INSULIN-STIMULATED HYDROLYSIS OF PHOSPHATIDYLCHOLINE BY PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D IN CULTURED RAT HEPATOCYTES

We have investigated the mechanism of action by which insulin increase s phosphatidate (PA) and diacylglycerol (DAG) levels in cultured rat h epatocytes. Insulin initially stimulated phosphatidylcholine-dependent phospholipase D (PC-PLD) with a significant increase in both PA and i ntracellular as well as extracellular choline. The involvement of phos pholipase D was confirmed by the formation of PC-derived phosphatidyle thanol in the presence of ethanol. The DAG increase appeared to be bip hasic. Only the early phase of DAG production was inhibited by propran olol, an inhibitor of the phosphatidate phosphatase (PAP) responsible for the conversion of PA into DAG, suggesting that initially the DAG i ncrease is due to the PLD-PAP pathway. The delayed DAG increase was in parallel with increased intracellular and extracellular phosphocholin e and probably derived directly from PC-PLC activity. Experiments perf ormed in the presence of 1 mu M phorbol 12-myristate 13-acetate (PMA) indicated that protein kinase C (PKC) mediated the insulin effect on P C-PLC, but not on PC-PLD. These findings were confirmed using the PKC inhibitors calphostin, H7 and staurosporine. The dual activation of th ese phospholipases with a biphasic elevation of DAG levels and activat ion of specific PKC isoenzymes could be necessary to elicit both early and delayed effects of insulin.