Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with metabolic syndrome Derrick M. Van Rooyen 1 , Lay T. Gan 1 , Matthew M. Yeh 2 , W. Geoffrey Haigh 3 , Claire Z. Larter 1 , George Ioannou 3 , Narci C. Teoh 1 , Geoffrey C. Farrell 1,⇑ 1 Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia; 2 Department of Pathology, University of Washington Medical Centre, Seattle, WA 98195, USA; 3 Department of Gastroenterology, University of Washington, Seattle, WA, USA Background & Aims: We have recently showed that hyperinsuli- nemia promotes hepatic free cholesterol (FC) accumulation in obese, insulin-resistant Alms1 mutant (foz/foz) mice with NASH. Here we tested whether cholesterol-lowering drugs reduce stress-activated c-Jun N-terminal kinase (JNK) activation, hepato- cyte injury/apoptosis, inflammation, and fibrosis in this meta- bolic syndrome NASH model. Methods: Female foz/foz and WT mice were fed HF (0.2% choles- terol) 16 weeks, before adding ezetimibe (5 mg/kg), atorvastatin (20 mg/kg), or both to diet, another 8 weeks. Hepatic lipidomic analysis, ALT, liver histology, Sirius Red morphometry, hepatic mRNA and protein expression and immunohistochemistry (IHC) for apoptosis (M30), macrophages (F4/80), and polymorphs (myeloperoxidase) were determined. Results: In mice with NASH, ezetimibe/atorvastatin combination normalized hepatic FC but did not alter saturated free fatty acids (FFA) and had minimal effects on other lipids; ezetimibe and atorvastatin had similar but less profound effects. Pharmacologi- cal lowering of FC abolished JNK activation, improved serum ALT, apoptosis, liver inflammation/NAFLD activity score, designation as ‘‘NASH’’, macrophage chemotactic protein-1 expression, reduced macrophage and polymorph populations, and liver fibrosis. Conclusions: Cholesterol lowering with ezetimibe/atorvastatin combination reverses hepatic FC but not saturated FFA accumula- tion. This dampens JNK activation, ALT release, hepatocyte apoptosis, and inflammatory recruitment, with reversal of steato- hepatitis pathology and liver fibrosis. Ezetimibe/statin combina- tion is a potent, mechanism-based treatment that could reverse NASH and liver fibrosis. Ó 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction While environmental and genetic factors interact to cause non- alcoholic fatty liver disease (NAFLD), more severe metabolic com- plications (hyperglycemia/diabetes, dyslipidemia/metabolic syn- drome, hypoadiponectinemia) are associated with development of non-alcoholic steatohepatitis (NASH) and liver fibrosis [1,2]. In NASH, hyperinsulinemia, adipose inflammation, and hypoa- diponectinemia, dysregulate turnover of one or more hepatic lipid pools, leading to accumulation of lipid molecules that dam- age hepatocytes and lead to inflammation, a process known as lipotoxicity [1,2]. Lipotoxicity is the mechanism of tissue injury in atherosclero- sis and pancreatic b-cell destruction in type 2 diabetes [1]. Its hallmarks include activation of the c-Jun N-terminal kinases (JNK) by the toxic lipid molecules (cholesterol in atheroma), mac- rophage infiltration, oxidative stress and apoptosis, all features of human NASH [1–4]. While formation of triglyceride (TG), a safe storage form of lipid, appears to be a protective pathway against tissue injury in NAFLD [4], there is conflicting evidence about which potentially lipotoxic lipid fractions cause NASH. Saturated free fatty acids (satFFA) are favoured [1,4] by the observation that palmitic acid is lipotoxic to cells of hepatic lineage in vitro, by a JNK-dependent mechanisms [5,6]. However, while FFAs accumu- late in livers of lean, insulin-sensitive mice with steatohepatitis induced by methionine and choline deficiency (MCD) [7], hepatic FFA levels are the same in human NAFLD livers with and without NASH pathology [8]. On the other hand, an increase in hepatic free cholesterol (FC) clearly distinguishes NASH from ‘‘not NASH’’ pathology [8,9]. Insulin-mediated upregulation of sterol-regulating element binding protein-2 (SREBP-2) is a potential mechanistic link between insulin resistance in NAFLD and hepatic cholesterol accumulation [9,10]. SREBP-2 regulates hepatic cholesterol Journal of Hepatology 2013 vol. xxx j xxx–xxx Keywords: Lipotoxicity; Atorvastatin; Ezetimibe; Inflammatory recruitment; Liver fibrosis. Received 6 November 2012; received in revised form 10 February 2013; accepted 25 February 2013 ⇑ Corresponding author. Address: Australian National University Medical School, Gastroenterology and Hepatology Unit, The Canberra Hospital, Yamba Drive, Garran, ACT 2605, Australia. Tel.: +61 2 6244 2473; fax: +61 2 6244 3235. E-mail address: geoff.farrell@anu.edu.au (G.C. Farrell). Abbreviations: ALT, alanine aminotransferase; FC, free cholesterol; FFA, free (un- esterified) fatty acid(s); H&E, haematoxylin and eosin; HF, high fat; IHC, immu- nohistochemistry; JNK, c-Jun N-terminal kinase; LDL, low density lipoprotein; MCP-1, macrophage chemotactic protein-1; M30, cytokeratin-18 fragment; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NPC1L1, Niemann Pick C1-like 1; SatFFA, saturated free fatty acids; SREBP-2, sterol-regulating-element binding protein-2; SS, simple steatosis. Research Article Please cite this article in press as: Van Rooyen DM et al. Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice with met- abolic syndrome. J Hepatol (2013), http://dx.doi.org/10.1016/j.jhep.2013.02.024