Mechanisms whereby nerve growth factor increases diacylglycerol levels in differentiating PC12 cells

We previously showed indirectly that the increase in diacylglycerol (DAG) levels caused by exposing differentiating PC12 cells to nerve growth factor (NGF) must derive mainly from de novo synthesis and, to a lesser and transient extent, from the hydrolysis of [H-3]phosphatidylinositol (PI). To explore further the biochemical mechanisms of this increase, we measured, in PC12 cells, DAG synthesis from glycerol or various fatty acids; its liberation from phosphatidylcholine (PC); and the activities of various enzymes involved in DAG producdon and metabolism. Among cells exposed to NGF (0-116 h): the labeling of DAG from [H-3]glycerol peaked earlier than that of [H-3]PC, and the specific radioactivity of [H-3]glycerol-labeled DAG was much higher than those of the [H-3]phospholipids, indicating that [H-3]DAG synthesis precedes [H-3]phospholipid synthesis. NGF treatment also increased (by 50-330%) the incorporation of monounsaturated ([H-3]oleic acid) and polyunsaturated ([C-14]linoleic acid or [H-3]arachidonic acid) fatty acids into DAG, and, by 15-70%, into PC. NGF treatment increased the activities of long chain acyl-CoA synthetases (LCASs), including oleoyl-CoA synthetase and arachidonoyl-CoA synthetase, by 150-580% over control, but cholinephosphotransferase activity rose by only 60%, suggesting that the synthesis of DAG in the cells was increased to a greater extent than its utilization. NGF did not promote the breakdown of newly formed [H-3]PC to [H-3]DAG, nor did it consistently affect the activities of phospholipase C or D. NGF did increase phospholipase A(2) activity, however the hydrolysis catalyzed by this enzyme does not liberate DAG. Hence the major source of the increased DAG levels in PC12 cells exposed to NGF appears to be enhanced de novo DAG synthesis, probably initiated by the activation of LCASs, rather than the breakdown of PC or PI. (C) 1999 Elsevier Science B.V. All rights reserved.