Abstract
Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 microM [1-(14)C]oleic acid. Metabolism of [1-(14)C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-(14)C]oleate and [1,2,3-(3)H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-(14)C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.
Highlights
To date, five isoforms of long chain acyl-CoA synthetase have been cloned and shown to possess activity toward long chain fatty acids
Since ACSL5 is only one of five known ACSL isoforms involved in long chain fatty acid activation, we used triacsin C, an inhibitor of ACSL1, ACSL3, and ACSL4, to better gauge the increase in activity due to ACSL5 overexpression
ACSL is the first enzyme required for fatty acid activation in almost all known pathways of fatty acid metabolism
Summary
Five isoforms of long chain acyl-CoA synthetase have been cloned and shown to possess activity toward long chain fatty acids. Because ACSL5 is the only ACSL isoform known to be located on mitochondria, it is logical to speculate that it has a role in the -oxidation of fatty acids. In support of this hypothesis, rat liver mitochondrial protein content of ACSL5 increases following a 48-h fast but declines when rats are refed a high sucrose diet for 24 h [6]. Hepatic ACSL5 message level increases 1.6-fold in zinc-deficient mice with fatty livers [17] and decreases 1.5-fold in estrogen-related receptor knock-out mice, which exhibit a reduction in lipogenic gene expression and reduced total body fat [18]. We ectopically overexpressed ACSL5 in rat hepatoma McArdle-RH7777 cells to determine its role in fatty acid activation and partitioning
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