Insect Biochem. Vol. 20, No. 8, pp. 859-863, 1990 0020-1790/90$3.00+ 0.00 Printed in Great Britain.All rights reserved Copyright © 1990PergamonPress plc ADIPOKINETIC HORMONE CAUSES FORMATION OF A LOW DENSITY LIPOPHORIN IN THE HOUSE CRICKET, ACHETA DOMESTICUS LISA M. STROBEL, MICHAELR. KANOST,ROLF ZIEGLERand MICHAEL A. WELLS* Department of Biochemistry and Center for Insect Science, Biosciences West, University of Arizona, Tucson, AZ 85721, U.S.A. (Received I May 1990; revised and accepted 20 August 1990) Abstract--In the house cricket, Acheta domesticus, injection of adipokinetic hormone induces the formation of a low density lipophorin via uptake of diacyglycerol from the fat body. Cricket low density lipophorin contains apolipophorin-III, which was purified and partially characterized. Cricket apolipophorin-III has a molecular weight of about 18,000, is not glycosylated,and has an isoelectricpoint of 4.8. Its amino acid composition is more similar to apolipophorin-IlI from Locusta migratoria than to that from Manduca sexta. The amino terminal sequence of cricket apolipophorin-III shows only limited homology to the amino terminal sequences of apolipophorin-III from L. migratoria and M. sexta. Key Word Index: lipophorin, apolipophorin-III, cricket, Acheta domesticus, adipokinetic hormone INTRODUCTION MATERIALS AND METHODS The role of adipokinetic hormone (AKH) in lipid mobilization during flight has been well studied in Locusta migratoria and Manduca sexta (see Shapiro et al. 1988; Kanost et al., 1990 for recent reviews). In these species AKH induces formation of diacyl- glycerol in the fat body which subsequently leaves the fat body and associates in hemolymph with high density lipophorin (HDLp) to form a low density lipophorin (LDLp). Concomitant with uptake of diacylglycerol, a small water-soluble apolipoprotein, apolipophorin-III (apoLp-III), binds to LDLp. ApoLp-III is thought to increase the lipid-carrying capacity of lipophorin by stabilizing the increment of lipid-water interface resulting from diacylglycerol uptake (Kawooya et al., 1986; Wells et al., 1987). LDLp is then carried by circulating hemolymph to flight muscle where fatty acids are released with reformation of HDLp and free apoLp-III. It appears that some insect species which have lost, during evolution, the ability to fly have retained components of the flight-related lipid transport sys- tem. In the flightless grasshopper, Barytettix psolus, AKH does not mobilize lipids in spite of the presence of HDLp, apoLp-III and AKH (Ziegler et al., 1988). It has been reported, however, that AKH causes lipid mobilization in the cricket, Acheta domesticus, which does not fly (Woodring et al., 1989), although the authors did not demonstrate the formation of LDLp or the presence of apoLp-III. HDLp has been iso- lated from A. domesticus and does not contain apoLp-III (Ryan et al., 1984). Since A. domesticus does not fly, yet responds to AKH with mobilization of lipid, we felt it worthwhile to investigate further its response to AKH, particularly the presence of apoLp-III and the formation of LDLp. *Author to whom all correspondence should be addressed. Insects Crickets were obtained from Fluker's Cricket Farm, Baton Rouge, La and were provided with water, dog food and potato slicesin a 20 gal plastic container. Both male and female adult insects were used in these experiments with no detectable difference in the results. Purification and characterization of apoLp-lll Crickets were bled by the flushing out method (Chino et al., 1987) using a 50 mM Tris buffer pH 7.5 containing 150 mM NaCI, 10mM glutathione and l mM diisopropyl- fluorophosphate. After low speed centrifugation to remove hemocytes, the hemolymph was heated in a boiling water bath for 5 min. Precipitated proteins were removed by centrifugation (5000g, 10rain), the supernatant was de- salted on a column of Sephadex G-25 (PD-10 column, Pharmacia) eluted with water and apoLp-III was purified by HPLC as described previously (Cole et aL, 1987). For amino acid analysis of apoLp-III, duplicate samples were hy- drolyzed in 6 N HCI at 110°C in vacuo for 24 h and analyzed on a Beckman 7300 amino acid analyzer. NH2-terminal sequencing was by automated Edman degradation as de- scribed by Cole et al. (1987). SDS-PAGE was carried out in 10% polyacrylamide gels (Laemmli, 1970). Isoelectric focusing was conducted in pH 3-9 gel (Phast system, Phar- macia). Carbohydrate was assayed by the phenol-sulfuric acid method (Ashwell, 1966). Isolation and characterization of lipophorins HDLp was prepared from hemolymph collected from resting insects as described above. LDLp was prepared from hemolymph collected 60 rain after injection of 100 pmol of Periplaneta MI (Penninsula, Belmont, Calif.) (O'Shea et aL, 1984) in 10#1 of water. MI was chosen because it was the most similar peptide to A. domesticus AKH that was commercially available. HDLp and LDLp were isolated by density gradient centrifugation and analyzed for protein and lipid as previously described (Prasad et al., 1986; Ryan et al., 1986). 859