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The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA1) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A1. The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent Km values of 152 ± 11 and 7.8 ± 1.1 μM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.

In this report we show the existence of a distinct pool of fat body diacylglycerol (DG) that can be distinguished from the bulk DG. This is a dynamic pool of DG that uses FA entering the fat body from the hemolymph, whereas the bulk DG uses the fatty acids stored in the fat body fat droplets. Using a dual labeling technique, it was possible to compare the effect of hormone-stimulated DG synthesis and secretion on the distribution of radiolabeled FA among the lipids of the dynamic pool (short-term radiolabeling), with the hormonal effect on the total complement of fat body lipids (long-term radiolabeling). We observed that, whereas DG represents 2% to 3% of the fat body lipid mass, about 20% of the short-term radiolabeled lipids are represented by DG. Stimulation of lipolysis produces a fast decrease in the fraction of short-term radiolabeled DG, whereas there is an increase in the mass of fat body DG. The subcellular distribution of bulk DG showed that its majority (62%) was in the fat cake whereas only 2.9% was in the cytosol. On lipolysis stimulation, the largest changes in specific activities of newly synthesized DG were detected in the cytosol and the fat cake, suggesting that newly synthesized DG localized in the lipid droplets and the cytosol is preferentially mobilized. —Arrese, E. L., J. L. Gazard, M. T. Flowers, J. L. Soulages, and M. A. Wells. Diacylglycerol transport in the insect fat body: evidence of involvement of lipid droplets and the cytosolic fraction.