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Physiological Significance of Lipid Transport by Lipophorin for Long-Distance Flight in Insects*
Comp. Biochem. Physiol. Vol. 117B, No. 4, pp. 455–461, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00183-1
Physiological Significance of Lipid Transport by Lipophorin for Long-Distance Flight in Insects* Haruo Chino Department of General Education, Hokkai-Gakuen University, Asahimachi, Toyohira-ku, Sapporo, Japan
KEY WORDS. Adipokinetic hormone, diacylglycerol, low- or high-density lipophorin, apolipophorin-III, fat body, hemolymph, solitary or gregarious locust, cockroach
INTRODUCTION In the early 1960s, a specific release of diacylglycerol from the fat body into the hemolymph was reported for the first time in the Cecropia silkworm (3,4). Subsequently, a unique lipoprotein was isolated from the pupal hemolymph of Philosamia silkworm and named ‘‘diacylglycerol-carrying lipoprotein’’ based on its function of taking up diacylglycerol from the fat body (5). Since then, it has generally been accepted that this lipoprotein exists in the hemolymph of perhaps all insect species, and in 1981 it was renamed ‘‘lipophorin’’ (lipid-bearing protein), based on its functional property to transport various lipids including diacylglycerol, hydrocarbons, free cholesterol and carotenoids, as a reusable shuttle, between tissues (7). Mayer and Candy (17,18) discovered a peptide hormone, in corpora cardiaca of locust, named adipokinetic hormone (AKH), which causes the elevation of diacylglycerol levels in the hemolymph. This peptide hormone was then purified from the locust corpora cardiaca and identified as a blocked decapeptide (26). It has naturally been assumed that AKH expresses its function via lipophorin, because one of the major functions of lipophorin is considered to be delivery of diacylglycerol from the fat body to the flight muscle as fuel for flight. In fact, a number of studies for elucidating the action of AKH on lipophorin were initiated in the late 1970s, and this research field achieved rapid development, particularly after synthetic AKH became commercially available in the early 1980s (8,19,25,27,29,31,32,34,35). This review describes the physiological significance of diacylglycerol transport by lipophorin under the action of
*Presented in part at the 4th International Congress of Comparative Biochemistry and Physiology, Birmingham, England, August 1995. Address reprint requests to: H. Chino, Department of General Education, Hokkai-Gakuen University, Asahimachi, Toyohira-ku, Sapporo, Japan. Abbreviations–AKH, adipokinetic hormone; HDLp, high-density lipophorin; LDLp, low-density lipophorin; apoLp-III, apolipophorin-III; LTP, lipid transfer particle. Received 29 December 1995; accepted 6 August 1996.
AKH, in relation to the mechanism of the fuel supply during insect flight. ACTION OF AKH ON LIPOPHORIN IN GEGARIOUS-PHASE LOCUSTS: SWITCHING THE FUEL FROM CARBOHYDRATE TO LIPID Transformation of High-Density Lipophorin to Low-Density Lipophorin Lipophorin particles usually exist as high-density lipophorin (HDLp, 1.12 g/ml) in the hemolymph of the resting adult male locusts, Locusta migratoria. A density gradient ultracentrifugation of the hemolymph collected 60–90 min after the injection of AKH demonstrates that HDLp is transformed to low-density lipophorin (LDLp, 1.065 g/ml) due to the increased loading with diacylglycerol. In fact, the determination of the diacylglycerol amount indicates that LDLp contains four to five times greater amounts of diacylglycerol than HDLp (8). However, 24 hr after the injection of AKH, LDLp disappears from the hemolymph, and all lipophorin particles are again found as HDLp, probably due to unloading of diacylglycerol at the flight muscle and/or other tissues. Morphological Change of Lipophorin Induced by AKH Electron micrographs of HDLp particles show a high homogeneity in size with a mean diameter of 14.5 6 1.6 nm (Fig. 1A). By contrast, the injection of AKH results in greater heterogeneity in size (Fig. 1B), and within 60–90 min of injection, the mean diameter of the LDLp particle increases to 21.7 6 3.0 nm. However, 24 hr after the injection of AKH, lipophorin particles return to a smaller size and become again homogeneous as observed for HDLp (Fig. 1C). The heterogeneity in size observed for LDLp particles could be interpreted to suggest that the amount of diacylglycerol loaded onto lipophorin particles is very variable. If this interpretation is correct, the LDLp particles should show variable densities. However, LDLp is almost equiva-
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FIG. 1. Electron micrographs
of high-density lipophorin (HDLp) and low-density lipophorin (LDLp) negatively stained with uranyl acetate. Magnification 3400,000. (A) 90 min after injection of saline as control (HDLp); (B) 90 min after injection of AKH (LDLp); (C) 24 hr after injection of AKH (HDLp). Modified from Chino et al. (8).
lent to HDLp in the homogeneity of density, as judged from the data of density gradient ultracentrifugation. Thus, there appears to be a discrepancy between the heterogeneity in size and the homogeneity of density of LDLp particles. To resolve this discrepancy, we have proposed an intermolecular fusion theory based on theoretical analyses: very large LDLp particles result from fusion of different numbers of
LDLp particles (8). For example, the largest LDLp particles observed in the electron micrograph theoretically result from fusion of about eight particles of LDLp primarily formed under the action of AKH. In fact, the electron micrograph shows some images that may represent the process of such fusion (Fig. 1B, arrow). Irrespective of the size of LDLp particles formed by intermolecular fusion, the ratio
The Physiological Significance of Lipid Transport by Lipophorin
FIG. 2. SDS-polyacrylamide gel electrophoresis of HDLp and
LDLp. Lane 1, HDLp prepared from resting locust by density gradient ultracentrifugation; lane 2, HDLp prepared from resting locust by precipitation method; lane 3, LDLp prepared by density gradient centrifugation 90 min after injection of AKH; lane 4, LDLp prepared by precipitation method 90 min after the injection of AKH; lane 5, HDLp prepared by density gradient centrifugation 24 hr after the injection of AKH. Modified from Chino et al. (8).
of protein to lipid should remain the same for each fused particle, and this results in the homogeneity of density. The above hypothesis has been supported by further analysis of the physicochemical nature of LDLp (20). At present, however, the physiological significance of intermolecular fusion of LDLp particles is unknown. Association of Apolipophorin III with Lipophorin Locust HDLp comprises two apoproteins, apolipophorin-I (mol wt 250,000) and apolipophorin-II (mol wt 85,000) (6) (Fig. 2, lanes, 1 and 2). The injection of AKH into adult locusts causes the association of a third low-molecularweight apoprotein, named apolipophorin-III (apoLp-III, mol wt 19,000) with lipophorin (Fig. 2, lanes 3 and 4), as initially reported for Manduca sexta (14,25). The association of apoLp-III with lipophorin is reversible; 24 hr after the injection of AKH, apoLp-III dissociates again (Fig. 2, lane 5). Further experiments have indicated that locust apoLpIII exists in the hemolymph of adult locust in free, and it becomes associated with lipophorin under the action of AKH, but apoLp-III dissociates itself again from lipophorin (8). The purification of locust apoLp-III has been achieved in
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our laboratory (9). Unlike apoLp-III from M. sexta, which is known not to contain carbohydrate (14), locust apoLp-III is a glycoprotein containing fucose and mannose. However, insect apoLp-III and lipophorin show no species specificity in function; M. sexta apoLp-III has the ability to associate with locust lipophorin under the action of AKH (33). Our subsequent experiments have indicated that in the case of locusts, on average, 9 mol (7–13 mol) apoLp-III associate with each mole of lipophorin; the amounts of apoLp-III binding with lipophorin vary, almost in parallel with the loading amount of diacylglycerol (20). The chemical and physical properties of locust apoLp-III have been studied extensively by many research groups and recently reviewed by Blacklock and Ryan (1). The complete amino acid sequence has been determined by cDNA cloning (13), and the three-dimensional structure of locust apoLp-III has been studied using X-ray crystallography (2). At present, however, the precise function of apoLp-III is not fully understood. For example, it is not clear whether apoLp-III associates with the lipophorin surface as a function for more diacylglycerol loading or if apoLp-III association allows the loading of more diacylglycerol, although apoLp-III is assumed to play an important role in stabilization of the LDLp structure (1). Locust apoLp-III is absent from the hemolymph throughout the larval stages (12). ApoLp-III and lipophorin are synthesized in the fat body, and the m-RNA for apoLp-III first appears in the fat body 3 days after the final molt and apoLpIII itself becomes detectable in the adult hemolymph on day 4. The concentration of apoLp-III in the hemolymph gradually increases and reaches a plateau about 2 weeks later, which is just when the locusts are ready to fly long distance. Therefore, apoLp-III is defined as a flight-specific protein, at least in locusts (12). Mechanism of Switching the Fuel for Flight from Carbohydrate to Lipid The effects of injected AKH on lipophorin described in the previous sections have all been fully demonstrated by experiments in vitro, in which the dissected fat body is incubated with hemolymph or lipophorin plus apoLp-III in the presence of AKH (10). The establishment of this incubation system has helped to further elucidate the role of apoLpIII, the mode of action of AKH and other related problems. The primary role of AKH in the formation of LDLp has been investigated by many researchers including our team [see the review of Van der Horst and Van Marrewijk in this series, and see also the review of Blacklock and Ryan (1)]. Based on these studies and related observations, we now propose the following mechanism of how the locusts switches the fuel from carbohydrate to lipid (mainly diacylglycerol). Under resting conditions, trehalose inhibits the loading process of diacylglycerol by lipophorin from fat body (15).
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This inhibition enables the locusts to save and store triacylglycerol in the fat body. When the locusts begin to fly, trehalose is consumed rapidly for the first 30 min (30), which then triggers the loading of diacylglycerol by lipophorin. Simultaneously, AKH is secreted from the corpora cardiaca. The released AKH acts on the plasma membrane of the fat body cells to open the calcium-channel, which, in turn, stimulates the influx of extracellular Ca 12 into the cells. The Ca12 then activates fat body lipase probably via calciumdependent protein kinase involving a cAMP-linked system, resulting in accelerated formation of diacylglycerol from the triacylglycerol pool. Thus, HDLp starts to load more and more diacylglycerol and associates with increasing amounts of apoLp-III and is transformed into LDLp. The LDLp moves to the flight muscle to unload diacylglycerol, dissociates apoLp-III and returns to HDLp. Thus, as long as AKH is continuously released from the corpora cardiaca, the locusts can keep flying until the triacylglycerol pool is exhausted. The above hypothesis has been strongly supported by our observation that the calcium ionophore A23187 is a perfect mimic of AKH in the formation of LDLp both in vivo and in vitro (16). LIPID TRANSPORT IN FLIGHT-CAPABLE AND FLIGHT-INCAPABLE INSECTS As mentioned in the previous sections, the transformation of HDLp to LDLp under the action of AKH seems essential for long-distance flight in insects. In addition, prolonged flight capability depends on morphological features such as wing, flight muscle and so on. However, many insects (e.g., the cockroaches and the solitary-phase locusts) possess such morphological features but are still unable to fly long distance. Therefore, comparison of lipid mobilization and transport in gregarious- and solitary-phase locusts and the cockroach should help to clarify the requirements for longdistance flight in insects. Thus, we have conducted the following experiments (11). Why the American Cockroach Lacks the Ability for Prolonged Flight In a preliminary experiment, AKH was injected into adult male American cockroaches, Periplaneta americana, to test if any LDLp would be formed in the hemolymph, but the results were completely negative. Locust apoLp-III together with AKH was then injected into cockroaches, because the formation of a hybrid LDLp consisting of locust lipophorin and M. sexta apoLp-III had been observed after the incubation of these components with locust fat body in the presence of AKH (33). However, the injection of AKH and locust apoLp-III into the cockroaches also gave completely negative results. These negative results suggest that not only does the cockroach lack its own apoLp-III, but its fat body may also lack the ability to respond to AKH. To resolve
the problem, the following experiments were carried out in vitro. When cockroach hemolymph is incubated with locust fat body in the presence of AKH, the increase of the diacylglycerol content of lipophorin is almost negligible (Fig. 3A), but if locust apoLp-III is added, the diacylglycerol level of lipophorin is elevated greatly. After 180 min incubation, the level was 14 times the original level (Fig. 3B). Essentially, similar results are obtained when purified cockroach lipophorin (HDLp) is incubated with locust fat body in the presence of AKH, without or with locust apoLp-III (Fig. 3, C and D). Further analyses by KBr density gradient ultracentrifugation of the above incubation media revealed that hybrid LDLp was formed after incubation, just as in the case of locust LDLp (8). These results indicate that the American cockroach lacks its own functional apoLp-III and that locust apoLp-III has the ability to associate with cockroach lipophorin. In fact, the association of locust apoLp-III with cockroach lipophorin was confirmed by immunoblotting analysis using anti-serum against locust apoLp-III, after SDS-PAGE of hybrid LDLp. In addition, electron micrographs of hybrid LDLp showed high similarity to locust LDLp in size and shape. It was also observed that no appreciable uptake of diacylglycerol by lipophorin occurred when locust hemolymph or cockroach hemolymph plus locust apoLp-III was incubated with cockroach fat body in the presence of AKH (Fig. 3, E and F). However, cockroach fat body responds to AKH, because diacylglycerol levels in the fat body after incubation with AKH was significantly elevated, as observed for locust fat body (16). Because cockroach fat body has the capacity to respond to AKH, the problem must lie elsewhere. The subsequent determination of the absolute amount of diacylglycerol in resting cockroach fat body showed that the diacylglycerol content was extremely low compared with that of resting locust fat body; locust fat body contains about 350 µg diacylglycerol per 100 mg tissue, whereas cockroach fat body contains only about 30 µg per 100 mg tissue. Therefore, even if cockroach fat body responded to AKH to nearly the same extent as locust fat body, it would still not reach even the resting level of locust fat body. This observation can explain, at least, the negative result that no LDLp is formed by incubating cockroach fat body with lipophorin plus locust apoLp-III in the presence of AKH. Why Solitary-phase Locusts Are Unable to Fly Long Distance Solitary-phase locusts (Locusta migratoria, adult male) were collected from a specific site (a small ski resort) near Sapporo from late August to early September and put on the same diet (pampas grass) as the gregarious locusts. They were used within 2 or 3 days after collection. Although it is difficult to tell the exact age of the collected solitary locusts, they were roughly estimated to be 2 or 3 weeks after
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FIG. 3. Diacylglycerol uptake by lipophorin from the fat body in vitro. Hemolymph or purified lipophorin was incubated with fat body from gregarious locust, Locusta migratoria or cockroach, Periplaneta americana, with or without locust apoLp-III, in the presence of synthetic locust AKH (100 nM) for 180 min. The diacylglycerol level of lipophorin is expressed as the relative (fold) increase compared with the amount at the start of incubation. Each histogram represents the mean of three replicates. Modified from Chino et al. (11).
the emergence, based on the first appearance of adult locusts in the above specific site. A preliminary experiment has revealed that the injection of AKH into solitary locusts causes no formation of LDLp at all. The lack of LDLp formation is not due to the absence of apoLp-III in the hemolymph; the SDS-PAGE of hemolymph from solitary locusts revealed the presence of enough apoLp-III in the hemolymph. Therefore, the inability in solitary locusts to form LDLp is assumed to be due to the nature of its fat body. Indeed, the solitary locusts have an extremely poor fat body (7–8 mg or less fresh tissue per adult male) compared with that (50–60 mg) of the gregarious locusts. In addition, as shown in Table 1, the content of triacylglycerol in the solitary locust fat body is only 1.1 6 0.3 mg/100 mg tissue, compared with that (25 6 5 mg) of the gregarious locust, and the diacylglycerol content is too small to be deTABLE 1. Amounts of acylglycerols in solitary locust fat
body and gregarious locust fat body Triacylglycerol (mg/100 mg tissue)
Diacylglycerol (mg/100 mg tissue)
Solitary
Gregarious
Solitary
Gregarious
1.1 6 0.3 (n 5 5)
25 6 5 (n 5 4)
Not detectable (n 5 4)
0.35 6 0.13 (n 5 14)
All data are means 6 SD; n, numbers determined. Modified from the data of Chino et al. (11).
tected. This result reveals that the absolute amount of triacylglycerol stored in the fat body of each solitary locust is equivalent to only 0.7% or less of that of gregarious locust fat body. Thus, it is concluded that the size of triacylglycerol pool in the fat body of solitary locust is quite insufficient to maintain prolonged flight.
CONCLUSION AND FUTURE PROSPECTS It has been generally accepted that in some migratory insects such as locust, the major fuel for flight is switched from carbohydrate to lipid soon after the commencement of prolonged flight, but the switching mechanism had remained unresolved until the mid-1960s. The discovery of locust AKH (17,18) and of specific release of diacylglycerol from insect fat body (3), and the subsequent isolation of lipophorin from hemolymph (5) have stimulated research for the elucidation of the above switching mechanism and resulted in the rapid development of the related research fields; this research field has yielded a number of new findings, including the discovery of apoLp-III, which is now considered to be a flight-specific protein. However, whether the accumulated knowledge concerning a typical migratory insect, the gregarious-phase locust, can be extended to other migratory insects such as some butterflies (Lepidoptera), dragonflies (Odonata) and leafhoppers (Hemiptera) remains a future problem.
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Despite the accumulated information on the physicochemical natures of apoLp-III, the precise role of this protein still remains unknown (1). In addition, a more basic and fundamental question remains unresolved; the mechanism of how lipophorin loads diacylglycerol from fat body cells and unloads this lipid at flight muscle cells is still a big and basic question, although the possibility has been proposed that a ‘‘lipid transfer particle’’ (LTP) may be involved in the loading process of diacylglycerol at fat body (28). In this connection, we should also note our early observation (4) that the uptake of diacylglycerol by lipophorin from fat body is an energy-dependent process. The insect LTP was first discovered by Ryan et al. (21) in the hemolymph of M. sexta. It is now known that LTP and lipophorin exist in the hemolymph of perhaps all insect species and that it has the function to catalyze the net transfer and/or exchange of various lipids, including diacylglycerol and hydrocarbons, between lipophorin particles [(22– 24), see also the review of Blacklock and Ryan (1)]. To understand more deeply the regulation of the lipid mobilization and transport during flight, it seems also very important to elucidate the physiological role of LTP. There are two possible explanations for the process of lipid transport by lipophorin from the sites of loading lipids (e.g., fat body) to the sites of unloading lipids (e.g., flight muscle). Lipophorin itself moving between two sites to load and unload lipids is one possibility. However, this seems unlikely, because, unlike vertebrates, insects have an open circulatory system without a definite and efficient bloodstream. The other more likely possibility is that lipids primarily taken up by lipophorin from the tissues are rapidly transferred and/or exchanged between lipophorin particles by the action of LTP, which eventually enables lipophorin to deliver the lipids from the loading sites to the unloading sites without lipophorin itself having to move between the two sites. If it is assumed that the primarily formed LDLp itself moves to the flight muscle to unload diacylglycerol and the resultant HDLp returns to the fat body to load diacylglycerol, both the LDLp and HDLp particles should be found in the hemolymph. However, this assumption quite disagrees with our observation that all lipophorin particles present in the hemolymph from a certain individual injected with AKH display homogeneity in density [(8); unpublished data]. What then is the real physiological role of LTP? This question also awaits further investigation. Why do solitary-phase locusts lack the ability to fly long distance? This question is now answered: solitary locusts do not store sufficient amounts of triacylglycerol in the fat body. This answer is very clear and simple. However, a basic question still remains as to why the solitary locusts do not store much triacylglycerol in the fat body. Our preliminary experiment in which a number of the newly collected adult solitary locusts were put in the same size cage with the same diet as gregarious locusts for 2 or 3 weeks has shown only a very slight increase in the size and the triacylglycerol content of the fat body even under such crowded conditions.
We now assume that the adult solitary locusts may be destined not to feed too much. Why is this? It would certainly be much more difficult to find a definite answer to this question than to resolve the molecular and metabolic mechanism of the regulation of the fuel supply system during flight. References 1. Blacklock, B.J.; Ryan, R.O. Hemolymph lipid transport. Insect Biochem. Mol. Biol. 24:855–873;1994. 2. Breiter, D.R.; Kanost, M.R.; Benning, M.M.; Wesenberg, G.; Law, J.H.; Wells, M.A.; Rayment, I.; Holden, H.M. Molecular structure of an apolipoprotein at 2.5 A resolution. Biochemistry 30:603–608;1991. 3. Chino, H.; Gilbert, L.I. Diglyceride release from insect fat body: A possible means of lipid transport. Science 143:359– 361;1964. 4. Chino, H.; Gilbert, L.I. Lipid release and transport in insects. Biochim. Biophys. Acta 98:94–110;1965. 5. Chino, H.; Murakami, S.; Harashima, K. Diglyceride-carrying lipoproteins in insect hemolymph: Isolation, purification and properties. Biochim. Biophys. Acta 176:1–26;1969. 6. Chino, H.; Kitazawa, K. Diglyceride-carrying lipoprotein of hemolymph of the locust and some insects. J. Lipid Res. 22: 1042–1052;1981. 7. Chino, H.; Downer, R.G.H.; Wyatt, G.R.; Gilbert, L.I. Lipophorin, a major class of lipoprotein of insect hemolymph. Insect Biochem. 11:491;1981. 8. Chino, H.; Downer, R.G.H.; Takahashi, K. Effect of adipokinetic hormone on the structure and properties of lipophorin in locusts. J. Lipid Res. 27:21–29;1986. 9. Chino, H.; Yazawa, M. Apolipophorin III in locusts: Purification and characterization. J. Lipid Res. 27:377–385;1986. 10. Chino, H.; Kiyomoto, Y.; Takahashi, K. In vitro study of the action of adipokinetic hormone in locusts. J. Lipid Res. 30: 571–578;1989. 11. Chino, H.; Lum, P.Y.; Nagao, E.; Hiraoka, T. The molecular and metabolic essentials for long-distance flight in insects. J. Comp. Physiol. B 162:101–106;1992. 12. Izumi, S.; Yamasaki, K.; Tomino, S.; Chino, H. Biosynthesis of apolipophorin-III by the fat body in locusts. J. Lipid Res. 28:667–672;1987. 13. Kanost, M.R.; Boguski, M.S.; Freeman, M.; Gordon, J.I.; Wyatt, G.R.; Wells, M.A. Primary structure of apolipophorin-III from the migratory locust, Locusta migratoria. Potential amphipathic structures and molecular evolution of an insect apolipoprotein. J. Biol. Chem. 263:10568–10573;1988. 14. Kawooya, J.K.; Keim, P.S.; Ryan, R.O.; Shapiro, J.P.; Samaraweera, P.; Law, J.H. Insect apolipophorin-III. J. Biol. Chem. 259:10733–10737;1984. 15. Lum, P.Y.; Chino, H. Trehalose, the insect blood sugar, inhibits loading of diacylglycerol by lipophorin from the fat body in locusts. Biochem. Biophys. Res. Commun. 172:588 –594; 1990. 16. Lum, P.Y.; Chino, H. Primary role of adipokinetic hormone in the formation of low density lipophorin in locusts. J. Lipid Res. 31:2039–2044;1990. 17. Mayer, R.J.; Candy, D.J. Changes in hemolymph lipoproteins during locust flight. Nature 215:987;1967. 18. Mayer, R.J.; Candy, D.J. Control of hemolymph lipid concentration during locust flight: An adipokinetic hormone from the corpora cardiaca. J. Insect Physiol. 15:611–620;1969. 19. Mwangi, R.W.; Goldsworthy, G.J. Diacylglycerol-transporting lipoproteins and flight in locusta. J. Insect Physiol. 27:47–50; 1981. 20. Nagao, E.; Chino, H. Further characterization of locust low
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ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00183-1
Physiological Significance of Lipid Transport by Lipophorin for Long-Distance Flight in Insects* Haruo Chino Department of General Education, Hokkai-Gakuen University, Asahimachi, Toyohira-ku, Sapporo, Japan
KEY WORDS. Adipokinetic hormone, diacylglycerol, low- or high-density lipophorin, apolipophorin-III, fat body, hemolymph, solitary or gregarious locust, cockroach
INTRODUCTION In the early 1960s, a specific release of diacylglycerol from the fat body into the hemolymph was reported for the first time in the Cecropia silkworm (3,4). Subsequently, a unique lipoprotein was isolated from the pupal hemolymph of Philosamia silkworm and named ‘‘diacylglycerol-carrying lipoprotein’’ based on its function of taking up diacylglycerol from the fat body (5). Since then, it has generally been accepted that this lipoprotein exists in the hemolymph of perhaps all insect species, and in 1981 it was renamed ‘‘lipophorin’’ (lipid-bearing protein), based on its functional property to transport various lipids including diacylglycerol, hydrocarbons, free cholesterol and carotenoids, as a reusable shuttle, between tissues (7). Mayer and Candy (17,18) discovered a peptide hormone, in corpora cardiaca of locust, named adipokinetic hormone (AKH), which causes the elevation of diacylglycerol levels in the hemolymph. This peptide hormone was then purified from the locust corpora cardiaca and identified as a blocked decapeptide (26). It has naturally been assumed that AKH expresses its function via lipophorin, because one of the major functions of lipophorin is considered to be delivery of diacylglycerol from the fat body to the flight muscle as fuel for flight. In fact, a number of studies for elucidating the action of AKH on lipophorin were initiated in the late 1970s, and this research field achieved rapid development, particularly after synthetic AKH became commercially available in the early 1980s (8,19,25,27,29,31,32,34,35). This review describes the physiological significance of diacylglycerol transport by lipophorin under the action of
*Presented in part at the 4th International Congress of Comparative Biochemistry and Physiology, Birmingham, England, August 1995. Address reprint requests to: H. Chino, Department of General Education, Hokkai-Gakuen University, Asahimachi, Toyohira-ku, Sapporo, Japan. Abbreviations–AKH, adipokinetic hormone; HDLp, high-density lipophorin; LDLp, low-density lipophorin; apoLp-III, apolipophorin-III; LTP, lipid transfer particle. Received 29 December 1995; accepted 6 August 1996.
AKH, in relation to the mechanism of the fuel supply during insect flight. ACTION OF AKH ON LIPOPHORIN IN GEGARIOUS-PHASE LOCUSTS: SWITCHING THE FUEL FROM CARBOHYDRATE TO LIPID Transformation of High-Density Lipophorin to Low-Density Lipophorin Lipophorin particles usually exist as high-density lipophorin (HDLp, 1.12 g/ml) in the hemolymph of the resting adult male locusts, Locusta migratoria. A density gradient ultracentrifugation of the hemolymph collected 60–90 min after the injection of AKH demonstrates that HDLp is transformed to low-density lipophorin (LDLp, 1.065 g/ml) due to the increased loading with diacylglycerol. In fact, the determination of the diacylglycerol amount indicates that LDLp contains four to five times greater amounts of diacylglycerol than HDLp (8). However, 24 hr after the injection of AKH, LDLp disappears from the hemolymph, and all lipophorin particles are again found as HDLp, probably due to unloading of diacylglycerol at the flight muscle and/or other tissues. Morphological Change of Lipophorin Induced by AKH Electron micrographs of HDLp particles show a high homogeneity in size with a mean diameter of 14.5 6 1.6 nm (Fig. 1A). By contrast, the injection of AKH results in greater heterogeneity in size (Fig. 1B), and within 60–90 min of injection, the mean diameter of the LDLp particle increases to 21.7 6 3.0 nm. However, 24 hr after the injection of AKH, lipophorin particles return to a smaller size and become again homogeneous as observed for HDLp (Fig. 1C). The heterogeneity in size observed for LDLp particles could be interpreted to suggest that the amount of diacylglycerol loaded onto lipophorin particles is very variable. If this interpretation is correct, the LDLp particles should show variable densities. However, LDLp is almost equiva-
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FIG. 1. Electron micrographs
of high-density lipophorin (HDLp) and low-density lipophorin (LDLp) negatively stained with uranyl acetate. Magnification 3400,000. (A) 90 min after injection of saline as control (HDLp); (B) 90 min after injection of AKH (LDLp); (C) 24 hr after injection of AKH (HDLp). Modified from Chino et al. (8).
lent to HDLp in the homogeneity of density, as judged from the data of density gradient ultracentrifugation. Thus, there appears to be a discrepancy between the heterogeneity in size and the homogeneity of density of LDLp particles. To resolve this discrepancy, we have proposed an intermolecular fusion theory based on theoretical analyses: very large LDLp particles result from fusion of different numbers of
LDLp particles (8). For example, the largest LDLp particles observed in the electron micrograph theoretically result from fusion of about eight particles of LDLp primarily formed under the action of AKH. In fact, the electron micrograph shows some images that may represent the process of such fusion (Fig. 1B, arrow). Irrespective of the size of LDLp particles formed by intermolecular fusion, the ratio
The Physiological Significance of Lipid Transport by Lipophorin
FIG. 2. SDS-polyacrylamide gel electrophoresis of HDLp and
LDLp. Lane 1, HDLp prepared from resting locust by density gradient ultracentrifugation; lane 2, HDLp prepared from resting locust by precipitation method; lane 3, LDLp prepared by density gradient centrifugation 90 min after injection of AKH; lane 4, LDLp prepared by precipitation method 90 min after the injection of AKH; lane 5, HDLp prepared by density gradient centrifugation 24 hr after the injection of AKH. Modified from Chino et al. (8).
of protein to lipid should remain the same for each fused particle, and this results in the homogeneity of density. The above hypothesis has been supported by further analysis of the physicochemical nature of LDLp (20). At present, however, the physiological significance of intermolecular fusion of LDLp particles is unknown. Association of Apolipophorin III with Lipophorin Locust HDLp comprises two apoproteins, apolipophorin-I (mol wt 250,000) and apolipophorin-II (mol wt 85,000) (6) (Fig. 2, lanes, 1 and 2). The injection of AKH into adult locusts causes the association of a third low-molecularweight apoprotein, named apolipophorin-III (apoLp-III, mol wt 19,000) with lipophorin (Fig. 2, lanes 3 and 4), as initially reported for Manduca sexta (14,25). The association of apoLp-III with lipophorin is reversible; 24 hr after the injection of AKH, apoLp-III dissociates again (Fig. 2, lane 5). Further experiments have indicated that locust apoLpIII exists in the hemolymph of adult locust in free, and it becomes associated with lipophorin under the action of AKH, but apoLp-III dissociates itself again from lipophorin (8). The purification of locust apoLp-III has been achieved in
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our laboratory (9). Unlike apoLp-III from M. sexta, which is known not to contain carbohydrate (14), locust apoLp-III is a glycoprotein containing fucose and mannose. However, insect apoLp-III and lipophorin show no species specificity in function; M. sexta apoLp-III has the ability to associate with locust lipophorin under the action of AKH (33). Our subsequent experiments have indicated that in the case of locusts, on average, 9 mol (7–13 mol) apoLp-III associate with each mole of lipophorin; the amounts of apoLp-III binding with lipophorin vary, almost in parallel with the loading amount of diacylglycerol (20). The chemical and physical properties of locust apoLp-III have been studied extensively by many research groups and recently reviewed by Blacklock and Ryan (1). The complete amino acid sequence has been determined by cDNA cloning (13), and the three-dimensional structure of locust apoLp-III has been studied using X-ray crystallography (2). At present, however, the precise function of apoLp-III is not fully understood. For example, it is not clear whether apoLp-III associates with the lipophorin surface as a function for more diacylglycerol loading or if apoLp-III association allows the loading of more diacylglycerol, although apoLp-III is assumed to play an important role in stabilization of the LDLp structure (1). Locust apoLp-III is absent from the hemolymph throughout the larval stages (12). ApoLp-III and lipophorin are synthesized in the fat body, and the m-RNA for apoLp-III first appears in the fat body 3 days after the final molt and apoLpIII itself becomes detectable in the adult hemolymph on day 4. The concentration of apoLp-III in the hemolymph gradually increases and reaches a plateau about 2 weeks later, which is just when the locusts are ready to fly long distance. Therefore, apoLp-III is defined as a flight-specific protein, at least in locusts (12). Mechanism of Switching the Fuel for Flight from Carbohydrate to Lipid The effects of injected AKH on lipophorin described in the previous sections have all been fully demonstrated by experiments in vitro, in which the dissected fat body is incubated with hemolymph or lipophorin plus apoLp-III in the presence of AKH (10). The establishment of this incubation system has helped to further elucidate the role of apoLpIII, the mode of action of AKH and other related problems. The primary role of AKH in the formation of LDLp has been investigated by many researchers including our team [see the review of Van der Horst and Van Marrewijk in this series, and see also the review of Blacklock and Ryan (1)]. Based on these studies and related observations, we now propose the following mechanism of how the locusts switches the fuel from carbohydrate to lipid (mainly diacylglycerol). Under resting conditions, trehalose inhibits the loading process of diacylglycerol by lipophorin from fat body (15).
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This inhibition enables the locusts to save and store triacylglycerol in the fat body. When the locusts begin to fly, trehalose is consumed rapidly for the first 30 min (30), which then triggers the loading of diacylglycerol by lipophorin. Simultaneously, AKH is secreted from the corpora cardiaca. The released AKH acts on the plasma membrane of the fat body cells to open the calcium-channel, which, in turn, stimulates the influx of extracellular Ca 12 into the cells. The Ca12 then activates fat body lipase probably via calciumdependent protein kinase involving a cAMP-linked system, resulting in accelerated formation of diacylglycerol from the triacylglycerol pool. Thus, HDLp starts to load more and more diacylglycerol and associates with increasing amounts of apoLp-III and is transformed into LDLp. The LDLp moves to the flight muscle to unload diacylglycerol, dissociates apoLp-III and returns to HDLp. Thus, as long as AKH is continuously released from the corpora cardiaca, the locusts can keep flying until the triacylglycerol pool is exhausted. The above hypothesis has been strongly supported by our observation that the calcium ionophore A23187 is a perfect mimic of AKH in the formation of LDLp both in vivo and in vitro (16). LIPID TRANSPORT IN FLIGHT-CAPABLE AND FLIGHT-INCAPABLE INSECTS As mentioned in the previous sections, the transformation of HDLp to LDLp under the action of AKH seems essential for long-distance flight in insects. In addition, prolonged flight capability depends on morphological features such as wing, flight muscle and so on. However, many insects (e.g., the cockroaches and the solitary-phase locusts) possess such morphological features but are still unable to fly long distance. Therefore, comparison of lipid mobilization and transport in gregarious- and solitary-phase locusts and the cockroach should help to clarify the requirements for longdistance flight in insects. Thus, we have conducted the following experiments (11). Why the American Cockroach Lacks the Ability for Prolonged Flight In a preliminary experiment, AKH was injected into adult male American cockroaches, Periplaneta americana, to test if any LDLp would be formed in the hemolymph, but the results were completely negative. Locust apoLp-III together with AKH was then injected into cockroaches, because the formation of a hybrid LDLp consisting of locust lipophorin and M. sexta apoLp-III had been observed after the incubation of these components with locust fat body in the presence of AKH (33). However, the injection of AKH and locust apoLp-III into the cockroaches also gave completely negative results. These negative results suggest that not only does the cockroach lack its own apoLp-III, but its fat body may also lack the ability to respond to AKH. To resolve
the problem, the following experiments were carried out in vitro. When cockroach hemolymph is incubated with locust fat body in the presence of AKH, the increase of the diacylglycerol content of lipophorin is almost negligible (Fig. 3A), but if locust apoLp-III is added, the diacylglycerol level of lipophorin is elevated greatly. After 180 min incubation, the level was 14 times the original level (Fig. 3B). Essentially, similar results are obtained when purified cockroach lipophorin (HDLp) is incubated with locust fat body in the presence of AKH, without or with locust apoLp-III (Fig. 3, C and D). Further analyses by KBr density gradient ultracentrifugation of the above incubation media revealed that hybrid LDLp was formed after incubation, just as in the case of locust LDLp (8). These results indicate that the American cockroach lacks its own functional apoLp-III and that locust apoLp-III has the ability to associate with cockroach lipophorin. In fact, the association of locust apoLp-III with cockroach lipophorin was confirmed by immunoblotting analysis using anti-serum against locust apoLp-III, after SDS-PAGE of hybrid LDLp. In addition, electron micrographs of hybrid LDLp showed high similarity to locust LDLp in size and shape. It was also observed that no appreciable uptake of diacylglycerol by lipophorin occurred when locust hemolymph or cockroach hemolymph plus locust apoLp-III was incubated with cockroach fat body in the presence of AKH (Fig. 3, E and F). However, cockroach fat body responds to AKH, because diacylglycerol levels in the fat body after incubation with AKH was significantly elevated, as observed for locust fat body (16). Because cockroach fat body has the capacity to respond to AKH, the problem must lie elsewhere. The subsequent determination of the absolute amount of diacylglycerol in resting cockroach fat body showed that the diacylglycerol content was extremely low compared with that of resting locust fat body; locust fat body contains about 350 µg diacylglycerol per 100 mg tissue, whereas cockroach fat body contains only about 30 µg per 100 mg tissue. Therefore, even if cockroach fat body responded to AKH to nearly the same extent as locust fat body, it would still not reach even the resting level of locust fat body. This observation can explain, at least, the negative result that no LDLp is formed by incubating cockroach fat body with lipophorin plus locust apoLp-III in the presence of AKH. Why Solitary-phase Locusts Are Unable to Fly Long Distance Solitary-phase locusts (Locusta migratoria, adult male) were collected from a specific site (a small ski resort) near Sapporo from late August to early September and put on the same diet (pampas grass) as the gregarious locusts. They were used within 2 or 3 days after collection. Although it is difficult to tell the exact age of the collected solitary locusts, they were roughly estimated to be 2 or 3 weeks after
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FIG. 3. Diacylglycerol uptake by lipophorin from the fat body in vitro. Hemolymph or purified lipophorin was incubated with fat body from gregarious locust, Locusta migratoria or cockroach, Periplaneta americana, with or without locust apoLp-III, in the presence of synthetic locust AKH (100 nM) for 180 min. The diacylglycerol level of lipophorin is expressed as the relative (fold) increase compared with the amount at the start of incubation. Each histogram represents the mean of three replicates. Modified from Chino et al. (11).
the emergence, based on the first appearance of adult locusts in the above specific site. A preliminary experiment has revealed that the injection of AKH into solitary locusts causes no formation of LDLp at all. The lack of LDLp formation is not due to the absence of apoLp-III in the hemolymph; the SDS-PAGE of hemolymph from solitary locusts revealed the presence of enough apoLp-III in the hemolymph. Therefore, the inability in solitary locusts to form LDLp is assumed to be due to the nature of its fat body. Indeed, the solitary locusts have an extremely poor fat body (7–8 mg or less fresh tissue per adult male) compared with that (50–60 mg) of the gregarious locusts. In addition, as shown in Table 1, the content of triacylglycerol in the solitary locust fat body is only 1.1 6 0.3 mg/100 mg tissue, compared with that (25 6 5 mg) of the gregarious locust, and the diacylglycerol content is too small to be deTABLE 1. Amounts of acylglycerols in solitary locust fat
body and gregarious locust fat body Triacylglycerol (mg/100 mg tissue)
Diacylglycerol (mg/100 mg tissue)
Solitary
Gregarious
Solitary
Gregarious
1.1 6 0.3 (n 5 5)
25 6 5 (n 5 4)
Not detectable (n 5 4)
0.35 6 0.13 (n 5 14)
All data are means 6 SD; n, numbers determined. Modified from the data of Chino et al. (11).
tected. This result reveals that the absolute amount of triacylglycerol stored in the fat body of each solitary locust is equivalent to only 0.7% or less of that of gregarious locust fat body. Thus, it is concluded that the size of triacylglycerol pool in the fat body of solitary locust is quite insufficient to maintain prolonged flight.
CONCLUSION AND FUTURE PROSPECTS It has been generally accepted that in some migratory insects such as locust, the major fuel for flight is switched from carbohydrate to lipid soon after the commencement of prolonged flight, but the switching mechanism had remained unresolved until the mid-1960s. The discovery of locust AKH (17,18) and of specific release of diacylglycerol from insect fat body (3), and the subsequent isolation of lipophorin from hemolymph (5) have stimulated research for the elucidation of the above switching mechanism and resulted in the rapid development of the related research fields; this research field has yielded a number of new findings, including the discovery of apoLp-III, which is now considered to be a flight-specific protein. However, whether the accumulated knowledge concerning a typical migratory insect, the gregarious-phase locust, can be extended to other migratory insects such as some butterflies (Lepidoptera), dragonflies (Odonata) and leafhoppers (Hemiptera) remains a future problem.
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Despite the accumulated information on the physicochemical natures of apoLp-III, the precise role of this protein still remains unknown (1). In addition, a more basic and fundamental question remains unresolved; the mechanism of how lipophorin loads diacylglycerol from fat body cells and unloads this lipid at flight muscle cells is still a big and basic question, although the possibility has been proposed that a ‘‘lipid transfer particle’’ (LTP) may be involved in the loading process of diacylglycerol at fat body (28). In this connection, we should also note our early observation (4) that the uptake of diacylglycerol by lipophorin from fat body is an energy-dependent process. The insect LTP was first discovered by Ryan et al. (21) in the hemolymph of M. sexta. It is now known that LTP and lipophorin exist in the hemolymph of perhaps all insect species and that it has the function to catalyze the net transfer and/or exchange of various lipids, including diacylglycerol and hydrocarbons, between lipophorin particles [(22– 24), see also the review of Blacklock and Ryan (1)]. To understand more deeply the regulation of the lipid mobilization and transport during flight, it seems also very important to elucidate the physiological role of LTP. There are two possible explanations for the process of lipid transport by lipophorin from the sites of loading lipids (e.g., fat body) to the sites of unloading lipids (e.g., flight muscle). Lipophorin itself moving between two sites to load and unload lipids is one possibility. However, this seems unlikely, because, unlike vertebrates, insects have an open circulatory system without a definite and efficient bloodstream. The other more likely possibility is that lipids primarily taken up by lipophorin from the tissues are rapidly transferred and/or exchanged between lipophorin particles by the action of LTP, which eventually enables lipophorin to deliver the lipids from the loading sites to the unloading sites without lipophorin itself having to move between the two sites. If it is assumed that the primarily formed LDLp itself moves to the flight muscle to unload diacylglycerol and the resultant HDLp returns to the fat body to load diacylglycerol, both the LDLp and HDLp particles should be found in the hemolymph. However, this assumption quite disagrees with our observation that all lipophorin particles present in the hemolymph from a certain individual injected with AKH display homogeneity in density [(8); unpublished data]. What then is the real physiological role of LTP? This question also awaits further investigation. Why do solitary-phase locusts lack the ability to fly long distance? This question is now answered: solitary locusts do not store sufficient amounts of triacylglycerol in the fat body. This answer is very clear and simple. However, a basic question still remains as to why the solitary locusts do not store much triacylglycerol in the fat body. Our preliminary experiment in which a number of the newly collected adult solitary locusts were put in the same size cage with the same diet as gregarious locusts for 2 or 3 weeks has shown only a very slight increase in the size and the triacylglycerol content of the fat body even under such crowded conditions.
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