Biosynthesis of apolipophorin-Ill by the fat body in locusts

Biosynthesis of locust apolipophorin-111 (apo-111) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-111 first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-I11 and released it into the medium. The developmental change in the apo-I11 synthesizing activity in the fat body reflected that of the apo-111 concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-111 as a major translation product in a cell-free system.u These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-111, and biosynthesis of apo-I11 is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.-Izumi, S., K. Yamasaki, S. Tomino, and H. Chino. Biosynthesis of apolipophorin-I11 by the fat body in locusts. J Lipid Res. 1987. 28: 667 -672. Supplementary key words lipophorin diacylglycerol insect adipokinetic hormone hemolymph Lipophorin is the major lipoprotein in the hemolymph of most insects (l), and serves as a reusable shuttle to transport various lipids including diacylglycerol, hydrocarbons, cholesterol, and carotenoids between tissues (2). Locust adipokinetic hormone (AKH), a blocked decapeptide (3), is released from the corpora cardiaca in response to flight (4) and promotes the association of non-lipidcontaining protein, named apolipophorin I11 (5), with lipophorin and the loading of diacylglycerol from the fat body by lipophorin to produce larger, lower density lipophorin particles (6). The purification of locust apolipophorin I11 (apo-111) from the hemolymph has recently been achieved in our laboratory and we have demonstrated the following points regarding the physicochemical properties of apo-I11 (7). I) There are three different molecular species of apo-I11 in the locust hemolymph. 2) The three apo-IIIs, apo-111a, apo-111-b, and apo-111-c, are indistinguishable with regard to the mobility on SDS-PAGE, amino acid composition, and immunodiffusion. However, they have different isoelectric points and, therefore, are separable on nativeand urea-PAGE. 3) The three apo-111s are glycoproteins containing fucose, mannose, and glucosamine. 4) The native molecular weight of apo-I11 (ca. 19,500) is almost equivalent to the molecular weight (ca. 20,000) determined by SDS-PAGE, thereby indicating that he locust apo-I11 exists in hemolymph as a monomeric form. 5) A total of 9 moles of apo-III(2 moles apo-111-a, 6 moles apo-111-b, and 1 mole apo-111-c) associate with each mole of lipophorin in response to the action of AKH. Lipophorin itself is synthesized by the fat body and released into hemolymph (8) and always exists in the hemolymph throughout the life of the insect from the larval stage to the adult stage (2). However, the question of which tissue is responsible for the synthesis of apo-I11 remains unanswered. The flight of the locust is restricted to the adult stage and, therefore, it is also important to determine when the synthesis of apo-I11 is initiated in relation to the flight activity and the mechanism of AKH action. In this report, we provide the evidence that, in locusts, the fat body is the tissue responsible for the synthesis of apo-111, and that the synthetic activity first appears at a certain stage of the adult, 3 days after the final molt. This study also reports the translation in a wheat germ cell-free system of mRNA for apo-I11 isolated from the fat body. MATERIALS AND METHODS