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-dependent Ca2+ entry. The insulin secretagogues D-glucose and the muscarinic agonist carbachol both increase unesterified arachidonic acid accumulation in isolated islets. We now show that diacylglycerol, a product of phospholipase C action, is a major source of free arachidonic acid in islets. Diacylglycerol hydrolysis in islets occurs through a two-step process. In the first step, the sn-1 bond of 1-stearoyl-2-arachidonyl-sn-glycerol is hydrolyzed by a diacylglycerol lipase, giving rise to 2-arachidonyl-sn-glycerol. Next, the sn-2 bond of 2-arachidonyl-sn-glycerol is hydrolyzed by a monoacylglycerol lipase, which is the rate-limiting step, releasing unesterified arachidonic acid. Both diacylglycerol lipase and monoacylglycerol lipase are highly enriched in the plasma membrane of beta-cells. Diacylglycerol lipase activity in islet homogenates is selectively inhibited in a dose-dependent manner by the compound RHC-80267, a specific diacylglycerol lipase inhibitor. RHC-80267 inhibits glucose- and carbachol-induced insulin release from intact islets in a dose-dependent manner that parallels its inhibition of diacylglycerol lipase activity. Importantly, RHC-80267, at concentrations that almost completely inhibit diacylglycerol lipase activity and glucose- and carbachol-induced insulin secretion by islets, markedly inhibits glucose- and carbachol-induced increases in islet arachidonic acid levels, as measured by gas chromatography with electron-capture detection of its pentafluorobenzyl esters. RHC-80267 did not significantly affect islet glucose oxidation, phospholipase C, monoacylglycerol lipase, or phospholipase A2. Since glucose and carbachol are known to stimulate phospholipase C, our observations indicate that diacylglycerol is an important source of arachidonic acid and other free fatty acids in islets. Furthermore, production of arachidonic acid from the hydrolysis of diacylglycerol is essential for glucose- and carbachol-induced insulin secretion.