Inhibition of hepatic bile salt uptake by Bulevirtide reduces atherosclerosis in Oatp1a1-/-Ldlr-/- mice.

Bile salts can strongly influence energy metabolism through systemic signaling, which can be enhanced by inhibiting the hepatic bile salt transporter Na+ taurocholate cotransporting polypeptide (NTCP), thereby delaying hepatic reuptake of bile salts to increase systemic bile salt levels. Bulevirtide is an NTCP inhibitor and was originally developed to prevent NTCP-mediated entry of Hepatitis B and D into hepatocytes. We previously demonstrated that NTCP inhibition lowers body weight, induces glucagon like peptide-1 (GLP1) secretion, and lowers plasma cholesterol levels in murine obesity models. In humans, a genetic loss-of-function variant of NTCP has been associated with reduced plasma cholesterol levels. Here, we aimed to assess if Bulevirtide treatment attenuates atherosclerosis development by treating female Ldlr-/- mice with Bulevirtide or vehicle for 11 weeks. Since this did not result in the expected increase plasma bile salt levels, we generated Oatp1a1-/-Ldlr-/- mice, an atherosclerosis-prone model with human-like hepatic bile salt uptake characteristics. These mice showed delayed plasma clearance of bile salts and elevated bile salt levels upon Bulevirtide treatment. At study endpoint, Bulevirtide-treated female Oatp1a1-/-Ldlr-/- mice had reduced atherosclerotic lesion area in the aortic root that coincided with lowered plasma LDL-c levels, independent of intestinal cholesterol absorption. In conclusion, Bulevirtide, which is considered safe and is EMA-approved for the treatment of Hepatitis D, reduced atherosclerotic lesion area by reducing plasma LDL-c levels. We anticipate that its application may extend to atherosclerotic cardiovascular diseases, which warrants clinical trials.

The Phospholipid Flippase ATP8B1 is Involved in the Pathogenesis of Ulcerative Colitis via Establishment of Intestinal Barrier Function.

AIMS: Patients with mutations in ATP8B1 develop progressive familial intrahepatic cholestasis type 1 [PFIC1], a severe liver disease that requires life-saving liver transplantation. PFIC1 patients also present with gastrointestinal problems, including intestinal inflammation and diarrhoea, which are aggravated after liver transplantation. Here we investigate the intestinal function of ATP8B1 in relation to inflammatory bowel diseases. METHODS: ATP8B1 expression was investigated in intestinal samples of patients with Crohn's disease [CD] or ulcerative colitis [UC] as well as in murine models of intestinal inflammation. Colitis was induced in ATP8B1-deficient mice with dextran sodium sulphate [DSS] and intestinal permeability was investigated. Epithelial barrier function was assessed in ATP8B1 knockdown Caco2-BBE cells. Co-immunoprecipitation experiments were performed in Caco2-BBE cells overexpressing ATP8B1-eGFP. Expression and localization of ATP8B1 and tight junction proteins were investigated in cells and in biopsies of UC and PFIC1 patients. RESULTS: ATP8B1 expression was decreased in UC and DSS-treated mice, and was associated with a decreased tight junctional pathway transcriptional programme. ATP8B1-deficient mice were extremely sensitive to DSS-induced colitis, as evidenced by increased intestinal barrier leakage. ATP8B1 knockdown cells showed delayed barrier establishment that affected Claudin-4 [CLDN4] levels and localization. CLDN4 immunohistochemistry showed a tight junctional staining in control tissue, whereas in UC and intestinal PFIC1 samples, CLDN4 was not properly localized. CONCLUSION: ATP8B1 is important in the establishment of the intestinal barrier. Downregulation of ATP8B1 levels in UC, and subsequent altered localization of tight junctional proteins, including CLDN4, might therefore be an important mechanism in UC pathophysiology.

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.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

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.

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.

Efficacy of AAV8-hUGT1A1 with Rapamycin in neonatal, suckling, and juvenile rats to model treatment in pediatric CNs patients.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation.

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

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.

The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state.

The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.

Fruticuline A, a chemically-defined diterpene, exerts antineoplastic effects in vitro and in vivo by multiple mechanisms.

Natural products have been recognized as important bioactive compounds on the basis of their wide biological properties. Here we investigated the antitumor effect and molecular mechanisms of the diterpene Fruticuline A (fruti) from Salvia lachnostachys, in human cancer cell lineages and Solid Ehrlich Carcinoma in mice. Fruti reduced MCF-7 and HepG2 proliferation by the reduction of Cyclin D1 levels and decreased NF-κB gene levels in both cell types. Furthermore, fruti also induced apoptosis in HepG2 cells, reduced Bcl-2 gene expression and induced necroptosis by increasing Ripk in MCF-7 cells. In mice, fruti prevented tumor development and reduced Cyclin D1, Bcl-2 and Rela gene levels, and reduced the p-NF-κB/NF-κB ratio in tumor tissue. Furthermore, fruti induced necrosis and apoptosis, increased N-acetyl-ß-D-glucosaminidase and TNF-α levels and reduced IL-10 and Vegf levels in tumor tissue. Collectively, fruti exerts antitumor effects through the inhibition of the NF-κB pathway, reducing Cyclin D1 and Bcl-2 levels. In vitro the apoptosis and necroptosis pathways are involved in the cellular death, whereas in vivo, cells undergo necrosis by increased tumor inflammation and reduction of angiogenesis. Thus, fruticuline A acts in tumor cells by multiple mechanisms and represents a promising molecule for drug development in cancer treatment.

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.

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.

Glyco-conjugated bile acids drive the initial metaplastic gland formation from multi-layered glands through crypt-fission in a murine model.

Bile acid reflux is known to be associated with the development of Barrett's esophagus and esophageal adenocarcinoma (EAC), yet the role of specific bile acids and the mechanism behind the metaplastic changes is unclear. Here, we demonstrate that multi-layered glandular structures at the squamo-columnar junction in mice contain multiple cell lineages, which resemble the human esophageal submucosal gland ducts. Exposing mice to patient's refluxates induced expansion of multi-layered glandular structures and development of columnar metaplasia at the squamo-columnar junction. The glycine conjugated bile acids induced an intestinal type of metaplasia more typical for Barrett's esophagus. Through lineage tracing, we excluded the involvement of K5+, DCLK1+, and LGR5+ progenitor cells as the primary source in the development of the glandular metaplastic epithelium. We show that the mechanism behind development of metaplasia involves crypt fission and may be independent of stem cell proliferation. Our findings support the hypothesis that in humans, BE arises from non-squamous cells residing in submucosal gland ducts and that induction of intestinal type of metaplasia is most effectively induced by glycine-conjugated bile acids. These novel insights may lead to more effective strategies to prevent development of Barrett's esophagus and esophageal adenocarcinoma.

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. METHODS: Ten-week-old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry. RESULTS: Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study. CONCLUSION: Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted. LAY SUMMARY: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. The proliferation of liver cells was also reduced, which contributes to long-term correction of the phenotype. Further studies are required to evaluate whether this approach can be applied to patients with PFIC3.

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity.

BACKGROUND/AIMS: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective. METHODS: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription. RESULTS: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible. CONCLUSION: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.

Intestinal Farnesoid X Receptor Activation by Pharmacologic Inhibition of the Organic Solute Transporter α-ß.

BACKGROUND & AIMS: The organic solute transporter α-ß (OSTα-OSTß) mainly facilitates transport of bile acids across the basolateral membrane of ileal enterocytes. Therefore, inhibition of OSTα-OSTß might have similar beneficial metabolic effects as intestine-specific agonists of the major nuclear receptor for bile acids, the farnesoid X receptor (FXR). However, no OSTα-OSTß inhibitors have yet been identified. METHODS: Here, we developed a screen to identify specific inhibitors of OSTα-OSTß using a genetically encoded Förster Resonance Energy Transfer (FRET)-bile acid sensor that enables rapid visualization of bile acid efflux in living cells. RESULTS: As proof of concept, we screened 1280 Food and Drug Administration-approved drugs of the Prestwick chemical library. Clofazimine was the most specific hit for OSTα-OSTß and reduced transcellular transport of taurocholate across Madin-Darby canine kidney epithelial cell monolayers expressing apical sodium bile acid transporter and OSTα-OSTß in a dose-dependent manner. Moreover, pharmacologic inhibition of OSTα-OSTß also moderately increased intracellular taurocholate levels and increased activation of intestinal FXR target genes. Oral administration of clofazimine in mice (transiently) increased intestinal FXR target gene expression, confirming OSTα-OSTß inhibition in vivo. CONCLUSIONS: This study identifies clofazimine as an inhibitor of OSTα-OSTß in vitro and in vivo, validates OSTα-OSTß as a drug target to enhance intestinal bile acid signaling, and confirmed the applicability of the Förster Resonance Energy Transfer-bile acid sensor to screen for inhibitors of bile acid efflux pathways.

Na+ -taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice.

Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+ -Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1-G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC-fed, Atp8b1-G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC-fed and Atp8b1-G308V mice. NTCP-inhibition increased plasma BS levels from 604±277 to 1746±719 µm in DDC-fed mice, 432±280 to 762±288 µm in Atp8b1-G308V mice and from 522±130 to 3625±378 µm in BDL mice. NTCP-inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP-inhibition reduced biliary BS output in DDC-fed and Atp8b1-G308V mice by ∼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP-inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP-inhibition were well tolerated. NTCP-inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).