Adipokinetic hormone-induced lipolysis in the fat

The pathway for the adipokinetic hormone-stiniulated synthesis of sn-1,2-diacylglyceroIs in the adult Manducu sexta fat body was studied. Adult fat body lipids were labeled by feeding 5th instar larvae either with [9,10(n)-3H]oleic acid or [ 1 (3)-3H]glycerol and after 32 days insects at the adult stage were used. This long-term prelabeling led to labeled fat body acylglycerols in which triacylglycerols comprised the main radioactive lipid component (95.5%), regardless of the radiolabeled compound used. Because the distribution of radioactivity among the lipid classes was very close to the mass distribution of the fat body lipid subspecies, it was concluded that homogeneous labeling of fat body lipids was obtained. After adipokinetic hormone treatment, an accumulation of radioactivity in the sn-1 ,2-diacylglycerol fraction was the only significant change found in the distribution of radioactivity among fat body lipids. The size of diacylglycerol pool increased 280% 60 min after adipokinetic hormone stimulation, whereas the fatty acid, monoacylglycerol and phosphatidic acid pool sizees remained constant.m These results support the hypothesis that adipokinetic hormone-stimulated synthesis of sn-1 ,2-diacylglycerol in the fat body involves stereospccific hydrolysis of the triacylglycerol stores.-Arrese, E. L., and M. A. Wells. Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manducn srxtn: synthesis of .sn-l,2diacylglycerols. ,I. Lipad Res. 1997. 38: 68-76. Supplementary key words diacylglycerol Manduca wxtn lipid mobilization fat bodv AKH The insect fat body, which combines many of the properties and functions of vertebrate liver and adipose tissue, is the principal site in insects for the storage of lipids ( l ) , and triacylglycerols (TG) constitute the main lipid storage form, representing about 90% of the total fat body lipid (2). The content of TG in the fat body is influenced by several factors, including development stage, nutritional state, and migratory flight (3) . In the tobacco hornworm, Manduca sex@ which is widely used as a model insect, the maximum content of fat body TG occurs at the end of larval development, as a consequence of the accumulation ofreserves during laxval feeding (4). Afterwards, as a result of lipolysis and the fatty acid oxidation required to sustain energy metabolism during the subsequent non-feeding pupal and adult periods, the TG stores decline (4, 5). Two factors of cephalic origin have been shown to activate lipolysis in the fat body: adipokinetic hormone (AKH) and octopamine. AKH, a peptide that is released from the corpora cardiaca into the hemolymph during flight in many insects, greatly stimulates the secretion of sn-1 ,2-diacylglycerol from the fat body (6) . Octopamine, a monohydroxyphenolic analogue of noradrenaline, whose secretion is stimulated by stress, was shown to induce lipid mobilization in Locusta migralom'a (7) and Acheta domesticus (8). Adult M. sexta show only a moderate response to octopamine compared to AKH (E. L. Arrese and M. A. Wells, unpublished results). Starvation also stimulates lipid mobilization by an 1111characterized mechanism that is independent of AKH ( 5 ) . Unlike vertebrates, where the fatty acids in the stored TG are niobilized as free fatty acids (FFA), i n insects, most, if not all, fatty acids are released from the fat body as sn-l,2-diacylglycerols (DG) (3) . The DGs are transported from the fat body to sites of utilization by lipophorin, the major lipoprotein present in the hetnolymph of most insects (9). The mechanisms of stereospecific synthesis and secretion of .sn-1,2-DG are unknown. Three different pathways for the formation of DG have been proposed: 1) the hydrolysis of TG into sn-2-monoacylglycerol Abbreviations: TG, triacylglycerol; DG, diacylglycerol; MC' , monwicylglycerol; ITA, free fatty acids; PL, phospholipid; PA, phosphatidic acid; AKH, adipokinetic hormone; TLC, thin-layer chromatography; GLC, gas-liquid chromatography: MGAT, morioacylglvcerol-~~cvltrdllSferASe 'To whom correspondcnrc should be adtlresscd. 68 Journal of Lipid Research Volume 38, 1997 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom (MG) followed by stereospecific acylation of sn-2-MG (10-12); 2) de novo synthesis of DG from sn-glycerol-3 phosphate via phosphatidic acid (PA) using the fatty acids produced by TG hydrolysis (13); and 3) the stereospecific hydrolysis of TG into sn-l,2-DG (14, 15). In the first step of lipid mobilization, TG must be hydrolyzed by the action of a lipase. Several investigators have studied insect fat body lipase activity (16) and recently we purified a TGlipase from M. sextu fat body ( 13). Like vertebrate hormone-sensitive lipase, which catalyzes the rate-limiting step in mobilization of adipose tissue fatty acids (17), the M. sexta fat body T G lipase is a phosphorylatable enzyme (13). In vitro, TG hydrolysis catalyzed by the TGlipase led to the accumulation of sn2-MG, which suggested that the monoacylglycerol pathway might be the major route for synthesis of snl,2-DG in M. sexta fat body. Such a pathway requires the activity of a monoacylglycerol-acyltransferase (MGAT). This enzyme is present in M. sexta fat body microsomes (18); however, we found no effect of AKH on MGAT activity, suggesting that AKH does not stimulate the synthesis of sn-l,2-DG through the activation of MGAT. The experiments described below were performed to gain more information about this fundamental metabolic pathway. We have studied the pathway for the synthesis of sn-l,2-DG from stored TG in the adult M. sexta fat body when lipid mobilization is stimulated by AKH. Our data indicated that the most likely origin of the sn1,2-DG is via stereospecific hydrolysis of TG. MATERIALS AND METHODS