Voluntary oral feeding of rats not requiring a very high fat diet is a clinically relevant animal model of non-alcoholic fatty liver disease (NAFLD)

Abstract

Animal models used to study the pathogenesis of non-alcoholic fatty liver disease (NAFLD) are, in general, either genetically altered, or fed with a diet that is extremely high in fat or carbohydrates. Recent findings support the role of oxidative stress, lipid peroxidation and inflammation as probable causative factors. We hypothesize that not only the amount of dietary fat, but the quality of fat is also important in inducing NAFLD. Based on previous observations that female rats fed a diet comprising unsaturated fatty acids are susceptible to liver injury, we proposed that female rats fed with a diet containing fish oil and dextrose would develop pathological and biochemical features of NAFLD. We fed a highly unsaturated fat diet (30% fish oil) to female Sprague-Dawley rats (180-200g), consumed ad libitum for 8 weeks (NAFLD; n=6-8). Control animals (CF; n=6-8) were fed with an isocaloric regular rat chow. At killing, blood and liver samples were collected for serum alanine aminotransferase (ALT), histology and molecular analysis. Each histological sample was evaluated for fatty liver (graded from 0 to 4+ according to the amount of fatty change), necrosis (number of necrotic foci (no./mm2) and inflammation (cells per mm 2). The amount of collagen formation was estimated based on the amount of Sirius Red staining. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out for tumor necrosis factor alpha (TNF- alpha;), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), adiponectin, glutathione peroxidase (GPx), superoxide dismutase (Cu/Zn SOD) and catalase (CAT). Western Blot analysis was done for cyclooxygenases-2 (COX-2), inducible nitric oxide synthase (iNOS) and nitrotyrosine. Electrophoretic mobility shift assay was performed for nuclear factor-kappa B (NF-kB) activity. NAFLD rats had a significantly higher serum ALT level, amount of collagen formation, fatty liver, necrosis and inflammation when compared with the chow-fed control rats. mRNA and protein levels of NF-kB regulated genes, which included TNF-alpha;, COX-2 and iNOS were also significantly (p<0.01; p<0.01; p<0.05 respectively) upregulated in the NAFLD group when compared with the chow-fed control rats. mRNA levels of antioxidants CAT and GPX were reduced by 35% and 50% respectively in the NAFLD group. However, Cu/Zn SOD mRNA was similar in both groups. The mRNA level of adiponectin was also reduced in NAFLD group. NF-kB activity was markedly increased in the NAFLD rats (p<0.01). The level of oxidative stress, representedby the formation of nitrotyrosine, was significantly elevated in the NAFLD rats (p<0.01). We conclude that NAFLD rats demonstrated several features of NAFLD, which included fatty liver, inflammation, necrosis, increased oxidative stress, an imbalance between pro and antioxidant enzymes mRNAs, reduced adiponectin levels and upregulation of pro-inflammatory mediators. We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD.