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Apolipophorin III has been isolated and characterized from two orthopteran species, Barytettix psolus and Melanoplus differentialis. Both apoLp-IIIs have Mr ∼ 20,000 and contain 5% covalently bound sugar. Compositional analysis of the carbohydrate component of these apoLp-IIIs, as well as Locusta migratoria apoLp-III, revealed the presence of manose, fucose and N-acetyl glucosamine. In all three proteins the ratios of these sugar residues was similar (∼2:2:1). Amino acid analysis of B. psolus and M. differentialis apoLp-IIIs was performed and the results compared with those from L. migratoria and M. sexta apoLp-IIIs. The three orthopteran apoLp-IIIs possessed very similar compositions that were distinct from that of M. sexta. Similarly, N-terminal sequence analysis revealed ∼50% sequence identity between the three orthopteran apoLp-IIIs but only 9–18% identity was observed when B. psolus or M. differentialis apoLp-IIIs were compared to M. sexta apoLp-III. The implications of these findings, with respect to flight-related physiology and adipokinetic hormone stimulated lipid mobilization in these different species, is discussed.

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.

On the initiation of flight activity in Locusta migratoria, the concentration of glycerol in the flight muscles is rapidly elevated to six times the basal level, while in the fat body the glycerol concentration is increased nearly 50-fold. In the haemolymph, a steady state concentration of glycerol is maintained at the resting level during the initial 20 min of flight. A second steady state is attained after a further 40 min of flight when the concentration increases to six times that of the resting value. Minimal turnover rates of the haemolymph glycerol pool at both steady state levels during flight are 6.3 and 18.7 μ-mole glycerol/ml haemolymph per hr, respectively, corresponding with a 2.5- and 7-fold acceleration of the glycerol turnover in unflown insects. In the haemolymph, the main products of injected [U-14C]-glycerol are diacylglycerol and trehalose, both during flight and at rest, as well as after injection of adipokinetic hormone, though the quantitative incorporation of radioactivity differs. In the fat body, a considerable proportion of the radiolabel is recovered in triacylglycerol as well. It is suggested that during prolonged flight, when diacylglycerol is the major fuel, the glycerol liberated by lipolysis in the flight muscles and re-esterified with free fatty acids in the fat body accounts for the balance between triacylglycerol mobilization and diacylglycerol oxidation. Conversion of glycerol to trehalose is only of limited quantitative importance for flight muscle energy supply.

Formation of lipoprotein A ÷ and elevation of lipoprotein fraction O in locust (Locusta migratoria migratorioides) haemolymph as induced by adipokinetic hormone (AKH) includes the participation of non-lipid carrying proteins (fraction C), which was examined in more detail. By using gel filtration chromatography, the rather heterogenous C-proteins were resolved into three protein fractions, only one of which (C2) appeared to be actually involved in the lipoprotein reassociation. The changes in amino acid composition of the elevated lipoprotein fractions as compared with those from the lipoproteins in the resting situation are accounted for by the contribution of the rather specific amino acid composition of this C2-fraction. Polyacrylamide gel electrophoresis (PAGE) indicates that the C2-protein is migrating as only one band; SDS-PAGE revealed that the C2-protein consists of one single polypeptide chain with an approximate molecular weight of 20,000. This chain is also recovered in the subunit structure of the lipoprotein fractions induced by AKH-injection (A ÷, OATH) in contrast with that of the lipoprotein fractions in resting haemolymph. Unlike the other C-proteins, protein C2 displayed immunoreactivity with antiserum raised against lipoprotein A +. From carbohydrate analyses, C 2 appeared to be a glycoprotein containing approx. 12.5% carbohydrate. In vivo pilot studies on the dynamics of C2-proteins using 3H-labelled glycoprotein C2 gave evidence for the incorporation of radiolabel into both A ÷ and OATH. Possible functions of the involvement of the glycoprotein to A ÷ formation are discussed. Key Word Index: Locusta migratoria migratorioides, lipoproteins, lipid transport, energy supply, adipokinetic hormone, glycoprotein

The constant, elevated diglyceride concentration in the haemolymph of adult male Locusta migratoria during sustained tethered flight (14.1 ± 1.2 mg/ml haemolymph) was established to be a steady state, in which the rate of diglyceride mobilization matched the rate of utilization. Pulse-labelling of the haemolymph diglycerides with [1- 14 C]-oleic acid in steady state flight conditions results in a turnover time of about 1 hr for the diglyceride pool, whereas in animals rested after flight values close to 8 hr were obtained. So, during flight there is a sharp increase in lipid transport, the initial turnover rate in the resting animal (0.3–0.5 mg diglyceride/hr) reaching 3.4 mg diglyceride/hr at the steady state flight level. As identical turnover rates were obtained using [1- 14 C]-palmitic acid or [1- 14 C]-linolenic acid, apparently there is no preferential utilization of diglycerides with a specific fatty acid composition in flight muscle energy metabolism. The characteristic diglyceride fatty acid spectrum at the steady state flight level remained remarkably constant during flight performances lasting for 6 hr. The above findings indicate homogeneity of the haemolymph diglyceride pool and suggest random utilization of fatty acids.

The diacylglycerol-carrying lipoproteins in the haemolymph of adult male Locusta migrator& appear to be dynamic structures which participate in exchange and transfer processes involving their lipid and protein moieties. Between the two lipoprotein fractions present in the resting condition (O and Am~ow), a rapid exchange of apoprotein is observed upon re-injection of isolated and (12 si)_labelled lipoprotein Aya~ow. Furthermore, a high exchange rate of diacylglycerol is inferred from the incorporation of (14C)-labelled diacylglycerol into the lipid components. Elevation of the haemolymph diacylglycerol level in response to injection of adipokinetic hormone is accompanied by the appearance of a high molecular weight lipoprotein A ÷ and an increase in lipoprotein O, originating from a transfer of apoprotein from (125I)-Ayello w to both lipoproteins. Simultaneously, radioiodinated non-lipid containing haemolymph protein C participates in their formation. In contrast, (12 si)_protei n B is not involved in the lipoprotein remodelling. Though the increased diacylglycerol moieties carried by A + and O are derived principally from the augmented release of lipid from the fat body, net transfer of (14C)-diacylglycerol from Ayenow to both higher molecular weight lipoproteins evidences participation of the diacylglycerol component as well. Key Word Index: Lipoproteins, diacylglycerol transport, lipid exchange, apoprotein transfer, radioiodination, adipokinetic hormone, Locusta migratoria

When the release of the adipokinetic hormone in Locusta migratoria during flight is prevented by high concentrations of haemolymph trehalose or sucrose, neither the characteristic elevation of the haemolymph diglyceride concentration nor the formation of the flight specific diglyceride-carrying haemolymph lipoproteins is observed. Nevertheless, although the transport capacity is restricted to the lipoproteins present in the resting condition, the transport rate of diglycerides through the haemolymph is elevated considerably. Continuation of flying activity in such locusts injected with extracts of corpora cardiaca and flying under trehalose or sucrose loads, resulted in a rapid increase of both the content and the turnover of the haemolymph diglycerides. This was accompanied by the formation of the specific haemolymph lipoprotein system capable of accepting the elevated lipid. It is suggested that the primary effect of the increasing adipokinetic hormone titre at the onset of normal flight is to elicit the changes in association of haemolymph protein components already present in the resting locust to form the specific lipoproteins which allow both the enhanced concentration and transport of diglycerides required for optimum energy supply for the flight muscles. In addition, the hormone may have a moderatory function in controlling fat body lipolytic processes.

The interaction of locust high density lipophorin (HDLp) with pieces of fat body tissue was studied at 33°C using a radiolabelled ligand binding assay. Under the assay conditions, binding of tritium-labelled HDLp ((3H)HDLp) was demonstrated to correlate linearly with tissue concentration up to ~ 7 mg of fat body protein per ml of incubation medium. The (3H)HDLp binding that was displace.able by a 20-fold excess of unlabelled HDLp (which is an approximation of the specific binding) reached equilibrium after ~ 2 h, whereas low levels of non-displaceable binding increased linearly during this time interval. Analysis of the concentration dependent total binding of (3H)HDLp revealed the presence of a specific binding site with an equilibrium dissociation constant of Ko = 3.1 ( + 0.5) x 10 -7 M and a maximal binding capacity of 9.8 (+0.5)ng/zg-~ tissue protein. Competition experiments demonstrated that the affinity of unlabelled HDLp for the binding site is similar to the affinity of (3H)HDLp. Unlabelled low density lipophorin (LDLp), however, was shown to have an approx. 20-fold lower affinity for the binding site. Key Word Index: Locusta migratoria, lipophorin0 lipophorin receptor, lipid transfer, fat body

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 (C 2 ), 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 C 2 by injection of isolated adult C 2 , 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.

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 C 2 was shown to be released upon hydrolysis of lipophorin A + -carried diacylglycerol by the flight muscle lipoprotein lipase. This in vitro released protein C 2 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 C 2 with lipophorin A y and support the shuttle function of the protein components of locust lipophorin A + in lipid transport.

In adult male Locusta changes in level and specific radioactivity of the haemolymph trehalose pool were studied during flight and rest after pulse-labelling with [U-14C]-trehalose. In the initial period of flight, consumption of haemolymph trehalose is high (about 7.3 mg/animal/hr), but there is little mobilization of trehalose from body carbohydrate stores. After 30 min of flight, however, steady state conditions in the haemolymph trehalose pool are reached in which the turnover rate of the (constant) pool amounts to 2.4 mg/animal/hr. At rest, turnover rate of the haemolymph trehalose pool is significantly lower (0.5 mg/animal/hr). It is concluded that during sustained tethered flight (when diglyceride is the predominant energy source) trehalose contributes substantially to flight muscle energy metabolism since 23% of the energy supply originates from carbohydrate oxidation.

The flight of an insect is of a very complicated and extremely energy-demanding nature. Wingbeat frequency may differ between various species but values up to 1000 Hz have been measured. Consequently metabolic activity may be very high during flight and the transition from rest to flight is accompanied by an increase of 50-100-fold in metabolic rate. Small mammals running at maximal speed and flying birds achieve metabolic rates exceeding resting levels by only 7-14-fold. The exaggerated metabolic rate during insect flight is not accompanied by an oxygen debt, which implies -apart from metabolic adaptations- ample availability of oxygen in the organs responsible for flight. Metabolic rate therefore can be estimated, apart from the depletion of fuel depots, by rates of oxygen consumption.