Functional proteomic analysis of nonalcoholic fatty liver disease in rat models: Enoyl-coenzyme a hydratase down-regulation exacerbates hepatic steatosis

Abstract

Nonalcoholic fatty liver disease (NAFLD) has emerged as a common public health problem that can progress to end-stage liver disease. A high-fat diet (HFD) may promote the development of NAFLD through a mechanism that is poorly understood. We adopted a proteomic approach to examine the effect ofHFDonthe liver proteome during the progression ofNAFLD.MaleSprague-Dawley rats fed an HFD for 4, 12, and 24 weeks replicated the progression of human NAFLD: steatosis, nonspecific inflammation, and steatohepatitis. Using two-dimensional difference gel electrophoresis (DIGE) combined with matrix-assisted laser desorption ionization time of flight/time of flight analysis, 95 proteins exhibiting significant changes (ratio≥1.5 or≤-1.5, P<0.05) during the development of NAFLDwere identified. Biological functions for these proteins reflected phase-specific characteristics during the progression of the disease.Thepotential role of enoyl-coenzymeAhydratase (ECHS1), an enzyme that catalyzes the second step of mitochondrial fatty acid beta-oxidation, received further investigation. First, the reduced protein level of ECHS1 was validated both in rat models and in patients with biopsy-proven hepatic simple steatosis via immunoblotting or immunohistochemical analysis. Then the small interfering RNA (siRNA)-mediated knockdown of ECHS1 in the murine hepatocyte cell line alpha mouse liver 12 (AML12) demonstrated increased cellular lipid accumulation induced by free fatty acid (FFA) overload. Furthermore, using a hydradynamic transfection method, the in vivo silencing effect of siRNA duplexes targeting ECHS1 was further investigated in mice. Administering ECHS1 siRNA specifically reduced the expression of ECHS1 protein in mice liver, which significantly exacerbated the hepatic steatosis induced by an HFD. Conclusion: Our results revealed that ECHS1 down-regulation contributed to HFD-induced hepatic steatosis, which may help clarify the pathogenesis of NAFLD and point to potential targets for therapeutic interventions. Copyright © 2010 by the American Association for the Study of Liver Diseases.