pyrimidinones and 1-stearoyl-2-arachidonoylglycerol

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.

Topics: Animals; Cerebral Cortex; Chromones; Cyclosporine; Diacylglycerol Kinase; Diglycerides; Genistein; Guanosine 5'-O-(3-Thiotriphosphate); Hippocampus; Insulin; Insulin Antagonists; Morpholines; Okadaic Acid; Phosphatidic Acids; Phosphoprotein Phosphatases; Piperidines; Pyrimidinones; Quinazolinones; Rats; Rats, Wistar; Sodium Fluoride; Synaptosomes; Thiazoles; Vanadates

Insulin stimulated protein synthesis in L6 myoblasts but did not increase the labelling of DAG or the release of phosphocholine from phosphatidylcholine. The DAG lipase inhibitor, RHC 80267, more than doubled the amount of label appearing in DAG but did not stimulate protein synthesis. Even in the presence of the DAG lipase inhibitor insulin failed to have any effect on DAG labelling, and conversely RHC 80267 did not modify the insulin-induced increase in protein synthesis. These results suggest that endogenous DAG production is not involved in the stimulation of protein synthesis by insulin. However, exogenous diacylglycerols (1-oleoyl-2-acetyl glycerol and 1-stearoyl-2-arachidonoyl glycerol) both stimulated protein synthesis in L6 myoblasts. The efficacy of the former (arachidonate-free) DAG suggested that their action was by activation of protein kinase C rather than by arachidonate release and prostaglandin formation. Ibuprofen, an inhibitor of cyclo-oxygenase failed to block the effects of insulin whereas a second cyclo-oxygenase inhibitor, indomethacin had only a partial inhibitory effect. The protein kinase C (PKC) inhibitor, RO-31-8220, totally blocked the effect of insulin. Since indomethacin is also recognised to inhibit phospholipase A2, the data suggests that insulin acts on protein synthesis in myoblasts by arachidonate activation of PKC.

Topics: Animals; Arachidonic Acid; Cell Line; Cyclohexanones; Cyclooxygenase Inhibitors; Diglycerides; Enzyme Activation; Indoles; Insulin; Lipoprotein Lipase; Muscle Proteins; Muscles; Protein Kinase C; Pyrimidinones; Rats; Thiazoles