1260 URI HEPATOCYTE-SPECIFIC OVEREXPRESSION IS SUFFICIENT TO INDUCE NON ALCOHOLIC STEATOHEPATITIS (NASH)

POSTERS Methods: 7 weeks-old ovariectomized swiss CD-1 mice (20–25 g) were used in this study. The results were compared to those obtained with control (sham-operated) mice. After 10 weeks, mice were fasted for 12 h, blood was taken by cardiac puncture and tissues were immediately harvested. Plasma glucose, total cholesterol, triglycerides, HDL-cholesterol, VLDL-cholesterol and LDL-cholesterol concentrations were analysed by standard methods. Hepatic mitochondrial function was measured by assessing oxygen consumption in the presence of fatty acids, succinate and b-hydroxybutyrate, as well as, the maximal respiration in the presence of FCCP, and reactive oxygen species (ROS) production. Results: Fasting plasma glucose levels were significantly elevated in the OVX mice (+31.5%±0.71, n = 8), as well as, total cholesterol (+86%±0.65, n = 8) and LDL cholesterol levels (+200%±0.63, n = 8). Average fasting triglyceride and HDL cholesterol levels were not significantly different between the groups. Oxygen consumption by intact mitochondria in b-oxidation was inhibited irrespective of the fatty acid used. Octanoyl-CoA (−56%±8.5, n = 6), palmitoylCoA (−45%±11.6 n = 6), and palmitoyl-carnitine (−45%±11.8 n = 6) oxidations were inhibited in OVX mice. With b-hydroxybutyrate and succinate as substrates the rates of oxygen consumption in the presence of ADP (state III) were decreased (−25.2%±3.1 and −55.2%±12.6, n = 8) in OVX mice. State IV of respiration was also significantly lower in OVX group with both substrates (−17.2%±0.84 and −27.6%±1.69, n = 8), as well as, the respiratory control ratio. In the presence of FCCP the rates of oxygen consumption were decreased in OVX mice with both substrates, b-hydroxybutyrate (−19.7%±2.4, n = 8) and succinate (−18.09%±3.46, n = 8). The OVX group exhibited increased (+94.42%±4.0 n = 6) ROS production compared to the SHAM group. Conclusion: These data indicate that OVX resulted in significant impairment in the mitochondrial carbohydrate and lipid oxidations. So, in a condition of oestrogen deficiency, these could be contributing effects to divert hepatic lipid metabolism from fatty acid oxidation to FFA synthesis, thereby favouring liver triglyceride accumulation. Financial support: CAPES. 1260 URI HEPATOCYTE-SPECIFIC OVEREXPRESSION IS SUFFICIENT TO INDUCE NON ALCOHOLIC STEATOHEPATITIS (NASH) A. Gomes, K. Tummala, N. Djouder. BBVA Foundation-Cancer Cell Biology Programme, CNIO, Madrid, Spain E-mail: [email protected] Background and Aims: Liver is the most essential metabolic organ. Nutrients overload, through elevated levels of insulin and related insulin-like growth factor (IGF) dysregulate hepatic function affecting whole body energy metabolism balance leading to severe hepatic disorders which can ultimately progress to hepatocellular carcinoma (HCC), supporting epidemiological studies indicating the strong correlation existing between obesity, type 2 diabetes (T2D) and HCC development. The well established nutrient sensing mTOR circuitry has been described to be a critical signalling pathway downstream of IGF and deregulated in both obesity-associated T2D and HCC and therefore linked to poor prognosis. However, downstream mTOR effectors remain elusive. In this regard, URI, identified as a downstream effector of mTOR/S6K1 pathway, was described to be an addictive oncogene amplified in human ovarian cancer. Moreover, data from our lab show URI is frequently up-regulated in human HCC and correlates with tumour cell proliferation and decreased patients survival. Additionally we gather evidence that URI is increased in livers from obese patients and high-fat diet-treated C57Bl6/J mice present high hepatic URI expression, contrariwise to fasted mice, placing URI as a nutrient sensor. Finally, mice generated in our lab and expressing S510

URI in hepatocytes display hepatic metabolic dysfunctions and spontaneous liver tumours. However, the underlying molecular mechanisms remain unknown. We therefore aim to a better understanding of the role of URI in hepatic metabolic dysfunctions that may progress to HCC. Methods: Transgenic mice have been generated in which, FLAG tagged hURI transgene is targeted to the Col1A1-locus and the rtTA transactivator is expressed under the LAP promoter. To follow disease progression, mice were sacrificed and analysed at different time points of hURI expression. Liver histopathological characterization, body densitometries, cytokine profile and glucose and insulin tolerance tests were performed. Results: Increased fibrosis, inflammation and finally hepatosteatosis were detected at early time points of hURI expression. Physiological mechanisms indicate insulin resistance (IR) of the white adipose tissue (WAT) leading to increased serum circulating free fatty acids accumulating into the liver as triglycerides and defining the presence of a NASH. Conclusions: URI induces NASH pinpointing URI to be a new therapeutic target in NASH treatment. 1261 DELETION OF CASPASE-8 IN HEPATOCYTES IMPROVES HEPATIC INFLAMMATION AND INJURY BY MODULATING THE EXPRESSION OF LIVER METABOLISM-RELATED GENES IN A MURINE MODEL OF NASH M. Hatting1 , F.J. Cubero1 , Z. Gang1 , F. Schumacher1 , G. Sellge1 , N. Gassler2 , M. Boekschoten3 , M. Mueller3 , C. Liedtke1 , C. Trautwein1 . 1 Medical Department III, 2 Department of Pathology, RWTH Aachen University, Aachen, Germany; 3 Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands E-mail: [email protected] Background: Hepatocyte injury in form of apoptosis is a prominent feature in human disease and murine models of non-alcoholic steatohepatitis (NASH). Caspase-8 (Casp8) is essential for the activity of death receptor signalling pathway and thus for the modulation of apoptosis. However, little is known on non-apoptotic functions of Casp8 in liver disease with the view to find a novel therapy for the treatment of NASH. Aim: To dissect the role of hepatocyte Casp8 in murine models of steatohepatitis. Methods: We generated hepatocyte-specific Casp8 knockout (Casp8Dhep ) mice by crossing Casp8f /f with transgenic Alb-cre mice. Animals were then fed with either a methionine-choline-deficient (MCD) diet, a high fat diet (HFD) or a chow diet for 10 weeks. Liver injury was evaluated by histo-pathological analysis, serum markers, apoptotic cell-death, FACS analysis of liver infiltration and inflammation and assessment of reactive oxygen species (ROS). Moreover, microarray analysis was performed on animals treated with or without TNFa. Results: Expectedly, MCD and HFD feeding triggered hepatosteatosis, lipid storage and accumulation of free fatty acids (FFA) in wildtype (WT) livers. In contrast, Casp8Dhep livers elicited significant reductions in these NASH-related hallmarks. Additionally, deletion of Casp8 in hepatocytes caused a reduction in apoptosis and decreased hepatic infiltration and expression of proinflammatory cytokines such as TNFa and IL-6 in the MCD feeding model. However, no differences in hepatocyte injury and inflammation were observed in HFD-fed mice. Furthermore, microarray analysis of animals challenged with TNFa revealed a profound change in gene expression of liver enzymes related to lipid metabolism (e.g. MCAD, ELOVL3). In order to further prove these results we analyzed these genes in the NASH-diet models at mRNA and protein levels and confirmed our previous findings in MCD and HFD fed mice. Conclusion: Our results demonstrate that selective ablation of Casp8 in hepatocytes ameliorates development of NASH by

Journal of Hepatology 2013 vol. 58 | S409–S566