Peroxisome proliferator-activated receptors (PPARs α, β, γ1, and γ2) are widely regarded as monitors of intracellular nonesterified fatty acid (NEFA) levels. As such, fatty acid binding to PPAR leads to changes in the transcription of many genes involved in lipid metabolism and storage. Although the composition of the intracellular NEFA pool is likely an important factor controlling PPAR activity, little information is available on factors affecting its composition. Accordingly, we have examined the effects of exogenous fatty acids on PPARα activity and NEFA pool composition in rat primary hepatocytes. Prior to the addition of fatty acids to primary hepatocytes, nonesterified unsaturated fatty acid levels are very low, representing ≤0.5% of the total fatty acid in the cell. The relative abundance of putative PPARα ligands in the NEFA pool is 20:4n-6 = 18:2n-6 = 18:1n-9 > 22:6n-3 > 18:3n-3/6 = 20:5n-3. Of these fatty acids, only 20:5n-3 and 22:6n-3 consistently induced PPARα activity. Metabolic labeling of primary hepatocytes indicated that both 14C. 18:1n-9 and 14C-20:5n-3 are rapidly assimilated into neutral and polar lipids. Although the addition of 18:1n-9 had no effect on NEFA pool composition, 20: 5n-3 mass increased > 15-fold within 90 min. Changes in NEFA pool 20:5n-3 mass correlated with dynamic changes in the PPARα-regulated transcript mRNACYP4. Metabolic labeling also indicated that a significant fraction of 14C-20:5n-3 was elongated to 22:5n-3. Cells treated with 22:5n-3 or 22:6n-3 led to a significant accumulation of 20:5n-3 in the NEFA pool through a process that requires peroxisomal β-oxidation and fatty acyl CoA thioesterase activity. Further analyses suggest that 20:5n-3 and 22:6n-3, but not 22:5n-3, are active ligands for PPARα. These studies suggest that basal fatty acid levels in the NEFA pool coupled with rates of fatty acid esterification, elongation, desaturation, peroxisomal β-oxidation, and fatty acyl thioestease activity are important determinants controlling NEFA pool composition and PPARα activity.
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