J Schmitt

Free fatty acids repress small heterodimer partner (SHP) activation and adiponectin counteracts bile acid‐induced liver injury in superobese patients with nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries and may proceed to steatohepatitis (NASH). Apoptosis and free fatty acid (FFA)‐induced lipotoxicity are important features of NASH pathogenesis. We have shown a hepatoprotective effect of adiponectin in steatotic livers of hepatitis C virus (HCV) patients and recent data links bile acid (BA) metabolism to the pathogenesis of NAFLD. The aim of this study was to identify potential interactions between BA and FFA metabolism in NAFLD. Liver biopsies and serum samples from 113 morbidly obese patients receiving bariatric surgery, healthy individuals, and moderately obese NAFLD patients were studied. Serum FFA, BA, and M30 were increased in NASH versus simple steatosis, while adiponectin was significantly decreased. The NAFLD activity score (NAS) score correlated with BA levels and reversely with adiponectin. Adiponectin reversely correlated with CD95/Fas messenger RNA (mRNA) and hepatocellular apoptosis. The BA transporter high‐affinity Na+/taurocholate cotransporter (NTCP) and the BA synthesizing enzyme cholesterol 7 alpha‐hydroxylase (CYP7A1) were significantly up‐regulated in obese patients and hepatoma cells exposed to FFA. Up‐regulation of NTCP and CYP7A1 indicate failure to activate small heterodimer partner (SHP) upon farnesoid X receptor (FXR) stimulation by increasing BA concentrations. In line with the NAS score, adiponectin levels were reversely correlated with BA levels. Adiponectin correlated with NTCP and affects Cyp7A1 expression both in vivo and in vitro. Conclusion: BA synthesis and serum BA levels correlated with disease severity in NAFLD, while adiponectin is reversely correlated. FFA exposure prevented SHP‐mediated repression of NTCP and Cyp7A1 expression, which lead to increased BA synthesis and uptake. In NASH, BA accumulation induced hepatocyte cell death and late FXR activation failed to prevent hepatocyte injury due to decreased adiponectin levels. Early treatment with FXR ligands and/or adiponectin‐receptor agonists might prevent NASH. (HEPATOLOGY 2013;57:1394–1406)

Combined effect of 25-OH vitamin D plasma levels and genetic Vitamin DReceptor (NR 1I1) variants on fibrosis progression rate in HCV patients

Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date.

Performance of Serum microRNAs -122, -192 and -21 as Biomarkers in Patients with Non- Alcoholic Steatohepatitis

Objectives Liver biopsies are the current gold standard in non-alcoholic steatohepatitis (NASH) diagnosis. Their invasive nature, however, still carries an increased risk for patients’ health. The development of non-invasive diagnostic tools to differentiate between bland steatosis (NAFL) and NASH remains crucial. The aim of this study is the evaluation of investigated circulating microRNAs in combination with new targets in order to optimize the discrimination of NASH patients by non-invasive serum biomarkers. Methods Serum profiles of four microRNAs were evaluated in two cohorts consisting of 137 NAFLD patients and 61 healthy controls. In a binary logistic regression model microRNAs of relevance were detected. Correlation of microRNA appearance with known biomarkers like ALT and CK18-Asp396 was evaluated. A simplified scoring model was developed, combining the levels of microRNA in circulation and CK18-Asp396 fragments. Receiver operating characteristics were used to evaluate the potential of discriminating NASH. Results The new finding of our study is the different profile of circulating miR-21 in NASH patients (p<0.0001). Also, it validates recently published results of miR-122 and miR-192 to be differentially regulated in NAFL and NASH. Combined microRNA expression profiles with CK18-Asp396 fragment level scoring model had a higher potential of NASH prediction compared to other risk biomarkers (AUROC = 0.83, 95% CI = 0.754–0.908; p<0.001). Evaluation of score model for NAFL (Score = 0) and NASH (Score = 4) had shown high rates of sensitivity (91%) and specificity (83%). Conclusions Our study defines candidates for a combined model of miRNAs and CK18-Asp396 levels relevant as a promising expansion for diagnosis and in turn treatment of NASH.

A common polymorphism in the ABCB11 gene is associated with advanced fibrosis in hepatitis C but not in non-alcoholic fatty liver disease

Chronic HCV (hepatitis C virus)-associated cirrhosis represents a major indication for liver transplantation. Bile acids contribute to hepatic stellate cell activation as a key event in fibrogenesis. The aim of the present study was to investigate the role of bile acids and polymorphisms in bile acid level-regulating genes on fibrosis progression. A total of 206 subjects with chronic HCV infection were included for ABCB11 (ATP-binding cassette, subfamily B, member II) 1331T>C and NR1H4 (nuclear receptor) -1G>T genotyping, 178 of which were analysed for fibrosis stage. Exclusion criteria were HBV (hepatitis B virus) or HIV coinfection, alcohol >40 g/day and morbid obesity. A total of 358 patients with NAFLD (non-alcoholic fatty liver disease) were genotyped for comparison with a non-viral liver disease. Caucasian individuals (n = 110), undergoing liver resection for focal hepatic metastasis, served as controls. The ABCB11 1331C allele was significantly overrepresented in HCV patients compared with controls {allelic frequency 62.9%; OR (odds ratio), 1.41 [95% CI (confidence interval), 1.012-1.965]}. Median plasma bile acid levels were not significantly increased in the CC compared with TT genotype [7.2 (1-110) μmol/l compared with 3.5 (1-61) μmol/l; values are medians (range). A significant association between the presence of cirrhosis and ABCB11 genotype (CC compared with CT or TT, P=0.047) was observed in the χ2 test and independent of other risk factors of age, gender, body mass index and disease duration in multivariate analysis (P = 0.010). No such association could be observed in fatty liver patients with regard to advanced fibrosis (F ≥ 2). The common ABCB11 1331CC genotype, which is present in 40% of HCV patients and renders the carrier susceptible to increased bile acid levels, is associated with cirrhosis.

Impact of genetic SLC28 transporter and ITPA variants on ribavirin serum level, hemoglobin drop and therapeutic response in patients with HCV infection

BACKGROUND & AIMS In the last decade, pegylated interferon-α (PegIFN-α) plus ribavirin (RBV) was the standard treatment of chronic hepatitis C for genotype 1, and it remains the standard for genotypes 2 and 3. Recent studies reported associations between RBV-induced anemia and genetic polymorphisms of concentrative nucleoside transporters such as CNT3 (encoded by SLC28A3) and inosine triphosphatase (encoded by ITPA). We aimed at studying genetic determinants of RBV kinetics, efficacy and treatment-associated anemia. METHODS We included 216 patients from two Swiss study cohorts (61% HCV genotype 1, 39% genotypes 2 or 3). Patients were analyzed for SLC28A2 single nucleotide polymorphism (SNP) rs11854484, SLC28A3 rs56350726, and SLC28A3 rs10868138 as well as ITPA SNPs rs1127354 and rs7270101, and followed for treatment-associated hemoglobin changes and sustained virological response (SVR). In 67 patients, RBV serum levels were additionally measured during treatment. RESULTS Patients with SLC28A2 rs11854484 genotype TT had higher dosage- and body weight-adjusted RBV levels than those with genotypes TC or CC (p=0.02 and p=0.06 at weeks 4 and 8, respectively). ITPA SNP rs1127354 was associated with hemoglobin drop ≥3 g/dl during treatment, in genotype (relative risk (RR)=2.1, 95% CI 1.3-3.5) as well as allelic analyses (RR=2.0, 95%CI 1.2-3.4). SLC28A3 rs56350726 was associated with SVR in genotype (RR=2.2; 95% CI 1.1-4.3) as well as allelic analyses (RR=2.0, 95% CI 1.1-3.4). CONCLUSIONS The newly identified association between RBV serum levels and SLC28A2 rs11854484 genotype, as well as the replicated association of ITPA and SLC28A3 genetic polymorphisms with RBV-induced anemia and treatment response, may support individualized treatment of chronic hepatitis C and warrant further investigation in larger studies.

Adaptation of iron transport and metabolism to acute high‐altitude hypoxia in mountaineers

Human iron homeostasis is regulated by intestinal iron transport, hepatic hepcidin release, and signals from pathways that consume or supply iron. The aim of this study was to characterize the adaptation of iron homeostasis under hypoxia in mountaineers at the levels of (1) hepatic hepcidin release, (2) intestinal iron transport, and (3) systemic inflammatory and erythropoietic responses. Twenty‐five healthy mountaineers were studied. Blood samples and duodenal biopsies were taken at baseline of 446 m as well as on day 2 (MG2) and 4 (MG4) after rapid ascent to 4559 m. Divalent metal‐ion transporter 1 (DMT‐1), ferroportin 1 (FP‐1) messenger RNA (mRNA), and protein expression were analyzed in biopsy specimens by quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry. Serum hepcidin levels were analyzed by mass spectrometry. Serum iron, ferritin, transferrin, interleukin (IL)−6, and C‐reactive protein (CRP) were quantified by standard techniques. Serum erythropoietin and growth differentiation factor 15 (GDF15) levels were measured by enzyme‐linked immunosorbent assay (ELISA). Under hypoxia, erythropoietin peaked at MG2 (P < 0.001) paralleled by increased GDF15 on MG2 (P < 0.001). Serum iron and ferritin levels declined rapidly on MG2 and MG4 (P < 0.001). Duodenal DMT‐1 and FP‐1 mRNA expression increased up to 10‐fold from baseline on MG2 and MG4 (P < 0.001). Plasma CRP increased on MG2 and MG4, while IL‐6 only increased on MG2 (P < 0.001). Serum hepcidin levels decreased at high altitude on MG2 and MG4 (P < 0.001). Conclusion: This study in healthy volunteers showed that under hypoxemic conditions hepcidin is repressed and duodenal iron transport is rapidly up‐regulated. These changes may increase dietary iron uptake and allow release of stored iron to ensure a sufficient iron supply for hypoxia‐induced compensatory erythropoiesis. (Hepatology 2013; 58:2153–2162)

Protective effects of farnesoid X receptor (FXR) on hepatic lipid accumulation are mediated by hepatic FXR and independent of intestinal FGF15 signal

There is a growing evidence that bile acids are involved in the regulation of triglyceride‐, cholesterol‐homoeostasis and fat absorption. In this study organ‐specific Fxr knockout mice were used to further investigate the influence of farnesoid X receptor FXR in lipogenesis.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients.

BACKGROUND Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients. METHODS A total of 155 chronic hepatitis C patients were recruited from the Swiss Hepatitis C Cohort Study for NR1I1 genotyping and plasma 25-OH vitamin D level measurement. NR1I1 genotype data and combined effects of plasma 25-OH vitamin D level were analysed regarding therapy response (sustained virological response). RESULTS A strong association was observed between therapy non-response and the NR1I1 CCA (bAt) haplotype consisting of rs1544410 (BsmI) C, rs7975232 (ApaI) C and rs731236 (TaqI) A alleles. Of the HCV patients carrying the CCA haplotype, 50.3% were non-responders (odds ratio [OR] 1.69, 95% CI 1.07, 2.67; P=0.028). A similar association was observed for the combinational CCCCAA genotype (OR 2.94, 95% CI 1.36, 6.37; P=0.007). The combinational CCCCAA genotype was confirmed as an independent risk factor for non-response in multivariate analysis (OR 2.50, 95% CI 1.07, 5.87; P=0.034). Analysing combined effects, a significant impact of low 25-OH vitamin D levels on sustained virological response were only seen in patients with the unfavourable NR1I1 CCA (bAt) haplotype (OR for non-SVR 3.55; 95% CI 1.005, 12.57; P=0.049). CONCLUSIONS NR1I1 vitamin D receptor polymorphisms influence response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C and exert an additive genetic predisposition to previously described low 25-OH vitamin D serum levels.

The role of bile acid retention and a common polymorphism in the ABCB11 gene as host factors affecting antiviral treatment response in chronic hepatitis C

Summary.  The outcome of hepatitis C virus (HCV) infection and the likelihood of a sustained virological response (SVR) to antiviral therapy depends on both viral and host characteristics. In vitro studies demonstrated that bile acids (BA) interfere with antiviral interferon effects. We investigate the influence of plasma BA concentrations and an ABCB11 polymorphism associated with lower transporter expression on viral load and SVR. Four hundred and fifty‐one Caucasian HCV‐patients treated with PEG‐interferon and ribavirin were included in the study. ABCB11 1331T>C was genotyped, and plasma BA levels were determined. The 1331C allele was slightly overrepresented in HCV‐patients compared to controls. In HCV‐patients, a significant difference between patients achieving SVR vs non‐SVR was observed for HCV‐2/3 (5 vs 9 μm; P = 0.0001), while median BA levels in HCV‐1 were marginally elevated. Normal BA levels <8 μm were significantly associated with SVR (58.3%vs 36.3%; OR 2.48; P = 0.0001). This difference was significant for HCV‐2/3 (90.7%vs 67.6%; P = 0.002) but marginal in HCV‐1 (38.7%vs 27.8%; P = 0.058). SVR rates were equivalent between ABCB11 genotypes for HCV‐1, but increased for HCV‐2/3 (TT 100%vs CC 78%; OR 2.01; P = 0.043). IL28B genotype had no influence on these associations. No correlation between BA levels and HCV RNA was detected for any HCV genotype. The higher allelic frequency of ABCB11 1331C in HCV‐patients compared to controls may indirectly link increased BA to HCV chronicity. Our data support a role for BA as host factor affecting therapy response in HCV‐2/3 patients, whereas a weaker association was found for HCV‐1.

Multikinase inhibitor sorafenib transiently promotes necrosis after radiofrequency ablation in rat liver but activates growth signals

AIM To investigate the effects of sorafenib when combined with radiofrequency ablation treatment in liver tissue, the necrosis volume, tissue repair and hepatocellular growth signals were analyzed in rats. Radiofrequency ablation (RFA) is a widely applied treatment for hepatocellular carcinoma (HCC). Radiofrequency ablation is combined with the multi-tyrosinkinase-inhibitor sorafenib in ongoing clinical trials. Whether this combination treatment affects liver tissue repair is unknown. MATERIALS AND METHODS Male Sprague Dawley (SD) rats received RFA or sham puncture with concomitant sorafenib (5mg/kg qd from day 2) or vehicle. Necrosis volume was calculated from resected specimens. Proliferation and micro vessel density were determined by Ki67 and CD31 immunofluorescence, respectively. mRNA expression of hepatocyte growth factor (HGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) was quantified. RESULTS While ablation size was identical in all treatment groups at day 1, sorafenib treated animals showed sustained necroses (219 ± 24 vs. 88 ± 52 mm(3) in controls; P = 0.03), elevated alanine aminotransferase (ALT) and elevated glutamate dehydrogenase (GLDH) (76 ± 37 vs. 47 ± 58 mm(3); P=0.50) at day 3. By day 7 necrosis volumes equalized for the treatment groups. Ki67 and CD31 staining showed reduced proliferation and micro vessel density at days 1 and 3 following sorafenib. Growth factors HGF and EGF were significantly overexpressed in liver tissue after sorafenib. CONCLUSION Sorafenib initially promotes necrosis after RFA in liver tissue. The delay in tissue repair is overcome at day 7 presumably by transient compensatory overexpression of growth signals. Based on these data from animal studies further investigation of adjuvant sorafenib in humans is warranted.