Mitochondrial glycerol phosphate acyltransferase directs the incorporationof exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), t he first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitoch ondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpres sed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cell s. After incubation with trace [C-14]oleate (similar to3 mum), control cell s incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phos pholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporati on of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that e xogenous oleate was directed primarily toward triacylglycerol synthesis. Tr ansiently transfected HEK293 cells that expressed a 4.4-fold increase in GP AT activity incorporated 9.7-fold more [C-14]oleate into triacylglycerol co mpared with control cells, showing that the effect of GPAT overexpression w as similar in two different cell types that had been transfected by differe nt methods. When the stable, GPAT-overexpressing CHO cells were incubated w ith 100 mum oleate to stimulate triacylglycerol synthesis, they incorporate d 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was n ot present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into tria cylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysoph osphatidic acid and phosphatidic acid that must be transported to the endop lasmic reticulum where the terminal enzymes of triacylglycerol synthesis ar e located, and W this pool remains relatively separate from the pool of lys ophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.