Abstract
Purpose: :
To maintain normal retinal function, the retinal pigment epithelium (RPE) cells engulf photoreceptor outer segments enriched in free fatty acids. FABPs are involved in intracellular transport of free fatty acids and their targeting to specific metabolic pathways. The present study was designed to elucidate the role of FABP5 in lipid metabolism in the human RPE cells using an siRNA approach.
Methods: :
The expression of the FABP5 gene in a human immortalized RPE cell line, ARPE-19, was knocked down with siRNA. The levels of FABP5 mRNA and protein were determined by real-time PCR and Western blot analysis, respectively. The influx of fatty acids into the siRNA-treated cells was measured by QBT fatty acid uptake assays, and lipid accumulation assessed with Nile Red staining. Enzymatic fluorimetric assays and electrospray tandem mass spectrometry were used to determine the lipid composition of the siRNA-treated cells. The secreted lipoprotein-like particles were visualized by negative-stain electron microscopy. The secretion of newly synthesized apoB was assessed by immunoprecipitation of 35S-radiolabeled apoB100.
Results: :
FABP5 gene knockdown effectively reduced the production of FABP5 protein, resulting in a decrease in fatty acid uptake. As a result, the levels of cholesterol and cholesterol ester were significantly decreased by about 40%, whereas FFAs and triglycerides were significantly increased by 18 and 67%, respectively after siRNA treatment. Some classes of phospholipids were significantly decreased. Cellular lipid droplets were evident and apoB100 secretion was decreased in these cells. Cellular lipid droplets were evident in these cells and increased dramatically in size after oleic acid challenge. Furthermore, ARPE-19 cells could secrete lipoprotein-like particles, but secretion of apoB100 decreased by 76% in the FABP5 siRNA-treated cells.
Conclusions: :
Our results indicate that the FABP5 gene knockdown interrupts free fatty acid transport and results in decreased cholesterol and phospholipid levels, which cause reduced secretion of apoB-containing lipoproteins and the accumulation of cellular free fatty acids and triglycerides. These observations indicated that FABP5 plays a critical role in lipid metabolism in RPE cells, suggesting that FABP5 down-regulation in the RPE/choroid complex in vivo may contribute to aging and early age-related macular degeneration.
Keywords: metabolism • retinal pigment epithelium • RNAi