSoft and
Western Balkans Business Group.
Front cover (growing tree) by Alain Eskinasi.
Layout design by Frank van Broekhoven & Davor Slijep
č
evi
ć
.
Printing by Guus Gijben, proefschrift-aio.nl.
-The role of NTCP
revisited-ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. ir. K.I.J. Maex
ten overstaan van een door het College voor Promoties ingestelde commissie,
in het openbaar te verdedigen in de Aula der Universiteit
op 10 mei 2019 te 11.00 uur
door
Davor Slijep
č
evi
ć
AMC-UvA
AMC-UvA
AMC-UvA
AMC-UvA
Universiteit Utrecht
AMC-UvA
Emory University, USA
AMC-UvA
Rijksuniversiteit Groningen
Promotores
Prof. dr. U.H.W. Beuers
Prof. dr. R.P.J. Oude Elferink
Co-promotor
Dr. K.F.J. van de Graaf
Overige leden
Dr. S.W.C. van Mil
Dr. K.J. van Erpecum
Prof. dr. P.L.M. Jansen
Prof. dr. P.A. Dawson
Prof. dr. A.K. Groen
Prof. dr. F. Kuipers
Faculteit der Geneeskunde
Chapter 1
General introduction
Chapter 2 Impaired Uptake of Conjugated Bile Acids and
Hepatitis B Virus PreS1-Binding in Na
+-Taurocholate
Cotransporting Polypeptide Knockout Mice
Chapter 3 Hepatic Uptake of Conjugated Bile Acids Is Mediated
by Both Sodium Taurocholate Cotransporting
Polypeptide and Organic Anion Transporting
Polypeptides and Modulated by Intestinal Sensing of
Plasma Bile Acid Levels in Mice
Chapter 4 NTCP inhibition has hepatoprotective effects in
cholestasis in mice
Chapter 5
Blocking sodium-taurocholate cotransporting
polypeptide stimulates biliary cholesterol and
phospholipid
excretion
Chapter 6 Summarizing discussion, Nederlandse samenvatting
Appendix
List of contributing authors and affiliations
PhD
portfolio
Acknowledgements,
dankwoord
About the author
9
31
61
91
119
137
151
Davor Slijepcevic and Stan F.J. van de Graaf
Adapted from Digestive Diseases 2017;35(3):251-258
General introduction
Chapter 1
Bile salt formation and the enterohepatic circulation
Bile is a highly complex secretory product of the liver that plays a pivotal role in the elimination of endogenous and exogenous (toxic) compounds, such as bilirubin, heavy metals and drug metabolites. Bile mainly consists of bile salts, which are amphipathic molecules that have the ability to form an interface between lipids and water in the form of micelles. Bile salts undergo a complex biosynthetic pathway starting from cholesterol, involving many different enzymes, which are responsible for modifications of the ring structure and oxidation/shortening of the side chain.
Synthesis of bile salts takes place in the pericentral hepatocytes via the rate-limiting conversion of cholesterol into 7α-hydroxycholesterol by the hepatic microsomal P450 enzyme cholesterol 7α-hydroxylase (CYP7A1), which gives rise to chenodeoxycholic acid (CDCA). (1). Sterol 12α-hydroxylase (CYP8B1) determines synthesis of cholate (CA) and thereby dictates the CA to CDCA ratio. Most bile acid species are formed in this classical pathway in the adult human liver. As a result, the main primary bile acids CDCA and CA are formed (~80% of the human bile acid pool) (2). Specifically in mice, CDCA is converted to muricholic acid (MCA) by a species specific 6β-hydroxylase (CYP2C70) (3). In the terminal step of biosynthesis, bile acids undergo conjugation with an amino acid group, either glycine (predominantly in humans) and/or taurine (the latter predominantly in rodents), which is controlled by the bile acid coenzyme A:amino acid N-acyltransferase (BAAT). When bile
acids exit the liver, they are in a hydrophilic, deprotonated state, and associate with Na+ in a
physiological environment (pH 7-8; hence the name bile salts). Upon arrival of bile salts in the distal intestine, conjugated primary bile acid species are (partially) deconjugated and dehydroxylated to secondary bile acids by bacterial biotransformation. The bacterial bile salt hydrolase deconjugates bile salts, while 7α-dehydroxylase converts CDCA and CA to lithocholic acid (LCA) and deoxycholic acid (DCA), respectively (4). Microbial metabolism of bile salts and their impact on the host are reviewed elsewhere (5).
Bile salts are biological detergents, since they have the unique ability to form small aggregates (mixed micelles) upon contact with other lipidic substances, in particular with phospholipids and cholesterol (6). Conjugated bile salts are excreted in millimolar concentrations in the bile canaliculi, generating an osmotic force that pulls water to the lumen, the so-called bile salt-dependent bile flow (7). Bile salts move from the small canaliculi through the biliary tree and are temporarily stored in the gall bladder (in most mammals). Eventually bile salts enter the common bile duct and are released into the duodenum, where they improve intestinal lipid digestion by forming micelles with various lipids and fat-soluble vitamins. Finally, bile salts efficiently return to hepatocytes by passing the enterocyte into the portal circulation. A hallmark of (hydrophilic) bile salts is their inability to passively diffuse across membranes, so active transport proteins are crucial to retain bile salts in an organism.
Ch
ap
ter 1
Bile salts are efficiently retained within the enterohepatic circulation by multiple specific transmembrane transporters, which were molecularly characterized in the 1990s. Bile salts are effectively reabsorbed in the terminal ileum by the apical sodium-dependent bile acid transporter (SLC10A2/ASBT), and excreted into portal blood by the heterodimeric organic solute transporter α/β (SLC51A/B/OSTα/β) together with other extrusion pumps (8). Ileocytes limit the loss of bile salts, as bile salts recycle 4-12 times per day and only 3-5% of the daily pool is lost via the feces (9). In the physiological situation, hepatic bile acid synthesis de novo equals fecal bile acid loss, and thereby boosts cholesterol elimination from the body (10).
The enterohepatic cycle exposes several tissues to fluctuating amounts of bile salts. Bile salts in the hepatobiliary system reach millimolar concentrations, whereas these levels are always relatively low (<10 µM) in blood. Bile salts are eventually recycled from portal blood at the hepatic basolateral membrane by two transport systems: the sodium-dependent taurocholate cotransporting polypeptide (SLC10A1/NTCP) and members of the sodium-independent organic anion transporting polypeptide (SLCO/OATP) transport family (11, 12). In humans, the physiological bile salt concentration ranges from <5 µM (fasting) to 3-7 µM (postprandial) in systemic blood. In portal blood, the post-prandial bile salt peak is more evident (from 4-27 µM to 22-55 µM) (13, 14). Thus, the hepatic uptake machinery efficiently limits escape of bile salts to the general circulation, with a first-pass extraction fraction ranging from 50% - 90% depending on the bile salt structure (15). Bile salts in peripheral plasma are basically the spillover of bile salts that escaped the first-pass extraction of the liver.
Hepatic basolateral uptake systems for bile salts
NTCP is a family member of the solute carrier 10 family (which consists of seven members) and is present on the basolateral membrane of hepatocytes. However, NTCP expression was also reported in pancreatic acini (16, 17), where it may reabsorb bile salts from the biliary tree. NTCP adopts a dimeric or even higher order quaternary structure, in which the individual subunits form functional units (18). Recently, the crystal structure of ASBT, the second member of the SLC10A family, was solved revealing a structure with 9 transmembrane domains with an exoplasmic N terminus and cytoplasmic C terminus (19), and a similar conformation might be expected for the homologous NTCP. NTCP-mediated uptake of
taurocholate was demonstrated with published Km values that vary from 5 – 84 µM for
human NTCP and 8 – 61 µM for rodent NTCP (20). Besides bile salts, NTCP is a transporter of steroidal hormones and a variety of drugs (21-23). In the liver, NTCP is distributed equally along all liver lobules, but uptake of conjugated bile salts occurs predominantly in
with a single homozygous NTCP mutation (p.R252H) was described in 2015, phenotypically characterized by high plasma conjugated bile salt levels without any signs of liver injury or pruritus (27). Currently, adults with SLC10A1 polymorphisms (e.g. p.S267F) are increasingly being detected and show persistent hypercholanemia (28).
The presence of additional Na+-dependent membrane transporters has been suggested,
in particular bile salt transport by microsomal epoxide hydrolase (mEH) (29), but little experimental evidence supports this notion (30). mEH-mediated bile salt transport in vitro was not verified yet by other groups and mEH-knockout mice do not show a bile salt related phenotype (31). Hepatic bile salt uptake can also be mediated by (one or more members of)
the Na+-independent OATP transporter family. All OATPs are 12 transmembrane domain
glycoproteins with broad substrate preference, such as (un)conjugated bile salts, bilirubin and numerous drugs and toxins (32). The most abundant hepatic OATP subfamilies are OATP1A and OATP1B, and are designated Oatp1a1, Oatp1a4 and Oatp1b2 in rodents (11). OATP1A1 in rodents is distributed homogenously along the liver acinus, while OATP1B2 and OATP1A4 exhibit a heterogeneous lobular expression, preferably localized in the perivenous area (33). It is difficult to estimate the role of each single OATP-isoform in
vivo, as there is large substrate overlap and rodent Oatps have no direct human orthologue
(34). In humans, two-gene biallelic human OATP1B1 and OATP1B3 deficiency is known as the Rotor syndrome, characterized by high conjugated plasma bilirubin levels (35). Evidence for human OATP polymorphisms affecting endogenous bile salt uptake is sparse. Interestingly, mice lacking all Oatp1a/1b-family members (Slco1a/1b knockout mice) display 13-fold elevated levels of unconjugated bile salts in blood (36), whereas conjugated bile salt levels remained mostly unchanged, and similar results were found in knockout mice lacking only Slco1b2 (37). The role of the rodent Oatp1a-isoforms in bile salt transport is less obvious, nevertheless, taurocholate uptake was reduced in primary hepatocytes isolated from Oatp1a4-null mice, and to less extent in Oatp1a1-null mice (38). Other studies suggest that Oatp1a1 and Oatp1a4 preferably transport secondary unconjugated bile salts, thereby altering intestinal bile salt metabolism (39).
Novel function of NTCP: the receptor of HBV/HDV
Beside the role of NTCP as major transporter for conjugated bile salts, NTCP was recently found to be the main receptor for hepatitis B and delta viral (HBV/HDV) particles (40), and the specific NTCP inhibitor myrcludex B is currently being tested in phase II trials as an HBV/HDV entry inhibitor (41, 42) (Figure 1). Myrcludex B is a synthetic lipopeptide based on the preS1 domain of the HBV envelop L-protein targeting NTCP, and effectively inhibits HBV entry in vitro and in vivo (43). This domain on the L-protein has diverse functions, most importantly binding of the nucleocapsid during virus envelopment and receptor binding during entry (44). Myrcludex B has a potent ability to block virus entry, which occurs at even lower (picomolar) concentrations than
Ch
ap
ter 1
those necessary to block bile salt uptake, creating a therapeutic window for virus restriction only (45). Pharmacokinetic studies with myrcludex B show rapid hepatic accumulation (within minutes) and found a half-life of ~12 hours. Liver-specific binding of myrcludex B is also observed in non-HBV susceptible animals (dogs, rats and mice) (46) and NTCP-specificity was confirmed using NTCP knockout mice (47 and chapter 2 of this thesis).
NTCP
SLC10A1
Bile acids, drugs(e.g. cyclosporin) HBV/HDV, peptide ligand (e.g. Myrcludex B)
L-protein preS1 domain O R OH OH OH 3 7 12 + taurine/glycine Cholic acid
Figure 1
ASBT
SLC10A2
luminalBile acids, drugs
(e.g. nifedipine) (e.g. A4250/SC-435/LUM001)Specific ASBT-inhibitors O R OH OH OH 3 7 12 + taurine/glycine Cholic acid sinusoidal NH2 COOH NH2 COOH
Figure 1: NTCP and ASBT: topology and ligands
Both NTCP (upper panel) and ASBT (lower panel) from the SLC10 family contain 9 transmembrane spanning domains. Conjugated bile acids are natural substrates for these transporters, and recently the preS1 domain of HBV was found to specifically bind to NTCP. Also, the HBV-derived lipopeptide myrcludex B strongly binds to and inhibits NTCP in vitro and in vivo. Many ASBT-specific inhibitors (e.g. A4250) were developed over the past decade to increase fecal bile acid excretion.
RNA levels towards undetectable levels upon 12 or 24 weeks of treatment especially using a combination of myrcludex B and interferon, although HBsAg did not show any reduction with this treatment duration (41). Myrcludex B treatment resulted in elevated plasma bile salts levels in humans (up to ~200 uM, mainly glycine-conjugated species), which was well tolerated and the drug showed no adverse effects (42, 48).
Inhibition of bile salt uptake to ameliorate cholestatic liver injury
Cholestatic liver damage occurs when bile flow is impeded, leading to accumulation of toxic bile salts within hepatocytes and causing liver damage, inflammation, fibrosis and ultimately cirrhosis. Debilitating symptoms of chronic cholestasis include fatigue and pruritus. Main causes are defects in genes encoding transporters in the canalicular membrane of hepatocytes, i.e. the class of progressive familial intrahepatic cholestasis (PFIC) (49-51), fibrosing inflammation of the small/large bile ducts or mechanical obstruction of bile flow (e.g. cholelithiasis or obstruction by a tumor). The hepatobiliary and/or intestinal secretory processes are great targets for pharmacotherapy in cholestasis, and novel molecular players are summarized elsewhere (52, 53).
Nuclear receptors (NRs), bile salt transporters and hepatic enzymes play a key-role in orchestrating bile salt metabolism to protect against accumulation of toxic bile salts (54). Classical intrahepatic bile salt sensing by farnesoid X receptor (FXR) (55-57) induces expression of the atypical NR that has only a ligand-binding domain, called short heterodimer partner (SHP) (58-59), which represses bile acid biosynthesis via downregulation of CYP7A1 expression. Hepatic FXR activation by (semi-) synthetic FXR agonists has been successful in cholestatic animal models (60-61) in order to induce hepatic bile salt efflux and reduce bile salt uptake, and such therapies showed promising results in phase II and III trials in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) (62-63). Patients with PBC showed an improvement of alkaline phosphatase and other liver enzymes after obeticholic acid treatment, and the FDA approved this drug in 2016 despite an increase in itch scores (63).
In several models of cholestasis, a common adaptive regulation of plasma membrane transporters protects from an overload of particularly bilirubin and bile salts. This protective response is not only restricted to the liver, but also occurs in kidney, intestine and bile ducts. In murine extrahepatic cholestasis, hepatic Ntcp, Oatp1a1 and Oatp1b2 mRNA levels are reduced (64). In contrast, Oatp1a4 is upregulated, and this protein is supposed to be a bidirectional transporter of organic anions operating as an exporter in cholestasis (65). The regulation of NTCP was shown to be FXR-dependent (66-67), but several other pathways have been implicated as well and appear different among species (68). In humans, NTCP and OATP1B1 decrease during primary sclerosing cholangitis (PSC) (69),
Ch
ap
ter 1
whereas OATP1B3 expression was transactivated by FXR (70).
Targeting FXR tissue-specifically during cholestasis attracted considerable attention since the discovery of fibroblast growth factor 15/19 (FGF15/19). This hormone, when released from the gut, binds to the tyrosine kinase receptor FGFR4/β-Klotho on hepatocytes, which activates the JNK1/2 signaling pathway (71). In humans, FGF19 is also found in bile duct epithelium and excreted in bile of patients with advanced cholestasis (72). Intestinal FXR activation (73) and the FGF19 mimetic M70 (74-75) dampen cholestatic liver injury by strongly reducing hepatic bile acid synthesis and the circulating bile salt pool. So, mouse studies clearly show hepatoprotection through (gut-specific) FGF15/19 signaling, primarily by reducing bile salt pools, by sharing its mode of action with ASBT-inhibition. NGM282 (a variant of human FGF19 engineered to lack the proliferative effects of FGF19) is now tested in a phase II trial in PBC patients unresponsive to ursodeoxycholic acid (UDCA) treatment (76).
When we focus on modulation of intestinal bile salt transport, most studies investigated intestinal bile salt sequestrants (e.g. cholestyramine and colesevelam) as first-line agents in the treatment of cholestatic pruritus. Colesevelam proved to be effective in reducing plasma bile salt levels by ~50%, but failed to reduce pruritus (77). Currently, clinical studies are evaluating ASBT inhibitors as a novel pharmacological treatment for cholestasis. The rationale of ASBT inhibition is based on an increase of fecal bile salt elimination and preventing bile salt return to the liver, thereby potentially reducing the bile salt pool by 80%. The ASBT inhibitor A4250, previously used in a clinical phase I study (78), showed improvement of cholestatic liver injury in mice deficient for multidrug resistance protein 2 (MDR2) (79). In a similar study, two week treatment with SC-435, a different ASBT inhibiting small molecule, demonstrated reduced bile salt pool size and attenuation of cholestasis in the same mouse model (80). At present, human studies (phase II/III) are being performed, demonstrating the ability of ASBT-inhibition to quickly reduce plasma bile salt pool and pruritus in PBC patients (81).
So far, the effectiveness of (further) inhibition of basolateral hepatic bile salt uptake in the context of cholestasis was only studied using OATP1A1 knockout mice, which were not protected against hepatic injury after bile-duct ligation (BDL) (82). The role of complete (pharmacological) NTCP-inhibition during cholestasis is not reported yet and will be discussed in chapter 4. Myrcludex B is well tolerated in humans, causes increased bile salt levels in plasma and induces normalization of ALT levels in HBV infected individuals (41). Such data calls for investigation of NTCP-inhibition during cholestasis.
of bile salts in glucose metabolism, inflammation and energy expenditure. Detection of bile salts is mostly mediated via the nuclear receptor FXR and transmembrane G protein coupled receptor 5 (TGR5), and not exclusively in the liver and intestine (83-84). Therefore, targeting bile salt signaling is appealing to treat metabolic diseases such as diabetes and cardiovascular disease. Existing pharmacological strategies entail the use of specific bile salts or synthetic agonists to effectuate metabolic signaling. Below we discuss the (possible) beneficial metabolic effects of modulation of endogenous bile salt dynamics by inhibition of ASBT and NTCP transport activity, which often has a direct relation with FXR/ TGR5 activity. Specific effects of NTCP-inhibition on cholesterol transport (focusing on biliary excretion) will be addresses in chapter 5 of this thesis.
I) Relationship of bile salts with cholesterol metabolism
Cholesterol is a water-insoluble molecule, crucial to increase integrity of cell membranes, serving as a substrate for steroid hormone synthesis and supply energy to the cell. Cholesterol circulates in plasma by associated lipoproteins in order to reach the liver, intestine and peripheral tissue. Fecal excretion determines removal of cholesterol from the body, to which both biliary excretion and transintestinal cholesterol excretion (TICE) contribute, relevant excretion routes in both rodents and humans (85). Importantly, bile acid synthesis is the major pathway for cholesterol catabolism, accounting for around one-third to 50% of cholesterol elimination in humans (86), the remaining elimination being in the form of cholesterol and steroids.
Upon intestinal cholesterol uptake, chylomicron (remnants) carry exogenous cholesterol esterified to fatty acids to the liver, while other lipoprotein classes mediate transport of endogenous cholesterol. Subsequently, cholesterol-esters enter hepatocytes (1) via the low-density lipoprotein (LDL)-receptor on the basolateral membrane and are hydrolyzed in the lysosomes to free cholesterol and partly excreted into bile. Alternatively, free cholesterol is taken up by (2) the scavenger receptor class B type 1 (SR-B1) (87-88), which is the pivotal player in reverse cholesterol transport of cholesterol-rich high-density lipoproteins (HDL). Cholesterol that is in excess can be incorporated in membranes or esterified by Acyl-CoA cholesterol acyltransferase (ACAT). If cholesterol levels are low, cholesterol synthesis is stimulated by induction of the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) in the endoplasmatic reticulum (89).
Both newly synthesized and absorbed cholesterol, possibly processed in two distinct pools, are directly secreted in bile in their unesterified free form (90-91). If not designated for direct excretion in bile or processing to bile acids, cholesterol(-esters) can be packed in intracellular lipid droplets or excreted in very-low density lipoproteins (VLDL). Biliary excretion of cholesterol across the canalicular membrane is controlled by the heterodimer
Ch
ap
ter 1
hepatocyte (92). Mutations in ABCG5/8 cause sitosterolemia due to insufficient (chole) sterol excretion. In contrast, excessive cholesterol excretion into bile predisposes to gall stone disease, where supersaturation of bile with free cholesterol, known as lithogenic bile, drives stone formation (93). Biliary cholesterol excretion is tightly coupled to secreted bile salts, where an increase in bile salt concentrations and hydrophobicity stimulate cholesterol extraction into the canalicular lumen (94-95). Biliary cholesterol excretion critically depends on functional phospholipid export by ATP-binding cassette subfamily B member 4 (ABCB4), because solubilization of cholesterol in bile salt micelles requires the presence of phospholipids (49, 96).
Understanding transport dynamics of cholesterol is therapeutically relevant as it may be exploited to protect from gall stone disease, non-alcoholic fatty liver disease (NAFLD) and cardiovascular complications. Hepatic accumulation of triglycerides, free cholesterol as well as disturbed lipoprotein transport are key features in the pathogenesis of NAFLD, reviewed elsewhere in detail (97-98). To counteract sterol overload, whole-body levels are closely sensed by three pivotal nuclear receptors, mainly along the gastrointestinal tract. The sterol-regulatory element binding proteins (SREBPs) (99), the bile acid receptor FXR and oxysterol liver X receptor (LXR) regulate downstream targets involved in cholesterol and lipid homeostasis. ABCG5/8-mediated cholesterol excretion is largely controlled by LXR (100), and ABCB4-mediated phospholipid excretion predominantly by FXR (101). Both FXR and LXR-deficient mice are characterized by hepatic cholesterol overload when fed on a high-cholesterol diet, caused by chronically impaired biosynthesis, altered bile acid pool composition/size and transport (66, 102). In contrast, the semi-synthetic bile acid analogue obeticholic acid, a potent FXR agonist that undergoes effective enterohepatic cycling, improved the histological NAFLD activity scores in humans (103). However, plasma LDL-cholesterol were elevated and HDL-cholesterol decreased, potentially limiting the use of this FXR-based therapy. Various (synthetic) FXR-agonists have differential capacities to stimulate transhepatic cholesterol flux and/or promote cholesterol efflux via TICE, the latter being mainly mediated by ABCG5/8 (104-105).
Inhibition of intestinal bile salt uptake specifically via ASBT is recognized for its LDL-cholesterol lowering effect (106), relevant for treatment of hyperLDL-cholesterolemia. Preclinical studies further showed that ASBT-inhibition reduces hepatic triglyceride and cholesterol accumulation in high fat diet-fed mice (107-108). ASBT-inhibition in steatohepatitis (NASH) patients is currently being investigated in phase II clinical trial (https://clinicaltrials.gov/ct2/ show/NTC02787304). Similarly, interrupting bile salt return to the liver by surgical creation of ileal bypass or by administration of bile salt binding resins, including cholestyramine
induced fecal bile salt excretion can provoke gastrointestinal side effects (i.e. bloating and diarrhoea) and cause non-compliance to therapy. Spillover of (more hydrophobic) bile acids might cause gut microbiome dysbiosis during ASBT-inhibition. ASBT-deficient mice treated with azoxymethane had a 2-fold increase in colonic adenocarcinomas (110), which should carefully be considered in long-term human studies.
II) Relationship of bile salts with glucose metabolism
The characterization of the bile salt receptors FXR and TGR5 boosted recognition of bile salts as hormones in the control of glucose metabolism. Both FXR and TGR5 are expressed in intestinal endocrine L-cells, but also in other metabolically active tissues (e.g. brown adipocytes, immune cells and myocytes). FXR-signaling and functional consequences are especially complex, and results depend on the use of various (endogenous) bile salt species, synthetic ligands, tissue-specific pharmacotherapy and the crosstalk between FXR and TGR5 (111-115). Bile salt binding resins have a beneficial effect on glucose handling, recognized several decades ago. For example, long-term treatment with the bile salt sequestrant Colestilan induces GLP1 release from the colon (116). GLP1 increases insulin secretion and improves insulin sensitivity. Similarly, colesevelam improved glucose homeostasis. This effect seems mostly mediated by TGR5 activation (116-118), and partially through inhibition of FXR signaling (119). Bile salt-induced intestinal GLP1 release occurs upon activation of TGR5 at the basolateral (blood) side of intestinal L-cells (120, 121). Nevertheless, ASBT inhibitors and bile salt binding resins both stimulate release of enterohepatic hormones, including GLP-1, and on the other hand inhibit the uptake of bile salts into the circulation. In our view, the most direct explanation for this apparent discrepancy is that TGR5-mediated effects occur after (passive) bile salt translocation to the basolateral side of the colonocyte (Figure 2). ASBT-inhibition and bile salt sequestration results in increased presence of bile salt in the colon. Bile salt sequestrants do not covalently bind bile salts, and some diffusion is still likely. The secondary bile acids lithocholic and deoxycholic acid, (and their corresponding taurine and glycine conjugates) are most potent activators of TGR5 in vitro, so local elevation of these bile salt species could stimulate GLP-1 release into the circulation.
At present, it is unclear whether elevated bile salt levels in the systemic circulation would stimulate GLP-1 secretion. Secondary bile acids, produced in the colon, potently boost TGR5-GLP1 signaling (5, 122). Thomas et al. demonstrated that the positive effects on glucose homeostasis of the CA-derived TGR5 agonist INT-777 were also mediated by intestinal TGR5 (117). However, INT-INT-777 was provided orally in this study, so TGR5 might have been activated from either the systemic circulation or by local diffusion across the colonic epithelium.
Ch
ap
ter 1
III) New perspective: systemic bile salts to improve whole-body metabolism
In general, little is known about the influence of endogenous circulating bile salts on metabolic processes, and whether inhibition of hepatic bile salt (re)uptake boosts energy expenditure and/or lipid metabolism. A pivotal study showed that cholate-feeding activates TGR5 and attenuates diet-induced obesity in mice (123). Activation of TGR5 by bile salts is linked to increased energy expenditure in brown adipose tissue and muscle (117). We postulate that such effects are mimicked by (transiently) increased bile salts levels that would occur with NTCP-inhibition, which is under investigation in our group (Donkers et al. unpublished data). This process could also play a role in the beneficial metabolic consequences of bariatric surgery. Bariatric procedures might induce bile salt signaling by increased circulating bile salt levels, as shown in several human studies (124-126). Previously, ileal interposition surgery in mice showed strongly elevated plasma bile salt levels (127), without changes in hepatic Ntcp mRNA. Beneficial effects of vertical sleeve gastrectomy (VSG) on body weight and glucose tolerance are dependent on both FXR (128) and TGR5 (129), suggesting that bile salt signaling is indeed relevant. Interestingly, mRNA-seq in VSG mice showed significant downregulation of hepatic Ntcp and Oatp1b2, possibly explaining the increase in bile salt levels in plasma (130).
What happens to the liver when NTCP is inhibited and/or bile salt levels in the systemic circulation increase? TGR5 is not detected in hepatocytes, but is expressed in sinusoidal endothelial cells where its activation has a hepatoprotective role by inducing nitric oxide synthase in a cAMP dependent manner (131). Furthermore, TGR5 activation in Kupffer cells decreases release of proinflammatory cytokines IL-6 and TNF-α (131-132) and (more generally) dampens macrophage-mediated inflammation by inhibiting the NFκB-pathway (133). A high-fat diet caused more liver steatosis in male TGR5-knockout mice (134), indicating that TGR5 activation prevents NAFLD. Indeed, INT-777 reduces liver fatty acid and triglyceride content as well as plasma triglycerides (117). A beautiful study using glucocorticoid receptor deficient mice pointed to a contribution of NTCP-governed bile salt dynamics to metabolism (135). These mice showed impaired hepatic bile salt uptake by downregulation of NTCP, which reduces dietary fat absorption and increases brown adipose tissue mitochondrial uncoupling. Reduced bile salt uptake via NTCP could also dampen hepatic FXR activation. The (metabolic) consequences of this are currently not clear, although some first insight were obtained using a mouse model, where human hepatocytes repopulated the liver of urokinase plasminogen activator/severe combined immunodeficiency mice. NTCP-inhibition using myrcludex B resulted in increased CYP7A1 expression, suggesting reduced FXR activity (136). Chronic HBV infection in these humanized mice had a similar effect. Further studies are required to assess the (metabolic) consequences of NTCP-inhibition, as effects on lipid,
Liver Intestine NTCP OATP BA BA BA Kidney BA CYP7A1 cholesterol
[BA]
Bsep OSTαβ ASBT Ileum Colon ASBT-inhibitors Gallbladder FGF15/19 BAT TGR5 GLP1 WAT FGFR1c Muscle TGR5 Gallbladder NTCP-inhibition: plasmaFigure 2
A
B
ASB T TGR5 BAFigure 2: Enterohepatic circulation of bile salts
(A) Overview of the distinct hepatic and intestinal bile salt transporters. The key-transporters are BSEP, ASBT, OSTα/β, OATP and NTCP (schematically depicted). Upon intracellular bile salt sensing, FXR/SHP and FGF15/19 become activated and regulate bile salt synthesis (i.e. pre-dominantly CYP7A1). Furthermore, spillover of bile salts into the systemic circulation might also activate the TGR5 receptor, present on the basolateral side in various tissues, such as brown adipose tissue (BAT), muscle and in the colon.
(B) Pharmacological inhibition of ileal bile salt uptake (by bile salt binding resins or ASBT inhibitors) induce the presence of bile salts in the colon and increase fecal bile salt loss. The latter contributes to cholesterol catabolism. Increased GLP1 release in the distal intestine improves systemic glucose handling and is likely induced by (passive) translocation of bile salts leading to basolateral stimulation of TGR5.
Ch
ap
ter 1
Aims and outline of the thesis
As stated before, conjugated bile salts are present in relatively low concentrations in blood and transiently peak when a meal is ingested. Efficient (re)absorption by NTCP in the liver prevents conjugated bile salt concentrations in blood to rise above 10 µM. To better understand the physiological function of NTCP within the enterohepatic circulation, we generated NTCP knockout mice and provided their initial phenotyping. This is described in Chapter 2. Plasma bile salt levels are elevated in a subset of adult mice, however bile salt clearance from the systemic circulation is clearly reduced in all NTCP knockout mice. We confirmed liver-specific pharmacological targeting of NTCP in mice using myrcludex B, a HBV-derived synthetic peptide that binds to NTCP. The interesting phenotype of elevated systemic bile salt levels only in a subset of NTCP-deficient mice led to a more detailed investigation of the role of OATPs in bile salt transport in mice. Myrcludex B was used as a potent NTCP-inhibitor in vivo, causing transient conjugated bile salts elevations in plasma when Oatp1a/1b-isoforms were lacking. This is presented in chapter 3, demonstrating conjugated bile salt uptake capacity of multiple murine OATPs. The high bile salts levels in blood and dynamic regulation of bile salt synthesis in our NTCP-inhibition mouse models prompted us to investigate bile salt transport and signalling in enterocytes. We found evidence that high levels of conjugated bile salts activate the intestinal Fxr-FGF15/19 axis, likely after transport across the basolateral side of the enterocyte. The endocrine hormone FGF19 directly modulates expression of hepatic bile salt transporters (chapter 3). In chapter 4, we propose NTCP-inhibition as a new strategy to reduce cholestatic liver injury. The benefits and limitations of this strategy were investigated in four distinct mouse models of cholestasis. Our results clearly show that NTCP-inhibition protects the hepatobiliary system by reducing intrahepatic bile salt accumulation. However, the effects were not evident during complete obstructive cholestasis (BDL) nor when phospholipid excretion was impaired (PFIC3). Chapter 5 shows how cholesterol transport in the liver is affected during NTCP-inhibition and which hepatic transporter(s) or mechanism(s) might be involved in this phenomenon. Our data suggest that NTCP-inhibition stimulates biliary phospholipid and cholesterol excretion. NTCP sits at the intersection of bile salt metabolism and HBV/ HDV virology. Chapter 6 is a general discussion of the major findings observed upon NTCP-inhibition in mice. We challenge the concept that elevated conjugated bile salt levels in blood are always harmful. Overall, the experiments improve our understanding of the physiological consequences of prolonged bile salt signalling, with a focus on hepatobiliary and intestinal system. Future research should focus on strategies to inhibit NTCP in defined settings of human cholestasis and/or metabolic dysfunction.
References
1. Jelinek DF, Andersson S, Slaughter
CA et al. Cloning and regulation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. J Biol Chem. 1990 May 15;265(14):8190-7.
2. Russell DW. The enzymes,
regulation, and genetics of bile acid synthesis. Annu Rev Biochem 2003;72:137-174.
3. Takahashi S, Fukami T, Masuo Y
et al. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans. J Lipid Res. 2016 Dec; 57(12): 2130–2137
4. Ridlon JM, Kang DJ, Hylemon
PB. Bile salt biotransformations by human intestinal bacteria. J Lipid Res. 2006 Feb;47(2):241-59.
5. Wahlström A, Sayin SI, Marschall
HUet al. Intestinal Crosstalk between Bile
Acids and Microbiota and Its Impact on Host Metabolism. Cell Metab. 2016 Jul 12;24(1):41-50
6. Esteller A. Physiology of bile
secretion. World J Gastroenterol 2008;14:5641-5649.
7. JL Boyer. Bile formation and
secretion. Compr Physiol. 2013 Jul;3(3):1035-78.
8. Dawson PA, Karpen SJ. Intestinal
transport and metabolism of bile acids. J Lipid Res 2015;56:1085-1099.
9. Hofmann AF. The enterohepatic
circulation of bile acids in mammals: form and functions. Front Biosci (Landmark Ed) 2009;14:2584-2598.
10. Vlahcevic ZR, Pandak WM, Stravitz
RT. Regulation of bile acid biosynthesis. Gastroenterol Clin North Am 1999;28:1-25, v.
11. Meier PJ, Stieger B. Bile salt
transporters. Annu Rev Physiol 2002;64:635-661.
12. Kullak-Ublick GA, Stieger B, Meier
PJ. Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 2004;126:322-342.
13. Angelin B, Bjorkhem I, Einarsson
K et al. Hepatic uptake of bile acids in man. Fasting and postprandial concentrations of individual bile acids in portal venous and systemic blood serum. J Clin Invest 1982;70:724-731.
14. Linnet K. Postprandial plasma
concentrations of glycine and taurine conjugated bile acids in healthy subjects. Gut 1983;24:249-252.
15. Meier PJ. Molecular mechanisms
of hepatic bile salt transport from sinusoidal blood into bile. Am J Physiol 1995;269:G801-812.
16. Kim JY, Kim KH, Lee
JA. Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells. Gastroenterology. 2002 Jun;122(7):1941-53.
17. Ferdek PE, Jakubowska
MA, Gerasimenko JV et al. Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium-driven bile uptake. J Physiol. 2016 Nov 1;594(21):6147-6164.
18. Bijsmans IT, Bouwmeester RA, Geyer
J et al. Homo- and heterodimeric architecture
of the human liver Na+-dependent taurocholate
cotransporting protein NTCP. The Biochemical journal 2012;441.
19. Hu NJ, Iwata S, Cameron AD et al.
Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT. Nature 2011;478:408-411.
20. Stieger B. The role of the
sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation. Handb Exp Pharmacol 2011:205-259.
Ch
ap
ter 1
Modulation by drugs of human hepatic sodium-dependent bile acid transporter (sodium taurocholate cotransporting polypeptide) activity. The Journal of pharmacology and experimental therapeutics 1999;291:1204-1209.
22. Kersseboom S, van Gucht
ALM, van Mullem Aet al. Role of the Bile
Acid Transporter SLC10A1 in Liver Targeting of the Lipid-Lowering Thyroid Hormone Analog Eprotirome. Endocrinology. 2017 Oct 1;158(10):3307-3318.
23. Donkers JM, Zehnder B, van Westen
GJPet al. Reduced hepatitis B and D viral
entry using clinically applied drugs as novel inhibitors of the bile acid transporter NTCP. Sci Rep. 2017 Nov 10;7(1):15307.
24. Groothuis GM, Hardonk MJ,
Keulemans KP et al. Autoradiographic and kinetic demonstration of acinar heterogeneity of taurocholate transport. Am J Physiol 1982;243:G455-462.
25. Ho RH, Leake BF, Roberts RL et
al. Ethnicity-dependent polymorphism in Na+-taurocholate cotransporting polypeptide (SLC10A1) reveals a domain critical for bile acid substrate recognition. J Biol Chem 2004;279:7213-7222.
26. Ho RH, Tirona RG, Leake BF et al.
Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression,
and pharmacogenetics. Gastroenterology 2006;130:1793–1806.
27. Vaz FM, Paulusma CC, Huidekoper
H et al. Sodium taurocholate cotransporting polypeptide (SLC10A1) deficiency: conjugated hypercholanemia without a clear clinical phenotype. Hepatology 2014.
28. Liu R, Chen C, Xia X et al.
Homozygous p.Ser267Phe in SLC10A1 is associated with a new type of hypercholanemia and implications for personalized medicine. Sci
hepatocytes is mediated by a protein similar to microsomal epoxide hydrolase. Am J Physiol 1993;264:G528-534.
30. Honscha W, Platte HD, Oesch F et al.
Relationship between the microsomal epoxide hydrolase and the hepatocellular transport of bile acids and xenobiotics. Biochem J 1995;311 ( Pt 3):975-979.
31. Miyata M, Kudo G, Lee YH Targeted
disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.. J Biol Chem. 1999 Aug 20;274(34):23963-8.
32. Kullak-Ublick GA, Stieger B,
Hagenbuch B et al. Hepatic transport of bile salts. Semin Liver Dis 2000;20:273-292.
33. Reichel C, Gao B, Van Montfoort J.
Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver. Gastroenterology. 1999 Sep;117(3):688-95.
34. Klaassen CD, Aleksunes LM.
Xenobiotic, bile acid, and cholesterol transporters: function and regulation. Pharmacol Rev 2010;62:1-96.
35. van de Steeg E, Stranecky V,
Hartmannova H et al. Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J Clin Invest 2012;122:519-528.
36. van de Steeg E, Wagenaar E, van der
Kruijssen CM et al. Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs. J Clin Invest 2010;120:2942-2952.
37. Csanaky IL, Lu H, Zhang Y, Ogura K
et al. Organic anion-transporting polypeptide 1b2 (Oatp1b2) is important for the hepatic uptake of unconjugated bile acids: Studies in
Characterization of organic anion-transporting polypeptide (Oatp) 1a1 and 1a4 null mice reveals altered transport function and urinary metabolomic profiles. Toxicol Sci 2011;122:587-597.
39. Zhang Y, Csanaky IL, Selwyn FP et
al. Organic anion-transporting polypeptide 1a4 (Oatp1a4) is important for secondary bile acid metabolism. Biochem Pharmacol 2013;86:437-445.
40. Yan H, Zhong G, Xu G et al. Sodium
taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 2012;1:e00049.
41. Bogomolov P, Alexandrov A,
Voronkova N, et al. Treatment of chronic hepatitis D with the entry inhibitor myrcludex B - first results of a Phase Ib/IIa study. J Hepatol 2016;65:490-498.
42. Blank A, Markert C, Hohmann N et
al. First-in-human application of the first-in-class hepatitis B and hepatitis D virus entry inhibitor myrcludex B. J Hepatol 2016.
43. Schulze A, Schieck A, Ni Y et al.
Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol 2010;84:1989-2000.
44. Urban S, Bartenschlager R, Kubitz
R et al. Strategies to inhibit entry of HBV and HDV into hepatocytes. Gastroenterology. 2014 Jul;147(1):48-64.
45. Nkongolo S, Ni Y, Lempp FA et al.
Cyclosporin A inhibits hepatitis B and hepatitis D virus entry by cyclophilin-independent interference with the NTCP receptor. J Hepatol. 2014 Apr;60(4):723-31.
46. Schieck A, Schulze A, Gahler C, et al.
Hepatitis B virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts. Hepatology 2013;58:43-53.
47. Slijepcevic D, Kaufman C, Wichers
CG et al. Impaired uptake of conjugated bile
acids and hepatitis b virus pres1-binding in na -taurocholate cotransporting polypeptide knockout mice. Hepatology 2015;62:207-219.
48. Haag M, Hofmann U, Mürdter TE
et al. Quantitative bile acid profiling by liquid chromatography quadrupole time-of-flight mass spectrometry: monitoring hepatitis B therapy by a novel Na(+)-taurocholate cotransporting polypeptide inhibitor. Anal Bioanal Chem. 2015 Sep;407(22):6815-25.
49. Smit JJ, Schinkel AH, Oude Elferink
RP et al. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993 Nov 5;75(3):451-62.
50. Wang R, Lam P, Liu L
et al. Severe cholestasis induced by cholic acid feeding in knockout mice of sister of P-glycoprotein. Hepatology. 2003 Dec;38(6):1489-99
51. Reichert MC, Hall RA, Krawczyk Met
al. Genetic determinants of cholangiopathies: Molecular and systems genetics. Biochim Biophys Acta. 2018 Apr;1864(4 Pt B):1484-1490.
52. Beuers U, Trauner M, Jansen P et
al. New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond. J Hepatol 2015;62:S25-37.
53. Trauner M, Fuchs CD, Halilbasic
E et al. New therapeutic concepts in bile acid transport and signaling for management of cholestasis. Hepatology. 2017 Apr;65(4):1393-1404.
54. Eloranta JJ, Kullak-Ublick GA.
Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch Biochem Biophys 2005;433:397-412.
55. Makishima M, Okamoto AY, Repa JJ
et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362-1365.
56. Parks DJ, Blanchard SG, Bledsoe RK
et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365-1368.
Ch
ap
ter 1
57. Wang H, Chen J, Hollister K et
al. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Molecular cell 1999;3:543-553.
58. Goodwin B, Jones SA, Price RR et al.
A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 2000;6:517-526.
59. Lu TT, Makishima M, Repa JJ et al.
Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 2000;6:507-515.
60. Pellicciari R, Fiorucci S, Camaioni
E et al. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J Med Chem 2002;45:3569-3572.
61. Liu Y, Binz J, Numerick MJ et al.
Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest 2003;112:1678-1687.
62. Hirschfield GM, Mason A, Luketic
V et al. Efficacy of obeticholic acid in patients with primary biliary cirrhosis and inadequate response to ursodeoxycholic acid. Gastroenterology 2015;148:751-761 e758.
63. Nevens F, Lindor KD, Jones DE.
Obeticholic Acid in Primary Biliary Cholangitis. N Engl J Med. 2016 Nov 17;375(20):e41.
64. Slitt AL, Allen K, Morrone J et
al. Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis. Biochim Biophys Acta. 2007 Mar;1768(3):637-47.
65. Li L, Meier PJ, Ballatori N. Oatp2
mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol Pharmacol. 2000 Aug;58(2):335-40.
66. Sinal CJ, Tohkin M, Miyata M
et al. Targeted disruption of the nuclear
W et al. The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp. Gastroenterology. 2001 Jul;121(1):140-7.
68. Jung D, Hagenbuch B, Fried M
et al. Role of liver-enriched transcription factors and nuclear receptors in regulating the human, mouse, and rat NTCP gene. Am J Physiol Gastrointest Liver Physiol. 2004 May;286(5):G752-61.
69. Oswald M, Kullak-Ublick
GA, Paumgartner G et al. Expression of hepatic transporters OATP-C and MRP2 in primary sclerosing cholangitis. Liver. 2001 Aug;21(4):247-53.
70. Jung D, Podvinec M, Meyer UA
et al. Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor. Gastroenterology. 2002 Jun;122(7):1954-66.
71. Inagaki T, Choi M, Moschetta A et
al. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell metabolism 2005;2:217-225.
72. Zweers SJ, Booij KA, Komuta M et
al. The human gallbladder secretes fibroblast growth factor 19 into bile: towards defining the role of fibroblast growth factor 19 in the enterobiliary tract. Hepatology. 2012 Feb;55(2):575-83.
73. Modica S, Petruzzelli M, Bellafante
E et al. Selective activation of nuclear bile acid receptor FXR in the intestine protects mice against cholestasis. Gastroenterology 2012;142:355-365 e351-354.
74. Luo J, Ko B, Elliott M et al. A
nontumorigenic variant of FGF19 treats cholestatic liver diseases. Sci Transl Med 2014;6:247ra100.
75. Zhou M, Learned RM, Rossi SJ et
76. Mayo MJ, Wigg AJ, Roberts SK et al. NGM282, A Novel Variant of FGF-19, Demonstrates biologic activity in primary biliary cirrhosis patients with an incomplete response to ursodeoxycholic acid: results of a phase 2 multicenter, randomized, double blinded, placebo controlled trial. Hepatology (Suppl 1)2015;62:74A.
77. Kuiper EM, van Erpecum KJ, Beuers
U et al. The potent bile acid sequestrant colesevelam is not effective in cholestatic pruritus: results of a double-blind, randomized, placebo-controlled trial. Hepatology
2010;52:1334-1340.
78. Graffner H, Gillberg PG, Rikner L
et al. The ileal bile acid transporter inhibitor A4250 decreases serum bile acids by interrupting the enterohepatic circulation. Aliment Pharmacol Ther 2016;43:303-310.
79. Baghdasaryan A, Fuchs CD,
Osterreicher CH et al. Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis. J Hepatol 2016;64:674-681.
80. Miethke AG, Zhang W, Simmons J et
al. Pharmacological inhibition of apical sodium-dependent bile acid transporter changes bile composition and blocks progression of sclerosing cholangitis in multidrug resistance 2 knockout mice. Hepatology 2016;63:512-523.
81. Hegade VS, Kendrick SF, Dobbins
RLet al. Effect of ileal bile acid transporter
inhibitor GSK2330672 on pruritus in primary biliary cholangitis: a double-blind, randomised, placebo-controlled, crossover, phase 2a study. Lancet. 2017 Mar 18;389(10074):1114-1123.
82. Zhang Y, Csanaky IL,
Lehman-McKeeman LD et al. Loss of organic anion transporting polypeptide 1a1 increases deoxycholic acid absorption in mice by increasing intestinal permeability. Toxicol Sci 2011;124:251-260.
83. Schaap FG, Trauner M, Jansen
PL. Bile acid receptors as targets for drug development. Nat Rev Gastroenterol Hepatol 2014;11:55-67.
84. de Aguiar Vallim TQ, Tarling EJ,
Edwards PA. Pleiotropic roles of bile acids in metabolism. Cell Metab 2013;17:657-669.
85. Jakulj L, van Dijk TH, de Boer JF
et al. Transintestinal Cholesterol Transport Is Active in Mice and Humans and Controls Ezetimibe-Induced Fecal Neutral Sterol Excretion. Cell Metab. 2016 Dec 13;24(6):783-794.
86. Grundy SM. Cholesterol metabolism
in man. West J Med. 1978 Jan;128(1):13-25.
87. Rigotti A, Trigatti BL, Penman M
et al. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. PNAS 1997 Nov 11;94(23):12610-5.
88. Krieger M. Scavenger receptor class
B type I is a multiligand HDL receptor that influences diverse physiologic systems. J Clin Invest. 2001 Sep;108(6):793-7.
89. Goldstein JL, Brown MS. Regulation
of the mevalonate pathway. Nature 1990; 343, 425-430.
90. Botham KM, Bravo E. The role
of lipoprotein cholesterol in biliary steroid secretion. Studies with in vivo experimental models. Prog Lipid Res. 1995;34(1):71-97.
91. Robins SJ, Fasulo JM. High density
lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile. J Clin Invest. 1997 Feb 1;99(3):380-4.
92. Berge KE, Tian H, Graf GA et
al. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science. 2000 Dec 1;290(5497):1771-5.
93. Portincasa P, Moschetta
A, Palasciano G. Cholesterol gallstone disease. Lancet. 2006 Jul 15;368(9531):230-9.
Ch
ap
ter 1
Bile salt hydrophobicity influences cholesterol recruitment from rat liver in vivo when cholesterol synthesis and lipoprotein uptake are constant. Gastroenterology. 1988 Sep;95(3):771-9.
95. Oude Elferink RP, Paulusma
CC, Groen AK. Hepatocanalicular transport defects: pathophysiologic mechanisms of rare diseases.
Gastroenterology. 2006 Mar;130(3):908-25.
96. Langheim S, Yu L, von Bergmann
K et al. ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile. J Lipid Res. 2005 Aug;46(8):1732-8.
97. Musso G, Gambino R, Cassader M.
Cholesterol metabolism and the pathogenesis of non-alcoholic steatohepatitis. Progress in Lipid Research 52 (2013) 175–191
98. Arguello G, Balboa E, Arrese M et
al. Recent insights on the role of cholesterol in non-alcoholic fatty liver disease. Biochim Biophys Acta. 2015 Sep;1852(9):1765-78.
99. Horton JD, Goldstein JL, Brown MS.
SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest. 2002 May;109(9):1125-31.
100. Repa JJ, Berge KE, Pomajzl C et
al. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem. 2002 May 24;277(21):18793-800.
101. Huang L, Zhao A, Lew JL et al.
Farnesoid X receptor activates transcription of the phospholipid pump MDR3. J Biol Chem. 2003 Dec 19;278(51):51085-90.
102. Peet DJ, Turley SD, Ma W et al.
Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 1998. 93:693-704.
103. Neuschwander-Tetri BA, Loomba R,
Sanyal AJ et al. Farnesoid X nuclear receptor
trial. Lancet. 2015 Mar 14;385(9972):956-65.
104. Hambruch E, Miyazaki-Anzai
S, Hahn U et al. Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (-/-) mice. J of Phar and Exp Ther 2012, 343 (3) 556-567.
105. de Boer JF, Schonewille M,
Boesjes M et al. Intestinal Farnesoid X Receptor Controls Transintestinal Cholesterol Excretion in Mice.
Gastroenterology. 2017 Apr;152(5):1126-1138. e6.
106. Rudling M, Camilleri M, Graffner
H et al. Specific inhibition of bile acid transport alters plasma lipids and GLP-1. BMC Cardiovasc Disord 2015;15:75.
107. Lundasen T, Andersson EM, Snaith
M et al. Inhibition of intestinal bile acid transporter Slc10a2 improves triglyceride metabolism and normalizes elevated plasma glucose levels in mice. PLoS One 2012;7:e37787.
108. Rao A, Kosters A, Mells J et al.
Administration of an Ileal Apical Sodium-dependent Bile Acid Transporter Inhibitor Protects Against Non-alcoholic Fatty Liver Disease in High Fat Diet-fed Mice. Hepatology 2015;62:217A.
109. Staels B, Fonseca VA. Bile acids and
metabolic regulation: mechanisms and clinical responses to bile acid sequestration. Diabetes Care. 2009 Nov;32 Suppl 2:S237-45.
110. Raufman JP, Dawson PA, Rao
A et al. Slc10a2-null mice uncover colon cancer-promoting actions of endogenous fecal bile acids. Carcinogenesis. 2015 October; 36(10):1193–1200.
Med. 2015 Feb;21(2):159-65.
112. Jiang C, Xie C, Li F et al. Intestinal
farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. J Clin Invest. 2015 Jan;125(1):386-402.
113. Trabelsi MS, Daoudi M, Prawitt J et
al. Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells. Nat Commun. 2015 Jul 2;6:7629.
114. Pathak P, Liu H, Boehme S et al.
Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism. J Biol Chem. 2017 Jun 30;292(26):11055-11069.
115. Pathak P, Xie C, Nichols RG et al.
Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism. Hepatology. 2018 Feb 27. doi: 10.1002/ hep.29857.
116. Harach T, Pols TW, Nomura M,
Maida A et al. TGR5 potentiates GLP-1 secretion in response to anionic exchange resins. Sci Rep 2012;2:430.
117. Thomas C, Gioiello A, Noriega
L et al. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell metabolism 2009;10:167-177.
118. Potthoff MJ, Potts A, He T et al.
Colesevelam suppresses hepatic glycogenolysis by TGR5-mediated induction of GLP-1 action in DIO mice. Am J Physiol Gastrointest Liver Physiol 2013;304:G371-380.
119. Nwose OM, Jones MR. Atypical
mechanism of glucose modulation by colesevelam in patients with type 2 diabetes. Clin Med Insights Endocrinol Diabetes 2013;6:75-79.
120. Ullmer C, Alvarez Sanchez R,
Sprecher U et al. Systemic bile acid sensing by G protein-coupled bile acid receptor 1 (GPBAR1) promotes PYY and GLP-1 release. Br J Pharmacol 2013;169:671-684.
121. Brighton CA, Rievaj J, Kuhre
RE et al. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid
Receptors. Endocrinology 2015;156:3961-3970.
122. Kuipers F, Bloks VW, Groen
AKBeyond intestinal soap--bile acids in
metabolic control. Nat Rev Endocrinol. 2014 Aug;10(8):488-98.
123. Watanabe M, Houten SM, Mataki
C et al. Bile acids induce energy expenditure by promoting intracellular thyroid
hormone activation. Nature. 2006 Jan 26;439(7075):484-9.
124. Pournaras DJ, Glicksman C, Vincent
RP et al. The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control. Endocrinology 2012;153:3613-3619.
125. Cummings BP, Bettaieb A,
Graham JL et al. Vertical sleeve gastrectomy improves glucose and lipid metabolism and delays diabetes onset in UCD-T2DM rats. Endocrinology 2012;153:3620-3632.
126. Risstad H, Kristinsson JA, Fagerland
MW et al. Bile acid profiles over 5 years after gastric bypass and duodenal switch: results from a randomized clinical trial. Surg Obes Relat Dis. 2017 Sep;13(9):1544-1553.
127. Kohli R, Kirby M, Setchell KD et al.
Intestinal adaptation after ileal interposition surgery increases bile acid recycling and protects against obesity-related comorbidities. Am J Physiol Gastrointest Liver Physiol 2010;299:G652-660.
128. Ryan KK, Tremaroli V, Clemmensen
C et al. FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature 2014;509:183-188.
129. Ding L, Sousa KM, Jin L, Dong B
et al. Vertical Sleeve Gastrectomy Activates GPBAR-1/TGR5 to Sustain Weight Loss, Improve Fatty Liver, and Remit Insulin Resistance in Mice. Hepatology 2016.
Ch
ap
ter 1
al. Vertical sleeve gastrectomy reduces hepatic steatosis while increasing serum bile acids in a weight-loss-independent manner. Obesity (Silver Spring) 2014;22:390-400.
131. Keitel V, Reinehr R, Gatsios P et al.
The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells. Hepatology 2007;45:695-704.
132. Calmus Y, Guechot J, Podevin P et
al. Differential effects of chenodeoxycholic and ursodeoxycholic acids on interleukin 1, interleukin 6 and tumor necrosis factor-alpha production by monocytes. Hepatology 1992;16:719-723.
133. Pols TW, Nomura M, Harach T et al.
TGR5 Activation Inhibits Atherosclerosis by Reducing Macrophage Inflammation and Lipid Loading. Cell metabolism 2011;14:747-757.
134. Vassileva G, Hu W, Hoos L, Tetzloff
G et al. Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice. J Endocrinol 2010;205:225-232.
135. Rose AJ, Diaz MB, Reimann A et
al. Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor. Cell metabolism 2011;14:123-130.
136. Oehler N, Volz T, Bhadra OD et
al. Binding of hepatitis B virus to its cellular receptor alters the expression profile of genes of bile acid metabolism. Hepatology 2014;60:1483-1493.
Davor Slijepcevic, Christina Kaufman, Catharina G.K. Wichers,
Eduardo H. Gilglioni, Florian A. Lempp, Suzanne Duijst, Dirk
R. de Waart, Ronald P.J. Oude Elferink, Walter Mier, Bruno
Stieger, Ulrich Beuers, Stephan Urban and Stan F.J. van de Graaf
Hepatology.
2015;62(1):207-19
Impaired uptake of conjugated
bile acids and Hepatitis B
Virus preS1-binding in
Na⁺-taurocholate cotransporting
polypeptide knockout mice
Chapter 2
Abstract
The Na+-taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated
bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. NTCP has recently been recognized as the receptor mediating hepatocyte-specific entry of Hepatitis B (HBV) and Hepatitis Delta viruses (HDV). Myrcludex B, a peptidic inhibitor of HBV entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary
Slc10a1-/- hepatocytes showed absence of sodium-dependent taurocholic acid (TCA) uptake,
whereas sodium-independent TCA uptake was unchanged. In vivo, this presented as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with UDCA. Biliary BA output remained intact, while fecal BA excretion was reduced in
hypercholanemic Slc10a1-/- mice, explained by increased Asbt and Ostα/β expression. These
mice further showed reduced Asbt expression in kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by downregulation of OATP1A1 and upregulation of OATP1A4. Furthermore, sodium-dependent TCA uptake was inhibited
by Myrcludex B in wild-type hepatocytes, while Slc10a1-/- hepatocytes were insensitive
to Myrcludex B. Finally, positron emission tomography showed a complete abrogation
of hepatic binding of labelled Myrcludex B in Slc10a1-/- mice.
Conclusion: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and HBV preS1/Myrcludex B binding in vivo. The NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally
Ch
ap
ter 2
Introduction
During and after a meal, BAs are released into the small intestine, where they facilitate the absorption of dietary fat and fat-soluble vitamins (1). The majority of BAs are reabsorbed in the terminal ileum and transported back to the liver via the portal circulation. At the basolateral membranes of the hepatocytes, BAs are taken up from the portal circulation, are extruded across the canalicular hepatocyte membrane and transported to the gallbladder to complete the enterohepatic circulation (2). Conjugated BAs are transported across the basolateral hepatic membrane via a sodium-dependent pathway, mainly mediated by the sodium taurocholate cotransporting polypeptide (NTCP; gene name SLC10A1) (3, 4), and a sodium-independent pathway. The latter is likely mediated by members of the organic anion transporter superfamily (OATPs), which are also responsible for the hepatocellular uptake of unconjugated BAs (5, 6). NTCP is a liver-specific dimeric transmembrane glycoprotein (7) responsible for the majority of glycine/taurine-conjugated BA uptake in primary hepatocytes (4). Besides its role in BA homeostasis, NTCP is able to transport sulphated steroids, thyroid hormones and xenobiotics (8, 9). Reducing hepatocellular BA uptake is considered as a hepatic defence mechanism to prevent accumulation of potentially toxic BAs within hepatocytes. NTCP is regulated at the transcriptional level by cellular signalling pathways directly or indirectly involving farnesoid X receptor (FXR) and small heterodimer partner (SHP) (10). NTCP is strongly downregulated in conditions that could lead to hepatocellular BA overload, such as cholestasis (11). Recently, interest in NTCP activity and regulation and its contribution to BA homeostasis was boosted immensely when Li and co-workers identified NTCP as the functional cellular receptor permitting HBV and HDV to enter primary human liver cells (12). Their findings were confirmed by others (13), who showed that HBV binding to NTCP is mediated by the myristoylated preS1-domain of the HBV large surface (L)-protein (14). NTCP-mediated virus entry is blocked by Myrcludex B, a synthetic preS1-lipopeptide derived from the HBV L-protein (15). The consequences of prolonged inhibition of NTCP-mediated BA transport in vivo are unknown. This is important to investigate, since Myrcludex B inhibits NTCP-mediated BA uptake at high concentrations (13). Here, we describe the first genetic knockout mouse model to study the consequences of prolonged and complete absence of NTCP. Our findings underscore that NTCP plays a pivotal role in hepatic uptake of conjugated BAs in vivo and is a crucial receptor for the myristoylated preS1-domain of the HBV L-protein.
Materials & methods
