Impact of dietary fatty acids on gene expression of liver-specific knockout of cytochrome P450 reductase

In the present study, we explored the hypothesis that the fatty liver phenotype and associated gene expression changes associated with the specific deletion of the POR gene in adult mouse liver could be abrogated by supplementation of the mouse diet with the very long chain highly unsaturated fatty acids, arachidonic acid (C20:4ω6), eicosapentaenoic acid (C20:5ω3) and docosahexaenoic acid (C22:6ω3). We expected the fatty liver phenotype would not be reduced by the polyunsaturated fatty acids linoleic (C18:2ω6) or linolenic acid (C18:3ω3), since these accumulated in the fatty livers of LivPORKO animals. This proved to be the case. However, we also made two surprising observations. First, control animals fed a diet enriched in PUFA had fatty livers and gene expression profiles similar to animals fed a lard diet, which was deficient in both PUFA and HUFA. Second, while a diet enriched in HUFA did result in reduced steatosis in livers of the LivPOKO animals, fat accumulation was still elevated relative to controls. Array analyses indicated most differences in gene expression were related to fatty acid metabolism and could explain differences in fat accumulation in LivPORKO livers with dietary treatment. Generation of the founder line with hepatic ablation of POR is described in Wu, et al. ((2003) Genesis 36, 177-181). Male test animals used in this study had the genotype Alb-Cre+/-/POR-lox+/+ and were called LivPORKOs. For each series of experiments, mice were placed on one of three synthetic diets upon weaning. Siblings of the same genotype were randomized between dietary groups. Upon weaning (four weeks after birth) male mice of each genotype were placed on one of the test diets for eight weeks. Synthetic diets used base diet AIN-93G and contained 18.7% protein, 64.7% carbohydrate, 5% fiber, 6% fat, 0.025% cholesterol, 0.008% sodium cholate, and 3.97 kcal per gram. The added fats were as follows: 6% lard (lard diet); 6% canola oil (canola diet); or 3.27% ARASCO oil (Martek Biosciences Corp.) and 2.73% menhadin oil (fish/fungal diet). After 8 weeks on the test diets (12 weeks of age) the animals were fasted for four hours prior to sacrifice. Liver samples were placed in mRNAlater® for mRNA analysis and immediately frozen and stored at -80°C. To reduce variability between arrays, three RNA samples from individual mice of the same genotype/diet group were pooled using equivalent RNA amounts for each animal. Three microarrays on three pools were done per genotype/diet. The quality of RNA was analyzed using an Agilent 2100 Bioanalyzer. Three micrograms of RNA was reverse transcribed to make cDNA. Hybridization of cDNA, washing, and scanning of the Affymetrix GeneChip Mouse Genome 430 2.0 (containing 45,101 probe sets representing over 34,000 mouse genes) was performed according to standard Affymetrix protocol.