Diacylglycerol generated in CHO cell plasma membrane by phospholipase C isused for triacylglycerol synthesis

The diacylglycerol (DAG) signal generated from membrane phospholipids by ho rmone-activated phospholipases is attenuated by mechanisms that include lip olysis or phospholipid resynthesis, To determine whether the DAG signal mig ht also be terminated by incorporation of DAG into triacylglycerol (TAG), w e studied the direct formation of TAG from endogenous DAG generated by bact erial phospholipase C (PLC), When Chinese hamster ovary (CHO) cells prelabe led with [C-14]oleate were treated with PLC from Clostridium perfringens fo r 6 h, [C-14]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of C-14 label was even greater when the cells were simultaneously exposed to PLC and 100 muM oleic acid. PLC as well as oleate treatment con comitantly increased the TAG mass within the cell. Moreover, when phospholi pids were prelabeled with [H-3]glycerol, a subsequent increase in [H-3]TAG indicated that an intact DAG moiety was channeled into the TAG structure. I ncubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhance d the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in th e presence of 100 muM oleate, but not by incubation with oleate alone, indi cating that the DAG released from plasma membrane phospholipids does not re quire the formation of a phosphatidic acid precursor for TAG synthesis. Sim ilarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG syn thesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG. These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a t ransport mechanism exists to move plasma membrane-derived DAG to the endopl asmic reticulum.