Clinical Nutrition Experimental Deleterious effect of n-3 polyunsaturated fatty acids in non-alcoholic steatohepatitis in the fat-1 mouse model  Diana Shefer-Weinberg, Shlomo Sasson, Betty Schwartz, Nurit Argov-Argaman, Oren Tirosh  Clinical Nutrition Experimental  Volume 12, Pages 37-49 (April 2017) DOI: 10.1016/j.yclnex.2016.12.003 Copyright © 2016 Terms and Conditions

Fig. 1 The effect of alpha linolenic acid (ALA) on total lipid and ROS accumulation in AML12 hepatocytes. AML12 cells were incubated for 18 h with different fatty acids mixtures (oleic acid (OA): palmitic acid (PA); 2:1, OA: PA: ALA 1:1:1 in complex with BSF), total concentration 1 mM. Steatosis, evaluated by Nile red (A), and ROS, evaluated by DCFH oxidation (B). Cells were analyzed by flow cytometry. Means with different letters in each group differ significantly at P < 0.05. n = 3. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Fig. 2 The effect of alpha linolenic acid (ALA) on lipid peroxidation evaluated by conjugated dienes formation. AML12 cells were incubated for 18 h with 1 mM of different kinds of fatty acid mixture as described in the legend to Fig. 1. At the end of incubation the cell fat was extracted using Folch reagent (methanol: chloroform; 1:2). Conjugated dienes levels were determined at 234 nm (ε = 28*103 M−1 cm−1). t-test at P = 0.1. n = 3. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Fig. 3 Increased fat accumulation in fat-1 mice: Evaluation of total fat in mice livers (fat percentage). Livers of fat-1 and C57BL/J6 mice treated or not with MCD diet were evaluated. Frozen liver tissues were weighed and extracted using Folch reagent dried and analyzed by weight (A), and by Gas chromatography and ratio of quantitative measurements of specific fatty acids was calculated (B). Means with different letters in each group statistically differ at p < 0.05. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Fig. 4 Increased fat accumulation in Fat-1 mice: liver histology and oil red staining. Images of liver sections stained with oil red. C57BL/J6 and fat-1 mice treated or not with MCD diet for 4 weeks were evaluated. Liver morphology and fat accumulation were evaluated by oil-red-O staining (red) for lipids at 40× magnification bars indicate 50 μm. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Fig. 5 Increased liver damage in fat-1 mice: serum hepatic enzymes activities. Blood was collected from the abdominal aorta of fat-1 and C57BL/J6 mice treated or not with MCD diet. Serum was separated and liver enzyme activity of alanine aminotransferase (ALT) (A), and aspartate aminotransferase (AST) (B) was determined. Means with different letters are statistically different at p < 0.05. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Fig. 6 Increased lipid peroxidation in fat-1 mice. Livers of fat-1 and C57BL/J6 mice treated or not with MCD diet were evaluated for lipid peroxidation. Conjugated dienes formation levels. Fat was extracted from frozen liver tissue and the protein concentration was determined using Bradford assay. Conjugated dienes levels were determined at 234 nm (ε = 28*103 M−1 cm−1). Means with different letters in each group differ at p < 0.05. (A), Determination of thiobarbituric acid reacting substances (TBARS) levels. Frozen liver tissues homogenized in 1 ml 10% TCA, centrifuged at 5000 RPM, for 10 min at 4 °C. 2 ml TBA (0.375 g %) were mixed with 500 μl of the upper phase and incubated for 15 min in boiling water. The absorbance was measured at 532 nm (ε = 1.56*105 M−1 cm−1) (B). Protein concentration was determined using Bradford assay. Means with different letters in each group differ at p < 0.05. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions

Scheme 1 Hypothesis scheme showing that oxidized omega 3 fatty acids are deleterious under non-alcoholic steatohepatitis conditions. Clinical Nutrition Experimental 2017 12, 37-49DOI: (10.1016/j.yclnex.2016.12.003) Copyright © 2016 Terms and Conditions