Reduction of Liver Steatosis and Fibrosis with an Ask1 Inhibitor in a Murine Model of Nash is Accompanied by Improvements in Cholesterol, Bile Acid and Lipid Metabolism

ORAL PRESENTATIONS triglycerides, adiposity index, insulin resistance and an increase of serum adiponectin. IVA337 also inhibited CCL4-induced fibrosis ( prophylactic and therapeutic), expression of TGF-β family members and extracellular matrix components. Conclusions: These findings demonstrate that simultaneous activation of the 3 PPAR isoforms by IVA337 exerts a beneficial effect on HSC proliferation and differentiation in vitro, and on liver fibrosis in vivo. Interestingly, selective PPARs agonists did not inhibit HSC proliferation, pointing out the benefit of using a pan-PPAR agonist for the treatment of liver fibrosis. In addition, IVA337 demonstrated positive effects on metabolic markers considered root causes for NASH. Together this data support the clinical investigation of IVA337 for the treatment of NASH patients. PS069 THE EFFECT OF MTORC1 INHIBITION ON NAFLD AND SUBSEQUENT HCC DEVELOPMENT A. Umemura1,2, Y. Itoh1, M. Karin2. 1Department of Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; 2Laboratory of Gene Regulation and Signal Transduction, Departments of Pharmacology and Pathology, UCSD, San Diego, United States E-mail: [email protected] Background and Aims: Since mTORC1 is activated in up to 50% of HCCs, there has been much interest in the use of mTORC1 inhibitors for HCC treatment. mTORC1 is also activated in response to obesity which greatly enhances HCC risk. So, we hypothesized that mTORC1 inhibition attenuates obesity-induced fatty liver and its subsequent HCC development. Methods: We conducted short-term mTORC1 inhibition by a mTORC1 inhibitor rapamycin (A). To determine the direct effect of long-term mTORC1 suppression in hepatocytes, we used hepatocytespecific Raptor-deficient (Rpt KO) mice (B). We also used Tsc1 knockout (TSC1 KO) mice, in which mTORC1 is constitutively activated (C). Results: (A) Rapamycin treatment improved fatty liver in HFD (highfat diet)-fed mice, surprisingly however, such mTORC1 inhibition also resulted in increased IL-6 production, and activation of STAT3, which is previously shown to enhance HCC development. (B) Unexpectedly, complete mTORC1 inhibition by Raptor ablation in hepatocytes resulted in not only increased IL-6 production and STAT3 activation, but also strongly potentiating DEN-induced HCC development in HFD-induced obese mice. Thus, mTORC1 activation does not play a crucial role in obesity-promoted HCC development, at least in this model. (C) On the other hand, it is reported that mTORC1 inhibition prevented spontaneous liver damage, fibrosis and tumorigenesis in TSC1KO mice, in which mTORC1 is hyperactivated. We then generated TSC1/ p62 double knock-out mice to investigate the role of p62, a multifunctional protein in TSC1 KO liver. In the absence of p62, liver damage and fibrosis observed in TSC1 KO mice were attenuated, and most importantly, the tumorigenesis was completely abolished. Conclusions: mTORC1 inhibition suppresses liver damage, fibrosis and tumorigenesis in a mTORC1-hyperactive state. However, in normal mice, even obese ones, the adverse effects of mTORC1 inhibition outweigh its benefits. Recently, increased liver damage was also observed during a clinical trial of a mTOR inhibitor everolimus (EVOLVE-1), and this trial did not meet the primary end point. Hyperactivation of AKT and aberrant liver regeneration, also seen after rapamycin treatment and in Rpt KO livers, may be reasons to be concerned about long-term mTORC1 inhibition as an HCC treatment. Of note that patients with HCC usually underlie a chronic liver disease, typically liver cirrhosis and require regeneration to maintain their liver functions. In conclusion, mTORC1 inhibition may not be a suitable treatment for HCC.

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PS070 REDUCTION OF LIVER STEATOSIS AND FIBROSIS WITH AN ASK1 INHIBITOR IN A MURINE MODEL OF NASH IS ACCOMPANIED BY IMPROVEMENTS IN CHOLESTEROL, BILE ACID AND LIPID METABOLISM G. Budas1, S. Karnik1, T. Jonnson2, T. Shafizadeh2, S. Watkins2, D. Breckenridge1, D. Tumas1. 1Gilead Sciences, Foster City; 2Metabolon, Durham, United States E-mail: [email protected] Background and Aims: GS-4997 is a first-in-class, small molecule inhibitor of ASK1 that is in clinical development for the treatment of NASH. ASK1 is a redox-sensitive kinase which is activated by various pathological stimuli including oxidative stress, TGF-b and hyperglycemia. Activation of ASK1 mediates activation of JNK and p38 MAP kinases which can induce hepatocyte apoptosis, hepatic inflammation and myofibroblast activation leading to fibrosis. We previously demonstrated that ASK1 inhibition reduces hepatic steatosis and fibrosis and improves insulin sensitivity in preclinical models of NASH. In the current study, we comprehensively characterized the effect of ASK1 inhibition on the metabolome in a murine model of NASH. Methods: Male C57BL/6 mice were fed a diet high in fat, cholesterol, and sugar (FF diet) for 330 days. A NASH phenotype was established by day 240, after which animals were treated for 90 days with a small molecule inhibitor of ASK1 (GS-444217) or with vehicle (n = 15/group). Animals fed a normal diet were used as control group. Endpoints included hepatic steatosis evaluated by morphometry, clinical pathology, liver hydroxyproline levels, an oral glucose tolerance test and mass spectrometric analysis of plasma and liver using the MetabolonÒ platform. Results: Chronic administration of the FF diet caused liver steatosis, fibrosis and insulin resistance, all of which were significantly reduced by treatment with an ASK1 inhibitor. The FF diet also significantly increased hepatic and serum cholesterol and serum bile acids. ASK1 inhibition reduced serum and liver cholesterol by 14% and 45%, respectively, and strongly reduced serum bile acids including cholate (91% reduction) and deoxycholate (90% reduction). FF diet induced alterations in hepatic fatty acid metabolism similar to that observed in human NASH. These alterations included increased hepatic levels of monounsaturated fatty acids produced by stearoyl CoA desaturases ( palmitoleic and oleic acids) and increased total hepatic diacylglycerol and triacylglycerol levels. These FF diet-induced alterations to the hepatic lipid profile were significantly reduced by ASK1 inhibition. Conclusions: Inhibition of ASK1 in a murine model of NASH reduced hepatic steatosis and fibrosis. This efficacy was also accompanied by improvements in cholesterol, bile acid and lipid metabolism as determined by unbiased metabolic profiling. PS071 HSP72 OVEREXPRESSION PROTECTS FROM LIVER INJURY VIA ATTENUATION OF JNK SIGNALLING K. Levada1, N. Guldiken1, G. Vella1, L.P. James2, J. Haybaeck3, A. K. Kiemer4, S.M. Kessler4, C. Trautwein1, P. Strnad1. 1Department of Medicine III and IZKF, University Hospital Aachen, Aachen, Germany; 2 Department of Pediatrics, Arkansas Children’s Hospital Research Institute, Little Rock, United States; 3Institute of Pathology, Medical University of Graz, Graz, Austria; 4Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbrücken, Germany E-mail: [email protected] Background and Aims: Heat shock protein (Hsp) 72 is a molecular chaperone that is upregulated in response to a variety of stress situations and possesses broad cytoprotective functions. To elucidate its hepatic function, we studied its expression in various human liver disorders and its biological significance in newly generated transgenic animals.

Journal of Hepatology 2016 vol. 64 | S159–S182