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1269

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Identification of cAMP kinase as a direct target of reactive aldehydes in a murine model of alcoholic liver disease

Colin T. Shearn, David J. Orlicky, Dennis R. Petersen;
Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO

Background: The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the patho-genesis in alcoholic liver disease (ALD). One consequence of ALD is altered cAMP kinase (AMPK) signaling resulting in dys-regulation of β-oxidation. Recently, we determined that in conjunction with a high fat diet, chronic ethanol consumption increased phosphorylation of AMPK but did not result in into significant changes in AMPK-dependent phosphorylation of Acetyl CoA Carboxylase (ACC) (Shearn et. alJour. Nutr. Bioch. 2013). The aim of the present study was to understand the effects of increased oxidative stress on AMPK signaling and activity in vitro as well as in a murine model of chronic ethanol consumption. Methods: Using recombinant protein, an in vitro human hepatocyte HepaRG cell culture system or a murine model of ALD, the direct effects of lipid peroxidation were examined with respect to AMPK phosphorylation, carbonyla-tion, enzymatic activity and phosphorylation of ACC. Results: In HepaRG cells, incubation with increasing concentrations of 4-HNE resulted in decreased phosphorylation of AMPK and ACC. Pretreatment of 4-HNE inhibited both hydrogen peroxide and adiponectin induced phosphorylation of AMPK and subsequent phosphorylation of ACC. Using biotin hydrazide capture, it was confirmed that exposure of HepaRG cells to 4-HNE resulted in carbonylation of AMPKα/β which was not observed in untreated control cells. Based on this data, mass spectral analysis of 4-HNE treated recombinant AMPKα identified Michael addition adducts of 4-HNE on Cys130, Cys174, Cys227 and Cys309. Global computational based molecular modeling analysis of AMPK following 4-HNE modification revealed no significant changes in secondary or tertiary structure. Molecular modeling analysis of 4-HNE adducted to Cys174 AMPKα suggest inhibition of AMPK activity by steric hindrance of the active site pocket. Using a murine model of alcoholic liver disease, chronic ethanol consumption resulted in an increase in carbonylated AMPKα despite increased phosphorylation of Thr172AMPK. There was no significant change in phosphorylation of ACC. Conclusions: Combined these data demonstrate for the first time that AMPK is a direct target of reactive aldehyde during conditions of increased oxidative stress in the liver. The inhibition of AMPK by reactive aldehydes provides a novel mechanism for defective AMPK signaling and β-oxidation in ALD. This work was funded by NIH 5F32 AA018613-03 (C.T.S.) and 5R37 AA009300-16 (D.R.P.).

Disclosures:

The following people have nothing to disclose: Colin T. Shearn, David J. Orlicky, Dennis R. Petersen

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A System Biology Approach Identifies Cytokeratin 23 as a Novel Marker of Progenitor Cell Expansion and Potential Molecular Driver of Alcoholic Hepatitis

Gemma Odena1, Juan José Lozano2,3, Jose Altamirano2,3, Daniel Rodrigo-Torres2,3, Oriol Morales-Ibanez2,3, Silvia Affò2,3, Malika Humphries1, Pau Sancho-Bru2,3, Vicente Arroyo2,3, Juan Caballe-ria2,3, Ramón Bataller1,3;
1 Departments of Medicine and Nutrition, University of North Carolina, Chapel Hill, NC; 2Liver Unit, Hospital Clinic. Centro de Investigacion Biomedica en red de Enfer-medades Hepaticas y Digestivas (CIBERehd), Barcelona, Spain; 3Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease and is one of the most deadly conditions in hepa-tology. There is a clear need to identify non-invasive biomarkers and molecular drivers in order to develop novel targeted therapies. We recently showed that progenitor cell expansion is a hallmark of severe AH and correlates with disease severity. Here, we performed a translational study including human and experimental data using a systems biology approach to identify new molecular biomarkers of AH. We first compared the whole transcriptome analysis of patients with alcoholic (n=15) and non-alcoholic steatohepatitis (NASH) (n=8), as well as normal livers (n=7). For the detection of differentially expressed genes, we used the Limma package. Adjustment of p-values was done by the determination of false discovery rates (FDR). Functional analysis was conducted using the R/Bioconductor package GOstats and the GO database. Unsupervised clustering analysis revealed a unique gene expression signature of livers with AH, which was markedly distant to NASH and control groups. We next identified the pathways that were only overexpressed in patients with AH compared to NASH and control livers. The “structural molecule activity” was the most significant up-regulated pathway in AH (p= 7.5 x10-9). Within this family, we identified cytokeratin 23 (KRT23), an intermediate filament, and one of the most up-regulated genes in the whole transcrip-tome (94-fold increased compared to normal livers). Next, we confirmed by qPCR that hepatic expression of KRT23 was markedly up-regulated in patients with AH compared to other liver disease such as HCV, compensated alcoholic cirrhosis and NASH. Serum levels of KRT23 were also found elevated only in patients with AH. Importantly, the baseline hepatic mRNA expression of KRT23 correlated with disease severity and 90-day survival (AUROC: 0.72). Immunohistochemistry studies showed that KRT23 was expressed at the areas of ductular reaction and progenitor cell expansion. Next, we explored the expression of KRT23 in experimental models of acute-on-chronic liver injury and in models progenitor cell expansion in mice. We found that hepatic KRT23 was induced by an acute injury (either by LPS orethanol) on a fibrotic liver. Interestingly, KRT23 was expressed in two models of progenitor cells expansion in liver injury (DDC and CDE diets) and was detected in progenitor cells. In summary, human and experimental data indicate that KRT23 is a novel marker of progenitor cell expansion and potential molecular driver of alcoholic hepatitis. Loss-of-function studies in animal models of AH should investigate the role of KRT23.

Disclosures:

Vicente Arroyo - Speaking and Teaching: GRIFOLS

The following people have nothing to disclose: Gemma Odena, Juan José Lozano, Jose Altamirano, Daniel Rodrigo-Torres, Oriol Morales-Ibanez, Silvia Affò, Malika Humphries, Pau Sancho-Bru, Juan Caballeria, Ramón Bataller

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Dual exposures to ethanol and nicotine-derived nitrosamine ketone (NNK) have additive effects on severity of steatohepatitis in Long Evans (LE) rats

Valerie Zabala1, Ming Tong1, Elizabeth Silbermann1, Teresa Ramirez1,2, Diana Lizarazo1,2, Rebecca Ducore1, Fusun Gundo-gan1, Suzanne M. de la Monte1,3;
1 Department of Medicine, Liver Research Center, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI; 2Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI; 3Department of Pathology, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI

Background: In our established chronic alcohol exposure model, progressive liver injury is associated with microvesicular steatohepatitis, early fibrosis, hepatic insulin resistance, and increased hepatic ER and oxidative stress. Previous studies showed that limited low-level exposures to dietary nitrosamines also cause steatohepatitis with hepatic insulin resistance and oxidative stress. Epidemiologic data indicate that in humans, heavy alcohol abuse that leads to alcoholic liver disease (ALD) is associated with binge drinking and cigarette smoking. Hypothesis: Combined injurious effects of chronic plus binge alcohol and tobacco nitrosamine (NNK) exposures mediate ALD pathogenesis. Methods: Adult male LE rats were chronically fed with isocaloric liquid diets containing 0% or 37% (caloric content) ethanol (EtOH) for 8 weeks. From Week 3 through 8, rats in each group were treated by i.p. injection of NNK 3x/week, and in Weeks 7 and 8, chronic EtOH-fed rats were also binge-administered EtOH. Upon sacrifice, livers were harvested for histological and biochemical studies. Results: Body weight was similar for all groups, but blood glucose was significantly elevated in NNK ± EtOH treated rats. Blood alcohol levels increased from 55%ndash;113 g/dL with chronic feeding, to 188%ndash;229 g/dL 30 minutes after binge exposures. Livers in the EtOH, NNK, and EtOH+NNK groups exhibited swelling and pallor by macroscopic examination, and steatohepatitis by H&E and Oil Red O staining of histological sections. Although NNK, EtOH, and EtOH+NNK treatments caused steatohepatitis, hepatocellular necrosis, disruption of hepatic cord architecture, focal ballooning degeneration, and early chicken-wire fibrosis (Sirius Red stain), the severity of lesions and extent of liver involvement were consistently highest in the EtOH+NNK group, followed by EtOH, and then NNK treatment alone. The histopathological abnormalities were associated with impairments in mitochondrial function (reduced expression of cytochrome oxidase, subunit IV) and reductions in actin cytoskeletal protein content. Conclusion: The findings in these studies demonstrate that chronic exposure to tobacco nitrosamines and ethanol can each cause steatohepatitis, but the combined exposures produce additive adverse effects with respect to steatohepatitis and hepatic fibrosis. This new model provides a tool for further investigating ALD pathogenesis and possibly strategies for treatment or prevention of ALD in humans.

Disclosures:

The following people have nothing to disclose: Valerie Zabala, Ming Tong, Elizabeth Silbermann, Teresa Ramirez, Diana Lizarazo, Rebecca Ducore, Fusun Gun-dogan, Suzanne M. de la Monte

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Analysis of abnormal iron metabolism in patients with nonalcoholic steatohepatitis

Koji Miyanishi, Toshifumi Hoki, Shingo Tanaka, Yutaka Kawano, Masayoshi Kobune, Kohichi Takada, Tsutomu Sato, Yasushi Sato, Rishu Takimoto, Junji Kato;
Medical Oncology and Hematology, Sapporo Medical University School of Medicine, Sapporo, Japan

Background and aim: In nonalcoholic steatohepatitis (NASH) patients, increased hepatic iron accumulation is thought to be involved in the pathogenesis. In NASH livers, hepatic iron accumulation as well as oxidative DNA damage significantly increased. However, the precise mechanism for iron accumulation in the NASH liver remains unclear. In this study, we evaluated iron absorption from the gastrointestinal tract in patients with NASH. The expression of a panel of molecules in association with iron absorption in the duodenum and the liver was measured to analyze the mechanism of iron accumulation in the NASH liver. Methods: Thirty-seven cases who had been diagnosed as NASH by liver biopsy were enrolled. To exam the iron absorption, 100 mg of sodium ferrous citrate was administered to each individual after an overnight fasting. Subsequently, blood samples for serum iron measurement were taken after 15, 30, 60, 120 and 180 min. Serum hepcidin concentration was measured by mass of the liquid chromatograph tandem method. mRNA levels of hepcidin, hemojuvelin, DMT1 and fer-roportin-1 were measured by the real-time PCR method. We examined the mRNA expression of DMT1, Ferroportin-1, trans-ferrin receptors and ferritin on differentiated Caco2 cells grown with NASH patients' serum in transwells. Activity of iron regulatory protein (IRP) on these cells was also analyzed by elec-trophoresis mobility shift assay (EMSA). Results: Absorption of iron from the gastrointestinal tract, the DMT1 mRNA levels of the duodenal mucosa, serum Hepcidin concentrations and Hepcidin mRNA levels of the liver and Hemojuvelin mRNA expression of the liver significantly increased in NASH patients, when compared with healthy subjects. The DMT1 mRNA levels of the Caco2 monolayer cultured with NASH patients' serum significantly increased. N-acetylcysteine or IRP-1 siRNA clearly inhibited the increment of DMT1 mRNA levels. EMSA showed the activation of IRP on Caco2 cells grown with NASH patients' serum. Conclusion: In patients with NASH, increased iron absorption from the gastrointestinal tract causes hepatic iron accumulation, resulting in hepatic oxidative damage. Humoral factor(s) which induce oxidative stress in NASH serum may upregulate DMT1 expression in small intestine through the activation of IRP-1.

Disclosures:

The following people have nothing to disclose: Koji Miyanishi, Toshifumi Hoki, Shingo Tanaka, Yutaka Kawano, Masayoshi Kobune, Kohichi Takada, Tsutomu Sato, Yasushi Sato, Rishu Takimoto, Junji Kato

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ASMase deficiency unmasks the dissociation between hyperhomocysteinemia and intrasgastric alcohol feeding-induced endoplasmic reticulum stress

Anna Baulies1,2, Laura Martinez1,2, Hidekazu Tsukamoto3, Neil Kaplowitz4, Carmen Garcia-Ruiz1,2, Jose Fernandez-Checa1,2;
1Instituto Investigaciones Biomedicas Barcelona, CSIC, Barcelona, Spain; 2Liver Unit-IDIBAPS, and CIBEREHD, Barcelona, Spain; 3Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine, University of Southern California,, Los Angeles, CA, CA; 4USC Research Center for Liver Disease Keck School of Medicine University of Southern California, Los Angeles, CA, CA

Alcohol induced liver disease (ALD) is a major health concern of alcohol abuse and a leading cause of liver-related morbidity and mortality. The pathogenesis of ALD is multifactorial and still ill characterized. Endoplasmic reticulum (ER) stress has emerged as an important player in alcohol-induced steatosis and liver injury. Alcohol-mediated hyperhomocysteinemia (Hcy) is considered a key mechanism in alcohol-induced ER stress and recent evidence described the correlation between Hcy and liver injury in mouse strains sensitive to ALD. Acid sphin-gomyelinase (ASMase) promotes hepatocellular apoptosis and liver fibrosis, and has been shown to play a key role in oral alcohol-induced ER stress independently of Hcy. However, the degree of Hcy differs between oral alcohol feeding and the intragastric alcohol infusion model, being significantly lower in the former, thus raising the possibility for a threshold phenomenon for Hcy to induce ER stress regardless of ASMase. To test this hypothesis, ASMase null mice were subjected to alcohol feeding using the intrasgastric infusion model to examine their susceptibility to steatosis, liver injury, inflammation, mitochon-drial cholesterol trafficking and Hcy. Methods: ASMase null mice were fed a high-fat ethanol containing diet intragastrically for 4 weeks. Serum and liver samples were processed for histology, ALT determination, homocysteine analyses (HPLC), expression of ER stress markers, cholesterol trafficking (confocal microscopy), inflammation markers (RT-PCR) and lysosomal per-meabilization monitored by the cytosolic release of N-acetyl-β-glucosaminidase (NAG). Results: Compared to wild type mice, ASMase-/- mice were resistant to alcohol induced steatosis, exhibiting 70%ndash;90% lower trigliceride levels and oil red staining. Consistent with these findings, alcohol-induced ER stress (Chop, Pdi) and liver injury (3%ndash;4 fold increase in ALT) were observed in wild type but not in ASMase null mice. Interestingly, increase in plasma Hcy levels was similar in wild type and ASMase null mice (5%ndash;7 fold). Wild type but not ASMase null mice exhibitied increased StARD1 overexpression and mitochondrial cholesterol trafficking by alcohol feeding. Moreover, evidence for Kupffer cell M1 /M2 polarization was similar for wild type and ASMase mice. Lysosomal permeabilization examined by NAG activation in the cytosol was lower in ASMase null mice compared to wild type mice. Conclusion: ASMase null mice are resistant to intragastric alcohol-induced ER stress, steatosis, and liver injury despite severe Hcy, implying that the ability of Hcy to induce ER stress in response to alcohol is dependent on ASMase.

Disclosures:

Hidekazu Tsukamoto - Consulting: Shionogi & Co., S.P. Pharmaceutics; Grant/Research Support: The Toray Co.

Neil Kaplowitz - Consulting: GlaxoSmithKline, JNJ, Merck, Novartis, Hepregen, Takeda, Otsuka; Independent Contractor: Acetaminophen Litigation

The following people have nothing to disclose: Anna Baulies, Laura Martinez, Carmen Garcia-Ruiz, Jose Fernandez-Checa

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Chronic ethanol-induced impairment in Wnt/β-catenin signaling is attenuated by PPARδ agonist

Chelsea Q. Xu1, Suzanne M. de la Monte1,2, Jack R. Wands1, Miran Kim1;
1Department of Medicine, Liver Research Center, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI; 2Department of Pathology, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI

Backgrounds: The Wnt/β-catenin pathway is important for the regulation of liver growth, repair, and regeneration. It has been previously shown that chronic ethanol consumption blunts normal liver regenerative responses, in particular by inhibiting insulin/IGF signaling. Treatment with PPARδ agonist restored hepatic insulin responsiveness and normalized liver histology. Accordingly, we hypothesized whether these effects are associated with improvements in Wnt signaling. In this study, we investigated the effects of chronic ethanol exposure and subsequent treatment with PPARδ agonist on the expression of Wnt pathway genes during a post-partial hepatectomy (PH) time course. Methods: Adult male Long Evans rats were fed with isocaloric liquid diets containing 0 or 37% ethanol for 8 weeks followed by 2/3 PH. During the last three weeks, a portion of rats was fed with PPARδ agonist. All animals were sacrificed at 0, 18, 24, 30, 48, 72 hour, and one week time points after PH. Total RNA was extracted from liver tissue. The expression of 19 genes involved in the Wnt pathway was quantified by reporter signal amplification using the Quantigene 2.0 Multiplex Assay (Affymetrix). Results: Chronic ethanol consumption led to expression changes in the 19 genes tested, demonstrating an inhibition of Wnt/β-catenin signaling. During the first peak of DNA synthesis at 24 hours post-PH, a suppression of Wnt pathway genes was observed for Wnt7a, c-Jun, Ccnd1, and Fgf4. Chronic ethanol exposure also led to an overall downward shift in the expression of Wnt 1, Fzd3, Lef1, Bcl9, Wisp 1, Sfrp5, and Wif1. The expression of Ccnd1, a major regulator of the cell cycle, was elevated in the control group at 24 hours. However, ethanol treatment caused a delayed response and peak expression occurred at 72 hours post-PH. Treatment with PPARδ agonist rescued the ethanol-induced depression of Wnt gene expression for genes including Wnt1, Wnt7a, Fzd3, Lef-1, Tcf7l2, Bcl9, Ccnd1, Axin2, Wif1, and Sfrp2. Conclusions: These observations demonstrate that long-term ethanol consumption inhibits Wnt signaling, leading to an impairment of liver regeneration. Treatment with PPARδ agonist ameliorated this effect, suggesting that improvement of liver function in chronic ALD requires the restoration of Wnt signaling in addition to insulin/IGF signaling.

Disclosures:

The following people have nothing to disclose: Chelsea Q. Xu, Suzanne M. de la Monte, Jack R. Wands, Miran Kim

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The Breathprints in Patients with Liver Disease Identify Novel Breath Biomarkers in Alcoholic Hepatitis

Ibrahim A. Hanouneh1, Nizar N. Zein1, Frank S. Cikach2, Luma Dababneh2, David Grove2, Rocio Lopez3, Naim Alkhouri1, Raed Dweik2;
1Gastroenterology and Hepatology, Cleveland Clinic, Cleveland, OH; 2Pulmonary, Allergy, and Critical Care Medicine, Cleveland Clinic, Cleveland, OH; 3Quantitative Health Science, Cleveland Clinic, Cleveland, OH

Using selected-ion flow-tube mass spectrometry (SIFT-MS), precise identification of trace gases in human breath can be achieved. Aim: To determine whether concentration of volatile compounds in breath correlates with diagnosis and severity of liver disease in pts with alcoholic hepatitis (AH). Methods: We prospectively recruited pts with liver disease into two groups: liver cirrhosis with AH (N=40) and liver cirrhosis with acute decompensation (AD) from etiologies other than alcohol (N=40). A healthy control group without liver disease was identified (N=43). Using SIFT-MS, precise identification of volatile compounds in breath in parts per billion ranges was achieved in fasting state. Results: Of 14 pre-selected breath compounds, we identified 6 compounds that were elevated in pts with liver disease compared to healthy control. Those include 2-propanol, acetaldehyde, acetone, ethanol, pentane and trimethylamine (TMA). The levels of TMA, acetone and pentane, in particular, in breath were remarkably higher in pts with AH compared to those with AD and to healthy volunteer (p<0.001). Using ROC curve, we developed model for diagnosis of AH that included breath levels of TMA, Acetone and Pentane (TAP model). TAP provided excellent prediction accuracy for diagnosis of AH (AUC=0.93) with 97% sensitivity and 72% specificity for TAP score of 28. The levels of breath TMA moderately correlated with severity of AH as presented by MELD score [rho (95%CI); 0.38 (0.07, 0.69),p=0.018]. Isoprene and ethanol in breath were associated with survival in AH. Conclusion: Breathprint may provide non-invasive method for diagnosis of AH and may provide independent prognostic value in patients with AH.

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The inhibitory effect of ethanol on Sestrin3 in the pathogenesis of ethanol-induced Hepatic Steatosis

Xinqin Kang1, Rongya Tao2, Xiwen Xiong2, X Charlie Dong2, Suthat Liangpunsakul1,2;
1 Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, IN;2Bio-chemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

Background: Sestrins (Sesns) are a small family of stress-sensitive genes that control lipid metabolism. Chronic alcohol feeding leads to the alteration in lipogenic genes; which are under the regulation of Sesns. The present study was designed to investigate the role of sesn3 in the pathogenesis of alcohol-induced hepatic steatosis. Methods: The in vitro experiments were performed in the VL-17A cells. For the in vivo experiment, we fed C57BL/6J mice with alcohol containing diet for 4 weeks. We used both gain-of-function- and loss-of-function-based approaches by transfecting VL-17A cells with AdSesn3 and shSesn3 or injecting these adenoviruses through tailed vein of mice 10 days before the sacrifice. Results: Ethanol inhibits the expression of Sesn3 in VL-17A cells. Over-expression of Sesn3 by AdSesn3 significantly ameliorates TG accumulation; whereas down regulation using shSesn3 significantly deteriorated TG accumulation in VL-17A cells. Ethanol feeding decreased the hepatic mRNA expression of all 3 sesns; however, its effect was most pronounced on Sesn3. Over expression of Sesn3 using AdSesn3 prevents hepatic steatosis whereas knock down of Sesn3 with shSesn3 worsened hepatic steatosis in alcohol-fed mice (Figure A and B). Over-expression of Sesn3 significantly abrogates ethanol's effect on AMPK phosphoryla-tion and reduced the expression of genes encoding for lipid synthesis. The effect of ethanol on AMPK phosphorylation was augmented by knocking down Sesn3. The levels of hepatic LC3 expression, the marker for autophagy, were significantly decreased in ethanol-fed mice injected with shSesn3 compared to controls. Conclusion: The role of Sesn3 in ethanol-induced hepatic steatosis is mediated in part through AMPK signaling which leads to the alteration in the set of genes involving in lipid synthesis.

Disclosures:

The following people have nothing to disclose: Xinqin Kang, Rongya Tao, Xiwen Xiong, X Charlie Dong, Suthat Liangpunsakul

Sesn3 regulates ethanol-induced hepatic steatosis in vivo. (A) Hepatic Sesn3 expression from mice in each group. (B) Liver Histology (H&E and Oil Red O stain). Overexpression of Sesn3 using AdSesn3 prevents ethanol-induced hepatic steatosis and knockdown of Sesn3 with shSesn3 significantly worsened hepatic steatosis in mice fed with ethanol.

Disclosures:

The following people have nothing to disclose: Ibrahim A. Hanouneh, Nizar N. Zein, Frank S. Cikach, Luma Dababneh, David Grove, Rocio Lopez, Naim Alkhouri, Raed Dweik

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The signature biomarkers serum lipids of alcoholic steatohepatitis

Cristina Alonso1, Javier Michelena2, Ibon Martinez-Arranz1, Jose Altamirano2, Rebeca Mayo1, Ramón Bataller3,4, Jose M. Mato5, Juan Caballeria2;
1OWL, Derio, Spain;2Liver Unit, Hospital Clinic, CIBERehd, IDIBAPS, Barcelona, Spain; 3Department of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NY; 4IDIBAPS, Barcelona, Spain; 5CIC bioGUNE, CIBERehd, Derio, Spain

Background and aims. Alcoholic steatohepatitis (ASH) is a severe form of alcoholic liver disease that usually occurs in patients with alcoholic cirrhosis. Although the presence of ASH can be suspected on clinical and biochemical grounds, it is difficult to distinct ASH from decompensated alcoholic cirrhosis (DC). Several studies have shown that without histological confirmation the diagnosis of ASH would be inaccurate in 10%ndash;30% of patients. Thus, there is a need for noninvasive biomarkers for the diagnosis of ASH especially in patients with acute deterioration of alcoholic cirrhosis. The aim of the study was to identify a metabolic signature that distinguishes ASH from decompensated alcoholic cirrhosis. Methods. An ultra-performance liquid chromatography/time-of-flight tandem mass spec-trometry (UPLC/TOF-MS/MS)-based metabolomics platform (Barret al. J. Proteome Res. 2012, 11, 2521) was used for the semi-quantitative determination of amino acids and different classes of lipids (fatty acyls, glycerophospholipids, glyc-erolipids, sphingolipids and sterol lipids) in serum from patients with biopsy proven alcohol-related liver diseases. Sera metabolite profile was determined in 179 patients diagnosed as mild liver disease (n=47), ASH (mild n=17; severe n=48, according to ABIC and Maddrey scores) and cirrhosis (compensated n=26; decompensated n=41). Results. As expected, patients with mild liver disease showed clear metabolic differences with those with more advanced liver diseases, having significant higher levels of diglycerols, ceramides or diacylphospho-cholines and lower bile acids concentration. Although differences were also found when ASH and cirrhosis were compared as a whole, we focused the study in the comparison of severe ASH and DC due to the important clinical implications. DC samples were characterized by increased levels of cholesteryl esters and decreased content of lysophosphatidylcholines, acyl carnitines and free fatty acids, mainly those involved in the biosynthetic pathway of omega-3 and omega-6 fatty acids. A linear discriminant analysis based on those serum metabolic profiles was applied to generate a model able to separate patients with severe ASH and DC. The area under the receiver operating characteristic curve was 0.97 ± 0.02 (AUC ± se), and 0.92 ± 0.03 in the leave-one-out cross-validation. Conclusions. We have identified a robust serum metabolomic signature that reliably/accurately distinguishes patients with severe alcoholic steatohepatitis from those with decompensated cirrhosis.

Disclosures:

Jose M. Mato - Advisory Committees or Review Panels: ABBOTT; Stock Shareholder: OWL METABOLOMICS

The following people have nothing to disclose: Cristina Alonso, Javier Michelena, Ibon Martinez-Arranz, Jose Altamirano, Rebeca Mayo, Ramón Bataller, Juan Caballeria

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Effects of alcohol on the circadian oscillation of serum and hepatic lipid levels and the rhythmic expression of liver clock related genes

Hiroyuki Tsuchiya, Sangmin Lee, Yuxia Zhang, Rana Smalling, Li Wang;
University of Utah School of Medicine, Salt Lake City, UT

[Purpose] Chronic alcohol consumption causes the development of steatosis and severely damages liver function. Emerging evidence suggests that hepatic lipid metabolism is regulated by the circadian clock. In the present study, we investigated changes in hepatic lipid metabolism throughout the circadian cycle in the liver of mice subject to chronic and binge ethanol feeding. [Methods] The chronic and binge ethanol-feeding model was established using 8 weeks old, male C57BL/6 mice according to the protocol developed by Dr. Bin Gao's laboratory (Nat Pro-toc 2013;8:627%ndash;637). Briefly, the mice were randomly assigned to either the control-fed group (CTRL) or ethanol-fed group (EtOH). After acclimatization to a control liquid diet (Lieber-DeCarli diet; Bio-Serv, Frenchtown, NJ) for 5 days, the mice in the EtOH group were fed the Lieber-DeCarli diet containing 5% ethanol (Bio-Serv) ad libitum while the mice in the CTRL group were pair-fed with the EtOH group. Ten days after consuming the experimental diets, the mice were orally administered maltose dextrin solution (9 g of maltose dextrin/kg of body weight; CTRL group) or ethanol solution (5 g of ethanol/kg of body weight; EtOH group) at zeitgeber time (ZT) 3 (9 am), and were sacrificed at ZT12, 18, 0, and 6. Serum and livers were collected at each time point. [Results] Serum ALT and AST levels were induced by alcohol at all time points, but with ALT showing a stronger oscillation, which was highest at ZT12 and lowest at ZT0. Serum triglyceride (TG) levels exhibited the highest induction by alcohol at ZT0, which declined to basal levels by ZT12. Interestingly, hepatic TG reached the highest levels in the EtOH group at ZT12, which was gradually decreased to the lowest levels by ZT6. Serum cholesterol levels did not show marked differences in CTRL and EtOH groups, whereas liver cholesterol content was constantly higher in the EtOH group with a moderate rhythm. Consistently, oil red O staining revealed the highest hepatic neutral lipid accumulation at ZT12 and lowest at ZT6 in the EtOH group. Gene expression analysis by qPCR uncovered a striking effect of alcohol on the alteration of rhythmic expression of transcription factors E2F1 and Egr-1, nuclear receptors SHP and RORγ, bile acid synthesis enzyme Cyp7a1, lipid metabolic gene VLDLR, and the key clock gene NPAS2. [Conclusions] The effect of alcohol consumption by chronic and binge ethanol feeding in mice on the disruption of serum and hepatic lipid metabolism is strongly associated with alterations in the expression of key liver circadian clock genes.

Disclosures:

The following people have nothing to disclose: Hiroyuki Tsuchiya, Sangmin Lee, Yuxia Zhang, Rana Smalling, Li Wang

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Ethanol reprograms the transcription factor FOXO3 converting it from an antioxidant to an apoptosis-inducing protein in primary human hepatocytes and human hepatoma cells

Zhuan Li1, Josiah Cox1, Irina Tikhanovich1, Sudhakiranmayi Kuravi1, Kenneth Dorko2, Steven A. Weinman1;
1Internal Medicine, University of Kansas Medical Center, Kansas City, KS; 2Phar-macology and Toxcology, University of Kansas Medical Center, Kansas City, KS

FOXO3 is a multifunctional transcription factor that initiates several different transcriptional programs including oxidative stress resistance, cell proliferation, apoptosis, autophagy, and metabolism. The mechanisms that regulate transcriptional specificity of FOXO3 are unknown. We have recently shown that ethanol and HCV infection each individually activate FOXO3 but they do so by different post-translational modifications. The AIM of this study was to determine the effects of ethanol on the transcriptional specificity and post-translational modifications of FOXO3 and their consequences. METHODS: Huh7.5 cells were transfected with HA-tagged FOXO3, treated with 50 mM ethanol for 48 h and/or infected with HCV strain JFH1. ChiP assays were performed with anti-HA or FOXO3 antibodies. A phospho-specific S574-P_FOXO3 antibody was generated by Epitomics. RESULTS: Ethanol treatment increased mRNA for the apoptotic FOXO3 target protein Bim but not the antioxidant target protein SOD2. HCV-infection, which similarly stimulated FOXO3 reporter activity, had the opposite effect activating SOD2 but not Bim. We performed ChIP assays on Huh7.5 cells overexpressing FOXO3 and determined promoter binding of nine FOXO3 target genes. EtOH resulted in a 5-fold increase in FOXO3 binding to the proapototic promoters (TRAIL and Bim), but not the antioxidant (SOD2 and PrxIII) promoters. The increased binding of FOXO3 to proapoptotic promoters was associated with an increase in mRNA and protein levels for TRAIL, activation of caspase 3, increased LDH release and cell death. FOXO3/ethanol induced caspase activation and cell death was completely prevented by either TRAIL receptor antagonists or caspase inhibitors. Ethanol caused rapid JNK dependent phosphorylation of FOXO3 at serine 574 in both Huh7.5 cells and primary human hepatocytes. FOXO3-S574-P was found exclusively in the nucleus and ChIP studies with an S574-P specific antibody showed binding of this form exclusively to the pro-apoptotic promoters BIM and TRAIL. Blocking FOXO3 phosphorylation at this site with an S574A mutant abolished ethanol-induced apoptosis and TRAIL promoter binding. A phos-phomimetic FOXO3_S574D mutant induced apoptosis even in the absence of ethanol. CONCLUSION: Ethanol causes a specific phosphorylation of FOXO3 that selectively activates binding to promoters for pro-apoptotic proteins and induces caspase and TRAIL-dependent cell death without activating antioxidant or cell cycle control genes. This novel mechanism may contribute to the phenotype of alcohol-induced liver disease and is a potential therapeutic target.

Disclosures:

The following people have nothing to disclose: Zhuan Li, Josiah Cox, Irina Tikhanovich, Sudhakiranmayi Kuravi, Kenneth Dorko, Steven A. Weinman

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Hepatocyte- and myeloid cell-specific deletion of Sirtuin-6 prevents alcohol-induced hepatocellular damage in mice

Adeline Bertola1, Ming-Jiang Xu1, Chuxia Deng2, Bin Gao1;
1 Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD;2Genetics of Development and Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD

Sirtuin-6 (SIRT6) is a member of the sirtuin family of NAD+-dependent deacetylases and has been implicated in a wide range of cellular processes including genomic stability, stress response, energy metabolism, inflammation, tumorigenesis and ageing. Recent studies have shown that hepatocyte-specific deletion of SIRT6 results in fatty liver formation and that myeloid cell-specific SIRT6 knockout mice develop chronic liver inflammation. Given that chronic alcohol consumption is associated with decreased cellular NAD+ levels, we hypothesized that decreased SIRT6 activity may contribute to alcoholic liver disease. To investigate the cell-specific role of SIRT6 in the patho-genesis of alcoholic liver disease, hepatocyte-specific SIRT6 knockout (L-SIRT6 KO) mice, myeloid cell-specific SIRT6 knockout (M-SIRT6 KO) mice, hepatocyte- and myeloid cell-specific double knockout (d-SIRT6 KO) mice and their wild-type (WT) lit-termates were fed a Lieber-DeCarli liquid diet containing 5% ethanol for 10 days then gavaged with a single dose of ethanol (5g/kg body weight) and sacrificed 9 hours later (NIAAA model). As expected, chronic plus binge ethanol feeding caused substantial liver injury in WT mice, as indicated by elevated serum ALT and AST levels. Surprisingly, L-SIRT6, M-SIRT6 and d-SIRT6 KO mice were resistant to chronic plus binge ethanol feeding-induced elevation of serum ALT and AST levels. However, the level of hepatomegaly and hepatic triglyceride accumulation was similar in ethanol-fed L-SIRT6, M-SIRT6 and d-SIRT6 KO mice compared with WT mice. The hepatic gene expression level of the proinflammatory cytokines TNF-alpha and interleukin (IL)-1 beta was similar in all groups of mice after chronic plus binge ethanol feeding. On the other hand, the expression level of the hepatoprotective cytokine IL-6 was higher in ethanol-fed L-SIRT6 KO mice and may protect these mice against alcoholic liver injury. Furthermore, the hepatic gene expression of the macrophage marker F4/80 was increased in ethanol-fed M-SIRT6 and d-SIRT6 KO mice compared with WT mice, suggesting that SIRT6 may regulate Kupf-fer cell functions. In conclusion, our findings indicate that SIRT6 in both hepatocytes and myeloid cells plays an important role in promoting hepatocellular damage induced by chronic plus binge ethanol feeding independently of liver steatosis and likely through modulation of inflammatory components.

Disclosures:

The following people have nothing to disclose: Adeline Bertola, Ming-Jiang Xu, Chuxia Deng, Bin Gao

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Absence of FN14 Ameliorates Acute Ethanol-Induced Steatohepatitis

Gamze Karaca1, Guanhua Xie1, Marzena Swiderska-Syn1, Gregory A. Michelotti1, Steve S. Choi1,2, Linda C. Burkly3, Anna Mae Diehl1;
1 Division of Gastroenterology, Duke University Medical Center, Durham, NC; 2Section of Gastroenterology, Durham Veterans Affairs Medical Center, Durham, NC; 3Departments of Exploratory Science, Discovery Biology, and Validation Biology, Biogen Idec Inc., Cambridge, MA

Background: Tweak and its receptor, fibroblast growth factor-inducible 14 (Fn14, a TNF receptor superfamily member) function as growth factors for bipotent liver progenitor cells. Accumulation of Fn14-positive progenitors occurs in severe acute alcoholic steatohepatitis and correlates with acute mortality in humans. This study examined whether Fn 14 deletion is beneficial in an acute ethanol (EtOH) induced steatohepatitis model in mice. Methods: Adult C57BL/6 (WT, n=16) or FN14 KO (n=16) male mice were treated with High Fat Lieber de Carli diet (HF), HF+ 2% EtOH Lieber deCarli diet (EtOH), HF + CCl4 (1 μl/g body weight i.p. twice per week), or HF+EtOH+CCl4 for 2 weeks, and sacrificed 72 h after the last CCl4 injection (n=4/group). Livers were analyzed for injury, fibrosis, progenitors, and inflammatory cytokines using qRT-PCR, biochemical assays, and immunohistochemistry. Results: Compared to each of the respective WT control groups, WT mice treated with HF+ETOH+CCl4 had significantly higher hepatic expression of Fn14 mRNAand protein, and developed more severe steatohepatitis and bridging fibrosis, as evidenced by H&E and Sirius red staining, induction of cytokines (TNFα, IL6 and IL4 mRNAs), up-regulation of myofibroblast markers (α-SMA, Desmin mRNAand protein), and increased collagen content quantified by hydroxyproline assay. The progenitor response (as assessed by changes in mRNA and protein levels of α fetoprotein, Sox9, CD24 and Lgr5) paralleled the severity of steatohepatitis in WT mice. In Fn14 KO mice, elevation of Fn14 did not occur, steatohepatitis severity was reduced, and all the inflammatory and fibrosis responses were inhibited (each p < 0.05 vs WT mice). Progenitor accumulation was also dramatically attenuated (>50% reduction; p<0.05). Conclusion: Fn14 expression is up-regulated during severe acute EtOH-induced steatohepatitis. Deleting Fn14 inhibits hepatic cytokine induction, reduces steatohepatitis severity, blocks accumulation of progenitors and myofibroblasts (a.k.a, the ductular reaction), and reduces liver fibrosis. This suggests that Fn14-positive progenitors promote fibro-inflammatory responses during acute alcoholic hepatitis and identifies Tweak/Fn 14 as a potential target in this disease.

Disclosures:

Linda C. Burkly - Employment: Biogen Idec, Inc.; Stock Shareholder: Biogen Idec, Inc.

Anna Mae Diehl - Consulting: Bristol Myers Squibb, Synergy, GlaxoSmithKline, Norgine; Grant/Research Support: GlaxoSmithKline

The following people have nothing to disclose: Gamze Karaca, Guanhua Xie, Marzena Swiderska-Syn, Gregory A. Michelotti, Steve S. Choi

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Increased Methylation Demand Exacerbates Alcohol-Induced Liver Injury

Kusum K. Kharbanda1,2, Sandra L. Todero1, David J. Orlicky3, Dean J. Tuma1,2;
1 Research Service, Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, NE; 2Internal Medicine, University Nebraska Medical Center, Omaha, NE; 3Pathology, University of Colorado Denver, Aurora, CO

We previously reported that chronic ethanol intake lowers hepatocellular S-adenosylmethionine (SAM) to S-adenosylho-mocysteine (SAH) ratios and significantly impairs many essential liver transmethylation reactions catalyzed by specific SAM-dependent methyltransferases. One such enzyme is guani-dinoacetate methyltransferase (GAMT) that catalyzes the transfer of a methyl group from SAM to guanidinoacetate (GAA) to form creatine. Hepatic GAMT is a major consumer of methyl groups and utilizes as much as 40% of the SAM-derived methyl groups. In the past few decades, ingestion of methyl-consuming compounds has substantially increased primarily due to pollution, food additives, niacin fortification and high meat consumption putting additional stresses on cellular methylation potential. The purpose of our study was to investigate the role that increased methyl consumption, either alone or combined with alcohol consumption, could play in the pathogenesis of liver injury. Because of our interest in GAMT-catalyzed reaction, we chose GAA as a potent methyl group consumer. Adult male Wistar rats were pair-fed the Lieber DeCarli control or ethanol diet in the presence or absence of 0.36% GAA in these respective diets for 2 weeks. At the end of the feeding regimen, the rats were sacrificed and blood and livers were collected and processed for biochemical and histological analyses. We observed microvescicular steatosis and a 7 fold-increased triglyceride accumulation in the livers of rats fed the ethanol-alone diet for 2 weeks as compared to controls. GAA administration alone resulted in similar changes as the ethanol fed group but to a lesser extent, only a 4-fold increased triglyceride accumulation compared to controls was observed. However, supplementing GAA in the ethanol diet produced a marked decrease in the methylation potential as evident from a significantly lower hepatocellular SAM:SAH ratio, panlobular macro-and micro-vesicular steatosis and a 28-fold increased triglyceride accumulation as compared to the control group. These GAA-supplemented ethanol diet-fed rats displayed inflammatory changes as indicated by the histological presence of lipogranulomas and microgranulomas. These rats also exhibited 2 to 10-fold increased hepatic GAA and serum AST, ALT, homocysteine and GAA levels compared to the rest of the groups. To summarize, increased methylation demand superimposed on chronic alcohol consumption causes hyperhomo-cysteinemia, steatohepatitis and more pronounced indices of liver injury. To conclude, chronic alcohol consuming patients should be cautioned for increased dietary intake of methyl-consuming compounds even for a short period of time.

Disclosures:

The following people have nothing to disclose: Kusum K. Kharbanda, Sandra L. Todero, David J. Orlicky, Dean J. Tuma

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The dysregulation of CD4+/CD8+ T cells is associated with expansion of myeloid derived suppressor cells in peripheral blood of alcoholic liver cirrhosis patients

Qifa Xie1, Mohammad K. Mohammad1, Matthew C. Cave1,2, Shirish Barve1, Craig J. McClain1,2;
1 Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY

We previously noted the accumulation of myeloid derived suppressor cells (MDSCs), mainly composed of monocytic MDSCs (M-MDSC) in mice fed a high-fat diet or administered chronic alcohol by gavage feeding. The M-MDSCs isolated from the steatotic livers of obese mice were functional MDSCs, readily regulating T cell responses and mediating chronic inflammation. Here, we evaluated the clinical relevance of MDSCs and MDSC-related dysregulation of lymphocytes in peripheral blood of alcoholic cirrhotic patients (Evidence of alcoholic cirrhosis: Child-Pugh score A or B; No HCV, HBV, HIV, history of recent infection, hospitalization within 28 days, suspicion of cancer, history of serve chronic disease, pregnancy, or hepatic encephalopathy; Creatinine > 1.5). The subpopulation of MDSCs, T cell subsets and NK cells were tested in peripheral blood from alcoholic cirrhotics (n=16) and healthy donors (n=12). The expressions of IFN-γ, IL-4, and IL-17 and proliferation were analyzed using anti-CD3/CD28-stimulated T lymphocytes. There was significant reduction of CD8+T cells (15.99 ± 1.6 vs 24.89 ± 2.25, p=0.0028) and the CD8+/CD4+ ratio (0.5806 ± 0.10 vs 0.9622 ± 0.12, p=0.0341) in peripheral blood of alcoholic cirrhotics compared with healthy controls. The CD8+ or CD4+ T cells from alcoholic cirrhotics also exhibited less proliferation potential and made more IFN-γ following anti-CD3/CD28 stimulation. This dysregulation of T cells in alcoholic cirrhotics was associated with total expansion of MDSCs, denoted here as CD33+ HLA-DR-. MDSCs were evaluated as a component of total blood leukocytes and as a component of PBMCs. An accumulation of MDSCs, mainly composed of CD33+HLA-DR-CD15+CD14-granulocytic-MDSCs (55.28 ± 9.00 vs 16.18 ± 5.16, p=0.0037, N=6) was observed in peripheral blood leukocytes of alcoholic cirrhotics. Moreover, an expansion of MDSCs, mainly composed of CD33+HLA-DR-CD15-CD14+ M-MDSCs (52.57 ± 5.56 vs 24.14 ± 7.44, p=0.0099) was seen in PBMCs in alcoholic cirrhotics. Conclusions: Our study provides evidence of an increased population of CD33+ HLA-DR-MDSCs in the peripheral blood of alcoholic cirrhotics. Our data also suggest that there is MDSC-related dysregulation of CD4+/CD8+ T cells in the peripheral blood of patients with alcoholic cirrhosis. The differentiation of M-MDSC or G-MDSC, and the ability to regulate T cell (CD4+/CD8+, Regulatory T cells), NKT and NK cell responses in alcoholic cirrhosis are under further investigation by our group.

Disclosures:

Shirish Barve - Speaking and Teaching: Abbott

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Qifa Xie, Mohammad K. Mohammad, Matthew C. Cave

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Long-term plus singe binge ethanol feeding induces severe steatohepatitis in mice: a novel model of human alcoholic hepatitis

Ming-Jiang Xu1, Yan Cai2, Hua Wang1, Adeline Bertola1, Jim Lu3, Ramón Bataller4, Bin Gao1;
1 National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD; 2National Cancer Institute, National Institutes of Health, Bethesda, MD; 3GoPath Diagnostics, LLC, Chicago, IL; 4Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC

Alcoholic hepatitis (AH) is one of the most deadly liver diseases. AH often occurs in patients who have a background of chronic drinking and a history of recent excessive drinking. The development of new therapies is hampered by lack of an animal model. We have recently developed 10-day chronic plus single binge model, which induces significantly elevation of serum ALT but mild steatosis and inflammation in C57BL/6J female mice (Bertola et al, Nature Protocols 2013). By using various combinations of long-term plus one or multiple binges of ethanol feeding, we identified that 8- to 12-week chronic plus single binge induced the most severe form of alcoholic steatohepatitis among the several other combinations. This model induced histological changes similar to AH in humans, which include severe steatosis with ∼10-fold increase in liver triglyc-eride, significant infiltration of neutrophils evidenced by MPO staining, significant elevation of serum ALT (∼230 U/L) and AST (AST/ALT>2), remarkable increase of TUNEL positive liver cells, and mild fibrosis identified by MASSON staining and increased expression of collagen genes (eg. Col1a1, col3a1, col4a2, col5a2, col12a 1). We next assessed whether this new model reproduces the changes in the hepatic transcriptome recently described in patients with AH (Affo et al, Gut 2013). Microarray and qPCR analyses revealed a marked up-regula-tion of key pro-inflammatory and pro-apoptotic genes (eg. Fn14, TRAIL-R2, CD137, TNFR1, TNFR2, DR6, CXCL1, CXCL2, CXCL4, LCN2, et al.), which were also found overexpressed in the livers from patients with AH. In conclusion, this novel model closely simulates the histological and molecular features of human alcoholic hepatitis.

Disclosures:

Jim Lu - Employment: GoPath Pathology Associates, SC; Independent Contractor: GoPath Laboratories LLC; Management Position: GoPath Global LLC

The following people have nothing to disclose: Ming-Jiang Xu, Yan Cai, Hua Wang, Adeline Bertola, Ramón Bataller, Bin Gao

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Upregulation of hepatic phosphodiesterase 4 (PDE4) by ethanol is involved in the development of alcoholic steatosis in mice

Leila Gobejishvili1, Shirish Barve1, Diana Avila1, Jingwen Zhang1, Craig J. McClain1,2;
1 Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY

Background: Steatosis is the initial, most frequent hepatic manifestation that occurs in response to acute as well as chronic ethanol consumption. Alcohol-induced hepatic steatosis is no longer considered to be a benign state; it is now regarded as a significant risk factor for more progressive disease. Steatotic hepatocytes have increased sensitivity to injury produced by inflammatory cytokines, particularly TNF. Cyclic adenosine monophosphate (cAMP) plays a significant role in the regulation of both hepatic lipogenesis and fatty acid oxidation. Also, cAMP protects hepatocytes from apoptosis induced by LPS,

FasL and TNF-α. cAMP levels are tightly regulated by their degradation by phosphodiesterase enzymes, among which the PDE4 family plays a major role. Our recent work demonstrated that PDE4 is expressed in the liver. The aim of this study was to examine the effect of alcohol on the expression of hepatic PDE4 and its potential role in the development of alcoholic steato-hepatitis in a mouse model of alcoholic liver disease. Methods: C57Bl/6 and pde4b knockout mice on the same background were fed Lieber DeCarli liquid diet for 4 weeks. One group of mice received rolipram (5 mg/kg body weight, intraperi-toneally) three time a week. Liver steatosis was evaluated by Oil-Red-O staining and confirmed by biochemical assessment of hepatic and serum triglyceride accumulation. Expression of hepatic PDE4 and proteins involved in lipid metabolism was evaluated at mRNA and protein levels. Results: Our data show that alcohol feeding of mice, which leads to fat accumulation in the liver and up-regulation of fatty acid synthase, is accompanied by a significant increase in hepatic PDE4 mRNA and protein expression. Treatment of mice with the highly specific PDE4 inhibitor, rolipram, prevents alcohol induced fat accumulation in the liver. Further, mice deficient in pde4b (pde4b-/-) are markedly protected from the development of alcoholic steatosis. Conclusion: These results suggest that ethanol can significantly influence hepatic PDE4 expression and subsequent cAMP metabolism, leading to increased lipid accumulation. These data also strongly imply that hepatic PDE4 expression is a clinically relevant target, and its inhibition can significantly attenuate the development of hepatic steatosis and injury.

Disclosures:

Shirish Barve - Speaking and Teaching: Abbott

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Leila Gobejishvili, Diana Avila, Jingwen Zhang

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Role of sumoylation in alcoholic liver disease

Maria Lauda Tomasi, Minjung Ryoo, Shelly C. Lu;
Division of Gas-troenterology and Liver Diseases, USC Research Center for Liver Diseases; Southern California Research Center for Alcoholic Liver and Pancreatic Diseases and Cirrhosis, Keck School of Medicine USC, Los Angeles, CA

Purpose of Study: Alcohol abuse is a leading factor in mortality from liver disease and increases the risk for a wide range of adverse health effects. The liver, as the primary site of alcohol metabolism, is a major target of injury. The spectrum of Alcoholic Liver Diseases (ALD) includes simple steatosis, alcoholic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Sumoylation is a post-translational modification that modulates multiple cellular processes such as signal transduction, stress responses, cellular trafficking, protein-protein interactions, pro-tein-DNA interactions and transcriptional activity. SUMO is comprised of four distinct proteins in humans (SUMO-1, -2, 3-and -4). Sumoylation is often increased under oxidative stress. We recently reported that ubiquitin conjugating enzyme 9 (Ubc9), the sole E2 enzyme of sumoylation, is induced in the livers of intragastric ethanol-infusion (EI) treated mice but the functional significance of this is unknown. Our aim was to examine whether the dysregulation in sumoylation contributes to the pathogenesis of ALD and elucidate the molecular mechanism(s). Methods: Studies were done using in vivo EI treated mice and mouse hepatocytes. Expression of genes and proteins were measured by real-time PCR and Western blot analyses, respectively. Reactive oxygen species (ROS) and triglyceride (TG) production were analyzed using a commercial kit. Results: We found SUMO-1, -2, -3 and Ubc9 mRNA levels are increased the livers of EI mice. Also, EI mice show an overall increase in protein sumoylation by SUMO-1 but only minor changes in sumoylation by SUMO-2/3. Ethanol treatment of primary mouse hepatocytes increased production of ROS and TG. In addition, we found increased expression of Ubc9 and SUMO genes, Cyp2e1 and an overall increase in SUMO-1 protein sumoylation like in EI livers. Silencing of Ubc9 prevented ethanol-induced fat accumulation, ROS production and the increase in Cyp2e1 expression in primary mouse hepatocytes. Conclusions: Ethanol-mediated sumoylation increased triglyceride and ROS production in livers of EI mice and primary hepatocytes. Ubc9 knockdown has protective effect against ROS production and fat accumulation in primary hepatocytes.

Disclosures:

The following people have nothing to disclose: Maria Lauda Tomasi, Minjung Ryoo, Shelly C. Lu

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Increase in Secondary Bile Acids are Associated with Colonic Inflammation in Active Alcohol Abuse in Cir-rhotic Patients Compared to Non-Alcoholic Cirrhotics: A Potential Mechanism for Alcohol-induced Gut Inflammation?

Genta Kakiyama1, Huiping Zhou3, Phillip Hylemon2, William M. Pandak1, Xuan Wang3, Emily C. Gurley3, Douglas M. Heuman1, Pamela Monteith1, Nicole Noble1, Melanie White1, Jasmohan S. Bajaj1;
1Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth University and McGuire VA Medical Center, Richmond, VA; 2Microbiology, Virginia Commonwealth University and McGuire VA Medical Center, Richmond, VA; 3Immunology, Virginia Commonwealth University and McGuire VA Medical Center, Richmond, VA

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier, bacterial translocation, inflammation & infections but the mechanism is not known. Gut microbiota can transform primary bile acids (BA) to secondary BAs which are toxic and can adversely impact the gut barrier. The interaction of secondary BAs and alcohol abuse as modulators of intestinal inflammation in cirrhosis is unclear. Aim: Define the effect of active alcohol intake on fecal BA levels, ileal & colonic inflammation in cirrhotics. Methods: Four age-matched groups; one control & three cirrhotic (NAlc:non-alcoholic (non-drinkers),AbsAlc: abstinent alcoholic for >6mths & Curralc:(cur-rently drinking without alc hepatitis) were included. Fecal BA analysis using HPLC & GC-MS were performed. Median primary, secondary BAs concentrations and the ratios were compared between groups. A subgroup of controls, NAlc and CurrAlc underwent colonoscopy with ileal and sigmoid colon Bx. mRNA expression of TNF-α, IL1 β,IL6 and Cox-2 were performed on the Bx & compared between groups. Results: 97 patients (19 healthy, 10 CurrAlc, 38 AbsAlc and 30 NAlc, age 56 yrs, median MELD:10.5, alcohol use in Alc groups:28 yrs) were included; 5 each of healthy, Currr Alc and NAlc underwent ileal & colonic Bx. Median MELD was similar between groups (CurrAlc:8.5, AbsAlc: 13 and NAlc:9,p=0.1). Total BAs and relative proportion of secondary BAs (DCA, LCA) compared to their primary counterparts (CA, CDCA) were significantly higher in CurrAlc pts compared to the remaining cirrhotics (Table). This secondary BA increase was also associated with a significantly higher mRNA expression of TNF-α, IL1 β,IL6 and Cox-2 in colonic Bx but not ileum in only CurrAlc compared to NAlc & controls. Conclusion: Active alcohol abuse in cirrhosis is associated with a significant increase in the secondary BA formation compared to abstinent alcoholic cirrhotics and non-alcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.

   Cirrhosis 
***p<0.001,*p<0.05(n=19)NAlc (n=30)AbsAlc (n=38)CurrAlc (11=10)
Total BAs5.42.92.28.9***
Cholic (CA)<0.050.10.12<0.05
Chenodexoycholic (CDCA)0.010.10.210.16*
Lithocholic (LCA)1.61.00.41.9*
Deoxycholic(DCA)2.30.40.433***
Total Primary BAs0.010.230.460.16*
Total Secondary BAs4.11.51.05.7***
Secondary/Primary BA Ratio19.57.51.123.7*

Disclosures:

William M. Pandak - Patent Held/Filed: Virginia Commonwealth University, Veterans Affairs Medical Center

Douglas M. Heuman - Consulting: Bayer, Grifols, Genzyme; Grant/Research Support: Exilixis, Novartis, Bayer, Bristol Myers Squibb, Scynexis, Ocera, Mannkind, Salix, Globeimmune, Roche, SciClone, Wyeth, Otsuka, Ikaria, UCB, Celgene, Centocor, Millenium, Osiris; Speaking and Teaching: Otsuka, Astellas

Jasmohan S. Bajaj - Advisory Committees or Review Panels: Salix, Merz, otsuka, ocera, grifols, american college of gastroenterology; Grant/Research Support: salix, otsuka, grifols

The following people have nothing to disclose: Genta Kakiyama, Huiping Zhou, Phillip Hylemon, Xuan Wang, Emily C. Gurley, Pamela Monteith, Nicole Noble, Melanie White

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Dysregulated Systemic Lipid Metabolism in Acute Alcoholic Hepatitis

Jaideep Behari1, Charles Gabbert1, Amit Raina2, Shahid M. Malik1;
1Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh, Pittsburgh, PA; 2Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA

Background & Aims: Liver steatosis is a characteristic feature of alcoholic liver disease. Experimental studies using rodent models have implicated adipose tissue lipolysis and liver fatty acid uptake as contributors to alcohol-induced hepatic steatosis. However, the relevance of these findings to patients with acute alcoholic hepatitis (AAH) is unknown. We sought to determine whether patients with AAH demonstrate evidence of increased lipolysis, insulin resistance and altered adipokine expression. Methods: We prospectively enrolled 56 patients admitted with severe AAH [Maddrey Discriminant Function score > 32] to a single tertiary care center. As control, we enrolled 25 patients with alcoholic cirrhosis without alcoholic hepatitis from our outpatient liver clinic. We determined serum cytokine and adipokine levels using the Luminex platform. We measured serum glycerol, fatty acid and glucose levels by colorimetric assays and serum insulin by ELISA. We used gas chromatogra-phy for serum lipidomic analysis. The University of Pittsburgh Institutional Review Board approved the study and all participants signed informed consent prior to enrollment. Results: At baseline, AAH patients had higher serum bilirubin, AST, pro-thrombin time, and Model for Endstage Liver Disease (MELD) scores. AAH patients demonstrated higher mean serum glycerol (23.4 vs 5 mg/L, p<0.001) and total free fatty acid levels (436.7 vs 289.4 μM, p<0.01) compared with patients with alcoholic cirrhosis, suggesting higher rates of adipose tissue lipolysis. Lipidomic analysis revealed significant changes in individual serum free fatty acid species. AAH patients demonstrated higher unsaturated free fatty acid (C16:144.2 vs16.2 μM, p<0.001; C18:1157.3 vs 95.8 μM, p<0.01) and palmi-tate (122 vs 79.4 μM, p<0.01) levels. AAH patients had similar serum glucose but lower serum insulin levels compared with controls. Insulin sensitivity, as measured by HOMA-IR, was similar between the two groups. AAH patients had higher serum adiponectin and lower serum leptin levels, a pattern opposite to that seen in animal models. Striking elevation in serum resistin level was found in AAH patients (21.7vs8.5 ng/ml, p<0.001) and serum resistin levels correlated with severity of liver injury (defined as MELD score on admission; r = .44, p<0.001). Conclusions: Our results support the hypothesis that severe AAH is associated with increased adipose tissue lipolysis. However, there are important differences in patterns of adipokine elevation between rodent models and AAH patients. Dysregulated systemic lipid homeostasis in AAH suggests important patho-genetic links between the liver and adipose tissue in this disor-der.

Disclosures:

The following people have nothing to disclose: Jaideep Behari, Charles Gabbert, Amit Raina, Shahid M. Malik

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Mouse model of alcoholic hepatitis: no role of Osteopontin

Raul G. Lazaro1, Akiko Ueno1, Rylee Do1, Nian-Ling Zhu1, Raymond Wu1, Jun Xu1, Samuel W. French2,1, Keigo Machida1, Hidekazu Tsukamoto1;
1Southern California Research Center for ALPD and Cirrhosis, Keck School of Medicine, University of Southern California, Los Angeles, CA; 2Anatomic Pathology, Harbor-UCLA Medical Center, Los Angeles, CA

Alcoholic hepatitis (AH) represents a distinct spectrum of alcoholic liver disease with intense neutrophilic inflammation and high mortality and is yet to be reproduced in animal models. Epidemiology suggests an association of AH with Western diet high in cholesterol and saturated fat (HCFD) and binge drinking concurrent to habitual daily drinking. [Aim] We tested the effects of HCFD plus intragastric (iG) alcohol intake without or with weekly binge alcohol intake on liver pathology in wild type (WT) and osteopontin deficient (Spp-/-) mice. [Methods] Male WT and Spp-/- mice were fed ad lib chow or HCFD (1% w/w cholesterol, 20%Cal lard, 17% corn oil) for 2 wk prior to implantation of iG catheters. The animals were then iG-fed for 8 wk high fat diet (35%Cal as corn oil) plus ethanol (27 g/kg/day) or isocaloric dextrose at 60% of total calories. Mice continued to consume HCFD or chow ad lib for remaining 40% calories. Weekly from 2nd wk, alcohol infusion was withdrawn for 5 hr and alcohol binge (3.5∼5g/kg) equivalent to the amount withdrawn was given. [Results] Hybrid model of HCFD ad lib and ethanol iG feeding, produces synergistic ASH with elevated plasma ALT (398+37U/L), mononuclear cell inflammation, perisinusoidal and pericellular fibrosis in 62% of mice. Weekly binge in this hybrid model, despite the identical overall alcohol intake, induces in 57% (15/26) of the mice, numerous foci of neutrophilic infiltration and aggregation around necrotic hepatocytes. Hybrid+binge mice exhibit clinical features of AH such as a 2-fold increase in AST/ALT ratio compared to Hybrid ASH model, hypoalbuminemia (2.3+0.4g/dl), splenomegaly, and a 3-fold increase in plasma bilirubin. Hepatic myeloperox-idase (Myo) mRNA is increased 45-fold and correlates with neutrophilic infiltration (r=0.80, p<0.001). Spp, Cxcl1 (Gro), and Il-17a implicated in inflammation, are induced 42, 86, and 6.5 fold, respectively while Cd68 and Il-22 are repressed more than 10 fold. Hepatic TLR4 upregulation and activation as assessed by TLR4 IB and TRAF6/TAK1 co-IP, are most conspicuous in the AH model. Ingenuity analysis of AH vs. ASH livers reveals clusters of upregulated neutrophil- and tumor-associated genes and profoundly repressed metabolic (drug, lipid) and transport genes in AH. Histological evidence of AH is evident in 50% (5/10) of Spp-/- mice subjected to the identical Hybrid+Binge regimen, and no differences are found in ALT and Myo, Cxcl1, Il-17a, and Il-22 expression compared to WT mice. [Conclusions] Alcohol binge in the hybrid mouse model which produces ASH, triggers histological and pathophysio-logical features of AH, and Osteopontin has no role in this pathology.

Disclosures:

Hidekazu Tsukamoto - Consulting: Shionogi & Co., S.P. Pharmaceutics; Grant/Research Support: The Toray Co.

The following people have nothing to disclose: Raul G. Lazaro, Akiko Ueno, Rylee Do, Nian-Ling Zhu, Raymond Wu, Jun Xu, Samuel W. French, Keigo Machida

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The CirCom comorbidity scoring system for liver cirrhosis: development and validation

Peter Jepsen1,2, Hendrik V. Vilstrup1, Timothy L. Lash2,3;
1Department of Medicine V (Hepatology and Gastroenterology), Aarhus University Hospital, Aarhus, Denmark;2Department of Clinical Epidemiology, Aarhus University Hospital, Aarhus, Denmark; 3Depart-ment of Epidemiology & Prevention, Wake Forest School of Medicine, Winston-Salem, NC

Background: Comorbidity increases the mortality of cirrhosis patients. We developed a cirrhosis-specific comorbidity score (CirCom) and compared it with the universal Charlson Comorbidity Index that includes seventeen diseases. Methods: We used data from nationwide healthcare registries to identify Danish citizens diagnosed with cirrhosis in 1999%ndash;2008 (N=13,455). The majority had a history of alcoholism. They were followed through 2010 and characterized by 34 comor-bidities. We used Cox regression to assign severity weights to comorbidities with a mortality hazard ratio ≥1.20 after adjustment for gender and age. Patients were subsequently characterized by their two most severe comorbidities which constituted their CirCom score. Discriminative ability was quantified with Harrell's c statistic. The score was validated in a cohort of 419 patients with chart-validated alcoholic cirrhosis, adjusting for gender, age, MELD score, and alcohol drinking status. Results: Nine comorbidities had a hazard ratio ≥1.20: chronic obstructive pulmonary disease (severity weight=1), acute myocardial infarction (1), peripheral arterial disease (1), epilepsy (1), substance abuse other than alcoholism (1), heart failure (1), non-metastatic or hematologic cancer (1), chronic kidney disease (3), and metastatic cancer (3); 24.5% of patients had one or more of these, and CirCom scores ranged from 1+0 (N=2,511) to 3+3 (N = 1). In both cohorts the CirCom score was strongly associated with mortality and added significantly to the ability to discriminate between patients with high or low mortality (nationwide cohort: 1.6% [95% CI 1.3% to 1.9%]; validation cohort: 0.9% [95% CI -0.1% to 8.6%]). Moreover, it added marginally more discriminative ability than did the Charlson index (nationwide cohort: 0.4% [95% CI 0.2% to 0.7%]; validation cohort: 0.2% [95% CI -0.9% to 1.2%]). Conclusions: Comorbidity is prevalent and increases mortality, so it must be described, quantified, and controlled for in studies of cirrhosis patients. The CirCom score is specifically designed for these tasks, and it is much simpler and slightly better than the Charlson index.

Comorbidities included in the final CirCom score.

ComorbidityAdjusted hazard ratioSeverity weight
Chronic obstructive pulmonary disease1.22 (1.13 to 1.32)1
Acute myocardial infarction1.26 (1.08 to 1.47)1
Peripheral arterial disease1.28 (1.15 to 1.44)1
Epilepsy1.32 (1.17 to 1.49)1
Substance abuse other than alcoholism1.38 (1.25 to 1.54)1
Heart failure1.39(1.28to 1.52)1
Non-metastatic or hematologic cancer1.43(1.31 to 1.55)1
Chronic kidney disease1.91 (1.49 to 2.45)3
Metastatic cancer1.99 (1.64 to 2.42)3

Disclosures:

Timothy L. Lash - Advisory Committees or Review Panels: European Crop Protection Agency

The following people have nothing to disclose: Peter Jepsen, Hendrik V. Vilstrup

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Abnormal Immune Response To Antigen Challenge In Subjects With Alcoholic Cirrhosis: Baseline Data From The Zinc In Alcoholic Cirrhosis (Zac) Trial

Mohammad K. Mohammad1, Keith C. Falkner1, Shirish Barve1, Zhanxiang Zhou3, Craig J. McClain1,2, Matthew C. Cave1,2;
1 Department of Medicine/GI, University of Louisville, Louisville, KY; 2Louisville VAMC, Louisville, KY; 3Nutrition, UNC-Greensboro, Greensboro, NC

Purpose: Immune dysfunction contributes to liver disease progression and infection risk in alcoholic cirrhosis (AC). The purpose of the study is to better characterize liver injury biomarkers, insulin resistance/adipokines, and immune function in subjects enrolled in an NIH-funded, placebo-controlled, clinical trial of zinc sulfate for alcoholic cirrhosis (ZAC). Methods: Baseline data and fasting blood samples of 17 consenting subjects with (Child-Pugh class A or B) AC were evaluated and compared to 8 non-drinking, healthy controls. Plasma adipokines and whole blood ex vivo lipopolysacharide-stimu-lated (LPS) and phytohemagglutinin-stimulated (PHA) cytokine production were measured by Luminex. Plasma cytokeratin 18 (CK18, M30 and M65) were measured by ELISA. Differences between the means (AC vs. controls) were evaluated by t-test using GraphPad-Prism and statistical significance was set at p<0.05. Results: The mean age (55.0±10.1 years) and BMI (26.2±3.9 kg/m2) in AC were similar to controls. The mean Child-Pugh and MELD scores in AC were (6.0±1.4 and 9.0±3.5). 6 AC subjects were still drinking alcohol and 3 had type 2 diabetes. Mean plasma CK18 M30 and M65 were significantly increased in AC compared to controls (p<0.05). Mean insulin levels were significantly increased in AC (p<0.05) while mean glucose levels were similar. There were non-significant trends towards higher adiponectin, leptin, PAI-1, and resistin in AC. Un-stimulated whole blood ex vivo production of IL-6, IL-8, IL-10, and TNF-α were significantly increased in AC (p<0.05). Mean un-stimulated IL-1β, IL-2, IL-4, IL-17a, MCP-1, and IFN-γ were not significantly different. Mean LPS-stimulated cytokine production of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17a, TNF-α, MCP-1, and IFN-γ were not significantly different between groups. Mean PHA-stimulated cytokine production of IL-1 β, IL-6, IL-10, and TNF-α were significantly decreased in AC (p<0.05). PHA-stimulated IL-2, IL-4, IL-17a, MCP-1, and IFN-γ were not different. Conclusions: The first 17 subjects in the ZAC trial had increased CK18, insulin resistance, and immune dysfunction. Un-stimulated IL-6, 8, 10 and TNF-α were increased in AC. LPS stimulation induced cytokine production to a similar degree in AC and controls indicating an absence of priming in AC. PHA stimulation failed to induce production of IL-1β, IL-6, IL-10, and TNF-α in AC, but not controls, suggesting abnormal T-cell function. The potential of zinc therapy to correct these biomarkers will be evaluated in the ZAC Trial.

Disclosures:

Shirish Barve - Speaking and Teaching: Abbott

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Mohammad K. Mohammad, Keith

C. Falkner, Zhanxiang Zhou, Matthew C. Cave

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Alcoholic hepatitis: Cytotoxic cells display decreased degranulation and increased secretion of tissue healing cytokines

Sidsel Støy1, Anders Dige1, Thomas D. Sandahl1, Tea L. Laursen1, Marianne Hokland2, Hendrik V. Vilstrup1;
1 Hepatology and Gas-troenterology, Aarhus University hospital, Aarhus C, Denmark; 2Institute of Biomedicine, Aarhus University, Aarhus C, Denmark

Background: Susceptibility to infection and progressive hepatic injury are hallmarks in alcoholic hepatitis (AH), and depressed functions of natural killer (NK) cells and cytotoxic (CD8+) T lymphocytes may be implicated. Both subsets of lymphocytes can directly kill infected or damaged cells through degranulation and/or the production of IFN-γ, and they may also secrete tissue healing cytokines such as IL-4 and IL-22. The cells are activated through stimulatory receptors e.g. NKG2D on their surface. We, therefore, hypothesized that the cytotoxic cells may be dysactivated or dysfunctional in AH. Methods: We analysed blood samples from 20 severe AH patients, 10 stable alcoholic cirrhosis patients (AC) and 10 healthy controls (HC). We assessed the functionality of NK (CD3-CD56+) and cytotoxic T lymphocytes (CD3+CD8+) in vitro by a flow cytometry based degranulation assay with the human chronic myeloid leukemia cell line K562. Additionally, we quantified the frequency of IFN-γ, IL-4 and IL-22 producing cells following stimulation and measured the expression of NKG2D. Results: The frequency of cytotoxic T lymphocytes was halved in AH compared with HC (median±IQR: 27.0%±29,9 vs. 56.6±28.1, p=0.005), but we observed no changes in NK cell frequency (10.4%±12.4vs. 14.0%±10.1). Functionally, the NK cells had markedly decreased ability to degranulate compared with both AC (9.4%±3.7 vs. 18.8%±17.3, p=0.04) and HC (14.1%±11.2, p=0.02). In the same way, we detected no up-regulation of the IFN-γ production in either cell subset. Nevertheless, we observed at least a doubling in the frequencies of both NK cells and cytotoxic T lymphocytes that produced IL-4 or IL-22 compared with HC (p<0.05). As for the activation state, the expression of the NKG2D receptor in the NK cells was lowered to 70% of that in both control groups (p<0.05) (fig. B). This was not evident in the population of cytotoxic T lymphocytes. Conclusion: Our results show a decreased ability to degranulate and a lower activation of the NK cell population in AH, which may contribute to the patients' susceptibility to infections. Furthermore, the cytotoxic cells' production of tissue healing instead of cytotoxic cytokines in AH suggests that these cells are not primarily involved in the hepatic destruction but may, conversely, be phenotypically adjusted to limit the tissue damage.

Disclosures:

The following people have nothing to disclose: Sidsel Støy, Anders Dige, Thomas

D. Sandahl, Tea L. Laursen, Marianne Hokland, Hendrik V. Vilstrup

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Natural killer T cells play an important role in the pathogenesis of alcoholic liver disease

Stephanie Mathews, Dechun Feng, Bin Gao;
NIAAA/NIH, Rockville, MD

Alcoholic liver disease (ALD) affects over 140 million people worldwide and is a major health concern. Despite years of ongoing research, the molecular mechanisms contributing to disease progression are only partially understood. The liver is enriched in natural killer T (NKT) cells, a heterogeneous group of T lymphocytes that recognize lipid antigens presented by CD 1d, which play an important role in host defense against hepatic viral infection and tumor transformation; however, the role of NKT cells in pathogenesis of ALD remains unknown. We used a mouse model of chronic plus binge ethanol (EtOH) feeding to determine how NKT cells affect EtOH-induced liver injury and inflammation. NKT deficient (Jalpha18 KO or CD1d KO) mice and their wild-type controls were fed a 5% EtOH liquid diet for 10 days, followed by single gavage of EtOH (5g/kg). Sera and liver tissue were collected 9 hours post-gavage and analyzed for markers of liver injury. Neutrophils and lymphocytes were isolated 3 hours post-gavage and subjected to flow cytometry. Histological and Oil Red O staining revealed that EtOH feeding-induced hepatic steatosis was lower in Jalpha18 KO and CD1d KO mice compared to WT mice; which correlated with a lower liver/body weight ratio and less hepatic triglyceride in EtOH-fed NKT deficient mice. Sera from EtOH-fed NKT deficient mice had significantly less ALT compared to WT mice. Immunological staining for cytochrome P4502E1 was comparable in all EtOH-fed mice, suggesting that the resistance of NKT-deficient mice to ALD was not due to changes in EtOH metabolism. Importantly, flow cytometric analyses revealed an increased number of activated NKT cells in the blood and livers of WT mice after chronic plus binge EtOH feeding; which correlated to increased neutrophil accumulation, thus supporting a role for NKT cells in alcohol-induced liver injury. Neutrophils were not increased in the blood or livers of Jalpha18 KO mice. Real-time PCR analysis showed that induction of the pro-inflammatory cytokines TNF-alpha and IL-1 beta was significantly blunted in EtOH-fed Jalpha18 KO and CD1d KO mice compared to that in WT. Finally, hepatic expression of the inflammation-associated genes for e-selectin and MCP-1 was drastically reduced in EtOH-fed NKT deficient mice compared to WT. Taken together, our data suggest NKT cells play a critical role in the development of early alcoholic liver injury, neutrophil recruitment, and inflammation. Future studies will be aimed at elucidating the mechanism(s) by which ethanol activates NKT cells and further investigating how activated NKT cells modify the critical inflammatory pathways involved in progression of ALD.

Disclosures:

The following people have nothing to disclose: Stephanie Mathews, Dechun Feng, Bin Gao

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The PNPLA3 G allele is associated with the development of acute alcoholic hepatitis and with more severe disease

Alison Jazwinski1, Amit Raina2, Charles Gabbert1, Shahid M. Malik1, Michael O'Connell1, David C. Whitcomb1, Jaideep Behari1;
1 Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh, Pittsburgh, PA; 2Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA

Background: A PNPLA3 gene polymorphism (rs738409) is associated with liver fat content and histological severity in nonalcoholic fatty liver disease and with alcoholic cirrhosis. However the relationship between PNPLA3 genotype and acute alcoholic hepatitis (AAH), a distinct form of severe alcoholic liver disease, is unknown. The goal of this study was to determine whether rs738409 is associated with AAH and with differences in disease severity. Methods: We prospectively enrolled 46 patients admitted with severe AAH [Maddrey Discriminant Function (DF) >32]. As controls, we used 204 patients with history of heavy alcohol use (>8 and 15 drinks per week for females and males, respectively) but no known liver disease enrolled in the North American Pancreatitis Study (NAPS2). Clinical and biochemical measures were recorded. Median values were reported and nonparametric statistical tests utilized. PNPLA3 genotype in cases and controls was determined by real-time PCR. We compared allele and genotype frequencies between patients with AAH and controls using Fisher's exact and Chi-Square tests. We compared markers of disease severity using Kruskal-Wallis test. Results: The G allele was more frequent in patients with AAH compared with controls (0.32 vs 0.19; p< 0.01). Of the patients with AAH, 46% had CC genotype, 43% had CG genotype, and 11% had GG genotype, compared with controls, of which 67% had CC genotype, 27% had CG genotype, and 5% had GG genotype (p =0.02). Since AAH patients were predominantly male and Caucasian, we stratified the analysis by race and gender. Amongst Caucasian patients, 47% of AAH patients had CC genotype, 42% had CG genotype and 11% had GG genotype compared with 66% CC, 29% CG, and 6% GG (p = 0.056). Male AAH patients (n=34) had genotype frequencies of CC 41%, CG 50%, GG 9% versus male controls (n=100) with frequencies of CC 67%, CG 25%, and GG 8% (p=0.02). Female AAH patients (n=11) had genotype frequencies of CC 64%, CG 18%, GG 18% compared with female controls (n=105) with CC 68%, CG 29%, GG 3% (p = 0.05). To determine whether PNPLA3 gene variants were associated with disease severity in AAH, we determined Model for Endstage Liver Disease (MELD) scores on the first day of admission. Patients with GG genotype had higher MELD scores (MELD 31 in GG genotype versus MELD 26 in CC genotype; p < 0.05). Conclusions: The PNPLA3 G allele is more common in patients with severe acute alcoholic hepatitis compared with controls that drink alcohol heavily but do not have hepatitis. This polymorphism is also associated with more severe disease as determined by MELD score on the day of admission.

Disclosures:

The following people have nothing to disclose: Alison Jazwinski, Amit Raina, Charles Gabbert, Shahid M. Malik, Michael O'Connell, David C. Whitcomb, Jaideep Behari

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Budesonide in acute alcoholic hepatitis treatment

Inna Komkova, Marina V. Maevskaya, Vladimir T. Ivashkin, Peter Tkachenko;
Clinic of Internal Disease, Gastroenterology and Hepatology n.a. Vasilenko University Hospital 2, The First Moscow State Medical University n.a. I. M. Sechenov Health Ministry of Russian Federation, Moscow, Russian Federation

Aim: to compare efficacy and safety of Budesonide and Pred-nisolone in treatment of acute alcoholic hepatitis (AAH). To determine predictors of non-response, predictors of short-time mortality. Methods: 35 patients with AAH were enrolled in the prospective trial and randomised in 2 groups. Group 1: 15 patients (7 men, 8 women), average age 46,53±11,01. Median alcohol daily intake - 77 g., lower and upper quartiles - 55 and 96 g. Duration of alcohol intake - 13,41+8,55 years. Discriminant function (DF) average value was 65,22 (from 37,2 to 145,4). Group 2: 20 patients (16 men, 4 women), average age 46,5±11,89. Median alcohol daily intake - 70,55 g., lower and upper quartiles - 37 and 88 g. Duration of alcohol intake - 16,85+13,32 years. The average value of DF - 58,11 (from 32,1 to 121,7). Groups were comparable in key features. In group 1 Budesonide was prescribed 9 mg/daily per os. In group 2 - Prednisolone 40 mg/daily per os. Treatment duration was 28 days. Response criteria - Lille model. Statistical analysis was performed using SPSS 17.0 statistical package (chi-squared, Mann-Whitney and Wilcoxon tests, Kaplan-Meier method and Cox regression model). Results: Efficacy (p = 0,810) and short-term survival (p = 0,857) in budesonide group are equal to prednisolone group. In group 2 adverse events (infections, hepatorenal syndrome, hyper-glycemia, upper gastrointestinal bleeding and Cushing's syndrome) were statistically more frequently than in group 1: 70% vs. 26,7% (p = 0,011). Hepatorenal syndrome occurred more frequently in group 2 (p = 0,033). Predictors of non-response are MELD score (p=0,009), ABIC score (p=0,011), hepatic encephalopathy level (p=0,035), total bilirubin level (p=0,016). Predictors of mortality are Lille score (p=0,018), serum glucose level (p=0,017), total bilirubin level at the 7th day of the therapy (p=0,030). There is a positive correlation between BMI and absence of therapy response (correlation coefficient 0,519 ) and short-time mortality (correlation coefficient 0,630). Conclusions: Short-time survival in budesonide group is equal to prednisolone group, so budesonide can be used in treatment of this disease. According to the data resulting from the study budesonide is the drug of choice in patients with concomitant infections, hepatorenal syndrome and glucose intolerance.

Disclosures:

The following people have nothing to disclose: Inna Komkova, Marina V. Maevskaya, Vladimir T. Ivashkin, Peter Tkachenko

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Aldehyde dehydrogenase 2 deficiency ameliorates alcoholic fatty liver but exacerbates liver inflammation and fibrosis in mice

Hyo-Jung Kwon1, Young-Suk Won1, Ogyi Park1, Michael J. Duryee2, Geoffrey Thiele2, Akiko Matsumoto3, Surendra Singh3, Toshihiro Kawamoto4, Mohamed A. Abdelmegeed5, Byoung-Joon Song5, Vasilis Vasiliou3, Geoffrey M. Thiele2, Bin Gao1;
1Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National institute of health, Bethesda, MD; 2Experi-mental Immunology Laboratory, Omaha VA Medical Center and the University of Nebraska Medical Center, Omaha, NE; 3Depart-ment of Pharmaceutical Sciences, University of Colorado, Aurora, CO; 4Department of Environmental Health, University of Occupational and Environmental Health, Kitakyushu, Japan; 5Section of Molecular Pharmacology and Toxicology, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD

BACKGROUND & AIMS: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes toxic acetaldehyde produced from alcohol metabolism. Approximately 40∼50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogene-sis of alcoholic liver injury remains obscure. METHODS: Wild-type (WT) and ALDH2-/- mice were subjected to ethanol feeding and/or carbon tetrachloride (CCl4) treatment, and liver injury was assessed. RESULTS: Compared with WT mice, ethanol-fed ALDH2-/- mice had higher levels of malondialde-hyde and acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin-6 (IL-6) expression but surprisingly lower levels of steatosis and serum alanine transaminase (ALT). Higher IL-6 levels were also detected in ethanol-treated, precision-cut liver slices from ALDH2-/- mice and in Kupffer cells isolated from ethanol-fed ALDH2-/- mice than those levels in WT mice. In vitro incubation with MAA enhanced the LPS-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2-/- mice than in WT mice. An additional deletion of hepatic STAT3 resulted in teatosis and hepatocellu-lar damage in ALDH2-/- mice. Finally, ethanol-fed ALDH2-/-mice were more prone to CCl4-induced liver inflammation and fibrosis than ethanol-fed WT mice. CONCLUSIONS: ALDH2-/-mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis via MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that individuals who have ALDH2 deficiency may be resistant to steatosis, but are more prone to liver inflammation and fibrosis following alcohol consumption.

Disclosures:

The following people have nothing to disclose: Hyo-Jung Kwon, Young-Suk Won, Ogyi Park, Michael J. Duryee, Geoffrey Thiele, Akiko Matsumoto, Surendra Singh, Toshihiro Kawamoto, Mohamed A. Abdelmegeed, Byoung-Joon Song, Vasilis Vasiliou, Geoffrey M. Thiele, Bin Gao

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Increasing serum Pre-adipocyte factor-1 (Pref-1) correlates with decreased body fat, increased free fatty acids, and amount of alcohol consumption in heavy drinkers

Suthat Liangpunsakul1, Rachel D. Bennett1, Chi Westerhold1, Ruth A. Ross1, David W. Crabb1, Xianyin Lai2, Frank Witzmann2;
1Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, IN; 2Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN

Background: Under physiological state, free fatty acids (FFA) enter adipocytes and stored in the adipose tissues in the form of triglycerides (TG). Patients with alcoholic liver disease have been shown to have significantly lower percentage of body fat (%BF). This results in reducing TG storage as reflected by increasing serum FFA. In adipose tissue, Pref-1 is specifically expressed in preadipocytes but not in adipocytes. Increasing Pref-1 leads to inhibition of adipogenesis and reduced adipose tissue mass. Our aim is to investigate the association between alcohol consumption, serum Pref-1, and %BF in heavy drinkers compared to controls. Methods: 97 chronic heavy drinkers (mean age 41.3 years/69% men/81% Caucasian) were enrolled from Fairbanks Alcohol Treatment Center. 51 non-heavy drinkers (mean age 31.8 years/88% men/84% Caucasian) were recruited from Roudebush VAMC. Time Line Follow-Back (TLFB) was used to quantify the amount of alcohol consumed in the past 30 days before enrollment. Anthropo-metric measurement was performed to calculate %BF. Serum Pref-1 and FFA were measured. Alcohol intake was considered as categorical variable (heavy/non-heavy) using NIAAA criteria. It was also modeled as a continuous variable and divided by quartiles by calculating the total number of drinks during the past 30 days from TLFB. Linear regression was used in the analyses. Results: Heavy drinkers had higher levels of Pref-1 (0.32±0.13 vs 0.13±0.06, p<0.01), FFA (2.31 ±0.78 vs 0.42±0.28,p<0.001), and lower %BF (29.7±4.7vs 31.7±5.7, p<0.03). There were no differences in the BMI and waist circumferences. Serum Pref-1 was significantly associated with the amount of alcohol consumption during the past 30 days (Fig A). There was the trend on the paradoxical relationship between %BF and the amount of alcohol consumed (Fig B). In the sub-analyses, %BF was significantly decreased with the increased amount of alcohol consumption, specifically drinking in the 3rd (r2=0.11,p=0.03) and 4th quartiles range (r2=0.32, p=0.005). Serum Pref-1 is negatively correlated with %BF (Fig C), but positively associated with serum FFA (Fig D). Summary: Our data suggest that Pref-1 might play a role in the inhibition of adipogenesis and thus decreasing %BF in alcoholics. Further work is needed to validate these findings and to better understand the role of Pref-1 and its clinical significance in subjects with heavy alcohol use.

Disclosures:

The following people have nothing to disclose: Suthat Liangpunsakul, Rachel D. Bennett, Chi Westerhold, Ruth A. Ross, David W. Crabb, Xianyin Lai, Frank Witzmann

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Moderate Alcohol Consumption and Metabolic Outcomes in At-Risk Latinos With and Without Hepatitis C (HCV) Infection

Blaire E. Burman1, Nicholas Gelman1, Claudia E. Ayala1, Man-dana Khalili1,2;
1Medicine, University of California San Francisco, San Francisco, CA; 2Liver Center, San Francisco, CA

Background/Aims: Moderate alcohol intake has favorable effects on insulin sensitivity and glucose metabolism. For individuals with chronic hepatitis C (HCV), the impact of moderate alcohol use on metabolic outcomes is not well understood. The aim of this study is to investigate the effect of graded alcohol consumption in a cohort of Latinos, a population with high rates of insulin resistance (IR) and impaired fasting glucose (IFG), with and without HCV infection. Methods: Cross-sectional analysis of 100 non-diabetic Latino adults with fasting glucose ≤126 mg/dL and normal glucose tolerance on OGTT. All subjects underwent medical interview and exam, anthropomorphic measures, and fasting laboratory evaluation including direct quantification of IR via steady-state plasma glucose (≥180 mg/dL) during the last 30 minutes of a 240-min continuous infusion of octreotide, glucose, and insulin. Alcohol use was graded as moderate (<4 drinks/day or 14/wk in men, <3 drinks/day or 7/wk in women) or heavy (>moderate or binge drinking). Results: Baseline characteristics were notable overall for mean age 42 ± 10 years, 64% male, median BMI 27 kg/m2, 40% HCV positive, 32% with IFG, and 26% with IR. By alcohol category, 40% were abstainers, 36% had moderate, and 24% had heavy alcohol use, and patient characteristics were similar except moderate drinkers had lower rates of HCV (22% vs 50%), IR (13% vs 43%), and IFG (22% vs 43%) as compared to abstainers. Multivariate analysis was performed to evaluate factors associated with IR or IFG and included a priori age, sex, waist circumference, and HCV status as covari-ates. Independent predictors of IR were: female sex (OR 6.1, 95%CI (CI) 1.7%ndash;22.6), waist circumference (OR 1.5 per 5cm, CI 1.1%ndash;2.0), HDL (OR 0.7 per 5 unit, CI 0.5%ndash;0.96) and moderate alcohol use (OR 0.1, CI 0.03%ndash;0.6). HCV and ALT were also associated with IR (OR 2.5 and 2.1, respectively) but did not reach statistical significance. Removing HCV from the model did not significantly alter the OR associated with moderate alcohol use. Independent predictors of IFG included age (OR 3.4 per decade, CI 1.6%ndash;7.1), ALT (OR 3.4 per doubling, CI 1.3%ndash;8.6), and US birth (OR 5.6, CI 1.5%ndash;21.2). Moderate alcohol use was also associated with lower rates of IFG (OR 0.2, CI 0.1%ndash;1.0), but this did not reach statistical significance. Conclusions: In this large non-diabetic Latino cohort, moderate alcohol use was associated with lower odds of insulin resistance and prediabetes. Although this effect was independent of HCV status in Latinos, future studies are warranted to optimally assess the impact of moderate alcohol use on glucose metabolism in an ethnically diverse HCV population.

Disclosures:

Mandana Khalili - Advisory Committees or Review Panels: Gilead Inc.; Grant/Research Support: Gilead Inc., BMS Inc, BMS Inc

The following people have nothing to disclose: Blaire E. Burman, Nicholas Gelman, Claudia E. Ayala

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Misoprostol as a modulator of cytokine activity: possible implications for therapy in alcoholic hepatitis

Leila Gobejishvili1, Shirish Barve1, Rehan A. Khan1, Diana Avila1, Craig J. McClain1,2, Daniell B. Hill1,2;
1Department of Medi-cine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY

Background: Alcoholic liver disease (ALD) remains an important health problem in the United States and worldwide. Unfortunately, there is no FDA approved therapy for any stage of ALD. Dysregulated cytokine metabolism plays a critical role in the pathogenesis of alcoholic liver disease. Misoprostol is an approved drug used in the prevention of NSAID induced gastric ulcers. It is a prostaglandin analog with the demonstrated anti-inflammatory effect and used by some hepatologists as an alternative to Pentoxifylline (due to side effects) to treat alcoholic hepatitis (AH). The overall aim of this pilot study was to assess the efficacy of Misoprostol, as a potential therapeutic agent, in the treatment of AH and investigate the underlying mechanisms of its anti-inflammatory action. Patients and Methods: Healthy volunteers were given different doses of Misoprostol and baseline and LPS-inducible cytokine levels were examined ex vivo. Additionally, human peripheral blood mononuclear cells and murine macrophage cell line 264.7 were used to investigate underlying mechanisms of misoprostol effect on cytokine expression. Results: Our results show that a lower dose of Misoprostol was well-tolerated with fewer GI side effects and was effective in modulating cytokine activity as demonstrated by the whole blood ex vivo analysis. Specifically, Misoprostol administration significantly decreased LPS-inducible TNF and enhanced IL-10 expression. Mechanistically, the anti-inflammatory effect of Misoprostol was mediated by epigenetic mechanisms involving promoter associated histone modifications that regulate cytokine gene expression. Specifically, chromatin immunoprecipitation (ChIP) analysis showed that Misoprostol modified transcriptionally permissive histone states, including histone H3 lysine 9 acetylation (H3K9Ac) and H3 serine 10 phosphorylation (H3S10ph) in the TNF and IL-10 promoter regions. Further, Misoprostol induced promoter-histone modifications affected the occupancy by the critical transcription factors NFκB and CREB which in turn influenced the recruitment of RNA polymerase II and mRNA expression. Conclusions: Human and ex vivo studies provide initial evidence that Misoprostol can effectively down-regulate LPS-inducible TNF expression which plays a significant etiologic role in AH. Importantly, the study also identifies the role of epigenetic mechanisms involved in the mode of action of Misoprostol. Further studies are needed to evaluate the effects of Misoprostol on the modulation of cytokine activity, liver function and clinical course in AH patients.

Disclosures:

Shirish Barve - Speaking and Teaching: Abbott

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Leila Gobejishvili, Rehan A. Khan, Diana Avila, Daniell B. Hill

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Serum And Hepatic Fibroblast Growth Factor 21 (FGF-21) Levels Are Increased In Subjects With Severe Acute Alcoholic Hepatitis And In Mice Exposed To Chronic-Binge Alcohol By Decreased Transcriptional Suppression

Wenke Feng1, Yanlong Liu1, Cuiqing Zhao1, Keith C. Falkner1, Mohammad K. Mohammad1, Matthew C. Cave1,2, Craig J. McClain1,2;
1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY

Purpose: Fibroblast growth factor 21 (FGF-21) is a novel metabolic regulator of glucose and lipid metabolism and has excellent potential in the treatment of obesity and type 2 diabetes in rodents and monkeys. Alcohol exposure affects lipid metabolism by increasing lipogenesis and decreasing fatty acid beta-oxidation. However, it is currently unknown whether alcohol exposure affects FGF-21 expression, which is the purpose of this study. Methods: Serum FGF-21 levels were measured in 25 consenting human subjects with severe acute alcoholic hepatitis and were compared to 17 healthy, non-drinking controls by ELISA. C57BL/6 mice were fed Lieber DeCarli diet containing 5% alcohol or maltose dextrin for 12 days, and were given one dose of alcohol at 6 g/kg by gavage 6 hours before sacrificing. Serum and hepatic tissues from alcohol-exposed and control mice were harvested. Serum and hepatic FGF-21 levels were measured by ELISA, and hepatic FGF-21 mRNA levels were measured by real-time PCR. Liver triglyceride and serum free fatty acids were also measured. H4IIE cells were cultured and exposed to ethanol for various times and at different concentrations. mRNA levels of FGF-21 were measured. The data were analyzed by one-way analysis of variance and Newman-Keuls multiple-comparison test. Differences between groups were considered significant at P < 0.05. Results: Serum levels of FGF-21 were markedly increased in both human subjects with alcoholic hepatitis and in mice exposed to alcohol administrated in chronic-binge pattern vs. non drinking controls. In ethanol-treated mice, the hepatic and adipose expression of FGF-21 were increased by both mRNA and protein levels. The increased FGF-21 expression was positively correlated with increased hepatic levels of triglyceride and serum levels of free fatty acids. Alcohol increased FGF-21 expression in hepato-cytes in a time- and dose-dependent manner. The expression of PGC-1α and Rev-Erbα, which are important transcription suppressors of FGF-21, were decreased in mouse livers exposed to alcohol. Conclusions: Alcohol exposure increased hepatic and circulating FGF-21 expression likely through an inhibition of transcriptional suppression mediated by the PGC-1α-Rev-Erbα pathway. The regulation of FGF-21 expression may be associated with hepatic lipid metabolism in alcoholic steatohepatitis. The observed increase in circulating FGF-21 was conserved between animal models and human subjects with alcoholic hepatitis. Altered FGF-21 metabolism plays an etiologic role in the development/progression of alcoholic liver disease.

Disclosures:

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

The following people have nothing to disclose: Wenke Feng, Yanlong Liu, Cuiqing Zhao, Keith C. Falkner, Mohammad K. Mohammad, Matthew C. Cave

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Alcoholic hepatitis induced by synergism between cholesterol and alcohol drinking

Laura Conde de la Rosa1,2, Carmen Garcia-Ruiz1,2, Jose Fernan-dez-Checa1,2;
1 Instituto Investigaciones Biomedicas Barcelona, CSIC, Barcelona, Spain; 2Liver Unit-IDIBAPS, and CIBEREHD, Barcelona, Spain

Alcohol induced liver disease (ALD) is a leading cause of chronic liver disease worldwide and its pathogenesis remains poorly understood. ALD comprises a spectrum of disorders starting from steatosis that can progress to alcoholic steatohepatitis (ASH), characterized by liver injury, inflammation and fibrosis. To better understand the transition from alcohol-induced steatosis to ASH adequate experimental models are needed. The Lieber-DeCarli (L-DC) ethanol liquid diet and binge drinking are among the most common approaches of chronic and acute alcohol drinking but their impact in ALD is moderate. Since cholesterol has emerged as a critical factor in the progression from steatosis to NASH, we hypothesized that cholesterol supplementation may synergize with alcohol to cause ASH. Thus, our aim was to test if cholesterol aggravates liver disease in chronic and binge models of ALD. Methods: Mice were fed a standard or modified L-DC ethanol liquid diet (Dyets, Inc) containing corn oil (Coil, 35% calories) with or without cholesterol (Chol, 0.2%) for 4 weeks. In addition, mice fed with a high cholesterol diet (HC, 1%) or Lombardi diet (LD, choline-deficient) for 2%ndash;3 days were gavaged with ethanol (3g/kg) every 12 hours for 3 days. Samples were processed to examine steatosis, liver injury, inflammatory and fibrotic markers by histology, IHC and RT-PCR. Results: Total serum cholesterol was significantly higher in EtOH+Chol and EtOH+Chol+Coil groups, while serum triglycerides were more elevated in Coil and ETOH+Coil groups, with similar findings observed in liver samples. Liver injury (serum ALT) and steatosis (H&E and oil red staining) were significantly greater in ETOH+Chol and ETOH+Chol+Coil compared to ETOH and ETOH+Coil groups. M1 proinflammatory macrophage markers, MCP-1, Ly6c, TNFα, NOS2 and F4/80, were higher in EtOH+Chol and EtOH+Chol+Coil groups. However, M2 anti-inflammatory markers, CD163 and Arg-1, were reduced in EtOH+Chol or EtOH+Chol+Coil fed groups. Furthermore, the inflammatory markers IL-6 and ICAM-1 were more elevated in EtOH+Chol and EtOH+Chol+Coil groups. Additionally, fribrosis examined by Sirius red staining and fibrotic markers, collagen, αSMA, osteopontin, desmin and hydroxyproline levels were highly increased in EtOH+Chol and EtOH+Chol+Coil groups. Alcohol binge drinking caused more injury (serum ALT and H&E) and inflammation in HC than in LD pretreated mice despite similar degree of steatosis. Conclusion: cholesterol intake has a synergic effect with alcohol aggravating liver injury and progression to alcoholic hepatitis, pointing that targeting cholesterol may be a valuable approach for future therapeutic interventions in ALD.

Disclosures:

The following people have nothing to disclose: Laura Conde de la Rosa, Carmen Garcia-Ruiz, Jose Fernandez-Checa

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A novel in vitro model for joint effects of alcohol and free fatty acids on hepatocellular steatosis and inflammation

Abdo Mahli1, Wolfgang E. Thasler2, Martina Müller1, Claus Heller-brand1;
1 University Hospital Regensburg, Regensburg, Germany; 2Ludwig Maximilians University, Munich, Germany

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepati-tis (NASH) are the most frequent conditions leading to elevated liver enzymes and liver cirrhosis, respectively, in the Western world. However, despite strong epidemiological evidence for combined effects on the progression of liver injury, the mutual interaction of the pathophysiological mechanisms is incompletely understood. The aim of this study was to establish an in vitro model for joint effects of alcohol and lipids on hepatic steatosis and inflammation. Methods and Results: Initially, we established the dose range in which neither alcohol nor incubation with the free fatty acid oleate affected viability or mito-chondrial activity in primary human hepatocytes (PHH). Subsequently, we assessed the combined effect of alcohol (1%ndash;2%) and oleate (0.2mM) on hepatocellular lipid accumulation in PHH. Under these conditions, alcohol significantly enhanced oleate induced expression of genes regulating lipogenesis (FASN, SCD-1) and lipid peroxidation (CPT-1) as well as cellular triglyceride content and free fatty acid (FFA) levels, while alcohol alone had only a minimal effect. Analysis of heme oxy-genase-1 (HMOX-1) expression and malondialdehyde levels revealed that the combination of alcohol and oleate caused significantly higher oxidative stress and lipid peroxidation than either of the two substances alone. Further, we observed a syn-ergistic effect of alcohol and FFA on JNK-activation and pro-inflammatory (IL-8 and ICAM-1) gene expression. HepG2 hepatoma cells stably overexpressing CYP2E1 but not control transfected HepG2 cells lacking CYP2E1 revealed similar syn-ergistic effects of alcohol and FFA as PHH. Also inhibition of CYP2E1 activity by chlormethiazole or incubation with ROS scavenger (NAC) blunted these synergistic effects on lipogenesis, TG accumulation, lipid peroxidation and inflammation response in PHH. Conclusion: Our new model allows the investigation of isolated or joint effects of alcohol and FFA on hepatocellular lipid metabolisms and inflammatory signaling. Our present findings indicate Cyp2e1 as critical mediator of a synergistic effect of alcohol and FFA on hepatic steatosis and inflammation.

Disclosures:

Martina Müller - Grant/Research Support: Novartis

The following people have nothing to disclose: Abdo Mahli, Wolfgang E. Thasler, Claus Hellerbrand

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The Antizyme Inhibitor 1 gene (AZIN-1) rs62522600_A allele is associated with risk of alcoholic liver cirrhosis

Alison Jazwinski, Nijole Pollock, Kimberly Stello, Michael O'Con-nell, David C. Whitcomb;
Gastroenterology, Hepatology, and Nutrition, University of PIttsburgh Medical Center, Pittsburgh, PA

Background: Polyamines are organic cations that promote cell growth/proliferation and are synthesized via a pathway that begins with the conversion of arginine to L-ornithine. Antizyme inhibitor 1 (AZIN1) regulates ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis. The AZIN1 gene Y342Y variant (rs62522600G/A) has been described to protective against hepatitis C-induced cirrhosis by inhibiting expression of fibrosis genes in hepatic stellate cells via a polyamine-independent mechanism. It is not known whether there is an association between rs62522600 and alcohol-induced cirrhosis. We tested for an association between AZIN1 rs62522600_A and risk of alcoholic cirrhosis. Methods: Patients with alcoholic cirrhosis were identified from a liver disease biorepository at the University of Pittsburgh Medical Center. Diagnosis was confirmed by retrospective chart review, and patients with known concurrent liver disease (including chronic hepatitis C) were excluded. As controls, we used 161 Caucasian patients with history of heavy alcohol use (>8 and 15 drinks per week for females and males, respectively) but no known liver disease enrolled in the North American Pancreatitis Study (NAPS2). Rs62522600 genotype was determined by real-time PCR. Allele and genotype frequencies were compared with Fisher's exact and Chi-Square tests. Results: The A (minor allele) was more frequent in patients with alcoholic cirrhosis compared with alcoholic controls (0.13 vs 0.05, p=0.02). The AG genotype was seen at higher frequency in patients with alcoholic cirrhosis compared with control patients (p=0.01, see table). There was no difference in genotype frequencies between males and females. Conclusions: The A allele of AZIN1 rs62522600 is more frequent in patients with alcoholic cirrhosis compared to control patients with heavy alcohol use. This is in contrast to the protective effect seen in patients with chronic hepatitis C infection, though the polyamine pathway has been described to be altered by ethanol. Additionally, in animal models, arginine reverses ethanol-induced inflammatory and fibrotic changes. Further work is needed to replicate the findings and to determine underlying mechanisms.

Genotype Frequencies Alcohol Cirrhosis vs Control

 AAAGGGP value
Alcohol Cirrhosis (n=34)0 (0%)9 (26.5%)25 (73.53%) 
Control (n= 161)1 (0.62'/;)14(8.7%)146(90.68%) 

Disclosures:

The following people have nothing to disclose: Alison Jazwinski, Nijole Pollock, Kimberly Stello, Michael O'Connell, David C. Whitcomb

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Aging aggravates alcoholic liver injury in mice by downregulating hepatic Sirtuin 1 and inhibiting autophagy

Yongmei Li1,2, Dechun Feng1, Ming-Jiang Xu1, Hua Wang1, Yan Wang1, Bin Gao1;
1 Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD; 2Department of Oncology, Changhai Hospital, the Second Military Medical University, Shanghai, China

Chronic alcohol consumption is a leading cause of chronic liver disease with broad spectrum of disorders including: fatty liver, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Accumulating evidence suggests that elderly people have more severe liver injury after excessive drinking than young people, but the underlying mechanisms are not clear. In the current study, 2-, 12-, and 18-month old female C57BL/6J mice were subjected to 10-day chronic plus single binge feeding (NIAAA model). Twelve- and 18-month old mice had higher levels of serum ALT and AST compared with 2-month old mice. Liver his-tological analysis revealed that there was greater degree of steatosis and bigger lipid droplets in the liver from ethanol-fed old mice than those from young mice. Hepatic expressions of several pro-inflammatory genes and CYP2E1 protein were comparable in ethanol-fed young and old mice. Interestingly, chronic plus binge ethanol feeding markedly increased autophagy in the liver in young mice, but to a lesser extent in old mice. Hepatic expression of Sirtuin 1 (Sirt 1) was markedly lower in old mice compared to young mice. Chronic plus binge ethanol feeding upregulated hepatic Sirt1 expression with two fold higher in young mice than in old mice. In vitro exposure of ethanol induced autophagosome in hepatocytes from young mice, but such induction was much weaker in hepatocytes from old mice. Overexpression of Sirt1 via the infection of aden-ovirus Sirt1 restored ethanol-induced autophagosome in these old mouse hepatocytes. These findings suggest that aging downregulates hepatic Sirt1 protein expression and consequently inhibits autophagy, thereby exacerbating alcoholic liver injury.

Disclosures:

The following people have nothing to disclose: Yongmei Li, Dechun Feng, Ming-Jiang Xu, Hua Wang, Yan Wang, Bin Gao

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Acrolein, a reactive aldehyde metabolite, is a major mediator of alcohol-induced endoplasmic reticulum stress and liver injury

Wei-Yang Chen1, Jingwen Zhang1, Mohammad K. Mohammad1, Craig J. McClain1,2, Shirish Barve1, Swati Joshi-Barve1;
1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Rob-ley Rex Veterans Administration Medical Center, Louisville, KY

Purpose: Alcohol is the most socially accepted addictive drug, and it can cause liver disease, which is a major cause of morbidity and mortality in the United States. Animal and human studies demonstrate that chronic alcohol consumption causes a pro-oxidant environment in the liver and increases hepatic lipid peroxidation. Acrolein is the most reactive and toxic aldehyde generated through lipid peroxidation. Also, acrolein is a major component of cigarette smoke, and there is increasing evidence that smoking negatively impacts the incidence, severity, and clinical course of chronic liver disease. Acrolein is known to form DNA and protein adducts, and can trigger endoplasmic reticulum (ER) stress. Notably, alcohol-induced perturbations in the ER have emerged as an important etiologic factor in alcoholic liver disease. This study investigates the role of acrolein as a mediator of hepatic ER stress and injury during alcohol consumption. Methods: In vitro: Human hepatic cells (HepG2) were exposed to different concentrations of acrolein for varying times. ER stress was monitored by changes in gene expression (mRNA and protein) of known ER-stress proteins (ATF3, ATF4, GADD153/CHOP, GRP78 and GRP94). Activation of JNK was assessed by immunoblotting. Cytotoxicity was evaluated by Cellomics-HCS analysis. In vivo: Two models of alcohol consumption were used in this study on male C57/BL6N mice. The acute or weekend binge model consisted of three gavages of ethanol (5g/kg), 12h apart. The chronic model was ten days feeding with Lieber De Carli-ethanol diet, followed by a single gavage of ethanol (5g/kg). The effects of alcohol consumption were assessed on hepatic (i) steatosis; (ii) injury/apoptosis; (iii) activation of JNK; and (iv) ER stress. Results and Conclusions: Acrolein activated the pro-apoptotic kinase, JNK, in HepG2 cells, and induced ER stress, without upregulation of ER chap-erones. Acrolein also caused hepatocyte cell death. Livers of mice administered alcohol exhibited a remarkable accumulation of acrolein adducts compared to control mice. This was accompanied by hepatic steatosis and mild liver injury. Alcohol exposure triggered ER stress, phospho-activated JNK and induced hepatocyte apoptosis. Similar to our in vitro results, minimal upregulation was seen in the ER chaperones, suggesting an inhibition of protective responses with alcohol feeding. Our study demonstrates that acrolein is likely to be a major culprit in the ER stress and hepatotoxicity associated with alcohol consumption. Acrolein scavengers may have therapeutic potential in alleviating the adverse effects of alcohol consumption, and we are actively investigating this concept.

Disclosures:

Craig J. McClain - Consulting: Vertex, Gilead, Baxter, Celgene, Nestle, Danisco, Abbott, Genentech; Grant/Research Support: Ocera, Merck, Glaxo SmithKline; Speaking and Teaching: Roche

Shirish Barve - Speaking and Teaching: Abbott

The following people have nothing to disclose: Wei-Yang Chen, Jingwen Zhang, Mohammad K. Mohammad, Swati Joshi-Barve

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Hepatic neutrophil migration imaged with indium-111 -(111In)-radiolabeled leukocytes correlates with histologi-cal severity in severe alcoholic hepatitis

Jonathan R. Potts1,2, Mark Howard3, Mark Taylor3, Neda Farahi4, Sarah Heard5, Arun N. Shankar4, Graeme J. Alexander4, Edwin R. Chilvers4, Sumita Verma1,2, Adrien M. Peters2;
1Department of Gastroenterology and Hepatology, Brighton and Sussex University Hospitals NHS Trust, Brighton, United Kingdom; 2Department of Medicine, Brighton and Sussex Medical School, Brighton, United Kingdom; 3Department of Histopathology, Brighton and Sussex University Hospitals NHS Trust, Brighton, United Kingdom; 4Depart-ment of Medicine, University of Cambridge, Cambridge, United Kingdom; 5Department of Nuclear Medicine, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom

Background/aims: We previously reported the use of autolo-gous indium-111-(111In)-radiolabeled leukocytes for imaging hepatic neutrophil migration in vivo in severe alcoholic hepatitis (SAH) [AASLD 2012 abstract #1690]. We hypothesized that abolition of physiological hepatic neutrophil destruction in SAH would permit detection of neutrophil migration from increasing liver 111In activity 24h after injection of radiolabeled cells. Our current aim was to correlate 111In-radiolabeled scintigraphy with histological severity of SAH. Methods: Patients with biopsy-proven SAH (DF >32), recruited from two centers, had abdominal gamma scintigraphy 30min and 24h after injection of mixed leukocytes radiolabeled in vitro with 111In-oxine or 111In-tropolone. Change in liver activity, corrected for isotope decay and background activity, was expressed as the ratio of 24h/30min count rates. Two blinded pathologists compared this to histological quantification of parenchymal granulocytes using the marker CD15. Semi-quantitative grading of steatohepatitis (SH) (mild, moderate or severe) was also assessed. Contemporaneous liver/spleen 99mTc-nanocolloid scintigraphy, in which hepatic activity was expressed relative to the spleen, was performed in a subset with SAH and an additional group with inactive alcohol related cirrhosis (ARC). Results: Seventeen patients with SAH (14 male, median DF 52, range 33%ndash;155; 14 studied with 111In-oxine radi-olabeling and 3 with 111In-tropolone) and 7 with ARC (all male, median MELD 9, range 7.5%ndash;13) were recruited. The 24h/30min activity ratios in SAH positively correlated with CD15 biopsy quantification (r=0.62, p=0.023). SH grading demonstrated good inter-observer agreement (Κ=0.886, p<0.001) and 24h/30min ratios were significantly higher in those with histologically severe vs moderate vs mild SH (3.85 ±1.12, 2.29 ±0.99 and 1.42 ±0.36, respectively; p=0.01). Imaging appearances in the SAH cohort receiving 111In-tropolone-labeled leukocytes were similar to the group studied with 111In-oxine. 99mTc-nanocolloid liver/spleen ratios were significantly lower in SAH (n=11) vs ARC (n=7; 0.80 ±0.59 vs 2.29 ±1.60, p=0.049) and did not correlate with histological CD15 quantification or SH grading. Conclusions: The 24h/30min hepatic activity ratio correlates with the histological severity of SAH, thereby validating 111 In-leukocyte scintigraphy as a non-invasive diagnostic tool in this condition. Additional corroborative data from 99mTc-nanocolloid scintigraphy suggest that liver activity in 111In-leukocyte scintigraphy reflects active neutrophil migration rather than physiological destruction of neutrophils or non-specific phagocytosis of free 111 In.

Disclosures:

Sumita Verma - Advisory Committees or Review Panels: Janssen; Grant/Research Support: Gilead, Roche, Janssen

The following people have nothing to disclose: Jonathan R. Potts, Mark Howard, Mark Taylor, Neda Farahi, Sarah Heard, Arun N. Shankar, Graeme J. Alexander, Edwin R. Chilvers, Adrien M. Peters