Steatohepatitis Mechanisms


663

Heterozygous Mutations Affecting the Sonic Hedgehog Pathway Predispose to Non-Alcoholic Fatty Liver Disease

Maria J. Guillen1, Ariel F. Martinez1, Robert Lipinski4, Benjamin Solomon1, Joshua L. Everson5, Niraj S. Trivedi2, Abdel G. Elkahloun3, Ramón Bataller6, Kathleen Sulik5, Maximilian Muenke1;

1Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD; 2Genome Technology Branch, National Human Genome Research Institute, Bethesda, MD; 3Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, MD; 4Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI; 5The Bowles Center for Alcohol Studies, University of North Carolina, Chapel Hill, NC; 6Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, NC

The Sonic Hedgehog (SHH) pathway is fundamental in early embryonic development and while relatively quiescent in the adult healthy liver, the SHH pathway becomes active in alcoholic and non-alcoholic fatty liver disease. In humans, heterozygous mutations in the SHH pathway cause non-syndromic holoprosencephaly (HPE), a rare disorder of forebrain development. In a cohort of patients with HPE studied in our center, we observed a 6-fold higher prevalence of fatty liver disease when compared to the general population (pediatric: p=0.0023; adult: p=0.0135). The majority of these patients have no environmental/medical risk factors for fatty liver. Steatosis was also seen in liver sections from affected individuals who succumbed in the fetal/neonatal period. To test the hypothesis that mutations in the SHH pathway predispose to steatohepatitis, we compared heterozygous knockout mice (Gli2+/-) bred back to the C57Bl/6J strain to their wild-type (WT) littermates. Animals were fed a high-fat (HF) diet for 12 weeks, after which their livers were harvested. Weight gain, gross liver morphology, and liver fat accumulation were analyzed. We then performed whole-transcript mRNA expression profiling of liver tissues. Gli2+/- mice showed increased susceptibility to fatty liver when fed HF diet compared to controls.

All mice fed the HF diet showed an increase in body mass, but Gli2+/- animals gained significantly more weight than WT. Gli2+/- mice fed control diet also demonstrated a differential expression of genes involved in carbohydrate and lipid metabolism, immune function, oxidative stress and cell cycle regulation, which suggests an inherent metabolic defect affecting liver function. The most dysregulated molecules include Ctse, Taf1d, Gadd45g, Snora74a, Lcn2, Hsph1, Sult2a1, Sult2a2, Cyp3a5, Cyp2c9, Rnf1 86, Pdk4 and Rsad2. SHH pathway genes did not show significant differences in gene expression. Although all animals exposed to the HF diet showed similar changes in genes involved in the above mentioned cellular processes, Gli2+/- mice had more genes with aberrant expression involved in these pathways and a specific fingerprint of molecules involved in liver cirrhosis. OurGli2+/- mouse model shows that SHH signaling defects predispose to the development of fatty liver disease causing a more severe phenotype when exposed to environmental factors (diet). We propose that mutations interfering with SHH signaling be considered a target for investigation as a risk factor for idiopathic fatty liver in the general population. We are currently working on a Shh+/-mouse model and using a different diet that induces fatty liver through an alternate pathway.

Disclosures:

The following people have nothing to disclose: Maria J. Guillen, Ariel F. Martinez, Robert Lipinski, Benjamin Solomon, Joshua L. Everson, Niraj S. Trivedi, Abdel G. Elkahloun, Ramón Bataller, Kathleen Sulik, Maximilian Muenke

664

Role of Integrated MicroRNA-mRNA Network in Human Hepatic Fat Accumulation and Non-Alcoholic Fatty Liver Disease

Rajneesh Srivastava1, Xiaoliang Wang2, Jingmei Lin3, Rongrong Wei2, Praneet Chaturvedi1, Naga P. Chalasani4,5, Sarath Chandra Janga1, Wanqing Liu2,4;

1School of Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN;2Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN; 3Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN; 4Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, IN; 5Indiana Fatty Liver Research Group, Indiana University School of Medicine, Indianapolis, IN

Background: Recent studies have suggested that microRNAs (miRs) play an important role in the pathogenesis of NAFLD and NASH but human studies especially those that integrate miRs with mRNAs are lacking. Aim: To identify miRs, mRNAs as well as the miR-mRNA regulatory network involved in hepatic fat accumulation and human NAFLD. Materials and Methods: This study consisted of 206 human liver samples obtained from organ donors on which transcriptome has been profiled and total hepatic fat content (HFC, mg fat/mg total protein) was measured. In a subset (n=73) we also measured genome-wide miRs; out of which 50 samples were characterized into normal (n=34) and NAFLD (n=16) based on their histology. Spearman correlation was calculated between miR/mRNA expression and HFC to identify miRs and mRNA significantly associated with HFC (p < 0.05 for miR and p< 0.001 for mRNA). Further, miR-mRNA association network was built separately for both NAFLD and normal groups based on miRs and mRNA exhibiting high level correlation (p<0.001). To build a post-transcrip-tional regulatory network associated with HFC, miRs and mRNAs strongly correlated with HFC were further integrated based on the potential miR-mRNA targeting by employing two miR target prediction tools (TargetScan and MiRanda). mRNAs targeted by miRNAs in this network (significantly correlated with HFC) were analyzed by pathway enrichment analysis tools, DAVID and Panther. Results: We identified 67 miRs significantly correlated with HFC (p<0.05), among which 16 miRs were strongly associated with NAFLD phenotype (p<0.05). MiR-34a and miR-19b were demonstrated to possess the most significant positive and negative correlation with HFC, respectively (p<0.001 for both). MiRs such as miR-122 and mir-34a that were previously associated with NAFLD/NASH were also observed (p=0.02 and 0.003, respectively). A number of genes (mRNAs) involved in lipid metabolism were correlated with HFC, e.g. ACADSB, ALDH6A1 and PPP1R9B (p<0.001). Pathway analyses of the mRNAs in the resulting miR/mRNA-HFC network revealed that inflammation pathways mediated by chemokine and cytokine signaling, Wnt signaling, Integrin signaling and MAPK signaling pathways were enriched (p<0.01) and are likely involved in hepatic fat accumulation. Integrated network analysis indicated that a few miRs 30, 26, 19, 215, 381, 195 and 497 controlled >60% of HFC-associated miRs in this network, and their expression was significantly different between NAFLD and normal groups as well. Conclusion: Our study for the first time identified miR-mRNA regulatory network that may contribute to the pathogenesis of NAFLD via modulating the hepatic lipid deposition.

Disclosures:

Naga P. Chalasani - Consulting: Salix, Abbott, Merck, Lilly, Enterome, Aegerion; Grant/Research Support: Intercept, Lilly, GenFit, Gilead, Enterome, Cumberland, Galectin

The following people have nothing to disclose: Rajneesh Srivastava, Xiaoliang Wang, Jingmei Lin, Rongrong Wei, Praneet Chaturvedi, Sarath Chandra Janga, Wanqing Liu

665

RING-domain Dependent Degradation of cIAP-1 Contributes to Hepatocyte Lipoapoptosis

Yuko Akazawa1,2, Maria Eugenia Guicciardi2, Steven Bronk2, Nathan Werneburg2, Kazuhiko Nakao1, Michael R. Charlton2, Gregory J. Gores2;

1Gastroenterology and Hepatology, Nagasaki University, Sakamoto, Japan;2Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN

Background and Aim: Elevated serum free fatty acid and hepatocyte apoptosis are features of nonalcoholic steatohepatitis (NASH). We have previously shown that saturated free fatty acids (FFAs) induce hepatocyte apoptosis by translational up-regulation of death receptor (DR) 5 (Cazanave et al, JBC, 2010). However, details regarding DR5-mediated lipoapoptosis are unexplored. Cellular inhibitor of apoptosis 1 protein is a potent inhibitor of DR5 mediated cell death (Guicciardi etal, Exp Cell Res. 2011). Previous studies have also demonstrated that proteasomal degradation of cIAP-1 occurs through their auto-ubiquitination, which is mediated by the E3 ubiquitin lig-ase activity of its RING domain. Aim of the current study was to determine the detailed role of cIAP-1 degradation during hepatocyte lipoapoptosis. Methods: Huh-7 cells, Hep3B cells, primary hepatocytes from wild type and DR5 -/- mice were employed for the study. Cells were treated with unsaturated free fatty acid, palmitate (200-800μM). Huh-7 cells were transiently transfected with constructs expressing cIAP-1 mutant deficient in their E3 ligase activity due to specific replacement of critical histidine residues in their RING domains. cIAPs protein was assessed by immunoblotting. Apoptosis was assessed by characteristic nuclear staining with DAPI and cleavage of PARP by immunoblotting. Results: cIAP-1 protein underwent cellular elimination following treatment with the 800μM palmitate (PA) in Huh7, Hep3B, and HepG2 cells. Huh-7 cells transfected with RING-mutant cIAP-1 did not degrade with PA treatment, indicating that cIAP-1 undergoes proteasomal degradation by PA.Furthermore, transfection of cells with RING-mutant cIAP-1 decreased PA-induced apoptosis. Incubation with the SMAC mimetic JP1584 (500 nM), which induces degradation of cIAPs, significantly enhanced PA- mediated apoptosis in Hep3B cells and Mouse primary hepatocytes. In contrast, JP 1584 failed to sensitize primary hepatocytes from DR5 -/- mouse to apoptosis, suggesting that degradation of cIAP-1 by PA enhances death receptor mediated pathway of lipoapoptosis. Conclusions: Collectively, these results implicate RING domain dependent degradation of cIAP-1 by FFA regulates lipoapoptosis via DR5-induced hepatocyte lipoapoptosis.

Disclosures:

Gregory J. Gores - Advisory Committees or Review Panels: Bayer, Chugia, Dai-ichi, Generon, Conatus, IntegraGen

The following people have nothing to disclose: Yuko Akazawa, Maria Eugenia Guicciardi, Steven Bronk, Nathan Werneburg, Kazuhiko Nakao, Michael R. Charlton

666

The caspase 8 homologue cFLIP contributes to hepatic lipogenesis and intrahepatic immune competent cells during the development of non-alcoholic fatty liver disease (NAFLD)

Nadine Gehrke1, Henrike Zinngrebe1, Amrit Mann1, Arno Schad2, Martin F. Sprinzl1, Marcus A. Woerns1, Peter R. Galle1, Marcus Schuchmann1, Jörn M. Schattenberg1;

1I.Department of Medicine, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; 2Department of Pathology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany

Introduction The caspase 8 homologue cellular FLICE inhibitory protein (cFLIP) is an important regulator of apoptosis- and stress-signaling pathways in the liver and contributes to tissue home-ostasis. The role of cFLIP in non-apoptotic pathways is less clear and its function in the pathophysiology of non-alcoholic fatty liver disease (NAFLD) has not been investigated. Method 8-12 week old male mice with a hepatocyte-specific deletion of cFLIP using the cre-loxP system under control of the albumin promoter (flip-/-) and corresponding wildtype mice were fed with a high fat diet (35.5% fat) or an appropriate control diet (5.4% fat), respectively (n=17-18 mice/group). Mice on HFD diet additionally received drinking water enriched with fructose and glucose (55%, resp. 45%). During the dietary period of 12 weeks, body weights and dietary consumption were measured weekly, blood samples were taken and after sacrifice further analysis were performed. Results Weight gain in mice on HFD diet was significantly increased compared to mice on control diet and more pronounced in flip-/- mice. Likewise, hepatomegaly and a significantly increased relative liver weight were observed compared to wildtype mice. Serological analysis revealed that serum ALT, glucose and insulin levels were not affected by dietary feeding at 12 weeks. Histological analysis showed an accumulation of hepatic, microvesicular steatosis and ballooning hepatocytes that was more pronounced in flip-/- mice. In line with this, the expression of regulators of hepatic lipogenesis including fatty acid synthase (FAS) and acetyl-CoA car-boxylase (ACC) were up-regulated in flip-/- mice on HFD diet, whereas expression of the transcription factor PPARγ, which improves glucohomeostasis and hepatic inflammation, was significantly less suppressed. Likewise, PPARα was significantly down-regulated in flip-/- mice by HFD diet. FACS analysis of intrahepatic immune cells demonstrated an infiltration of natural killer-cells and cytotoxic T-cells but a significant loss of B-cells in the liver of flip-/- mice compared to wildtype on HFD diet. Conclusion In a murine model of NAFLD, hepatocyte-specific deletion of cFLIP contributes to obesity and hepatic steatosis,involving the regulation lipogenesis-related proteins. In addition, alterations of intrahepatic immune competent cell populations with a pro-inflammatory phenotype occur depending on the expression of cFLIP. This implicates a role of cFLIP in the development and progression of NAFLD and could be an underlying factor in patients that progress to a more severe phenotype, including non-alcoholic steatohepatitis (NASH).

Disclosures:

Marcus A. Woerns - Advisory Committees or Review Panels: Bayer, Bayer

Peter R. Galle -Advisory Committees or Review Panels: Bayer, BMS, Lilly, Daiichi, Jennerex; Consulting: Medimmune; Grant/Research Support: Roche, Lilly; Speaking and Teaching: Bayer, BMS

Marcus Schuchmann - Advisory Committees or Review Panels: Roche, BMS, Norgine, Boehringer; Speaking and Teaching: Gilead, Merck, Falk

The following people have nothing to disclose: Nadine Gehrke, Henrike Zinngrebe, Amrit Mann, Arno Schad, Martin F. Sprinzl, Jörn M. Schattenberg

667

A Novel Mechanism by which Anti-diabetic Drug, Metformin, Attenuates Hepatic Steatosis and Insulin Resistance in Ob/ob Mice: Coordinated Regulation of LKB1 /AMPK and Autophagy

Jong Woo Lee, Yu Li, Mengwei Zang;

Department of Medicine, Boston University School of Medicine, Boston, MA

The biguanide metformin was introduced clinically in the 1950s and is widely used as the first line treatment for patients with type 2 diabetes, but its underlying mechanism remains elusive. The serine/theronine kinase LKB1, a tumor suppressor gene mutated in the familiar cancer condition Peutz-Jeghers syndrome, has emerged as a unique energy-state sensitive regulator of metabolic reprogramming and cell growth via AMPK. We have discovered that AMPK activation by metformin directly phosphorylates and inactivates the transcription factor SREBP to attenuate obesity-induced hepatic steatosis. Ser-372 phosphorylation of SREBP-1 by AMPK is required for metformin and AICAR to reduce lipogenesis in hepatocytes. More recently we have found an additional connection between LKB1 /AMPK signaling and the control of autophagy. Our studies showed that LKB1 regulation of AMPK was essential for metformin to induce autophagic signaling, as indicated by p62 degradation and LC3-II accumulation, in human HepG2 cells under conditions of excess amino acids. Enhanced autophagic signaling by metformin was diminished by adenovirus-mediated overexpression of dominant-negative forms of either LKB1 or AMPK as well as abrogated in cells lacking LKB1, suggesting that metformin induced autophagy via an LKB 1-AMPK dependent mechanism. Adenovirus-mediated overexpression of the LKB1 complex, which consists of LKB1, STRADα and MO25α, induced an autophagic process in HepG2 cells through AMPK activation. Moreover, administration of metformin (350mg/kg/day) in a liquid diet to 8-week-old genetically obese ob/ob mice for 4 weeks significantly lowered hyperglycemia, improved glucose tolerance, and ameliorated hepatic steatosis and insulin resistance. These salutary effects of metformin were associated with stimulation of AMPK and restoration of reduced autophagy-associated proteins such as Atg7, Beclin-1, and Atg5-Atg12 conjugation seen in the liver of diabetic mice. Hepatic activation of SREBP-1, elevation of Ser-1101 phosphorylation of IRS-1, and gene expression of gluconeogenesis caused by obesity were also suppressed in metformin-treated diabetic mice. Furthermore, AMPKα isoform strongly interacted with Atg7 protein, an important autophagic mediator. These findings provide mechanistic insight into that the integrated activation of LKB 1 -AMPK and Atg7-mediated autophagy may contribute to the therapeutic effect of metformin on hepatic steatosis and glucose intolerance in diabetes. Collectively, these new drug targets of metformin may sever to mediate autophagy and metabolic homeostasis with important implications for obesity-related fatty liver disease, insulin resistance, and cancer.

Disclosures:

The following people have nothing to disclose: Jong Woo Lee, Yu Li, Mengwei Zang

668

Fatty acids induce the expression of TLRs as the proinflammatory state in NAFLD mice liver

Koji Sawada1, Takaaki Ohtake1, Takumu Hasebe1, Shunsuke Nakajima1, Masami Abe1, Hiroki Tanaka2, Yutaka Kohgo1;

1Division of Gastroenterology and Hematology/Oncology Department of Medicine, Asahikawa Medical University, Asahikawa, Japan; 2Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Asahikawa, Japan

Background: There is considerable evidence that intestinal microbiota are involved in the development of metabolic syndromes and consequently with that of nonalcoholic fatty liver disease (NAFLD). Toll-like receptors (TLRs) are essential for the recognition of microbiota. However, the induction mechanism of TLR signals through the gut-liver axis for triggering the development of nonalcoholic steatohepatitis (NASH) or NAFLD remains unclear. In this study, we investigated the role of fatty acids on the formation of proinflammatory state of NAFLD. Methods: C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks. Antibiotics treatment was followed by feeding a HFD for further 8 weeks. The mice were sacrificed and histopathologi-cal evaluation was performed. The expressions of TLRs, tumor necrosis factor (TNF), interleukin 1β (IL-1β), and phospho-inter-leukin-1 receptor-associated kinase 1 (pIRAK1) in the liver and small intestine were assessed. In addition, Huh7 and THP-1 cells, both of which are representatives of hepatocytes and Kupffer cells, respectively, were treated with fatty acids, and the direct effects of fatty acids on TLR induction by these cells were evaluated. Results: Histopathological evaluation showed intra-cellular fat droplets and ballooning degeneration of hepatocytes, but no obvious infiltration of inflammatory cells was noted. The expressions of inflammatory cytokines such as TNF, IL-1β, and TLR2, TLR4, TLR5, and TLR9 and pIRAK1 were increased in the liver, but decreased in the small intestine of HFD mice in vivo. Antibiotics treatment improved steatosis, serum ALT, serum free fatty acids, and TLR expressions in the liver. In addition, the expression of carbohydrate response element binding protein (ChREBP) and sterol response element binding protein 1 (SREBP1) were decreased in the liver. The expressions of TLRs in Huh7 and THP-1 cells were increased by treatment with fatty acids. Conclusion: Our data suggests that dietary fatty acids trigger the expressions of TLRs, which contribute to the proinflammatory state of NAFLD. Furthermore intestinal microbiota of HFD develops it via induction of hepatic de novo lipogenesis.

Disclosures:

Yutaka Kohgo - Grant/Research Support: Novartis, Chugai-Roche

The following people have nothing to disclose: Koji Sawada, Takaaki Ohtake, Takumu Hasebe, Shunsuke Nakajima, Masami Abe, Hiroki Tanaka

669

Mice deficient in CD18 are protected against diet-induced steatohepatitis, but at the expense of increased hepatic steatosis

Andrew Pierce, Kevin Siao, Aras N. Mattis, Jacquelyn J. Maher;

Liver Center and Department of Medicine, University of California, San Francisco, San Francisco, CA

CD18, or β2-integrin, is a leukocyte adhesion molecule that promotes neutrophil and macrophage invasion into sites of tissue injury. Mice lacking CD18 have impaired neutrophil migration and activation. Studies have shown that CD18-deficient mice are protected from some toxic and cholestatic liver diseases; this underscores the potential for inflammatory cells to exaggerate liver injury by releasing oxidants and causing secondary damage to hepatocytes. The degree to which leukocyte invasion accentuates liver injury in the setting of non-alcoholic steatohepatitis (NASH) is incompletely understood. The objective of this study was to determine whether CD18 deficiency protects mice from hepatic inflammation and liver injury in a dietary model of NASH. Methods: Wild-type (WT) and CD18-deficient (KO) mice were fed methionine-choline-deficient (MCD) or control diets for 21 days. At the end of the experiment, mice were killed for biochemical and molecular analyses of blood and liver tissue. Results: WT and KO mice ate similar amounts of food and exhibited similar changes in body weight on the experimental diets. MCD feeding induced steatohepatitis in both WT and KO mice, but the degree of liver injury was significantly lower in KO mice (ALT 607.1 ± 29.8 vs. 347.2 ± 24.4 IU/L in WT vs. KO, P < 0.01). Hepatic neutrophil infiltration was also significantly milder in KO than WT mice after MCD feeding (neutrophil counts increased 14.7-fold vs. 2.0-fold in WT vs. KO, P < 0.01). Chlorotyrosine staining confirmed reduced oxidant production in the livers of KO mice. Despite these differences, KO mice developed nearly twice as much hepatic steatosis as WT mice after MCD feeding (triglyc-eride 99.4 ± 7.0 vs. 179.9 ± 9.2 mg/g tissue in WT vs. KO, P < 0.01). The enhanced steatosis in KO mice coincided with a 30% reduction in hepatic mRNA encoding adipose triglyc-eride lipase (P = 0.02). Other measures of hepatic lipid uptake and metabolism were unchanged. In addition, KO mice exhibited significantly greater hepatic mRNA encoding the M1 cytokines TNF and IL-1 after MCD feeding, while showing no change in mRNA encoding the M2 cytokine IL-10. Summary: CD18 deficiency reduces hepatic inflammation and liver injury in response to MCD feeding, but does so at the expense of increased hepatic steatosis. The latter may be due to enhanced local production of M1 cytokines and reduced triglyceride hydrolysis. Conclusion: CD18 has the capacity to accentuate fatty liver disease by promoting hepatic invasion and activation of leukocytes. By contrast, CD18 does not appear necessary to influence macrophage polarization toward M1, which can promote fat accumulation in the liver.

Disclosures:

Jacquelyn J. Maher - Consulting: Sanofi Aventis

The following people have nothing to disclose: Andrew Pierce, Kevin Siao, Aras N. Mattis

670

Compromised intestinal epithelial barrier integrity in Junctional Adhesion Molecule (JAM-A) knockout mice regulates the severity of diet-induced non-alcoholic steatohepatitis (NASH) but not features of metabolic syndrome or steatosis

Khalidur Rahman1, Pradeep Kumar1, Asma Nusrat2, Charles A. Parkos2, Frank A. Anania1;

1Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA; 2Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can progress to more severe lesions including nonalcoholic steatohepatitis (NASH) and cirrhosis. A diet rich in fat along with host environmental factors, including gut-flora, play a central role in NASH pathogenesis. Investigation for the molecular basis of gut-permeability in NAFLD progression has not been previously evaluated. We obtained knockout mice for Junctional Adhesion Molecule A (JAM-A-/-) which have a 10 fold increase in intestinal permeability as previously assessed by dextran absorption. The AIM of the present study was to delineate the respective contribution of intestinal epithelial barrier integrity to the pathogenesis of diet-induced steatosis and its contribution to the severity of NASH. METHODS: Male C57BL/6j (WT) or JAM-A-/- mice were fed ad libitum either normal chow or a high fat, high fructose corn syrup, high cholesterol diet (HFD). Mouse cohorts were pair-fed and subjected to metabolic testing at 0, 4, and 8 wks post feeding. Liver histology was assessed with stains for fat, collagen, and α-smooth muscle actin. Furthermore, serum transaminase and leptin levels were measured. Liver RNA and protein were harvested and analyzed to determine changes in key pro-inflammatory markers. RESULTS: Within 8 wks of HFD feeding, JAM-A-/- mice developed severe histologic features of NASH including ballooning degeneration, inflammatory cell infiltration, and extensive pericellular fibrosis; while only modest NASH-related histologic findings were observed in the HFD fed WT mice. Liver injury in the HFD fed JAM-A-/- mice was also associated with a significant increase in serum transaminases and leptin levels (p<0.05). RT-qPCR of liver mRNA and immunoblot revealed that HFD fed JAM-A-/- mice also had significantly higher levels of TLR4 expression and expression of TNF-α, IL-1 β, and IL-6, compared to HFD fed WT mice. Conversely, both HFD fed WT and JAM-A-/- developed identical features of metabolic syndrome including histologic grade of steatosis as well as measured parameters associated with metabolic syndrome {e.g. serum triglycerides} (p>0.05). Taken together, the rapid progression of liver injury in the HFD fed JAM-A-/- can be attributed to an intestinal permeability defect in JAM-A-/- mice since both this cohort and the WT mice consumed the same diet and had similar weight gain. CONCLUSION: These data suggest that a major factor in the severity of NASH may be directly related to impaired gut permeability, implicating the critical need for further mechanistic investigation of the gut-liver axis in NASH pathogenesis. Therapies to restore gut integrity in NASH patients may be beneficial.

Disclosures:

The following people have nothing to disclose: Khalidur Rahman, Pradeep Kumar, Asma Nusrat, Charles A. Parkos, Frank A. Anania

671

Decaffeinated coffee reduces gut leakiness in rats fed with high-fat diet. The role of Occludin and Zonulin-1

Giovanna Mazzone1, Vincenzo Lembo1, Giuseppe D'Argenio1, Paola Vitaglione2, Maria Guarino1, Silvia Camera1, Eduardo Clery1, Carmine Ferraiuoli1, Vincenzo Fogliano2, Nicola Caporaso1, Filomena Morisco1;

1Department of Clinical Medicine and Surgery, Federico II University of Naples, Naples, Italy; 2Department of Agricolture, Federico II University of Naples, Portici, Italy

Background & Aims: Exposure to bacterial products of intestinal origin, leads to liver inflammation, hepatocyte injury and hepatic fibrosis. Impaired gut epithelial integrity due to alterations in tight junction (TJ) proteins may be the pathological mechanism underlying bacterial translocation. It is well known that gut permeability plays a crucial role in the pathogenesis of non-alcoholic steatohepatitis (NASH) and that some nutrients and food components may modulate intestinal TJ. Many epidemiological, experimental and clinical studies demonstrate that coffee beverage reduces liver damage caused by high fat diet (HFD). Aims of the study were to evaluate the effect of coffee on the HFD-induced damage of intestinal mucosal barrier and on proinflammatory TLR-4 in a rat model of NASH. Methods: Male Wistar rats were fed with a HFD for 5 months and divided in two groups: one group drunk simple water while the other group drunk water added with 1.2 mL of decaffeinated coffee per die starting from the 4th month. Contemporarily, another group of rats fed a standard diet and was used as control. Protein and mRNA expression levels of TLR-4, Occludin and ZO-1 were examined from proximal jejunum of rats fed with standard diet, HFD + water and HFD + coffee. Results: A significant reduction of TJ proteins Occludin and Zo-1 in HFD fed rats was observed; it was partially reverted by the addition of coffee to the HFD (0.83±0.27 vs 0.14±0.07, p<0.05 and 0.85±0.12 vs 0.57±0.14, p<0.05 respectively). Coffee also reduced TLR-4 expression up-regulated in the HFD + water group. Conclusion: These preliminary data confirmed that HFD impairs the intestinal TJ barrier integrity and that coffee is able to partially revert it by increasing ZO-1 and Occludin and reducing TLR4 expression.

Disclosures:

The following people have nothing to disclose: Giovanna Mazzone, Vincenzo Lembo, Giuseppe D'Argenio, Paola Vitaglione, Maria Guarino, Silvia Camera, Eduardo Clery, Carmine Ferraiuoli, Vincenzo Fogliano, Nicola Caporaso, Filomena Morisco

672

Role of miRNA-21, miRNA-22 and miRNA-451 in modulating gluconeogenesis, and the role of high glucose and free fatty acids in regulating the expression of these miRNAs in hepatic cells

Sweta Khanal, Aashirwad Shahi, Satendra Kumar, Parul Gupta, Preeti Damania, Senthil K. Venugopal;

Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India

Background: NAFLD is associated with obesity and insulin resistance. It affects one-third of adults and 10-15% of these patients will develop NASH and sometimes to hepatocellular carcinoma (HCC). The pathogenesis of NAFLD is associated with an increased fat deposition in liver, which alters the normal metabolic pathways including gluconeogenesis. miRNAs are small non-coding RNAs which regulate the cell functions. We hypothesized that dysregulation of miRNAs might modulate gluconeogenesis, thereby allowing NAFLD progression towards NASH. Methods: Three miRNAs (miRNA-21, miRNA-22 and miRNA-451) were over-expressed in Huh7 cells and their effect on modulating gluconeogenesis was evaluated. miRNAs wereover-expressed using siPORT NeoFX transfection reagent. After 72 hours, the cells were collected either for RNA or protein isolation. Total RNA enriched with miRNAs was isolated, cDNA was synthesized and real time PCR for these miRNAs were performed. The cellular protein was isolated and Western blots were performed for SIRT-1, PGC-1α and PEPCK and β-actin. Effect of high glucose alone (25 mM), palmitic acid alone (200 μM), oleic acid alone (150 μM), linoleic acid alone (100 μM) or in combination with high glucose was evaluated on the expression of these miRNAs using real time RT-PCR and gluconeogenesis by Western blots. Results: Over-expression of these miRNAs led to increased intracellular levels of each miRNA in hepatic cells. Among these miRNAs, miRNA-21 increased SIRT-1 levels to 4-fold in Huh7 cells, while miRNA-22 and miRNA-451 inhibited the SIRT-1 levels to 3-fold and 2-fold respectively. Expression of PGC-1 α was determined and was found that only miRNA-451 inhibited significantly (n=3; p<0.01). One of the key regulatory enzymes of gluconeogenesi, PEPCK, was determined in miRNA over-expressing cells and the results showed that miRNA-21 significantly increased PEPCK expression (10-fold increase; n=3; p<0.01), while both miRNA-22 and miRNA-451 inhibited the expression of PEPCK (5-fold and 15-fold respectively; n=3; p<0.01). Both oleic acid and oleic acid plus high glucose showed significant increase in the expression of miRNA-21. Palmitic acid increased the expression of SIRT-1 to 4-fold compared to control cells. Conclusion: These data demonstrate that over-expression of miRNA-21 expression increased the gluconeogenesis while both miRNA-22 and miRNA-451 inhibited gluconeogenesis. Some of the free fatty acids alone or in combination with high glucose modulated the expression of these miRNAs and regulate the gluconeogenesis in hepatic cells.

Disclosures:

The following people have nothing to disclose: Sweta Khanal, Aashirwad Shahi, Satendra Kumar, Parul Gupta, Preeti Damania, Senthil K. Venugopal

673

CCAAT/Enhancer binding protein homologous protein deficiency attenuates progression from steatohepatitis to liver fibrosis and carcinogenesis in mice treated with MCD diet

Kan Toriguchi, Etsuro Hatano, Kazutaka Tanabe, Kenji Takemoto, Satoru Seo, Kojiro Taura, Shinji Uemoto;

Surgery, Kyoto university, Graduate school of medicine, Kyoto, Japan

Background & Aims: Nonalcohlic steatohepatitis (NASH) is a worldwide rapidly increasing metabolic liver disease, gaining much attention because of its potential to progress cirrhosis and hepatocellular carcinoma (HCC). CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional factor induced by DNA damage, which plays an essential role in endoplasmic reticulum (ER) stress-induced apoptosis and has been reported to be involved in the pathogeneses of malignancies widely. The aim of this study was to clarify the impact of CHOP in the development of NASH and HCC. Methods: Wild type (WT) and CHOP knockout (CHOP-/-) mice aged 8 weeks old were fed a normal diet or methionine-choline deficient (MCD) diet. Mice were sacrificed after 3 weeks of feeding, and then assessments of steatosis, inflammation, apoptosis and liver damage were conducted. We also evaluated fibrosis after 6 weeks of feeding. Furthermore, to explore the role of CHOP in liver carcinogenesis, 25 mg/kg of diethylnitrosamine (DEN) was injected intraperitoneally at 2 weeks old, then treated with respective food from 8 to 24 weeks old. Expressions of CHOP in the liver from HCC patients were also evaluated. Results: CHOP deficiency did not affect steatosis, but significantly reduced apoptosis cells, inflammation scores and serum liver enzymes. It also significantly suppressed serum total bilirubin levels, sirius red positive fibrosis area and mRNA expression of pro-fibrotic cytokine. DEN-initiated carcinogenesis was promoted by MCD diet, while CHOP deficiency significantly attenuated total numbers and maximum diameters of tumors and Ki-67 labeling index. In human liver, CHOP expression was enhanced in parallel with NASH to HCC progression. Conclusions: CHOP deficiency attenuated apoptosis, inflammation, fibrosis and promotoion of carcinogenesis under fat loading circumstances, indicating that therapeutic strategy targeting CHOP might be effective for preventing NASH and subsequent HCC.

Disclosures:

The following people have nothing to disclose: Kan Toriguchi, Etsuro Hatano, Kazutaka Tanabe, Kenji Takemoto, Satoru Seo, Kojiro Taura, Shinji Uemoto

674

β-catenin Antisense Oligonucleotides Protect Mice Against Diet-Induced Insulin Resistance and Regulate Liver Lipid Metabolism

Violeta Popov1, Francois R. Jornayvaz1, Emin O. Akgul1, Michael J. Jurczak1, Dongyan Zhang1, Sachin Majumdar1, Kitt Petersen1, Vara Prasad Manchem2, Sanjay Bhanot2, Gerald I. Shulman1, Var-man Samuel1;

1Medicine, Yale University, New Haven, CT; 2Isis Pharmaceuticals, Carlsbad, CA

Genetic studies have linked mutations in the Wnt pathway with type 2 diabetes. However, the cellular mechanism by which these pathways affect insulin sensitivity is unclear. β-catenin is the final downstream mediator of canonical Wnt signaling. To examine the link between β-catenin and insulin action, we treated for four weeks high fat fed (HFF) male C57BL/6 mice with an antisense oligonucleotide (ASO) to decrease hepatic and adipose expression of β-catenin. β-catenin mRNA was decreased by =80% in the liver and =70% in white adipose tissue, and β-catenin protein was decreased by =65% in liver cytosol relative to control ASO treated HFF mice. HFF β-catenin ASO and control ASO-treated mice had similar weight and body composition and whole body energy balance, however, β-catenin ASO improved the plasma lipid profile with a 17% decrease in plasma cholesterol, and a 37% decrease in triglyc-eride (TG), and 20 % decrease in free fatty acids (FFA), (p<0.05). β-catenin knockdown protected mice from lipid-induced insulin resistance, reflected by 50% higher glucose infusion rate (p<0.001) required to maintain euglycemia during hyperinsulinemic-euglycemic clamp compared to HFF control ASO mice. Insulin suppressed hepatic glucose production by 82% in the β-catenin ASO group vs. 31% in the control ASO group(p<0.05). Peripheral insulin response was also improved, with 30% increase in peripheral glucose uptake (p<0.01). The improvement in hepatic insulin sensitivity was associated with 44% reduction in hepatic TG and 60 % reduction in diacyl-glycerol (DAG) content(p<0.01). In contrast, hepatic ceramide content was increased by 30%(p<0.001). Gene expression analysis showed 45% lower expression in the liver of key genes (mitochondrial GPAT and DGAT2) regulating lipid re-esterifica-tion in the β-catenin knockdowns (p<0.05). The expression of CPT1 and PPARα in the liver was reduced by 40% (p<0.05), consistent with previous reports of impaired FA oxidation (FAO) with loss of β-catenin. Liver fatty acyl CoA content was similar between the groups, suggesting redirection of liver FA metabolism from FAO and DAG and TG synthesis towards ceramide synthesis in the β-catenin ASO mice. The reduction in liver DAG was associated with a 33% decrease in PKCε activation (p<0.05) and improved insulin signaling with a 64% increase in insulin-stimulated Akt2 phosphorylation (p<0.05). Conclusion: Reducing β-catenin expression in liver and adipose tissue decreases expression of enzymes involved in FA esterification, decreases hepatic steatosis in HFF mice and protects from lipidinduced insulin resistance. These studies provide evidence fora novel role of β-catenin in liver lipid metabolism.

Disclosures:

The following people have nothing to disclose: Violeta Popov, Francois R. Jornayvaz, Emin O. Akgul, Michael J. Jurczak, Dongyan Zhang, Sachin Majumdar, Kitt Petersen, Vara Prasad Manchem, Sanjay Bhanot, Gerald I. Shulman, Varman Samuel

675

Macrophage migration inhibitory factor (MIF) attenuates hepatic steatosis and fibrosis but accelarates liver tumors development in murine NAFLD model

Norio Horiguchi1, Daichi Takizawa1, Satoru Kakizaki1, Hiroki Tojima1, Hiroaki Hashizume1, Yuichi Yamazaki1, Ken Sato1, Bin Gao2, Masatomo Mori1;

1Department of Medicine and Molecular Sciense, Gunma Graduate University School of Medicine, Maebashi, Gunma, Japan;2Laboratory of Liver Diseases, NIAAA/NIH, Bethesda, MD

Background: Macrophage activation plays a crucial role in the development of metabolic syndrome. Non-alcoholic fatty liver disease (NAFLD) is known as a phenotype of metabolic syndrome in the liver. Macrophage migration inhibitory factor (MIF) is involved in both innate and adaptive inflammatory response and regulates macrophage accumulation/activation. We have previously reported MIF-knockout mice (MIF-KO) is resistant against T-cell mediated (concanavalin A-induced) hepatitis. Recently, anti-fibrogenic effects of MIF has been reprted in chronic CCL4 and TAA-induced liver injury. However, the precise role of MIF in NAFLD is not clear. Methods: MIF-KO and wild-type (WT) were fed either a control or a high-fat diet for 24-weeks and 72-weeks. Hepatic inflammation, steatosis, fibrosis, and liver tumor development were examined. Results: MIF-KO showed more incresed body weight, liver/bodyweight ratio, and ALT elevation than WT after 24-weeks high fat feeding and this phenomena got more evident at 72-weeks. With regard to steatosis, oil-red-O staining and triglyceride levels showed MIF-KO is more susceptibele to high-fat-diet induced steatosis. In addition, MIF-KO showed significantly higher insulin levels than WT, suggesting insulin resistance partly accounts for the progression of steatosis in MIF-KO. Regarding to fibrosis, MIF-KO showed severe fibrosis compared with WT, which was confirmed by sirius-red staing and collagen 1 mRNA expression at 24-weeks, and this progression of fibrosis in MIF-KO was pronounced at 72-weeks. In terms of inflammation, no difference was found in inflammatory cell marker (F4/80, CCR2, and MPO) mRNA expression between WT and MIF-KO in the liver at 24-weeks and 72-weeks. Interestingly, WT developed higher number of liver tumors compared with MIF-KO at 72 weeks with high-fat diet feeding. Conclusions: We have shown MIF-KO is more susceptible to hepatic steatosis and fibrosis but resistant against liver tumor development in murine NAFLD model. These results suggest MIF has double swords in the development of NAFLD.

Disclosures:

The following people have nothing to disclose: Norio Horiguchi, Daichi Takizawa, Satoru Kakizaki, Hiroki Tojima, Hiroaki Hashizume, Yuichi Yamazaki, Ken Sato, Bin Gao, Masatomo Mori

676

Ceramide as a mediator of NAFLD induced atherosclerosis

Takhar Kasumov, Ling Li, Belinda Willard, Min Li, Arthur J. McCullough;

Gastroenterology, Cleveland Clinic, Cleveland, OH

Background: Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Insulin resistance (IR)-induced hyperlipidemia and hyperlipoproteinemia are risk factors of atherosclerosis and CVD. We have reported plasma ceramides and the ApoB100/ApoAI ratio are elevated in obese NAFLD patients and are reduced after gastric bypass surgery. Based on these data and that sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, we hypothesized that activation of ceramide mediates both liver injury and atherosclerosis by regulating dyslipidemia. Aim: To assess the role of hepatic ceramides in NAFLD induced atherosclerosis. Methods: Low density lipoprotein receptor (LDLR)-/-mice, a diet induced mouse model of obesity and atherosclerosis, were fed a low fat chow diet or a western diet (WD, high fat with 15% cholesterol) for 12 weeks with or without myriocin - a ceramide inhibitor. We used a lipidomics approach and a novel metabolic 2H2O-labeling based “lipoproteome dynamics“ technique for comprehensive profiling of ceramides and kinetic analyses of plasma lipids and proteins. Insulin resistance (IR), hepatic and serum lipids, and lipoproteins were measured. Liver histology and a Tunnel stain for apoptosis were reviewed by a liver pathologist. Aortic root lesions were quantified to assess atherosclerosis. One week prior to sacrifice, mice were given 2H2O in the drinking water and serial blood samples were taken. 2H-labeling of lipoprotein derived pep-tides were analyzed by high resolution mass spectroscopy. The spectral index was used for relative changes in protein expression. Results: WD caused IR, increased hepatic and serum triglycerides and atherosclerosis. WD caused hepatic steatosis, inflammation, apoptosis and mild fibrosis (F1-2). Hepatic levels of long chain ceramides (C16 & C18), involved in apoptosis, were increased by 80% and 27%, respectively. The plasma ratio of ApoB100/ ApoA1, principle proteins of VLDL/LDL and HDL was increased more than 2 fold. Kinetic analysis revealed that changes in plasma levels of these proteins were due to increased production of ApoB100 and increased clearance of ApoA1. Pharmacologic inhibition of hepatic ceramide production in WD mice reduced hepatic ceramides, prevented atherosclerosis and decreased hepatic steatosis, fibrosis and apoptosis. Conclusion: Modulation of ceramide synthesis may lead to the development of novel therapeutic strategies for the treatment of both NAFLD and its associated atherosclerosis. Lipidomics and “lipoproteome dynamics“ methods provide powerful tools for the characterization of novel mechanisms involved in the lipotoxicity of NAFLD.

Disclosures:

The following people have nothing to disclose: Takhar Kasumov, Ling Li, Belinda Willard, Min Li, Arthur J. McCullough

677

A Lipidomic Readout of Disease Progression in Nonalcoholic Fatty Liver Disease (NAFLD)

Tommy Pacana1, Hae K Min1, Vaishali Patel1, Riikka Katainen2, Terhi Vihervaara2, Faridoddin Mirshahi1, Puneet Puri1, Arun J. Sanyal1;

1Div. of Gastroenterology, Hepatology and Nutrition, Dept. of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA; 2Zora Biosciences, Espoo, Finland

BACKGROUND: NAFLD is an increasingly common cause of end-stage liver disease and indication for liver transplantation. The mechanisms underlying disease progression are not fully understood. NAFLD is associated with generation of biologically active lipid metabolites. It is not known which changes are related to disease progression. AIM: To perform a lipidomic analysis of early versus advanced stage NAFLD to identify changes associated with disease progression. METHODS: A diet-induced obesity mouse (129SI/SvlmJ;B6 female) model of NAFLD which progressed to steatosis and inflammation by week 16 and extensive pericellular fibrosis with bridging by week 52 was used. Chow- or high fat-diet (HFD) was given for 16 or 52 weeks (n=5 per group). Shotgun lipidomics, sphingolipid and eicosanoid profiling with identification of unique molecular lipid species was performed. Inter-group comparisons were performed using a T-test or ANOVA as appropriate.RESULTS: The mice fed HFD for 16 weeks developed extensive steatosis with scattered inflammation while mice on chow diet had normal histology. In contrast, macrovesicular steatosis remained unchanged and there was extensive small droplet steatosis and pericellular fibrosis with bridging in mice fed HFD for 52 weeks. There was a marked increase in monounsaturated fatty acid (MUFA) containing diacyl-glycerols (DAG) by week 16 which trended down by week 52 but remained significantly higher than in chow-fed mice. Similarly, there was an almost 700-fold increase in cholesterol esters enriched with saturated fatty acids (SFA) by week 16 which decreased significantly by week 52 in HFD-fed mice. Phosphatidic acid (PA) and its downstream product phosphatidylglycerol (PG) increased at weeks 16 and 52 in HFD-fed mice. Disease progression was characterized by decreasing phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Palmitate containing ceramides increased while total ceramides and sphingomyelin (SM) decreased at weeks 16 and 52 in HFD-fed mice. Arachidonic acid (AA) and eicosapentanoic acid also decreased. Progression to advanced disease was associated with increase in several lipoxygenase (LOX) metabolites (e.g.hydoxyeicosatetraenoic acids) and decreased PGD2. Several Cyp metabolites of AA also decreased at week 16, but trended back up at week 52. CONCLUSIONS: NAFLD was associated with increased cholesterol esters, PA, PG, and MUFA content of several lipid classes and decreased ceramides and SM. Disease progression was associated with decreasing PE and PC and increasing LOX activity.

Disclosures:

Riikka Katainen - Employment: Zora Biosciences Oy

Terhi Vihervaara - Employment: Zora Biosciences

Puneet Puri - Advisory Committees or Review Panels: Health Diagnostic Laboratory Inc.; Consulting: NPS Pharmaceuticals Inc.

Arun J. Sanyal - Advisory Committees or Review Panels: Gore, Gilead, Abbott, Ikaria; Consulting: Salix, Immuron, Exhalenz, Bayer-Onyx, Genentech, Norgine, GalMed, Novartis, Echosens, Takeda; Grant/Research Support: Salix, Genentech, Genfit, Intercept, Ikaria, Takeda, Gilead; Independent Contractor: UpToDate

The following people have nothing to disclose: Tommy Pacana, Hae K Min, Vaishali Patel, Faridoddin Mirshahi

678

Lipidomic insights into actions of vitamin E in a dietinduced mouse model of NAFLD

Vaishali Patel1, Tommy Pacana1, Puneet Puri1, Haeki Min1, Riikka Katainen2, Reijo Laaksonen2, Kim Ekroos2, Terhi Vihervaara2, Faridoddin Mirshahi1, Arun J. Sanyal1;

1Gastroenterology, Hepatology and Nutrition, VCU, Richmond, VA; 2Zora Biosciences, Espoo, Finland

Background: Vitamin E (VitE) has been shown to improve nonalcoholic steatohepatitis (NASH). While VitE is widely believed to work through its anti-oxidant effects, this has not been experimentally verified. Specifically, the effect of VitE on various lipid classes and their relationship to disease phenotype remain unknown. Aim: To use an unbiased lipidomic approach to evaluate the impact of VitE on hepatic lipid metabolism. Methods: 129SI/SvlmJ;B6 female mice were fed chow diet (n=5), high-fat diet (HFD) alone (n=5) or HFD with vitE (n=5) supplementation (0.1 IU/gm/day) for the last 6 weeks of a 52 week feeding regimen. At the end of the study period individual molecular species level lipidomic analysis was performed on liver tissue using mass spectrometry. Students T-test was used to assess significance (p-value <0.05). Results: HFD led to development of steatosis with inflammation and fibrosis in all mice. Compared to HFD alone, VitE administration produced a modest non-significant decrease in total fat, diacylglycerol (DAG) and cholesterol esters (CE) while increasing total phospholipid (PL).The MUFA:PUFA ratio decreased with VitE+HFD compared to HFD alone. VitE also tended to reverse HFD-associated increase in lysophospholipids. VitE decreased several ceramide (Cer) classes with the greatest effect on lactosylceramide (LacCer) and globotriaosylceramide (Gb3). Sphingomyelins(SM) increased in VitE+HFD vs HFD mice. VitE also tended to reverse HFD-associated increase in 12-hydroperoxyecosapentaenoic acid(12-HETE) while increasing PGD2 and 6keto-PGF1α, a marker of PGI2 activity. Results are summarized in the table below. Conclusions: VitE has a widespread impact on lipid metabolism in the liver. It decreases pro-apoptotic ceramides and pro-inflammatory Gb3. It decreases pro-inflammatory-while increasing anti-inflammatory-eicosanoids.

image

Disclosures:

Puneet Puri - Advisory Committees or Review Panels: Health Diagnostic Laboratory Inc.; Consulting: NPS Pharmaceuticals Inc.

Riikka Katainen - Employment: Zora Biosciences Oy Reijo Laaksonen - Employment: Zora Bioscience Terhi Vihervaara - Employment: Zora Biosciences Arun J. Sanyal - Advisory Committees or Review Panels: Gore, Gilead, Abbott, Ikaria; Consulting: Salix, Immuron, Exhalenz, Bayer-Onyx, Genentech, Norgine, GalMed, Novartis, Echosens, Takeda; Grant/Research Support: Salix, Genentech, Genfit, Intercept, Ikaria, Takeda, Gilead; Independent Contractor: UpToDate

The following people have nothing to disclose: Vaishali Patel, Tommy Pacana, Haeki Min, Kim Ekroos, Faridoddin Mirshahi

679

Coagulation as an inducible modifier of fatty liver pathogenesis: Effects of thrombin inhibition

Anna K. Kopec1, Karen M. Kassel3, Nikita Joshi2, Bradley Sullivan1, Keara Towery1, Holly M. Cline1, Matthew J. Flick4, James P. Luyendyk1;

1 Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI; 2Pharmacology/Toxicology, Michigan State University, East Lansing, MI; 3Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 4Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital, Cincinnati, OH

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes significantly to liver-related morbidity. Robust blood coagulation cascade activation, marked by thrombin generation and hepatic fibrin deposition, is a common pathological feature of obese patients with NAFLD, and in mouse models of high fat diet (HFD) challenge. Our previous studies demonstrated that the thrombin receptor protease activated receptor-1 (PAR-1) is a critical driver of HFD-induced NAFLD in mice. To discern the temporal connection between thrombin generation and the development of hepatic steatosis and inflammation, wild type C57BL/6J mice were fed a HFD (40% calories from fat) or a control diet (10% calories from fat) for 1, 2 or 3 months. Significant time-dependent increases were noted in body weight gain, liver weight, serum cholesterol levels, and hepatic lipogenic and proinflammatory gene expression. Serum alaine aminotransferase (ALT) levels increased only at 3 months in HFD-fed mice, which coincided with histological evidence of widespread macrovesicular steatosis and inflammatory cell infiltration. Of importance, an increase in plasma thrombin-antithrombin levels, an indicator of coagulation cascade activation, was temporally associated with these severe changes. To assess the impact of thrombin inhibition on the progression of NAFLD in mice on a HFD, we fed C57BL/6J mice a HFD formulated to contain 10 g/kg dabigatran etexilate (DE) or HFD without drug for 3 months. Dabigatran treatment significantly reduced hepatic fibrin deposition, steatosis, inflammation, and serum ALT activity in mice fed a HFD. Of interest, dabigatran treatment also reduced body weight gain in HFD-fed mice, consistent with recent and similar observations in tissue factor-deficient mice. Hepatic whole-genome sequencing identified novel shifts in liver metabolism directed by thrombin inhibition, suggesting thrombin targets promote NAFLD by altering expression profiles of genes associated with fatty acid metabolism and storage, de novo lipogenesis, and bile acid synthesis. Taken together, the results indicate that thrombin generation promotes the pathogenesis of HFD-induced NAFLD in mice. Defining the mechanism whereby thrombin and thrombin targets promote NAFLD pathogenesis may lead to new treatments for this disease and lead to identification of novel benefits of direct thrombin inhibitors in obese patients.

Disclosures:

The following people have nothing to disclose: Anna K. Kopec, Karen M. Kassel, Nikita Joshi, Bradley Sullivan, Keara Towery, Holly M. Cline, Matthew J. Flick, James P. Luyendyk

680

TIGAR is an insulin-regulated bisphosphatase that promotes hepatic insulin resistance and ChREBPβ-mediated steatosis in the fatty liver

Zoltan Derdak, Aryanna M. Sousa, Dante M. Votolato, Rafael Gonzalez, Jack R. Wands;

Department of Medicine, Liver Research Center, RI Hospital and the Warren Alpert Medical School of Brown University, Providence, RI

Background and aims: Tp53-Induced Glycolysis and Apoptosis Regulator (TIGAR) is a bisphosphatase that hydrolyzes fructose-2,6-bisphosphate (F26BP), and thus inhibits glycolysis, activates the pentose-phosphate shunt (PPS) and gluconeogenesis. We previously linked TIGAR to diminished insulin signaling, steatosis and hepatocellular apoptosis in rodent models of alcoholic and non-alcoholic fatty liver disease. The aim of this study was to demonstrate the causative role of TIGAR in hepatic insulin resistance and steatosis. We hypothesized that the metabolites (xylulose-5-phosphate (X5P), NADPH), which are increasingly produced in PPS as a result of TIGAR activation, may promote insulin resistance and steatosis. Methods: Human HepaRG, HepG2, Hep3B and FOCUS cells were employed in this study. TIGAR overexpression, biochemical and functional assays were carried out in low TIGAR expressing HepaRG cells. The other cell lines were used to further characterize the correlation between TIGAR and ChREBP-β (carbohydrate response element binding protein-beta) expression as well as AKT phosphorylation. Results: TIGAR overexpression in HepaRG cells was confirmed by real-time PCR, Western-blotting, immunofluorescence, F26BP, X5P and NAD(P)H/NAD(P)+ measurements. Overexpression of TIGAR significantly decreased insulin-mediated phosphorylation of AKT at the Ser473 and Thr308 sites. The mechanisms of TIGAR-induced insulin resistance involved reductive stress-mediated suppression of mTORC2 function (phos-phorylates Ser473) as well as increased X5P formation followed by the activation of PP2A (dephosphorylates Thr308), since the reductive stress inhibitor β-lapachone and PP2A inhibitor okadaic acid improved AKT phosphorylation, respectively. The activation of PP2A due to increased X5P production was also followed by the upregulation of ChREBP-β transcription factor, which had been linked to de novo fatty acid synthesis. More importantly, insulin stimulation dose-dependently decreased the abundance of both endogenous and exogenous TIGAR by promoting the proteasomal degradation of this protein, which was prevented by the proteasome inhibitor MG132. In the other investigated cell lines we found an inverse correlation between TIGAR expression and AKT phosphorylation and a positive correlation between TIGAR and ChREBP-β expression. Conclusions: The upregulation of p53 and mounting insulin resistance may increase the abundance of TIGAR by transcriptional and post-translational mechanisms in the fatty liver, which in turn enhances hepatic insulin resistance and steatosis. Therefore, targeting TIGAR may alleviate these biochemical abnormalities in fatty liver disease.

Disclosures:

The following people have nothing to disclose: Zoltan Derdak, Aryanna M. Sousa, Dante M. Votolato, Rafael Gonzalez, Jack R. Wands

681

Selective Induction of the Nrf2-Antioxidant Response Element (ARE) Pathway in Hepatocytes Decreases the Development of Non-Alcoholic Steatohepatitis and is Associated with Augmented Expression of β-Oxidation and Triglyceride Export Genes in Mice

Lung-Yi Lee1, Drew Roenneburg1, Li Zhang1, Jeffrey A. Johnson2, David Foley1;

1Surgery, University of Wisconsin School of Medicine and Public Health, Madison, WI; 2Pharmaceutical Sciences, University of Wisconsin-Madison School of Pharmacy, Madison, WI

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-ARE pathway protects cells from oxidative damage. We have demonstrated that the absence of Nrf2 exacerbates NASH progression. The purpose of this study was to determine whether hepatocyte-specific over-expression of Nrf2 mitigates the progression of NASH. Trans-genic mice with hepatocyte-specific overexpression of Nrf2 (AlbCre/caNrf2, n=16) and littermate controls (n=13) were fed either standard chow or methionine-choline-deficient (MCD) diet. After 28 days, livers and serum were obtained for analyses. Serum ALT and AST were measured using the IDEXX VetTest Chemistry Analyzer. Oil Red O staining of liver tissue was performed to evaluate for area of steatosis. Staining was quantified using ImageJ. Hepatic triglyceride was extracted using the Folch method, and concentration determined enzymatically. qRT-PCR was used to assess mRNA abundance of Nrf2-dependent and lipid metabolism genes. Comparisons between the groups were performed with one-way ANOVA followed by Fisher's LSD post hoc test. p<0.05 was used for statistical significance. Compared with standard chow, both AlbCre/caNrf2 and control mice on MCD diet had elevated serum transaminases, area of steatosis, and liver tissue triglyceride. However, among the MCD diet groups, AlbCre/caNrf2 animals had significantly decreased serum transaminases, area of steatosis, and liver tissue triglyceride as compared to the control animals. There are significant increases in the baseline mRNA abundance of Nrf2-dependent genes in AlbCre/caNrf2 mice as compared to the control mice. When fed MCD diet, Nrf2 induction did not alter the expression of genes involved in de novo fatty acid synthesis. However, compared to control mice on MCD diet, AlbCre/caNrf2 mice on MCD diet demonstrated significantly increased expression of CPT2, a critical gene required for β-oxidation of fatty acid, and MTTP, an important gene regulating the assembly of VLDL and export of hepatic triglyceride. In addition, AlbCre/caNrf2 mice on MCD diet have increased expression of genes involved in fatty acid transport (CD36, FATP1, and FATP4) and decreased expression of genes involved in intracellular fatty acid binding (FABP1 and FABP5). Selective overexpression of Nrf2 in hepatocytes decreases the development of steatosis in a dietary model of NASH. These findings are associated with increased mRNA abundance of genes involved in fatty acid transport, β-oxida-tion, and triglyceride export. The current study suggests novel Nrf2-dependent mechanisms involving lipid metabolism in NASH. Future studies are warranted to elucidate these interactions.

Disclosures:

The following people have nothing to disclose: Lung-Yi Lee, Drew Roenneburg, Li Zhang, Jeffrey A. Johnson, David Foley

682

Aroclor 1260 Exposure Worsens Hepatic And Systemic Inflammation In An Animal Model Of Diet-Induced Obesity And Nonalcoholic Fatty Liver Disease

Banrida Wahlang4, Ming Song1, Juliane I. Beier4, Laila Al-Eryani4, Heather B. Clair1,JohnJ. Guardiola1, Keith C. Falkner1, Russell A. Prough3, Matthew C. Cave1,2;

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY; 3Biochemistry, University of Louisville, Louisville, KY; 4Pharmacology, University of Louisville, Louisville, KY

Purpose: Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with nonalcoholic fatty liver disease (NAFLD) in epidemiologic studies. Our previous work demonstrated that PCB 153 worsened diet-induced obesity (DIO) and hepatic steatosis in mice fed high fat diet (HFD) (PMID: 23618531). Because highly chlorinated PCB mixtures, rather than single congeners, have bio-accumulated in humans, the purpose of this study is to evaluate the effects of Aroclor 1260 (Ar), in a mouse model of DIO and NAFLD. Methods: Male C57Bl/6 mice (8 weeks old, n=10) were fed control diet (CD, 10% kCal fat) or HFD (42% kCal fat). Ar (20 mg/kg or 200 mg/kg in corn oil) was administered by oral gavage. Serum, liver and fat samples were taken for immunohistochem-istry, RT-PCR, lipid analysis and adipocytokine measurements. Results: In mice fed HFD, Ar co-exposures were associated with increased lean mass (20 mg/kg) and decreased body fat mass (200 mg/kg). Blood glucose/lipid levels and insulin resistance were higher in HFD groups, but this was not affected by Ar exposure. Mice fed HFD developed hepatic steatosis by histology and hepatic triglyceride measurement, but the degree of steatosis was not affected by Ar co-exposures. However, Ar worsened liver necro-inflammation in mice fed HFD. AST and ALT were increased in HFD+Ar (20 mg/kg); and more inflammatory foci and hepatocellular death were observed histologi-cally in HFD+Ar animals than HFD. In contrast, hepatocyte hypertrophy and karyomegaly without steatosis or necro-inflammation were noted in CD+Ar treated mice. Expression of several pro-inflammatory hepatic TLR4 target genes, including IL-6 and TNFα, were increased in HFD+Ar (20 mg/kg). Likewise, plasma IL-6 and tPAI-1 levels were increased in HFD+Ar (20 mg/kg). Hepatic gene expression of P450s including Cyp3a11 (PXR target) and Cyp2b1 0 (CAR target) was upregulated by Ar exposure in both CD and HFD-fed mice. Interestingly, Cyp1a2 (AhR target gene) was upregulated only in groups exposed to Ar (200 mg/kg) suggesting dose-dependent activation of this receptor. Hepatic expression of PPARα and LXR target genes was not affected by Ar exposure. Conclusion: Aroclor 1260 worsened hepatic and systemic inflammation in the DIO model of NAFLD consistent with a transition from steatosis to steatohepatitis. While Aroclor 1260 did not worsen insulin resistance or hepatic steatosis, it was paradoxically associated with an increase in lean body mass at low-dose exposure. These effects may be driven by dose and congener-dependent receptor activation. Environmental pollution may be a relevant “second hit“ in the transformation of steatosis to steatohepatitis.

Disclosures:

The following people have nothing to disclose: Banrida Wahlang, Ming Song, Juliane I. Beier, Laila Al-Eryani, Heather B. Clair, John J. Guardiola, Keith C. Falkner, Russell A. Prough, Matthew C. Cave

683

Anti-Adipocyte Antibodies and Their Role in Non Alcoholic Fatty Liver Disease (NAFLD)

Azza Karrar1, Li Zheng1, Khatera Salam Malik1, Sharon L. Hunt1, Zachary D. Goodman1, Zobair M. Younossi1,2;

1Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA; 2Center for Liver Diseases, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA

Background and Aim: Previous studies have shown that 21 % of NAFLD patients have auto-antibodies without any evidence for autoimmune liver disease. Our aim was to assess the role of disease and cell-specific antibodies, namely Anti-Adipocyte Antibodies (AdAb) in patients with NAFLD and Non Alcoholic Steatohepatitis (NASH). Methods: Sera samples from patients with biopsy-proven NAFLD were reacted with cultured adipocytes followed by florescent secondary antibodies of the IgM or IgG class. Flow Cytometry was then used to detected the presence of the AdAb IgM and IgG. Cut-off values of 75 percentile of the control were used to determine positive and negative values. Statistical analysis was performed to assess associations between AdAb levels and the different variables using uni- and multivariate analysis. P-values <0.05 were considered to be potentially significant. Results: The study cohort (N = 172) included 98 patients with biopsy-proven NAFLD [Age=44.89±10.81; Female=74 (75.5%); NASH=49 (50%); Diabetes Mellitus=30 (30.6%); Hyperlipidaemia= 57 (59.4%); hypertension=57 (58.2%)] and 74 controls without liver disease [Age= 43.24+/-13.13; Female =35 (47.3%). Diabetes Mellitus=9 (12.2%); Hyperlipidaemia= 14(19.2%)]; hypertension=10 (13.7%)]. Univariate analysis showed that AdAb IgM and IgG were associated with NAFLD [AdAb IgM: Odds Ratio (OR): [2.003(1.077-3.725)] and AdAb IgG: [OR: 0.098 (0.021-0.448)]. AdAb IgM was more likely to be found in patients with histologic NASH (59.00% vs. 44.00%, P=0.0441). AdAb IgG was likely to be found in NAFLD with hyperlipidaemia and hypertension [(1.4% vs. 14.29%, P= 0.0002 and 1.49% vs. 13.46%, P= 0.0239)]; respectively. Interestingly, AdAb IgM was more likely to be present in NAFLD with fibrosis, (57.14% vs. 41.96%, P= 0.05). After adjusting for important confounders, multivariate analysis showed that increased levels of AdAb IgG was protective against NAFLD [OR: 0.191 (0.038-0.968)]. On the other hand, increased level of AdAb IgM was an independent risk factor for the presence of histologic NASH [OR: 2.530(1.007-6.356)]. Conclusions: AdAb IgM and IgG may serve as disease specific biomarkers for NAFLD. They may have diagnostic value playing a role in the pathogenesis of NAFLD and NASH.

Disclosures:

Zachary D. Goodman - Grant/Research Support: Gilead Sciences, Fibrogen, Galectin Therapeutics, Merck, Vertex

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J and J; Consulting: Gilead Sciences

The following people have nothing to disclose: Azza Karrar, Li Zheng, Khatera Salam Malik, Sharon L. Hunt

684

Loss of Liver Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1) Causes Insulin Resistance and NASH Pathogenesis in Mice

Lucia Russo1, Sumona Ghosh Lester1, Saja S. Khuder1, Terry D. Hinds1, Scott L. Friedman2, Sonia M. Najjar1;

1Physiology and Pharmacology, University of Toledo, Toledo, OH; 2Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY

Nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH), are a global health concern. Whether insulin resistance, commonly associated with obesity, is an obligate metabolic manifestation of the disease is still debated. We have demonstrated that mice with transgenic liver-specific inactivation and with global null deletion of Ceacam1 (Cc1) gene are characterized by impaired insulin clearance in liver, which causes hyperinsulinemia followed by insulin resistance, liver steatosis and visceral obesity. These mice have spontaneous liver fibrosis with a NASH-characteristic chickenwire collagen deposition pattern. Moreover, high-fat feeding provoked progression to the full spectrum of NASH in both Cc1 mutants. This prompted us to test whether liver-specific loss of CEACAM1 causes insulin resistance and NASH, both being driven by hyperinsulinemia. Clinically, this is bolstered by findings that hepatic CEACAM1 levels are markedly decreased in patients with NAFLD/NASH. Thus, we aimed to determine whether specific deletion of Ceacam1 gene (Cc1) in liver causes insulin resistance and the pathological abnormalities of NAFLD/NASH in L-Cc1fl/fl mice. Conversely, we tested whether exclusive rescuing of CEACAM1 in liver protects Cc1-/-xliver+ mice against NAFLD/NASH. L-Cc1fl/fl null mice exhibited a higher body weight beginning at 2 months of age that was associated with an increase in total fat mass and lower lean mass as compared to their littermate controls starting at 4 months of age. These mice also developed hyperinsulinemia and insulin resistance, as demonstrated by insulin intolerance testing. Conversely, liver-rescued Cc1-/-xliver+ mice (2 months old) exhibited lower body weight and total fat mass, a higher total lean mass and lower levels in almost all the metabolic parameters compared to Cc1-/- littermates. They also exhibited restored insulin sensitivity. We are currently feeding mice with a high fat diet to examine whether null mice are predisposed while rescued mice are protected against NASH-characteristic pathological abnormalities. Furthermore, lentiviral-mediated ShRNA loss of CEACAM1 in immortalized human LX2 stellate cells decreased cellular fat content with a parallel increase in its mobilization into the medium, associated with increased cell proliferation and expression of smooth muscle actin, all consistent with increased stellate cell activation. These data support a key role for CEACAM1 in attenuating hepatic steatosis and stellate cell activation. These findings uncover an important novel role for CEACAM1-dependent pathways in regulating key features of NASH.

Disclosures:

Scott L. Friedman -Advisory Committees or Review Panels: Pfizer Pharmaceutical, Sanofi-Aventis; Consulting: Abbott Laboratories, Conatus Pharm, Exalenz, Genenetch, Glaxo Smith Kline, Hoffman-La Roche, Intercept Pharma, Isis Pharmaceuticals, Melior Discovery, Nitto Denko Corp., Debio Pharm, Synageva, Gilead Pharm., Ironwood Pharma, Alnylam Pharm, Tokai Pharmaceuticals, Bristol Myers Squibb, Takeda Pharmaceuticals, Nimbus Discovery, Isis Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira Pharm, Vaccinex Therapeutics; Stock Shareholder: Angion Biomedica

The following people have nothing to disclose: Lucia Russo, Sumona Ghosh Lester, Saja S. Khuder, Terry D. Hinds, Sonia M. Najjar

685

A novel GPR120 agonist, LC540449 reduced steatosis and insulin resistance in the animal model of NAFLD

Min Kyung Yoon, Jung Gyu Park, Sang Yong Hong, Sang Dae Lee, Young Kwan Kim, Hee Dong Park;

R&D Center, LG Life Sciences, Daejeon, Republic of Korea

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease associated with metabolic syndrome. GPR120 has been suggested to be a sensor of unsaturated long chain fatty acids that have been shown to prevent NFALD. The objective of the current investigation was to examine the efficacy of LC540449, a novel small-molecule agonist of GPR120, in the animal model of NAFLD. NAFLD was induced in C57/BL6 mouse by high-fat diet (HFD) feeding for 16 weeks. After that, LC540449 was administered once a day for 7 weeks at a dose of 30 mg/kg. Treatment of LC540449 attenuated an increase in liver weight (18.8±3.1%, p < 0.05) and liver TG level (39±4.7%, p < 0.01) compared to the non-treated group despite comparable intake amount of HFD between two groups. Though plasma ALT level was in a normal range in the LC540449 treated group, it increased twofold in the non-treated group by HFD. Other metabolic parameters were also improved in the LC540449 treated group; body weight was decreased (8.8±1.6%, p < 0.05) and non-fasting glucose level was decreased throughout the treatment period. In the insulin tolerance test, the LC540449 treated group showed higher insulin response compared to the non-treated group. The basal insulin level was decreased in the LC540449 treated group which indicates improved insulin sensitivity. Furthermore, the symptoms of steatosis were ameliorated in the LC540449 treated group. In conclusion, LC540449 reversed the pathological progression of NAFLD and the relevant metabolic syndrome in the animal model, replicating the beneficial effects of long-chain free fatty acid, the natural agonist of GPR120.

Disclosures:

Min Kyung Yoon - Employment: LG Life Sciences

Jung Gyu Park - Employment: LG Life Sciences

The following people have nothing to disclose: Sang Yong Hong, Sang Dae Lee, Young Kwan Kim, Hee Dong Park

686

Osteopontin enhances liver progenitor cell responses in progressive Nonalcoholic Steatohepatitis

Jason Coombes1, Lee C. Claridge2, Marzena Swiderska-Syn3, Marco A. Briones-Orta1, Rasha Younis1, Husnain Shah1, Salvatore Papa1, Anna Mae Diehl3, Bertus Eksteen4, Ali Canbay5, Wing-Kin Syn1;

1Regeneration and Repair, The Institute of Hepatology, London, United Kingdom; 2Hepatology, Leeds Hospital NHS Trust, Leeds, United Kingdom; 3Medicine and Gastroenterology, Duke University, Durham, NC; 4Medicine and Gastroenterology, University of Calgary, Calgary, AB, Canada; 5Gastroenterology and Hepatology, University Hospital Essen, Essen, Germany

Background/Aim: Chronic liver disease progression is associated with a robust ductular reaction / liver progenitor cell (LPC) response. Previously, we showed that Hedgehog (Hh), a morphogen responsible for tissue construction during fetal development, promotes LPC expansion and reprogramming (epithelial-mesenchymal transition, EMT) during progressive nonalcoholic steatohepatitis (NASH). We also reported that Osteopontin (OPN), a matrix protein and cytokine, is upregulated in mice and human with NASH, is Hh-regulated, and is a proximal effector of the Hh pathway. On the basis of these findings, we hypothesized that OPN overexpression contributes to LPC response in NASH. Methods: Human tissue was obtained from explanted NASH and healthy donor livers. C57BL/6 mice were fed control chow or methionine-choline deficient (MCD) diet for 5 weeks, and treated with sham or OPN-neutralizing aptamers. Liver tissues from human and mice were assessed by Sirius Red (SR) staining, OPN and Sox9 immunohistochemistry, and qRT-PCR for collagen, aSMA and TGFβ mRNA. In vitro, 603B immature murine cholangiocytes (which are Sox9+ LPC) were treated with recombinant (r)OPN ligands (0-500ng/ml) or TGFβ (5ng/ml); effect of OPN neutralization was assessed under TGFβ-stimulated conditions. Cell responses to OPN neutralization were assessed by wound healing assay, western blot (for pSmads, MEK, ERK1/2) and qRT-PCR (for EMT changes). Results: MCD-fed mice developed NASH-fibrosis, upregulated whole liver OPN mRNA and protein, and show accumulation of Sox9+ cells. A similar induction of OPN and Sox9 (mRNA and cells) occurred in human NASH. Conversely, livers from MCD-fed mice that received OPN-neutralizing aptamers exhibit reduced levels of fibrogenic genes (TGFβ and αSMA), less fibrosis (collagen mRNA and SR staining) and fewer Sox9+ cells and mRNA (all p<0.05 vs. sham-treated MCD-fed mice). In vitro, TGFβ-treated 603B undergo EMT changes by upregulating mesenchymal genes (Snail, Vimentin), while repressing epithelial markers (E-Cadherin, BMP7). OPN neutralization under TGFβ-stimulated conditions attenuated EMT changes, and inhibited cell migration in the wound healing assay. OPN effects were mediated, in part, by modulating the MEK-ERK and TGFβ pathway. Conclusions: OPN is overexpressed during NASH and enhances LPC EMT. OPN neutralization abrogates the LPC response, and leads to an attenuated fibrogenic outcome. OPN neutralization is a promising anti-fibrotic strategy in NASH

Disclosures:

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

The following people have nothing to disclose: Jason Coombes, Lee C. Claridge, Marzena Swiderska-Syn, Marco A. Briones-Orta, Rasha Younis, Husnain Shah, Salvatore Papa, Bertus Eksteen, Ali Canbay, Wing-Kin Syn

687

miRNAs are Differentially Expressed in Skeletal Muscle from Non-Alcoholic Fatty Liver Disease Patients and Targeted by Tauroursodeoxycholic Acid in vitro

Duarte M. Ferreira1, Pedro M. Borralho1,2, Mariana V. Machado3, Helena Cortez-Pinto3, Cecília M. Rodrigues1,2, Rui E. Castro1,2;

1 Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Lisboa, Portugal; 2Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisboa, Portugal; 3 Institute of Molecular Medicine (IMM), Faculty of Medicine, University of Lisbon, Lisboa, Portugal

microRNAs (miRNAs or miRs) are being increasingly associated with the pathogenesis of human liver diseases. In particular, human non-alcoholic fatty liver disease (NAFLD) severity is associated with activation of the miR-34a pro-apoptotic pathway, targeted by ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids in primary rat hepatocytes. In addition, we have demonstrated that intramyocellular lipids are associated with muscle insulin resistance (IR) and NAFLD in morbid obese patients. Recent studies suggest that miRNAs may also regulate IR in skeletal muscle. The aim of this study was to profile miRNA expression in the skeletal muscle of patients at different NAFLD stages, trying to correlate them with IR, and ascertain the potential therapeutic use of TUDCA in such settings. Muscle and matching liver biopsies were obtained from morbid obese patients undergoing bariatric surgery and classified as simple steatosis, less severe and more severe non-alcoholic steatohepatitis (NASH). Muscle RNA was run in TaqMan MicroRNA arrays. C2C12 cells were incubated with or without 200 and 400 microM of palmitic acid (PA), in the presence or absence of TUDCA. The insulin-signalling pathway was evaluated by Western blot. Our results show a progressive and significant increase in the expression of 45 muscle miRNAs from steatosis to more severe NASH (p < 0.05), including muscle specific miRNAs miR-133a and b, as well as miR-34a, miR-10b, miR-29a, let-7a, among others. C2C12 cells exposed to PA also displayed increased miR-34a, let-7a, and miR-133a expressions (p < 0.05), as well as apoptosis (p < 0.05). PA additionally induced IR in C2C12 cells, as evidenced by the inhibition of the insulin signalling pathway (p < 0.05), suggesting that miR-34a and let-7a might mediate free fatty acid-induced cytotoxicity and IR in the muscle. Finally, incubation of C2C12 cells with TUDCA reduced miR-34a induction, IR and apoptosis (p < 0.05). Altogether, our results indicate that miRNAs are differently modulated with NAFLD severity in the muscle, correlating with changes in liver. In particular, miR-34a is increased in the muscle of NAFLD patients and targeted by TUDCA in vitro, which further inhibits evidence of IR and apoptosis. A better understanding of the overlapping roles of miRNAs in different tissues during the metabolic syndrome, and suitable targeting options, may help in establishing new therapeutic options (Supported by PTDC/SAU- OSM/102099/2008, PTDC/SAU-OSM/100878/2008, PTDC/SAU-ORG/111930/2009, Pest- OE/SAU/UI4013/2011 and SFRH/BD/60521/2009).

Disclosures:

Helena Cortez-Pinto - Advisory Committees or Review Panels: Norgine, Lund-beck; Speaking and Teaching: Janssen

The following people have nothing to disclose: Duarte M. Ferreira, Pedro M. Borralho, Mariana V. Machado, Cecília M. Rodrigues, Rui E. Castro

688

High fat diet decreases activity of the mitochondrial respiratory chain in mice

Jose A. Solís-Herruzo, Pablo Solís-Muñoz, Daniel Fernández-Mor-eira, Teresa Muñoz-Yagüe, Inmaculada García-Ruiz;

Research Institute, University Hospital “12 de Octubre“, Madrid, Spain

In previous studies, we have reported that mitochondrial respiratory chain (MRC) activity is decreased in the liver of patients with non-alcoholic steatohepatitis (Hepatology 2003; 38:999-1007) and in ob/ob mice (J Prot Res 2010;9:2450-2459). The aims of this study were to determine whether (1) feeding C57BL/6J mice with a high-fat diet (HFD) induces any change in the MRC activity and (2) nitro-oxidative stress plays any role in the pathogenesis of these changes. Material and Methods. Thirty mice were distributed in five groups: (1) Six mice fed a standard chow diet. (2) Six mice on HFD (Harlan Lab., Madison, WI) treated with 100 μL 0.8% saline solution i.p. for 28 weeks. (3) Six HFD mice treated with 10 mg/Kg/day melatonin, i.p. for 28 weeks. (4) Six HFD mice treated with 10 mg/Kg/day MnTBAP (a superoxide dismutase analog). (5) Six HFD mice receiving 20 mg/day uric acid. In the liver of these mice, we studied liver histology, MRC activity (spectrophotometry), fully assembled MRC complexes (BN-PAGE), subunits of the MRC complexes (BN/SDS-PAGE), gene expression of these subunits, as well as of TNFα, IFNγ, MCP-1, Caspase-1, Collagen α1 (I) (RT-PCR), oxidative (thiobarbituric acid-reactive substances, glutathione, aldehyde-protein adducts) and nitrosative (immunohistochemistry, nitration of MRC proteins; iNOS) stress, and oxidative DNA damage [8-hydroxy-2'-deoxyguanosine (8-OHdG)]. Results: In HFD mice, we found: (1) severe steatosis(almost 100% of hepatocytes), mild inflammatory infiltrates, ballooning and eosinophilic degeneration, pericellular fibrosis, 3-tyrosine nitrated proteins, and marked increased gene expression of TNFα, IFNγ, MCP-1, caspase-3, and collagen α1 (I). (2) Activity of all complexes of the MRC was decreased to about 50-60% of control activity. (3) Fully assembled complexes were reduced to 50% to 60% of that found in control mice. (4) The amount of all studied subunits was markedly decreased, particularly, the mitochondrial DNA-encoded subunits. (5) Gene expression of mitochondrial, but not of genomic, DNA-encoded subunits was decreased to about 60% of control gene expression. (6) 8-OHdG was increased in mitochondrial, but not in genomic DNA. (7) Treatment of HFD mice with melatonin, MnTBAP or uric acid for 28 weeks prevented all changes observed in untreated HFD mice. Conclusion: HFD decreases MRC enzymatic activity, which can be ascribed to a decreased amount of fully-assembled complexes caused by a reduced synthesis of its subunits. Antioxidants and antiperoxynitrite prevents all these changes suggesting that nitro-oxidative stress plays a key role in the pathogenesis of these alterations.

Disclosures:

The following people have nothing to disclose: Jose A. Solís-Herruzo, Pablo Solís-Muñoz, Daniel Fernández-Moreira, Teresa Muñoz-Yagüe, Inmaculada García-Ruiz

689

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) deficient mice protect against apoptotic hepatocyte cell death and permits ER-stress related adaptation via GRP78 (BiP) expression

Pradeep Kumar, Khalidur Rahman, Tekla Smith, Frank A. Anania;

Department of Medicine, Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA

BACKGROUND: Injury from non-alcoholic steatohepatitis (NASH) is becoming one of the most common etiologies of chronic liver disease. A key feature in the pathogenesis of fatty liver diseases is the accumulation of hepatocyte free fatty acids (FFA) which may invoke lipotoxicity and hepatocyte apoptosis. Excess accumulation of FFA is central to endoplasmic reticulum stress (ER), which if unabated leads to CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) mediated hepatocytes apoptosis. AIM: To investigate the role of CHOP-related apoptosis in a diet-induced model of NASH. METHODS: Wild type (WT) male C57BL/6J and CHOP deficient mice (CHOP-/-) were pair fed or fed ad libitum either standard chow (CD) or a diet high in fat, high fructose corn syrup and cholesterol (HFHC). During the feeding period, and at 8 weeks, metabolic parameters (e.g. glucose tolerance) were measured; and serum transaminases values, triglycerides and cholesterol were measured. At necropsy liver sections were used for immunohistochemical, immunofluorescence and TUNEL assay. Liver total RNA and protein were harvested for qRT-PCR and Western blot analysis respectively. RESULTS: There was no difference in caloric intake between the WT and CHOP-/- mice, and both cohorts gained identical weight. There was no significant difference with the parameters related to key metabolic parameters or parameters related to fatty liver. CHOP-/- mice exhibited significantly lower apoptotic bodies when assessed by TUNEL and immunofluorescence compared to WT mice at both 4 and 8 weeks of HFHC diet. GRP78 (BiP) mRNA and protein were expressed in both WT and CHOP-/- but expression in the CHOP-/- mice was significantly higher (p<0.05). Despite identical histology, activation of JNK was significantly increased in the WT mice fed the HFHC diet compared to the CHOP-/- mice, as was cleaved caspase 3. Conversely Bcl-2 sur-vival proteins were suppressed and the X-box protein XBP1S was significantly increased. CONCLUSION: While the HFHC diet induced similar metabolic and histological changes in either the WT or CHOP-/- mice associated with the metabolic syndrome and NASH, the CHOP-/- deficient mediated-ER stress attenuates apoptosis. Further, these data also suggest that GRP78 may serve as a key player in the adaptive role of the ER stress response.

Disclosures:

The following people have nothing to disclose: Pradeep Kumar, Khalidur Rahman, Tekla Smith, Frank A. Anania

690

The crosstalk between apoptosis and autophagy in the pathogenesis of non-alcoholic fatty liver disease

Satoshi Tanaka, Hayato Hikita, Tasuku Nakabori, Yoshinobu Saito, Satoshi Shimizu, Wei Li, Ryotaro Sakamori, Takuya Miyagi, Naoki Hiramatsu, Tomohide Tatsumi, Tetsuo Takehara;

Gastroenterology & Hepatology, Osaka University, Suita, Japan

Background and Aim: Free fatty acids play a critical roles in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis. Recent researches have shown that apoptosis is a characteristic feature of hepatocytes in NAFLD. Meanwhile, a complex relationship is reported between apoptosis and autophagy in several disease models but not well established in NAFLD. In this study, we investigated the interplay between apoptosis and autophagy in NAFLD. Methods: Murine non-transformed hepatocytes, CL2 cells, were cultured with saturated palmitic acid (PA). For in vivo studies, male C57BL/6J mice or Mx1-Cre mediated Atg7 knockout mice (Mx1-Cre Atg7fl/fl), were fed high fat diet (HFD). Results: PA increased caspase 3/7 activity and induced apoptosis in CL2 cells with JNK activation in a dose dependent manner. The expression levels of LC3-II and P62 were increased by PA, suggesting that PA inhibits the autophagic process after autophago-some formation. The inhibition of autophagy by PA was observed earlier (4h) than PA-induced apoptosis (8h). To examine the effect of PA's autophagy inhibition on apoptosis, PA-treated CL2 cells were cultured with chloroquine, a lysosomal inhibitor, or transfected with siRNA against Rubicon, a negative regulator of autophagosome maturation. As expected, autophagic flux in PA-treated CL2 cells was further suppressed by chloroquine, and was promoted by siRNA mediated knockdown of Rubicon. Interestingly, while autophagy inhibition by chloroquine exacerbated PA-induced apoptosis, autophagy promotion by Rubicon knockdown ameliorated PA-induced apoptosis with a decrease of JNK activation. Consistent with in vitro findings, mice on HFD for 3 months or more showed increased hepatocyte apoptosis compared with mice on control diet, evidenced by serum ALT, caspase 3/7 levels and TUNEL staining of liver tissues. The expression levels of LC3-II and P62 were increased in murine livers on HFD for 2 months or more. Electron microscopy analysis revealed increased autophagosomes but few autolysosomes in their livers. Although there was no difference in serum ALT levels and the number of TUNEL positive hepatocytes between mice on HFD for 1 month and those on control diet, polyI:C-induced knockout of Atg7, which impaired autophagy in their livers, significantly increased ALT levels and TUNEL positive hepatocytes in Mx1-Cre Atg7 fl/fl mice on HFD for 1 month compared with those on control diet. Conclusion: HFD, as well as PA, suppresses autophagic flux in hepatocyte, which promotes hepatocyte apoptosis. Enhancement of autophagy may provide new approaches for treatment of NAFLD.

Disclosures:

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.

The following people have nothing to disclose: Satoshi Tanaka, Hayato Hikita, Tasuku Nakabori, Yoshinobu Saito, Satoshi Shimizu, Wei Li, Ryotaro Sakamori, Takuya Miyagi, Naoki Hiramatsu, Tomohide Tatsumi

691

Highly Active Anti-Retroviral Therapy (Haart) Induced Hepatic Steatosis And Injury Is Markedly Enhanced By Ethanol

Hridgandh Donde1, Smita Ghare1, Jingwen Zhang1, Irina Kirpich1, Swati Joshi-Barve1, Leila Gobejishvili1, Craig J. McClain1,2, Shirish Barve1;

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex VA Medical Center, Louisville, KY

Background: Use of Highly Active Antiretroviral Therapy (HAART) has led to a significant increase in the life expectancy of HIV patients; however, there can be significant associated side effects including lipodystrophy and hepatotoxicity. Alcohol abuse is highly prevalent in HIV infected individuals and hence may be a significant negative modifying factor in HAART induced hepatotoxicity. The present study examines the mechanisms underlying HAART and alcohol induced hepatic steatosis and injury. Methods: The effects of HAART drugs [azidothymidine (AZT), Indinavir sulphate (IDV)] in combination with alcohol were examined both in vitro and in vivo. In vivo alcohol and HAART drug interactions and hepatotoxicity were assessed in an animal model (C57BL/6) of chronic alcohol feeding using Lieber DeCarli liquid diet for 5 weeks. HAART treatment groups received AZT (30mg/kg BW) and IDV (50mg/kg BW) by oral gavage for 5d/week for 5 weeks. In vitro studies were carried out by employing the H4IIE-rat hepatoma cell line. Results: In comparison to individual alcohol and HAART (AZT+IDV) treatment, animals exposed to alcohol and HAART combination showed increasing visceral adiposity and systemic endotoxemia which suggests altered intestinal permeability. Moreover, animals treated with alcohol and HAART combination developed increased micro- and macro-vesicular liver steatosis as evaluated by histological examination; significantly increased fat accumulation was demonstrated by Oil-Red-O staining and confirmed by biochemical assessment of hepatic triglyceride accumulation. Also, the animals receiving the combination treatment exhibited increased inflammation and greater hepatic neutrophil infiltration In vitro studies demonstrated that in comparison to alcohol and AZT alone their combination was able to significantly suppress Cpt1a gene expression, a critical rate-determining regulator of fatty acid β-oxidation in hepatocytes. Moreover, autophagy related genes Atg 5, 7 and 14 that are known to regulate lipid metabolism were also suppressed to a greater extent by the combined alcohol and AZT treatment. In relation to hepatotoxicity, the combination of AZT and alcohol further exacerbate the decreased in cell viability in H4IIE cells. Conclusion: Overall, these studies show that alcohol can be a significant pathogenic co-factor in HAART hepatotoxicity by deregulating hepatic autophagic and β-oxidation mechanisms that are relevant to lipid metabolism, leading to hepatic steatosis and injury.

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: Hridgandh Donde, Smita Ghare, Jingwen Zhang, Irina Kirpich, Swati Joshi-Barve, Leila Gobejishvili

692

CYP2E1-dependent and Leptin-induced CD57-positive cytotoxic T cells are crucial for progression of nonalcoholic steatohepatitis

Ratanesh K. Seth1, Suvarthi Das1, Ashutosh Kumar2, Anindya Chanda1, Maria B. Kadiiska2, Gregory A. Michelotti3, Anna Mae Diehl3, Saurabh Chatterjee1;

1ENVIRONMENTAL HEALTH SCIENCES, UNIVERSITY OF SOUTH CAROLINA, COLUMBIA, SC; 2Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC; 3Division of Gastroenterology, Duke University, Durham, NC

The progression of nonalcoholic steatohepatitis (NASH), a 'silent' liver disease, in steatotic liver is believed to require second hit. Metabolic oxidative stress following CYP2E1 activation and ensuing tissue damage and cytokine release can serve as the second or multiple hits. However, molecular mechanisms linking to CYP2E1 induced-metabolic oxidative stress, adipocytokine leptin and liver lymphocytes have remained unclear. The present study explored the role of CD57+ T lymphocytes in aiding inflammatory pathophysiology in NASH. Two mouse models of NASH were used in the study. Diet induced obese (DIO) mice, exposed to a chronic dose of CYP2E1 activator bromodichloromethane (BDCM) and mice fed with methyl choline deficient diets (MCD) were used as rodent models of NASH. Spontaneous knockout mice for CYP2E1, Leptin gene, and double knockout mice for Pfp/Rag2 genes were used to estimate their role in metabolic oxidative stress-induced T-cell activation and development of full blown NASH. Metabolic oxidative stress, inflammatory response and fibrosis were evaluated by gene expression, western blot, immunohistochemistry, confocal microscopy and histopathology. We found that the levels of lipid peroxidation and tyrosine nitration were increased following reductive metabolism of CYP2E1. They also show elevated hepatic leptin level. Metabolic oxidative stress and increased leptin levels caused CD57+ T-cell proliferation. There was a significant increase in the levels of T-cell mediated proinflammatory cytokines such as IL-2, IL-β, IFN-γ, TNF-α in both BDCM exposed DIO mice and MCD diet-fed mice but not in mice that lacked T and B cells. Similarly there was a significant increase in NASH progression markers α-SMA, TGF- β and Collagen 1-α-1, and was dependent on gene knockout mice that lacked T cells and B cells. NASH indicators, CD57 positive cytoxic T cell proliferation but not CD57/CD4 positive cells were significantly decreased in mice lacking CYP2E1 and/or leptin. Finally the CYP2E1, Leptin and Pfp/Rag2 (mice lacking both T and B cells) gene deleted mice were protected from NASH. The results described above suggest that higher levels of CYP2E1 and leptin mediated CD57 expressing cytotoxic T cells play a key role in the development of NASH and provide a novel insight of immune dysregulation in NASH.

Disclosures:

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

The following people have nothing to disclose: Ratanesh K. Seth, Suvarthi Das, Ashutosh Kumar, Anindya Chanda, Maria B. Kadiiska, Gregory A. Michelotti, Saurabh Chatterjee

693

Purinergic Receptor X7 is a Key Modulator of Metabolic Oxidative Stress - Mediated Autophagy and Inflammation in Experimental Nonalcoholic Steatohepatitis

Suvarthi Das1, Ratanesh K. Seth1, Ashutosh Kumar2, Maria B. Kadiiska2, Gregory A. Michelotti3, Anna Mae Diehl3, Saurabh Chatterjee1;

1Environmental Health Sciences, University of South Carolina, Columbia, SC; 2Free Radical Metabolism Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC; 3Division of Gastroenterology, Duke University, Durham, NC

Recent studies indicate that metabolic oxidative stress, autophagy and inflammation are hallmarks of nonalcoholic steatohepatitis (NASH) progression. However the molecular mechanisms that link these important events in NASH remain unclear. In this study we investigated the mechanistic role of purinergic receptor X7 (P2X7) in modulating autophagy and resultant inflammation in NASH in response to metabolic oxidative stress. We used two rodent models of NASH. In one of them, we used a CYP2E1 substrate bromodichloromethane to induce metabolic oxidative stress and NASH. For the second NASH model, methyl choline deficient diet feeding was used. CYP2E1 and P2X7 receptor gene deleted mice were used to establish their roles in regulating metabolic oxidative stress and autophagy. We studied the expression level changes of autophagy genes and corresponding proteins. Additionally, we studied confocal microscopy based immunolocalization of chaperone-mediated autophagy (CMA) marker LAMP2A, and histopathological slides. CYP2E1-dependent metabolic oxidative stress resulted in increase of protein levels of P2X7 receptor, CMA-markers: LAMP2A and HSC 70; and caused depletion of early autophagy marker protein: LC3B. P2X7 receptor gene deletion significantly decreased expressions of LAMP2A and inflammatory indicators while significantly increasing LC3B protein levels as compared to wild type mice. P2X7 receptor gene-deleted mice were also protected from NASH pathology as evidenced by decreased inflammation and fibrosis. Our study establishes for the first time that P2X7 receptor is a key regulator of autophagy induced by metabolic oxidative stress in NASH, and it modulates hepatic inflammation. We demonstrate that P2X7 receptor augments lysosomal fusion of the CMA-markers: LAMP2A and HSC 70 and depletes the level of early autophagy marker: LC3B. Further, our findings presented here, form a basis for establishing P2X7 receptor as a potential therapeutic target in the treatment for NASH.

Disclosures:

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

The following people have nothing to disclose: Suvarthi Das, Ratanesh K. Seth, Ashutosh Kumar, Maria B. Kadiiska, Gregory A. Michelotti, Saurabh Chatterjee

694

Nonalcoholic steatohepatitis is associated with decreased hepatic ABCA1 protein levels

Joel Vega-Badillo1, Roxana Gutierrez-Vidal1, Hugo Villamil-Ramirez1, Paola Leon-Mimila1, Hugo A. Hernández-Pérez1, Francisco J. Campos-Pérez4, Rogelio Hernández-Pando2, Carlos A. Aguilar-Salinas3, Samuel Canizales-Quinteros1;

1Unidad de Genómica de Poblaciones Aplicada a la Salud, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Instituto Nacional de Medicina Genómica, Mexico City, Mexico; 2Departamento de Patología, Instituto Nacional de Ciencias Médi-cas y Nutrición Salvador Zubirán (INCMNSZ), Mexico City, Mexico; 3Departamento de Endocrinología y Metabolismo, INCMNSZ, Mexico City, Mexico; 4Clínica Integral de Cirugía para la Obesi-dad y Enfermedades Metabólicas, Hospital General Dr. Rubén Leñero, Mexico City, Mexico

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease with a high incidence in Hispanic populations. The clinical-histologic spectrum of NAFLD extends from a nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). NASH is distinguished from NAFL by the presence of cytologic ballooning and lobular inflammation, with a greater risk of developing complications such as cirrhosis and hepatocellular carcinoma. Several recent lipidomic NAFLD analyses have demonstrated the presence of free cholesterol (FC) hepatic accumulation in both NAFL and NASH, and several studies have analyzed gene and protein expression involved in hepatic cholesterol homeostasis. However, although ABCA1 is known to have a crucial role in cholesterol homeostasis, its role in human NAFLD is not fully understood. AIM: The aim of this study was to determine if ABCA1 expression is associated with NAFLD in subjects with morbid obesity. METHODS: Three groups of subjects were studied: Twelve morbidly obese subjects with normal liver histology, twenty NAFL cases without inflammation, and forty-one NASH cases. Total lipids were extracted from liver biopsies, and triglyceride (TG), total cholesterol (TC), free cholesterol (FC) and phospholipid (PL) contents were measured by colorimetric assays. ABCA1 protein levels were measured in liver extracts by Western blotting and hepatic mRNA levels were determined by quantitative real-time RT-PCR analyses. RESULTS: Hepatic TG content was significantly increased in subjects with NAFL and NASH (1.72±1.5 mgTG/mgPT and 3.37±1.88, respectively) as compared to obese controls without steatosis (0.58±0.44; p<0.002). Similarly, hepatic TC and FC contents were higher in NASH (0.146±0.096 mgTC/mgPT and 0.108±0.075 mgFC/mgPT respectively) than in not NASH (0.102±0.047 and 0.080±0.312; P=0.017and P=0.045, respectively). Interestingly, although ABCA1 mRNA levels were not significantly different in subjects with NASH (0.29±0.06) and controls (0.33±0.11; P=0.182); there was a significant inverse association of ABCA1 protein levels with the severity of hepatic steatosis (P=0.035) and with lobular inflammation (P=0.016). The NAFLD activity score (NAS) was also significantly related to ABCA1 protein levels (r=-0.274, P=0.007). Furthermore, NASH biopsies showed a 2-3 fold decrease of ABCA1 protein levels (0.343±0.295) as compared to obese controls (0.713±0.486; P=0.002). CONCLUSIONS: To our knowledge, this is the first study in humans liver biopsies showing decreased ABCA1 protein levels associated with fatty liver progression. These findings suggest that ABCA1-mediated cholesterol efflux has a role in NAFLD.

Disclosures:

The following people have nothing to disclose: Joel Vega-Badillo, Roxana Gutierrez-Vidal, Hugo Villamil-Ramirez, Paola Leon-Mimila, Hugo A. Hernández-Pérez, Francisco J. Campos-Pérez, Rogelio Hernández-Pando, Carlos A. Aguilar-Salinas, Samuel Canizales-Quinteros

695

A novel Sirt1 agonist (MC2799) inhibits both lipids and ROS accumulation and reverts oleate-dependent activation of lipid metabolism and inflammatory genes in an in vitro model of steatosis in differentiated HepaRG cells

Natalia Pediconi1,2, Silvia Di Cocco1,3, Silvia Piconese1, Sergio Valente4, Dante Rotili4, Vincenzo Barnaba1, Antonello Mai4, Massimo Levrero1,2;

1Dept. Internal Medicine (DMISM), Sapienza University of Rome, Rome, Italy; 2Life Nanosciences Laboratory, Sapienza University of Rome, Rome, Italy; 3EAL Inserm U785, Sapienza University of Rome, Rome, Italy; 4Dip. di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy

Background and aim. Excessive accumulation of triglyceride-containing lipid droplets (LDs) within hepatocytes (steatosis) is a potentially reversible process leading, in patients, to NAFLD, that may evolve into a steato-hepatitis (NASH), and eventually into cirrhosis and hepatocellular carcinoma (HCC). The NAD+ -dependent deacetylase SIRT1 controls metabolic processes in response to restriction calories and administration of the synthetic SRT1720 activator has been shown to protect from diet-induced obesity and its negative consequences on glucose homeostasis by primarily promoting fat consumption in liver, skeletal muscle and adipose tissue in mice. Recent data have also shown that lipid loaded HCC cells up-regulate inflammatory pathways and genes. Here, we evaluated the effects of a specific Sirt1 agonist (MC2791) in a cellular model of steatosis in a non-transformed well-differentiated human hepatic cell line. Methods. DMSO-differentiated human HepaRG cells (dHep-aRG) were treated with mono-unsaturated oleic acid for 2-4 days. The expression of liver-specific markers (Aldolase B, cytochromeP450-CYP2E1, albumin and PPARG) was used to monitor HepaRG cells differentiation. Neutral lipids accumulation in cytoplasmic droplets and intracellular reactive oxygen species (ROS) generation were quantitated by flow cytometry analisys using a FACS-Canto (Becton-Dickinson) using the neutral lipid marker BODIPY 505/515 and with the non-fluorescent probe H2DCFDA, respectively. Epigenetic histone marks changes were monitored by chromatin immunoprecipitation (ChIP) assays. Results. Oleic acid-treated (4 days) dHepaRG cells showed a significantly higher BODIPY and H2DCFDA fluorescent signals as compared to untreated dHepaRG cells. Lipid accumulation in small to medium size lipid droplets and oxidative stress were paralleled by a transcriptional deregulation of lipid metabolism (i.e. LSS, PLIN4), ISGs (i.e. USP18, OAS1, ISG15) and NF-KB controlled (i.e. IL8) genes. Treatment of dHepaRG cells with the Sirt1 agonist MC2791 alone didn't affect liver-specific markers expression or basal TG levels in dHepaRG cells. The co-treatment (4 days) with oleic acid together with MC2791 strongly inhibited both lipids and ROS accumulation and reverted oleate-dependent activation of lipid metabolism and inflammatory genes. MC2791 transcriptional effects expression were accompanied by specific histone modifications changes at the promoter level of target genes. Conclusions. Pharmacological modulation of hSirt1 has positive effects, mediated by epigenetic changes, on both lipid and reactive oxygen species accumulation in response to excess lipid exposure.

Disclosures:

Massimo Levrero - Advisory Committees or Review Panels: BMS, Jansen, Gilead; Speaking and Teaching: MSD, Roche

The following people have nothing to disclose: Natalia Pediconi, Silvia Di Cocco, Silvia Piconese, Sergio Valente, Dante Rotili, Vincenzo Barnaba, Antonello Mai

696

Characterization of microRNA-21 and Toll-like receptor 4 in Alcoholic Liver Diseases

Phillip Levine1, Kelly McDaniel1, Yuyan Han1, Heather L. Francis1,2, Shannon S. Glaser1,2, Julie Venter1, Taylor Francis1, Chang-Gong Liu3, Hidekazu Tsukamoto4, Gianfranco Alpini1,2, Fanyin Meng1,2;

1Digestive Disease Research Center, Scott & White Hospital, Texas A&M HSC College of Medicine, Temple, TX; 2Research, Central Texas Veterans Health Care System, Temple, TX; 3Experimental Therapeutics, Division of Cancer Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX; 4Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Background: microRNAs are small non-coding RNAs which regulate hepatic cell proliferation and tissue repair through its ability to switch off specific target genes. New evidence indicates miR-21 as a regulatory master-switch during liver injuries. The current study aimed to characterize the functional role of miR-21 and its upstream modulators during alcoholic-induced hepatitis using human hepatic cell culture and transgenic animal models. Methods: miRNA expression was assessed using a hybridization based microarray and Taqman miRNA real-time PCR analysis in ethanol- and LPS-treated normal human hepa-tocytes (N-Heps) and human hepatic stellate cells (HHSCs), as well as in liver specimen from mice fed alcohol intragastrically for 4 weeks relative to control liver tissue. Cellular apoptosis/proliferation was measured by MTS assay, and real-time PCR analysis through specific markers such as active caspase 3. The upstream modulators of miR-21 were defined in TLR-4 knockout mice in vivo. Results: We identified that 4 wk of ethanol feeding significantly increased total liver histopathology score and hepatocellular apoptosis. The up-regulation of miR-21 (3.72±0.43-fold, p=0.008), the important miRNA for liver pathology, was further verified by Taqman real-time PCR assays. Treatment of N-Heps and HHSCs with ethanol (86 mM) and LPS (20 ng/ml) for 72 hr significantly increased miR-21 as well as active caspase 3, the key marker for apoptosis. Over-expression of miR-21 decreased ethanol and LPS-induced apoptosis in both N-Heps and HHSCs, whereas up-regulation of miR-122 only increased the survival in N-Hep cells. Interestingly, transfection of miR-21 precursors increased the pro-fibrotic markers, α-SMA, MMP-2 and MMP-9 in both N-Hep and HHSC cells, whereas silencing of LPS receptor TLR4 decreased these markers in the same group of hepatic cell lines, suggesting LPS-mediated miR-21 expression during alcohol-induced liver fibrosis. Furthermore, the expression of TLR4 and the verified target proteins of miR-21, including RECK1, PDCD4, PTEN, IGF-1 R and TIMP3, were significantly altered in ethanol-fed mouse liver specimens compared to controls. TLR4 knockout mice displayed less sensitivity to alcoholic injury, along with reduced miR-21 levels and recovered expressions of RECK1, PDCD4 and IGF-1 R. Summary and Conclusion: Our results show that miR-21 is a critical regulator of hepatic cellular apoptosis and alcoholic hepatitis. The findings provide new insight into the function of LPS regulated miR-21 and increase opportunities for the development of novel treatment paradigms for the management of alcoholic liver diseases.

Disclosures:

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

The following people have nothing to disclose: Phillip Levine, Kelly McDaniel, Yuyan Han, Heather L. Francis, Shannon S. Glaser, Julie Venter, Taylor Francis, Chang-Gong Liu, Gianfranco Alpini, Fanyin Meng

697

Liver selective acetyl-CoA carboxylase inhibition by ND-654 and related analogs inhibits hepatic fatty acid synthesis, stimulates hepatic fatty acid oxidation, reduces hepatic steatosis, and modulates dyslipidemia in diet-induced obese rats

Geraldine Harriman1, Jeremy R. Greenwood2, Sathesh P. Bhat2, Liang Tong3, Debamita Paul3, Ruiying Wang3, Rosana Kapeller1, H James Harwood1;

1Nimbus Discovery, Cambridge, MA; 2Schrodinger, New York, NY; 3Columbia University, New York, NY

Simultaneous inhibition of the acetyl-CoA carboxylase isozymes ACC1 and ACC2 in the liver results in concomitant inhibition of hepatic fatty acid synthesis (FASyn) and stimulation of hepatic fatty acid oxidation (FAOxn), reduces hepatic steatosis, and may favorably affect fatty liver disease. Our efforts to discover hepatoselective ACC inhibitors have focused on interaction with the subunit dimerization site on the biotin carboxylase domain of the enzyme to which the phosphopeptide of AMPactivated protein kinase-phosphorylated ACC binds to prevent dimerization and to which the fungal metabolite Soraphen A interacts. Using state-of-the-art structure-based drug design and crystal structures of the human ACC2 biotin carboxylase domain, we have identified a unique series of hepatoselective allosteric inhibitors that bind to the dimerization site, inhibit the enzymatic activity of both human ACC1 and ACC2, reduce FASyn and stimulate FAOxn in cultured cells, and exhibit acute and chronic in vivo efficacy. For example, ND-654 inhibits human ACC1(IC50 = 3 nM) and ACC2 (IC50 = 8 nM), demonstrates a 2700-fold selectivity for liver versus skeletal muscle), inhibits HepG2 cell FASyn (EC50 = 14 nM), inhibits rat liver FASyn (ED50 = 0.3 mg/kg), and stimulates rat whole body FAOxn (MED = 30 mg/kg). When administered orally once daily for 14 days to high fat-fed diet-induced obese rats at doses of 3-30 mg/kg, the related series analog ND-630, which exhibits a 273-fold selectivity for liver versus skeletal muscle and similar in vitro and acute in vivo efficacy parameters, reduced in a dose-dependent manner the hepatic steatosis and hepatic cholesterol elevation produced by the high-fat diet, with normalization of hepatic triglycerides to chow-fed levels and 50% normalization of hepatic cholesterol by a dose of 30 mg/kg. Similarly, when given as above to high sucrose-fed diet-induced obese rats, ND-630 reduced in a dose-dependent manner the hepatic steatosis and elevated plasma triglycerides and free fatty acids produced by the high-sucrose diet, with normalization of all parameters to chow-fed levels by doses between 10-30 mg/kg. ND-630 also markedly reduced plasma cholesterol levels at all doses evaluated. Taken together, these observations suggest that hepatoselective allosteric ACC inhibition may favorably affect the hepatic steatosis of fatty liver diseases.

Disclosures:

Geraldine Harriman - Employment: Nimbus Discovery

Jeremy R. Greenwood - Employment: Schrodinger Inc.; Patent Held/Filed: Nimbus Discovery; Stock Shareholder: Schrodinger Inc.

Liang Tong - Consulting: Nimbus Discovery; Grant/Research Support: Nimbus Discovery

H James Harwood - Consulting: Nimbus Inc

The following people have nothing to disclose: Sathesh P. Bhat, Debamita Paul, Ruiying Wang, Rosana Kapeller

698

High intrinsic aerobic capacity protects against 3-day high fat diet induced hepatic steatosis through increased fat utilization and energy expenditure

E. Matthew Morris1, Matthew R. Jackman5,7, Ginger C. Johnson5,7, Tzu-Wen Liu2, Jordan Houser5,7, Lauren G. Koch4, Steven L. Brit-ton4, R. S. Rector1,3, Jamal A. Ibdah1,3, Paul S. MacLean6,7, John P. Thyfault1,2;

1Internal Medicine - Gastroenterology, Univ Missouri, Columbia, MO; 2Nutrition & Exercise Physiology, University of Missouri, Columbia, MO; 3Research, Truman VA Hospital, Columbia, MO; 4Anesthesiology, University of Michigan, Ann Arbor, MI; 5Center for Human Nutrition, University of Colorado-Denver, Denver, CO; 6Physiology & Biophysics, University of Colorado-Denver, Denver, CO; 7Medicine - Endocrinology, Diabetes and Metabolism, University of Colorado-Denver, Denver, CO

Short-term increase in energy balance due to consumption of a high fat diet (HFD) is associated with increased adiposity and the induction of hepatic steatosis and hepatic insulin resistance. In a unique rat model of divergent intrinsic aerobic capacity, sedentary high capacity runner (HCR) rats have been observed to have decreased adiposity and hepatic triglyceride (TAG) content, increased liver and skeletal muscle fatty acid oxidation, and are protected from chronic HFD-induced insulin resistance compared to sedentary low capacity runner (LCR). Herein, we examined the hypothesis that increased energy expenditure and whole-body fat utilization would protect sedentary HCR rats from hepatic steatosis following a 3-day HFD. Indirect calorimetry was utilized to calculate energy expenditure, respiratory quotient (RQ), and substrate utilization. HCR and LCR rats were fed a low-fat, open source diet (LFD, 1 0% fat, Research Diets) prior to beginning the 3-day HFD (45%, Research Diets). Short-term HFD significantly increased daily energy intake relative to lean mass (LM) (HCR: 19%, LCR: 50%), daily weight gain (HCR: 70%, LCR: 2.5-fold), and fat mass (HCR: 13%, LCR: 26%) compared with LF feeding. HCR rats had 70% less liver TAG than the LCR on a LFD and were completely protected against the 50% HFD-induced increase in hepatic TAGs induced by HFD in the LCRs. Both total and resting energy expenditure per LM were higher in HCR than LCR regardless of diet. The increased energy intake following the 3-day HFD resulted in significantly higher positive energy balance per LM in both groups (HCR: 2-fold, LCR: 3-fold), but was 67% higher in LCR over HCR. No difference between phenotypes was observed in RQ on LFD. Acute HFD resulted in a significant reduction in RQ in both groups, but HCR RQ was significantly lower than the LCR. Also, HFD resulted in a dramatic 20-fold increase in lipid energy utilization in the HCR compared to the smaller 34% increase in LCR rats. In conclusion, sedentary HCR rats show protection against hepatic steatosis following acute HF feeding compared with LCR rats. These data suggest that higher intrinsic aerobic capacity may protect against hepatic steatosis following a high fat diet challenge due to increased energy expenditure and greater adaptability for increasing lipid utilization.

Disclosures:

The following people have nothing to disclose: E. Matthew Morris, Matthew R. Jackman, Ginger C. Johnson, Tzu-Wen Liu, Jordan Houser, Lauren G. Koch, Steven L. Britton, R. S. Rector, Jamal A. Ibdah, Paul S. MacLean, John P. Thyfault

699

Regulatory lymphocytes' subpopulations mismatch between visceral adipose tissue and peripheral blood in patients with non-alcoholic fatty liver disease (NAFLD) -A novel tissue specific aspect of NAFLD

Tomer Adar1, Ram M. Spira2, Shimon Shteingart1, Samer Abu-Kha-laf1, Ellen Broide3, Ariella B. Shitrit1, Mahmud Mahamid1, Eran Goldin1;

1Department of Gastroenterology, Digestive disease institute, Shaare Zedek Mdical Center, Jerusalem, Israel; 2Department of General Surgery, Digestive Diseases Institute, Shaare Zedek Medical Center, Jerusalem, Israel;3Flow cytometry unit, Department of immunology, Shaare Zedek Medical Center, Jerusalem, Israel

INTRODUCTION Adipose tissue has been recognized as an inflammatory active organ, which plays an important role in local and systemic inflammation. In obesity, the link between inflammatory and metabolic signaling is altered and hepatic lipid accumulation may be associated with inflammatory disarrangement. Regulatory (Tregs) cells are key players in the regulation of the adipose tissue inflammation. AIM To evaluate the correlation and mismatch in lymphocytes' subpopulations distribution between the visceral adipose tissue and the peripheral blood in patients with and without non alcoholic fatty liver disease (NAFLD). METHODS Peripheral blood and intra-operative visceral adipose tissue biopsies were collected from twelve consenting patients undergoing elective abdominal laparoscopic surgery (1 1 sleeve gastrectomy, 1 cholecystec-tomy). Patients were divided to NAFLD and non-NAFLD groups according to hepatic steatosis, as was determined by routine non invasive imaging. Lymphocytes were isolated from each sample, counted and stained for flow cytometry analysis (FACS). CD4+CD25+FOXP3+ markers and CD4+IL17+ markers were used for Tregs and TH17 identification respectively. FACS parameters were compared between the NAFLD and non-NAFLD groups. Pearson correlation test was used to determine the correlation between the visceral adipose tissue and peripheral blood for each group. RESULTS Hepatic steatosis was demonstrated in 8 patients (66%). Mean age and BMI for the NAFLD and non-NAFLD groups were 36.8 and 40 years and 42 and 41.7 respectively (both NS). A strong correlation was demonstrated between the peripheral blood and adipose tissue's CD3+CD4+ lymphocytes in both NAFLD and non-NAFLD patients (r= 0.84 and 0.85 respectively) . However, a mismatch was demonstrated in CD4/CD8 ratio, where a correlation was found only in non-NFLAD patients r=0.86 vs. 0.34 in NAFLD patients, and a greater mismatch was demonstrated for the Treg/TH17 ratio, where a strong positive correlation between visceral adipose tissue and peripheral blood was found in NAFLD patients (r=0.9) compared with a near-significant negative correlation (r = -0.77) in non-NAFLD patients. CONCLUSION Correlation and mismatch of regulatory lymphocytes population between the peripheral blood and visceral adipose tissue are different in patients with or without NAFLD. This tissue specific aspect of NAFLD may offer new understanding to the pathophysiology of liver steatosis, and mark potential future therapeutic targets.

Disclosures:

The following people have nothing to disclose: Tomer Adar, Ram M. Spira, Shimon Shteingart, Samer Abu-Khalaf, Ellen Broide, Ariella B. Shitrit, Mahmud Mahamid, Eran Goldin

700

Effect of Liver X receptor in liver lipid metabolism in patients with non-alcoholic fatty liver disease

Sang Bong Ahn1, Byoung Kwan Son1, Dae Won Jun2;

1Internal Medicine, Eulji General Hospital, Seoul, Republic of Korea; 2Internal Medicine, Hanyang University, Seoul, Republic of Korea

Background: Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by accumulation of fat in the liver accompanied by necroinflammation and hepatocellular injury. The mechanisms involved in the pathogenesis of NAFLD in humans have not been thoroughly investigated. Liver X receptor (LXR) is a nuclear receptor that regulates the metabolism of cholesterol and fatty acids. To understand the mechanisms involved in the pathogenesis of NAFLD, we investigated the transcriptional factors and lipogenic genes activated in the liver with NAFLD. Methods: We evaluated clinical characteristics including sex, age, BMI, and laboratory findings from 40 NAFLD and 1 6 control patients. Immunohistochemical stain was carried out on liver biopsy samples from all patients. We studied the degree of inflammation and fibrosis (NAS score) and expression of the target genes (LXR, ABCA1, ABCG5/8, SREBP-1C, NPC1L1) from liver biopsy samples. Results: In patients with NAFLD, a significant positive relationship between AST, ALT, BMI, hepatic steatosis and lobular inflammation was seen (p<0.001). Expression level of LXR intensity and extent was 4 and 150 times greater than those of the controls. Expression of hepatic LXR is associated with a degree of hepatic steatosis and inflammation (p<0.001). NAS score is correlated with LXR, ALT, ABCG5/8. LXR expression is also associated with expression of ABCG5/8, SREBP-1C (p<0.001), but not associated with NPC1L1. Conclusion: LXR acts as one of the main regulators of liver lipid metabolism by regulating SREBP-1C, ABCG5/8 in NAFLD patients.

Disclosures:

The following people have nothing to disclose: Sang Bong Ahn, Byoung Kwan Son, Dae Won Jun

701

FXR controlled CHOP as novel key player in NAFLD progression

Claudia D. Fuchs1, Thierry Claudel1, Emina Halilbasic1, Pooja Jha1, Walter Spindelboeck2, Tatjana Stojakovic3, Michael Trauner1;

1 Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria; 2Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; 3Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria

Background: The farnesoid X receptor (FXR) is the key bile acid (BA)-activated nuclear receptor and plays an important role in the regulation of hepatic BA and lipid homeostasis. We aimed to explore the role of FXR and BAs in the control of Chop (which was recently suggested to impact on lipid homeostasis via regulation of C/ebpa expression) as potential regulators of hepatic lipid metabolism and inflammation in patients and mice with nonalcoholic fatty liver disease (NAFLD). Methods: Candidate genes (such as Srebp1c; PPARa; sXbp1 and their downstream targets) which may contribute to the pathogenesis of NAFLD were investigated in human control and NAFLD samples by RT-QPCR. Female C57BL/6 wild type (WT) mice and FXR knockout (KO) mice were fed a MCD diet for 5 weeks to induce NASH. RNA was extracted from liver and gene-expression profiled by RT-QPCR for markers of hepatic triglyceride (TG) and lipoprotein (LP) metabolism as well as inflammation. Moreover, serum biochemistry, liver histology and hepatic TG content were assessed. Finally, HepG2 cells were treated with low and high glucose concentration, with or without FXR and RXR agonists. Gene-expression was analysed by RT-QPCR and chro-matin-immuno-precipitation (Chip) was performed. Results: Human liver samples with simple steatosis displayed 1,6-fold (p=0,029 ) increased Chop mRNA expression compared to control and NASH samples. WT and FXR KO mice on MCD diet displayed increased serum liver enzymes and BA levels while glucose and cholesterol serum levels were decreased. Chop mRNA expression was up-regulated in MCD fed WT but not FXR KO mice. Moreover, mRNA levels of C/ebpa - a master regulator of hepatic lipid homeostasis - were down-regulated only in MCD challenged WT but not FXR KO mice. In addition, WT MCD mice display markedly increased VLDL receptor mRNA levels consistent with elevated hepatic TG content and development of steatosis. Notably, genes involved in de novo lipogenesis and b-oxidation were repressed by MCD challenge independent of the genotype. Hepatic inflammation in response to MCD (reflected by F4/80 and TNFa mRNA expression) was aggravated by absence of FXR. High BA and low glucose levels increased Chop and subsequently repressed C/ebpa expression in a FXR/RXR dependent fashion in HepG2 cells. Finally, a FXR/RXR binding site was identified in the human promoter of Chop demonstrating a highly conserved regulated pathway in mouse and human. Conclusion: These findings demonstrate that glucose and BAs control Chop expression via FXR/RXR therefore providing novel insights into pathogenesis and treatment of NAFLD. Supported by the project FLIP (HEALTH-F2-2009-241762).

Disclosures:

Michael Trauner - Advisory Committees or Review Panels: MSD, Janssen; Consulting: Falk Pharma, Phenex, Amgen; Grant/Research Support: Intercept; Patent Held/Filed: Med Uni Graz (norUDCA); Speaking and Teaching: Falk Foundation, Roche, Gilead

The following people have nothing to disclose: Claudia D. Fuchs, Thierry Claudel, Emina Halilbasic, Pooja Jha, Walter Spindelboeck, Tatjana Stojakovic

702

Steatohepatitits-associated Hepatocellular Carcinoma: Evidence of a Keratinopathy

Kira Bettermann1, Anita Kuldeep Mehta1, Eva Lederer1, Christina Ernst1, Sonja M. Kessler1,2, Xintong Chen3, Yujin Hoshida3, Nabeel Bardeesy4, Bryan C. Fuchs4, Kenneth K. Tanabe4, Heimo Müller1, Vendula Svendova5, Michael G. Schimek5, Monika Mach6, Michael R. Speicher6, Vineet Mahajan1, Cornelia Stumpt-ner1, Andrea Thueringer1, Tatjana Stojakovic7, Thomas Longerich8, Peter Schirmacher8, Thomas M. Magin9, Pavel Strnad10, Claudia D. Fuchs11, Michael Trauner11, Rita Spilka12, Alexandra K. Kiemer2, Andreas Teufel13, Thorsten Maass13, Richard Moriggl14, Jean S. Campbell15, Snorri S. Thorgeirsson16, Jim Stauffer18, Michael Karin19, Josep M. Llovet3,20, Kurt Zatloukal1, Carolin Lackner1, Johannes Haybäck1;

1Institute of Pathology, Medical University Graz, Graz, Austria; 2Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbruecken, Germany; 3Liver Cancer Program, Tisch Cancer Institute, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY; 4Center for Cancer Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 5Institute for Medical Informatics, Statistics and Documentation, Medical University Graz, Graz, Austria; 6Institute of Human Genetics, Medical University Graz, Graz, Austria; 7Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, Graz, Austria; 8Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 9Translational Centre for Regenerative Medicine Leipzig, University of Leipzig, Leipzig, Germany; 10Department of Internal Medicine III, University Hospital RWTH Aachen, Aachen, Germany; 11 Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University Vienna, Vienna, Austria; 12Laboratory of Pathology, General Hospital Zams, Zams, Austria; 13Clinics and Polyclinics for Internal Medicine I, University Clinics Regensburg, Regensburg, Germany; 14Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria; 15Department of Medicine Pathology, University of Washington, Seattle, WA; 16Laboratory of Experimental Carcinogenesis, Center for Cancer Research NCI, NIH, Bethesda, MD; 17Institute for Genetics, University of Cologne, Cologne, Germany; 18 Mouse Cancer Genetics Program, Genetic Modifiers of Tumorigenesis Section, Center of Cancer Research, NCI, Frederick, MD; 19Department of Pharmacology, Laboratory of Gene Regulation and Signal Transduction, School of Medicine, University of California San Diego, La Jolla, La Jolla, CA; 20Hepatocellular Carcinoma Transla-tional Research Laboratory, Barcelona Clínic Liver Cancer Group, Liver Unit, CIBERehd, Institut d'Investigacions Biomèdiques, August Pi i Sunyer, Hospital Clínic Barcelona, Barcelona, Spain

Steatohepatitis (SH)-driven liver tumorigenesis is becoming increasingly important in clinical medicine. SH is morphologically characterized by fatty liver change, ballooning of hepa-tocytes, hepatocyte cell death, accumulation of cytoplasmic aggregates termed Mallory-Denk bodies (MDBs) and inflammation. Aggregation of keratins (Ks) plays a pivotal role in MDB formation linking Ks to SH and SH-associated HCC. Analysis of 3, 6, 12 and 18 month-old Krt 18-/-(129P2/OlaHsd background) mice revealed typical histopatho-logical features of SH accompanied by HCC development in 18 month-old livers of Krt1 8-/- mice. We examined micro-dissected wt, Krt18+/-, Krt1 8-/- HCCs and lung metastases in Krt18-/- mice for chromosomal aberrations by array comparative genomic hybridization (aCGH) analysis. aCGH analysis revealed many chromosomal aberrations in all HCCs. All tested lung metastases and the respective HCCs displayed significantly overlapping chromosomal aberrations throughout the entire genome, suggesting a clonal relationship between the primary and metastatic cancer. The altered chromosomes comprised loci with known oncogenes, tumor suppressors and liver cancer associated genes. Moreover, we compared Krt18-/-mice with 26 HCC cell lines, 44 established SH-, HCC- or SH induced HCC-mouse models and with data sets of >300 patients with HCC, where Krt18-/- gene signatures faithfully matched human HCC. Remarkably, the hepatic lipid profile in Krt18-/- mice was similar to human SH demonstrated by qRT-PCR and fatty acid (FA) analysis by gas chromatography coupled with mass spectrometry (GC-MS). A relative excess of K8 over K18 seems to determine the likelihood to develop SH and SH-associated HCC. We demonstrate that 18 month-old Krt18-/- mice spontaneously developed the entire morphological spectrum of features resembling human SH paralleled by a significant change in their hepatic lipid profile making Krt18-/-mice a useful tool for studying SH and SH-linked HCC. Our studies shed new light on the function of Ks which seems not to be only the backbone of the cells but rather play also important roles in cellular signaling cascades.

Disclosures:

Kenneth K. Tanabe - Patent Held/Filed: EGF SNP and risk for HCC, EGFR inhibition and HCC, gene signature for prognosis in cirrhosis

Michael Trauner - Advisory Committees or Review Panels: MSD, Janssen; Consulting: Falk Pharma, Phenex, Amgen; Grant/Research Support: Intercept; Patent Held/Filed: Med Uni Graz (norUDCA); Speaking and Teaching: Falk Foundation, Roche, Gilead

Josep M. Llovet - Consulting: Bayer Pharmaceutical, Bristol Myers Squibb, Imclone, Biocompatibles, Novartis; Grant/Research Support: Bayer Pharmaceutical, Bristol Myers Squibb, Boehringer-Ingelheim

The following people have nothing to disclose: Kira Bettermann, Anita Kuldeep Mehta, Eva Lederer, Christina Ernst, Sonja M. Kessler, Xintong Chen, Yujin Hoshida, Nabeel Bardeesy, Bryan C. Fuchs, Heimo Müller, Vendula Svendova, Michael G. Schimek, Monika Mach, Michael R. Speicher, Vineet Mahajan, Cornelia Stumptner, Andrea Thueringer, Tat ana Stojakovic, Thomas Longerich, Peter Schirmacher, Thomas M. Magin, Pavel Strnad, Claudia D. Fuchs, Rita Spilka, Alexandra K. Kiemer, Andreas Teufel, Thorsten Maass, Richard Moriggl, Jean S. Campbell, Snorri S. Thorgeirsson, Jim Stauffer, Michael Karin, Kurt Zatloukal, Carolin Lackner, Johannes Haybäck

703

Protective effects of plant sterol and stanol esters in dietinduced non-alcoholic steatohepatitis in hyperlipidemic mice

Jogchum Plat1, Tim Hendrikx1, Veerle Bieghs1, Mike Jeurissen1, Sofie Walenbergh1, Patrick van Gorp1, Els De Smet1, Maurice Konings1, Anita Vreugdenhil2, Ger H. Koek2, Yasmin Dias-Guichot1, Dieter Luetjohann3, Ronald Mensink1, Ronit Shiri-Sverdlov1;

1Maastricht University, Maastricht, Netherlands; 2Maastricht University Medical Center, Maastricht, Netherlands; 3Institute of Clinical Chemistry and Clinical Pharmacology, Bonn, Germany

Background: Non-alcoholic steatohepatitis (NASH) is generally recognized as the hepatic event of the metabolic syndrome. As pharmacological possibilities to interfere with NASH are hardly available, there is an urgent need to identify dietary interventions. In mice, omitting cholesterol from the diet resulted in reduced hepatic inflammation. Considering the beneficial effects of plant sterol and stanol esters on cholesterol metabolism, we hypothesized that plant sterol and stanol ester consumption reduces hepatic inflammation. Methods: Thirty low-density lipoprotein receptor deficient (LDLr-/-) mice consumed a plant sterol poor high fat diet (HFD) for 3 weeks. Hereafter, the mice were randomly allocated to one of the 3 experimental groups (n=10), i.e. HFD, HFD enriched with plant sterol (2%) esters or HFD with plant stanol (2%) esters for 3 weeks. As control, an additional group consumed a plant sterol poor chow diet. Results: Adding plant sterol or stanol esters to the HFD reduced hepatic inflammation as indicated by immunohistochemical stainings for inflammatory markers and the hepatic expression of pro-inflammatory genes Cd-68, Mcp-1, IL-1β, Tnf-α and Icam. Additionally, reduced lipid levels in Kupffer cells were found in the plant sterol or stanol groups as compared to the HFD group. Importantly, although hepatic cholesterol concentrations were lowered in the sterol and stanol groups, liver triacylglycerol concentrations were unchanged. Conclusion: Plant sterol or stanol ester consumption leads to a complete reversal of liver inflammation. This highly significant effect is of great interest since it can open a new venue in the dietary treatment and prevention of NASH.

Disclosures:

The following people have nothing to disclose: Jogchum Plat, Tim Hendrikx, Veerle Bieghs, Mike Jeurissen, Sofie Walenbergh, Patrick van Gorp, Els De Smet, Maurice Konings, Anita Vreugdenhil, Ger H. Koek, Yasmin Dias-Guichot, Dieter Luetjohann, Ronald Mensink, Ronit Shiri-Sverdlov

704

Olanzapine-induced liver injury: direct metabolic effects on liver and exacerbation by high-fat diet

Robin H. Schmidt1, Veronica L. Massey1, Jenny Jokinen1, Keith C. Falkner2, Juliane I. Beier1, Gavin E. Arteel1;

1 Pharmacology & Toxicology, University of Louisville, Louisville, KY; 2Medicine, University of Louisville, Louisville, KY

Background. Olanzapine (OLZ) is an effective first-line treatment for schizophrenia and bipolar disorder. The benefits of OLZ are countermanded by side effects such as weight gain, glucose intolerance, dyslipidemia, and liver injury. These effects impact not only patient compliance, but also increase the health risks to patients. Most studies to date have focused on potential effects of OLZ on the CNS (e.g., satiety); however, peripheral changes in key metabolic organs (e.g., the liver) may also play a critical role. The obesity rates in the US are now at epidemic levels and obesity-induced liver disease (i.e., NAFLD) is on the rise. It is now understood that obesity is a significant risk factor for a myriad of drug-induced liver injuries. Given that the obesity incidence in the psychiatric population is even higher than the US population as a whole, the effects of OLZ may exacerbate an underlying condition in these patients. The purpose of the current study was to determine the mechanisms of OLZ-induced hepatic dysmetabolism, and to test the hypothesis that obesity enhances OLZ-induced hepatic injury. Methods. 8-week old female C57BL/6 mice were fed either a high-fat ('Western' diet; HFD) or isocaloric low-fat diet (LFD) for 4 wks. Mice also received either OLZ (8 mg/kg/d) or vehicle (saline) subcutaneously via osmotic minipumps. Results. OLZ alone (LFD) increased body weight, and caused mild glucose intolerance, without a commensurate increase in food consumption. OLZ alone also caused hepatic steatosis and injury. Interestingly, although OLZ increased hepatic triglyceride synthesis and storage, it did not increase in hepatic free fatty acids synthesis or levels. OLZ administration appeared to cause a pseudo-fasted state and dramatically depleted hepatic glycogen reserves. AMPK and mTOR are generally differentially activated, and mediate opposing metabolic functions; interestingly, OLZ administration simultaneously activated both AMPK and mTOR. When OLZ and HFD were combined, there was a synergistic increase in weight gain and glucose intolerance. Furthermore, liver damage in the HFD/OLZ was synergistically enhanced compared to either alone. Conclusions. Taken together, these data show that OLZ dysregulates glucose and lipid metabolism and exacerbates hepatic changes caused by HFD exposure. The outcomes of OLZ administration on hepatic metabolism may reflect, in part, the contradictory inputs of simultaneously AMPK and mTOR activation. These data indicate that the hepatic metabolic changes caused by OLZ may sensitize the liver to injury caused by HFD and that underlying obesity/liver disease may synergize OLZ-induced side effects.

Disclosures:

The following people have nothing to disclose: Robin H. Schmidt, Veronica L. Massey, Jenny Jokinen, Keith C. Falkner, Juliane I. Beier, Gavin E. Arteel

705

Oxidative stress is a critical mediator of irinotecan induced steatohepatitis

Abdo Mahli1, Michael Saugspier1, Wolfgang E. Thasler2, Martina Müller1, Claus Hellerbrand1;

1University Hospital Regensburg, Regensburg, Germany; 2Ludwig-Maximilians-University Munich, Munich, Germany

Preoperative chemotherapy with irinotecan is associated with the development of chemotherapy-associated steatohepatitis (CASH). This increases the risk of perioperative morbidity and mortality, however, the underlying mechanisms of irinotecan induced hepatic steatosis and inflammation are still unknown. The aim of this study was to establish in vitro and in vivo models of irinotecan induced CASH and to unravel the molecular mechanisms of this phenomenon. Methods and Results: Initially, we determined the dose-range in which irinotecan treatment did not affect the viability of hepatoma cells and primary human hepatocytes. In this dose-range (up to 50μM) irinotecan induced dose-dependently cellular lipid accumulation as assessed by oil-red O staining and analysis of the triglyceride content. Moreover, irinotecan induced the accumulation of free fatty acids (FFA) and caused enhanced beta-oxidation and the formation of reactive oxygen species (ROS), ERK-activation and the expression of proinflammatory genes (IL-8 and ICAM-1). Time-course experiments indicated that ERK-activation and pro-inflammatory gene expression proceed hepatocellular lipid accumulation. Pre-incubation with ROS-scavengers or ERK-inhibition diminished both irinotecan induced lipid accumulation and inflammatory gene expression. To establish an in vivo CASH model, mice were injected with a dose of 50mg/kg irinotecan every 3 days for 2 weeks. Also in vivo we observed that irinotecan induced hepatic ERK-activation, inflammatory gene expression and enhanced oxidative stress and triglyceride levels compared to control mice. Conclusion: Our novel in vitro and in vivo models indicate oxidative stress and ERK-activation as critical mechanisms of irinotecan induced steatohepatitis.

Disclosures:

Martina Müller - Grant/Research Support: Novartis

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

706

T-box-1 (Tbx1) is a Phosphatidylcholine Transfer Protein (PC-TP) Interacting Protein in Subcutaneous White Adipose Tissue: Implications for Hepatic Insulin Sensitivity

Tibor Krisko, David E. Cohen;

Medicine/Gastroenterology, Brigham and Women's Hospital, Boston, MA

Aim: PC-TP is a specific phosphatidylcholine binding protein that is highly expressed in liver and oxidative tissues. By incompletely understood mechanisms, genetic ablation or small molecule inhibition of PC-TP increases both hepatic insulin sensitivity and energy expenditure in mice. The transcription factor Tbx1 has recently been identified as a cellular marker of beige fat progenitor cells, which reside within subcutaneous white adipose tissue depots. Upon differentiation, beige adipocytes adopt the phenotype of brown adipocytes and contribute to non-shivering thermogenesis in the animal. Using yeast two-hybrid screening, we have identified Tbx1 as a putative PC-TP interacting protein. The aim of this study was to validate the PC-TP-Tbx1 interaction and garner evidence for its functional role in thermogenesis. Methods: Endogenous expression of Tbx1 in adult mouse tissues was determined by immunoblot analysis and densitometry. PC-TP-Tbx1 interactions were assessed by co-immunoprecipitation in both mouse tissues and HEK293E cells, which express both proteins. Subcellular colocalization of PC-TP and Tbx1 within HEK 293E cells was determined by cellular fractionation followed by immunoblot analysis. Results: Tbx1 expression was 7- and 10-fold higher in subcutaneous (inguinal) and visceral (epididymal) white adipose tissue, respectively, than in liver. Consistent with restricted expression in a subpopulation of beige progenitor cells, PC-TP expression was detected in subcutaneous but not visceral white adipose tissue, and to a much lesser extent than in liver or brown adipose tissue. Tbx1 expression was increased 2-fold in the subcutaneous white adipose of Pctp-/- compared to wild type mice. Tbx1 co-immunoprecipitated with PC-TP in both subcutaneous white adipose tissue and liver lysates from wild type mice, with the interaction being more pronounced in subcutaneous white adipose tissue. Antibodies to PC-TP did not precipitate Tbx1 in subcutaneous white adipose tissue from Pctp-/-mice. Co-immunoprecipitation of PC-TP and Tbx1 was confirmed in HEK293E cells in which both proteins were identified in the nuclear fraction. Conclusions: PC-TP and Tbx1 are interacting proteins that colocalize within subcutaneous white adipose tissue and co-reside in the nucleus. The increase in Tbx1 expression in subcutaneous white adipose tissue of Pctp-/- mice suggests that PC-TP may function to suppress differentiation of beige adipocyte precursors and thereby limit energy expenditure. We speculate that increased energy expenditure by beige adipose tissue in response to targeted inhibition of PC-TP contributes to improving insulin sensitivity in the liver.

Disclosures:

David E. Cohen -Advisory Committees or Review Panels: Merck, Genzyme; Consulting: Novartis, Aegerion, Dignity Sciences; Speaking and Teaching: Merck

The following people have nothing to disclose: Tibor Krisko

707

Elevated levels of Serum Alanine affect NK function and Predict Severity of Liver Fibrosis in NAFLD patients

Johnny Amer1, Mahamid Mahmud2,3, Lina F. Abu Tair1, Sarit Doron1, Rami Ghantous2, Rifaat Safadi1,2;

1Hadassah University Hospital, Jerusalem, Israel; 2Holy Family Hospital, Nazareth, Israel; 3Shaare Zedek Medical Center,, Jerusalem, Israel

Background/aims: Alanine is a non-essential amino acid required for glucose metabolism. Obesity and the metabolic syndrome cause non-alcoholic fatty liver disease (NAFLD). The anti-fibrotic effect of NK-cells may impair and cause NAFLD progress to cirrhosis and hepatocellular cancer. We investigated a potential role of Alanine levels in NK impairment via the specific nMDAR (N-methyl-D-aspartate receptor) in NAFLD patients. Methods/Results: Non metabolic syndrome NAFLD adult patients with different histological fibrosis-scoring were investigated. CD107a (lysosomal-associated membrane protein-1), a marker of NK activation, decreased in F3-4 derived NK-cells, suggesting NK impairment. In healthy donors serum alanine levels were 2.8±0.4ng/ul. These levels were elevated in F0 and F1 to 4.3±0.5ng/ul and 8.1 ±0.5 ng/ul, respectively (p<0.02). Alanine levels showed to be significantly higher in serums of F1 patients compared to their F0 counterparts (p=0.0005). Moreover, serum alanine levels showed to increase in F2 patients to 12.9±3.9ng/ul and to 14.8±2.9ng/ul in F3-4 scored patients (p=0.01 and p=0.006, respectively). These results were also well correlated with levels of CRP and HOMA-score in these patients (p<0.05). In-vitro incubation of Alanine (15 uM) with isolated peripheral blood NK-cells (10*6 cells/ml) from healthy donors revealed a significant NK impairment as CD1 07a was reduced from 36±7 to 25±10.6% (p=0.004). Incubation of NK-cells with Ketamin (400 nM), an nMDAR antagonist, increased CD107a to 40.5±0.7% (p=0.01). nMDAR was expressed on 8.6±0.4% of NK-cells cultured with medium and was not affected by Alanine and Ketamin incubations. Conclusion: Elevated serum Alanine levels correlate linearly with Fibrosis-scoring and predict liverinjury. High Alanine exposure in NAFLD impairs NK function and its anti-fibrotic ability through its nMDA receptor.

Disclosures:

The following people have nothing to disclose: Johnny Amer, Mahamid Mahmud, Lina F. Abu Tair, Sarit Doron, Rami Ghantous, Rifaat Safadi

708

Developing a novel screening platform for liver fibrosis using integrative network biology approach

Anup M. Oommen, S. Satish, S. Nethra, A. N. Yatheesh, M. Pallavi, M. K. Khaiser, R. Mathiyazhagan, V. Jisha, Mahesh K. Verma, Raghavendra Pralhada Rao, M. R. Jagannath;

biology, Connexios life sciences, Bangalore, India

Background: Liver injury mediated by various etiologies can cause liver fibrosis. Despite extensive research efforts to identify and understand causal mechanisms underlying the disease pathology, a reliable model system that represents an agglomeration of disease mechanisms is lacking. Using our network biology approach we identified key signaling pathways that drive liver fibrosis and established a liver slice model that captures the fibrotic phenotype induced by these signaling pathways. Methods: Precision cut liver slices (PCLS) of 200 micron meter thickness were obtained from C57B6/J mice aged about 8 weeks. PCLS were cultured in Williams E media containing 25mM glucose and 10% fetal calf serum. We used a cocktail comprising of inflammation mediators, growth factors and lipid derivatives (hereafter referred to as IGL cocktail) to induce fibrotic phenotype associated with inflammation and steatosis. After 24 hours of culture, expression levels of key markers of inflammation, stellate cell activation and fibrosis were measured using Q-PCR. ATP, ROS and triglyceride levels were measured in the liver slices using commercially available kits. Results: Liver slices when cultured with IGL cocktail remained viable during the treatment period as measured by ATP levels. IGL cocktail treatment resulted in significant increase in expression of key markers of inflammation [IL-6(1.6+/-0.092), TNF-a(2.7+/-0.113), CRP(2.57+/-0.13)], stellate cell activation [aB-Crystallin(1 .9+/-0.06), a-SMA(1 .9+/-0.14)] and fibrosis [fibulin2(10+/-0.24), Col1a1 (3+/-0.26), Col3a1 (1.74+/-0.26)] and also showed increase in triglyceride (38%) and ROS levels (50%) and total collagen level (30%) compared to control conditions. Conclusion: The existing in vitro platforms for studying anti-fibrotic targets relies on non-physiological inducers like CCL4 and this approach might not capture multiple aspects of disease pathology. In our study we have used a unique cocktail activating several signaling pathways, to establish a platform capturing multiple aspects of NASH, such as steatosis, steatohepatitis and fibrosis. This provides a reliable platform for evaluating anti-fibrotic targets and for screening NCEs. Disclosure: None

Disclosures:

The following people have nothing to disclose: Anup M. Oommen, S. Satish, S. Nethra, A. N. Yatheesh, M. Pallavi, M. K. Khaiser, R. Mathiyazhagan, V. Jisha, Mahesh K. Verma, Raghavendra Pralhada Rao, M. R. Jagannath

709

Hepatic regulatory T cells contribute to the development of dietary steatohepatitis in mice

Junko Yokokawa, Kenichi Ikejima, Kumiko Arai, Kazuyoshi Kon, Shunhei Yamashina, Sumio Watanabe;

Department of Gastroen-terology, Juntendo University Graduate School of Medicine, Tokyo, Japan

Background: The hepatic immune system play a pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH); however, the pathophysiological role of CD4+Foxp3+ regulatory T (Treg) cells remains unclear. We have recently demonstrated that expression levels of anti-inflammatory/regulatory cytokines in hepatic Treg cells are diminished in obese diabetic KK-Ay mice. In this study, therefore, we investigated the development of dietary-induced steatohepatitis in mice selectively depleted Treg cells. Methods: Male, 8-week-old C57Bl/6 mice were fed a high-fat (HF) diet for 4 weeks. Some mice were given inter-peritoneal injections of anti-CD25 Ab (50 μg/body) or control IgG twice a week for 4 weeks simultaneously. Hepatic mononu-clear cells were collected by differential centrifugations using Percoll gradient, and CD4+Foxp3+ Treg cells were detected by flowcytometry. Liver histology was assessed, and hepatic triglyceride contents were measured colorimetrically. Hepatic mRNA levels for inflammatory cytokines and regulators of lipid metabolism were measured by real time RT-PCR. Results: Regardless of anti-CD25 Ab injections, mice fed a HF diet for 4 weeks gained body weight nearly 26% over basal values, which were almost equivalent to those in mice received control IgG. Following a single injection of anti-CD25 Ab, the fraction of hepatic CD4+Foxp3+ cells was depleted to the levels nearly 1/5 over controls, and the levels in HF diet-fed mice following repeated injections of anti-CD25 Ab remained to the levels nearly 1/3 of those in mice given control IgG. Mice given control IgG showed trivial hepatic steatosis following HF diet feeding for 4 weeks as expected. In contrast, anti-CD25 Ab-treated mice fed a HF diet exhibited overt hepatic steatosis. Indeed, liver triglyceride levels were 35% higher in mice given anti-CD25 Ab as compared to the control IgG-treated mice following HF diet feeding. HF diet-induced increases in hepatic mRNA levels of fatty acid synthase (FAS) and acetyl-CoA car-boxylase alpha (Acaca), as well as stearoyl-CoA dehydroge-nase-1 (SCD1), were significantly enhanced in mice treated with anti-CD25 Ab. Conclusions: These observations clearly indicated that depletion of Treg cells enhances hepatic steatosis induced by HF diet feeding, most likely through modulation of hepatic lipid metabolism. It is hypothesized that dysfunction of Treg cells leads to enhanced pro-inflammatory immune responses in the liver, thereby exacerbating steatohepatitis in these mice. Thus, alterations in the hepatic immune system involving Treg cells most likely contribute to the development of metabolic syndrome-related NASH.

Disclosures:

The following people have nothing to disclose: Junko Yokokawa, Kenichi Ikejima, Kumiko Arai, Kazuyoshi Kon, Shunhei Yamashina, Sumio Watanabe

710

Influence of PNPLA3 genotypes on histological features and adiponutrin serum levels in patients with NAFLD

Rocío Gallego-Durán1, Isidora Ranchal1, Lourdes Rojas1, María J. Pareja1, Carmelo García-Monzón2, Javier Crespo3, Maria Teresa Arias-Loste3, Inmaculada Moreno-Herrera4, Raul J. Andrade4, Manuel Romero-Gomez1;

1Valme University Hospital, Sevilla, Spain; 2Santa Cristina University Hospital, Madrid, Spain; 3Mar-qués de Valdecilla University Hospital, Santander, Spain; 4Virgen de la Victoria University Hospital, Málaga, Spain

Aim: To investigate the relationship between genotypes of Patatin like Phospholipase-3 (PNPLA3) gene and histopathological findings in non-alcoholic fatty liver disease (NAFLD). Material and methods: One-hundred and thirty-three patients with histologically confirmed NAFLD disease were included. Liver biopsies were reviewed by a single pathologist and classified according to Kleiner score. Lobular inflammation, ballooning, steatosis degree and fibrosis stage were assessed. The single-nucleotide polymorphism (SNP) of PNPLA3 gene (rs738409) was genotyped using Taqman probe (Applied Byosistems, Barcelona, Spain). Adiponutrin levels were measured in twelve hours-fasting serum of patients using a commercialized available ELISA kit (Uscn, Life science Inc., Wuhan, China). Statistical analysis was performed using ANOVA and U-Mann Whitney tests by SSPS v20.0. Results: In NAFLD patients GG genotype was overrepresented in comparison with general population (1 6.5% vs 3.3%;p<0.0001). Genotype GG of PNPLA3 was found in (13/35; 37%) patients with severe steatosis versus 9/94 (9.5%) with mild steatosis;p<0.001. Steatohepatitis (NASH) was detected in 40/133 (30%). GG genotype was present in 12/40 (30%) patients with NASH versus 11/93 (12%) in non-NASH (p<0.01). No impact of GG genotype was seen on ballooning, lobular inflammation, significant fibrosis and serum adiponutrin levels. Conclusions: Genotype GG of PNPLA3 was strongly associated with steatosis degree and steatohepatitis but not with ballooning, lobular inflammation or fibrosis. Functional studies are warranted to elucidate if this genotype is related to disease progression or could be a bias linked to raise fat-deposition.

Results of PNPLA3 genotype and adiponutrin levels in NAFLD patients

Table 1. 
  GGnonGGp
Severe SteatosisYes (n=35)13(37%)22(63%)0.001
 No(n=94)9(9.5%)85(90.5%)0.001
SteatohepatitisYes (n=40)12(30%)28(70%)0.01
 No (n=93)11(12%)82(88%)0.01
BallooningYes(n=15)3(20%)12(80%)0.7
 No(n=50)12(24%)38(76%)0.7
Lobular InflammationYes (n=55)14(25.5%)41(74.5%)0.2
 No(n=10)1(10%)9(90%)0.2
Significant FibrosisYes (n=26)6(23%)20(77%)0.3
 No(n=107)17(16%)90(84%)0.3
Adiponutrin Levelsn=350.0+0.00.28+1.080.5

Disclosures:

Javier Crespo - Board Membership: MSD, Roche, Janssen, Gilead

Manuel Romero-Gomez - Advisory Committees or Review Panels: Roche Farma,SA., MSD, S.A., Janssen, S.A., Abbott, S.A.; Grant/Research Support: Ferrer, S.A.

The following people have nothing to disclose: Rocío Gallego-Durán, Isidora Ranchal, Lourdes Rojas, María J. Pareja, Carmelo García-Monzón, Maria Teresa Arias-Loste, Inmaculada Moreno-Herrera, Raul J. Andrade

711

L-Fabp deletion attenuates both hepatic steatosis and fibrosis resulting from hepatic microsomal triglyceride transfer protein (Mttp) deletion

Elizabeth P. Newberry, Susan M. Kennedy, Victoria Cooke, Joshua N. Cohen, Yan Xie, Nicholas O. Davidson;

Gastroenterology, Washington University School of Medicine, St Louis, MO

Pharmacologic and genetic studies have shown that loss of hepatic microsomal triglyceride transfer protein (Mttp) function blocks VLDL secretion and causes hepatic steatosis, but not inflammation, insulin resistance or steatohepatitis. Here we asked if liver specific deletion of Mttp (Mttp-LKO) promotes hepatic stellate cell activation and hepatic fibrosis and whether this phenotype is altered by simultaneous deletion of L-Fabp (liver fatty acid binding protein), a cytosolic lipid binding protein involved in FA trafficking. Mttp-LKO mice were bred with germline L-Fabp-/- mice to generate mice lacking both hepatic L-Fabp and Mttp (DKO mice). At 12 weeks, Mttp-LKO mice fed chow diet showed ∼10 fold increased hepatic triglyceride (TG) vs age-matched C57BL/6 controls, with increased expression of fibrotic genes, including Col1a1, Ctgf and Krt19. Hepatic steatosis was significantly attenuated in DKO mice (223 ± 13 μg TG/mg protein vs 331 ± 36, Mttp-LKO, n=6/group), with reduced expression of fibrogenic mRNAs. To examine whether a high fat diet exacerbated fibrosis in Mttp-LKO mice, we fed a high trans fat, high fructose diet (TFF) for 16 weeks. Hepatic lipid was increased ∼2x in TFF-fed Mttp-LKO vs C57BL/6 controls (783 ± 71 μg TG/mg protein, C57BL/6; 1346 ± 108, Mttp-LKO, n=5-8/group), with a dramatic increase in fibrosis, as shown by both Sirius Red staining (5.3 ± 0.7 fibrotic foci/field, C57BL/6; 18.4 ± 3, Mttp-LKO) and gene expression. In contrast, DKO mice showed decreased steatosis and fibrosis (8.6 ± 0.5 foci/field), with hepatic TG levels in DKO mice (632 ± 88 μg TG/mg protein, n=5) similar to C57BL/6 controls. We next sought to distinguish the effects of a lipogenic diet on hepatic steatosis and fibrosis in mice fed a high fructose diet. Hepatic TG was ∼3-fold greater in both fructose-fed Mttp-LKO and DKO mice (558 ± 43 μg TG/mg protein, Mttp-LKO; 592 ± 33, DKO, n=7-9) compared to C57BL/6 controls (154 ±11). These finding show that L-Fabp deletion impairs hepatic TG accumulation derived from dietary fatty acids but not the accumulation of TG derived from de novo synthesized fatty acids. More importantly, the findings demonstrate that hepatic steatosis alone resulting from blocking VLDL secretion is a driving force in the development of fibrosis. Our findings show that L-Fabp deletion may prevent this transition as a result of decreased storage of dietary TG. These data are relevant in light of Phase III clinical trials of pharmacologic Mttp inhibitors and their potential to promote hepatic steatosis and fibrosis, particularly in the setting of a high fat diet.

Disclosures:

The following people have nothing to disclose: Elizabeth P. Newberry, Susan M. Kennedy, Victoria Cooke, Joshua N. Cohen, Yan Xie, Nicholas O. Davidson

712

Effects of andrographolide in experimental non-alcoholic steatohepatitis

Daniel Cabrera1, Margarita Pizarro1, Nancy Solis1, Javiera Torres3, Enrique Brandan2, Marco Arrese1;

1 Department of Gastroenterology, School of Medicine, Catholic University of Chile, Santiago, Chile; 2Department of Cell and molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Santiago, Chile; 3 Department of Pathology, School of Medicine, Catholic University of Chile, Santiago, Chile

Background: Currently, the therapeutic armamentarium to treat Non-alcoholic fatty liver disease (NAFLD) and its progressive form Non-alcoholic steatohepatitis (NASH) is limited. Although pathogenetic details are unknown insulin resistance and lipotoxicity-related hepatic inflammation are key for disease progression. Andrographolide (AP), a diterpenoid lactone of the plant Andrographis paniculata, is a safe botanical drug with strong anti-inflammatory effects in experimental models of asthma, stroke, and arthritis. Aim: To evaluate the effects of AP administration in experimental NASH. Methods: C57bl6 mice were fed a choline-deficient amino acid-defined (CDAA) diet for 12-22 weeks to induce NASH. Choline-suplemmented deficient amino acid-defined (CSAA) was used as control diet. Mice were intraperitonally injected with AP (1mg/Kg), three times per week. Hepatic steatosis, inflammation, and fibrosis were scored histologically (hematoxylin-eosin and Sirius red staining). Also, hepatic triglyceride content (HTG) and mRNA expression of inflammatory markers such as IFN-γ, TNF-α and MCP-1 were assessed. Results: treatment with AP reduced inflammatory foci evaluated through hematoxilin/eosin staining. Also, total macrophages (F4/80 positive cells), specially M1 macrophages (CD68 positive cells) were reduced by AP administration as evaluated by immunofluorescence staining. In addition, total liver mRNA levels of IFN-γ, TNF-α and MCP-1 were reduced by AP treatment. Finally, AP slightly but significably reduced fibrosis scores and collagen protein levels. Total hepatic triglycerides levels and the number of oil red positive cells were not affected by AP treatment. Conclusions: AP reduced inflammation and fibrosis in NAFLD with no effect on steatosis. AP could be a novel agent to test for NASH treatment. (Support from FONDECYT Project 1110455) and Conicyt, project ACT79/Basal Project CARE).

Disclosures:

The following people have nothing to disclose: Daniel Cabrera, Margarita Pizarro, Nancy Solis, Javiera Torres, Enrique Brandan, Marco Arrese

713

Increased eNOS Activity After Short-term Chronic Intermittent Hypoxia Prevents Intrahepatic Endothelial Dysfunction in NAFLD Rats

Manuel Hernandez-Guerra1,2, Francisco Javier Gonzalez-Pare-des2, Beatriz Abrante2, Alejandro Hernandez-Camba2, Felicitas Diaz-Flores3, Raquel Marcelino2, Yanira González2, Cecilia Fumero2, Enrique Quintero1,2; 1 Liver Unit, Canary Island University Hospital, La Laguna, Spain; 2Research Unit, Canary Island University Hospital, La Laguna, Spain;

3Central laboratory, Canary Island University Hospital, La Laguna, Spain

Background: Intrahepatic endothelial dysfunction occurs early after induction of non-alcoholic fatty liver disease (NAFLD) and cirrhosis in experimental animal models. Chronic intermittent hypoxia (CIH) is a feature of obstructive sleep apnea and has recently shown to aggravate endothelial dysfunction in cirrhotic rats by means of increased oxidative stress and eNOS uncoupling. Whether CIH triggers intrahepatic endothelial dysfunction and thereafter liver inflammation and fibrosis in NAFLD is unknown. Aim: To investigate whether a short term course of CIH impairs the intrahepatic vasodilator response to acetylcholine (ACh) in NAFLD compared with cirrhotic rats and to explore mechanisms involved. Material and methods: NAFLD (Harlan TD.06414 diet; 60% fat, n=6) and cirrhotic-induced (after carbon tetrachloride inhalation, n=10) male Sprague-Dawley rats were exposed during the diurnal sleep period, for 14 days to repetitive cycles of CIH (nadir O2 7-1 0% with return to 21% for 1 min at 3-min intervals, 12 h/day). Another group of NAFLD (n=5) and cirrhotic (n=8) rats were caged in similar conditions but with air room (Handled controls;HC). After this period livers were perfused and portal pressure response-curves to methoxamine (10-4) followed by ACh (10-8, 10-7, 10-6 mol/L) were obtained. Blood was analysed for haematocrit, ALT/AST and lipids. Liver tissue was obtained for eNOS, p-eNOS, GMPc, nitrotyrosine, α-TNF, myeloperoxidase activity, hydroxyproline and histological evaluation to assess inflammation and fibrosis. Results: Cirrhotic livers showed a significantly attenuated vasodilatory response to ACh aggravated by CIH (max. -25±6% vs. -49±9%, p=0.03). In contrast, NAFLD compared to cirrhotic rats did not show significant differences in ACh responses after CIH (ACh10-6: -21 ±4% vs. -22±3%, p=0.8). This response was associated with increased oxidative stress but increased eNOS activity, contrary to cirrhotics. As a result, GMPc was not different between CIH and HC NAFLD rats. In addition, NAFLD rats induced to CIH did not show more inflammation or fibrosis. Conclusions: Short term exposure to CIH in NAFLD rats is associated with oxidative stress and compensatory increased eNOS activity. This may contribute to prevent intrahepatic endothelial dysfunction and histological damage. Whether long-term exposure to CIH provokes an unbalance and further endothelial impairment remains to be studied.

Disclosures:

The following people have nothing to disclose: Manuel Hernandez-Guerra, Francisco Javier Gonzalez-Paredes, Beatriz Abrante, Alejandro Hernandez-Camba, Felicitas Diaz-Flores, Raquel Marcelino, Yanira González, Cecilia Fumero, Enrique Quintero

714

Lipid Biomarkers in Liver Cancer Derived from Diet-induced Non-alcoholic Fatty Liver Disease

Maria Rivera1, Puneet Puri1, Tommy Pacana1, Vaishali Patel1, Riikka Katainen2, Terhi Vihervaara2, Faridoddin Mirshahi1, ArunJ. Sanyal1;

1Gastroenterology, VCU Medical Center, Richmond, VA; 2Zora Biosciences, Espoo, Finland

BACKGROUND: Non-alcoholic Fatty Liver Disease (NAFLD) spectrum extends from hepatic steatosis to nonalcoholic steato-hepatitis (NASH), which can progress to cirrhosis and liver cancer. NASH increases the risk for liver cancer development; however, the underlying biologic mechanisms to cancer progression are not entirely clear. The aim of this study is to identify lipid biomarkers associated with liver cancer derived from diet-induced NAFLD. METHODS: Female 129S1/SvlmJ:B6 mice (n=1 5) were fed a high-fat diet for 52 weeks. Cholesterol esters, phosphatidic acid, diacylglycerol, glycerophospho-lipids, sphingolipids, and eicosanoids and the distribution of fatty acids within lipid classes were quantified by mass spec-trometry. The lipid changes were analyzed and compared between mice with liver cancer (n=6) and mice without liver cancer (n=9) using unpaired student's T-test. RESULTS: Prolonged HFD led to marked hepatic steatosis, inflammation, and extensive fibrosis. 40% (6/15) of the NAFLD mice developed liver cancer. There was a 2-fold increase in cholesterol ester 22:1 (p=0.043) in the liver cancer group compared to the group without liver cancer. There was a significant decrease in phosphatidylcholine 17:0/20:3 (p=0.0329) concentrations in the liver cancer group. Total phosphatidylinositol (PI) was also significantly decreased (p=0.0084). Particular fatty acid composition of importance include PI 16:0/20:3, PI 18:0/20:4, PI 18:0/20:4, and PI 18:0/20:4 (p=0.0493, 0.0128, and 0.0412 respectively). In the liver cancer group, ceramide d1 8:1/26:0 (p=0.0038) was depleted, whereas several sphin-gomyelin (SM) molecules, which include SM (d18:1/26:0) (d1 8:1/25:1-OH), SM (d: 18:1/26:1) (d1 8:1/25:2-OH), and SM (d1 8:1/26:2) (p= 0.0496, 0.0445, and 0.03 respectively), were decreased significantly. CONCLUSIONS: Lipid biomarkers were identified and associated with liver cancer derived from diet-induced NAFLD. Further investigation of these markers may reveal new insights for improved diagnosis, prognosis, and personalized therapy.

Disclosures:

Puneet Puri - Advisory Committees or Review Panels: Health Diagnostic Laboratory Inc.; Consulting: NPS Pharmaceuticals Inc.

Riikka Katainen - Employment: Zora Biosciences Oy Terhi Vihervaara - Employment: Zora Biosciences

Arun J. Sanyal - Advisory Committees or Review Panels: Gore, Gilead, Abbott, Ikaria; Consulting: Salix, Immuron, Exhalenz, Bayer-Onyx, Genentech, Norgine, GalMed, Novartis, Echosens, Takeda; Grant/Research Support: Salix, Genentech, Genfit, Intercept, Ikaria, Takeda, Gilead; Independent Contractor: UpToDate

The following people have nothing to disclose: Maria Rivera, Tommy Pacana, Vaishali Patel, Faridoddin Mirshahi

715

Characteristics of hepatic fatty acid compositions and the gene expression levels of enzymes involved in fatty acid metabolism in patients with nonalcoholic steatohepatitis

Kazutoshi Yamada1, Eishiro Mizukoshi1, Kiichiro Kaji1, Hidetoshi Nakagawa1, Masaaki Kitahara1, Hajime Sunagozaka1, Kuniaki Arai1, Tatsuya Yamashita1, Yumie Takeshita2, Hirofumi Misu2, Toshinari Takamura2, Seiko Sawada-Kitamura3, Yoh Zen3, Yasuni Nakanuma4, Masao Honda1, Shuichi Kaneko1;

1Department of Gastroenterology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan; 2Department of Disease Control and Homeostasis, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan; 3Division of Pathology, Kanazawa University Hospital, Kanazawa, Japan; 4Department of Human Pathology, Graduate School of Medicine, Kanazawa University, Kanazawa, Japan

Background: Nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and lipid metabolism. Recent studies have suggested that the quality of fat accumulated in the liver is associated with the development of nonalcoholic steatohepatitis (NASH). In this study, we measured the fatty acid contents of liver tissue, clarified the characteristics of the composition and composition ratio of these fatty acids, and investigated their association with the disease state and pathological changes. Methods: The subjects were 103 patients diagnosed with NAFLD based on liver biopsy (simple steatosis (SS): 63, NASH: 40). Fatty acids were extracted from liver tissue and measured using gas chromatography. Samples of 65 (SS: 35, NASH: 30) patients were subjected to RTD-PCR, and the gene expression levels of enzymes involved in fatty acid metabolism were measured. In addition, relationships between the composition and composition ratios of various fatty acids and patient backgrounds, laboratory test values, histology of the liver, and expression of fat metabolism-related enzymes were investigated. Results: The content of many fatty acids was significantly higher in the SS and NASH than in the Control(p<0.05). The C1 6:1 n7 content was significantly higher in the NASH group than in the SS group. Regarding the composition ratio, the C18:0/C16:0 ratio was decreased and the C16:1n7/C16:0and C1 8:1 n9/C1 8:0 ratios were increased in the NASH group. Fatty acid composition ratios were associated with liver histology: the C18:0/C16:0 and C1 8:1 n9/C1 8:0 ratios were associated with the steatosis score, and the C1 6:1 n7/C 16:0 ratio was associated with the lobular inflammation scores. The expressions levels of genes: SCD1, Elovl6, SREBP1c, FAS, and PPARγ, were enhanced in the NASH group (p<0.05). Regarding the association of the expressions levels of genes with the histological features of the liver, no significant correlation was noted between the steatosis score and the expression of these genes. On the other hand, a significant correlation was observed between the lobular inflammation score and SCD1 expression (p<0.01). In multi-variate analysis, the C18:0/C16:0 ratio was the most important factor that was correlated with the steatosis score in liver tissue. In contrast, the C1 6:1 n7/C1 6:0 ratio was correlated with lobular inflammation in liver tissue. Conclusion: The composition of fatty acids in liver tissue and the expression of genes related to fatty acid metabolism were different between the SS and NASH groups, which suggests that the acceleration of fatty acid metabolism is deeply involved in pathogenesis of NASH.

Disclosures:

Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Aji-nomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Kazutoshi Yamada, Eishiro Mizukoshi, Kiichiro Kaji, Hidetoshi Nakagawa, Masaaki Kitahara, Hajime Sunagozaka, Kuniaki Arai, Tatsuya Yamashita, Yumie Takeshita, Hirofumi Misu, Toshinari Takamura, Seiko Sawada-Kitamura, Yoh Zen, Yasuni Nakanuma, Masao Honda

716

Increased accumulation of 4-hydroxynonenal adducts in male GSTA4/PPARα double knockout mice enhances injury during early stages of alcoholic liver disease

Kelly E. Mercer1,2, Neha Sharma2, Colin T. Shearn3, Jamie Vantrease2, Emanuele Albano4, Thomas Badger1,2, Martin J. Ronis1,2, Dennis R. Petersen3;

1Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR; 2Arkansas Children's Nutrition Center, Little Rock, AR; 3University of Colorado Anschutz Medical Campus, Aurora, CO; 4University A Avogadro of East Piedmont, Novara, Italy

Hepatic lipid peroxidation and accumulation of aldehydeadducted proteins occur early in alcohol-mediated injury and are postulated to mediate the subsequent pro- inflammatory and fibrotic responses observed in alcoholic liver disease. To test the significance of lipid peroxidation formation in the development of early-stage liver injury, we fed a Lieber-DeCarli ethanol (EtOH) liquid diet to male 129/SvJ glutathione S-transferase A4 knockout mice (GSTA4-/-) mice for 40 d. These mice lack the ability to metabolize lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE). At sacrifice, we observed marked increases in malondialdehyde (MDA) and 4-HNE adducts in liver sections, increased lipid accumulation, and mRNA expression of molecular markers of inflammation (TNFα, CD14) and fibrosis (ColA1, αSMA) in the EtOH-treated GSTA4-/- mice compared to EtOH-treated wild type controls (p<0.05). Crossing the GSTA4-/- mice with the peroxisome proliferator-activated receptor-α null mice (PPARα-/-), which are predisposed to hepatic steatosis, had a significant impact on the degree of EtOH-mediated liver injury in the resulting double knockout (dKO) strain. Serum alanine aminotransferase (ALT) concentrations were significantly higher in the EtOH-treated dKO mice compared to similarly EtOH-treated GSTA4-/- and PPARα-/- mice and wild type controls (p<0.001). EtOH-feeding of the dKO mice also resulted in significant elevation of hepatic lipid peroxidation adducts and auto-antibodies directed against these adducts when compared to EtOH-feeding of GSTA4-/-, PPARα-/-and wild type control mice (p<0.05). Yet, while markers of oxidative stress and lipid accumulation were significantly elevated in the EtOH-treated dKO mice compared to the GSTA4-/-and control mice, we observed no significant differences in these markers when compared to the EtOH-treated PPARα-/-mice, suggesting that the PPARα-/- phenotype is the major driver in these cellular events. However, in the EtOH-treated dKO mice, we did see a more robust inflammatory response as measured by increased mRNA expression of TNFα and CD14, a marker for Kuffercell activation, and increased expression of matrix remodeling markers, αSMA, PDGFR, MMP9, when compared to either the GSTA4-/- or the PPARα-/- genotype alone (p<0.05). These findings highlight the importance of lipid peroxidation in mediating alcohol-induced liver injury, and provide model system to study role of lipid peroxidation products in the progression of alcohol liver disease from steatosis to steatohepatitis and fibrosis. Funded in part by R01 AA009300 (DRP).

Disclosures:

The following people have nothing to disclose: Kelly E. Mercer, Neha Sharma, Colin T. Shearn, Jamie Vantrease, Emanuele Albano, Thomas Badger, Martin J. Ronis, Dennis R. Petersen

717

Increased accumulation of 4-hydroxynonenal adducts in female GSTA4/PPARα double knockout mice enhance steatosis and inflammation in a model of pediatric nonalcoholic fatty liver disease

Kelly E. Mercer1,2, Neha Sharma2, Colin T. Shearn3, Jamie Vantrease2, Emanuele Albano4, Thomas Badger1,2, Dennis R. Petersen3, Martin J. Ronis1,2;

1 Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR; 2Arkansas Children's Nutrition Center, Little Rock, AR; 3University of Colorado Anschutz Medical Campus, Aurora, CO; 4University A Avogadro of East Piedmont, Novara, Italy

Hepatocellular injury resulting from increased lipid peroxidation products and oxidative stress is considered a potential mechanism driving the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitsis (NASH). To test the significance of lipid peroxidation and protein adduct formation in the development of pediatric NASH, we crossed 129/SvJ gluthatione S-transferase alpha 4 (GSTA4-/-) knockout mice which lack the ability to metabolize lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE), with peroxisome proliferator-activated receptor-α null mice (PPARα-/-) to produce a double knockout (dKO) strain. At PND21, wild type, GSTA4-/-, PPARα-/- and dKO female mice were fed a 70% corn oil high fat (HF) liquid diet for 12 weeks. In response to the HF diet, we observed larger increases in body wt., liver and visceral fat in the PPARα-/- null mice compared to HF-treated dKO mice (p<0.05). However in the HF-fed dKO mice, hepatic lipid accumulation and lipid droplet size were significantly increased when compared to either the GSTA4-/- or PPARα-/- fed mice. Hepatomegaly in the HF-fed dKO mice also corresponded to marked increases in 4-HNE adducts and a decrease in the GSH:GSSG ratio (p<0.05) when compared to the PPARα-/- null mice, suggestive of increased oxidative stress. Expression of pro-inflammatory cytokines, TNFα and IL-6 mRNA were up-regulated to a greater extent in the livers of HF-fed dKO mice compared to HF-fed GSTA4-/-, PPARα-/- mice (p<0.05), despite no observable differences in immune cell infiltration. Development of NASH pathology occurred in both HF-fed PPARα-/- and dKO mice but not in HF-fed wild type or GSTA4-/- mice. However, there were no overall changes in expression of fibrotic markers (αSMA, ColA1) or extracellular matrix remodeling (MMP9, TIMP-1, MMP13) between the HF-fed dKO and PPARα-/- mice, suggesting that the PPARα-/- phenotype is the predominant driver of fibrosis in this model of pediatric NAFLD. In conclusion, we developed an animal model by which HF feeding of weanling GSTA4/PPARα dKO mice resulted in a pathological progression of NAFLD to NASH, supporting the role of lipid peroxidation in development of steatosis and inflammation in pediatric NAFLD. Funded in part by USDA ARS CRIS 6251-51000-007-00D-4 and R01 AA009300 (DRP).

Disclosures:

The following people have nothing to disclose: Kelly E. Mercer, Neha Sharma, Colin T. Shearn, Jamie Vantrease, Emanuele Albano, Thomas Badger, Dennis R. Petersen, Martin J. Ronis

718

Stress influences on bile acid homeostasis in development of diet-induced steatohepatitis

Tsutomu Matsubara1, Kenji Kitamura1, Yuga Teranishi2, Norifumi Kawada2, Kazuo Ikeda1;

1 Department of Anatomy and Regenerative Biology, Graduate School of Medicine, Osaka City University, Osaka, Japan; 2Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan

Background: Stress can affect our body, and is known to lead to some diseases. But the influence on development of non-alcohol steatohepatitis (NASH) remains unknown. In this study, influence of stress on the development of NASH was investigated to further understand molecular alteration in the liver. Methods: NASH was generated in C57/B6 mice by feeding methionine-and choline-deficient diet (MCD). One week after treatment with MCD or methionine- and choline-supplement diet (MCS) as control, the mice were given restraint stress (1 hour twice a day) for 1 or 3 weeks, and sacrificed. The mouse samples were subject to assessment of the stress. Results: The stress did not influence on changes in body weight, liver and adipose tissue mass. The stress enhanced serum ALT level in MCD mice one week after stress challenge (the value was 208 and 421 U/L in control and stress, respectively), while the stress did not in MCS mice. Serum and hepatic bile acid, triglyceride, and non-ester-ified fatty acid levels were not changed. Three weeks after stress challenge, the stress increased both serum and hepatic bile acid levels [up to 38.8 μM (1.6-fold) and 0.740 μmol/g liver (2.2-fold), respectively], and decreased both serum and hepatic non-esterified fatty acid levels [down to 1.34 mEq/L (0.74-fold) and 0.212 μEq/g liver (0.63-fold), respectively], although the stress-enhancement of serum ALT level was not observed. Interestingly, the stress also elevated hepatic bile acid levels in MCS group [control and stress was 0.033 and 0.137 μmol/g liver (6.0-fold), respectively]. Of the bile acid-related gene, hepatic CYP7A1, rate-limiting enzyme of de novo bile acid synthesis, was clearly induced in both the MCS and MCD (4.0- and 2.2-fold in MCS and MCD, respectively). Hepatic alteration of fatty acid-related genes (Fasn, Acc1, Acadm, Acadl, Acox1 and Cd36) was not observed. Furthermore, the stress for 2 days also increased both CYP7A1 mRNA and protein level (2.5-fold) under condition of standard chow diet. Glucocorticoid-induced CYP7A1 expression was observed in primary hepatocyte (2.1-fold). Conclusion: The present study demonstrated that the stress could stimulate derangement of bile acid homeostasis in a mouse model of steatohepatitis induced by methionine- and choline-deficient diet. Bile acid homeostasis may be also regulated via interplay of adrenal gland and liver.

Disclosures:

The following people have nothing to disclose: Tsutomu Matsubara, Kenji Kitamura, Yuga Teranishi, Norifumi Kawada, Kazuo Ikeda

719

Immunological amplification of CXCL1-S100A8 loop via CXCR2-expressing CD11b+ Gr-1high cells in the development of nonalcoholic fatty liver disease

Kaori Mukai, Takuya Miyagi, Kumiko Nishio, Yoshinobu Yokoyama, Teppei Yoshioka, Yoshinobu Saito, Tsukasa Kawaguchi, Satoshi Aono, Satoshi Tanaka, Satoshi Shimizu, Hayato Hikita, Ryotaro Sakamori, Yoshihiro Kamada, Tomohide Tatsumi, Naoki Hiramatsu, Tetsuo Takehara;

Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan

Background and Aim: Nonalcoholic fatty liver disease (NAFLD) has become one of the most common liver diseases worldwide. The factor which promotes progression of NAFLD, however, remains unclear. S100A8, an endogenous Toll-like receptor 4 agonist released from myeloid lineage cells, has been attracting attention because the protein can play a pivotal role in inflammatory diseases. The aim of this study is to investigate the involvement of S100A8 in the progression of NAFLD. Method: We utilized a lithogenic diet (LD) model of NAFLD. Six-week-old male C57/BL6 mice were fed with the LD or normal diet (ND) for 3 weeks. We also used liver tissue from the patients with NAFLD. Results: Immunohistochemical analyses showed that S100A8-positive area was observed in a part of non-parenchymal cells but not in hepatocytes. Total positive-area was significantly greater in the liver tissue from the patients with nonalcoholic steatohepatitis (n=41) than those from the patients with nonalcoholic fatty liver (n=7). LD-fed mice, but not ND-fed mice, displayed hepatitis with steatosis. S100A8-positive cells were observed in a part of hepatic leukocytes, and significantly greater in number in the livers of LD-fed mice compared with ND-fed mice. Flow cytometric analyses revealed that CD11b+ Gr-1high myeloid lineage cells significantly increased in the livers of LD-fed mice compared with ND-fed mice. Around 75% of CD11b+ Gr-1 high cells in the livers of LD-fed mice or ND-fed mice expressed intracellular S100A8. Collectively, S100A8-expressing CD11b+ Gr-1high cells accumulated in the livers of LD-fed mice. We next examined the role of S100A8 in our model. S100A8 significantly stimulated the production of CXCL1, a chemokine for myeloid lineage cells, as well as TNF-α from hepatic leukocytes in vitro. Hepatic leukocytes from the LD-fed mice, but not those from the ND-fed mice, spontaneously produced substantial amounts of CXCL1 as well as TNF-α in vitro. We then examined the expression of CXCR2, a chemokine receptor for CXCL1, in hepatic leukocyte. More than 75% of CXCR2-expressing cells in the livers of LD-fed mice or ND-fed mice were CD11b+ Gr-1 high cells. The number of CXCR2-expressing CD11b+ Gr-1high cells was significantly increased in the livers of LD-fed mice. Conclusion: The present study suggested that the accumulation of CD11b+ Gr-1 high cells in the livers of LD-fed mice led to the increased production of S100A8 that stimulates the production of CXCL1, which would result in the further accumulation of CD11b+ Gr-1 high cells via CXCR2. The amplification of S100A8-CXCL1 loop via CXCR2-expressing CD11b+ Gr-1high cells might be involved in the development of NAFLD.

Disclosures:

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.

The following people have nothing to disclose: Kaori Mukai, Takuya Miyagi, Kumiko Nishio, Yoshinobu Yokoyama, Teppei Yoshioka, Yoshinobu Saito, Tsukasa Kawaguchi, Satoshi Aono, Satoshi Tanaka, Satoshi Shimizu, Hayato Hikita, Ryotaro Sakamori, Yoshihiro Kamada, Tomohide Tatsumi, Naoki Hiramatsu

720

Protective effect of glutamine on the development of a Western style diet-induced non alcoholic steatohepatitis (NASH) in mice

Cathrin Sellmann1, Chengjun Jin1, Jean-Pascal De Bandt2, Ina Bergheim1;

1SD Modelsystems of molekular Nutrition, Friedrich-Schiller-University, Jena, Germany; 2Laboratoire de Biologie de la Nutrition, Université Paris Descartes, Paris, France

Background and aims: By now, non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases with still increasing prevalence in Westernized countries. Apart from genetic factors, possible risk factors for the development and progression of NAFLD include changes in the nutritional pattern e.g. a diet rich in fat and sugar like fructose and an impaired intestinal barrier function. Several studies have shown that a dietary supplementation of amino acids such as glutamine (Gln) may have positive effects on intestinal barrier function. The aim of the present study was thus to investigate the effect of an oral glutamine supplement on the development of a Western style diet (WSD)-induced non-alcoholic steatohepatitis (NASH) in a mouse model. Methods: C57BL/6J mice (n=6-8) were pair-fed either a modified standard liquid diet (control) or a liquid Western style diet (fortified with fructose, fat and cholesterol) for 6 weeks. Some of the mice were additionally fed Gln (2.09 g/kg bodyweight per day) in the diet. Indices of liver damage, hepatic inflammation (e.g. number of neutrophils, TNF-α and active PAI-1 protein levels) and parameters of glucose metabolism as well as portal endotoxin levels were measured. Results: As expected feeding a WSD leads to the development of steatosis and inflammation in the liver. Whereas Gln supplementation had only limited effects on hepatic fat accumulation and fasting glucose metabolism, it markedly suppressed inflammation (e.g. less inflammatory foci and lower number of neutrophils). Protein levels of TNF-α and active PAI-1 were significant lower in WSD fed mice treated with Gln when compared to mice only fed the WSD. Portal endotoxin levels did not differ between the two WSD groups. Conclusion: Taken together these data suggest that oral Gln supplementation has a protective effect against hepatic inflammation and thus on the development of WSD-induced NASH in our mouse model.

Disclosures:

The following people have nothing to disclose: Cathrin Sellmann, Chengjun Jin, Jean-Pascal De Bandt, Ina Bergheim

721

P53 Activity Controls Copper Homeostasis in Experimental Model of Hepatic Steatosis

Alessia Longo1, Mario Arciello2,1, Manuele Gori1, Barbara Bar-baro1, Carmela Viscomi1, Roberta Maggio1, Emidio Scarpellini1, Balsano Clara3,1;

1Laboratory of Molecular Virology and Oncology, Andrea Cesalpino Foundation, Roma, Italy; 2Dept. Internal Medicine and Medical Specialties, “Sapienza“ University of Rome, Roma, Italy; 3Institute of Molecular Biology and Pathology (IBPM) -CNR, Roma, Italy

Purpose: Nonalcoholic fatty liver disease (NAFLD) is a pathological condition that from simple steatosis can progress to Non-Alcoholic Steatohepatitis (NASH), which may lead to cirrhosis and hepatocarcinoma (HCC). The molecular mechanisms underlying NAFLD development have not been completely elucidated. Recently, p53 protein has been proposed as new player in NAFLD pathogenesis, because growing evidences highlight its relevance as metabolic modulator. One of its known target is Synthesis of Cytochrome c Oxidase 2 (SCO2). SCO2 is a copper (Cu) chaperone involved in the assembly of the mitochondrial complex IV and in the maintenance of copper homeostasis, and it is implicated in the cellular secretory pathway of this metal. Cu is essential for aerobic respiration and its altered balance may affect lipid metabolism and causes oxidative stress. Our study was performed in an in vitro model of steatosis in order to investigate the role of p53 in the modulation of Cu homeostasis in NAFLD, since it seems to play a role in NAFLD progression. Methods: HepG2 (wild-type p53, wt) and Huh7.5.1 cells (Y220C mutant p53) were cultured for 14 and 24 hours in a medium containing a solution of Free Fatty Acids (FFAs), oleic and palmitic acids in a 2:1 ratio (final concentration 0,5 mM). Intracellular lipids and cytotoxic effects were evaluated by AdipoRed and AlamarBlue assays. Intracellular Cu content was measured by Atomic Absorption Spectrometry. mRNA and protein expression of p53, its target genes and some genes involved in copper trafficking were analysed by qRT-PCR and Western blotting (WB), respectively. Results: In both cell lines, FFAs treatment produced an accumulation of intracellular lipid content. FFAs induced an up-regulation of p53 in HepG2 and Huh 7.5.1 after 14 hrs, whereas at 24 hrs we appreciated an up-regulation of the wt p53 but a down-regulation of the mutated one. Phosphorylated form of p53 and its target p21 have the same trend of p53 in both cell lines. In HepG2 cells FFAs did not altered the intracellular Cu content. On the contrary, the Huh 7.5.1 Cu amount was progressively reduced. The genes involved in Cu trafficking were also differentially modulated by FFAs. In HepG2, in fact, treatment induced an up-regulation of Cu secretory pathway at 14 hrs that is counter-balanced by the up-regulation of the gene responsible of Cu import in the cells, Ctr1, and a parallel down-modulation of the secretory genes at 24 hrs. This cellular response fails in Huh 7.5.1. Conclusions: Our data provide new insights into the mechanisms underlying p53 involvement in NAFLD pathogenesis, highlighting its role in the modulation of copper homeostasis.

Disclosures:

The following people have nothing to disclose: Alessia Longo, Mario Arciello, Manuele Gori, Barbara Barbaro, Carmela Viscomi, Roberta Maggio, Emidio Scarpellini, Balsano Clara

722

Dissociation of Hepatic Insulin Resistance with Severity of Steatosis in Diet-Induced Mice Model of Nonalcoholic Fatty Liver Disease

Akihiro Asai1,3, Pauline M. Chou2, Heng-Fu Bu1,3, Xiao Wang1,3, Xiao-Di Tan1,3;

1Pediatrics, Ann & Robert H. Lurie Children's Hospital of Chicago Research Center, Chicago, IL; 2Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL; 3Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, IL

Background: Insulin resistance (IR) plays a fundamental role in pathogenesis of non-alcoholic fatty liver disease (NAFLD). Systemic IR comprises hepatic (central) IR and muscle/adipose tissue (peripheral) IR. Fat deposition in hepatocytes is a primary pathological feature of NAFLD. It remains a major debate whether fat accumulation in hepatocyte interferes with insulin signaling pathway or intrinsic hepatic IR induces fat overstock by derangement of lipid metabolism. In the present study, therefore, we examined two common mouse strains for obesity, systemic and hepatic IR, and hepatic steatosis in response to high fat and high carbohydrate diets (HFHC diet). Method: C57BL/6J (B6) and DBA/2J (D2) mice were fed with HFHC diet for 1 6 weeks. Control groups of B6 and D2 mice were fed with regular chow diet. Liver tissues were processed for routine histology at 8, 12 and 16 weeks of feeding. The level of hepatic steatosis was assessed with Oil Red O staining of histological slides and measurement of triglyceride (TG) in liver tissues. After 16 weeks of HFHC diet, systemic IR was assessed with intraperitoneal glucose/insulin tolerance test and analyzing fasting serum glucose/insulin levels. Hepatic IR was determined by measuring levels of insulin-induced AKT phosphorylation in the liver with Western blotting. Results: After feeding with HFHC diet for 8, 12 and 16 weeks, both B6 and D2 mice developed obesity at similar degrees. Histological examination showed no inflammatory changes in livers of HFHC diet-fed mice in both strains. However, B6 mice exhibited severe pan-lobular steatosis at 12, and 16 weeks after HFHC diet. In contrast, D2 mice developed only mild peri-central steatosis throughout the treatment course. B6 mice with 16 weeks of HFHC diet showed profound hepatic TG level (3729.5 ± 304 mg/dl per 100mg tissue vs. D2: 1144.7 ± 90 mg/dl). Furthermore, HFHC diet-fed B6 mice showed severe fasting hyperglycemia with elevated serum insulin level. They showed greater HOMA-IR (B6:140 ± 16 vs. D2:78 ± 13) and glucose&insulin intolerance compared to HFHC diet-fed D2 mice. In contrast, the response to insulin-triggered hepatic AKT phosphorylation was strongly suppressed in both B6 (71 ± 7% reductions compared to chow fed control) and D2 (89 ± 5% reductions). Conclusion: Both strains developed severe hepatic IR after fed with HFHC diet for 16 weeks, despite B6 showing severe steatosis with greater peripheral IR and D2 exhibiting mild steatosis with minimal peripheral IR. Results suggest dissociation of hepatic IR from severity of hepatic steatosis. This model has an important potential to discover key regulatory factors in NAFLD.

Disclosures:

The following people have nothing to disclose: Akihiro Asai, Pauline M. Chou, Heng-Fu Bu, Xiao Wang, Xiao-Di Tan

723

Mouse non-alcoholic steatohepatitis livers up-regulate expression of T-cell regulatory gene PD-1 and LAG3

Kanji Yamaguchi1, Takeshi Nishimura1, Yuya Seko1, Hiroshi Ishiba1, Akira Okajima1, Yoshio Sumida1, Hironori Mitsuyoshi1, Kohichiroh Yasui1, Masahito Minami1, Takeshi Okanoue2, Yoshito Itoh1;

1 Gastroenterology, Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Hepatology Center, Saiseikai Suita Hospital, Osaka, Japan

Background: Recent reports have demonstrated the important role of the immunoreceptor, such as programmed death (PD)-1, in regulating immune responses in the liver. Here, to study the effect of T-cell immunodepression in senescence on the development of non-alcoholic liver disease (NAFLD) to more advance stage, the expression of T-cell regulatory molecules in murine non-alcoholic steatohepatitis (NASH) were examined. Methods: Eight-week-old male C57/BL6 mice were fed chow, high fat (HF) and high fat high cholesterol (HFHC) diets for 24 weeks. Hepatic and splenic lymphocytes were isolated by density gradient centrifuge with 35% isotonic Percoll solution after perfusion with 10 ml PBS via portal vein in each group of mice. Expression of PD-1, lymphocyte-activation gene (LAG) 3, cyto-toxic T-lymphocyte antigen (CTLA) 4 and CD25 on lymphocytes was analyzed by FACS at the time points of 8, 16, and 24 weeks in the livers, spleens and peripheral bloods. We also evaluated the markers of hepatic inflammation, steatosis and fibrosis at 24 weeks. Results: As evidenced by liver-body weight ratio, histological grading, serum transaminase levels, hepatic proinflammatory gene expression (TNF-alpha, Osteopontin, MIP1-alpha, MIP1-beta, MCP-1, RNATES, F4/80, CD11c and IP-10) and profibrogenic gene expression (Collagen I, alpha-SMA, TGF-beta), NASH was more severe in HFHC diet-fed mice compared to chow and HFD-fed mice. While CTLA4 and CD25 expression levels were no different between each group of mice, the number of PD-1 and LAG3 expressing T-cells was highest in HFHC diet-fed mouse livers and increased in a time and the degree of hepatic inflammation dependent manner. Furthermore, both HF and HFHC diets significantly induced hepatic PD-L1 mRNA at 24 weeks as previously reported. Conclusion: PD-1 and LAG3 expressing T-cells were increased in murine NASH livers with the elevation of hepatic PD-L1 expression. These regulatory signals may play a protective role against developing to NASH.

Disclosures:

The following people have nothing to disclose: Kanji Yamaguchi, Takeshi Nishimura, Yuya Seko, Hiroshi Ishiba, Akira Okajima, Yoshio Sumida, Hironori Mitsuyoshi, Kohichiroh Yasui, Masahito Minami, Takeshi Okanoue, Yoshito Itoh

724

Circulating miRNA in Coronary Artery Disease (CAD) and associated Non-Alcoholic Fatty Liver Disease (NAFLD)

Rohini Mehta1, Zahra Younoszai1, Heshaam M. Mir1, Bryan Raybuck2, Tadesse Gebreab1, Zobair M. Younossi1,2;

1Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA; 2Center for Liver Disease, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA

NAFLD is the hepatic manifestation of metabolic syndrome (MetS). CAD is the cardiac manifestation of MetS. In NAFLD, an increase in proinflammatory markers may contribute to the development of CAD. miRNAs are small non-coding RNAs constituting a new layer of regulatory control. The concept of miRNA's being shed into circulation with systemic effect is appealing. Circulating miRNAs may be probed and can serve as novel diagnostic/prognostic markers. Aim:To assess the expression profile of selected miRNA's in the plasma of patients undergoing cardiac catheterization for suspicion of CAD with or without NAFLD. Methods: Plasma samples were obtained after informed consent from 54 patients (BMI:29.0±4.74;Age:63.2±10.3) undergoing cardiac catheterization.Clinical data and liver ultrasound at the time of cardiac catheterization were used for the diagnosis of NAFLD. All coronary angiographies were read systematically and graded as normal, moderate and severe CAD.42 miRNA's were shortlisted.Total RNA was extracted from plasma, converted to cDNA and qPCR was performed using custom plates.Normalization was achieved by global normalization. Groupwise comparison of overall gene expression was carried out.Statistical analysis was done using Mann-Whitney and Spearman's correlation analysis.Results: Cardiac catheterization documented that CAD was present in 35 patients while 1 9 patients have normal coronary angiography. As compared to those without CAD, patients with CAD had downregulation of miR-195 (-1.6,p=0.03), while miR-29c (1.4,p=0.03) and miR30a (1.09,p=0.02) were upregulated. Subjects with CAD who also had NAFLD by ultrasound and clinical data were compared to those patients with CAD without NAFLD.This comparison showed that miR-122 (2.21,p=0.03) was upregulated, while miR-143 (-2.1,p=0.02) was downregulated. Spearman correlation analysis showed miR-143 (r=0.41 ,p=0.001), miR-199a-5p (r=0.3,p=0.02), miR-29c (r=0.3,p=0.02) and miR-30a (r=0.28,p=0.03) to be positively correlated with severity of CAD.miR-423-5p,miR-425,miR-451, miR-519d and miR-93 were not detectable (Ct>45) in the circulation. Conclusions: miR-195,miR-29c and miR-122 have been previously shown to be altered in tissue specific manner in CAD and steatohepatitis, respectively.miR-195 has been shown to be upregulated in cardiac hypertrophy and miR-122 to be downregulated in steatohepatitis. In our study circulating miRNAs show an opposite trend in expression compared to previously reported tissue specific expression patterns. While, additional studies are needed to determine the source and function of circulating miRNA, our findings indicate a system-wide deregulation in miRNA expression.

Disclosures:

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J and J; Consulting: Gilead Sciences

The following people have nothing to disclose: Rohini Mehta, Zahra Younoszai, Heshaam M. Mir, Bryan Raybuck, Tadesse Gebreab

725

Oleic Acid and Induction of Autophagy in Hepatocyte Model of Steatosis

Rohini Mehta1, Maria Keaton2, J. Michael Estep1, Aybike Birerdinc1,2, Ancha Baranova2, Zobair M. Younossi1,3;

1Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA; 2Center for the Study of Chronic Metabolic Diseases, George Mason University, Fairfax, VA; 3Center for Liver Disease, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA

Background: Steatosis, the hallmark feature of NAFLD, occurs with the accumulation of lipid droplets (LD) into the hepatocytes. Under conditions of high circulating levels of free fatty acid (FFA), an imbalance in lipid uptake, beta-oxidation and LD-dependent autophagy (lipophagy) develops. Inhibition of lipophagy in hepatocytes may trigger steatosis. So far, studies reporting the relationship between FFA and lipophagy induction are conflicting.Aim: To assess the relationship between lipid storage and lipophagy induction in the presence of high levels of FFA in cultured human hepatocytes.Methods: Primary human hepatocytes (ZenBio) were exposed to 500uM oleic acid for different time intervals (30min,2hrs, 12hrs and 24hrs).Total RNAs were extracted and cDNAs were synthesized. mRNA expression levels were assessed by qPCR using custom designed PCR panel.LD accumulation was monitored by fluorescence assay.Apoptosis was monitored using Caspase-Glo 3/7 assay.Fold changes were calculated relative to untreated cells.Results: LPL,a triglyceride hydrolase with a role in receptor-mediated lipid uptake,showed upregulation immediately after the treatment with FFA (fold upregulation range: 2.9 to 57.7),followed by downregulation at 24hrs (-1.3 fold). Notably,mRNAs encoding two other lipases,LIPC and LIPE,were suppressed at all timepoints with ranges:-1.3 to -5.5 and -1.6 to -4.1, respectively.mRNA encoding the lipid droplet associated protein PLIN1 was upregulated at time points up to 12hrs after treatment (range: 1.6 to 4.9); PLIN5 was upregulated at 2hrs (1.1) and 12hrs (2.2) post-exposure; an increase in expression of PLIN3 was observed only at 24hrs (3.6) post-exposure. Lipophagy specific marker LC3A was upregulated at 12hrs 4.93 folds, while LC3B was initially downregulated at 30 min (-1.93), followed by its upregulation at 2hrs-24hrs timepoints post-exposure (range:1.8 to 45.89).Among autophagy induction genes, ULK1 showed upregulation only at 2hrs (1.09) and 12hrs (1.64) timepoints, while ULK2 showed upregulation at 24hrs (2.61).mRNAs of autophagosome formation and elongation genes ATG13 and ATG5 were consistently downregu-lated post-exposure (range:-1.1 to -5.4). Assays for caspases 3 and 7 confirmed the absence of apoptosis.Conclusions: The increase in LPL and PLIN1 expression as early as 30 min post-exposure to FFA indicates immediacy of hepatocyte response to increase in FFA levels.This early response is not accompanied by simultaneous increase in lipophagy and apoptosis.The increase in LC3B 12hrs-24hrs post-exposure,but absence of upregulation of ATGs suggests that oleic acid is not an inducer of hepatocytic lipophagy, at least at the transcriptional level.

Disclosures:

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J and J; Consulting: Gilead Sciences

The following people have nothing to disclose: Rohini Mehta, Maria Keaton, J. Michael Estep, Aybike Birerdinc, Ancha Baranova

726

Different toll like receptors functionality and expression profiles in the development and course of NAFLD in morbidly obese patients

Maria Teresa Arias-Loste1, Paula Iruzubieta1, Emilio Fábrega1, Fernando Casafont1, Joaquin Cabezas1, Marcos López-Hoyos2, Lorena Álvarez2, David San Segundo2, Angel Estebanez-Gallo1, Agustín Dominguez-Diez3, Antonio López-Useros3, Javier Crespo1;

1Digestive, Marques de Valdecilla University hospital, Santander, Spain; 2Immunology, Marques de Valdecilla University hospital, Santander, Spain; 3General Surgery, Marques de Valdecilla University hospital, Santander, Spain

Background and aims Obesity has been associated with a low-grade chronic inflammatory state that leads to obesity-related comorbidity. This inflammation is mediated by different factors of innate immunity such as Toll-like receptors (TLR). Therefore, the aim of our study is to assess the different expression profiles and functionality of TLR in peripheral blood mononuclear cells (PBMCs) in morbidly obese (MO) patients before and after bariatric surgery (BS) and to correlate these results with the results of liver histology. Patients and methods Prospectively we have included 21 MO patients undergoing BS and 8 healthy controls. Blood samples have been collected immediately before BS and six months later and in 19 patients, liver biopsies were taken during the operation. TLR expression in PBMCs was analysed by flow cytometry and TLR function was determined by stimulating PBMCs in culture with specific ligands. The diagnosis of NAFLD was established according to Brunt classification. Results According to liver histology, 4 cases (21.0%) displayed a normal liver histology, 6(31.6%) simple steatosis (SS) and 9(47.4%) showed an inflammatory infiltrate (NASH). Significant differences were observed in the expression of different TLRs in B cells of MO patients compared to HC. TLR4 expression was lower in MO (p=0.02) whereas expression of TLR7 was higher (p=0.01). When patients were stratified according to liver histology, we found a higher expression of TLR4 in both B (p=0.05) and T cells (p=0.02) in patients with a histologic diagnosis of NASH, compared to those with SS. Circulating monocytes from MO patients showed significant differences compared to HC according to in vitro response to TLR4, with a decreased production of TNF (p=0.04) and IL1 (p=0.03), as well as to TLR2, with a decreased production of IL1 (p=0.009). Although differences did not reach statistical significance, there was a trend towards a higher production of TNF and IL6 in monocytes of NASH patients when compared to patients with SS after stimulation with specific TLR2 and TLR4 agonists. Expression profile of TLR changed overtime after bariatric surgery. In this sense, TLR4 expression in B cells was significantly higher 6 months after surgery (p=0.03), and the rate of increase was higher in patients with NASH(1 32%) com-pared to those with SS(14%). Functionality also changed over time and monocytes showed an increased production of IL6 (P=0.001) and IL6 (p=0.02) in response to TLR4 agonist after surgery. Conclusions These findings support a key role of TLR4 in PBMCs, and to some extent of TLR2 and 7, in both the patho-genesis and the disease evolution and response to treatment in NAFLD.

Disclosures:

Javier Crespo - Board Membership: MSD, Roche, Janssen, Gilead

The following people have nothing to disclose: Maria Teresa Arias-Loste, Paula Iruzubieta, Emilio Fábrega, Fernando Casafont, Joaquin Cabezas, Marcos López-Hoyos, Lorena Álvarez, David San Segundo, Angel Estebanez-Gallo, Agustín Dominguez-Diez, Antonio López-Useros

727

MicroRNAs as Mediators in the Pathogenesis of Non- Alcoholic Fatty Liver Disease and Steatohepatitis

Philip P. Becker1,6, Johannes Schmitt2, Beate Niesler3, Oliver Tschopp4, Thomas Dandekar5, Beat Mullhaupt1, Andreas Geier1,2;

1Department of Gastroenterology and Hepatology, University Hospital Zurich (USZ), Zurich, Switzerland; 2Department of Hepatology, University Hospital Wurzburg, Wurzburg, Germany; 3Institute of Human Genetics, University Hospital Heidelberg, Heidelberg, Germany; 4Department of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Zurich (USZ), Zurich, Switzerland; 5Bioinformatics, University of Wurzburg, Biocenter, Wurzburg, Germany; 6Zurich Center for Integrative Human Physiology (ZIHP), University Zurich (UZH), Zurich, Switzerland

Objectives: The biological mechanisms of non-alcoholic fatty liver disease (NALFD) and non-alcoholic steatohepatitis (NASH) are not entirely understood. Since MicroRNAs (miRNAs) have been discovered, scientists proofed a regulative function in various gene expression pathways. Aim of this study is to identify miRNAs that function as biomarkers and functionally relevant mediators of fatty liver disease. Methods: Liver tissue and clinical data of 19 patients with NAFLD/NASH and five healthy controls from the local Zurich biobank were assembled. A detailed functional metabolic characterization of the entire cohort was performed, to obtain homogeneous groups for further comparison. After isolation of total RNA, a miRNA expression assay was performed using a newly developed method in which 800 miRNAs are directly measured in a multiplexed assay without previous amplification. After normalization of data, statistical analysis was performed using Wilcoxon test for different group comparisons. Newly described miRNA candidates as well as those already associated with fatty liver disease were replicated by quantitative rtPCR. At the same time, a human gene expression array to investigate mRNA expression changes in miR target genes and involved signaling cascades was performed. Finally, expression changes and molecular interactions between miRNA and target genes are analyzed by mathematical network modeling. Results: Primary analysis showed significant p-values (p<0.05) after correction for almost 250 miRNAs in all calculations. 34 miRNAs from the patient group showed a significant difference of the mean compared to control (log2 expression (±0.8 to ±3.264)). Comparing the two different stages respective to signs of hepatic inflammation (NAFLD vs. NASH), a total number of 71 miRNAs with a significant difference were (0.0001 <=p>=0.042) found. Replication of quantification of newly assigned miRNAs with a distinct expression between NAFLD and NASH (e.g. miR-223-3p, 21-5p, 1915) and established reference miRNAs (e.g. miR-802, 122, 33a, 200a, 103, 107, 155) could be reproduced by rtPCR. Similarly, respective miRNAs were also analyzed in depth with regard to the development of type II diabetes. Patho-

physiological consequences on central metabolic and inflammatory signaling pathways are obtained by mathematical network modeling. Conclusions: Our study identifies functionally relevant miRNAs in liver tissue as mediators of central signaling pathways and clinically relevant pathophysiological events in fatty liver disease. The data point towards a role of certain miRNAs as potential prognostic biomarkers to monitor the progression of fatty liver disease.

Disclosures:

Beat Mullhaupt - Consulting: MSD, Novartis, Gillead, Janssen; Grant/Research Support: Bayer, Roche, MSD, Boehringer

The following people have nothing to disclose: Philip P. Becker, Johannes Schmitt, Beate Niesler, Oliver Tschopp, Thomas Dandekar, Andreas Geier

728

Wasabi derivative 6-methylsulfinylhexyl isothiocyanate, a potent Nrf2 activator, prevents the fatty liver produced by a high-fat diet but did not attenuate hepatic iron overload in mice

Yuji Tanaka, Toshinori Kamisako;

Dept. of Clinical Laboratory Medicine, Kinki University Faculty of Medicine, Osakasayama, Japan

The Nuclear factor E2-related factor 2 (Nrf2) transcription factor serves as a cellular sensor for oxidative and electrophilic stress. We have shown that Nrf2 deletion dysregulates hepatic mRNA expression of lipid and iron metabolism related genes, leading to increased hepatic triglyceride and iron levels. 6-methylsulfinylhexyl isothiocyanate (6-MSITC) is a bioactive ingredient present in Japanese horseradish, wasabi and has been shown as a potent activator of Nrf2 signaling. The purpose of this study was to determine whether 6-MSITC ameliorates hepatic steatosis and iron accumulation in mice fed a high-fat diet (HFD) with or without iron. Seven-week-old male wild-type mice (W), heterozygous Nrf2 mice (He), and Nrf2-null mice (K) were fed the following diets. Experiment 1; 1) control diet (containing 4% soybean oil) for 12 weeks (C) 2) HFD (containing 4% soybean oil and 31% lard) for 12 weeks (H) 3) HFD for 12 weeks plus 6-MSITC (10mg/Kg/day ip; 4 times/week) for the last 4 weeks (HM). Experiment 2; 1) HFD for 6 weeks followed by a HFD containing 1 % carbonyl iron for 6 weeks (HI) 2) HFD for 6 weeks followed by a HFD containing 1 % carbonyl iron for 6 weeks plus 6-MSITC for the last 4 weeks (HIM). After 12 weeks, blood samples and livers were obtained. In wild-type mice, UIBC tended to be decreased in the HWIM group compared to the HWI group. In Nrf2-null mice, serum total cholesterol, HDL, and LDL were increased and ALT was decreased in HKIM compared to HKI. In wild-type mice, hepatic triglycerides were higher in HW than CW and 6-MSITC inhibited the increase in hepatic triglycerides. Similar to wild-type mice, hepatic triglycerides were higher in HHe than CHe, and in HK than CK, but were not reduced by 6-MSITC, indicating that 6-MSITC reduced hepatic triglycerides via the Nrf2 pathway. In heterozygous mice, 6-MSITC decreased hepatic total cholesterol in HHeM compared to HHe. In Experiment 2, 6-MSITC did not block hepatic iron accumulation and malondialdehyde in any genotype. In wild-type mice, mRNA expression of the Nrf2 target genes NQO1 and GCLC were higher in HW than CW and were attenuated in HWM compared to HW, but were unchanged in heterozygous and Nrf2-null mice. The PPARγ target gene CD36 is involved in fatty acid uptake. PPARγ and CD36 mRNA were higher in HW than CW and tended to be attenuated in HWM, but were not changed in other genotypes. Interestingly, the pattern of gene regulation of PPARγ and CD36 is similar to that of NQO1. In Experiment 2, TfR1 and ferroportin1 mRNA were not altered by 6-MSITC. In conclusion, 6-MSITC decreased hepatic triglyceride levels by possibly inhibiting PPARγ and CD36, but did not reduce hepatic iron overload.

Disclosures:

The following people have nothing to disclose: Yuji Tanaka, Toshinori Kamisako

729

Integrated Omics Analysis of Human Liver Tissue Revealed a Causal Mechanism Underlying the Role of FADS1 in Hepatic Lipid Accumulation

Libo Wang1, Shaminie Athinarayanan2, Naga P. Chalasani3, Min Zhang1, Wanqing Liu2,3;

1Department of Statistics, Purdue Uni-veristy, West Lafayette, IN; 2Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN; 3Division of Gastroenterology and Hepatology, Indiana University School of Medicine, Indianapolis, IN

Background: Several recent genome-wide association studies (GWAS) have revealed that multiple polymorphisms located at the fatty acid desaturase gene 1 (FADS 1) locus were associated with various metabolic phenotypes including hepatic function, altered serum lipid levels and metabolic perturbations. We have also demonstrated in our previous study that increased hepatic FADS1 gene expression was negatively associated with hepatic fat content. FADS1 gene encodes an important enzyme delta-5 desaturase involved in the elongation and desaturation reaction cascade of polyunsaturated fatty acid (PUFA). However, questions remain unaddressed regarding whether these polymorphisms as well as the FADS1 gene are causal to the phenotypes, and if so what mechanism is underlying these significant genotype-phenotype associations. Aim: To explore the potential causal role of FADS1 genetic polymorphisms in hepatic lipid accumulation by integrating genomic, transcriptomic and lipidomic data obtained from human liver samples. Method and Materials: We performed an integrated eQTL (expression quantitative trait loci) and lipidomic analysis focused on the potential function of GWAS-identified FADS1 polymorphisms and/or their linkage disequilibrium proxies (n=6). These 6 SNPs (rs1 74576, rs1535, rs1 74546, rs1 02275, rs1 74537 and rs1 74556) were correlated with hepatic FADS1 gene expression in 206 human liver tissue samples from organ donors that were utilized previously in a genome-wide eQTL study. These SNPs were further correlated with levels of 169 individual lipid components and also with ratio of their saturated and unsaturated fractions. Results: We found that these SNPs (tagged by rs1 74556) across the FADS1 locus were significantly associated with decreased hepatic FADS1 gene expression (p=9.2E-4). These SNPs were also significantly associated with increased level of a phosphatidylinositol (C47H85O13P) (rs1 74556, p=2.7E-7). When the analysis was focused on the pair-wise ratios between hepatic lipids, each SNP was significantly correlated with increased ratios particularly between the more saturated phospholipids and relatively less saturated forms (e.g. rs1 74556 vs C47H85O13P/C45H79O13P, p=2E-8). Meanwhile, these SNPs were also associated with the total hepatic fat content measured in the livers (p=0.019 for rs1 74556). Conclusion: Our findings suggest a functional role of FADS1 SNPs in modulating hepatic fat accumulation by regulating the FADS1 activity in desaturating long-chain phospholipids. Our study highlights the power of systems biology to better understand causal mechanisms behind the genotype-phenotype associations noted by GWAS.

Disclosures:

Naga P. Chalasani - Consulting: Salix, Abbott, Merck, Lilly, Enterome, Aegerion; Grant/Research Support: Intercept, Lilly, GenFit, Gilead, Enterome, Cumberland, Galectin

The following people have nothing to disclose: Libo Wang, Shaminie Athinarayanan, Min Zhang, Wanqing Liu

730

Dietary iron overload results in hepatic reticuloendothelial system cell iron accumulation, oxidative stress, mitochondrial dysfunction, immune cell activation, and nonalcoholic steatohepatitis in obese, diabetic mice

Priya Handa1,2, Vicki Morgan-Stevenson1,2, James E. Nelson1,2, Bryan D. Maliken1,2, Matthew M. Yeh3, Kris V. Kowdley1,2;

1Liver Center of Excellence, Digestive Disease Institute, Seattle, WA; 2Benaroya Research Institute, Seattle, WA; 3University of Washington, Seattle, WA

Aim: The goal of this study was to determine the effect of dietary iron overload on development of nonalcoholic steatohepatitis (NASH) in an obese and diabetic mouse model. Methods: Leptin-receptor deficient mice (leprdb/db) were fed a normal or an iron- supplemented chow for 8 weeks. They were then switched to a normal chow diet for 8 more weeks. After 16 weeks, blood and liver tissue were collected. Histological features of NASH and iron deposition were graded by a hepatopathologist following the NASH CRN scoring criteria. Liver transaminase levels, glucose and iron parameters were measured in serum; hepatic triglyceride levels, malondialde-hyde (MDA) and iron were assessed in liver tissue. Liver expression of genes related to lipid metabolism, macrophage and T-lymphocyte signatures, oxidative stress and inflammation were determined by qRT-PCR, normalized to GAPDH. Results: After 16 weeks, body mass was significantly lower in the dietary iron (DI) fed mice compared to the chow fed group. Liver histology in the dietary iron group (DI) revealed a high degree of ballooning (88%), and preferential reticuloendothelial system (RES) iron deposition pattern. 75% of the DI mice developed NASH relative to chow-fed controls (20%). Serum levels of glucose, AST, ALT, ferritin, hepatic iron and hepcidin mRNA (3 fold) were significantly increased in the DI group compared to chow fed controls (p<0.05). Hepatic MDA levels (p<0.03) and HO-1 mRNA (p<0.05) were elevated in the DI group, while gene expression levels of the anti-oxidant SOD1 (p=0.06) were diminished. Hepatic gene expression levels of lipolytic genes such as LPL (p=0.01), ACOX (p=0.02) and CPT1α (p<0.05) showed a marked decrease in DI mice compared to controls. Gene expression levels of PGC1α (p=0.01) and NRF1 (p=0.03) were also lower in the DI mice, suggesting diminished mitochondrial biogenesis. Hepatic gene expression levels of CD11c, CD4, CD8, IFN-gamma, INOS, IL-6, TNF-α and TLR4 were upregulated in the dietary mice (all p<0.05), while levels of the M2 macrophage marker Mgl1 (p=0.03) were significantly reduced. Conclusions: Dietary iron excess frequently leads to NASH in leprdb/db mice and is associated with increased inflammatory activation of hepatic immune cell populations, increased oxidative stress, impaired mitochondrial biogenesis and preferential trafficking of the iron to the hepatic macrophages. These data provide evidence for a role of RES iron loading in the pathogenesis of NASH associated with obesity and diabetes via oxidative stress, immune activation and impaired mitochondrial gene expression.

Disclosures:

Kris V. Kowdley - Advisory Committees or Review Panels: Abbott, Gilead, Merck, Novartis, Vertex; Grant/Research Support: Abbott, Beckman, Boeringer Ingel-heim, BMS, Gilead Sciences, Ikaria, Janssen, Merck, Mochida, Vertex

The following people have nothing to disclose: Priya Handa, Vicki Morgan-Stevenson, James E. Nelson, Bryan D. Maliken, Matthew M. Yeh

731

Comparison of hepatic and peripheral blood lymphocyte gene expression in non-alcoholic fatty liver disease

Samer Gawrieh1, Eugene Drigalenko2, Jack Littrell3, Richard Komorowski4, James R. Wallace5, Matthew I. Goldblatt5, Harald H. Goring2, Michael Olivier3;

1Gastroenterology and Hepatology, Medical College of Wisconsin, Milwaukee, WI; 2Genetics, Texas Biomedical Research Institute, San Antonio, TX; 3Physiology, Medical College of Wisconsin, Milwaukee, WI; 4Pathology, Medical College of Wisconsin, Milwaukee, WI; 5Surgery, Medical College of Wisconsin, Milwaukee, WI

Background: Significant changes in hepatic gene expression are noted in patients with non-alcoholic fatty liver disease (NAFLD). We aimed to determine if significant changes in gene expression also exist in peripheral blood lymphocytes (PBL) of obese patients with NAFLD, and to describe patterns of gene expression changes associated with NAFLD in both liver and PBL (concordant/discordant). Methods: PBL were collected prior to subjects undergoing bariatric surgery. Fresh frozen liver tissue was obtained by intraoperative liver biopsy. Liver biopsies for standard pathology evaluation were read by an expert pathologist to define the histological phenotype and grade the severity of NAFLD. The Illumina Human-6 Expression Bead-Chips which allow interrogation of almost 48,000 transcripts, were used to detect gene expression in both tissues. After standardization of data, gene expression profiles were compared between NAFLD patients and controls in liver and PBL. Results: 54 Caucasian subjects were included (26 NAFLD, 28 obese controls with normal liver histology). No significant differences were noted between NAFLD and control in age (45 vs 41 years), gender (3 vs 0 males), BMI (49 vs 48 kg/m2), TNF-α (8.7 vs 8.2 pg/ml), and IL-6 (11 vs 8.5 pg/ml) but ALT (29 vs 18 U/L), triglycerides (170vs 124 mg/dL), and HOMA (12.2 vs 5.9) were significantly different (p<0.05). In total, 869 transcripts showed significant differences between NAFLD and control, 717 in liver and 159 in PBL (p<0.01). Of these 7 were significant in both tissues (table), and 5 of these (SERPINE2, NT5DC3, BBS2, MTHFD1L, SPON1) had expression changes in the same direction (upregulated in NAFLD in both tissues). Overall, for transcripts significant in at least 1 tissue, direction (increase or decrease in NAFLD) agreed between tissues for 436. Conclusions: NAFLD is associated with significant changes in PBL gene expression. Interestingly, many transcripts demonstrate concordant changes in levels both in liver and PBL. Effects of interventions on hepatic gene expression may be inferred in a less invasive way by assessing corresponding changes in PBL transcript levels for genes that consistently show concordant or discordant changes between the two tissues.

Table 2. 
GeneLiverPBL
 pRegression coefficientpRegression coefficient
SERPINE20.00090.2250.0030.204
NT5DC30.0050.1910.0050.196
BBS20.00050.2280.0020.206
MTHFD1L0.00030.2500.0030.201
SPON10.0010.2150.0070.194
AGPAT4-IT10.00050.2350.004-0.201
MAMDC40.00010.2510.0009-0.23

Disclosures:

The following people have nothing to disclose: Samer Gawrieh, Eugene Drigalenko, Jack Littrell, Richard Komorowski, James R. Wallace, Matthew I. Goldblatt, Harald H. Goring, Michael Olivier

732

Crosstalk between the liver and intestine contributes to NASH pathogenesis

Jay Luther1, John Scichilone2,3, John Garber1, Hamed Khalili1, Lee F. Peng1, Stephane Chevaliez1, Raymond T. Chung1, Suraj J. Patel2,3;

1Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA; 2Surgery, Massachusetts General Hospital, Boston, MA; 3Research, Shriner's Hospital, Boston, MA

While the simple steatosis seen in non-alcoholic fatty liver disease (NAFLD) is benign, progression to non-alcoholic steato-hepatitis (NASH) increases morbidity and mortality. Accordingly, understanding of the dysregulated inflammatory response seen in NASH is critical. Emerging data suggest a role for the gut-liver axis in NASH pathogenesis (Henao-Mejia J et al., Nature 2012); however, the extent and mechanism of this interaction is uncertain. To further examine the extent of intestinal changes seen in NAFLD patients, we performed a meta-analysis of the literature to compare the rates of increased intestinal permeability in patients with versus without NAFLD. For inclusion, a study had to multiple criteria set a priori by the study team. Overall, four clinical studies (N=162 patients) were included in the analysis. In total, 41.9% of patients with NAFLD had evidence for increased intestinal permeability, whereas 10.1 % of healthy controls had increased intestinal permeability. To further understand this clinical observation, we studied changes in intestinal permeability induced in a NASH murine model. Mice fed a methionine-choline-deficient (MCD) diet exhibited more severe hepatic inflammation, as evidenced by significantly increased levels of serum ALT/AST and NAFLD-activity score on histology, compared to standard chow (SC)-fed mice. MCD-fed mice also had higher levels of TNF-α and IL-6 (cytokines known to increase intestinal permeability) in the liver and serum. The differences in ALT/AST and cytokine profile appeared as early as 6 days into the 21 -day diet. Furthermore, MCD-fed mice had higher levels of FITC-dextran (orally gav-aged prior to sacrifice) in their portal vein serum as compared to SC-fed mice, reflecting increased paracellular permeability. MCD-fed mice also had higher levels of lipopolysaccrhide in the portal vein serum as compared to SC-fed mice, suggesting increased intestinal microbial translocation. Last, IHC staining for tight junction proteins revealed a higher degree of tight junction disruption in the MCD-fed mice compared to the SC-fed mice. Interestingly, the difference in these markers for intestinal permeability did not appear until 14 days into the diet, well after significant liver injury was noted. Taken together, these data suggest the changes in intestinal permeability seen in NASH follow liver injury and maybe the result of a liver-driven increase in TNF-α and IL-6. Moreover, intestinal permeability changes may further contribute to worsening liver injury via increased microbial product translocation. Further investigation into this liver-gut crosstalk in NASH pathogenesis is needed.

Disclosures:

Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead

Suraj J. Patel - Stock Shareholder: Heprotech

The following people have nothing to disclose: Jay Luther, John Scichilone, John Garber, Hamed Khalili, Lee F. Peng, Stephane Chevaliez

733

Multiple organ crosstalks for hepatic steatosis: roles of myokine (FGF2, myostatin, and irisin)

Motoyuki Kohjima1, Tsuyoshi Yoshimoto1, Tsukasa Nakamura1, Shinichi Tsuruta1, Tomoko Ohashi1, Kunitaka Fukuizumi1, Nao Fujimori1 , Ken Kawabe1, Kazuhiro Haraguchi1, Akira Aso1, Yorinobu Sumida1, Naohiko Harada1, Tomoki Ryu2, Yoshiyuki Wada2, Yuko Takami2, Hideki Saitsu2, Tohru Utsunomiya3, Mitsuo Shimada3, Kazufumi Dohmen4, Hideyuki Nomura5, Munechika Enjoji6, Makoto Nakamuta1;

1 Gastroenterology, Clinical Research Center, Kyushu Medical Center, National Hospital Organization, Fukuoka, Japan; 2Hepato-Biliary-Pancreatic Surgery, Clinical research center, Kyushu Medical Center, National Hospital Organization, Fukuoka, Japan; 3Digestive and Pediatric Surgery, Tokushima University, Tokushima, Japan; 4Internal Medicine, Chihaya Hospital, Fukuoka, Japan; 5Internal Medicine, Shin-Kokura Hospital, Kitakyushu, Japan; 6Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan

Multiple organ crosstalks for hepatic steatosis: roles of myokine (FGF2, myostatin, and irisin) Background: The liver has a critical role in metabolism. Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver function tests, and it is characterized by lipid accumulation in hepatocytes. NAFLD is often accompanied with obesity, hyper-lipidemia, diabetes, and/or, insulin resistance, which are simultaneously affect multiple organs. Multiple organ relationship such as between visceral fat, subcutaneous fat, muscle, and liver could be a key for lipid accumulation in the hepatocyte. Here we focused on myokines, hormones or cytokines secreted from muscle cells, and examined the organ crosstalk between muscles and other tissue. Methods: We collected visceral fat, subcutaneous fat, muscle, and liver tissue from surgery patients granted permission (n=139) and extracted mRNA from the tissue. HepG2 cells were cultured in medium supplemented with FGF2 and lipid metabolism-associated genes were evaluated after 24 h. Expression of the genes was analyzed using quantitative PCR analysis. All experimental procedures were carried out under a protocol approved by ethical committee at Kyushu Medical Center. Results: Genes involved in lipid metabolism in each organ had positive relationship with the genes with different tissue. FGF2 expression in rectus muscle was significantly correlated with FAS and SREBP1c expression in the liver (r =0.4421, p <0.0001, and r =0.5799, p <0.0001, respectively). Furthermore, the expression of CD36 and PPARγ in the liver was strongly associated with muscle FGF2 expression (r =0.1991, p =0.0482, and r =0.3513, p =0.0005, respectively). The expression of myostatin and FNDC5 (a precursor protein of irisin) in muscle had strong relationship with adipose mass volume (r =0.2184, p =0.046 and r =0.2586, p =0.0059, respectively), while no mutuality could be detected with the lipid metabolism-related genes in the liver. Patients with liver steatosis had higher serum FGF2 level than patients without steatosis, and serum FGF2 correlated significantly with Wnt2a mRNA expression in visceral and subcutaneous fat. Furthermore, FGF2 showed stimulative effect on the hepatic fatty acid synthesis and enhanced expression of ROR2 in HepG2 cells. Conclusion: It has been recently reported that up-regulated muscle FGF2 expression by aging results in breaking the quiescence of muscle stem cells and taking away their self-renewing capacity, leading age-related sarcopenia. It is possible muscle FGF2 is involved in Wnt signaling pathway and directly promote fatty acid synthesis in the liver.

Disclosures:

Mitsuo Shimada - Grant/Research Support: TSUMURA & CO, Japan

The following people have nothing to disclose: Motoyuki Kohjima, Tsuyoshi Yoshimoto, Tsukasa Nakamura, Shinichi Tsuruta, Tomoko Ohashi, Kunitaka Fukuizumi, Nao Fujimori, Ken Kawabe, Kazuhiro Haraguchi, Akira Aso, Yorinobu Sumida, Naohiko Harada, Tomoki Ryu, Yoshiyuki Wada, Yuko Takami, Hideki Saitsu, Tohru Utsunomiya, Kazufumi Dohmen, Hideyuki Nomura, Munechika Enjoji, Makoto Nakamuta

734

Simvastatin decreases fibrosis in a new rodent model of NASH due to Ras/ERK-inhibition

Robert Schierwagen1, Lara Maybüchen1, Sabine Klein1, Kanishka Hittatiya5, Jogchum Plat4, Dieter Luetjohann3, Tilman Sauerbruch1, Sebastian Zimmer2, Jonel Trebicka1;

1Internal Medicine I, University of Bonn, Bonn, Germany; 2Department of Internal Medicine II, University of Bonn, Bonn, Germany; 3Department of Clinical Pharmacology, University of Bonn, Bonn, Germany; 4Department of Human Biology, University of Maastricht, Maastricht, Netherlands; 5Institute of Pathjology, Unviersity of Bonn, Bonn, Germany

Introduction: Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum from simple steatosis to steatosis with necroin-flammatory changes (non-alcoholic steatohepatitis, NASH). The latter may lead to progressive fibrosis and cirrhosis. Due to their pleiotropic effects (inhibition of small GTPases RhoA, Ras and Rac1) statins beneficially influence vascular dysfunction in metabolic syndrome besides lowering cholesterol. Since the role of small GTPases in NASH is still unknown, we elucidated their effects and tested Simvastatin (SMV) as a potential therapy. Methods: ApoE-/- and wild type mice were fed for 7 weeks with diet rich in fat and cholesterol (western diet; WD), whereas control groups received normal chow. Activated SMV (inhibits RhoA/Ras/Rac1), NSC23766 (Rac1-inhibitor) or Clostridium sordellii lethal toxin (RhoA-/Rac1-inhibitor) were administered using subcutaneous osmotic mini pumps over 6 weeks. Steatosis was analyzed using Oil-Red-O-staining and by determination of hepatic cholesterol in analogue liver segments. Hepatic fibrosis was analyzed by αSMA-immunohistochemistry, Sirius-red-staining and hydroxyproline determination. Protein expression and phosphorylation was assessed by Western blot. Results: ApoE-/- mice receiving WD showed, besides metabolic syndrome, full-blown NASH including increased steatosis, fibrosis and inflammation in the liver, which was not found in wild type animals. SMV treatment in ApoE-/- mice significantly decreased fibrosis, without any effect on inflammation and steatosis of the liver. Total cholesterol remained unchanged in the liver after SMV treatment. SMV elicited these effects, at least in part, due to decreased ERK phosphorylation. Interestingly, the inhibition of Rac1 and RhoA or Rac1 alone could not decrease fibrosis to the same extent as simvastatin, which inhibits Ras, RhoA and Rac1. Discussion: Here we describe a NASH model in rodents with distinct steatosis, inflammation and fibrosis. Simvastatin decreased fibrosis probably mainly due to inhibition of Ras/ERK pathway. Patients receiving statins should be tested for concomitant benifical effects on fatty liver disease.

Disclosures:

The following people have nothing to disclose: Robert Schierwagen, Lara Maybüchen, Sabine Klein, Kanishka Hittatiya, Jogchum Plat, Dieter Luetjohann, Tilman Sauerbruch, Sebastian Zimmer, Jonel Trebicka

735

Oral treatment with PBI-4050, a novel first-in-class antifibrotic compound, reduces hepatic steatosis, and improves kidney function in the diabetic db/db mouse model

Lyne Gagnon, Lilianne Geerts, François Sarra-Bournet, Mikaël Tremblay, Kathy Hince, Shaun Abbott, Jean-Simon Duceppe, Bou-los Zacharie, Christopher Penney, Pierre Laurin, Brigitte Grouix;

ProMetic BioSciences Inc., Laval,QC,Canada

Introduction and Aims: Type 2 diabetes is strongly associated with nonalcoholic steatohepatitis (NASH). Also, severity of NASH histology (i.e., fibrosis stage) is associated with decreased kidney function. Obese and diabetic db/db mice develop hepatic steatosis, and exhibit a consistent and robust increase in albuminuria and mesangial matrix expansion in the kidney. PBI-4050, a potential first-in-class treatment for fibrotic diseases, possesses a pleiotropic mechanism of action with anti-inflammatory, anti-oxidant and anti-fibrotic properties. The aim of this study was to investigate the effect of PBI-4050 on hepatic steatosis and on inflammatory/fibrotic markers in liver and kidney in uninephrectomized (NX) diabetic (db/db) mice. Methods: Total nephrectomy of the right kidney was performed on day 0 and animals were treated with vehicle or PBI-4050 (1 00 mg/kg, oral once a day) from day 1 through 130. Hepatic steatosis and liver fibrosis were examined. Liver and kidney fibrosis were assessed by qPCR of inflammatory/fibrotic and remodeling markers (IL-6, TGF-β1, collagen I, TIMP-1 and MMP-2). Results: Hepatic steatosis, observed in NX-db/db mice, was significantly reduced (50%) with PBI-4050-treatment. Furthermore, qPCR analysis of NX-db/db mice indicated that PBI-4050 treatment resulted in a significant reduction of the expression of TGF-β1, collagen I, TIMP-1 and MMP-2 in liver tissue. Kidney function was measured by albuminuria and pro-teinuria, waminotransferasehich were increased by 1.5 to 2 fold in NX-C57BL/6 and NX-db/db compared to sham C57BL/6 mice. Treatment with PBI-4050 reduced albuminuria and proteinuria to the sham C57BL/6 mice level. NX-db/db mice demonstrated a significant increase in GFR (hyperfiltration), which was significantly reduced by treatment with PBI-4050. Mesangial lesion scores were also reduced in NX-db/db mice treated with PBI-4050. Lipid peroxidation (TBARS) in kidney was increased in NX-db/db mice and was reverted to the control level by treatment with PBI-4050. IL-6, collagen I, TIMP-1 and MMP-2 in kidney tissue were reduced in PBI-4050-treated NX-db/db mice. Conclusions: Taken together, these results suggest that PBI-4050 offers the potential as a novel therapy for hepatic steatosis and diabetic nephropathy.

Disclosures:

Lyne Gagnon - Management Position: ProMetic BioSciences Inc.

Lilianne Geerts - Employment: Prometic Biosciences inc; Stock Shareholder: Prometic Biosciences inc

François Sarra-Bournet - Employment: ProMetic BioSciences Inc.; Stock Shareholder: ProMetic BioSciences Inc.

Mikaël Tremblay- Employment: Prometic Biosciences Inc. Kathy Hince - Employment: ProMetic BioSciences Inc Shaun Abbott -Employment: ProMetic BioSciences Inc. Jean-Simon Duceppe - Employment: ProMetic BioSciences Inc.

Boulos Zacharie - Management Position: ProMetic BioSciences Inc.; Stock Shareholder: ProMetic Life Sciences Inc.

Christopher Penney - Advisory Committees or Review Panels: ProMetic BioSciences Inc.

Pierre Laurin - Management Position: ProMetic BioSciences Inc.; Stock Shareholder: ProMetic Life Sciences Inc.

Brigitte Grouix - Employment: ProMetic BioSciences

736

The association between serum levels of apoptosis inhibitor of macrophage and the clinical features of nonalcoholic fatty liver disease

Kohei Oda1, Hirofumi Uto1, Seiichi Mawatari1, Kazuaki Tabu1, Kaori Ohno1, Eriko Toyokura1, Akihiko Oshige1, Dai Imanaka1, Kotaro Kumagai1, Tsutomu Tamai1, Akihiro Moriuchi1, Makoto Oketani1, Akio Ido1, Yoshio Sumida2,3, Hirohito Tsubouchi4;

1Digestive and Lifestyle Diseases, Department of Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; 2Center for Digestive and Liver Diseases, Nara City Hospital, Nara, Japan; 3Department of Gastroenterology and Hepatology, Kyoto Prefec-tual University of Medicine, Kyoto, Japan; 4Department of HGF Tissue Repair and Regenerative Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

Objective: Apoptosis inhibitor of macrophage (AIM) was initially identified as supporting the survival of macrophages against various apoptosis-inducing stimuli, and has been reported to decrease lipid droplets in adipocytes via inhibition of fatty acid synthase activity. In addition, increases in the blood levels of AIM have been associated with insulin resistance and AIM is thought to be involved in the progression of metabolic syndrome. While nonalcoholic fatty liver disease (NAFLD) is closely related to insulin resistance and metabolic syndrome, the impact of AIM on the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has not been investigated. The aim of this study was to elucidate the association between AIM and NAFLD. Methods: Eighty-four patients with biopsy-proven NAFLD, including 67 patients with nonalcoholic steatohepatitis (NASH) and 17 with nonalcoholic fatty liver (NAFL), were enrolled in this study. Serum levels of AIM were determined by ELISA. Results: Fifty patients were female, and the total group had a mean age of 61 years. Serum aspartate aminotransferase (NASH vs. NAFL: 61 vs. 48 IU/L; P=0.02), serum hyaluronic acid (104 vs. 42 ng/mL; P<0.001) and type IV collagen 7S <i>(5.5</i> vs. 3.8 ng/mL; P<0.001) were higher and platelet counts (19.6x104 vs. 24.4x104 <i>/μL,</i> P<0.01) were lower in patients with NASH vs. those with NAFL. Furthermore, serum AIM levels were significantly higher in patients with NASH compared with those with NAFL (1437 vs. 1006 ng/mL; P=0.03). Serum AIM levels also correlated significantly with hepatic fibrosis markers, i.e., platelet counts (r=-0.40, P<0.001), hyaluronic acid (r=0.33, P<0.01) and type IV collagen 7S (r=0.25, P=0.03), and tended to correlate with the degree of hepatic fibrosis, as assessed by histological examination (r=0.20, P=0.08). By contrast, there was a significant negative correlation between serum AIM levels and the degree of hepatic steatosis (r=-0.28, P=0.01). Conclusions: Serum AIM levels were higher in the cases of advanced hepatic fibrosis in patients with NAFLD, but they were lower in the case of severe hepatic steatosis. These results indicate that AIM plays important roles in the development of NAFLD/NASH in both advancing and inhibiting disease progression.

Disclosures:

Makoto Oketani - Grant/Research Support: Bristol-Myers Squibb

Hirohito Tsubouchi -Grant/Research Support: MSD, Chugai Pharmaceutical, Kan Research Institute, Daiichi-Sankyo, Eisai, Tanabe Mitsubishi; Speaking and Teaching: MSD, Tanabe Mitsubishi

The following people have nothing to disclose: Kohei Oda, Hirofumi Uto, Seiichi Mawatari, Kazuaki Tabu, Kaori Ohno, Eriko Toyokura, Akihiko Oshige, Dai Imanaka, Kotaro Kumagai, Tsutomu Tamai, Akihiro Moriuchi, Akio Ido, Yoshio Sumida

737

Kupffer Cell Derived MCP-1 Induces Hepatic Steatosis by Up-regulation of SREBP1 in High Fructose and Marginal Copper Deficient Diet Fed Rats

Ming Song1, Dale Schuschke2, Zhanxiang Zhou5, Jiayuan Zhang4, Xiang Zhang4, Yuhua Wang1, Wenke Feng1, William M. Pierce3, Craig J. McClain1,6;

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Physiology, University of Louisville, Louisville, KY; 3Pharmacology, University of Louisville, Louisville, KY; 4Chemistry, University of Louisville, Louisville, KY; 5Nutrition, UNC-Greensboro, Greensboro, NC; 6Robley Rex VA Medical Center, Louisville, KY

Background/Aims: The interaction of dietary marginal copper deficiency and fructose consumption leads to liver injury and fat accumulation, likely due to hepatic iron overload. Excess iron in the liver secondary to copper deficiency distributes throughout hepatocytes and Kupffer cells (KCs). The aim of this study was to examine the role of KC in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), and the role of cell type-specific iron deposition on disease progression. Methods: Male weanling Sprague-Dawley rats were fed either an adequate copper or a marginally copper deficient diet for 4 weeks. Deionized water or deionized water containing 30% fructose (w/v) was also given ad lib. KCs were depleted by intravenous administration of gadolinium chloride (GdCl3) before and/or in the middle of experiment. Results: Hepatic triglyceride accumulation, but not liver injury, was completely eliminated with KC depletion in rats with marginal copper deficiency plus a high fructose diet (CuMF). However, hepatic copper and iron content was not significantly affected with KC depletion. Increased hepatic sterol regulatory element binding protein-1 (SREBP-1) expression, as well as elevated plasma monocyte chemoattractant protein-1(MCP-1), were abolished by KC depletion in CuMF rats. Moreover, MCP-1 was robustly increased in the culture medium when isolated KCs from CuMF rats were treated with LPS, and this increase was significantly reversed by pretreatment with a lysosomal iron chelator. In addition, increased hepatocyte iron and decreased spleen iron were observed to be associated with hepcidin deficiency in CuMF rats, whereas KC iron was not significantly changed. Conclusion: Our data suggest that KC/MCP-1 signaling mediated hepatic lipogenesis likely contributes to fat accumulation, whereas hepatocyte iron overload contributes to liver injury in this CuMF-induced NAFLD model.

Disclosures:

William M. Pierce - Board Membership: Pradama, Inc.

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: Ming Song, Dale Schuschke, Zhanxiang Zhou, Jiayuan Zhang, Xiang Zhang, Yuhua Wang, Wenke Feng

738

Serum Endocan is Associated with Histologic Severity in Patients Nonalcoholic Fatty Liver Disease

Elzafir Elsheikh1,2, Zahra Younoszai1,2, Munkhzul Otgonsuren1 ,2 Sharon L. Hunt1,2, Zachary D. Goodman1, Zobair M. Younossi1 ,2;

1 Center for Liver Disease, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA; 2Betty and Guy Beatty Center for Integrated, Inova Health System, Falls Church, VA

Background: The spectrum of nonalcoholic fatty liver disease (NAFLD) is associated with both progressive liver disease and cardiovascular diseases (CVD). Endocan is an anti-inflammatory adipokine that inhibits leukocyte adhesion and migration through the endothelium. Aim: To assess the relationship between severity of liver disease and endocan levels in well-characterized group of patients with biopsy-proven NAFLD. Methods: The study cohort consisted of consecutive patients with biopsy-proven NAFLD. Endocan (ng/ml) concentrations were determined in the serum collected at the time of liver biopsy using ELISA technique. Results: Of the study cohort (N=93), 51% had histologic NASH and 1 1% had advanced fibrosis. Furthermore, 33% had type-2 diabetes (DM). Serum endocan level increased with grade of hepatocyte ballooning (none: 0.59 ± 0.07; few: 0.68 ±0.14; and many ballooning: 1.33 ± 0.44; p=0.002) and portal inflammation (no inflammation: 0.52 ± 0.11; moderate inflammation: 0.66 ± 0.08; and severe inflammation: 1.04 ± 0.29; p=0.01 9). More importantly NAFLD patients with advance fibrosis had higher serum endocan level as compared to NAFLD patients with minimal or no fibrosis (1.23±0.28 vs. 0.59±0.07, p= 0.014). In fact, levels of serum endocan was significantly associated with the risk of advance fibrosis [OR: 2.57 (95% CI: 1.20-5.49)]. There were no associations with DM. Conclusions: In patients with NAFLD, the histologic severity as indicated by highere stages of fibrosis and higher grade of inflammation is associated with elevated level of serum endocan. This data implicates serum endocan as a possible biomarker in patients with NAFLD.

Disclosures:

Zachary D. Goodman - Grant/Research Support: Gilead Sciences, Fibrogen, Galectin Therapeutics, Merck, Vertex

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J and J; Consulting: Gilead Sciences

The following people have nothing to disclose: Elzafir Elsheikh, Zahra Younoszai, Munkhzul Otgonsuren, Sharon L. Hunt

739

The prebiotic oligofructose protects against enhanced liver injury caused by arsenic in a model of NASH

Veronica L. Massey1,3, Robin H. Schmidt1,3, Min Tan2,3, Walter Watson2,3, Gavin E. Arteel1,3;

1 Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY; 2Medicine, University of Louisville School of Medicine, Louisville, KY; 3Univer-sity of Louisville Health Sciences Center, Louisville, KY

Background. Arsenic (As), a ubiquitous drinking water contaminant, tops the ATSDR list of hazardous environmental chemicals and is known to cause liver injury. Although the concentrations of As found in the US water supply are generally too low to directly damage the liver, this group recently showed that sub-hepatotoxic doses of As sensitize the liver to experimental NAFLD caused by a high fat diet (HFD). It is now strongly suspected that an altered GI tract microbiome plays an important role in development of NALFD. Indeed, prebiotics (oligofructose; OFC), protect against experimental NAFLD, and correlate with repletion of commensal bacteria (e.g., Bifidobacteria spp.) in the GI tract. Arsenic has been shown to be bacteriostatic in vitro, and may therefore also affect GI tract bacteria. The purpose of the current study was to determine the effect of As exposure on key commensal bacteria in the GI tract, and to test the hypothesis that the OFC protects against enhanced liver injury caused by As in experimental NAFLD. Methods. Male C57Bl6/J mice were fed low fat diet (LFD), high fat diet (HFD) or HFD containing oligofructose (OFC) during concomitant exposure to either tap water or As-containing water (4.9 ppm as sodium arsenite) for 1 0 wks. Abundance of select commensal bacteria in cecal contents was determined by qPCR. Results. HFD significantly increased body mass and caused fatty liver injury, as characterized by an increased liver weight-to-body weight ratio, histologic changes and transaminases. In line with previous work from this group, As synergistically enhanced HFD-induced liver damage, and was characterized by enhanced inflammation. HFD and As alone both altered content of cecal bacteria, and both decreased the abundance of Bifi-dobacterium spp. OFC supplementation protected against this effect of HFD and As on the abundance of Bifidobacterium spp. OFC supplementation also protected against the enhanced liver damage caused by the combination of HFD and As; indeed liver injury in this group was even improved compared to animals receiving HFD alone. Conclusions. These data support previous findings that low levels of As enhance liver damage caused by high fat diet, suggesting that As exposure may be an underlying factor that increases the risk of obesity-induced liver disease. Furthermore, these results indicate that these effects of arsenic may be mediated, at least in part, by GI tract dysbiosis and that prebiotic supplementation may confer significant protective effects. Together, these data support our hypothesis that As-mediated changes in the GI tract flora play an important factor in sensitizing the liver to injury.

Disclosures:

The following people have nothing to disclose: Veronica L. Massey, Robin H. Schmidt, Min Tan, Walter Watson, Gavin E. Arteel

740

UCP1 Expression Levels are Higher in Females and Negatively Correlated with Inflammation in the Liver

Aybike Birerdinc1,2, Maria Keaton2, Katherine Doyle2, Lei Wang2, Zahra Younoszai1, Rohini Mehta1,3, Vikas Chandhoke2, Ancha Baranova1,2, Zobair M. Younossi1,3;

1Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA; 2Center for the Study of Chronic Metabolic Diseases, George Mason University, Fairfax, VA; 3Center for Liver Disease, Department of Medicine, Inova Fairfax Hospital, Falls Church, VA

Previous studies have documented the expression of brown adipose biomarkers UCP1 and PRDM16 in visceral adipose tissue of obese NAFLD patients. In the current study, we aim to see the correlation of the expression levels of these genes with clinical variables as well as expression levels of known regulators of inflammation and liver histology. Methods: Primers for UCP1 and PRDM16 were validated using commercial RNA samples from various human tissues. The Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue Kits were used to extract RNA from frozen visceral fat (VAT) samples from patients with liver biopsy proven NAFLD. RNA from each sample was converted to cDNA using SABiosciences' RT2 First Strand Kit followed byq-PCR reactions in a 96 well plate format. Group differences in expression levels were assessed using Mann-Whitney tests and chi-square test. Co-expression patterns of the cytokines and other pertinent genes were analyzed using Spearman's ranksum correlation. Results: A total of 241 biopsy-proven NAFLD patients were selected (39.1% NASH, 20.8% with type 2 diabetes, 100% obese with BMI>30, age 43.61 +/-11.41 years, AST 29.1 8 +/- 41.01 U/L, ALT 34.67 +/- 29.05U/L). Group comparisons revealed UCP1 to be significantly higher in females than in males (p<0.002). In the correlation analysis UCP1 and PRDM16 were tightly correlated with each other (r=0.6032, p<4.035e-009), but only UCP1 levels were positively correlated with gender (r= 0.3426, p<0.002). Both PRDM16 and UCP1 expression levels were negatively correlated with histopathological inflammatory scores of NAFLD patients that reflect infiltration of lymphocytes in the hepatic parencyma (r=-0.2809 p<0.01162 and r=-0.326, p<0.002799). Neither UCP1 nor PRDM1 6 levels were influenced by BMI. Conclusion: In obese females with NAFLD, the levels of UCP1 gene expression in VAT is higher than in BMI and disease stage matched males, possibly reflecting the larger amounts of interspersed brown adipose (BAT) cells in females. The negative correlation of PRDM16 and UCP1 expression levels with inflammatory scores in the liver may indicate molecular crosstalk between brown adipose tissue and liver tissue.

Disclosures:

Zobair M. Younossi - Advisory Committees or Review Panels: Merck, Vertex, Tibotec/J and J; Consulting: Gilead Sciences

The following people have nothing to disclose: Aybike Birerdinc, Maria Keaton, Katherine Doyle, Lei Wang, Zahra Younoszai, Rohini Mehta, Vikas Chandhoke, Ancha Baranova

741

Sonoelastographic Dispersion Correlates with Hepatic Triglyceride Content and Histology

Christopher T. Barry1, Alexander Partin2, Zaegyoo Hah2, KuangH-siang Chuang1, Robert Mooney3, Wenqing Cao3, Anthony Almudevar4, Deborah Rubens5, Kevin J. Parker2;

1Transplant Surgery, University of Rochester, Rochester, NY; 2Biomedical Engineering, University of Rochester, Rochester, NY; 3Pathology, University of Rochester, Rochester, NY; 4Biostatistics, University of Rochester, Rochester, NY; 5Imaging Sciences, University of Rochester, Rochester, NY

Accurate noninvasive assessment of hepatic steatosis is clinically desirable. We employ an ultrasound based elastography method using mechanically generated vibration sources to measure shear wave dispersion in fatty livers. Dispersion is the slope or increase of shear speed with increasing frequency and is a consequence of adding a viscous element (i.e., fat) to a tissue. We demonstrate that dispersion correlates with triglyceride content and histologic findings over a wide range of steatosis. Seventy C57BL/6J mice were fed high fat diets and sacrificed for hepatectomy at weeks 0,4,8,11,12,13,14,15,20, and 25 to capture a range of hepatic steatosis. Livers were subjected to triglyceride (TG) assay, H&E analysis, histomorphometry of adipophilin immunostained sections, and ex vivo sonoelastog-raphy of gelatin suspended livers. Group analysis of livers containing <0.1 mg TG/mg liver (“low fat”, n=30), 0.1-0.25 mg TG/mg liver (“moderate fat”, n=21), and >0.25 mg TG/mg liver (“high fat”, n=17) revealed mean dispersion values of 0.05 cm/s/100 Hz, 0.28 cm/s/100 Hz, and 0.35 cm/s/100 Hz respectively. These triglyceride groupings corresponded to visual H&E assessments of 1 9%, 69%, and 78%. Using a linear fixed effects model, dispersion slope curves are statistically different between low and high fat groups (p=0.01). The average dispersion for livers with 0-10% fat = 0, 15-40% fat = 0.25, and 40-70% fat = 0.43. Preliminary analysis of individual his-tomorphometric fat area measurements versus dispersion revealed a high correlation (R2=0.90). Sonoelastographic dispersion measurements correlate with intrahepatic fat content as measured biochemically and histologically. The wide dynamic range allows distinction of clinically relevant degrees of steatosis. These results provide impetus to further study this technique in larger animals, in vivo, and in humans.

Disclosures:

The following people have nothing to disclose: Christopher T. Barry, Alexander Partin, Zaegyoo Hah, KuangHsiang Chuang, Robert Mooney, Wenqing Cao, Anthony Almudevar, Deborah Rubens, Kevin J. Parker

742

L-carnitine prevents progression of non-alcoholic steato-hepatitis with regulation of mitochondrial β-oxidation and redox system in NASH model Mice

Hisashi Ishikawa, Akinobu Takaki, Kazuhide Yamamoto;

Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

(Purpose) Nonalcoholic steatohepatitis (NASH) is a common liver disease characterized by hepatic steatosis, lobular inflammation, and hepatocellular ballooning with its inherent risk for progression to cirrhosis and hepatocellular carcinoma. In the development of NASH, a two-hit theory has been considered. The first hit is lipid accumulation in hepatocytes which increases the sensitivity of the liver to the second hit. The second hit includes several cell stresses such as apoptosis, inflammation and oxidative stress. Oxidative stress such as reactive oxygen species (ROS) appears to be responsible for the initiation of necroinflammation. Increased ROS generation is key feature of mitochondrial dysfunction, with a central role in the pathophys-iology of NASH. L-Carnitine (LC) is an endogenous mitochondrial membrane compound. The main physical function of LC in human body is facilitating the transport of long chain fatty acids into mitochondria in order to enter the β-oxidation cycle. Recent years LC has been proposed for treatment of various kinds of diseases, including liver injury. The aim of the present study was to explore the preventive and therapeutic effect of LC on oxidative stress conditions such as ROS production, lipid peroxida-tion and redox system in NASH mouse model to provide theoretical evidence for LC as a clinical therapy for NASH. (Methods) We used STZ-treated mouse diabetes model with high fat diet supplementation, known as the NASH hepatocar-cinogenesis model (STAM mice). Eight-week-old STAM mice were divided into 3 experimental groups and fed for 4 weeks as follows:(1)high fat diet(S group) (2)high fat diet + LC(SC group) (3)high fat diet + vitaminE(SE group). After 4 weeks mice were sacrificed. Following data were compared between these three groups; hepatic histological findings, hepatic 8-OHdG concentration, expression of hepatic inflammatory and hepatic lipogenic genes, and redox system genes by real time PCR. (Results) In histological analysis, SC group and SE group showed low inflammation and fibrosis compared with S group. The concentration of 8-OHdG was reduced in SC and SE group compared with S group. The hepatic gene expression of inflammatory cytokine TNF-α was down-regulated in SC and SE group. The beta oxidation pathway related genes AOX and MCAD, the peroxisomal proliferator-activated receptor PPAR-α and PPAR-γ were up-regulated in SC and SE group. The redox system related gene Mn-SOD and GPx4 were up-regulated in SCgroup compared with S and SE group. (Conclusion) L-carnitine prevents progression of non-alcoholic steatohepatitis with regulation of mitochondrial β-oxidation and redox system.

Disclosures:

Kazuhide Yamamoto - Advisory Committees or Review Panels: Shionogi Pharmaceutical Co; Grant/Research Support: Tanabe Mitsubishi Co, MSD, Chugai Pharmaceutical Co, Esai Co

The following people have nothing to disclose: Hisashi Ishikawa, Akinobu Takaki

743

Effect of chronic psychosocial stress on nonalcoholic steatohepatitis in mice

Barbara Czech, Martina Müller, Claus Hellerbrand;

University Hospital Regensburg, Regensburg, Germany

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which may progress towards inflammation (nonalcoholic steatohepatitis [NASH]). NAFLD is regarded as a consequence of a sedentary, food-abundant lifestyle which, in the modern world, often coincides with chronically high levels of perceived psychosocial stress. Here, we aimed to characterize the effect of chronic psychosocial stress on the development of NAFLD/NASH in male mice either fed with standard chow or NASH-inducing high fat diet. Methods and Results: Chronic psychosocial stress was induced by chronic subordinate colony housing (CSC), a pre-clinically validated paradigm relevant for human affective and somatic disorders. Single housed (SHC) mice served as controls. Under standard chow conditions CSC mice revealed lower hepatic triglyceride levels but higher hepatic TNF, MCP-1 and HMOX mRNA expression, while serum transaminase levels did not significantly differ from SHC mice. Under the NASH-inducing high-fat diet CSC and SHC mice showed similar body weight-gain and serum levels of glucose and adiponectin. Moreover, liver histology as well as TNF, MCP-1 and HMOX expression were similar in CSC and SHC mice fed with HFD. Surprisingly, CSC showed even significantly lower transaminase levels than SHC mice fed with the same NASH-inducing diet. Summary and Conclusion: Under normal dietary conditions the CSC model induces noticeable hepatic oxidative stress and inflammation without causing manifest hepatocellular injury. In contrast, CSC exhibited a protective effect on hepatocellular injury in a dietary NASH-model. Identification of the underlying mechanisms of this phenomenon may lead to novel therapeutic strategies to prevent progression of NAFLD.

Disclosures

Martina Müller - Grant/Research Support: Novartis

The following people have nothing to disclose: Barbara Czech, Claus Hellerbrand

744

The effect of peroxisome proliferator-activated receptor-delta activator on fatty acid-induced inflammasome in human liver carcinoma cell line

Hyun Jung Lee, Jong Eun Yeon, Eileen L. Yoon, Sang Jun Suh, Keun-hee Kang, Hae Rim Kim, Seong Hee Kang, Yang Jae Yoo, Ji Hye Je, Ji Hoon Kim, Yeon Seok Seo, Hyung Joon Yim, Soon Ho Um, Kwan Soo Byun;

Korea University College of Medicine, Seoul, Republic of Korea

Background: Non-alcoholic steatohepatitis (NASH) is progressive form of nonalcoholic fatty liver disease and can lead to cirrhosis and even hepatocellular carcinoma (HCC). The inflammasome, a multiprotein complex formed by nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family members, has been recently shown to be activated in NASH. This study was designed to evaluate the inflmammasome activation induced by palmitic acid (PA) and the effects of the per-oxisome-proliferator activated receptor (PPAR)-δ agonist GW501516 in this condition. Methods: Human HCC cell line (HepG2) was used in this study. PA (0.2 mM) and lipopolysac-charide (LPS) were used to induce inflammation. The cells were treated with PA (0.2 mM) and/or lipopolysaccharide (LPS) for 16 hours, along with PPAR-δ agonist (GW501516, 1 and 10 μM). We measured the mRNA expression levels of inflamm-some-related molecules using real time PCR analysis. Protein levels of inflammatory cytokines were determined by western blotting. Results: In the group treated with PA plus LPS, there was a significantly increase in several inflammasome gene expression (including NLRP3, NLRP6, NLRP10, caspase-1) versus the control, PA or LPS-treatment group (P < 0.05). NLRP1 and NLRC4 mRNA expression levels were not affected by such stimulation. Inflammasome activation was indicated by higher mature IL-1 β and caspase-1 protein levels in the PA plus LPS treatment group but not in the control group. All of the above effects of PA and LPS were attenuated by the PPAR-δ agonist GW 50151 6 in a dose dependent manner. Conclusions: These results demonstrate that PPAR-δ agonist attenuates PA-induced inflammation through suppressing several inflammasome activation, caspase-1 activation and IL-1 β cleavage. PPAR-δ agonist may serve as a potential therapeutic agent for NASH. Keywords: Inflammasome, Non-alcoholic steatohepatitis, Palmitic acid, Peroxisome proliferator-activated receptor-delta

Disclosures:

Hyung Joon Yim - Grant/Research Support: GSK Korea, Handok Pharm, Gilead Korea; Speaking and Teaching: BMS Korea

The following people have nothing to disclose: Hyun Jung Lee, Jong Eun Yeon, Eileen L. Yoon, Sang Jun Suh, Keunhee Kang, Hae Rim Kim, Seong Hee Kang, Yang Jae Yoo, Ji Hye Je, Ji Hoon Kim, Yeon Seok Seo, Soon Ho Um, Kwan Soo Byun

745

Links between non alcoholic steato-hepatitis and psoriasis in a murine model

Philippe Vasseur1,2, Hanitriniaina Rabeony2, Isabelle Paris2, Jean-Françcois Jegou2, Franck Morel2, Pierre Levillain3, Jean-Claude Lecron4,2, Christine Silvain1,2;

1Hepato-Gastroenterology unit, Poitiers University Hospital, Poitiers, France; 2LITEC, Pôle Biologie Santé, Poitiers, France; 3Pathology unit, Poitiers University Hospital, Poitiers, France; 4Immunology and Inflammation Laboratory, Poitiers University Hospital, Poitiers, France

Introduction Psoriasis is a T-cell mediated disease in which T helper 1 7 lymphocytes cells play a crucial role. Psoriasis is associated with systemic manifestations including arthritis and atherosclerosis. Psoriasis and non alcoholic fatty liver disease are associated, but the link between these two diseases remains unclear. Material and methods We fed male C57BL/6 mice for 1 1 weeks with a high fat diet enriched in cholic acid. From week 9 to week 1 1, psoriasis-like inflammation was induced by topical application of imiquimod, a toll like receptor 7/8 agonist. Skin and liver inflammation were studied by histology and RT-qPCR. Spleen cellular composition was characterized by flow cytometry. Results Imiquimod induced features resembling psoriasis with red and scaly plaques, acanthosis and thickening of epidermis, overexpression of interleukine-1 7A (IL-1 7A), IL-22 and antimicrobial peptide S100A9. Skin inflammation was associated with an increase of splenic natural killer cells and T lymphocytes. High fat diet induced non alcoholic steato-hepatitis (NASH) as shown by steatosis, cellular ballooning, poly-morphonuclear cells infiltrates and overexpression of IL-6. Induction of psoriasis in NASH mice did not aggravate liver histology severity but potentialized hepatic gene expression of the macrophagic chemokine CCL3, accompagnied by an overexpression of the neutrophilic marker Gr1. Conversely, high fat diet worsened psoriasis-like inflammation with an increase of epidermal thickening and higher expressions of IL-17A, IL-22 and S1 00A9. Interestingly, NASH was associated with an over-expression of IL-1 7A in the skin of untreated mice at week 5. Conclusion In this murine model, both NASH and psoriasis-like skin inflammation display systemic manifestations and aggravate each other. Psoriasis may aggravate inflammatory cell recruitment into the liver whereas NASH may be associated with a pro-inflammatory state of the skin.

Disclosures:

The following people have nothing to disclose: Philippe Vasseur, Hanitriniaina Rabeony, Isabelle Paris, Jean-Françcois Jegou, Franck Morel, Pierre Levillain, Jean-Claude Lecron, Christine Silvain

746

Identification Of Pesticides And Other Environmental Chemicals Associated With The Development Of Nonalcoholic Fatty Liver Disease In Rodents

Laila Al-Eryani1, Banrida Wahlang1, Heather B. Clair1, John J. Guardiola1, Keith C. Falkner1, Russell A. Prough3, Matthew C. Cave1,2;

1Department of Medicine/GI, University of Louisville, Louisville, KY; 2Robley Rex Veterans Administration Medical Center, Louisville, KY; 3Biochemistry, University of Louisville, Louisville, KY

Purpose: Environmental chemical exposures may contribute to the genesis and progression of non-alcoholic fatty live disease (NAFLD). However, a comprehensive list of these chemicals has not been generated. This study evaluates two large rodent exposure study databases compiled by the federal government for environmental chemicals which caused NAFLD in animal models. Methods: ToxRefDB is a database designed by the National Center for Computational Toxicology (NCCT) and Environmental Protection Agency's (EPA) Office of Pesticide Programs (OPP) containing animal toxicity testing filed during pesticide registration. 474 rat/mouse studies (chronic, subchronic and multigeneration reproductive) were queried for histologic NAFLD descriptors including fatty change, lipid deposition, steatosis and Oil red O positivity. Chemical Effects in Biological systems (CEBS) is a data repository developed by the National Toxicology Program (NTP) warehousing about 9000 rodent toxicology studies. Chemical selection was based on histopatho-logical observations in the liver (fatty changes and the toxic hepatopathy). Chemicals excluded from the CEBS search were prescription medications and dietary supplements while cosmetics, fragrances and food additives were included. Results: From the TOXRefDB, 42 unique chemicals were associated with NAFLD pathologic descriptors. The 42 compounds included 22 fungicides, 13 herbicides, 6 insecticides, and 1 miticide. Thus, nearly 1 0% of archived pesticide registration studies were associated with the development of NAFLD. From CEBS, 329 studies of 91 unique chemicals reported positive NAFLD descriptors. These chemicals included 14 dyes, 8 plasticizers, 7 pesticides, 7 paints and polishes, 6 cosmetic additives, 4 food additives, and 3 fragrances. Several chemicals produced NAFLD in more than one study increasing the likelihood for a true causal effect. Conclusion: 371 studies archived in federal (U.S.) databases linked 1 33 unique environmental chemicals to NAFLD in rodents. Pesticides composed 37% of these chemicals and nearly 10% of pesticide registration toxicity studies reported the development of NAFLD. The potential role of the identified environmental chemicals, and pesticides in particular, should be evaluated in human NAFLD.

Disclosures:

The following people have nothing to disclose: Laila Al-Eryani, Banrida Wahlang, Heather B. Clair, John J. Guardiola, Keith C. Falkner, Russell A. Prough, Matthew C. Cave

747

Resveratrol improves pathogenesis of nonalcoholic steatohepatitis through inhibition of endotoxin-induced liver damage

Takaomi Kessoku1, Kento Imajo1, Wataru Tomeno1, Yuji Ogawa1, Hironori Mawatari1, Masato Yoneda1, Hiroyuki Kirikoshi1, Satoru Saito1, Koichiro Wada2, Atsushi Nakajima1;

1 Gastroenterology Division, Yokohama City University, Yokohama, Japan; 2Pharma-cology DIvision, Osaka University Graduate School of Dentistry, Osaka, Japan

Aim: Although gut-derived endotoxin (ET), such as lipopolysac-charide (LPS), plays a key role in the pathogenesis of nonalco-holic steatohepatitis (NASH), detailed mechanisms of this pathogenesis becomes clear. We reported that overexpression of CD14 via activation of leptin-STAT3 signaling in Kupffer cells induced hyper-inflammatory response to low-dose ET, resulting in progression from simple steatosis to steatohepatitis with liver fibrosis. Therefore, inhibition of leptin-STAT3 signaling in Kupffer cells may lead to attenuate the progression of steatohepatitis via inhibition of CD1 4 expression. The aim of this study was to investigate whether the resveratrol which is known to inhibit activation of STAT3, improves the pathogenesis of steatosis or steatohepatitis in murine model. Material and methods: Male C57BL/6J mice were initially divided into a high fat diet (HFD) group (HFD-fed group for 12 weeks) or resveratrol groups (group fed a HFD mixed with resveratrol of 2 mg/kg/day or 20 mg/kg/day for varying periods of time). In this study, E. coli-derived LPS (0.25 mg/kg) was used. 1) We investigated whether the resveratrol attenuates HFD-induced steatosis. 2) We investigated whether the resveratrol attenuates ET-induced liver damage via inhibition of response to ET. 3) We investigated whether the resveratrol improves the pathogenesis of long-term exposed ET-induced steatohepatitis with liver fibrosis. Result: 1 )Compared with HFD group, the resveratrol-treated mice for 28 days showed the improvement of HFD-induced obesity and steatosis. The signals of lipogenesis, such as SREBP1 c, were significantly decreased. 2)Compared with HFD group, the resveratrol-treated mice for 14 days showed significant inhibition of hepatic CD14 expression through suppression of STAT3 activity in Kupffer cells, following inhibition of a single low-dose LPS-induced liver damage. 3)Compared with HFD group, the resveratrol-treated mice for 28 days showed inhibition of long-term low-dose LPS-induced liver fibrosis. Conclusion: This is a first report that the resveratrol improves not only the pathogenesis of steatosis thorough inhibition of lipogenesis but also steatohepatitis through inhibition of endotoxin-induced liver damage via suppression of STAT3-CD14 signaling in Kupffer cells. The resveratrol may have application for the treatment of nonalcoholic fatty liver disease.

Disclosures:

The following people have nothing to disclose: Takaomi Kessoku, Kento Imajo, Wataru Tomeno, Yuji Ogawa, Hironori Mawatari, Masato Yoneda, Hiroyuki Kirikoshi, Satoru Saito, Koichiro Wada, Atsushi Nakajima

748

Elucidation of the role of microRNA-155 in a murine model of non-alcoholic steatohepatitis

Tasneem Motiwala, Mufaddal Mustafa, Huban Kutay, Rachael Sullivan, Kun-Yu Teng, Vivek K. Chowdhary, Thomas M. Kaffenberger, Lianbo Yu, Vincenzo Coppola, Jianhua Yu, Kalpana Ghoshal, Samson T. Jacob;

Ohio State University, Columbus, OH

Purpose of the study Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide with increasing incidence and mortality. Risk factors include acquisition of the Hepatitis B orC virus, alcohol use, and non-alcoholic steatohepatitis (NASH). We have previously demonstrated consistent upregu-lation of miR-155, a pro-inflammatory microRNA, in a diet-induced mouse model of HCC that mimics the progression of steatohepatitis to HCC in humans (B.Wang et.al, Hepatology. 2009). Importantly, increased miR-1 55 level in humans is an independent predictor of poor prognosis and survival and also correlates with invasion in HCC patients. The involvement of miRs in malignancies has become an increasingly important aspect in the field of oncology due to the potential for novel therapeutics directed at these small non-coding RNAs that can regulate a number of genes and play a role in oncogenesis. We hypothesize that upregulation of miR-155 plays a causal role in NASH and HCC and that the absence of miR-155 will result in decreased inflammation, fibrosis, and eventually, development of HCC. The overall goal of this study is to establish the role of miR-155 in the pathogenesis of HCC for future development of anti-miR therapy. Methods Male C57BL6 mice and miR-1 55KO mice were fed the CHOW diet (control) or the CDAA diet for duration of either 4 or 8 weeks. Blood and liver were then harvested for serologic, pathologic, and biochemical studies. Additionally, body and liver weight were also noted at the time of sacrifice. From the hepatic tissues, gene expressions at the protein and mRNA levels were quantified using Western blot analysis and qRTPCR. Results & Conclusions miR-155KO mice exhibited increased liver to body weight ratio as compared to control mice in the absence of increased body weight. As expected, reduced inflammation was seen in the miR-155KO mice that correlated with decreased NF-κB activity. Histopatho-logical analysis also revealed increased steatosis in the KO mice suggesting that miR-155 plays a role in hepatic lipid metabolism. Global gene expression analysis was consistent with reduced inflammation and enhanced steatosis in the miR-155KO mice. Importantly, changes in expression of genes associated with hepatic necrosis, fibrosis and cancer indicate reduced liver damage in miR-155KO mice suggesting that they will be less prone to tumorigenesis. Reduced expression of the p65 subunit of NF-κB in the miR-155KO mice was identified as one of the mechanisms of decreased NF-κB activity. Currently studies are underway to explore further the mechanism of regulation of p65 expression/NF-κB activity and lipid metabolism by miR-155.

Disclosures:

The following people have nothing to disclose: Tasneem Motiwala, Mufaddal Mustafa, Huban Kutay, Rachael Sullivan, Kun-Yu Teng, Vivek K. Chowdhary, Thomas M. Kaffenberger, Lianbo Yu, Vincenzo Coppola, Jianhua Yu, Kalpana Ghoshal, Samson T. Jacob

Ancillary