DIACYLGLYCEROL HYDROLYSIS TO ARACHIDONIC-ACID IS NECESSARY FOR INSULIN-SECRETION FROM ISOLATED PANCREATIC-ISLETS - SEQUENTIAL ACTIONS OF DIACYLGLYCEROL AND MONOACYLGLYCEROL LIPASES

Arachidonic acid has been implicated as a second messenger in insulin secretion on the basis of (1) mobilization of intracellular Ca2+ from the endoplasmic reticulum of islets and (2) amplification of voltage-d ependent Ca2+ entry. The insulin secretagogues D-glucose and the musca rinic agonist carbachol both increase unesterified arachidonic acid ac cumulation in isolated islets. We now show that diacylglycerol, a prod uct of phospholipase C action, is a major source of free arachidonic a cid in islets. Diacylglycerol hydrolysis in islets occurs through a tw o-step process. In the first step, the sn-1 bond of 1-stearoyl-2-arach idonyl-sn-glycerol is hydrolyzed by a diacylglycerol lipase, giving ri se to 2-arachidonyl-sn-glycerol. Next, the sn-2 bond of 2-arachidonyl- sn-glycerol is hydrolyzed by a monoacylglycerol lipase, which is the r ate-limiting step, releasing unesterified arachidonic acid. Both diacy lglycerol lipase and monoacylglycerol lipase are highly enriched in th e plasma membrane of beta-cells. Diacylglycerol lipase activity in isl et homogenates is selectively inhibited in a dose-dependent manner by the compound RHC-80267, a specific diacylglycerol lipase inhibitor. RH C-80267 inhibits glucose- and carbachol-induced insulin release from i ntact islets in a dose-dependent manner that parallels its inhibition of diacylglycerol lipase activity. Importantly, RHC-80267, at concentr ations that almost completely inhibit diacylglycerol lipase activity a nd glucose- and carbachol-induced insulin secretion by islets, markedl y inhibits glucose- and carbachol-induced increases in islet arachidon ic acid levels, as measured by gas chromatography with electron-captur e detection of its pentafluorobenzyl esters. RHC-80267 did not signifi cantly affect islet glucose oxidation, phospholipase C, monoacylglycer ol lipase, or phospholipase A(2). Since glucose and carbachol are know n to stimulate phospholipase C, our observations indicate that diacylg lycerol is an important source of arachidonic acid and other free fatt y acids in islets. Furthermore, production of arachidonic acid from th e hydrolysis of diacylglycerol is essential for glucose- and carbachol -induced insulin secretion.