The mechanism by which insulin increases diacylglycerol kinase (DAGK) activity has been studied in cerebral cortex (CC) synaptosomes from adult (3-4 months of age) rats. The purpose of this study was to identify the role of phospholipases C and D (PLC and PLD) in DAGK activation by insulin. Neomycin, an inhibitor of PLC phosphatidylinositol-bisphosphate (PIP2) specific; ethanol, an inhibitor of phosphatidic acid (PA) formation by the promotion of a transphosphatidyl reaction of phosphatidylcholine phospholipase D (PC-PLD); and DL propranolol, an inhibitor of phosphatidate phosphohydrolase (PAP), were used in this study. Insulin (0.1 microM) shielded an increase in PA synthesis by [32P] incorporation using [gamma-32P]ATP as substrate and endogenous diacylglycerol (DAG) as co-substrate. This activated synthesis was strongly inhibited either by ethanol or DL propranolol. Pulse chase experiments also showed a PIP2-PLC activation within 1 min exposure to insulin. When exogenous unsaturated 18:0-20:4 DAG was present, insulin increased PA synthesis significantly. However, this stimulatory effect was not observed in the presence of exogenous saturated (di-16:0). In the presence of R59022, a selective DAGK inhibitor, insulin exerted no stimulatory effect on [32P]PA formation, suggesting a strong relationship between increased PA formation by insulin and DAGK activity. These data indicate that the increased synthesis of PA by insulin could be mediated by the activation of both a PC-PLD pathway to provide DAG and a direct DAGK activation that is associated to the use of 18:0-20:4 DAG species. PIP2-PLC activation may contribute at least partly to the insulin effect on DAGK activity.

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The highly efficient formation of phosphatidic acid from exogenous 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) in rat brain synaptic nerve endings (synaptosomes) from cerebral cortex and hippocampus is reported. Phosphatidic acid synthesized from SAG or 1,2-dipalmitoyl-sn-glycerol (DPG) was 17.5 or 2.5 times higher, respectively, than from endogenous synaptosomal diacylglycerides. Insulin increased diacylglycerol kinase (DAGK) action on endogenous substrate in synaptic terminals from hippocampus and cerebral cortex by 199 and 97%, respectively. Insulin preferentially increased SAG phosphorylation from hippocampal membranes. In CC synaptosomes insulin increased phosphatidic acid (PA) synthesis from SAG by 100% with respect to controls. Genistein (a tyrosine kinase inhibitor) inhibited this stimulatory insulin effect. Okadaic acid or cyclosporine, used as Ser/Threo protein phosphatase inhibitors, failed to increase insulin effect on PA formation. GTP gamma S and particularly NaF were potent stimulators of PA formation from polyunsaturated diacylglycerol but failed to increase this phosphorylation when added after 5 min of insulin exposure. GTP gamma S and NaF increased phosphatidylinositol 4,5 bisphosphate (PIP2) labeling with respect to controls when SAG was present. On the contrary, they decreased polyphosphoinositide labeling with respect to controls in the presence of DPG. Our results indicate that a DAGK type 3 (DAGKepsilon) which preferentially, but not selectively, utilizes 1-acyl-2-arachidonoyl-sn-glycerol and which could be associated with polyphosphoinositide resynthesis, participates in synaptic insulin signaling. GTP gamma S and NaF appear to be G protein activators related to insulin and the insulin receptor, both affecting the signaling mechanism that augments phosphatidic acid formation.