Use of cytosolic triacylglycerol hydrolysis products and of exogenous fatty acid for the synthesis of triacylglycerol secreted by cultured rat hepatocytes.

The fatty acyl moieties incorporated into triacylglycerol (TAG) secreted by rat hepatocytes are derived from diacylglycerol (DAG) that is synthesized de novo through the phosphatidate pathway or derived from endogenous, cytosolic TAG after hydrolysis to DAG and re-esterification. We have used a dual-labeling technique (overnight labeling of cell TAG with [3H]oleate, followed by 3 h incubation with [14C]oleate) to quantify the contributions of the two sources towards TAG secretion by cultured rat hepatocytes. A wide range of TAG secretion rates was achieved by short-term incubation of the cells under a variety of conditions. There was no correlation between the overall amount of exogenous 14C-labeled fatty acid metabolized and the rate of either [14C]- or [3H]TAG secretion. By contrast, there was a strong positive correlation between the fraction of newly synthesized [14C]TAG that was secreted (the fractional secretion rate, FSR) and the absolute rate of TAG secretion. This suggests that the partitioning of DAG between (re)synthesis of cytosolic TAG and synthesis of secreted TAG is an important locus for the control of the rate of TAG secretion. Comparison of the ratio: oxidation/TAG secretion for 3H- and 14C-labeled acyl moieties indicated that, for all conditions studied, approximately half the acyl moieties already esterified to the glyceroyl backbone within cytosolic TAG remain unavailable for oxidation when this pool of TAG is mobilized for the synthesis of secreted TAG. The data provide evidence that hydrolysis of cytosolic triacylglycerol (TAG) does not proceed fully to the constituent fatty acids and glycerol, but only to the level of diacylglycerol, followed by re-modelling of approximately half of its acyl chains, before re-esterification to form secretory TAG.