Coordination of peroxisomal beta-oxidation and fatty acid elongation in HepG2 cells.
A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mm [U-(13)C(18)]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mm [U-(13)C(16)]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal beta-oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.
Department of Pediatrics, Harbor-UCLA Research and Education Institute, UCLA School of Medicine, Torrance, California 90502, USA.
Derek A
DA
Wong
Sara
S
Bassilian
Shu
S
Lim
Wai-Nang
WN
Paul Lee
D000105:Acetyl Coenzyme A / Q000378:metabolism; D002247:Carbon Isotopes; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D045744:Cell Line, Tumor; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000032:analysis / Q000096:biosynthesis; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction; D010169:Palmitic Acids / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D013229:Stearic Acids / Q000378:metabolism
D002247:Carbon Isotopes; D005227:Fatty Acids; D008055:Lipids; D010169:Palmitic Acids; D013229:Stearic Acids; D000105:Acetyl Coenzyme A