cells isolated from WT mice were stimulated with palmitic acid and/or a synthetic TLR2ligand. IL-1β and inflammasome components were measured from the cells and supernatant.TLR2 deficient mice were fed a CDAA diet to assess the requirement of TLR2 in NASH.(Results) The CDAA diet feeding for 22 weeks induced steatohepatitis, obesity and insulinresistance. Hepatic and blood FFAs were increased in WT mice fed the CDAA diet comparedwith those fed a standard diet. Hepatic and blood IL-1β levels were also increased in WTmice fed the CDAA diet. CDAA diet feeding increased in expression of inflammasomecomponents including nalp3, nalp1, ASC, caspase-1, and AIM2. InWTKupffer cells, palmiticacid did not increase expression of IL-1β and inflammasome components. In contrast, asynthetic TLR2 ligand increased gene expression of IL-1β as well as an inflammasomecomponent nalp3. Although a synthetic TLR2 ligand increased mRNA expressions of IL-1βand nalp3, active form of IL-1β was not detected from the supernatant. Interestingly, palmiticacids treatment induced secretion of active IL-1β form in Kupffer cells primed with TLR2ligand. Finally, we tested the role of TLR2 in murine NASH model. TLR2 deficient micefed the CDAA showed less steatohepatitis and lower expression of inflammasome componentsthan WT mice. (Conclusion) TLR2 signaling induces expression of inflammasome compon-ents and the subsequent treatment with palmitic acid induces activation of IL-1β. Thus,activation of inflammasome and IL-1β require both FFA stimulation and TLR2 signalingin NASH.

691

Cell-Specific Roles of miR-155 and miR-122 in Inflammation, Immunity andFibrosis in Non-Alcoholic SteatohepatitisTimea Csak, Shashi Bala, Dora Lippai, Gyongyi Szabo

Background/Aims: Liver MicroRNA (miRNA) expression is altered in patients with non-alcoholic steatohepatitis (NASH). Steatosis, inflammation and cell death are targets of miRNAsand contribute to the pathogenesis of NASH. MiRNA-155 is a master regulator of inflamma-tion while miR-122, the most abundant miRNA in hepatocytes, regulates lipid metabolism.We hypothesized that NASH-related alterations in miR-155 and miR-122 have specificpathogenic roles in NASH. Methods: MiRNA-155 and -122 and their target genes wereevaluated in livers and isolated cell populations (hepatocytes, liver mononuclear cells-LMNCsor Kupffer cells-KCs) in mice fed with a methionine-choline-deficient (MCD) or -supple-mented (MCS) control diet for 6 weeks. Results: MCD diet caused steatohepatitis andincreased miR-155 expression in the livers, isolated LMNCs, KCs and hepatocytes. LPSinduced significantly higher miR-155 expression and TNFα production in LMNCs and KCswith steatohepatitis than in controls. MiRNA-155 is a positive regulator of TNFα and wefound a linear correlation between miR-155 and TNFα mRNA increases in steatohepatitis.IKKε, a target of miR-155, is critical in TLR-induced Type I IFN production. IKKε levelswere decreased in hepatocytes and livers after MCD diet. This was associated with decreasedIFNβ induction by LPS in hepatocytes and not in LMNCs and KCs suggesting a cell-specific effect. Using over-expression of miR-155 in isolated hepatocytes, we confirmedposttranscriptional suppression of IKKε by miR-155. We found that both TLR9 (CpG) andTLR4 (LPS) ligands induced miR-155 in the liver suggesting potential mechanisms for miR-155 upregulation in NASH. MiRNA-122 is decreased in human livers with NASH. We founddecreased liver miR-122 levels with a complementary increase in serum miR-122 in micewith MCD versus MCS diet feeding. Reduced miR-122 levels in MCD diet feeding wereassociated with liver fibrosis and increased Sirius red staining, collagen and αSMA mRNAlevels. The mRNA levels of miR-122 targets, hypoxia-inducible factor 1α (HIF1α) and iNOSwere increased along with increased HIF1α nuclear binding. HIF1α is pro-fibrotic throughits target gene, lysil oxidase, which was increased in steatohepatitis. We also demonstratedregulation of HIF1α by miR-122 as miR-122 overexpression downregulated but miR-122knockdown increased HIF1α mRNA levels. Conclusions: Our results demonstrate a cell-specific mechanistic role for increased miR-155 and decreased miR-122 expression in thepathogenesis of NASH. We show for the first time that increased miR-155 is linked to TNFαproduction in liver MNC/KCs while in hepatocytes it contributes to decreased IKKε levelsand impaired IFNβ induction in NASH. Our data also imply that decreased liver expressionof miR-122 has a causal role in increased HIF1α expression in steatohepatitis with fibrosis.

692

Macrophage Migration Inhibitory Factor (MIF) Attenuates Liver Inflammation,Steatosis, and Fibrosis in Murine Non-Alcoholic Liver Disease ModelNorio Horiguchi, Daichi Takizawa, Satoru Kakizaki, Hiroki Tojima, Hiroaki Hashizume,Yuichi Yamazaki, Ken Sato, Bin Gao, Masatomo Mori

Backgroud: Emerging evidence revealed the importance of macrophage in metabolic syn-drome. Non-alcoholic fatty liver disease (NAFLD) is known as a phenotype of metabolicsyndrome in the liver. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine,which is involved in inflammatory response and regulates macrophage accumulation. How-ever, the precise role of MIF in NAFLD is not clear. Methods: MIF-knockout mice (MIF-KO) and wild-type (WT) were fed either a control or a high fat diet for 24weeks. Hepaticinflammation, steatosis, and fibrosis were examined. Results: High fat diet feeding inducedmore MIF mRNA expression compared to control diet feeding in WT. In high fat feedinggroup, MIF-KO showed significantly higher ALT levels than WT. Although no differencewas found in F4/80, CCR2, andMPO expression betweenWT andMIF-KO, pro-inflammatorycytokine (TNF-α and MCP1) mRNA expression were significantly increased in MIF KO.With regard to steatosis, oil-red O staining and triglyceride levels in the liver showed MIFKO is more susceptible to high-fat-induced steatosis. Moreover, MIF-KO showed severefibrosis by sirius-red staining and αSMA staining, while no fibrosis was found in high-fat-fedWT. This progression of fibrosis was also confirmed by elevation of collagen1,TIMP1,TGF-B and αSMA mRNA. Conclusions: We have shown MIF KO is more susceptible to liverinflammation, steatosis, and fibrosis in murine NAFLD model. These results suggest MIFmight be the therapeutic target for non-alcoholic liver diseases.

S-921 AASLD Abstracts

693

Extracellular ATP and Activation of JNK1 Signaling Play Key Roles in theInduction of Adipose Differentiation-Related Protein (ADFP) in HepaticSteatosisBryan Tackett, Arunmani Mani, Benny H. Chang, Sundararajah Thevananther

Background: Adipose differentiation-related protein (ADFP) is a lipid droplet associatedprotein expressed in hepatocytes, essential for lipid droplet formation and accumulationduring hepatic steatosis. Efficient hepatocyte proliferation and liver regeneration in responseto partial hepatectomy (PH) is critically dependent on the induction of transient steatosisin the remnant livers, which coincides with the peak proliferative phase. Extracellular ATPvia P2 purinergic receptor activation induces c-Jun N-terminal kinase (JNK) signaling inhepatocytes. However, the functional significance of P2 purinergic receptor-mediated JNKsignaling in hepatic steatosis remains unknown. Therefore, the purpose of this study wasto test the hypothesis that extracellular ATP signaling via JNK activation plays a criticalrole in the induction of ADFP expression in hepatic steatosis in regenerating livers. Methods:PH (70%) was performed on adult male wild-type (WT), JNK1, and JNK2 knockout (KO)mice. The remnant livers harvested at 24hr post-PH were analyzed for ADFP protein andmRNA expression and lipid droplet formation within hepatocytes. Primary hepatocytes (WT,JNK1, and JNK2 KO mice) were treated with ATP (100μM) for 48 hrs with or without pre-treatment with JNK, p38, or PI3 Kinase inhibitors for 30 min. Hepatocyte lysates wereanalyzed by Western blotting for the induction of ADFP (48 hrs). Results: Induction ofhepatic steatosis in response to PH was attenuated in the remnant livers of JNK1 KO.Conversely, JNK2 KO had increased hepatic steatosis (lipid droplets) in response to PH, ascompared to WT. PH induced ADFP protein (3.5 fold) and mRNA expression (4.0 fold) inthe remnant livers of WT. ADFP protein (0.6 fold) and mRNA expression (0.3 fold) wereseverely impaired in the JNK1 KO mice remnant livers, as compared to the WT (1.0). ATPtreatment alone was sufficient to induce ADFP expression in WT hepatocytes In Vitro (6fold). Correspondingly, ATP-induced ADFP protein expression was significantly impairedin the JNK1 KO hepatocytes (0.40 fold), and enhanced in the JNK2 KO hepatocytes (4.7fold). Inhibition of JNK and p38 signaling prior to ATP treatment resulted in an attenuationof ADFP protein expression in WT hepatocytes (0.6, 0.5 fold respectively). Interestingly,inhibition of PI3 Kinase signaling prior to ATP treatment resulted in an increase in ADFPprotein expression in WT hepatocytes (1.5 fold). Conclusions: Our findings suggest thatextracellular ATP treatment alone is sufficient to induce ADFP expression in hepatocytes InVitro, and hepatic steatosis and ADFP induction in response to PH In Vivo is criticallydependent on intact JNK1 signaling, but not JNK2. These results highlight a novel role forextracellular ATP as a potential humoral mediator necessary for the induction of hepaticsteatosis in regenerating livers.

694

Palmitate Induced Suppression of Macrophage THP-1 (Cd68+) Production ofIGFBP-3: A Novel Mechanism of Lipotoxicity in the LiverHae-Ki Min, Faridoddin Mirshahi, Hitoshi Maruyama, Byoung Kuk Jang, MasahikoShimada, Tommy Pacana, Michael Fuchs, Youngman Oh, Arun J. Sanyal

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is associated with increased lipolysisand the net delivery of free fatty acids (FFA) to the liver. Saturated FFA e.g. palmitate havebeen shown to induce inflammation and apoptosis in the liver. It has also been recentlyshown that NASH is associated with low levels of insulin-like growth factor binding protein-3 (IGFBP-3). It is however not known whether this is a function of underlying obesity andthe potential role of IGFBP-3 in the pathogenesis of NASH is unknown. AIMS: To clarifythe relationship between IGFBP-3 and NASH and define the mechanisms by which IGFBP-3 modulates pro-inflammatory, pro-apoptotic pathways in the liver. METHODS: Humanstudies were performed in subjects with biopsy-proven fatty liver (FL) n=12, NASH (n=12)and compared to lean normal (n=10) and obese subjects without liver disease (n=10). IGFBP-3 production was assessed in hepatocyte (Huh-7 cells) and in Macrophage THP-1 (CD68+)(Kupffer cell-like) cells in the absence and presence of palmitate. The role of IGFBP-3 inactivation of inflammatory pathways was assessed by gain of function (over-expression) andexposure to conditioned media. Specific blockers of the JNK, NFκB, ERK and MAP kinasepathways were used determine the involvement of these pathways. IL-8 expression was usedas the endpoint of interest in defining a pro-inflammatory state. RESULTS: There was astep-wise increase in circulating IL-8 levels and decrease in IGFBP-3 from lean normal, toobese controls to FL to NASH. Palmitate produced a dose-related increase in IL-8 expressionby hepatocytes. This was associated with increased JNK and NFκB activation and could beblocked by JNK or IKK inhibitors but not ERK inhibitors. There was no basal or palimitate-induced expression of IGFBP-3 in hepatocytes. However, Macrophage THP-1 (CD68+) cellsdemonstrated IGFBP-3 expression which was suppressed by palmitate. Overexpression ofIGFBP-3 in hepatocytes suppressed palmitate-induced hepatocyte IL-8 expression (mRNAand protein). These effects were associated with decreased JNK and NFκB activation. Com-pared to Macrophage THP-1 (CD68+) cell conditioned media in basal state, media obtainedafter palmitate exposure (with low IGFBP-3) increased palmitate-induced IL-8 expressionin hepatocytes. CONCLUSIONS: IGFBP-3 inhibits IL-8 synthesis in hepatocytes by sup-pressing JNK and NFκB but not ERK. Macrophage THP-1 (CD68+) cells and not hepatocytesexpress IGFBP-3; palmitate suppresses Macrophage THP-1 (CD68+) cell IGFBP-3 expressionwhich disinhibits JNK and NFκB thereby promoting inflammation and palmitate-inducedIL-8 expression.

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