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J. Biol. Chem., Vol. 279, Issue 40, 41302-41309, October 1, 2004

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Coordination of Peroxisomal -Oxidation and Fatty Acid Elongation in HepG2 Cells^*

Derek A. Wong, Sara Bassilian, Shu Lim, and Wai-Nang Paul Lee

From the Department of Pediatrics, Harbor-UCLA Research and Education Institute, UCLA School of Medicine, Torrance, California 90502

A major product of mitochondrial and peroxisomal -oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal -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 -oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-¹³C₁₈]stearate or [U-¹³C₁₆] 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-¹³C₁₈]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-¹³C₁₆]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 -oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.

Received for publication, June 17, 2004 , and in revised form, July 22, 2004.

^* This work was supported by National Institutes of Health Grant DK56090–04 (to W.-N. P. L.). The GC/MS facility is supported by Public Health Service Grants P01-CA42710 (to the UCLA Clinical Nutrition Research Unit, Stable Isotope Core) and M01-RR00425 (to the General Clinical Research Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Derek Wong is supported by Public Health Service Training Grant GM-08243.

To whom correspondence should be addressed: Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502. Tel.: 310-222-6729; Fax: 310-222-3887; E-mail: lee{at}gcrc.rei.edu.

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