ratio (1.7-fold less; P<0.01). Conclusion: Aquaporin-1 contributes to angiogenesis, fibrosis,and portal hypertension after bile duct ligation and could represent an important therapeutictarget in chronic liver disease and accompanying portal hypertension.

313

The Hepatic Inflammsome Has an Increased NK-T and CD8+ T- Cell Signaturein a Murine Model of Fibrotic Nonalcoholic Steatohepatitis (NASH)Pranav Shivakumar, Samir Softic, Michelle Kirby, Rohit Kohli

High fat diet induced models of simple hepatic steatosis have been shown to result in adecreased hepatic natural killer- T (NKT) cell population. Interestingly, NKT cells are knownto correlate positively with the development of adipose tissue inflammation and glucoseintolerance in diet-induced obesity. Recent data implicates refined carbohydrates (Fructose)in the progression of hepatic steatosis to advanced fibrotic NASH in humans. This has alsobeen replicated in a novel model of obesity and NASH with fibrosis in mice. We thereforehypothesized that NKT cells populate the livers in a murine model of advanced NASH withfibrosis. Methods: Adult male C57BL6 mice were randomly assigned to control (C) or highfat high carbohydrate diet (HFHC). The C group received regular chow and water whilethe HFHC group received diet with Medium Chain Triglycerides (MCT; 58% kcal) andwater supplemented with 55% fructose & 45% sucrose. Mice were sacrificed at 16 wks ofage and underwent hepatic mononuclear cell phenotyping by flow cytometry. Results: Micereceiving HFHC diet gained significantly more weight than C fed mice starting at 4 wk(p<0.01), with a Mean ± SE weight of 45.84±1.4g at 16 wk compared to 31.33±1.0g forC fed mice (p<0.0001). Fasting glucose was higher in HFHC mice at 16 wks (176.3±9.2mg/dl; p=0.001). Liver weight at 16 weeks was higher in the HFHC group (2.44±0.2g)compared to the C fed mice (1.47±0.03g; p<0.01). Steatosis as measured by liver triacylgly-cerol (TG) content was also higher in HFHC mice at 16 wks (2354±487.8 mg/dl/100 mgwet liver) compared to the C fed mice (p= 0.001). Inflammation as measured by serumalanine aminotransferase (ALT) levels was higher in HFHC mice (135.8±24.28 IU/L) at 16wks compared to the C fed mice (p=0.019). Flow cytometric analysis identified increasedNK-T cell (CD3+NK1.1+) signature in mice receiving HFHC (3.7±2.3x104 cells) comparedto C fed mice (1.1±0.6x104 cells; p=0.02). While hepatic CD1d-restricted invariant NK-TCD4+ and CD8+ cell populations remained unchanged, total CD3+CD8+ T-cells significantlyincreased in the HFHC group (1.93±0.79x105) vs. C fed (1.0±0.7x105; p=0.04). A corres-ponding increase in CD8+ T-cells expressing the activation marker CD69 was also seen[(2.02±1.01x104 (C) vs 6.85±2.6x104 (HFHC); p<0.01]. Liver fibrosis was visible at sacrificein majority of the HFHC fed mice (Stage 1c and 2) while none was seen in C diet fed mice.Conclusion: HFHC diet produces a relatively rapid and progressive liver injury resulting inNASH with fibrosis in a background of diet-induced obesity. In this system, which closelyresembles the human phenotype of advanced NASH with fibrosis, we observed a significantincrease in hepatic NK-T and CD8+ T-cells. These cell types may potentially typify thehepatic inflammasome in the progression of human disease to NASH with fibrosis.

314

IL-1 Receptor Antagonist Treatment Attenuates Liver Steatosis andInflammation in Alcoholic Liver Disease: The Role of InflammasomeActivation and IL-1β-Mediated Hepatocyte Steatosis and CytotoxicityJan Petrasek, Angela Dolganiuc, Karen Kodys, Evelyn A. Kurt-Jones, Gyongyi Szabo

Background: Alcohol-induced liver damage (ALD) is characterized by steatosis and upregul-ation of pro-inflammatory cytokines, including interleukin 1-beta (IL-1β). Inflammasome,an intracellular complex of NALP3 and ASC activates caspase-1 that converts pro-IL-1β intomature IL-1β and perpetuates inflammation. The significance of inflammasome-activatedIL-1β in ALD is unknown. Interleukin (IL)-1β, IL-1receptor (IL1r) and IL-1 receptor antagon-ist (IL-1ra) are all part of IL-1 superfamily and play a role in regulation of inflammation.We hypothesized that inflammasome activation in the liver may contribute to inflammationand steatosis in alcoholic liver disease. Aim: To investigate the role of inflammasome-dependent IL-1β in regulation of ALD. Methods: Wild-type (WT) and interleukin-1 receptor(IL-1r)-deficient (IL1r-KO) mice were fed with Lieber-DeCarli diet (5% v/v ethanol) orcontrol diet for 4 weeks. Liver histology, triglycerides, cytokines and serum ALT wereassessed. Principal findings were validated In Vitro using primary murine hepatocytes andIn Vivo using recombinant IL-1ra, Anakinra. Results: Alcohol feeding resulted in liver injury(ALT), steatosis (triglycerides) and induction of inflammatory cytokines in WT mice. Therewas a significant upregulation of inflammasome components in the liver, including NALP-3, ASC and procaspase-1 at mRNA levels. There was increased Caspase-1 cleavage andactivity, corresponding with increased IL-1β in alcohol-fed compared to pair-fed WT mice.In contrast, alcohol-fed IL1r-KO mice showed lower levels of serum ALT, inflammatorycytokines (TNF-α, IL-1β, MCP-1) and liver triglycerides compared to WT mice, suggestingthat functional impairment of IL-1 pathway is protective against ALD. We determined that InVitro, treatment of primary hepatocytes with IL-1β induced diacylglycerol O-acyl transferase 2(DGAT2), a key enzyme involved in triglyceride synthesis, and resulted in lipid accumulation.Furthermore, pretreatment with recombinant IL-1β sensitized primary WT hepatocytes tothe cytotoxic effect of TNF-α and hepatocyte survival was significantly increased by blockingof IL-1r with rIL-1ra, Anakinra, suggesting a role for IL-1 in induction of steatosis. In Vivotreatment with Anakinra decreased hepatic steatosis and inflammatory cytokine production,and protected WT mice from ALT elevation induced by chronic ethanol. In addition, liversteatosis and serum ALT increase induced by a single alcohol gavage were also amelioratedby rIL-1ra treatment. Conclusions: Our novel data demonstrate inflammasome activationin ALD and support an indispensable role for IL-1β in the pathogenesis of hepatocytesteatosis, cytotoxicity and liver inflammation. The therapeutic potential of IL-1ra in ALDneeds further investigation. (Supported by NIAAA 1RO1AA017729).

S-887 AASLD Abstracts

392

Hippo Signaling Pathway in Liver Development and RegenerationHaibo Bai, Nailing Zhang, Yang Xu, Karen K. David, Duojia Pan, Robert A. Anders

Introduction: The Hippo signaling pathway is a potent regulator of organ size. Originallyuncovered in Drosophila, it is now being explored in vertebrates. In mammals, the yesassociated protein (YAP) controls the output from the Hippo signaling cascade. We've shownconditional liver YAP expression expands the liver to 5 times its expected size.We hypothesizethat YAP is an essential regulator of liver development and regeneration. Methods: Sincecomplete YAP deficiency is lethal, a floxed YAP allele that deletes its first 2 exons wasconstructed. After ES cell injection and selection, homologous recombination was confirmed.YAP f/f mice were crossed with Albumin-Cre (AlbC), MX1-Cre (MxC) or injected with anadenovirus associated virus (AAV) expressing Cre (AvC). MxC;YAPf/f mice were treated with3 every other day 600 ug poly (I:C) doses. In all 3 genotypes, the deleted allele was confirmedwith PCR. YAP mRNA and protein expression was determined. AlbC;YAPf/f mice wereharvested at embryonic (E) day 18.5 and 4 weeks (w) post-partum, and MxC;YAPf/f andAvC;YAPf/f mice were harvested 2 and 8 w after poly (I:C) treatment or viral infection,respectively. Bile ducts were examined with pan-cytokeratin (CK) immunohistochemistry.Hepatocyte proliferation was examined with BrdU at 42 hrs and liver/body weight ratio (Lv/Body) was determined 14 days after partial hepatectomy (PhX). Results: YAPf/f mice crossedwith AlbC or MxC mice were born in the expected Medelian frequencies with no grossabnormalities. AvC infected YAPf/f mice showed no excess mortality or abnormalities com-pared to control infection. Deletion efficiencies were 95% AlbC;YAPf/f, 85% MxC;YAP f/fand 75% AvC;YAP f/f and unlike control mice YAP protein was not detected in the liver ofany of the genotypes. At 4 w (Lv/Body) was 2% greater in AlbC;YAPf/f mice compared tocontrol. There was no significant difference in the Lv/Body of MxC;YAPf/f and AvC;YAPf/fmice. AlbC;YAPf/f mice had a significant 3 fold decrease in CK positive periportal cells atE18.5. There was no difference in the CK positive cells in MxC;YAPf/f and AvC;YAPf/f miceat 2 w and 8 w. Following PhX both MxC;YAPf/f and AvC;YAPf/f showed no significantdifference in the number of BrdU positive hepatocyte nuclei at 42 hr and the Lv/Body wasno different at14 days compared to control. Conclusions: The Hippo signaling pathwayshows an important role in embryonic bile duct development, but it has yet to demonstratea role in adult bile duct homeostasis. Inactivation of Yap in the adult shows no obviousdefect. Liver regeneration is not severely impaired following partial hepatectomy in Yap-deficient mice although we can't exclude a kinetic delay in proliferation. These data beginto build the foundation for the uncovering this pathway in liver biology.

393

Modulation of the Biliary Expression of Arylalkylamine N-Acetyltransferase(AANAT, the Key Enzyme Regulating Melatonin Synthesis) Alters theProliferative/Apoptotic Responses of Cholangiocytes to Liver Injury In Vivoand In VitroAnastasia Renzi, Sharon DeMorrow, Shannon Glaser, Paolo Onori, Julie Venter, RominaMancinelli, Antonio Franchitto, Mellanie White, Heather Francis, Yoshiyuki Ueno, FanyinMeng, Wendy Butler, Eugenio Gaudio, Gianfranco Alpini

After bile duct ligation (BDL), there is enhanced biliary growth and bile duct mass (IBDM).Conversely, acute administration of CCl4 induces the functional damage of cholangiocytesby apoptosis and reduction of IBDM. Cholangiocytes secrete several neuroendocrine factorsregulating biliary functions by autocrine/paracrine mechanisms. We have previously shownthat melatonin inhibits both In Vitro and In Vivo the growth of cholangiocytes from BDLrats by modulation of clock genes. We aim to demonstrate that: (i) the key enzyme involvedin melatonin synthesis, AANAT, is present in cholangiocytes; and (ii) In Vivo and In Vitromodulation of biliary AANAT expression alters normal biliary growth and the proliferative/apoptotic responses of cholangiocytes to CCl4. Methods: Normal rats were treated for 1 wkwith AANATmorpholino (to reduce the biliary expression of AANAT) or scrambledmorphol-ino (1 mg/kg BW/day) by a portal vein catheter before receiving a single dose of mineraloil or CCl4 (0.4 ml by gavage 1:1 in mineral oil/100 gm BW). Two days later, in liversections we evaluated: (i) the hepatic expression of AANAT; and (ii) the percentage ofTUNEL-positive cholangiocytes, and IBDM. In total liver tissue, we studied the quantitativeexpression of AANAT, PCNA, Bax and Bcl-2 by qPCR and immunoblots. In mouse cholangi-ocyte lines (NMC) stably overexpressing AANAT we studied the expression of AANAT andCLOCK genes, melatonin secretion and biliary growth after 48 hr incubation. Results: Inliver sections, we found that: (i) AANAT was expressed by cholangiocytes and hepatocytes;(ii) the expression of AANAT decreased in cholangiocytes from AANAT morpholino-treatedrats receiving vehicle or CCl4; and (iii) in rats treated with morpholino there was enhancedIBDM compared to controls, and absence of biliary apoptosis in response to CCl4. We founddecreased AANAT and increased PCNA expression in liver tissue from normal rats receivingAANAT morpholino compared to scrambled morpholino. In liver tissue from rats treatedwith scrambled morpholino + CCl4 there was enhanced Bax and decreased Bcl-2 and PCNAexpression. CCl4-induced changes in Bax, Bcl-2 and PCNA expression were prevented bythe In Vivo administration of AANAT morpholino. In Vitro overexpression of the AANATgene in NMC increased melatonin secretion, decreased PCNA and Per 1 expression butincreased the expression of BMAL 1, Cry 1 and Clock compared to mock-transfectedNMC. Conclusion: We found that: (i) In Vivo downregulation of biliary AANAT stimulatescholangiocyte proliferation and prevents CCl4-induced ductal apoptosis by an autocrineloop; and (iii) In Vitro overexpression of AANAT in NMC decreases the proliferation andmodulates clock gene biliary expression In Vitro. Local targeting of AANAT in cholangiocytesmay be important for the management of cholangiopathies.

AA

SL

DA

bst

ract

s