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Schreuder T.C.M.A.,VU University Amsterdam | Lenicek M.,Charles University | Jansen P.L.M.,Liver Center | Schaap F.G.,Liver Center
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2010

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 ≥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 (7α-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 7α-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 ≥2.5). This impaired hepatic response to FGF19 may contribute to the dysregulation of lipid homeostasis in NAFLD. Copyright © 2010 American Physiological Society.


Schreuder T.C.M.A.,Medical Center | Jansen P.L.M.,Liver Center
European Journal of Radiology | Year: 2010

Rationale and objective: Non-alcoholic fatty liver disease (NAFLD) is related to the metabolic syndrome and obesity. Proton magnetic resonance spectroscopy ( 1H MRS) is a non-invasive technique to assess hepatic triglyceride content (HTGC) and allows assessment of unsaturated fatty acids (UFA). There is increasing evidence that hepatic UFA are associated with the development of NAFLD. Therefore the objective of this study was to assess hepatic UFA in patients with NAFLD using 1H MRS. Materials and methods: We included 26 consecutive patients with deranged liver enzymes, with and without type 2 diabetes mellitus (DM2), suspected for NAFLD. Liver function and metabolic parameters were assessed. 1H MRS measurements were performed at 3.0 T. From the 1HMR spectra two ratios were calculated: Ratio 1 (UFA); unsaturated fatty acid peak vs. reference water peak and ratio 2 (HTGC); total fatty acid peak vs. reference water peak. Results: Twenty-six patients were included. In these patients hepatic UFA (ratio 1) correlated with AST/ALT ratio (r =-0.46, p = 0.02), glucose levels (r = 0.46, p = 0.018), HOMA-IR (r = 0.59, p = 0.004) and HTGC (r = 0.81, p < 0.001). In diabetic patients (n = 12) hepatic UFA correlated with alkaline phosphatase levels (r = 0.72, p = 0.01), HOMA-IR (r = 0.73, p = 0.01) and HTGC (r = 0.83, p = 0.002). Compared to nondiabetic patients with NAFLD, hepatic UFA levels were increased in patients with DM2 and NAFLD (0.032 vs. 0.014, p = 0.03). Conclusion: Hepatic UFA can be assessed with 1H MRS. 1H MRS determined hepatic UFA correlate with clinical and metabolic parameters associated with NAFLD. Hepatic UFA are increased in patients with DM2. This study provides evidence for the use of non-invasive 1H MRS to assess hepatic UFA in vivo. © 2009 Elsevier Ireland Ltd.


Scaglioni F.,Liver Center | Ciccia S.,Liver Center | Marino M.,Liver Center | Bedogni G.,Liver Research Center | And 2 more authors.
Digestive Diseases | Year: 2011

Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) have a similar pathogenesis and histopathology but a different etiology and epidemiology. NASH and ASH are advanced stages of non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD). NAFLD is characterized by excessive fat accumulation in the liver (steatosis), without any other evident causes of chronic liver diseases (viral, autoimmune, genetic, etc.), and with an alcohol consumption ≤20-30 g/day. On the contrary, AFLD is defined as the presence of steatosis and alcohol consumption >20-30 g/day. The most common phenotypic manifestations of primary NAFLD/NASH are overweight/obesity, visceral adiposity, type 2 diabetes, hypertriglyceridemia and hypertension. The prevalence of NAFLD in the general population in Western countries is estimated to be 25-30%. The prevalence and incidence of NASH and ASH are not known because of the impossibility of performing liver biopsy in the general population. Up to 90% of alcoholics have fatty liver, and 5-15% of these subjects will develop cirrhosis over 20 years. The risk of cirrhosis increases to 30-40% in those who continue to drink alcohol. About 10-35% of alcoholics exhibit changes on liver biopsy consistent with alcoholic hepatitis. Natural histories of NASH and ASH are not completely defined, even if patients with NASH have a reduced life expectancy due to liver-related death and cardiovascular diseases. The best treatment of AFLD/ASH is to stop drinking, and the most effective first-line therapeutic option for NAFLD/NASH is non-pharmacologic lifestyle interventions through a multidisciplinary approach including weight loss, dietary changes, physical exercise, and cognitive-behavior therapy. Copyright © 2011 S. Karger AG, Basel.


Lee S.-T.,University of California at San Francisco | Lee S.-T.,Sungkyunkwan University | Xiao Y.,University of California at San Francisco | Muench M.O.,Blood Systems Research Institute | And 11 more authors.
Nucleic Acids Research | Year: 2012

The epigenetic changes during B-cell development relevant to both normal function and hematologic malignancy are incompletely understood. We examined DNA methylation and RNA expression status during early B-cell development by sorting multiple replicates of four separate stages of pre-B cells derived from normal human fetal bone marrow and applied high-dimension DNA methylation scanning and expression arrays. Features of promoter and gene body DNA methylation were strongly correlated with RNA expression in multipotent progenitors (MPPs) both in a static state and throughout differentiation. As MPPs commit to pre-B cells, a predominantly demethylating phenotype ensues, with 79% of the 2966 differentially methylated regions observed involving demethylation. Demethylation events were more often gene body associated rather than promoter associated; predominantly located outside of CpG islands; and closely associated with EBF1, E2F, PAX5 and other functional transcription factor (TF) sites related to B-cell development. Such demethylation events were accompanied by TF occupancy. After commitment, DNA methylation changes appeared to play a smaller role in B-cell development. We identified a distinct development-dependent demethylation signature which has gene expression regulatory properties for pre-B cells, and provide a catalog reference for the epigenetic changes that occur in pre-B-cell leukemia and other B-cell-related diseases. © 2012 The Author(s).


Lim Y.-S.,Liver Center | Han S.,Asan Medical Center | Heo N.-Y.,Inje University | Shim J.H.,Liver Center | And 2 more authors.
Gastroenterology | Year: 2014

Background & Aims Little is known about whether the antiviral agent entecavir is more effective than a less potent drug, lamivudine, in reducing the risk of death and hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. Methods We performed a retrospective analysis of data from 5374 consecutive adult patients with chronic hepatitis B, treated with entecavir (n = 2000) or lamivudine (n = 3374), at a tertiary referral hospital in Seoul, Korea, from November 1, 1999, through December 31, 2011. Data were collected from patients for up to 6 years and analyzed by a multivariable Cox proportional hazards model for the entire cohort and for propensity score-matched cohorts. Results During the study period, 302 patients (5.6%) died, 169 (3.1%) received a liver transplant, and 525 (9.8%) developed HCC. Multivariable analyses showed that compared with lamivudine, entecavir therapy was associated with a significantly lower risk of death or transplantation (hazard ratio [HR], 0.49; 95% confidence interval [CI], 0.38-0.64), but a similar risk of HCC (HR, 1.08; 95% CI, 0.87-1.34). In the 1792 overall propensity-matched pairs, entecavir again was associated with a significantly lower risk of death or transplantation (HR, 0.49; 95% CI, 0.37-0.64) and a similar risk of HCC (HR, 1.01; 95% CI, 0.80-1.27). Entecavir also reduced the risk of death or transplantation, compared with lamivudine, in 860 pairs of patients with cirrhosis (HR, 0.42; 95% CI, 0.31-0.57) but there were no differences in risk for HCC (HR, 1.00; 95% CI, 0.78-1.28). However, entecavir and lamivudine did not have significantly different effects on clinical outcome in 878 pairs of patients without cirrhosis. Conclusions In a retrospective study of 5374 patients with chronic hepatitis B virus infection, entecavir therapy was associated with a significantly lower risk of death or transplantation than lamivudine. However, the drugs did not have different effects on HCC risk. © 2014 by the AGA Institute.


Wang H.H.,Liver Center | Portincasa P.,University of Bari | Liu M.,Genome Research Institute | Tso P.,Genome Research Institute | And 2 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids | Year: 2010

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth. © 2009 Elsevier B.V. All rights reserved.


Pauli-Magnus C.,University of Basel | Meier P.J.,University of Basel | Stieger B.,University of Zürich | Stieger B.,Liver Center
Seminars in Liver Disease | Year: 2010

Intrahepatic cholestasis of pregnancy and drug-induced cholestasis are two clinically important forms of acquired cholestatic liver disease. The understanding of the underlying mechanisms of acquired cholestasis has recently made considerable progress by the identification of canalicular ATP-binding cassette (ABC) transporters as likely targets for these forms of cholestasis. Cholestasis of pregnancy is linked to estrogen and progesterone metabolites. These metabolites have been shown to impair the bile salt export pump (BSEP) function by an indirect mechanism. In addition, genetic variants (as well as mutants) of the genes coding for the phosphatidylcholine translocator MDR3 and BSEP and for the farnesoid X receptor, which is critical in the transcriptional activation of MDR3 (ABCB4) and BSEP (ABCB11) have been associated with intrahepatic cholestasis of pregnancy. The pathogenesis of drug-induced liver injury encompasses a wide spectrum of mechanisms, some of which are still poorly understood. BSEP is now known to be subject to drug inhibition in susceptible patients. Information on genetic factors rendering individuals susceptible to inhibition of BSEP by drugs or their metabolites is still scarce. Besides rare mutations that have been linked to drug-induced cholestasis, the common p.V444A polymorphism of BSEP has been identified as a potential risk factor. In this review, the authors summarize key concepts of physiology of bile formation, diagnostic principles to indentify these forms of acquired cholestasis, as well as pathogenetic mechanisms leading to intrahepatic cholestasis of pregnancy or drug-induced cholestasis. In addition, they review the current knowledge on genetic susceptibility factors for these two forms of cholestasis. © 2010 by Thieme Medical Publishers, Inc.


De Waart D.R.,Liver Center | Hausler S.,University of Zürich | Vlaming M.L.H.,Netherlands Cancer Institute | Kunne C.,Liver Center | And 4 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2010

Cholyl-L-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(-/-), and Abcc3(-/-) mice. Human Na+-dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with Km values of 4.6 ± 2.7, 3.3 ± 2.0, and 3.7 ± 1.0 μM, respectively. In Abcc2(-/-) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(-/-) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.


Uriarte I.,University of Navarra | Banales J.M.,University of Navarra | Saez E.,University of Navarra | Arenas F.,University of Navarra | And 3 more authors.
Hepatology | Year: 2010

Bicarbonate secretion from cholangiocytes is required for appropriate adjustment of primary canalicular bile along the biliary tract. In human and rat cholangiocytes, bicarbonate secretion is mediated by anion exchanger (AE) 2, an electroneutral Na-independent Cl/HCO3 AE also involved in intracellular pH (pHi) regulation. In Ae2a,b-deficient mice, pHi is increased in lymphocytes and fibroblasts, whereas it is surprisingly normal in cholangiocytes. Here, we analyze the mechanisms for HCO3 secretion in cultured Ae2a,b /and Ae2a,b /mouse cholangiocytes by microfluorimetric measurement of pHi changes upon established perfusion maneuvers. Cl withdrawal by isethionate-based perfusions showed that Ae2a,b /but not Ae2a,b /mouse cholangiocytes can display Cl/HCO3 exchange, which is therefore entirely mediated by Ae2. Nevertheless, simultaneous withdrawal of Cl and Na revealed that mouse cholangiocytes possess an additional transport activity for HCO3 secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na-driven HCO3-secreting activity is Cl-independent, consistent with a Na-HCO3 cotransport (NBC). NBC activity is greater in Ae2a,b /than Ae2a,b /mouse cholangiocytes, and membrane-depolarization experiments showed that it is electrogenic. Consistent with the potential role of Slc4a4/Nbc1 as the involved transporter, Ae2a,b /mouse cholangiocytes exhibit up-regulated expression of this electrogenic NBC carrier. Whereas Ae2-mediated Cl/HCO3 exchange in Ae2a,b /mouse cholangiocytes is stimulated by cyclic adenosine monophosphate (cAMP) and acetylcholine, the NBC activity is down-regulated by cAMP and adenosine triphosphate (ATP) in Ae2a,b /mouse cholangiocytes. Polarized Ae2a,b /mouse cholangiocytes placed in Ussing chambers show decreased (but not abolished) cAMP-dependent Cl current and increased ATP-dependent/Ca2-activated Cl secretion, which run in parallel with decreased cystic fibrosis transmembrane conductance regulator messenger RNA expression and increased intracellular Ca2 levels. Conclusion: Bicarbonate secretion in mouse cholangiocytes involves two differentially regulated activities: Ae2-mediated Cl/HCO3 exchange and Na-HCO3 cotransport. Copyright © 2010 by the American Association for the Study of Liver Diseases.


Markusic D.,Liver Center
Methods in molecular biology (Clifton, N.J.) | Year: 2010

Lentiviral vectors are a powerful tool to achieve regulated expression of transgenes in vivo and in vitro. The doxycycline-inducible system is well characterized and can be used to regulate expression mediated by lentiviral vectors. Because many different doxycycline-inducible lentiviral vectors have been described, choosing the best vector system can be difficult. This chapter can be used as a guide to select the optimal system for a particular application.

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