Role of lipophorin in lipid transport to the insect egg

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. IS,Issue of June 26, pp. 87484763,lSSg Printed in U.S.A. 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Role of Lipophorin in Lipid Transportto the Insect Egg* (Received for publication, September 8, 1987) John K. Kawooya and JohnH. Law From the Department of Biochemistry, Biological Sciences West,University of Arizona, Tucson, Arizona 85721 Lipid accounts for 40%of the dryweight of a mature phorin (HDLp-A, p = 1.078 g/ml, M , = 7.63 X lo6) (Kawooya Manduca sexta egg. Less than 1% of the totalegg lipid et al. (1988)). HDLp-A consists of 52% lipid and 48% protein is derived from de novo synthesis by the follicles. The (Ryan et al., 1986). The protein moiety of HDLp-A is made remaining egg lipid originates in the fat body and is up of three immunologically distinct apoproteins, apolipotransported to the ovary by lipoproteins. Vitellogenin, phorin I, apolipophorin 11, and apolipophorin 111, that occur the major egg yolk lipoprotein, accounts for 6%of the in a stoichiometric ratio of 1:1:2 (Kawooyaet al., 1984; Shapiro 95%lipid is attributable et al., 1984). During the conversion of HDLp-A to VHDLp-E total egg lipid. The remaining to the hemolymph lipophorins, adult high density liin the egg, 60% of the lipid is stripped from HDLp-A and pophorin (HDLp-A) and low density lipophorin (LDLp).When HDLp-A that is dual labeledwith ‘H in 68% of the diacylglycerol fraction is converted to triacylglycerol. In addition, the two apolipophorin I11 molecules of thediacylglycerolfractionand “S intheprotein moiety is incubated with follicles in vitro, the ratio of HDLp-A dissociate from the particle, but the two larger SH:S6Sin the incubation medium does not vary and is apoproteins (apolipophorin I and apolipophorin 11) remain as integral components of the newly formed VHDLp-E and are similar to the ratio of the labels that are associated (Ka- not degraded as a result of the HDLp-A transformation (Kawith the follicles. In anaccompanyingpaper wooya, J. K., Osir, E. O., and Law, J. H. (1988) J. wooya et al., 1988). Chino et al. (1977) addressed the problem Biol. Chem. 263,8740-8747), we show that HDLp-A of lipid accumulation in the egg of the silkmoth, Philosamia is sequestered by the follicles without subsequent hy- Cynthia, and concluded that there was more lipid in the egg drolysis ofits apoproteins. These results, together with than could be accounted for by the egg lipophorin and vitelthose presented in this paper, support our conclusion logenin. They proposed that the extraegg lipid was shuttled that HDLp-A is not recycled back into the hemolymphfrom the fat body to theovary by the hemolymph lipophorin. after it is internalized by the follicles and, therefore, Although such a process of lipid transport to the egg is does not function as a reusable lipid shuttle between plausible, it has never been verified. The present study adthe fat body and the ovary. When follicles are incu- dresses two possible ways by which lipid may find its way to bated with dual labeled LDLp, the diacylglycerol comthe ovary during egg development. 1)Egg lipid may bederived ponent of the particle is internalized by the follicles from de mu0 synthesis by the oocyte. Such synthesis hasbeen without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by reported in isolated oocytes of the annelid, Perinereis cultrifera (Dhainaut and Belhamra, 1986) in those of the amphibwhich lipidis delivered to theovary. ian, Xenopus Iaevis (Alonso et al., 1987), and in those of Locusta migratoria (Lubzens et aL, 1981; Ferenz, 1985). 2) Lipid may be transported from the fat body to the ovary Vitellogenin, the precursor of the insect egg yolk protein, through the putative recycling process of lipophorin between and lipophorin, the major insect hemolymph lipid-carrying the fat body and theoocytes, as suggested previously by Chino protein, accumulate in large amounts in insect eggs during et al. (1977). oogenesis, These proteins aresynthesized in the fat body and EXPERIMENTAL PROCEDURES are secreted into the hemolymph from which they are specifMaterials-The animals were raised as described by Prasad et al. ically internalized by the maturing follicles (Telfer, 1960, 1961; Telfer et al., 1981; Kunkel and Nordin, 1985; Rohrkas- (1986). Hemolymph lipophorins were isolated according to the method of Shapiro et al. (1984). [2-’H]Glycerol and tritiated water ten and Ferenz, 1985; Osir and Law, 1986; Kawooya et al., (3H20)were purchased from Amersham Corp.; [%]methionine was 1988).Both proteinscarry lipid and are,therefore, considered from ICN (Irvine, CA); Protosol tissue solubilizer was obtained from to be the source of the egg lipid (Chino et al., 1977). Du Pont-New England Nuclear. The BCA protein assay reagent was In Manduca sexta, egg lipophorin (VHDLp-E)* is a very purchased from Pierce Chemical Co. Triacylglycerol assay reagents high density lipoprotein ( p = 1.238,g/ml, M , = 4.14 X lo6) were purchased from Sigma. Lipid internal standards for gas-liquid that is derived from the adult high density hemolymph lipo- chromatography were obtained from Nu-Check Prep, Inc. (Elysian, * This work was supported by Grant GM 29238 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: VHDLp-E, very high density lipophorin from the egg; HDLp-A, adult high density lipophorin; LDLp, low density lipophorin; [3H]DG-[36S]-HDLp-A,dual labeled adult high density lipophorin; [3H]DG-[36S]LDLp,dual labeled low density lipophorin; [3H]DG,diacylglycerol of lipophorin labeled with 3H;%protein, protein moiety of lipophorin labeled with 36S;SDS, sodium dodecyl sulfate. MN). Antibodies against lipophorin were raised in New Zealand Whiterabbits according to the method of Shapiro et al. (1984). Antibodies against vitellogenin were prepared as described by Osir and Law (1986)and were a gift from Dr. Robert 0. Ryan, Department of Biochemistry. Protein Assay-Except where indicated, protein was assayed according to themethod of Smith et a l , 1985. Polyacrylamide Gel Electrophoresis-Electrophoresis under denaturing conditions (SDS-polyacrylamide gel electrophoresis) was performed on 4-15% gradient acrylamide gel slabs under the conditions described by Laemmli (1970). Properties of the Egg-Eggs were isolated from the abdomens of moths as described previously (Kawooya et al., 1986). Egg sizes were 8748 This is an Open Access article under the CC BY license. 8749 Lipid in the Insect Egg measured with a dissecting microscope that was equipped with a micrometer eyepiece. The average dry weight of the egg was determined on individual lyophilized eggs. Egg wet weight was measured from eggs within 5 min of isolation from the abdomens. Lipid Analysis-Lipids were extracted from 5 mgof lyophilized eggs according to the procedures of Bligh and Dyer (1959) and were analyzed by gas-liquid chromatography as described by FernandoWarnakulasuriya et al. (1981). Carbohydrate Analysis-Carbohydrate from 10 mgof lyophilized mature eggs was extracted and was analyzed as described by Grimes and Greegor (1976). Extraction of Protein from the Egg-Ten mature eggs (1.64-mm size) were homogenized in 500 pl of phosphate-buffered saline (0.1 M sodium phosphate, 0.15 M NaCl, 0.05 M EDTA, pH 7.0) using a ground glass tissue homogenizer in the presence of 200 pg of fine sand. The homogenate was sonicated for 1 min on a Bronson water bath, centrifuged at 12,000 X g for 10 min, and the supernatant solution was transferred to a separatevial. Three hundred p1 of buffer were then added tothe pellet, andthe pellet washomogenized followed by centrifugation and removal of the supernatant. This procedure was repeated (3X) in order to extract buffer-soluble protein from the pellet. The supernatant solutions were pooled and used to measure the total amount of protein, lipophorin, and vitellogeninl egg. At the end of extraction the pellet was boiled in 100 pl of 10 mM Tris-HC1, pH 8.4, containing 1% SDSand centrifuged so asto measure any residual lipophorin and vitellogenin. Total protein in the supernatantsolution was assayed as described by Peterson (1983). Included on the gelwas 10 pgof protein from buffer-soluble egg protein solution. The separated protein on the gel slab was transferred electrophoretically to nitrocellulose paper. The amount of vitellogenin and lipophorin on the nitrocellulose paper was measured immunologically as described previously for the measurement of microvitellogenin (Kawooya et al., 1986). Antibodies against vitellogenin and those againstlipophorin were used to probe these lipoproteins on the nitrocellulose sheet. The amount of lipophorin and vitellogenin in the pellet was expressed as a percentage of the total SDS-soluble protein fraction of the pellet. Measurements of Lipophorin and Vitellin in the Buffer-solubleEgg Protein Fraction-Single radial immunodiffusion analyses (Oudin, 1980) of vitellin and lipophorin were performed according to the method of Bailey (1984). A protein standard curve was constructed from known concentrations of vitellogenin and was used to determine the amount of protein inthe supernatantsolution of egghomogenates. Between 1-12 pg of VHDLp-E and 1-16 pg of vitellin were applied to 1% agar gel plates. Each gel contained the antibodies that were raised against the respective antigen (vitellogenin and lipophorin). Also applied to each gel slab were seven samples (4-20 pg) of buffersoluble egg protein. Plots of antigen concentration versus the square of the diameter of the precipitin reaction were linear and were, therefore, used to measure the total amount of lipophorin and vitellogenin in the egg. Preparation of Dual Labeled PH]DG-lj6S]HDLp-A and f'H]DGrj6SILDLp"wo one-day-old male moths were each injected with 20 pCi of [36S]methionine.After 14 h, each animal was injected with 20 pCi of [2-'H]glycerol. Two hours later, both [3H]DG-[SSS]HDLp-A and ['H]DG-[%3]LDLp were isolated from the hemolymph of these animals as described in the accompanying paper for the isolation of the single labeled [%S]HDLp-A(Kawooya et al., 1988). Specific Activities of PHIDG and 36S-Protein in Dual Labeled pH] DG-P'SIHDLp-A and rHlDG-r6S]LDLp-Total lipid was extracted from 200 pg of each of the dual labeled lipophorins, and its Components were separated as described previously (Kawooya et aL, 1988). The diacylglycerol fraction was extracted from the silica gel of the TLCplate according to themethod of Fernando-Warnakulasuriya et al. (1981). The amount of 'H that was associated with the diacylglycerol fraction was measured by liquid scintillation spectrometry. The total amount of diacylglycerol was measured colorimetrically as described by Carlson (1963) for the assay of triacylglycerol. The delipidated protein pellet was rinsed (5 X) with distilled water and was dissolved in 200 pl of 20 mM Tris-HC1, pH 8.4, that contained 6 M guanidinium hydrochloride. The total protein in this solution was assayed. The data collected from the above measurements were used to calculate the specific activities of [3H]DG and "S-protein in the labeled lipophorins. Incubations of Follicles with Dual Labeled Lipophorins-Pairs of follicles were incubated with dual labeled lipophorins in lepidopteran saline buffer (Jungreis et al., 1973) (0.005 M K2P0,, 0.1 M KC!, 0.004 M NaC1, 0.015 M MgClZ, 0.002 M CaClZ,pH 6.5) at 27 "C for 4 h. At selected intervals, the amount of each radiolabel in the incubation medium was measured. At the same time the follicles were removed from the incubation medium, rinsed (5 X ) with 10 mM Tris-HC1, pH 8.4 buffer that contained 100 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100.The radiolabel that was associated with the follicles was measured. The ['H]DG:%S-protein ratios in the incubation rnedium and in the follicles were calculated from these measurements. De Novo Lipid Synthesis by the Follicles-Approximately 10 follicles (0.6-0.8 mm) were incubated with 2 pCi of 3H20(specific activity, 5 mCi/ml) in 200 p1 of lepidopteran saline at 27 "C. After 4 h, the follicles were removed from the medium and were rinsed (5 X) with distilled HzO. Lipid was extracted from the follicles, and the organic phase was rinsed (8 X) with distilled HzO to remove free 'HzO. After the final rinse, 0.5 mg of anhydrous CaCL was added to the organic phase. The extracted lipid components were separated by TLC, and the distribution of the 3H label in the various lipid fractions was analyzed as described in the accompanying paper (Kawooya et al., 1988). Distribution of 'H from 3H20 in Oleic Acidand Linoleic Acid-Two mg (wet weight) of fat body and 10 follicles (0.6-0.8-mm size) were incubated separately a t 27 "C with 500 p1 of lepidopteran saline ~ After 6 h, the lipid containing 2 pCi of 3Hz0 and5 r n glutathione. was extracted from these tissues as described above and was methylated according to the procedure of Fernando-Warnakulasuriyaet al. (1981). The fatty acid methyl esters were separated by TLC according to themethod of Shibahara et al. (1986) and were assayed for 'H by liquid scintillation spectrometry. RESULTS AND DISCUSSION During egg maturation, large stores of lipid accumulate in the insect follicles (Wiemerslage, 1976; Chino et al., 1977). The results presented below describe how such lipid accumulation is achieved during this process. In addition, the various components of a mature egg were analyzed. Properties of the Insect Egg-Table I shows that 70% by weight of a mature insect egg is water. The remaining 30%is protein, lipid, and carbohydrate. Glucose accounts for 80% of the sugars in the M. sextu egg and has been reported as the major carbohydrate component in other insect eggs, where it is stored inthe form of glycogen granules (Sander et al.,1985; Yamashita and Hasegawa, 1985). In addition to glucose, a small amount of mannose and N-acetylglucosamine is found in M. seztu egg (Table I). Since these sugars occur in vitellogenin (Osir et ul., 1986a) and lipophorin (Nagao and Chino, 1987), their presence in the egg may be attributable to the TABLE I Properties of a mature M. sexta egg The properties were determined as described under "Experimental Procedures." Measurements of egg size were taken at themajor axis of the egg within 5 min of isolation from the abdomen. Average egg size was 1.64 f 0.46 mm. All values are expressed in pg. The mean values were calculated from 3 to 15 determinations. 1565 f 50 Fresh weight Dry weight 470 f 16 Total buffer-soluble protein 148 f 13 Vitellogenin 86 f 5 Lipophorin 20 f 3 Lipid 182.68 f 3.06 Lipid components 15.89 f 3.46 Phospholipid Diacylglycerol 6.16 f 1.32 Sterol 7.20 f 0.16 Free fatty acid 21.15 f 4.05 Triacylglycerol 115.02 f 1.82 Hydrocarbon 17.26 f 0.65 Carbohydrate 10.25 f 2.32 Carbohydrate components 1.20 k 0.31 Mannose Glucose 7.95 f 1.82 N-Acetylglucosamine 0.07 f 0.02 Unidentified 1.03 f 0.12 Buffer-insoluble material 129 F 24 Lipid in the Insect Egg 8750 abundance of these proteins in the eggyolk (Table I). As shown in Table I, lipid accounts for 40% of the dry weight of a mature M. sexta egg, and more than half of the total lipid in the eggs is triacylglycerol. In Hyalophora cecropia, 60% of the egg lipid, mostly triacylglycerol, is stored as discrete lipid droplets within the egg, and the remaining 40% lipid is associated with lipoproteins that are found in the yolk spheres (Wiemerslage, 1976). It is probably that the large amount of triacylglycerol in M. sexta eggs is also stored in lipid droplets, while the vitellin- and lipophorin-associated lipid is found in the yolk spheres. Thus, as in theinsect fat body (Beenakkers et al., 1986), lipid may be stored in the insect egg, predominantly as triacylglycerol. Contributions of HDLp-A and Vitellogeninto the Lipid Stores in the Egg-HDLp-A contains 53% lipid (Ryan et al., 1986), and 13% by weight of vitellogenin is lipid (Osir et al., 1986b). Studies on lipophorin presented inthe accompanying paper (Kawooya et al., 1988) as well as those on vitellogenin reported by Osir and Law (1986), show that the apoproteins of these lipoproteins are not degraded when the particles are internalized by the follicles. Although vitellogenin and lipophorinare major components of the egg (TableI),these lipoproteins accounted for <2% of the SDS-soluble protein fraction in the residual pellet. Therefore, the amount of vitellogenin and lipophorin we measured in the buffer-soluble fraction of a mature egg homogenate (Table I) is fairly representative of the total sum of these lipoproteins in the egg. From these values we calculated that the vitellogenin and lipophorin of the egg account for only 20 pg (-10%) of the total (182 eg) egg lipid. The metabolic source of the estimated 90% of the egg lipid and how it accumulates in the egg are described below. Metabolic Source of the Egg Lipid-The egg lipid may have been derived from de MUO synthesis by the oocyte. Lubzens et al. (1981) and Ferenz (1985) reported that isolated follicles h 150 S 6oC I DG Distance, ( m m ) FIG. 1. De nouo lipid synthesis by the ovary. A, lipid components of follicles that were incubated with 3H20 in vitro. Follicles were incubated with 3H20 as described under “Experimental Procedures.” The lipid components from the follicles were separated by TLC in a hexane:ether:acetic acid (60401)solvent system. The plate was developed with iodine vapor. PL, phospholipid; DG, diacylglycerol; TG,triacylglycerol; HC, hydrocarbon; 0,origin; sf, solvent front. B, distribution of 3Hlabel in lipid components of the eggs. The separated lipids on the TLC plate from the above experiment were measured for the distribution of the 3Hlabel by means of a TLC isotope analyzer. The plate was scanned at the rateof 1.24 cm/min. of L. migratoria have the capacity to incorporate the I4Clabeled acetateorpalmitateinto the egg lipid, butthese studies did not address the question as to how much of the total egg lipid is derived from de mu0 synthesis. We addressed in this question by incubating isolated follicles with 3H20 vitro. Tritiated water has been proven to be more reliable than I4C-labeled substrates for measuring rates of fatty acid synthesis. Unlike I4C-labeledsubstrates, the incorporation of the 3H from 3H20into fatty acids is independent of the large metabolic pools of carbon substrates that are generally associated with tissues (Windmueller and Spaeth, 1966; Jungas, 1968; Lowenstein, 1972). Fig. lA shows triacylglycerol as the major labeled lipid component of the follicles that were incubated with 3H20. Analysis of the distribution of 3H in the lipid (Fig. LA) fractions shows that 48% of the label was incorporated into phospholipid, 45% was in triacylglycerol, and 7% was in diacylglycerol (Fig.1B). Although the 3H label was incorporated into triacylglycerol and phospholipid in the ratio of 0.9:1, the datapresented in Table I andFig. L4 show that there is 7-fold more triacylglycerol than phospholipid in the egg. These results suggest that egg triacylglycerol is derived predominantly from an exogenous source rather than from de MUO synthesis by the follicles. From the dataof Ryan et al. (1986) for HDLp-A and that of Osir et al. (1986a) for vitellogenin, we calculated that there is twice as much sterol and 10 times more hydrocarbon in the egg than is attributable to the combined total egg lipophorin and vitellogenin (Table I). When follicleswere incubated with 3H20, only trace amounts of 3Hwere measured in sterol and hydrocarbon fractions. Unlike mammals, insects in general do not have the capacity to synthesize sterols; instead, they obtain themfrom the diet (Beenakkers et al., 1986). In M.sexta, active feeding is confined to the larval stage, and in our laboratory feeding ends at about 23 days before egg maturation. Therefore, the lipid that accumulates in the egg could not have been transported directly from the midgut. During insect feeding, dietary fat that is absorbed at the midgut is transported to the fat body where most of it is stored in the form of triacylglycerol (Chino, 1985; Beenakkers et al., 1986). With a few exceptions (Blomquist et al., 1982;De Renobales et al., 1986)) most insects, like mammals, do not have the capacity to synthesize polyunsaturated fatty acids such as linoleic acid and linolenic acid, but they synthesize mono-unsaturated fatty acids such as oleic acid (Beenakkers et al., 1986). When we incubated thefat body tissue and follicles separately ina medium containing 3H20 and analyzed the distribution of the label between oleic acid and linoleic acid, in both cases we found over 99% of the label in oleic acid and (0.1% in linoleic acid. Therefore, neither the fat body nor the follicles of M. sexta have the capacity to synthesize linoleic acid, but both tissues can synthesize oleic acid. Prior to the transportof lipid from the fatbody to the sites of lipid utilization, triacylglycerol is converted to diacylglycerol. The diacylglycerol is then transported through the hemolymph by lipophorin (Chino, 1985; Beenakkers et al., 1986). The diacylglycerol that is transported to theegg by lipophorin is processed and stored as triacylglycerol (Kawooya et al., 1988). In order to determine just how much of the egg triacylglycerol originates in the fat body, we analyzed the fatty acid compositions of the triacylglycerol fractions from the egg and from the fat body, and the diacylglycerol fraction from the hemolymph. The results are presented in Table 11. In general, the three components have similar fatty acid composition. Noteworthy are thesimilarities in the ratios of oleic acid to linoleic acid from the fat body, hemolymph, and the egg. Such similarities in the ratios could have occurred only Lipid in the Insect Egg 8751 TABLEI1 Fatty acid composition of diacylglycerol andtrhylglycerolfractions Fatty acids were analyzed as described under "Experimental Procedures." Fatty acidcomposition is expressed as relative percentage. Values were calculated from averages of three determinations (S.D. f 0.1-1.5). Fatty acid Fat bodp Hemolymphb Egg" 140 160 16:l 180 181 182 18:3 0.20.3 27.4 26.8 1.61.1 3.81.2 32.1 33.1 33.2 35.0 2.5 0.4 26.5 1.5 2.6 32.7 34.0 2.3 98 96 94 0 1.7 181/18:2 0.97 ratio0.96 0.95 Values were calculated from triacylglycerol fractions. * Values were calculated from diacylglycerolfractions. if the hemolymph and egg lipids were derived from the lipid stores in the fat body. Calculations that were based on the analyses presented above led us to conclude that 51%of the total lipid in a mature egg were synthesized by the follicle in situ. Retention of HDLp-A by the Follicles-From the results presented above and in theaccompanying paper (Kawooya et al., 1988), we infer that during egg development, fat body triacylglycerol is mobilized, transported through the hemolymph as diacylglycerol, and is delivered to thematuring eggs where it is converted to triacylglycerol. Chino et al. (1977) proposed that lipophorin was the major vehicle bywhich lipid was transported from the fat body to the egg and that the lipophorin particles were continuously exocytosed back into the hemolymph after they unloaded their lipid cargo into the oocyte. We examined this proposal by incubating a series of follicles in a medium that contained biologically dual labeled high density hemolymph lipophorin, [3H]DG-[35S]HDLp-A. In thisparticle, the 3H label was in thediacylglycerol fraction and the 35Slabel was in the protein moiety. The [3H]DG:3sS protein ratio of this particlewas 0.49. At specific intervals we measured the total amount of [3H]DG and "S-protein in the incubation medium and the radiolabels that were associated with the follicles. As shown in Fig. 2 A , there was a continuous depletion of both [3H]DGand 35S-proteinfrom the incubation medium and acorresponding accumulation of the radiolabeled components in the follicles (Fig. 2B). In an accompanying paper we have presented evidence that when [=S]HDLp-A is sequestered by the follicles in vivo or in vitro it ends upin the egg yolk (Kawooya et al., 1988). Therefore, the depletion of [3H]DG-[35S]-HDLp-Afrom the incubation medium (Fig. 2 A ) was due to a net uptake and retention of the particle in the follicles (Fig. 2B). During the 4-h incubation period, a total of 3.70 pg of [3H]DG and 7.66 pg of 35S-proteinwas sequestered by each pair of follicles. The net accumulation of [3H] DG-[35S]-HDLp-A by the follicleswas similar to the net depletion of the particle from the incubation medium (Fig. 2, A and B ) . Calculations based on the results presented inFig. 2A show that the [3H]DG:35S-proteinratio was 0.48 in the incubation medium during the course of the experiment. This ratio was similar to that we calculated from the radiolabels that were associated with the follicles at specific intervals of the experiment. Thus, during the 4-h incubation period the [3H]DG:35S-proteinratio varied neither within the incubation medium nor within the follicles.We conclude from these results that there was no recycling of [3H]DG-[35S]-HDLp-A back into theincubation medium once the particle was internalized by the follicles. If such recycling had occurred, we would have observed a decrease in 3H:35Sratio in the medium '1 B t 0 0 O I L - " 40 80 1 2 0 160 TI me, ( mln) FIG. 2. Lipid transport to the egg by HDLp-A. A, depletion of [3H]DG-[35S]-HDLp-A from the incubation medium. Ten pairs of follicles (0.6-0.8 mm) were dissected from animals 12 h after adult emergence. Each pair was incubated with 100 pg (protein) of ['HI DG-[36S]-HDLp-A(specific activities are 8.4 X lo5 cpm/mg for [3H] DG and 1.76 X 10' cpm/mg for 36S-protein)in 200 pl of lepidopteran saline at 27 "C for 4 h. At selected intervals, the follicleswere removed from the incubation medium. The amounts of 3H and 35Slabels in the medium were measured as described under "Experimental Procedures." The values from these measurements, together with the specific activities presented under "Results and Discussion," were used to calculate the total amount of [3H]DGand 36S-proteinthat remained in medium. Each data point was calculated from values measured inthree separate experiments. B , accumulation of t3H]DG[%]-HDLp-A in follicles. The follicles from the above experiment (A) were rinsed and solubilized as described under "Experimental that was associated with Procedures" and the amount of 3H and the follicleswas measured. Each data point represents an averageof values from these separate experiments. and a corresponding increase in the ratio of the labels in the egg yolk. Similar conclusive experiments could not be performed to study the process by which lipid is transported to the ovary by labeled HDLp-A in uiuo, because Ryan et al. (1986) have shown that when such a particle is injected into the hemolymph, its labeled diacylglycerol moiety is rapidly exchanged with the large pool of the unlabeled diacylglycerol that is associated with the lipophorin of the hemolymph. Furthermore, labeled diacylglycerol from HDLp-A may not only be utilized by the ovary, but itmay also be depleted from the hemolymph by other peripheral tissues such as muscle (Chino, 1985; Beenakkers et al., 1986). However,the progressive accumulation of HDLp-A that occurs in the follicles during egg development as reported in the accompanying paper (Kawooya et al., 1988) would suggest that lipophorin is not recycled back into the hemolymph once it is internalized by the follicles in uiuo. Therefore, lipophorin found in the mature insect egg (Kawooya et al., 1988) is the result of the cumulative internalization of HDLp-A without concomitant exocytosis of the particle after it unloads its lipid in the egg. Role of LDLp in Lipid Transport to the Egg-Wells et al. (1987) have shown that 20% of the total hemolymph lipophorin from moths that have not flown or moths not treated with adipokinetic hormone is LDLp; the remaining 80% is HDLp-A. In thepresent study we found that thehemolymph of females at the peak of egg maturation (1 day after emer- 8752 Lipid in the Insect Egg the delivery of cholesterol esters to mammalian cells by high density lipoprotein. Like insect LDLp, mammalian high density lipoprotein has been reported by Pittman et aL (1987) to deliver lipid to adrenal cortical tumor cells without the internalization of its associated apoprotein. Calculations based on ” the results presented in Figs. 2B and 3B show that LDLp delivers 10 times more lipid to the follicles than HDLp-A. 98 This diacylglycerol-enriched particle accounts for the extra lipid in the egg that is not attributable to egg lipophorin and vitellogenin. Forty percent of the dry weight of a mature M . sexta egg is lipid (Table I). The delivery of lipid to the ovary by LDLp may be of great significance in theproduction of viable mature insect eggs. Like most other insects, a single M. sexta female produces large numbers of eggs. Thus, egg production in insects may impose tremendous energy demands on the lipid stores in the fat body. Lipid may not only be mobilizedin the fat body at a fast rate, butmay it also have to be transported rapidly in large quantities through the hemolymph to the ovary in order to support the accelerated egg maturation. The high lipid carrying capacity of LDLp (Ryan et al., 1986)would make this lipoprotein a more suitable particle than HDLp-A to perform this role. The fate of LDLp after it unloads lipid to the ovary remains to be determined. In M. sexta, the FIG. 3. Lipid transport to the egg by LDLp. A, depletion of [3H]DGof [3H]DG-[36S]LDLpfrom the incubation medium. Pairs of formation of LDLp from HDLp-A can be mediated by the action of the peptide hormone, adipokinetic hormone (Shapfollicles were incubated with 100 pg (protein) of [’HIDG-[”S]LDLp (specific activities are 9.83 X LO6 cpm/mg for [‘HIDG and 1.07 X lo6 iro and Law, 1983; Beenakkers et aL, 1986),which is secreted cpm/mg for ’%-protein). The rest of the experiment and calculations from the corpus cardiacum (Beenakkers, 1969; Mayer and were as described under Fig. 2A and under “Experimental Proce- Candy, 1969). Whether the small amount of LDLp normally dures.” B , accumulation of [3H]DG from [3H]DG:36S-protein-LDLp present inresting animals is also dependent upon adipokinetic in the follicles. At the end of each incubation in A, the amount of [3H]DG and 36S-protein that was associated with the follicles was hormone secretion remains unknown. determined in the same manner as described in Fig. 2 A . gence) contained 23% LDLp and 77% HDLp-A. Since 90% of the lipid that accumulates in theinsect egg is neither due to de novo synthesis nor to thesequestered HDLpA, we explored the possible role of LDLp asthe carrier of the extra lipid into the egg. We incubated [3H]DG-[36S]LDLp with isolated follicles and followed the destinations of its radiolabeled components. The [3H]DG:36S-proteinratio in the dual labeled LDLp particle was 1.26. Fig. 3A shows that there was a progressive decrease in the amount of [3H]DG in the medium and a corresponding increase of [3H]DGin the follicles (Fig. 3B) during 4 h of incubation. Thus, unlike in the case of [3H]DG-[36S]-HDLp-A(Fig. 2, A and B ) , incubation of the follicles with [3H]DG-[36S]LDLpresulted neither in a decrease in the amount of 35S-proteinwithin the incubation medium nor an accumulation of the 35S-protein within the follicles. Calculations based on the results presented in Fig. 3, A and B, show that the (3H]DG:35S-proteinratios within the follicles increased from 0 to 46 during the course of the experiment, while those in the incubation medium decreased from 1.26 to 0.93. We conclude from these resultsthat LDLpassociated [3H]DG is taken up into the follicles without internalization of the LDLp-associated apoprotein moiety. Significance of LDLp in the Transport of Lipid to the EggSimilarities exist inthe presence by which lipid is transported to thefollicles byinsect lipophorin and thedelivery of cholesterol esters to cells by mammalian lipoproteins. Mammalian LDL delivers lipid to cells by receptor-mediated endocytosis (Brown and Goldstein, 1986). The uptake of HDLp-A into the insect follicle may involvea similar mechanism. However, unlike mammalian LDL, the apoprotein moiety of insect HDLp-A is not hydrolyzed within the egg cell cytoplasm (Kawooya et al., 1988). 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