Targeting bile salt homeostasis in biliary diseases.

PURPOSE OF REVIEW: Advances in the understanding of bile salt synthesis, transport and signalling show the potential of modulating bile salt homeostasis as a therapeutic strategy in cholestatic liver diseases. Here, recent developments in (pre)clinical research in this field is summarized and discussed. RECENT FINDINGS: Inhibition of the apical sodium-dependent bile salt transporter (ASBT) and Na + -taurocholate cotransporting polypeptide (NTCP) seems effective against cholestatic liver diseases, as well as Farnesoid X receptor (FXR) agonism or a combination of both. While approved for the treatment of primary biliary cholangitis (PBC) and intrahepatic cholestasis of pregnancy (ICP), ursodeoxycholic acid (UDCA) has retrospectively shown carefully promising results in primary sclerosing cholangitis (PSC). The side chain shortened derivate norUDCA is of further therapeutic interest since its mechanisms of action are independent of the bile salt transport machinery. In the pathogenesis of sclerosing cholangiopathies, a skewed T-cell response with alterations in gut microbiota and bile salt pool compositions are observed. In PSC pathogenesis, the bile salt receptor Takeda G-protein-coupled receptor 5 (TGR5) in cholangiocytes is implicated, whilst in immunoglobulin G4-related cholangitis the autoantigens annexin A11 and laminin 511-E8 are involved in protecting cholangiocytes. SUMMARY: Modulating bile salt homeostasis has proven a promising treatment strategy in models of cholestasis and are continuously being further developed. Confirmatory clinical studies are needed in order to assess the proposed treatment strategies in patients allowing for a broader therapeutic arsenal in the future.

Combined inhibition of bile salt synthesis and intestinal uptake reduces cholestatic liver damage and colonic bile salts in mice.

Background & Aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis. Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control. Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis. Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load. Impact and Implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.

IgG4-related cholangitis - a mimicker of fibrosing and malignant cholangiopathies.

IgG4-related cholangitis (IRC) is the major hepatobiliary manifestation of IgG4-related disease (IgG4-RD), a systemic fibroinflammatory disorder. The pathogenesis of IgG4-RD and IRC is currently viewed as multifactorial, as there is evidence of a genetic predisposition while environmental factors, such as blue-collar work, are major risk factors. Various autoantigens have been described in IgG4-RD, including annexin A11 and laminin 511-E8, proteins which may exert a partially protective function in cholangiocytes by enhancing secretion and barrier function, respectively. For the other recently described autoantigens, galectin-3 and prohibitin 1, a distinct role in cholangiocytes appears less apparent. In relation to these autoantigens, oligoclonal expansions of IgG4+ plasmablasts are present in patients with IRC and disappear upon successful treatment. More recently, specific T-cell subtypes including regulatory T cells, follicular T helper 2 cells, peripheral T helper cells and cytotoxic CD8+ and CD4+ SLAMF7+ T cells have been implicated in the pathogenesis of IgG4-RD. The clinical presentation of IRC often mimics other biliary diseases such as primary sclerosing cholangitis or cholangiocarcinoma, which may lead to inappropriate medical and potentially invalidating surgical interventions. As specific biomarkers are lacking, diagnosis is made according to the HISORt criteria comprising histopathology, imaging, serology, other organ manifestations and response to therapy. Treatment of IRC aims to prevent or alleviate organ damage and to improve symptoms and consists of (i) remission induction, (ii) remission maintenance and (iii) long-term management. Glucocorticosteroids are highly effective for remission induction, after which immunomodulators can be introduced for maintenance of remission as glucocorticosteroid-sparing alternatives. Increased insight into the pathogenesis of IRC will lead to improved diagnosis and novel therapeutic strategies in the future.

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice.

Background & Aims: Non-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues. Methods: Bile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL. Results: After BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology. Conclusions: A reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion. Lay summary: Novel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

Differential and organ-specific functions of organic solute transporter α and ß in experimental cholestasis.

Background & Aims: Organic solute transporter (OST) subunits OSTα and OSTß facilitate bile acid efflux from the enterocyte into the portal circulation. Patients with deficiency of OSTα or OSTß display considerable variation in the level of bile acid malabsorption, chronic diarrhea, and signs of cholestasis. Herein, we generated and characterized a mouse model of OSTß deficiency. Methods: Ostß -/- mice were generated using CRISR/Cas9 and compared to wild-type and Ostα -/- mice. OSTß was re-expressed in livers of Ostß -/- mice using adeno-associated virus serotype 8 vectors. Cholestasis was induced in both models by bile duct ligation (BDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding. Results: Similar to Ostα -/- mice, Ostß -/- mice exhibited elongated small intestines with blunted villi and increased crypt depth. Increased expression levels of ileal Fgf15, and decreased Asbt expression in Ostß -/- mice indicate the accumulation of bile acids in the enterocyte. In contrast to Ostα -/- mice, induction of cholestasis in Ostß -/- mice by BDL or DDC diet led to lower survival rates and severe body weight loss, but an improved liver phenotype. Restoration of hepatic Ostß expression via adeno-associated virus-mediated overexpression did not rescue the phenotype of Ostß -/- mice. Conclusions: OSTß is pivotal for bile acid transport in the ileum and its deficiency leads to an intestinal phenotype similar to Ostα -/- mice, but it exerts distinct effects on survival and the liver phenotype, independent of its expression in the liver. Our findings provide insights into the variable clinical presentation of patients with OSTα and OSTß deficiencies. Lay summary: Organic solute transporter (OST) subunits OSTα and OSTß together facilitate the efflux of conjugated bile acids into the portal circulation. Ostα knockout mice have longer and thicker small intestines and are largely protected against experimental cholestatic liver injury. Herein, we generated and characterized Ostß knockout mice for the first time. Ostα and Ostß knockout mice shared a similar phenotype under normal conditions. However, in cholestasis, Ostß knockout mice had a worsened overall phenotype which indicates a separate and specific role of OSTß, possibly as an interacting partner of other intestinal proteins.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection.

BACKGROUND & AIMS: Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. METHODS: Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. RESULTS: Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. CONCLUSION: Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. LAY SUMMARY: We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Applicability of different cell line-derived dendritic cell-like cells in autophagy research.

BACKGROUND AND AIMS: Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy. METHODS: Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres. RESULTS AND DISCUSSION: All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research. CONCLUSION: The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.

Thiopurines correct the effects of autophagy impairment on intestinal healing - a potential role for ARHGAP18/RhoA.

The ATG16L1 T300A single-nucleotide polymorphism (SNP) is associated with Crohn's disease and causes an autophagy impairment. We have previously shown that this SNP is involved in the migration and hyperactivation of Rac1 in dendritic cells. Mucosal healing, currently the main target for inflammatory bowel disease treatment, depends on restoration of the epithelial barrier and requires appropriate migration of epithelial cells towards and over mucosal lesions. Therefore, we here further investigated the impact of autophagy on epithelial migration. ATG16L1 knockdown was established in the HT29 human colonic epithelial cell line using lentiviral transduction. Migratory capacity was evaluated using scratch assays and RhoAGTP was measured using G-LISA. Immunofluorescent ARHGAP18 and sequestome 1 (SQSTM1; also known as p62) staining was performed on HT29 cells and primary colonic tissue of Crohn's disease patients. We observed that ATG16L1 knockdown cells exhibited decreased autophagy and decreased migration capacity. Furthermore, activity of RhoA was decreased. These characteristics were phenocopied using ATG5 knockdown and pharmacological inhibition of autophagy. The migration defect was dependent on accumulation of SQSTM1 and was alleviated upon SQSTM1 knockdown. Strikingly, thiopurines also mitigated the effects of impaired autophagy. RhoA dysregulation appeared mediated through accumulation of the upstream regulator ARHGAP18, which was observed in cell lines, human foetal organoids and primary colonic tissue. Our results indicate that the ATG16L1 T300A Crohn's disease-associated SNP causes a decrease in migration capacity in epithelial cells, mediated by an increase in SQSTM1 and ARHGAP18 protein and subsequent reduced RhoA activation.

Molecular regulation of the hepatic bile acid uptake transporter and HBV entry receptor NTCP.

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.

Secondary (iso)BAs cooperate with endogenous ligands to activate FXR under physiological and pathological conditions.

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. AIM: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. METHODS: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. RESULTS: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt-/-, Ostα-/-), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. CONCLUSION: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.

Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium-dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na+ -taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na+ -taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium-dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5' splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity.

BACKGROUND & AIMS: Bile acids are important metabolic signaling molecules. Bile acid receptor activation promotes body weight loss and improves glycemic control. The incretin hormone GLP-1 and thyroid hormone activation of T4 to T3 have been suggested as important contributors. Here, we identify the hepatic bile acid uptake transporter Na+ taurocholate co-transporting polypeptide (NTCP) as target to prolong postprandial bile acid signaling. METHODS: Organic anion transporting polypeptide (OATP)1a/1b KO mice with or without reconstitution with human OATP1B1 in the liver were treated with the NTCP inhibitor Myrcludex B for 3.5 weeks after the onset of obesity induced by high fat diet-feeding. Furthermore, radiolabeled T4 was injected to determine the role of NTCP and OATPs in thyroid hormone clearance from plasma. RESULTS: Inhibition of NTCP by Myrcludex B in obese Oatp1a/1b KO mice inhibited hepatic clearance of bile acids from portal and systemic blood, stimulated GLP-1 secretion, reduced body weight, and decreased (hepatic) adiposity. NTCP inhibition did not affect hepatic T4 uptake nor lead to increased thyroid hormone activation. Myrcludex B treatment increased fecal energy output, explaining body weight reductions amongst unaltered food intake and energy expenditure. CONCLUSIONS: Pharmacologically targeting hepatic bile acid uptake to increase bile acid signaling is a novel approach to treat obesity and induce GLP1- secretion.

The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake.

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein-protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.

A Compensatory U1snRNA Partially Rescues FAH Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I.

The elucidation of aberrant splicing mechanisms, frequently associated with disease has led to the development of RNA therapeutics based on the U1snRNA, which is involved in 5' splice site (5'ss) recognition. Studies in cellular models have demonstrated that engineered U1snRNAs can rescue different splicing mutation types. However, the assessment of their correction potential in vivo is limited by the scarcity of animal models with the targetable splicing defects. Here, we challenged the U1snRNA in the FAH5961SB mouse model of hepatic fumarylacetoacetate hydrolase (FAH) deficiency (Hereditary Tyrosinemia type I, HT1) due to the FAH c.706G>A splicing mutation. Through minigene expression studies we selected a compensatory U1snRNA (U1F) that was able to rescue this mutation. Intriguingly, adeno-associated virus-mediated delivery of U1F (AAV8-U1F), but not of U1wt, partially rescued FAH splicing in mouse hepatocytes. Consistently, FAH protein was detectable only in the liver of AAV8-U1F treated mice, which displayed a slightly prolonged survival. Moreover, RNA sequencing revealed the negligible impact of the U1F on the splicing profile and overall gene expression, thus pointing toward gene specificity. These data provide early in vivo proof-of-principle of the correction potential of compensatory U1snRNAs in HTI and encourage further optimization on a therapeutic perspective, and translation to other splicing-defective forms of metabolic diseases.

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium-taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B-mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP-binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8-/- mice. Stimulatory effects of NTCP inhibition were maintained in Sr-b1-/- mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high-density lipoprotein-derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

FDA-drug screening identifies deptropine inhibiting hepatitis E virus involving the NF-κB-RIPK1-caspase axis.

Hepatitis E virus (HEV) infection is the leading cause of acute hepatitis worldwide and can develop into chronic infection in immunocompromised patients, promoting the development of effective antiviral therapies. In this study, we performed a screening of a library containing over 1000 FDA-approved drugs. We have identified deptropine, a classical histamine H1 receptor antagonist used to treat asthmatic symptoms, as a potent inhibitor of HEV replication. The anti-HEV activity of deptropine appears dispensable of the histamine pathway, but requires the inhibition on nuclear factor-κB (NF-κB) activity. This further activates caspase mediated by receptor-interacting protein kinase 1 (RIPK1) to restrict HEV replication. Given deptropine being widely used in the clinic, our results warrant further evaluation of its anti-HEV efficacy in future clinical studies. Importantly, the discovery that NF-κB-RIPK1-caspase pathway interferes with HEV infection reveals new insight of HEV-host interactions.

Identification of FDA-approved drugs targeting the Farnesoid X Receptor.

The farnesoid X receptor (FXR) belongs to the nuclear receptor family and is activated by bile acids. Multiple, chemically rather diverse, FXR agonists have been developed and several of these compounds are currently tested in clinical trials for NAFLD and cholestasis. Here, we investigated possible FXR-agonism or antagonism of existing FDA/EMA-approved drugs. By using our recently developed FRET-sensor, containing the ligand binding domain of FXR (FXR-LBD), 1280 FDA-approved drugs were screened for their ability to activate FXR in living cells using flow cytometry. Fifteen compounds induced the sensor for more than twenty percent above background. Real-time confocal microscopy confirmed that avermectin B1a, gliquidone, nicardipine, bepridil and triclosan activated the FRET sensor within two minutes. These compounds, including fluticasone, increased mRNA expression of FXR target genes OSTα and OSTß in Huh7 cells, and in most cases also of MRP2, SHP and FGF19. Finally, avermectin B1a, gliquidone, nicardipine and bepridil significantly increased IBABP promoter activity in a luciferase reporter assay in a dose-dependent manner. In conclusion, six FDA/EMA-approved drugs currently used in the clinical practice exhibit moderate agonistic FXR activity. This may on the one hand explain (undesired) side-effects, but on the other hand may form an opportunity for polypharmacology.