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We have previously shown that stereospecific hydrolysis of stored triacylglycerol by a phosphorylatable triacylglycerol-lipase is the pathway for the adipokinetic hormone-stimulated synthesis of sn-1, 2-diacylglycerol in insect fat body. The current series of experiments were designed to determine whether cAMP and/or calcium are involved in the signal transduction pathway for adipokinetic hormone in the fat body, After adipokinetic hormone treatment, cAMP-dependent protein kinase activity in the fat body rapidly increased and reached a maximum after 20 min, suggesting that adipokinetic hormone causes an increase in cAMP, Forskolin (0.1 mu M), an adenylate cyclase activator, induced up to a 97% increase in the secretion of diacylglycerol from the fat body. 8Br-cAMP (a membrane-permeable analog of cAMP) produced a 40% increase in the hemolymph diacylglycerol content. Treatment with cholera toxin, which also stimulates adenylate cyclase, induced up to a 145% increase in diacylglycerol production. Chelation of extracellular calcium produced up to 70% inhibition of the adipokinetic hormone-dependent mobilization of lipids, Calcium-mobilizing agents, ionomycin and thapsigargin, greatly stimulated DG production by up to 130%. Finally, adipokinetic hormone caused a rapid increase of calcium uptake into the fat body.jlr Our findings indicate that the action of adipokinetic hormone in mobilizing lipids from the insect fat body involves both cAMP and calcium as intracellular messengers.