Docosahexaenoic acid membrane content and mRNA expression of acyl-CoA oxidase and of peroxisome proliferator-activated receptor-delta are modulated in Y79 retinoblastoma cells differently by low and high doses of alpha-linolenic acid.

ABSTRACT

The mRNA expression levels of acyl-CoA oxidase (AOX), a key enzyme in very-long-chain fatty acid peroxisomal oxidation, and of peroxisome proliferator-activated receptor-delta (PPAR-delta), a nuclear receptor possibly involved in the gene regulation of brain lipid metabolism, were determined in human Y79 retinoblastoma cells by using real-time quantitative polymerase chain reaction. Cells were dosed with alpha-linolenic acid (183n-3), the essential metabolic precursor of the n-3 polyunsaturated fatty acid series that normally gives rise through terminal peroxisomal oxidation to the synthesis of membrane docosahexaenoic acid (226n-3, or DHA). The AOX and PPAR-delta relative expression levels increased 2.3 and 3.4 times, respectively, upon dosing of cells with 7 microM 183n-3, whereas AOX cDNA abundance decreased by 50% upon dosing with 70 microM 183n-3. Concurrently, the DHA content increased by 23% in the membrane ethanolamine-phosphoglycerides from cells dosed with 7 microM 183n-3, whereas it decreased by 38% upon dosing with 70 microM 183n-3. The DHA's upstream precursors (205n-3 and 225n-3) both accumulated in cells dosed with 7 or 70 microM 183n-3. The 183n-3-induced changes in membrane phospholipid fatty acid composition support the hypothesis that the terminal peroxisomal step of n-3 conversion is rate limiting in the Y79 line. The concurrent 7 microM 183n-3-induced increase of mRNAs encoding for AOX and for PPAR-delta suggests that 183n-3 (or its metabolites) at low concentration could trigger its proper conversion to DHA, possibly through activation of PPAR-delta-mediated transcription of AOX. Decreased membrane DHA content and mRNA expression level of AOX in 70-microM 183n-3-dosed cells corroborated the relationship between AOX expression and DHA synthesis and suggested that simultaneous down-regulating events occurred at high concentrations of 183n-3.