Cytochrome P450 reductase (POR) is a microsomal electron transport
protein essential to
cytochrome 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. To evaluate the role of
cholesterol and
bile acid synthesis in this hepatic
lipidosis, as well as the possible role of
lipid transport from peripheral tissues, we developed a stable,
small interfering RNA (
siRNA)-mediated cell culture model for the suppression of POR. POR
mRNA and
protein expression were decreased by greater than 50% in McArdle-RH7777 rat
hepatoma cells 10 days after transfection with a POR-
siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular
triglyceride content, replicating the
lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (
lanosterol demethylase) did not result in
lipid accumulation, indicating that loss of
cholesterol synthesis is not the basis for this
lipidosis. Indeed, addition of
cholesterol to the medium appeared to augment the
lipidosis in POR-suppressed cells, whereas removal of
lipids from the medium reversed the
lipidosis.
Oxysterols did not accumulate in POR-suppressed cells, discounting a role for
liver X receptor in stimulating
triglyceride synthesis, but addition of
chenodeoxycholate significantly repressed
lipid accumulation, suggesting that the absence of
bile acids and loss of farnesoid X receptor stimulation lead to excessive
triglyceride synthesis.