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Due to the obesity epidemic, non-alcoholic steatohepatitis (NASH) is a prevalent liver disease, characterized by fat accumulation and inflammation of the liver. However, due to a lack of mechanistic insight, diagnostic and therapeutic options for NASH are poor. Recent evidence has indicated cathepsin D (CTSD), a lysosomal enzyme, as a marker for NASH. Here, we investigated the function of CTSD in NASH by using an in vivo and in vitro model. In addition to diminished hepatic inflammation, inhibition of CTSD activity dramatically improved lipid metabolism, as demonstrated by decreased plasma and liver levels of both cholesterol and triglycerides. Mechanistically, CTSD inhibition resulted in an increased conversion of cholesterol into bile acids and an elevated excretion of bile acids via the feces, indicating that CTSD influences lipid metabolism. Consistent with these findings, treating Wt BMDMs with PepA in vitro showed a similar decrease in inflammation and an analogous effect on cholesterol metabolism., Conclusion: CTSD is a key player in the development of hepatic inflammation and dyslipidemia. Therefore, aiming at the inhibition of the activity of CTSD may lead to novel treatments to combat NASH.

Background & Aims: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We aimed to identify novel protein biomarkers of disease severity and prognosis in primary sclerosing cholangitis (PSC)., Methods: Using a bead-based array targeting 63 proteins, we profiled a derivation panel of Norwegian endoscopic retrograde cholangiography bile samples (55 PSC, 20 disease controls) and a Finnish validation panel (34 PSC, 10 disease controls). Selected identified proteins were measured in serum from two Norwegian PSC cohorts (n=167 [1992-2006] and n=138 [2008-2012]), inflammatory bowel disease (n=96) and healthy controls (n=100)., Results: In the bile derivation panel, the levels of 14 proteins were different between PSC patients and controls (p<0.05); all were confirmed in the validation panel. Twenty-four proteins in the bile derivation panel were significantly (p<0.05) different between PSC patients with mild compared to severe cholangiographic changes (modified Amsterdam criteria); this was replicated for 18 proteins in the validation panel. Interleukin (IL)-8, matrix metallopeptidase (MMP)9/lipocalin (LCN)2-complex, S100A8/9, S100A12 and tryptophan hydroxylase (TPH)2 in the bile were associated with both a PSC diagnosis and grade of cholangiographic changes. Stratifying PSC patients according to tertiles of serum IL-8, but not MMP9/LCN2 and S100A12, provided excellent discrimination for transplant-free survival both in the serum derivation and validation cohort. Furthermore, IL-8 was associated with transplant-free survival in multivariable analyses in both serum panels independently of age and disease duration, indicating an independent influence on PSC progression. However, the Enhanced Liver Fibrosis (ELF®) test and Mayo risk score proved to be stronger predictors of transplant-free survival., Conclusions: Based on assaying of biliary proteins, we have identified novel biliary and serum biomarkers as indicators of severity and prognosis in PSC., Lay Summary: Prognostic biomarkers are lacking in primary sclerosing cholangitis, hampering patient care and the development of therapy. We have identified inflammatory proteins including calprotectin and IL-8 as important indicators of disease severity and prognosis in bile and serum from patients with primary sclerosing cholangitis., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: The bile salt-activated transcription factor farnesoid X receptor (FXR) is a key mediator of proliferative bile salt signalling, which is assumed to play a role in the early phase of compensatory liver growth. The aim of this study was to evaluate the effect of a potent FXR agonist (obeticholic acid, OCA) on liver growth following portal vein embolization (PVE)., Methods: Rabbits were allocated to receive daily oral gavage with OCA (10 mg/kg) or vehicle (control group) starting 7 days before PVE (n = 18 per group), and continued until 7 days after PVE. PVE of the cranial liver lobes was performed using polyvinyl alcohol particles and coils on day 0. Caudal liver volume (CLV) was analysed by CT volumetry on days -7, -1, +3 and +7. Liver function was determined by measuring mebrofenin uptake using hepatobiliary scintigraphy. Additional parameters analysed were plasma aminotransferase levels, and histological scoring of haematoxylin and eosin- and Ki-67-stained liver sections., Results: Three days after PVE of the cranial lobes, the increase in CLV was 2·2-fold greater in the OCA group than in controls (mean(s.d.) 56·1(20·3) versus 26·1(15·4) per cent respectively; P < 0·001). This increase remained greater 7 days after PVE (+1·5-fold; P = 0·020). The increase in caudal liver function at day +3 was greater in OCA-treated animals (+1·2-fold; P = 0·017). The number of Ki-67-positive hepatocytes was 1·6-fold higher in OCA-treated animals 3 days after PVE (P = 0·045). Plasma aminotransferase levels and histology did not differ significantly between groups., Conclusion: OCA accelerated liver regeneration after PVE in a rabbit model. OCA treatment might increase the efficacy of PVE and, thereby, resectability. Surgical relevance Liver failure is the most feared complication after liver surgery, with no effective treatment options. Liver regeneration is essential to avoid liver failure, and recently bile acid signalling was implicated in the initiation of liver regeneration through the nuclear bile acid receptor farnesoid X receptor (FXR). In this study, the potent FXR agonist obeticholic acid accelerated liver regeneration following portal vein embolization in a rabbit model, in terms of liver volume, liver function and proliferation. Obeticholic acid treatment could enhance the efficacy of portal vein embolization, thereby increasing resectability, and could reduce the interval to surgery. In addition, obeticholic acid might have a place in the prevention of liver failure after liver surgery., (© 2017 BJS Society Ltd Published by John Wiley & Sons Ltd.)

In this review we develop the argument that cholestatic liver diseases, particularly primary biliary cholangitis and primary sclerosing cholangitis (PSC), evolve over time with anatomically an ascending course of the disease process. The first and early lesions are in "downstream" bile ducts. This eventually leads to cholestasis, and this causes bile salt (BS)-mediated toxic injury of the "upstream" liver parenchyma. BS are toxic in high concentration. These concentrations are present in the canalicular network, bile ducts, and gallbladder. Leakage of bile from this network and ducts could be an important driver of toxicity. The liver has a great capacity to adapt to cholestasis, and this may contribute to a variable symptom-poor interval that is often observed. Current trials with drugs that target BS toxicity are effective in only about 50%-60% of primary biliary cholangitis patients, with no effective therapy in PSC. This motivated us to develop and propose a new view on the pathophysiology of primary biliary cholangitis and PSC in the hope that these new drugs can be used more effectively. These views may lead to better stratification of these diseases and to recommendations on a more "tailored" use of the new therapeutic agents that are currently tested in clinical trials. Apical sodium-dependent BS transporter inhibitors that reduce intestinal BS absorption lower the BS load and are best used in cholestatic patients. The effectiveness of BS synthesis-suppressing drugs, such as farnesoid X receptor agonists, is greatest when optimal adaptation is not yet established. By the time cytochrome P450 7A1 expression is reduced these drugs may be less effective. Anti-inflammatory agents are probably most effective in early disease, while drugs that antagonize BS toxicity, such as ursodeoxycholic acid and nor-ursodeoxycholic acid, may be effective at all disease stages. Endoscopic stenting in PSC should be reserved for situations of intercurrent cholestasis and cholangitis, not for cholestasis in end-stage disease. These are arguments to consider a step-wise pathophysiology for these diseases, with therapy adjusted to disease stage. An obstacle in such an approach is that disease stage-defining biomarkers are still lacking. This review is meant to serve as a call to prioritize the development of biomarkers that help to obtain a better stratification of these diseases. (Hepatology 2017;65:722-738)., (© 2016 by the American Association for the Study of Liver Diseases.)

This study explored whether bacterial endotoxins, in the form of lipopolysaccharides (LPS), could have an injurious effect on the biliary tract in conjunction with ischemia. A total of 64 rats were randomly assigned to 4 groups: sham operation (sham group), 1 mg/kg LPS intraperitoneal (LPS group), hepatic ischemia/reperfusion (IR; IR group), and IR combined with LPS (IR+LPS group). Following 1 or 6 hours of reperfusion, serum liver tests, bile duct histology, immunofluorescence microscopy (zonula occludens-1 [ZO-1]), bile composition (bile salts, phospholipids, lactate dehydrogenase), hepatic gene expression (bile salt transporters and inflammatory mediators), as well as serum and biliary cytokine concentrations were quantified and compared between the study groups. In addition, the integrity of the blood biliary barrier (BBB) was assayed in vivo using horseradish peroxidase (HRP). LPS administration induced severe small bile duct injury following 6 hours of reperfusion. Furthermore, total bile salts and bilirubin concentrations in serum were increased in the LPS groups compared with sham controls (LPS, + 3.3-fold and +1.9-fold; IR+LPS, + 3.8-fold and +1.7-fold, respectively). The BBB was impaired in the LPS groups as evidenced by elevated levels of HRP in bile (+4.9-fold), and decreased expression of claudin 1 (-6.7-fold) and claudin 3 (-3.6-fold). LPS was found to be a potent inducer of small bile duct injury following hepatic ischemia and 6 hours of reperfusion. This injury was associated with increased permeability of the BBB and impaired hepatic bile salt clearance. Liver Transplantation 23 194-206 2017 AASLD., (© 2016 by the American Association for the Study of Liver Diseases.)

Background: Bile salts likely contribute to liver injury in patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Fibroblast growth factor 19 (FGF19) is a bile salt-induced enterokine with hepatoprotective potential as it suppresses de novo bile salt synthesis. Here, we evaluated the bile salt receptor FXR/FGF19 gut-liver axis in PSC and PBC patients., Methods: Fasted patients with PSC (n = 12) and PBC (n = 10), and healthy controls (HC; n = 10) were orally challenged with the natural FXR agonist chenodeoxycholic acid (CDCA 15 mg/kg). Blood was sampled hourly until 8 h afterwards. Serum FGF19 and bile salt excursions were determined. Serum levels of 7α-hydroxy-4-cholesten-3-one (C4), reflecting bile salt synthesis, were measured as a biomarker of FGF19 response., Results: Baseline serum FGF19 levels were comparable between groups, while fasted bile salt levels in PSC patients were elevated. Upon CDCA challenge, HC and PBC patients showed a serum FGF19 peak after 4 h followed by a decline. PSC patients showed a prolonged and elevated serum FGF19 response up to 8 h, combined with a sustained serum elevation of CDCA and other bile salts. In general, C4 levels declined following FGF19 elevation. In PSC patients with less favorable prognosis, baseline C4 levels were drastically suppressed and did not further decline., Conclusion: Following an oral CDCA challenge, PSC patients showed an impaired clearance of CDCA and a prolonged serum FGF19 response. FXR agonist therapy in PSC could cause prolonged exposure to elevated levels of FGF19, and we propose careful monitoring for detrimental side effects in patient studies., Competing Interests: Serge Zweers, Elisabeth de Vries, Martin Lenicek, Dagmar Tolenaars, Rudi de Waart, Kiran Koelfat, Albert Groen, Steven Olde Damink, Ulrich Beuers, Cyriel Ponsioen, Peter Jansen, Frank Schaap declare that they have no conflict of interest. This study was supported by grants from the Dutch Digestive Diseases Foundation (WO#08-69), Dutch Society for Gastroenterology (Gastrostart 2012-10), Norwegian PSC Consortium (to Peter Jansen and Frank Schaap) and the German Crohn’s and Ulcerative Colitis Association (to Ulrich Beuers). Informed consent in studies with human subjects All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study.

Cyclosporine A (CsA) is an undecapeptide with strong immunosuppressant activities and is used a lot after organ transplantation. Furthermore, it may induce cholestasis in the liver. In general, the drug-induced cholestasis (DIC) pathway includes genes involved in the uptake, synthesis, conjugation, and secretion of bile acids. However, whether CsA-induced changes in the cholestasis pathway in vitro are persistent for repeated dose toxicity has not yet been investigated. To explore this, primary human hepatocytes (PHH) were exposed to a subcytotoxic dose of 30 μM CsA daily for 3 and 5 days. To investigate the persistence of induced changes upon terminating CsA exposure after 5 days, a subset of PHH was subjected to a washout period (WO-period) of 3 days. Multiple -omics analyses, comprising whole genome analysis of DNA methylation, gene expression, and microRNA expression, were performed. The CsA-treatment resulted after 3 and 5 days, respectively, in 476 and 20 differentially methylated genes (DMGs), 1353 and 1481 differentially expressed genes (DEGs), and in 22 and 29 differentially expressed microRNAs (DE-miRs). Cholestasis-related pathways appeared induced during CsA-treatment. Interestingly, 828 persistent DEGs and 6 persistent DE-miRs but no persistent DMGs were found after the WO-period. These persistent DEGs and DE-miRs showed concordance for 22 genes. Furthermore, 29 persistent DEGs changed into the same direction as observed in livers from cholestasis patients. None of those 29 DEGs which among others relate to oxidative stress and lipid metabolism are yet present in the DIC pathway or cholestasis adverse outcome pathway (AOP) thus presenting novel findings. In summary, we have demonstrated for the first time a persistent impact of repeated dose administration of CsA on genes and microRNAs related to DIC in the gold standard human liver in vitro model with PHH.

Hepatic failure is a feared complication that accounts for up to 75% of mortality after extensive liver resection. Despite improved perioperative care, the increasing complexity and extensiveness of surgical interventions, in combination with an expanding number of resections in patients with compromised liver function, still results in an incidence of postresectional liver failure (PLF) of 1-9%. Preventive measures aim to enhance future remnant liver size and function. Numerous non-invasive techniques to assess liver function and predict remnant liver volume are being developed, along with introduction of novel surgical strategies that augment growth of the future remnant liver. Detection of PLF is often too late and treatment is primarily symptomatic. Current therapeutic research focuses on ([bio]artificial) liver function support and regenerative medicine. In this review we discuss the current state and new developments in prediction, prevention and management of PLF, in light of novel insights into the aetiology of this complex syndrome., Lay Summary: Liver failure is the main cause of death after partial liver resection for cancer, and is presumably caused by an insufficient quantity and function of the liver remnant. Detection of liver failure is often too late, and current treatment focuses on relieve of symptoms. New research initiatives explore artificial support of liver function and stimulation of regrowth of the remnant liver., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Fibroblast growth factor 19 (FGF19) is an ileum-derived endrocrine factor that is produced in response to transepithelial bile salt flux. FGF19 represses bile salt synthesis in the liver. Despite the general assumption that FGF19 signals to the liver via portal blood, no human data are available to support this notion. The aim was to study portal FGF19 levels, and determined bile salt and FGF19 fluxes across visceral organs in humans. Bile salt and FGF19 levels were assessed in arterial, portal, and hepatic venous blood collected from fasted patients who underwent partial liver resection for colorectal liver metastases (n = 30). Fluxes across the portal-drained viscera (PDV), liver, and splanchnic area were calculated. Portal bile salt levels (7.8 [5.0-12.4] μmol/L) were higher than levels in arterial (2.7 [1.7-5.5] μmol/L, P < 0.0001) and hepatic venous blood (3.4 [2.5-6.5] μmol/L, P < 0.0001). Bile salts released by the PDV (+1.2 [+0.7-+2.0] mmol kg -1  h -1 , P < 0.0001) were largely taken up by the liver (-1.0 [-1.8 to -0.4] mmol kg -1  h -1 , P < 0.0001). Portal levels of FGF19 (161 ± 78 pg/mL) were higher than arterial levels (135 ± 65 pg/mL, P = 0.046). A net release of FGF19 by the PDV (+4.0 [+2.1 to +9.9] ng kg -1  h -1 , P < 0.0001) was calculated. There was no significant flux of FGF19 across the liver (-0.2 [-3.7 to +7.4] ng kg -1  h -1 , P = 0.93). In conclusion, FGF19 levels in human portal blood are higher than in arterial blood. FGF19 is released by the portal-drained viscera under fasted steady state conditions., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Introduction: Zellweger spectrum disorders (ZSDs) are characterized by a failure in peroxisome formation, caused by autosomal recessive mutations in different PEX genes. At least some of the progressive and irreversible clinical abnormalities in patients with a ZSD, particularly liver dysfunction, are likely caused by the accumulation of toxic bile acid intermediates. We investigated whether cholic acid supplementation can suppress bile acid synthesis, reduce accumulation of toxic bile acid intermediates and improve liver function in these patients., Methods: An open label, pretest-posttest design study was conducted including 19 patients with a ZSD. Participants were followed longitudinally during a period of 2.5 years prior to the start of the intervention. Subsequently, all patients received oral cholic acid and were followed during 9 months of treatment. Bile acids, peroxisomal metabolites, liver function and liver stiffness were measured at baseline and 4, 12 and 36 weeks after start of cholic acid treatment., Results: During cholic acid treatment, bile acid synthesis decreased in the majority of patients. Reduced levels of bile acid intermediates were found in plasma and excretion of bile acid intermediates in urine was diminished. In patients with advanced liver disease (n = 4), cholic acid treatment resulted in increased levels of plasma transaminases, bilirubin and cholic acid with only a minor reduction in bile acid intermediates., Conclusions: Oral cholic acid therapy can be used in the majority of patients with a ZSD, leading to at least partial suppression of bile acid synthesis. However, caution is needed in patients with advanced liver disease due to possible hepatotoxic effects., Competing Interests: Compliance with ethics guideline The study was approved by the Ethical Committee of the Academic Medical Center (AMC), Amsterdam, The Netherlands and took place between 2011 and 2014. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Conflict of interest Kevin Berendse, Femke C. C. Klouwer, Bart G.P. Koot, Elles M. Kemper, Sacha Ferdinandusse, Kiran V.K. Koelfat, Martin Lenicek, Frank G. Schaap, Hans R. Waterham, Frédéric Vaz, Marc Engelen, Peter L.M. Jansen, Ronald J.A. Wanders and Bwee Tien Poll-The declare that they have no conflicts of interest. Funding This work was supported by grants from Metakids, Hersenstichting, Axel Foundation and Stichting Steun Emma Kinderziekenhuis AMC, The Netherlands Informed consent Individual written informed consents were obtained from patients and/or patients’ parents.

Introduction: Postoperative liver failure (PLF) is a dreaded complication after partial hepatectomy. The peak bilirubin criterion (>7.0 mg/dL or ≥120 μmol/L) is used to define PLF. This study aimed to validate the peak bilirubin criterion as postoperative risk indicator for 90-day liver-related mortality., Methods: Characteristics of 956 consecutive patients who underwent partial hepatectomy at the Maastricht University Medical Centre or Royal Free London between 2005 and 2012 were analyzed by uni- and multivariable analyses with odds ratios (OR) and 95% confidence intervals (95%CI)., Results: Thirty-five patients (3.7%) met the postoperative peak bilirubin criterion at median day 19 with a median bilirubin level of 183 [121-588] μmol/L. Sensitivity and specificity for liver-related mortality after major hepatectomy were 41.2% and 94.6%, respectively. The positive predictive value was 22.6%. Predictors of liver-related mortality were the peak bilirubin criterion (p < 0.001, OR = 15.9 [95%CI 5.2-48.7]), moderate-severe steatosis and fibrosis (p = 0.013, OR = 8.5 [95%CI 1.6-46.6]), ASA 3-4 (p = 0.047, OR = 3.0 [95%CI 1.0-8.8]) and age (p = 0.044, OR = 1.1 [95%CI 1.0-1.1])., Conclusion: The peak bilirubin criterion has a low sensitivity and positive predictive value for 90-day liver-related mortality after major hepatectomy., (Copyright © 2016 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: To better understand the pathogenesis of primary sclerosing cholangitis, anti- and pro-inflammatory factors were studied in bile., Methods: Ductal bile of PSC patients (n = 36) and controls (n = 20) was collected by endoscopic retrograde cholangiography. Gallbladder bile was collected at liver transplantation. Bile samples were analysed for cytokines, FGF19 and biliary lipids. Hepatobiliary tissues of PSC and non-PSC patients (n = 8-11 per patient group) were collected at transplantation and were analysed for IL8 and FGF19 mRNA expression and IL8 localization. The effect of IL8 on proliferation of primary human cholangiocytes and expression of pro-fibrotic genes was studied., Results: In PSC patients, median IL8 in ductal bile was 6.6 ng/ml vs. 0.24 ng/ml in controls. Median IL8 in gallbladder bile was 7.6 ng/ml in PSC vs. 2.2 and 0.3 ng/ml in two control groups. IL8 mRNA in PSC gallbladder was increased and bile ducts stained positive for IL8. In vitro, IL8 induced proliferation of primary human cholangiocytes and increased the expression of pro-fibrotic genes., Conclusion: Elevation of IL8 in bile of PSC patients, collected at different stages of disease, indicates an ongoing inflammatory stimulus that drives IL8 production. This challenges the idea that advanced PSC is a burned-out disease, and calls for reconsideration of anti-inflammatory therapy in PSC., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background Data: Hepatic sinusoidal dilatation (SD) is a histopathological entity that occurs in up to 75% of patients undergoing oxaliplatin-based chemotherapy for colorectal liver metastases (CRLM)., Objective: To study the influence of SD on outcome after partial hepatectomy in patients with CRLM., Methods: Medline, Embase, CENTRAL, LILACS and CINAHL were searched for studies published between 01.01.2004 and 09.06.2015 with keywords: "sinusoidal obstruction syndrome", "hepatic veno-occlusive disease", and "Stuart-Bras syndrome". Studies comprising adults who underwent partial hepatectomy for CRLM with grading of SD and registration of postoperative morbidity and/or mortality were included. Risk of bias and quality of studies were evaluated with the Quality In Prognosis Studies Instrument (QUIPS) and modified GRADE framework., Results: Search strategies produced 2007 hits from which 23 and 13 articles were extracted for qualitative and quantitative analyses, respectively. Meta-analysis on the influence of SD grade 2-3 vs. SD grade 0-1 on postoperative overall morbidity showed an odds ratio (OR) of 1.26 [95% CI 0.74, 2.15](p = 0.40), an OR of 1.03 [0.15, 6.89](p = 0.98) for liver failure, an OR of 1.21 [0.23, 6.35](p = 0.82) for overall mortality, and an OR of 3.52 [0.31, 39.91](p = 0.31) for liver-related morbidity. QUIPS showed a low to high risk of bias for studies, and GRADE showed very low quality of evidence per outcome., Conclusions: No significant effect of SD grade 2-3 on short-term outcome after partial hepatectomy was found. However, the data on which this conclusion was based were not very robust and therefore no solid conclusions could be drawn., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A growing body of evidence has demonstrated that bile salts are important for liver regeneration following partial hepatectomy. The relative bile salt overload after partial liver resection causes activation of bile salt receptors in non-parenchymal (viz. the plasma membrane receptor TGR5) and parenchymal (viz. the intracellular receptor FXR) cells in the liver, thus, providing signals to the regenerative process. Impaired bile salt signaling in mice with genetic deficiency of Tgr5 or Fxr results in delayed liver regeneration after partial hepatectomy, and is accompanied by mortality in case of Fxr knock-out mice. Conversely, compensatory liver re-growth in hepatectomized mice can be stimulated by feeding of bile salts or alisol B 23-acetate, a natural triterpenoid agonist of Fxr. A large number of animal studies underscore the importance of strict maintenance of bile salt homeostasis for proper progression of liver regeneration. Both ileal and hepatic Fxr play a key role in regulation of bile salt homeostasis and, thus, preventing hepatotoxicity caused by excessive levels of bile salts. They further contribute to liver regeneration by induction of mitogenic factors. Agents that target bile salt receptors hold promise as drugs to stimulate liver regeneration in selected patients., Competing Interests: Compliance with ethical standards All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Conflict of interest Liyanne van de Laarschot, Peter Jansen, Frank Schaap, and Steven Olde Damink declare that they have no conflict of interest.

Objective: Pruritus is a common symptom of cholestatic liver disorders. The present study aimed at evaluating autotaxin (ATX), a lysophospholipase recently identified as potential cause for cholestatic pruritus, in pediatric cholestatic diseases presenting with or without itching., Methods: A cohort of 45 children consisting of 14 patients experiencing itching (Alagille syndrome [n = 10], complete extrahepatic biliary atresia [n = 2], neonatal sclerosing cholangitis (n = 1), progressive familial intrahepatic cholestasis type 2 [n = 1]), 9 patients with bile acid synthesis defects (3β-hydroxy-C27-steroid-oxidoreductase [n = 7] and Δ-3-oxosteroid-5β-reductase deficiency [n = 2]), and 22 healthy children were studied. Serum ATX activity and total serum bile salt were determined enzymatically, ATX protein content was semiquantified by Western blotting. Using real-time polymerase chain reaction, ATX mRNA expression was studied in HepG2 cells treated with farnesoid-X-receptor agonists or vehicle., Results: Serum ATX activity was increased in pruritic children with Alagille and other cholestatic syndromes (mean ± standard deviation: 16.1 ± 4.3 nmol · mL · min) compared with children with nonpruritic cholestatic diseases with bile acid synthesis defects (10.4 ± 4.7 nmol · mL · min; P < 0.01) and healthy controls (7.6 ± 2.3 nmol · mL · min; P < 0.001). ATX protein levels closely correlated with serum ATX activity. Serum ATX activity and total serum bile salt showed a linear correlation with itch intensity (r = 0.66, P < 0.001 and r = 0.80, P < 0.001, respectively). No correlation was observed between ATX activity and bilirubin. ATX mRNA expression in HepG2 cells was not induced by farnesoid-X-receptor ligands., Conclusions: Serum ATX activity correlated with itch intensity in children with cholestatic diseases. Bile salts did not increase ATX expression in vitro. ATX inhibitors may be useful antipruritic agents in pediatric cholestatic disorders.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

Unlabelled: The enterohepatic circulation of bile salts is an important physiological route to recycle bile salts and ensure intestinal absorption of dietary lipids. The Na(+)-taurocholate cotransporting polypeptide SLC10A1 (NTCP) plays a key role in this process as the major transporter of conjugated bile salts from the plasma compartment into the hepatocyte. Here we present the first patient with NTCP deficiency, who was clinically characterized by mild hypotonia, growth retardation, and delayed motor milestones. Total bile salts in plasma were extremely elevated (up to 1,500 μM, ref. <16.3) but there were no clinical signs of cholestatic jaundice, pruritis, or liver dysfunction. Bile salt synthesis and intestinal bile salt signaling were not affected, as evidenced by normal plasma 7α-hydroxy-4-cholesten-3-one (C4) and FGF19 levels. Importantly, the presence of secondary bile salts in the circulation suggested residual enterohepatic cycling of bile salts. Sequencing of the SLC10A1 gene revealed a single homozygous nonsynonymous point mutation in the coding sequence of the gene, resulting in an arginine to histidine substitution at position 252. Functional studies showed that this mutation resulted in a markedly reduced uptake activity of taurocholic acid. Immunofluorescence studies and surface biotinylation experiments demonstrated that the mutant protein is virtually absent from the plasma membrane., Conclusion: We describe the identification of NTCP deficiency as a new inborn error of metabolism with a relatively mild clinical phenotype. The identification of NTCP deficiency confirms that this transporter is the main import system for conjugated bile salts into the liver but also indicates that auxiliary transporters are able to sustain the enterohepatic cycle in its absence., (© 2014 by the American Association for the Study of Liver Diseases.)

Background: Infectious complications often occur in acute pancreatitis, related to impaired intestinal barrier function, with prolonged disease course and even mortality as a result. The bile salt nuclear receptor farnesoid X receptor (FXR), which is expressed in the ileum, liver and other organs including the pancreas, exhibits anti-inflammatory effects by inhibiting NF-κB activation and is implicated in maintaining intestinal barrier integrity and preventing bacterial overgrowth and translocation. Here we explore, with the aid of complementary animal and human experiments, the potential role of FXR in acute pancreatitis., Methods: Experimental acute pancreatitis was induced using the CCK-analogue cerulein in wild-type and Fxr-/- mice. Severity of acute pancreatitis was assessed using histology and a semi-quantitative scoring system. Ileal permeability was analyzed in vitro by Ussing chambers and an in vivo permeability assay. Gene expression of Fxr and Fxr target genes was studied by quantitative RT-PCR. Serum FGF19 levels were determined by ELISA in acute pancreatitis patients and healthy volunteers. A genetic association study in 387 acute pancreatitis patients and 853 controls was performed using 9 tagging single nucleotide polymorphisms (SNPs) covering the complete FXR gene and two additional functional SNPs., Results: In wild-type mice with acute pancreatitis, ileal transepithelial resistance was reduced and ileal mRNA expression of Fxr target genes Fgf15, SHP, and IBABP was decreased. Nevertheless, Fxr-/- mice did not exhibit a more severe acute pancreatitis than wild-type mice. In patients with acute pancreatitis, FGF19 levels were lower than in controls. However, there were no associations of FXR SNPs or haplotypes with susceptibility to acute pancreatitis, or its course, outcome or etiology., Conclusion: We found no evidence for a major role of FXR in acute human or murine pancreatitis. The observed altered Fxr activity during the course of disease may be a secondary phenomenon.

Objective: To study the effect of different weight loss strategies on levels of the metabolic regulator FGF21 in morbidly obese females with normal glucose tolerance (NGT) or type 2 diabetes mellitus (T2DM)., Design: Observational intervention trial., Patients and Measurements: Weight reduction was achieved by Gastric Banding (GB, n = 11) or Roux-en-Y Gastric Bypass (RYGB, n = 16) in subjects with NGT, and by RYGB (n = 15) or a very-low-calorie diet (VLCD, n = 12) in type 2 diabetics. Fasted and/or postprandial levels of FGF21, FGF19 (an FGF21-related postprandial hormone) and bile salts (implicated in regulation of FGF21 and FGF19 expression) were measured before, and 3 and 12 weeks after intervention., Results: Fasted FGF21 levels were elevated in T2DM subjects. Calorie restriction by either GB or VLCD lowered bile salt and FGF21 levels. In contrast, RYGB surgery was associated with elevated bile salt and FGF21 levels., Conclusions: Calorie restriction and RYGB have opposite effects on serum bile salt and FGF21 levels. Calorie restriction results in FGF21 approaching nonobese control levels, suggesting that this intervention is effective in reducing the "nutritional crisis" that appears to underly FGF21 elevation in obesity. FGF21 elevation after RYGB may contribute to the beneficial effect of this procedure., (© 2014 John Wiley & Sons Ltd.)

The intracellular nuclear receptor farnesoid X receptor and the transmembrane G protein-coupled receptor TGR5 respond to bile acids by activating transcriptional networks and/or signalling cascades. These cascades affect the expression of a great number of target genes relevant for bile acid, cholesterol, lipid and carbohydrate metabolism, as well as genes involved in inflammation, fibrosis and carcinogenesis. Pregnane X receptor, vitamin D receptor and constitutive androstane receptor are additional nuclear receptors that respond to bile acids, albeit to a more restricted set of species of bile acids. Recognition of dedicated bile acid receptors prompted the development of semi-synthetic bile acid analogues and nonsteroidal compounds that target these receptors. These agents hold promise to become a new class of drugs for the treatment of chronic liver disease, hepatocellular cancer and extrahepatic inflammatory and metabolic diseases. This Review discusses the relevant bile acid receptors, the new drugs that target bile acid signalling and their possible applications.

Purpose of Review: The liver adaptively responds to extra-intestinal and intestinal inflammation. In recent years, the role of the autonomic nervous system, intestinal failure and gut microbiota has been investigated in the development of hepatic, intestinal and extra-intestinal disease., Recent Findings: The autonomic nervous system can be stimulated via enteral fat leading to cholecystokinin release, stimulating receptors in the gut and in the brain. This promotes bowel integrity, dampening the inflammatory response to food antigens. Consensus exists that intravenously administered long-chain fatty acids can cause liver damage but randomized-controlled trials are lacking. Disruption of the enterohepatic circulation of bile salts can give rise to cholestasis and nonalcoholic fatty liver disease, which may progress to fibrosis and cirrhosis. Reduced intestinal availability of bile salts reduces stimulation of the farnesoid X receptor. This may induce hepatic bile salt overload and associated hepatotoxicity through reduced action of intestinal fibroblast growth factor 19. Evidence is put forward to suggest that the intestinal microbiota is associated with liver abnormalities., Summary: Enteral lipids reduce inflammation and liver damage during stress or systemic inflammation, whereas parenteral lipid is associated with liver damage. Maintaining the enterohepatic circulation of bile salts limits hepatic cholestasis through an farnesoid X receptor feedback pathway. Changes in gut microbiota composition may induce liver disease.

Mutations in apolipoprotein A5 (APOA5) have been associated with hypertriglyceridemia in humans and mice. This has been attributed to a stimulating role for APOA5 in lipoprotein lipase-mediated triglyceride hydrolysis and hepatic clearance of lipoprotein remnant particles. However, because of the low APOA5 plasma abundance, we investigated an additional signaling role for APOA5 in high-fat diet (HFD)-induced obesity. Wild-type (WT) and Apoa5(-/-) mice fed a chow diet showed no difference in body weight or 24-h food intake (Apoa5(-/-), 4.5±0.6 g; WT, 4.2±0.5 g), while Apoa5(-/-) mice fed an HFD ate more in 24 h (Apoa5(-/-), 2.8±0.4 g; WT, 2.5±0.3 g, P<0.05) and became more obese than WT mice. Also, intravenous injection of APOA5-loaded VLDL-like particles lowered food intake (VLDL control, 0.26±0.04 g; VLDL+APOA5, 0.11±0.07 g, P<0.01). In addition, the HFD-induced hyperphagia of Apoa5(-/-) mice was prevented by adenovirus-mediated hepatic overexpression of APOA5. Finally, intracerebroventricular injection of APOA5 reduced food intake compared to injection of the same mouse with artificial cerebral spinal fluid (0.40±0.11 g; APOA5, 0.23±0.08 g, P<0.01). These data indicate that the increased HFD-induced obesity of Apoa5(-/-) mice as compared to WT mice is at least partly explained by hyperphagia and that APOA5 plays a role in the central regulation of food intake.

Unlabelled: The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)(2) diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C0]/[C16+C18] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma-glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C16:1n-7]/[C16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB)., Conclusion: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Increased hepatic expression is held responsible for elevated serum levels of fibroblast growth factor 21 (FGF21) in non-alcoholic fatty liver disease (NAFLD) but the underlying molecular mechanism is unclear. In the present study we tested the postulate that the metabolic hormone FGF21 is regulated by endoplasmic reticulum (ER) stress, a condition that is observed in a number of diseases including NAFLD and results in activation of an adaptive response known as the unfolded protein response (UPR). ER stress stimuli were found to induce expression of Fgf21 mRNA in H4IIE hepatoma cells and in isolated rat hepatocytes. Moreover, intraperitoneal injection of the ER stressor tunicamycin induced hepatic Fgf21 expression in mice and resulted in marked elevation of serum FGF21 levels. The effect of ER stress on FGF21 expression could be mimicked by overexpression of ATF4, a transcriptional effector of the PERK-branch of the UPR. In silico analysis revealed the presence of two binding sites for ATF4 in the FGF21 promoter region. Combined disruption of these elements, abrogated FGF21 promoter activity induced by ER stress or ATF4 overexpression. These findings implicate the PERK/eIF2alpha/ATF4 cascade in ER stress regulation of FGF21. A consequence of this notion is that other intracellular stress signaling pathways that converge at eIF2alpha, can regulate FGF21 expression. Indeed, both nutrient (amino acid deprivation) and oxidative stress (arsenite) were found to induce Fgf21 expression in hepatoma cells and isolated rat hepatocytes. In conclusion, FGF21 expression is regulated by ER stress and additional intracellular stress signaling pathways. Our findings suggest that increased cellular stress in fatty livers may underlie the elevated FGF21 levels observed in patients with NAFLD., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Accurate prediction scores for liver steatosis are demanded to enable clinicians to noninvasively screen for nonalcoholic fatty liver disease (NAFLD). Several prediction scores have been developed, however external validation is lacking., Objective: The aim was to determine the diagnostic accuracy of four existing prediction scores in severely obese children, to develop a new prediction score using novel biomarkers and to compare these results to the performance of ultrasonography., Design and Results: Liver steatosis was measured using proton magnetic resonance spectroscopy in 119 severely obese children (mean age 14.3 ± 2.1 years, BMI z-score 3.35 ± 0.35). Prevalence of steatosis was 47%. The four existing predictions scores ("NAFLD liver fat score," "fatty liver index," "hepatic steatosis index," and the pediatric prediction score) had only moderate diagnostic accuracy in this cohort (positive predictive value (PPV): 70, 61, 61, 69% and negative predictive value (NPV) 77, 69, 68, 75%, respectively). A new prediction score was built using anthropometry, routine biochemistry and novel biomarkers (leptin, adiponectin, TNF-alpha, IL-6, CK-18, FGF-21, and adiponutrin polymorphisms). The final model included ALT, HOMA, sex, and leptin. This equation (PPV 79% and NPV 80%) did not perform substantially better than the four other equations and did not outperform ultrasonography for excluding NAFLD (NPV 82%)., Conclusion: The conclusion is in severely obese children and adolescents existing prediction scores and the tested novel biomarkers have insufficient diagnostic accuracy for diagnosing or excluding NAFLD., (Copyright © 2012 The Obesity Society.)

Heterozygous hotspot mutations in isocitrate dehydrogenases (IDH) IDH1 or IDH2 are frequently observed in specific types of cartilaginous tumors, gliomas, and leukemias. Mutant IDH enzyme loses its normal activity to convert isocitrate into α-ketoglutarate (αKG) and instead acquires the ability to reduce αKG to D-2-hydroxyglutarate. Through direct competition with αKG, accumulation of the oncometabolite D-2-hydroxyglutarate in IDH mutated tumors results in inhibition of αKG-dependent dioxygenases involved in DNA and histone demethylation. Apart from epigenetic alterations, perturbations in the tricarboxylic acid cycle (depletion of intermediates) and activation of the intricately linked hypoxia signaling pathway are apparent in IDH mutated cells. As molecular details are being unraveled, the emerging concept is that IDH mutations result in tumor formation by epigenetic alterations that affect gene expression and result in inhibition of cellular differentiation. Activation of hypoxic stress signaling reprograms cellular energy metabolism and promotes anabolic processes and angiogenesis, thus, providing an advantage to promote neoplastic growth.

Background: Portal vein embolization (PVE) is employed to increase future remnant liver (FRL) volume through induction of hepatocellular regeneration in the nonembolized liver lobe. The regenerative response is commonly determined by CT volumetry after PVE. The aim of the study was to examine plasma bile salts and triglycerides in the prediction of the regenerative response following PVE., Methods: PVE of the cranial liver lobe was performed in 15 rabbits, divided into three groups: NaCl (control), gelatin sponge (short-term occlusion), and polyvinyl alcohol particles with coils (PVAc, long-term occlusion). In all rabbits CT volumetry and blood sampling were performed prior to PVE and on days 3 and 7. Plasma bile salts and triglycerides were correlated with volume increase of the nonembolized liver lobe., Results: After 3 and 7 d, respectively, FRL volume was increased in both embolized groups, with the largest hypertrophy response observed in the PVAc group. Plasma bile salt levels were increased after PVE, especially in the PVAc group at day 3 (P < 0.01 compared to gelatin sponge). Plasma bile salts at day 3 predicted FRL volume increase at day 7 showing a positive correlation of 0.811 (P < 0.001). Levels of triglycerides were not significantly altered in either of the PVE procedures., Conclusions: Plasma bile salt levels early after PVE strongly correlated with the regenerative response in a rabbit model of PVE, showing more pronounced elevation with larger volume increase of the nonembolized lobe. Therefore, plasma bile salts, but not triglycerides, can be used in the prediction of the regenerative response after PVE., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Preoperative portal vein embolization (PVE) is used to increase the future remnant liver (FRL) in patients requiring extensive liver resection. Computed tomography (CT) volumetry, performed not earlier than 3-6 weeks after PVE, is commonly employed to assess hypertrophy of the FRL following PVE. Early parameters to predict effective hypertrophy are therefore desirable. The aim of the present study was to assess plasma bile salt levels, triglycerides (TG), and apoA-V in the prediction of the hypertrophy response during liver regeneration., Methods: Serum bile salt, TG, and apoA-V levels were determined in 20 patients with colorectal metastases before PVE, and 5 h, 1, and 21 days after PVE, as well as prior to and after (day 1-7, and day 21) subsequent liver resection. These parameters were correlated with liver volume as measured by CT volumetry (%FRL-V), and liver function was determined by technetium-labeled mebrofenin hepatobiliary scintigraphy using single photon emission computed tomography., Results: Triglyceride levels at baseline correlate with volume increase of the future remnant liver (FRL-V) post-PVE. Also, bile salts and TG 5 h after PVE positively correlated with the increase in FRL volume (r=0.672, p=0.024; r=0.620, p=0.042, resp.) and liver function after 3 weeks (for bile salts r=0.640, p=0.046). Following liver surgery, TG levels at 5 h and 1 day after resection were associated with liver remnant volume after 3 months (r=0.921, p=0.026 and r=0.981, p=0.019, resp). Plasma apoA-V was increased during liver regeneration., Conclusions: Bile salt and TG levels at 5 h after PVE/resection are significant early predictors of liver volume and functional increase. It is suggested that these parameters can be used for early timing of volume assessment and resection after PVE.

Unlabelled: Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkin's disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values., Conclusion: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR-dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Purpose of Review: Fibroblast growth factor 19 (FGF19) is a postprandial hormone released from the small intestine. FGF19 improves glucose tolerance when overexpressed in mice with impaired glucose tolerance or diabetes. This review summarizes the recent advances in our understanding of the biology of FGF19 and its role in glucose homeostasis, with emphasis on publications from 2010 to 2012., Recent Findings: Protein engineering was used to generate FGF19 protein variants that allowed the separation of its mitogenic and metabolic functions. Its cognate receptor in the liver (FGFR4) mediated the effects of FGF19 on proliferation and bile salt synthesis, while this receptor was dispensable for its effects on glucose homeostasis. New metabolic activities of FGF19 were uncovered. FGF19 signaling was shown to stimulate glycogen and protein synthesis, and inhibit gluconeogenesis. FGF19 employed signaling routes distinct from those used by insulin to regulate these pathways. Mice with genetic disruption of Fgf15 (the mouse FGF19 ortholog) were glucose intolerant but had normal insulin levels and normal insulin sensitivity. Reduced hepatic glycogen stores and elevated hepatic gluconeogenesis were observed in the knock-out mice under the conditions in which insulin signaling was active., Summary: FGF19 signaling regulates glucose homeostasis in mice. The (patho)physiological role of FGF19 in glucose homeostasis in humans remains to be determined. Its novel insulin-mimetic actions, combined with the elimination of its mitogenic activity by protein engineering, make FGF19 an attractive candidate for the treatment of type 2 diabetes.

Background & Aims: Ursodeoxycholic acid (UDCA) exerts anticholestatic, antifibrotic and antiproliferative effects in primary biliary cirrhosis (PBC) via mechanisms not yet fully understood. Its adequate biliary enrichment is considered mandatory for therapeutic efficacy. However, precise determination of biliary enrichment of UDCA is not possible in clinical practice. Therefore, we investigated (i) the relationship between biliary enrichment and plasma pharmacokinetics of UDCA, (ii) the effect of UDCA on plasma and biliary bile acid composition and conjugation patterns, and (iii) on the intestinal detoxification machinery in patients with PBC and healthy controls., Methods: In 11 PBC patients and 11 matched healthy subjects, cystic bile and duodenal tissue were collected before and after 3 weeks of administration of UDCA (15 mg/kg/day). Extensive pharmacokinetic profiling of bile acids was performed. The effect of UDCA on the intestinal detoxification machinery was studied by quantitative PCR and Western blotting., Results: The relative fraction of UDCA and its conjugates in plasma at trough level[x] correlated with their biliary enrichment[y] (r=0.73, p=0.0001, y=3.65+0.49x). Taurine conjugates of the major hydrophobic bile acid, chenodeoxycholic acid, were more prominent in bile of PBC patients than in that of healthy controls. Biliary bile acid conjugation patterns normalized after treatment with UDCA. UDCA induced duodenal expression of key export pumps, BCRP and P-glycoprotein., Conclusions: Biliary and trough plasma enrichment of UDCA are closely correlated in PBC and health. Taurine conjugation may represent an adaptive mechanism in PBC against chenodeoxycholic acid-mediated bile duct damage. UDCA may stabilize small intestinal detoxification by upregulation of efflux pumps., (Copyright © 2012. Published by Elsevier B.V.)

Unlabelled: Fibroblast growth factor 19 (FGF19) plays a crucial role in the negative feedback regulation of bile salt synthesis. In the postprandial state, activation of ileal farnesoid X receptor (FXR) by bile salts results in transcriptional induction of FGF19 and elevation of circulating FGF19 levels. An intestinal-liver axis of FGF19 signaling results in down-regulation of bile salt synthesis. The aim of this study was to explore a broader signaling activity of FGF19 in organs engaged in the enterohepatic circulation of bile salts. For this aim, FGF19 expression and aspects of FGF19 signaling were studied in surgical specimens and in cell lines of hepatobiliary and intestinal origin. FGF19 messenger RNA was found to be abundantly expressed in the human gallbladder and in the common bile duct, with only minor expression observed in the ileum. Interestingly, human gallbladder bile contains high levels of FGF19 (21.9 ± 13.3 versus 0.22 ± 0.14 ng/mL in the systemic circulation). Gallbladder explants secrete 500 times more FGF19 than FXR agonist-stimulated ileal explants. Factors required for FGF19 signaling (i.e., FGFR4 and βKlotho) are expressed in mucosal epithelial cells of the gallbladder and small intestine. FGF19 was found to activate signaling pathways in cell lines of cholangiocytic, enteroendocrine, and enterocytic origin., Conclusion: The combined findings raise the intriguing possibility that biliary FGF19 has a signaling function in the biliary tract that differs from its established signaling function in the portal circulation. Delineation of the target cells in bile-exposed tissues and the affected cellular pathways, as well as a possible involvement in biliary tract disorders, require further studies., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Background: The bile acid-activated nuclear receptor Farnesoid X Receptor (FXR) is critical in maintaining intestinal barrier integrity and preventing bacterial overgrowth. Patients with Crohn's colitis (CC) exhibit reduced ileal FXR target gene expression. FXR agonists have been shown to ameliorate inflammation in murine colitis models. We here explore the feasibility of pharmacological FXR activation in CC., Methods: Nine patients with quiescent CC and 12 disease controls were treated with the FXR ligand chenodeoxycholic acid (CDCA; 15 mg/kg/day) for 8 days. Ileal FXR activation was assessed in the fasting state during 6 hrs after the first CDCA dose and on day 8, by quantification of serum levels of fibroblast growth factor (FGF) 19. Since FGF19 induces gallbladder (GB) refilling in murine models, we also determined concurrent GB volumes by ultrasound. On day 8 ileal and cecal biopsies were obtained and FXR target gene expression was determined., Results: At baseline, FGF19 levels were not different between CC and disease controls. After the first CDCA dose, there were progressive increases of FGF19 levels and GB volumes during the next 6 hours in CC patients and disease controls (FGF19: 576 resp. 537% of basal; GB volumes: 190 resp. 178% of basal) without differences between both groups, and a further increase at day 8. In comparison with a separate untreated control group, CDCA affected FXR target gene expression in both CC and disease controls, without differences between both groups., Conclusions: Pharmacological activation of FXR is feasible in patients with CC. These data provide a rationale to explore the anti-inflammatory properties of pharmacological activation of FXR in these patients., Trial Registration: TrialRegister.nl NTR2009.

Thirty-five morbidly obese patients underwent Roux-en-Y gastric bypass surgery (RYGB). In addition to weight loss, these patients showed significant improvement of insulin resistance and a reduction of hepatic fat content. Three months after surgery, the serum bile salts were slightly but significantly elevated, and the levels of the endocrine-acting fibroblast growth factor 19 (FGF19) and FGF21 were increased. FGF19 and FGF21 play a role as regulators of hepatic lipid and glucose metabolism. These results show that RYGB surgery improves metabolism and that this improvement is still apparent 3 months after surgery. Bile salts may play a key role in the improvement of metabolism after RYGB. Why serum bile salt concentrations are elevated after RYGB needs to be investigated., (Copyright © 2011 S. Karger AG, Basel.)

Intestinal FGF19 has emerged as a novel endocrine regulator of hepatic bile salt and lipid metabolism. In patients with nonalcoholic fatty liver disease (NAFLD) hepatic lipid metabolism is deranged. A possible role of FGF19 in NAFLD has not been reported yet. In this study, we assessed intestinal FGF19 production and the hepatic response to FGF19 in NAFLD patients with and without insulin resistance [homeostasis model of assessment (HOMA) score > or =2.5 (n = 12) and HOMA score <2.5 (n = 8), respectively]. To this end, NAFLD patients received a standardized oral fat challenge. Postprandial excursions of triglycerides, bile salts, and FGF19 were monitored, and plasma levels of a marker for bile salt synthesis (7alpha-hydroxy-4-cholesten-3-one) were determined. Fasted FGF19 levels were comparable in a control group of healthy volunteers (n = 15) and in NAFLD patients (0.26 +/- 0.28 vs. 0.18 +/- 0.09 ng/ml, respectively, P = 0.94). Postprandial FGF19 levels in both controls and NAFLD patients peaked between 3-4 h and were three times higher than baseline levels. The areas under the postprandial FGF19 curve were similar in controls and in the HOMA score-based NAFLD subgroups. In NAFLD patients with HOMA score <2.5, the postprandial increase in plasma FGF19 was accompanied by a lowering of plasma levels of 7alpha-hydroxy-4-cholesten-3-one (-30%, P = 0.015). This anticipated decline was not observed in insulin-resistant NAFLD patients (+10%, P = 0.22). In conclusion, patients with NAFLD show an unimpaired intestinal FGF19 production. However, the hepatic response to FGF19 is impaired in NAFLD patients with insulin resistance (HOMA score > or =2.5). This impaired hepatic response to FGF19 may contribute to the dysregulation of lipid homeostasis in NAFLD.

Apolipoprotein AV (apoAV) overexpression causes a decrease in plasma triglyceride (TG) levels, while deficiency of apoAV causes hypertriglyceridemia in both men and mice. However, contrary to what would be expected, plasma apoAV and TG levels in humans are positively correlated. To address this apparent paradox, we determined plasma apoAV levels in various mouse models with median TG levels ranging from 30 mg/dl in wild-type mice to 2089 mg/dl in glycosylphosphatidylinositol-anchored HDL binding protein 1-deficient mice. The data show that apoAV and TG levels are positively correlated in mice (r = +0.798, P < 0.001). In addition, we show that LPL gene transfer caused a simultaneous decrease in TG and apoAV in LPL-deficient mice. The combined data suggest that apoAV levels follow TG levels due to an intimate link between the apoAV molecule and TG-rich lipoproteins, comprising both secretion and removal of these lipoproteins. Taken together, the data suggest that higher plasma apoAV levels reflect an increased demand for plasma TG hydrolysis under normal physiological conditions.

Unlabelled: Fibroblast growth factor 19 (FGF19) is an endocrine factor produced by the small intestine in response to uptake of luminal bile salts. In the liver, FGF19 binds to FGF receptor-4, resulting in down-regulation of cytochrome P (CYP) 7A1 and reduced bile salt synthesis. Down-regulation of CYP7A1 under cholestatic conditions has been attributed to bile salt-mediated induction of the transcriptional repressor short heterodimer partner (SHP), because the interrupted enterohepatic cycle of bile salts is thought to abrogate intestinal FGF19 production and thus result in lowering of plasma FGF19 levels. Unexpectedly, we observed marked elevation of plasma FGF19 in patients with extrahepatic cholestasis caused by a pancreatic tumor (2.3 +/- 2.3 in cholestatic versus 0.40 +/- 0.25 ng/mL and 0.29 +/- 0.12 ng/mL in postcholestatic patients who received preoperative drainage by biliary stenting, P = 0.004, and noncholestatic control patients, P = 0.04, respectively). Although FGF19 messenger RNA (mRNA) is virtually absent in normal liver, FGF19 mRNA was strongly increased (31-fold to 374-fold, P < 0.001) in the liver of cholestatic patients in comparison with drained and control patients. In the absence of changes in SHP mRNA, CYP7A1 mRNA was strongly reduced (7.2-fold to 24-fold, P < 0.005) in the liver of cholestatic patients in comparison with drained and control patients, indicating an alternative regulatory pathway. Alterations in transcripts encoding hepatobiliary transporters [adenosine triphosphate-binding cassette, subfamily C, member 3 (ABCC3)/multidrug resistance protein 3 (MRP3), organic solute transporter alpha/beta (OSTalpha/beta), organic anion-transporting polypeptide (OATP1B1)] further suggest that bile salts are secreted via a nonbiliary route in patients with extrahepatic cholestasis., Conclusion: The liver expresses FGF19 under conditions of extrahepatic cholestasis. This is accompanied by a number of adaptations aimed at protecting the liver against bile salt toxicity. FGF19 signaling may be involved in some of these adaptations.

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.

Objective: Apolipoprotein A-V (apoAV) contributes to the regulation of triglyceride metabolism, which plays a role in the pathogenesis of atherosclerotic diseases. We therefore ascertained determinants of hepatic APOA5 transcript and apoAV plasma levels in humans., Design: We determined influences of anthropometric variables, biochemical factors related to lipid and glucose metabolism, hepatic mRNA levels transcribed from the APOA1/C3/A4/A5 cluster and transcription factor genes implicated in the regulation of APOA5 as well as common single nucleotide polymorphisms (SNPs) at the APOA5 locus on APOA5 expression in 89 obese patients and 22 non-obese controls., Results: Mean, age and sex adjusted, hepatic APOA5 mRNA or apoAV plasma levels did not differ by obesity status, homoeostasis model assessment insulin resistance or inflammatory markers. In multivariate regression models, the c56C > G SNP, plasma apoCIII, plasma nonesterified fatty acids, hepatic APOA5 transcripts, sex and a weak association with obesity status explained 61% of the variance in apoAV plasma levels. Hepatic transcript levels of carnitine palmitoyltransferase 1 (CPT1A1) and peroxisome proliferator-activated receptor alpha (PPARA), plasma nonesterified fatty acids and the c56C > G SNP explained 48% of the variance in hepatic APOA5 transcript levels., Conclusion: Apolipoprotein A-V plasma levels are independently associated with plasma free fatty acid and hepatic APOA5 mRNA levels. Associations of APOA5 transcripts with PPARA and CPT1A1 transcripts suggest that APOA5 expression is intimately linked to hepatic lipid metabolism.

Objective: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible., Methods and Results: We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant., Conclusions: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.

Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.