Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

Lipid transfer particle in locust hemolymph: purification and characterization

A lipid transfer particle (LTP) from the hemolymph of adult male locusts, Locusta migratoria, was isolated and purified. The locust LTP exhibited its capacity to catalyze the exchange of diacylglycerol between low density lipophorin (LDLp) and high density lipophorin (HDLp). Contrary to the LTP reported for the tobacco hornworm, M. sexta, the locust LTP appeared to lack the capacity to promote net transfer of diacylglycerol to form an intermediate density lipophorin, although it seems premature to conclude the complete lack of such a capacity in locust LTP. The original concentration of LTP in hemolymph is assumed to be extremely low compared to that of lipophorin; only a catalytic amount of LTP may be present in the hemolymph (e.g., only 160 micrograms of LTP was obtained from the original hemolymph containing 400 mg protein). The molecular weight of intact LTP was estimated to be about 600,000 and the LTP was comprised of three glycosylated apoproteins, apoLTP-I (mol wt 310K), apoLTP-II (mol wt 89K), and apoLTP-III (mol wt 68K). The locust LTP contained significant amounts of lipids; the total lipid content amounted to 14.4% and the lipids were comprised of 17% hydrocarbons, 44% diacylglycerol, 8% cholesterol, 13% free fatty acid, and 18% phospholipids. The above molecular properties of locust LTP are essentially similar to those reported for M. sexta LTP.     

The recycling of protein components of the flight-specific lipophorin in Locusta migratoria

The metabolism of locust lipophorin A⁺ during lipid delivery to the flight muscle and lipid loading at the fat body was studied in vitro. Protein C₂ was shown to be released upon hydrolysis of lipophorin A⁺-carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C₂ was shown to reassociate with lipophorin A_y upon hormone-induced lipid mobilization from fat body in vitro. These results demonstrate the reversibility of the association of protein C₂ with lipophorin A_y and support the shuttle function of the protein components of locust lipophorin A⁺ in lipid transport.     

In vivo and in vitro loading of lipid by artificially lipid-depleted lipophorins: evidence for the role of lipophorin as a reusable lipid shuttle.

Lipid transport in the hemolymph of Manduca sexta is facilitated by a high density lipophorin in the resting adult insect (HDLp-A, d approximately 1.109 g/ml) and by a low density lipophorin during flight (LDLp, d approximately 1.060 g/ml). Lipophorin presumably shuttles different lipids between sites of uptake or storage, and sites of utilization. In order to shuttle lipid, a lipid-depleted lipophorin should be able to reload with lipid. To test this hypothesis, we used HDLp-A particles that were artificially depleted of either phospholipid (d approximately 1.118 g/ml) or diacylglycerol (d approximately 1.187 g/ml) and subsequently radiolabeled in their protein moiety. Upon injection into adult moths, both particles shifted their density to that of native HDLp-A, indicating lipid loading. Also, upon subsequent injection of adipokinetic hormone, both particles shifted to a lower density (d approximately 1.060 g/ml) indicating diacylglycerol loading and conversion to LDLp. Both phospholipid and diacylglycerol loading were also studied using an in vitro system. The lipid-depleted particles were incubated with fat body that had been radiolabeled in either the phospholipid or the triacylglycerol fraction. Transfer of radiolabeled phospholipid and diacylglycerol from fat body to lipophorin was observed. During diacylglycerol loading, apoLp-III associated with lipophorin, whereas phospholipid loading occurred in the absence of apoLp-III. The results show the ability of lipid-depleted lipophorins to reload with lipid and therefore reaffirm the role of lipophorin as a reusable lipid shuttle.     

Lipophorin conversions during flight of the death's-head hawkmoth Acherontia atropos

Flight activity or injection of the death's-head hawkmoth Acherontia atropos with locust synthetic adipokinetic hormone (AKH I) results in a dramatic increase in the concentration of hemolymph diacylglycerol which is carried by specific lipophorins. In resting hawkmoths diacylglycerols are associated with a high-density lipophorin (HDLp, density ∼1.13 g/ml) consisting of two major apolipophorins (apoLp-I and -II, mol. wt ∼240,000 and 70,000, respectively). During flight or after AKH injection the formation of a new low-density lipophorin is induced (LDLp, density ∼1.03 g/ml), exhibiting a much higher lipid loading and consisting of HDLp subunits and an additional subunit (apoLp-III, mol. wt approx. 20,000). This subunit is a regular constitutent of hemolymph proteins in resting hawkmoths and consists of two protein components with slightly different molecular weights. The component with the lowest molecular weight seems to be preferentially incorporated into the newly generated LDLp. In the resting situation the HDLp already contains some apoLp-III.In spite of some minor differences, the overall mechanism of lipophorin rearrangements upon flight activity in the hawkmoth appears to be very similar to the known systems established for both Locusta migratoria and Manduca sexta.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Inhibition of diacylglycerol uptake from the fat body by a monoclonal antibody against apolipophorin II in Locusta migratoria

Twenty monoclonal antibodies raised against locust native lipophorin were screened by testing their capacity to inhibit diacylglycerol (DG) uptake from fat body by lipophorin in vitro. One of the monoclonal antibodies clearly inhibits the loading of DG by lipophorin from the fat body. This antibody cross reacts only with apolipophorin-II(apoLp-II), one of the two apoproteins of lipophorin. By using proteolytic apoLp-II fragments, we have shown that the epitope for the antibody against apoLp-II contains lysine. Furthermore, both the apoproteins, apoLp-I and apoLp-II, were almost equally labeled with biotin when the native lipophorin was incubated with modified biotin-reagent. These observations strongly suggest that apoLp-II, at least in part, is localized on the outer surface of lipophorin and may contribute to the lipid loading process from fat body.     

An insect lipid transfer particle promotes lipid loading from fat body to lipoprotein

The role of Manduca sexta lipid transfer particle (LTP) in the transport of lipid from fat body to lipophorin was investigated in vitro. Fat body that contained radiolabeled lipid was incubated with either high density lipophorin or low density lipophorin, and it was shown that lipid was transferred from fat body to lipophorins. The transfer of diacylglycerol was blocked by preincubating fat body with LTP antibody. Furthermore, transfer was restored by the addition of LTP, indicating that LTP promotes the transfer of lipid from fat body to lipophorins. Using lipophorins radio-labeled in their lipid moiety, transfer of lipid from lipophorin to fat body was demonstrated. This transfer was not mediated by LTP. The adipokinetic hormone induced diacylglycerol mobilization from the fat body and the concomitant interconversion of high density lipophorin to low density lipophorin were performed in vitro and were shown to require the presence of LTP.     

Effect of adipokinetic hormone on the structure and properties of lipophorin in locusts

The reversible association of a low molecular weight hemolymph protein (mol wt 20,000 estimated by SDS-polyacrylamide gel electrophoresis) with lipophorin, following treatment with adipokinetic hormone (AKH), was demonstrated by density gradient ultracentrifugation and by specific precipitation of lipophorin from the hemolymph of resting and AKH-injected locusts. The injection of AKH also stimulated the loading of diacylglycerol from fat body by lipophorin and resulted in a lower density lipophorin ("activated lipophorin"). The activated lipophorin particles (diameter 21.7 +/- 3.0 nm, 15.8 to 33.6 nm) were larger and more heterogeneous in size than those of resting lipophorin (14.5 +/- 1.6 nm, 11.9 to 19.2 nm). A theoretical analysis based on the experimental data (e.g., density gradient profile, electron microscopic observation, and diacylglycerol content) suggests that very large lipophorin particles result from intermolecular fusion of the lipophorin molecules that are activated by AKH. Attempts to demonstrate the effect of AKH on the structure of lipophorin, in vitro, were unsuccessful.     

The role of apolipophorin III in in vivo lipoprotein interconversions in adult Manduca sexta

Sustained flight in the moth, Manduca sexta, necessitates lipid mobilization and transport to flight muscle, a process mediated by the adipokinetic hormone. An adult specific high density lipophorin (lipoprotein, HDLp-A, Mr = 7.68 X 10(5)) accepts diacylglycerol from the fat body, increasing in size and decreasing in density, to give a low density lipophorin (lipoprotein, LDLp, Mr = 1.56 X 10(6)). During this process, several molecules of the small apolipoprotein, apolipophorin III (apoLp-III), are added to the two molecules originally present in HDLp-A. A study of the time course of adipokinetic hormone-induced loading of diacylglycerol onto HDLp-A, using the analytical ultracentrifuge and gel filtration, suggests that a lipoprotein of density intermediate between HDLp-A and LDLp was formed transiently. Analysis of lipoproteins separated by density gradient ultracentrifugation in the course of the loading process indicates that apoLp-III is added more rapidly than diacylglycerol and that it changes its conformation on the surface as more diacylglycerol is added. Taken together with the known properties of apoLp-III, a prolate ellipsoid with an axial ratio of 3, we suggest that initially apoLp-III adds to the expanded hydrophobic surface of the lipoprotein with its short axis parallel to the surface and that apoLp-III subsequently unfolds to cover a greater area of hydrophobic surface. Exchange experiments with labeled apoLp-III showed that the two apoLp-III molecules in HDLp-A do not exchange with free apoLp-III, even when the lipoprotein passed through a loading and unloading cycle, suggesting a structural role for apoLp-III in HDLp-A.     

Lipoprotein interconversions in an insect, Manduca sexta. Evidence for a lipid transfer factor in the hemolymph

Hemolymph lipoproteins (lipophorins) of adult Manduca sexta are disinct from larval forms in density, lipid content, composition, and the presence of a third, low molecular weight apoprotein. Generally, only one lipoprotein species exists in M. sexta hemolymph during any given life stage. Progression through the life cycle results in alterations of existing lipoproteins to produce new forms, without new protein synthesis. The observed alterations in lipoprotein density could result from facilitated lipid transfer in insect hemolymph. An in vitro assay of facilitated lipid transfer was developed which employs a high density lipophorin from the wandering larva (density = 1.18 g/ml) as acceptor and adult low density lipophorin (density = 1.03 g/ml) as donor. Adult lipophorin-deficient hemolymph was shown to catalyze a time-dependent equilibration of the starting lipoproteins to produce a new intermediate lipophorin, Lp-I. Hydrodynamic experiments on the donor, acceptor, and product lipoproteins excluded fusion as the mechanism whereby Lp-I is produced. Thus, it is concluded that Lp-I results from facilitated net lipid transfer from low to high density lipoprotein. Furthermore, experiments conducted with radioiodinated donor and radioiodinated acceptor lipoproteins demonstrated that apoprotein exchange does not occur during the lipid transfer reaction. When donor lipoprotein was labeled in the lipid moiety with carbon-14, evidence of diacylglycerol and phospholipid exchange was obtained. Partial characterization of the lipid transfer factor revealed a relationship between incubation time, donor concentration, acceptor concentration, lipophorin-deficient hemolymph concentration, and transfer activity, as measured by Lp-I production. It is concluded that lipophorin-deficient hemolymph contains one or more factor(s) that catalyze net lipid transfer as well as diacylglycerol and phospholipid exchange between lipophorins to produce a single form at equilibrium.     

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca²⁺. The hormones stimulate both the influx and the efflux of Ca²⁺, the higher influx probably causing an increase in intracellular [Ca²⁺]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca²⁺ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.     

Locust flight activity as a model for hormonal regulation of lipid mobilization and transport

Flight activity of insects provides a fascinating yet relatively simple model system for studying the regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a long-standing favored model insect, which as one of the most infamous agricultural pests additionally has considerable economical importance. Remarkably many aspects and processes pivotal to our understanding of (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity have been discovered in the locust; among which are the peptide adipokinetic hormones (AKHs), synthesized and stored by the neurosecretory cells of the corpus cardiacum, that regulate and integrate lipid (diacylglycerol) mobilization and transport, the functioning of the reversible conversions of lipoproteins (lipophorins) in the hemolymph during flight activity, revealing novel concepts for the transport of lipids in the circulatory system, and the structure and functioning of the exchangeable apolipopotein, apolipophorin III, which exhibits a dual capacity to exist in both lipid-bound and lipid-free states that is essential to these lipophorin conversions. Besides, the lipophorin receptor (LpR) was identified and characterized in the locust.In an integrative approach, this short review aims at highlighting the locust as an unrivalled model for studying (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity, that additionally has offered a broad and profound research model for integrative physiology and biochemistry, and particularly focuses on recent developments in the concept of AKH-induced changes in the lipophorin system during locust flight, that deviates fundamentally from the lipoprotein-based transport of lipids in the circulation of mammals. Current studies in this field employing the locust as a model continue to attribute to its role as a favored model organism, but also reveal some disadvantages compared to model insects with a completely sequenced genome.     

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.     

Lipophorin density variation during oogenesis in Rhodnius prolixus

The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations of lipophorins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophoin of different densities. Arch. Insect Biochem. Physiol. 35:301-313, 1997.© 1997 Wiley-Liss, Inc.     

In vitro study of the action of adipokinetic hormone in locusts

The in vitro study was performed in order to demonstrate the structural changes of lipophorin induced in vivo by the injection of adipokinetic hormone (AKH) into adult locusts. After many unsuccessful attempts, we have established the reconstructed incubation system in which purified lipophorin and apolipophorin-III (9 mol/mol lipophorin) are incubated with the fat body in the presence of AKH under a supply of excess oxygen. In this system, high density lipophorin (HDLp) originally present in the incubation medium can be transformed entirely into low density lipophorin (LDLp) due to the loading of an increased amount of diacylglycerol from the fat body. The LDLp formed in this incubation system was exactly the same as the LDLp formed in vivo by the injection of AKH, in terms of density, particle size, diacylglycerol content, and the association with apolipophorin-III (apoLp-III). In the absence of apoLp-III, AKH did not exhibit its function to any extent. It was also demonstrated that the transformation of HDLp to LDLp requires calcium ions. Moreover, it appears that, up to a certain limit, the increase of diacylglycerol content of lipophorin and the amount of apoLp-III associated with lipophorin is nearly proportional to the amount of apoLp-III added to the incubation medium.     

Changes in lipophorin composition during the fourth to fifth instar molt of Manduca sexta

During the feeding stage of the fourth instar of the tobacco hornworm, Manduca sexta, lipophorin has a density of 1.144 g/ml and is comprised of 55% protein and 45% lipid, mainly diacylglycerol and phospholipid. These values are similar to those found during the corresponding period of the fifth instar. However, during the intermolt period the lipid composition of lipophorin changes to 45% protein and 55% lipid and the density decreases to 1.112 g/ml. The onset of these changes in lipid content correspond to the increase in ecdysteroid titer which signals the beginning of molting. At the end of molting both the lipid content and density return to those characteristic of the fifth instar. These data further confirm the complex nature of lipophorin metabolism in M. sexta larva and suggest that ecdysone may be the trigger for lipid mobilization during the intermolt period.     

Lipoprotein lipase activity in the flight muscle of Locusta migratoria and its specificity for haemolymph lipoproteins

Lipoprotein lipase activity in flight muscle homogenates of Locusta migratoria was measured, using natural radiolabelled lipoproteins as substrates. The flight specific lipoprotein A⁺ (or low density lipophorin) stimulated lipoprotein lipase activity several-fold compared to the resting lipoprotein A_y (or high density lipophorin). However, with the high mol. wt lipoprotein fraction O_AKH as a substrate, lipase activity was even doubled compared to lipoprotein A⁺. Lipase activity was not increased in flight muscle homogenates of insects which had flown. Neither adipokinetic hormone, nor octopamine had any direct effect on lipoprotein lipase activity. Aspects of hormonal regulation and apoprotein activation of the locust flight muscle lipoprotein lipase are discussed and compared with the model for vertebrate lipoprotein lipase.     

Characterization of Colorado potato beetle lipophorin: A hydrocarbon-rich diacylgycerol-poor lipophorin

• 1.1. The lipid composition of lipophorin from the Colorado potato beetle, Leptinotarsa decemlineata Say, was analyzed.
• 2.2. This insect lipophorin contains 44% lipid and is characterized by large amounts of hydrocarbons and small amounts of diacylglycerol.
• 3.3. This is the first observation of a diacylglycerol-poor insect lipophorin in haemolymph.
• 4.4. Since the main energy source for flight in the Colorado potato beetle is proline, the low diacylglycerol content in lipophorin must be related to its peculiar flight metabolism.
• 5.5. This lipophorin, however, can still take up appreciable amounts of diacylglycerol from the locust fat body. Hydrocarbon uptake by this lipophorin was also demonstrated.
• 6.6. The main function of this lipophorin therefore seems to be transport of hydrocarbons from oenocytes to the cuticle.