494A AASLD ABSTRACTS HEPATOLOGY, October 2003 693 OVEREXPRESSION OF PEROXISOME PROLIFERATOR- ACTIVATED RECEPTORS GAMMA AND UNCOUPLING PROTEIN 2 IN LIVER OF RATS WITH NONALCOHOLIC STEATOHEPATITIS. fian-Gao Fan, Xiao-Dong Ding, Guo-Lian Wang, Xiao-Ying Zheng, Zeng-Jie Xu, Li-Yan Tian, Shanghai First People's Hospital, Shanghai, China Aims & Background:The peroxisome proliferator-activated recep- tors gamma (PPAR-3') are ligand-activated transcription factors that play an important role in modulate enery homeostasis(espe- cially lipid homeostasis), also they are the key regulator of adipo- cyte differentiation. Uncoupling protein 2(UCP2), which is located in the inner-membrane of mitochondria, can mediate proton leak across the mitochondria and uncouple fuel oxidation from aden- osine triphosphate(ATP) synthesis. UCP2 therefore may be ideally suited for the task of abolishing high proton gradients built across the mitochondria in hepatocyte exposed to large amouts of fatty acids. In this study, we investigated the expression and possible roles of PPAR-3' isoforms of 3'~, 3'2 and UCP2 in a rat model of nonalcoholic steatohepatitis(NASH). Methods: NASH was in- duced in SD rats by fat-rich diet for 24weeks.The expression of PPAR-3' and UCP2 in the liver were assessed by immunohisto- chemistry and semi-quantitative RT-PCR. Results: oth immuno- histochemistry and semi-quantitive RT-PCR showed the up- regulated expression of PPAR3'~, 3'2 and UCP2 in the liver of NASH. Compared with control group fed with normal diet, mRNA levels of both PPAR3'~ and UCP2 in liver of rats of NASH group were increased gradually with time from 8, 12,16, to 24 weeks, and peaked at 24wk by 3.5 and 4.2 folds respectively, however, PPAR-3'2 mRNA peaked in 16wk by 5.8 folds. A positive correlation was seen between the mRNA expression of PPAR3'~, 3'2 and UCP2(r-0.79, 0.85, P<0.05, respectively), and a negative cor- relation between the UCP2 mRNA expression and the liver ATP content (r--0.77, P<0.05) in NASH group. Furthermore, a positive correlation between the mRNA expression of PPAR3'2 and the degree of liver steatosis(r-0.89, P<0.05) was seen in NASH group. Conclusion: The study suggested the expression of PPAR3' and UCP2 in the liver of NASH group were up-regulated. PPAR3' could stimulate the hepatocyte differentiate to have some charac- ters of adipocyte and lead to the development of steatosis which named "adipogenic hepatic steatosis'. It can also up-regulate the expression of UCP2 in liver. The increasing UCP2 may play a role in the reduction of ATP content in liver of NASH rats. Disclosures: Xiao-Dong Ding - No relationships to disclose Jian-Gao Fan - No relationships to disclose Li-Yan Tian - No relationships to disclose Guo-Lian Wang - No relationships to disclose Zeng-Jie Xu - No relationships to disclose Xiao-Ying Zheng - No relationships to disclose 694 LONG TERM EXPOSURE TO LOW LEVELS OF BACTERIAL DERIVED CELL WALL PRODUCTS ACTIVATES MURINE HEPATIC STELLATE CELLS. Paola Brun, Ignazio Castagliuolo, University of Padua, Padua, Italy; Massimo Pinzani, University of Florence, Florence, Italy; Giorgio Palfi, Diego Martines, University of Padua, Padua, Italy Non-alcoholic steatohepatitis (NASH) is an acquired metabolic disease of the liver associated with obesity that eventually progress to necro-inflammation, fibrosis and cirrhosis. Although activation of hepatic stellate cells (HSC) is recognised as the initiating event leading to fibrosis, the factor(s) responsible for HSC activation in NASH are not completely known. Since iper- glycemia, hyperinsulinemia and increased plasma levels of in- flammatory cytokines, all described in obesity, can increase intestinal permeability we tested the hypothesis that bacterial products may induce a fibrogenetic phenotype in HSC. First passage murine HSC were used in all the experiments. Serum starved HSC were cultured for 24 hrs in medium alone or containing lipopolysaccharide (LPS), lipoteichoic acid (LTA) or muramic acid (MurA) (10-0.01 /~g/ml). In different experiments, HSC were incubated for 6 days, with 10 ng/ml (an ineffective dose to activate HSC following 24 hrs stimulation) of LPS, LTA, MurA either alone or in association. At the end of the experiments TGF-/31, IL-6 and fibronectin mRNA transcripts and peptide levels were determined by quantitative RT-PCR and ELISA, respec- tively. We first showed that low passage murine HSC expressed specific mRNA transcripts (identified by RT-PCR) encoding mCD14, TLR4, MD2 (receptor system for LPS and LTA) and peptidoglycan recognition proteins (PGRP) long and short. In addition, mCD14 and TLR4 proteins were also detected by immunohistochemistry. Incubation of HSC for 24 hrs with LPS (1 /~g/ml) significantly increased TGF-/31 (by 1,8 + 0,5-fold) and IL-6 (by 12,45 + 5,2-fold) mRNA transcripts levels over controls, but had no effect on fi- bronectin mRNA levels. LTA (10 /~g/ml) increased only IL-6 (by 4,98 + 2,3-fold) mRNA transcripts over controls, whereas, MurA had no significant effects on any of the tested parameters. Similar results were obtained also by ELISA measurements of TGF-/31 and IL-6 in the conditioned medium. Six days exposure of HSC to 10 ng/ml of LPS alone increased TGF-/31 and IL-6 mRNA transcripts by 1,9 + 0,88 and 3,9 + 1,28 fold vs controls, respectively, but still had no effect on fibronectin mRNA levels. Similarly, LTA and MurA by themselves following 6 days exposure significantly increased only IL-6 mRNA tran- scripts by 2,1 + 0,24 and 1,9 + 0,56 fold, respectively, as compared to controls. However, simultaneous exposure of HSC to LPS and LTA significantly increased fibronectin (by 1,95+0,3 fold), IL-6 (by 4,2+2,2 fold) and TGF-/31 (by 1,7+0,4 fold) mRNA levels over controls. In summary, here we report that first passage murine HSC ex- press functional receptors for bacterial derived cell wall products. Furthermore persistent exposure to low levels of bacterial prod- ucts, a condition eventually mimicking the situation of NASH patients, triggers a pro-inflammatory and fibrogenetic phenotype in HSC. We speculate that intestinal lumen-derived bacterial cell wall fragments may contribute to the activation of HSC that even- tually will cause the development of fibrosis in NASH patients. Disclosures: Paola Brun - No relationships to disclose Ignazio Castagliuolo - No relationships to disclose Diego Martines - No relationships to disclose Giorgio Palfl - No relationships to disclose Massimo Pinzani - No relationships to disclose 695 LIPOPHILIC BUT NOT HYDROPHILIC ANTIOXIDANTS IN COMBINATION WITH URSODEOXYCHOLIC ACID PROTECT MOUSE HEPATOCYTES AGAINST AMIODARONE TOXICITY. Amine Ouazzani-Chahdi, Allal Chabli, Aziz~ limadi, University of Montreal, Montreal, PQ, Canada; Patrick Collin, Axcan Pharma, Mont Saint-Hilaire, PQ, Canada; Pierre S Haddad, University of Montreal, Montreal, PQ, Canada Non alcoholic steatohepatitis (NASH) is a common and poten- tially severe form of liver disease caracterized by lipid deposits (steatosis), inflammation (hepatitis) and fibrosis within the liver. The cause of NASH is not yet defined but is intensely investigated. The 'two hit' model of NASH pathogenesis implicates steatosis as a first hit that increases the sensitivity of the liver to the second hit, which is oxidative stress induced by reactive oxygen species (ROS). Lipid peroxidation ensues and leads to cell injury in the fatty liver. It is now established that the antiarrythmic drug ami- odarone (AMD) causes a NASH-Iike syndrome in mice via lipid peroxidation. At present time, there is no clear means for treat- ment of NASH. Ursodeoxycholic acid (UDCA) and antioxidants, such as a-tocopherol (vitamin E), are among the promising phar- macological therapies that may be of potential benefit for patients with NASH. Thus, the objective of this project was to determine the effect of UDCA alone or in combination with different anti- oxidants on amiodarone-induced hepatotoxicity in an in vitro model. Isolated mouse hepatocytes were incubated with or with- out UDCA (0-100/~M) associated or not to butylated hydroxytolu- ene (BHT, 0-100/~M) ascorbic acid (vitamin C, 0-100/~M) or to the lipophilic antioxidant a-tocopherol (vitamin E, 0-500/~M) 15 rain before adding AMD (50/~M) to the culture medium. Twenty hours