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Suppression of cytochrome P450 reductase expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic reductase‐null mice
Author(s) -
Porter Todd D,
Banerjee Subhashis,
Stolarczyk Elzbieta I
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.967.3
Subject(s) - oil red o , lipid droplet , reductase , lipid metabolism , cytochrome p450 , biology , hepatocyte , drug metabolism , transfection , nile red , staining , microbiology and biotechnology , gene expression , chemistry , metabolism , endocrinology , biochemistry , enzyme , gene , in vitro , adipogenesis , physics , genetics , quantum mechanics , fluorescence
Cytochrome P450 reductase (POR) is a microsomal electron transport protein that is essential to P450‐mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. It is unclear why lipids accumulate when POR expression is extinguished, as POR is not known to be involved in lipid processing in cells. Moreover, it has not been determined if this hepatic lipidosis is dependent on the transport of lipids from peripheral tissues to the liver. To address these questions, we have developed a stable, siRNA‐mediated cell culture model for the suppression of POR. McARH7777 rat hepatoma cells were transfected with a POR siRNA‐producing plasmid and grown in standard media under continuous neomycin selection for up to 20 days. Real‐time PCR analysis revealed less than 20% of normal POR mRNA levels by day 5, and less than 2% by day 20. Immunoblot analyses demonstrated a greater than 50% reduction in POR protein expression by day 10, and nearly complete loss by day 20. Lipid accumulation was evident by Oil Red O staining by day 15. Immunofluorescence studies demonstrated a marked decrease in POR expression in treated cells at day 20, and a marked accumulation of lipid droplets by Nile Red staining. These results demonstrate that the suppression of POR expression in hepatoma cells results in the accumulation of lipid droplets similar to that seen in the livers of mice in which POR expression is extinguished. This cell culture model should facilitate investigation of the mechanism by which POR contributes to lipid processing in hepatocytes.