Insect lipids and lipoproteins, and their physiological ...

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Insect lipids and lipoproteins, and their physiological processes ARTICLE in PROGRESS IN LIPID RESEARCH · FEBRUARY 1985 Impact Factor: 10.02 · DOI: 10.1016/0163-7827(85)90007-4 · Source: PubMed

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Prog. Lipid Res. Vol. 24, pp. 19-67, 1985 Printed in Great Britain. All rights reserved

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INSECT LIPIDS A N D LIPOPROTEINS, A N D THEIR ROLE IN PHYSIOLOGICAL PROCESSES AD M. TH. BEENAKKERS, DICK J. VAN DER HORST a n d WIL J. A. VAN MARREWIJK Department of Experimental Zoology, University of Utrecht, Utrecht, The Netherlands CONTENTS L INTRODUCTION II. FAT BODY AND H~MOLYMPH LIPIDS A. Fat body lipids B. Haemolymph lipids III. NtrrmTlON AND LIPID STORAGE A. Lipid requirements 1. Fatty acids 2. Sterols B. Lipid digestion and absorption C. Lipid storage in the fat body 1. Biosynthesis of fatty acids 2. Conversion of carbohydrate to fatty acids 3, Biosynthesis of triacylglycerols IV. LIPID METABOLISMAND FLIGHT A. Lipid and flight economy B. Lipid release from fat body 1. Stereospecificity of released diacylglycerols 2. Diacylglycerol production in the fat body 3. Diacylglycerol dynamics during flight C. Hormonal control of lipid release 1. Adipokinetic hormone 2. Octopamine D. Haemolymph proteins and lipid transport 1. Diacylglycerol-transporting hpoproteins 2. General characteristics of lipophorins 3. Structural organization of lipophorins 4, Lipophorin complexes E. Lipoprotein dynamics during flight 1. Hormone-induced lipophorin rearrangements 2. Occurrence and amino acid composition of apolipophorin--IIl 3. Function and nature of lipophorin-protein interactions F. Lipid utilization in flight muscles I. Flight muscle lipoprotein lipase 2. General characteristics of flight muscle lipoprotein lipase 3. Fatty acid oxidation V. LIPOPRO~INSAt~D VIrELLO~E~rmlS A. General aspects B. Survey of the yolk proteins C. Vitellogenins and vitellins 1. Molecular weights and subunit composition 2. Lipids 3. Carbohydrates 4. Phosphate D. Egg-specific protein 1. General characteristics 2. Site of synthesis E. Vitellin and egg-specific protein during embryonic development F. Hormonal control of vitellogenesis 1. Juvenile hormone 2. Ecdysone I~FEa~EHCES

19 20 20 21 24 24 24 27 29 31 32 33 33 35 35 36 36 36 37 38 39 40 40 40 40 42 43 43 43 46 47 48 48 49 50 51 51 52 52 53 54 55 56 56 56 56 57 58 58 59 60

I. I N T R O D U C T I O N T h e i m p o r t a n c e o f lipids to insects h a s b e e n r e c o g n i z e d b y several a u t h o r s . T h u s , G i l b e r t a n d C h i n o 123 r e m a r k e d that, to a g r e a t e x t e n t , the o b v i o u s success o f insects o n this p l a n e t h a s b e e n their a b i l i t y to utilize lipids efficiently as s u b s t r a t e for r e p r o d u c t i o n , era19

20

Ad M. Th. Beenakkerset al.

bryogenesis, metamorphosis and flight. As the reader will perceive, though many processes in lipid metabolism apparently are equal to those in vertebrates, in evolutionary periods over millions of years, insects have developed specific systems for lipid mobilization, transport and utilization deviating from mammalian metabolism. Often the rationale for these deviations remains enigmatic; their effectiveness, however, is beyond doubt. Particularly these specific metabolic pathways may provide valuable possibilities to interfere with normal development and to control distribution of insects. The classical treatment of insect lipids by Fast ~2 in this series inevitably being rather analytical and descriptive, the appearance since that time of numerous extensive biochemical and physiological studies on lipids in insects evidence insect lipid research to be an important and rapidly expanding field. The following review primarily proposes to provide the reader with an integrated view on the biochemistry of insect lipids and lipoproteins in coherence with the various important processes in insects in which lipids are used (e.g. nutrition, flight, reproduction and development). Reviews covering several topics in insect lipid biochemistry have appeared in recent years, and will be referred to where relevant to the discussion. In many instances, our lack of knowledge will become apparent, and the need for further investigation will be stressed. II. FAT BODY AND HAEMOLYMPH LIPIDS The amount of lipids in insects varies considerably and is influenced by many factors including stage of development, nutritional state, sex, environmental temperature, diapause and migratory flight. In general, female insects contain more lipid than males, the lipid reserve probably serving for egg production. However, the reverse may be true for many insects, as has been described for various families of Lepidoptera. 93"122'126Among species, the lipid content can vary from 1-50% of the wet weightf127The total lipid content and the fatty acid composition of a large number of whole insects of different orders at various stages of development has been described by Fast. 1~'~j2 Furthermore, Thompson, 3°° using data available from literature, has made a comparative characterization of the fatty acid compositions of total insects from seven orders. Total lipid contents and fatty acid compositions of aquatic insects have been reported very recently by Hanson et al.,143 who examined 58 genera from seven orders. The principal storage site for insect lipids is the fat body, and the lipid composition of the whole insect probably reflects the lipid composition of the fat body. ~84 This organ consists of aggregates of cells forming lobes or sheets of tissue, which is spread throughout the body and invests the internal organs. Its spatial arrangement in the abdomen, where large fat body deposits are found in close association with the gut, facilitates the uptake of dietary nutrients. The fat body may be considered functionally analogous to both vertebrate liver and adipose tissue, serving not only for the storage of carbohydrate, lipid and protein, but also as the major center for intermediary metabolism. Insects are known to possess only one extracellular fluid, which serves not only as a circulating medium, analogous to the blood of vertebrates, but also as a bathing medium for the tissue cells, analogous to the lymph of vertebrates. Because of this dual function, the term 'haemolymph' has been introduced, which is more accurate than 'blood' to designate this body fluid. The haemolymph transports nutrient substrates from the sites of absorption directly to sites of tissue respiration or synthesis, or to storage organs and, subsequently, to sites of utilization. Evidently, the fat body and the haemolymph occupy a pivotal position in the metabolism of insects; therefore, emphasis will be given to the role of these tissues in the different aspects of lipid metabolism that are discussed in this review. A. Fat Body Lipids

Lipid content and lipid composition of the fat body are the result of various processes, including storage of dietary lipids, de novo synthesis, degradation and modification of fat

Insect lipids and lipoproteins

21

body lipid and subsequent release for transport to sites of utilization. Usually the fat body accumulates large amounts of lipids and in some insects its fat content may exceed 50% of the total dry weight of the animal. 41 During the adult life of an insect, the amount of fat body lipid varies considerably but can be particularly high in reproducing female insects, as has been demonstrated in the blowfly, Phormia r e g i n a , 237 the desert locust, Schistocerca gregaria, TM and the cockroach, Leucophaea maderae. ~22 In the female bug Pyrrhocoris apterus, lipid accounts for 75-84% of the fat body dry weight, and the amount of lipid lost by the fat body during the first reproductive cycle was shown to be equal to the concomitant increase in ovarian lipid. 2°5 Also, male insects may contain large amounts of fat body lipid. In adult male locusts, for example, lipid contents have been reported of 10-30% of the fat body wet weight for S. gregaria and 23% (76% on a dry weight basis) for Locusta migratoria. ~72'348After adult emergence, locusts undergo a period of intensive feeding activity and lipid accumulates in the fat body prior to sexual maturation. ~5~'348 A positive correlation between the lipid reserves in the fat body and the inclination of the insect towards flight was observed in the Australian plague locust, Chortoicetes terrninifera. 16° Available data on the composition of fat body lipids in a variety of insects have been summarized by Bailey. 9The major lipid component in all insects examined is triacylglycerol (TG) which, in most insects, comprises more than 90% of total fat body lipid. The contributions of monoacylglycerol (MG), diacylglycerol (DG) and free fatty acids (FFA) vary among insects but are always small. Sterols and sterol esters constitute, if detected, also only minor components of total lipid. The effect of age on the fat body lipid composition was studied by Walker e t al., 34s who showed that in the male desert locust the contribution of TG to total lipid decreases from 95% after adult emergence to 78% in 20-day-old locusts, with a concomitant increase in particularly the phospholipid (PL) fraction (between 2.5-12%). In L. migratoria, the effect of age differs between sexes; H during adult development of male locusts, a decrease in the relative contribution of TG to total fat body lipid coincided with an increase of DG and PL, whereas in female locusts age dependent changes in the relative contributions of TG, DG and PL occurred in the opposite direction. Analyses of the fatty acid composition have been carried out in a large number of whole insects, ~2'3°° whereas only a few studies have been made of individual organs. In the fat body of adult female P. apterus, a fatty acid composition was found which was very similar to that of the diet. 2°6 The unsaturated fatty acids, oleate (20%), linoleate (55-60%) and linolenate (3%), comprised about 80% of total fatty acids, while palmitate (10%) and stearate (8% in fat body, 4% in diet) constituted the saturated fraction. In the neutral lipids of adult Hyalophora cecropia fat body, the C~8 unsaturated fatty acids also account for 80% of total fatty acids. 278 Oleate (45%) and linolenate (32%) are the predominant fatty acids, followed by palmitoleate (15%). Palmitate is the major saturated acid (4.5%). Thomas 294found a similar fatty acid profile in the fat body of the milkweed bug, Oncopeltus fasciatus. Since the relative amounts of oleate and linoleate were equally high in all of the lipid fractions of the fat body (except for DG) and in the diet, it was suggested that most of the unsaturated fatty acids in the fat body might originate from the diet. A clear effect of the fatty acid composition of the diet on the fatty acid profile of the fat body was demonstrated in L. migratoria by Beenakkers and Scheres. ~8 When diets were used with different amounts of unsaturated C~8 acids, a fair correlation was found between oleate and linolenate contents in diet and fat body (Fig. 1). A similar correlation did not exist, however, for linoleate.

B. Haemolymph Lipids The lipid profile of insect haemolymph reflects the role of this tissue as a transport medium of nutrient substrates from sites of absorption to sites of storage and utilization. Consequently, haemolymph lipid content and composition vary with the physiological state of the animal. Wide variations have been reported during development, metamor-

22

Ad M. Th. Beenakkers et al. feeding on:

a

50-

percentage of total

W

40-

reed endive pear

i Ho,. O.o_lOt0l

3020I0-

C18:0 fatty acids 50- percentage of total 40-

'°-

I1~

,,1~_ - ~ _

,,~m I

m .,~

C18:0 fatty acids

FiG. 1. Fatty acid composition of (a) fat body triacylglycerols from locusts fed on the diets indicated, and (b) the neutral lipids of the diets. After Beenakkers and Scheres. ~s

phosis, oogenesis and exercise (flight). 97 Age-dependent differences in haemolymph lipid levels were found in adult L. migratoria, variations being more pronounced in females than in males.l~ The total lipid content of insect haemolymph generally lies below 3%, though higher values have been reported in a few species. 9 Extremely large changes in haemolymph lipid levels have been observed in the tobacco hornworm, Manduca sexta. 368,369Larvae contain about 2 mgml-~; the lipid level increases to 8-10 mgm1-1 at the end of larval life when the animals stop feeding and prepare for pupation. After the final moult, the lipid concentration increases within a period of 8 hr from 8 to 40 mg ml-~. In starved adults, lipid levels increase further and may even exceed 100 mg ml-~. In L. migratoria, starvation provokes a three- to four-fold increase in haemolymph lipid concentration, paralleled by a decrease in fat body lipid. J72Concomitant with these changes haemolymph carbohydrate is reduced to about 10% of the control value. After feeding, the total lipid concentration returns rapidly to the normal resting level. 22t Injection of trehalose, the major sugar in insect haemolymph, also reduces the haemolymph lipid concentration in locusts, and this effect is dose-related. It has been suggested that an inverse relationship exists in the haemolymph between the concentration of lipid and that of trehalose, although the mechanism for such a relationship remains obscure. During flight of the locust, haemolymph lipid concentration increases by the action of adipokinetic hormone (AKH) from the glandular lobes of the corpora cardiaca, as will be discussed in Section IV.C. Unlike flight-induced lipid mobilization, the increase in haemolymph lipid during starvation does not depend upon AKH, as it is unaffected by removal of the glandular lobes. ~72 In M. sexta, haemolymph lipid accumulation during starvation is also independent of AKH, since this process occurs in cardiacectomized starved individuals a s well. 369 In Oxya japonica, which belongs to the same family as L. migratoria (Acrididae), starvation results in a decrease of haemolymph lipid, probably due to the small lipid reserves in the fat body of this non-migratory form. 2°° Data on the composition of haemolymph lipid in a number of insects have been compiled by Bailey9 and by Downer and Matthews. 97 Neutral lipids (NL) constitute the major part of haemolymph lipid. In the wax moth, Galleria mellonella, neutral acyl-


23

glycerols represent about 55% of the total lipid content of haemolymph, FFA 8%, sterols 15% and PL 22%. 358 In O. fasciatus, NL accounts for about 89% of haemolymph lipid, while PL constitutes the remaining 11%. About 65% of NL is acylglycerol, 15% FFA, 8% sterols and 10% sterol esters. 294 In all but a few species of insects, DG is the major lipid component of haemolymph, which may account for 80% or even more of the NL fraction. TG, FFA and MG are also present, in many species decreasing in that order. No DG could be, however, detected in haemolymph of the termite, Macrotermes goliath, 7° whereas in P. apterus TG is the major haemolymph lipid component due to the presence of a large number of TG-containing adipocytes in the haemolymphfl °6 Age and sex related fluctuations in the haemolymph lipid composition have been reported in the cockroach, Periplaneta americana, 224 and in L. migratoria. ~ In locusts, the elevated haemolymph lipid levels induced by starvation, ~72'2z°'22~ by flight 15'3°3'3°4as well as by the injection of corpus cardiacum extracts o r AKH 14'21°'28°are due almost exclusively to increases in the concentration of DG. The significance of haemolymph DG in relation to the mobilization of fat body lipid for flight will be discussed in Section IV.B. Analyses of the fatty acid composition of haemolymph lipids have been performed only in a few insect species. In haemolymph of Acheta domesticus, linoleate, oleate and palmitate are the major fatty acids, accounting for almost 90% of total fatty a c i d s . 349 Oleate (55%) and palmitate (23%) are the principal fatty acids in haemolymph of M. goliath. 7° Age-related variations occur in the fatty acid composition of acylglycerol and FFA fractions of P. americana haemolymph, changes being more pronounced in females than in malesf124 Oleate, palmitate and linoleate are the predominant fatty acids. Haemolymph lipids of H. cecropia contain oleate, linolenate and palmitate as the major fatty acids, although age- and sex-related variations in the fatty acid profile O c C u r . 17'278 The fatty acid composition of acylglycerols appeared to be different in fat body and haemolymph of the silkmoth, the DG, particularly, containing relatively more palmitate and less linolenate in haemolymph than in fat body. ~7 Differences in the fatty acid composition between fat body and haemolymph lipids have also been reported for L. migratoria ~5 and O. fasciatus. 294 The implications of these differences for the mechanism by which fat body lipid is released into the haemolymph will be discussed in Section IV.B. Data on insect phospholipids have recently been reviewed by Bridges. 35 Phosphatidylcholine is the principal PL component in haemolymph of haemolymph lipoprotein of G. mellonella, 358 H. cecropia, 296 Philosamia cynthia, 65 S. gregaria, 213 L. migratoria 242 and M. sexta, 216 whereas phosphatidylethanolamine was shown to be the major PL component in haemolymph of Musca domestica larvae ~°° and in the high density lipoprotein from larval haemolymph of M. sexta. 239 Sterols in insect haemolymph consist mainly of cholesterol, which either is absorbed directly from the diet or may be derived from dietary phytosterols, as will be discussed in Section III.A. Sterols are present mainly in a free form in haemolymph of Sitotroga cerealella, 69 M. domestica, 99"1°° M. goliath and M. natalensis. 7° In haemolymph of A. domesticus, free cholesterol is present as the sole sterol, sterol ester being totally absent? 49 In contrast, sterol esters constitute the major part (about 60%) of total sterols in haemolymph of O. f a s c i a t u s . 294 Other lipids that have been detected in insect haemolymph are hydrocarbons, which occur in large amounts in haemolymph of M. goliath and M. natalensis 7° and of S. cerealella, 69 ketone bodies 9'1°,15° and long-chain alcohols. 7° Ecdysteroids (Fig. 2), known as moulting hormones during insect development, have been detected in the haemolymph of adult insects as well, where they may play a role in the hormonal control of vitellogenesis (see also Section V.F), and possibly also in the induction of adult diapause. 9° Juvenile hormone, another lipoidal insect hormone which is synthesized and released by the corpus allatum, also occurs in haemolymph of both developing and adult insects (Fig. 3). During development, this hormone maintains the juvenile stage and prevents metamorphosis, while in adults juvenile hormone is involved in the control of reproduction, as will be discussed in Section V.F. Due to its lipophilic

24

Ad M. Th. Beenakkers et al. 22

24

24

STIGlt.~STEROL

5,2Z24"CHOLESTATRIENO// DESMOSTEROL r

~

~

.....

1 1. I1

CAMPESTEROL

ERGOSTEROL

24"METHYLE NECHOLESTEROL H

CHOLESTEROL

OHOH

20-HYDROXYECDYSONE """

OHH~O

HO

STEROL O (z'ECDY$ONE

FIG. 2. Metabolic pathways in the conversion of major phytosterols to cholesterol and therefrom to the ecdysteroids in many phytophagous insects. From Dadd, 83 reproduced with permission of the author.

nature, juvenile hormone is transported in haemolymph associated to specific binding proteins ~36 (see Section V.F.). III. N U T R I T I O N

AND

LIPID STORAGE

A. Lipid Requirements l. Fatty Acids Insects are able to synthesize the common C~6 and C~8 saturated and monounsaturated fatty acids from nonlipid precursors or may derive them from pre-existing fatty acids (Section III.C). They generally are, however, believed to be unable to add a second of third double bond into a fatty acid, and thus most insects display a dietary requirement for polyunsaturated fatty acids, especially linoleic and linolenic acids. 7~8t'83'94,158The methods used to assess the essentiality of nutrients have been surveyed by Dadd. 83 Primarily, the essential nature of polyunsaturated fatty acids has been demonstrated by nutritional studies using dietary techniques, which revealed as the main features of deficiency impaired growth and development, including failure at adult emergence and of wings to expand, and reduced reproductive capacity. In addition, metabolic studies using radiolabeled precursors have confirmed that most insects are unable to synthesize polyunsaturated fatty acids. Although incorporation of ~4C-acetate into linoleic and/or linolenic acids has been reported

H

JHIII

H

JH I

H

JH II

H

JH O

FIG. 3. Chemical structures of the juvenile hormones JH 0, JH I, JH II and JH III.


25

in the mosquito, Aedes sollicitans, in the aphid, Myzus persicae and in the cockroach, P. americana, evaluation of these data is difficult because of technical problems associated with the identification of both fatty acids and the possible contribution to the fatty acid pool by polyunsaturated acids synthesized by bacteria found in mycetocytes.94More recent studies, however, have demonstrated a substantial de novo biosynthesis of linoleic acid from [1-~4C]-acetate both in vivo and in isolated tissue preparations of P. americana, the termite, Zootermopsis angusticollis and the cricket, A. domesticus. 29'~°1 Experimental evidence indicated that the contribution of microorganisms was negligible, while linoleic acid synthesis was not correlated with the presence or absence of symbionts. These data suggest that the inability of de novo synthesis of polyunsaturated fatty acids might be less universal among insects than is generally accepted. The presence of biosynthetic ability as such does not necessarily exclude the component concerned as an indispensable nutrient; this will be true only when biosynthetic capacity is sufficient to meet the metabolic demand. The fatty acids required specifically for adult emergence may differ substantially among insect species. For example, nutritional studies with 18 lepidopteran species revealed that three of them had no apparent fatty acid requirement for pupal/adult ecdysis, nine utilized either linoleic or linolenic acids, five required linolenic acid specifically, and one species required both fatty acids. 8° Apart from these different needs for adult emergence, many Lepidoptera have been shown to require both linoleic and linolenic acids for normal growth and development. 8~ Polyunsaturated fatty acids have not been found essential for any of the several species of Diptera studied, except for the mosquitoes. 79For six mosquito species, an essential fatty acid requirement has been demonstrated that can be satisfied by arachidonic acid, while linoleic and linolenic acids are ineffective in this respect. 8°'84In the waxmoth, G. mellonella, linolenic acid appeared to be 10-fold more potent in alleviating faulty adult emergence than linoleic acid. 82 The C20 and C22 trienoic analogues (20:3093 and 22:309 3) of linolenic acid were as effective as linolenic acid itself, whereas 22:609 3 was totally ineffective. The Cz0 analogue of linoleic acid, 20:2096, was also adequate for normal emergence, though with the same low potency as was found for linoleic acid. The nutritional adequacy of polyunsaturated fatty acids in G. mellonella appeared to be associated with the presence of particular double bond sequences in their carbon chains, while the inadequacy of others with these same double bonds could be related to the presence of further unsaturation to the carboxyl side of the 099 and 096 double bonds common to all active fatty acids (Fig. 4). 274 It was proposed that a C~8 polyunsaturated fatty acid is physiologically required by Galleria and can be derived from various longer-chained analogues by simple chain shortening at the carboxyl end so long as there are no additional double bonds carboxylwards of an active di- or trienoic sequence. 82 The fatty acids that could fulfil the essential requirement of the mosquito, Culex pipiens, included both 093 and 096 polyunsaturates, each of them containing a group of at least three double bonds in divinyl methane rhythm terminating on carbon 6 from the methyl end of the chain. Additional double bonds on either side of this group had no further effect on activity.8° It has been suggested that the special dietary requirement for arachidonic acid or structurally related analogues may have evolved in mosquitoes as they normally have access to plentiful arachidonic acid during adult blood feeding and, as larvae, from animal-like protists, and have lost the ability to elongate and further desaturate di- and trienoic Cj8 fatty acids to arachidonic acid. 84 While activity of arachidonic acid as an essential fatty acid seemed to be restricted to mosquitoes, it has been reported very recently that also in the tufted apple budmoth, Platynota idaeusalis, arachidonic acid shows essential fatty acid activity, similar to linoleic and linolenic acids except in correcting wing deformity]56 This also lends support to the view of Dadd 82 that Lepidoptera may have multiple essential fatty acid requirements. All insect species recently examined contain C20 and/or C22 polyunsaturates in their tissues, 8~which led Dadd 83to propose that, if searched for by appropriate techniques, most insects will be found to contain higher polyunsaturates. The presence of such long-chain

A d M. T h . B e e n a k k e r s et al.

26

GALLERIA

C A R B O X Y L END

18:3o)3 (ot-linoleruc) 20:3o)3

e--•~e~e--e--e--e~e~e--e-

22:3o)3

iiiiiiiiiiitii

I Active

. . . . . . . . . . . . . . .

22:6o)3 ( d o c o s a h e x a e n o i c )

......

•~ C , - , - ' ~ ........

20:5o)3 ( e i c o s a p e n t a e n o i c ) 20:4o)6

METHYL END

(arachidonic)

.......

,-- ..... ..,-- ....

,-, ............

20:3o)6 (homo-3' -linolenic)

..........

18:3o)6 (')'-Iinolenic)

....

O--'-'~

--,_ ,-,_~....

,-- ....

-~'~

,_~. . . . .

Not Active

--__o-o..-,~ . . . . .

--° . . . . . . . . .

--._,_,~_ ....

~ ...........

-- . . . . . -- . . . . . --, r ......

20:2t~6

°l

Slightlyactive Semi-active Not-active

•t " ~ I ! +C_._-.2_=.j . . . . . . . .

..............

18:2o)6 (linoleic)

.....

18:1o)9 {oleic]

•= - o

e...e

~6

oJ3

CULEX 18:3o)3

(~-Iinole~c)

20:3o) 3

. . . . . . . . . . . . . .

_

. . . . . . . . . . . . . . . . . .

22:30~3

I

,--,--,-.,~.j,..,-,_~, .,--,_~,--,~

22:6¢~3 ( d o c o s a h e x a e n o i c )

,--~

20:5o)3 ( e i c o s a D e n t a e n o i c )

._,~_._

.--,~_..,--,~ ....

,'-o_,--,~o . . . . - - . . . . . . . . . . .

20:4o)6 ( a r a c h i d o n i c )

,-- .....

18:3o)6 (')'- linolenic) 20:2o)6

--. ....

Active

- - , .,._, . . . . .

I Se~-ac~ve

I I

..............

~J-,-- . . . . . L . . . . . . .

18:lo)9 (oleic)

.

,_--._._._--._.-.~-- . . . . . . . . . . .

20:.3~6 (homo-'?-Iinolenic)

18:2o)60inoleic)

Semi-active

. . . . .

--_.4- . . . . . . . . I

Not-ac~e

FIG. 4. P o s i t i o n s o f cis d o u b l e b o n d s in the c a r b o n c h a i n s o f essential f a t t y a c i d s f o r C. pipiens a n d G. mellonella. Solid a n d d a s h e d line b o x e s enclose fully active a n d p a r t i a l l y active s t r u c t u r e s , respectively. F r o m D a d d , 8~ r e p r o d u c e d w i t h p e r m i s s i o n o f the a u t h o r .

fatty acids in phytophagous species suggests that these insects, like vertebrates, are able to elongate and further desaturate essential dietary Cj8 fatty acids. Polyunsaturated fatty acids are sequestered preferentially into phospholipids and in particular into membranerich tissues, probably serving structural and physiological roles as components of cellular and intraceUular biomembranes. 273 275.342In the phospholipids of spermatophores of the criquet, Teleogryllus commodus, 18% of total fatty acids was accounted for by arachidonic acid, while an even higher proportion of arachidonic acid (24%) was found in the phosphatidylcholine fraction. 2v5 A possible role of C20 polyunsaturates, mainly arachidonic acid, as precursors for the synthesis of prostaglandins in insects has received attention only in the last few years. A prostaglandinogenic function for arachidonic acid in C. pipiens has been postulated by Dadd, s~ who demonstrated that a flight-inducing effect of dietary arachidonic acid in mosquitoes was inhibited if prostaglandin synthetase inhibitors were included in the rearing media. Prostaglandins have been demonstrated in about a dozen insect species. 34,219 They appear to be implicated in insect reproduction, 2°~.275but their occurrence in muscle tissue of several insects and in the brain and central nervous tissues of criquets 8~ suggests that they may have other physiological functions as well.


27

ACETYL- CoA(C2)-~ -'~'HYDROXYMETHYLGLUTARYL'CoA(C6 ) "-="MEVALONATE (Ce) "~"~"~ ISOPENTENYL PYROPHOSPHATE(Cs) '-=' DIMETHYLALLYL PYROPHOSPHATE (C s} ..-~,.-t. FARNESYL PYROPHOSPHATE (Cls } --e-~-~-~--~ J H I

FIG. 5. Simplified scheme for biosynthesis of juvenile hormone III by the sesquiterpenoid pathway.

In a large number of aquatic insects, all individuals examined contained arachidonic and eicosapentaenoic (20:5) acids in quantities up to 7.2 and 24.7%, respectively, of total fatty acids. 143The presence of large amounts of these acids cannot be attributed solely to dietary intake as, for example, late instar Clistoronia magnifica showed an order of magnitude increase in tissue levels of arachidonic and eicosapentaenoic acids on a diet with no detectable amounts of either acid) 42 Their specific physiological role in aquatic insects is unknown, but it has been hypothesized that, apart from a function as precursors for prostaglandins, high levels of both acids may be essential for proper membrane function in aquatic insects adapted to a cold, running water environment) 43 2. Sterols It is generally accepted that no insect devoid of symbionts can synthesize sterol. 94322`2ss'2s9 Insects have been shown to be incapable of de novo biosynthesis of the steroid nucleus, 2s32 implicating that dietary sterol is required to achieve normal growth, development and reproduction. No need for dietary sterols was found in several species that harbor intracellular symbionts within mycetocytes and are able to utilize the sterol biosynthetic abilities of their symbionts. 72'229 Usually insects can utilize a range of sterols, but the ability to use sterols of different structures varies among species. 72,78,79,lSs'28s'289Nevertheless, in general, cholesterol remains by far the major sterol in insect tissues, which indicates that insects are capable of converting other sterols to cholesterol. The cholesterol biosynthetic pathway including the conversion of acetate to mevalonate as the precursor for isoprenoid biosynthesis is present in insects up to farnesyl pyrophosphate. 132a332b:34,214 However, reductive condensation of farnesyl pyrophosphate to squalene appeared to be blocked in the blowfly, Sarcophaga bullata, which suggests that squalene synthetase is lacking or non-operative, m Although the blowfly is able to epoxidize administered squalene to squalene 2,3-0xide, this intermediate is not cyclized to lanosterol) 33 An interesting view on the possible significance of both cholesterogenic blocks for the evolution of juvenile hormone in insects was presented by D o w n e r . 94 Juvenile hormone III (Fig. 3), the most widespread form (juvenile hormones 0, I and II exist only in Lepidoptera,261), is biosynthesized via the sesquiterpenoid pathway, ~85'3j~and thus farnesyl pyrophosphate is an important intermediate in its production (Fig. 5). The absence of squalene synthetase activity would ascertain that farnesyl pyrophosphate remains available for the synthesis of juvenile hormone. The second cholesterogenic block at the level of squalene cyclization was suggested to protect the juvenile hormone molecule, which possesses a 2,3-epoxide linkage (Fig. 3), from possible cyclization. Sterols in insects are mainly used as components of biological membranes. In addition to this structural role, they have an important metabolic function as precursors of ecdysteroids such as ecdysone and 20-hydroxyecdysone (Fig. 2). Ecdysone is synthesized and released by the prothoracic glands (or homologous structures like the ring glands or the ventral glands) during postembryonic development, converted into 20-hydroxyecdysone (ecdysterone) probably in the fat body, and latter steroid plays, as moulting hormone, a major role in the control of moulting and development. Ecdysteroid biosynthesis also occurs in ovaries of several maturing adult female insects, where they may be involved in the control of vitellogenesis; however, in L. migrator&, the bulk of ovarian ecdysteroids was recovered from newly laid eggs, which led to the proposal that ovarian ecdysteroids play a role in the control of e m b r y o g e n e s i s . 194~197

28

Ad M. Th. Beenakkers et

al.

Ecdysone and 20-hydroxyecdysone differ strongly from vertebrate steroid hormones: the ecdysteroids (Fig. 2) have retained the entire carbon skeleton of the precursor molecule cholesterol; they bear several hydroxyl groups that make them rather hydropbylic, and the AB ring junction is of the cis type. Ecdysteroid biosynthesis and metabolism, including inactivation, have been the subject of several recent reviews 156"193'249,25°and will not be discussed here. In all but two known exceptions, the dietary sterol needs of insects can be satisfied by cholesterol. The exceptions are Drosophila pachea and Xyleborusferrugineus, which require a dietary sterol containing either a A7-bond or a As'7-diene system for normal development and reproduction. 7H4v Apparently, these species are unable to introduce a AT-bond into the steroid nucleus, which is essential for the synthesis of ecdysteroids. In insects that have a dietary requirement for cholesterol, other sterols may have a 'sparing' role. For example, in the carnivorous hide beetle, Dermestes maculatus, cholesterol is the only sterol that can fulfill the sterol requirement alone, but the normal dietary cholesterol requirement can be replaced by phytosterols provided that some cholesterol is included in the diet. Apparently only a small amount of cholesterol is needed for the specific function(s), while for the nonspecific function(s) sparing sterols can be used equally well. 72~77

Many phytophagous and omnivorous insects, though not all, are able to dealkylate the C24 position of the phytosterol side chain, thereby converting the phytosterois to cholestane derivatives. In L. migratoria, for example, fl-sitosterol (29A 5) is converted to cholesterol, 5 while in locusts reared on a diet of reed [sterols: 87% campesterol (28A5); 11.4% brassicasterol (28A5'22); 1.6% cholesterol], the haemolymph lipoprotein involved in the uptake and transport of sterol from the gut contained 82.9% cholesterol (Fig. 6), indicative of phytosterol dealkylation (Van der Horst and Van Doorn, unpublished results). Metabolic pathways for the various conversions of the three most common phytosterols, sitosterol, campesterol and stigmasterol (29A 5'22) to cholesterol and other tissue sterols are included in the reviews of Svoboda et al., 285 Downer 94 and Svoboda and Thompson 28s'289 and will be discussed only briefly here. A common intermediate in the conversions of C28 and C29 phytosterols to cholesterol is As'Za-desmosterol (Fig. 2). Other intermediates identified include As'24(28)-fucosterol and fucosterol-24,28-epoxide involved in the con-

Locust lipoprotein A yellow

2x

1

Reed A4 (Glyceria maxima) / l

FIG. 6. Sterol composition of the locust major sterol transporting haemolymph lipoprotein (Ayellow) and that of the diet (reed). Sterol fractions were analyzed by GLC without prior derivatization using a 3% OV 225 column at 190°C. Peak numbers indicate: 1. cholestane reference; 2. cholesterol (27A5); 3. brassicasterol (2845.22);4. campesterol (2845); 5. ~-sitosterol (2945). Van der Horst and Van Doorn, unpublished results.


29

version of sitosterol, AS'24~2S)-methylenecholesterol in the conversion of campesterol, and As'22'24-cholestatrien-3fl-ol in the conversion of stigmasterol to cholesterol. Phytosterol conversions led to very high levels of 7-dehydrocholesterol in the flour beetles, Tribolium confusum and Tenebrio molitor; 50% and 15-20% of total tissue sterols, respectively. 287"2ss The function of the large proportion of 7-dehydrocholesterol is unknown; only a small fraction will satisfy the need for ecdysteroid synthesis. Interestingly, in another insect found in product stores, the khapra beetle, Trogoderma granarium, no 7-dehydrocholesterol was detected. This insect is unable to dealkylate phytosterols, but shows selective uptake of cholesterol and campesterol. 286 Highly unusual results were obtained for the Mexican bean beetle, Epilachna varivestis. The tissue sterols of this insect, reared on soybean leaves (sterols consisting for over 98% of campesterol, stigmasterol and sitosterol), contained 77% saturated sterols (stanols: cholestanol, campestanol and stigmastanol) and 12% lathosterol (AT-cholesten-3fl-ol). It was proposed that AS-dietary phytosterols are first reduced to stanols, which are dealkylated to produce cholestanol; the AT-bond is then introduced to yield lathosterol. 29° The milkweed bug, O. fasciatus, is another example of a phytophagous species that does not dealkylate C28 and C29 phytosterols. Plant sterols were shown to be incorporated into the insect tissues and subsequently also into the eggs with only little modification. 284 It has been suggested that campesterol thus could well be the precursor for makisterone A (a C28 ecdysteroid with a 24-methyl group), which is the major ecdysteroid in eggs of the milkweed bug. 176

B. Lipid Digestion and Absorption Studies on the digestion and absorption of dietary lipids in insects are limited to only a few species and apply almost exclusively to neutral lipids (particularly to triacylglycerols and fatty acids), as shown by several reviews covering this subject. 77"94'122'159In the last ten years, research of Turunen and co-workers has also provided some information on the possible role of the more complex polar dietary lipids. 31~-317 Triacylglycerols were, and probably still are, considered as the principal lipid class that is digested in the gut. Hydrolysis of TG proceeds by the action of lipases, enzymes defined as long-chain fatty acid ester hydrolases, with the alcohol moiety of the ester being glycerol) 8't22 Digestive lipase of P. americana has been shown to be secreted exclusively by the epithelium of the midgut and caeca, which indicates that the lipolytic activity found in the foregut also originates from the midgut. 1°2 This seems to apply also to Blaberus craniifer, another cockroach with lipolytic activity in the foregut. 3° Lipase activity was higher in Periplaneta than in Blaberus, possibly because of the much higher rate of food flow in the former species. Midgut lipase of Periplaneta displays dual pH optima (pH 5.0 and pH 7.2) corresponding to the pH found in fore- and midgut, u2 Available data on the digestive lipase systems of some other species of insects including Aedes aegypti, Dysdercus fasciatus, G. mellonella, M. domestica and Reticulitermes flavipes are discussed in several p r e v i o u s reviews. 7"94't22'159 By the use of double labeled 1,3-dipalmitoyl, 2-01eoyl glycerol (3H-glycerol and ~4C-oleic acid), Bollade et al. 3° could demonstrate that the digestive lipase of the cockroach [ Mot~acylghtcgtolpathway]

2-MG [] ~

Glycerob3-phosphate Fa.y~c~-CoA

I ~ ' ~ A ~

t,2:~j-"

[]TQ~"~CoA

lar-~ycerol0hos0hate laathwayI

_.--1[] CoA Phosphatidicacid

~,2-,~

CoA..--~Ta[]

FIG. 7. Alternative pathway for acylglycerol synthesis. Numbers refer to the enzymes involved: I. monoacyglycerol acyltransferase; 2. diacylglycerol acyltransferase; 3. glycerol phosphate acyltransferase; 4. phosphatidate phosphohydrolase.

30

Ad M. Th. Beenakkers et al.

preferentially liberates the fatty acids located at the 1- and 3-positions of the TG molecule, leading to the formation of 1,2 (2,3)-DG and 2-MG; part of the 2-MG is further hydrolyzed, as indicated by the presence of free ~4C-oleic acid in the crop. These results were confirmed by Hoffman and Downer, 154who demonstrated by stereospecific analysis of the DG formed upon incubation of midgut homogenates with trioleoyl 3H-glycerol that TG hydrolysis results in the production of a racemic mixture of sn-l,2- and sn-2,3enantiomers. As the midgut also appeared to contain monoacylglycerol acyltransferase activity (Fig. 7) with DG as the major product, ~s5 and evidence has been presented that DG is the principal form in which acylglycerol is taken up from the midgut by haemolymph,58 it was proposed that the uptake of dietary TG in the cockroach requires prior hydrolysis to MG and FFA in the gut lumen with subsequent resynthesis of DG occurring in the intestinal mucosa. ~54On the other hand, Reisser-BolladeTM claimed that dietary TG are absorbed into the haemolymph of cockroaches as TG, unchanged or resynthesized from the products of their hydrolysis. This finding has been disputed, however, by Chino and Downer. 5s In the intestine of L. migratoria, hydrolysis of dietary TG proceeding to completion was demonstrated by the presence of free 3H-glycerol in the intestinal content of locusts fed tri-l-~4C-oleoyl 2-3H-glycerol. 35° Glycerol was rapidly absorbed across the intestinal wall, as indicated by the large amount of radioactivity associated with free glycerol in the haemolymph of locusts in vivo, and in the incubation medium of intestinal preparations isolated from locusts fed l-t4C-trioleoyl glycerol with trioleoyl 2-3H-glycerol. 35°'35~The fatty acids were incorporated into intestinal wall PL, DG and TG, and into haemolymph lipoproteins mainly as DG and to a much lesser extent as FFA. A positive correlation was found between PL synthesis in the midgut wall and DG release into the haemolymph. Furthermore, the 3H/t4C-ratio was similar in intestinal PL and DG and haemolymph DG, providing evidence that the intestinal wall is the direct source of haemolymph DG upon lipid absorption. Membrane formation was considered as the most likely explanation for the high labeling of PL in the intestinal wall, although the possibility that PL synthesis is part of the fatty acid absorption process, PL cleavage yielding DG which are released into the haemolymph, was not excluded. 35° Similar results were obtained with larvae of Bombyx mori fed mulberry leaves to which tri-lnC-oleoyl-3H-glycerol had been applied: rapid and almost complete lipid hydrolysis, recovery of 3H-glycerol and 14C-DG from the haemolymph and of ~4C-PL, DG and TG from the intestinal wall. TM These data are strongly indicative of acylglycerol synthesis in the intestinal wall of L. migratoria and B. mori through the ~-glycerophosphate pathway (Fig. 7). On the other hand, the above mentioned activity of monoacylglycerol acyltransferase in the midgut of P. americana provides evidence that in the cockroach intestine the monoacylglycerol pathway is operative. In no insect species studied, however, the relative contributions of the ~-glycerophosphate pathway and the monoacylglycerol pathway to acylglycerol synthesis during lipid absorption have been established. In larvae of Pieris brassieae, intestinal hydrolysis of TG also resulted in the release of DG into the haemolymph. A substantial proportion of dietary TG, however, was excreted unchanged. 3~4 The utilization of neutral, phospho- and glycolipids in larvae reared on cabbage leaves was demonstrated to increase in that order. ~TsSuch an adaptation as in P. brassieae to absorb the more polar lipid component of the diet may be of more general importance to phytophagous insects, since the majority of plant leaf acyl lipids consists of glycolipids (galactosyl DG), sulfolipids and phospholipids. 3~5-3~6 Furthermore, the essential linoleic and linolenic acids accumulate preferentially in plant galactosyl DG, which may also contribute to the nutritional significance of these glycolipids.3j5 Little information is available on the absorption of phospholipids in insects. Phospholipase activity has been reported in the digestive juice of three lepidopteran species, P. brassicae, Spodoptera exiguens and Triehoplusia h i . 269 In P. brassicae dietary phosphatidylcholine, a PL common in plant and animal tissues, was hydrolyzed to the corresponding monoacyl compound, lysophosphatidylcholine3~9 It has been proposed by Turunen 3~5 that in insects, which lack lipid emulsifiers comparable to the bile salts in

Insect lipids and lipoproteins Lumen TG •-.--~ 1,2-DG 2-MG ~ Glycerol

Synthesis of 1,2-DG TG ,,

Incorporation into lipoprotein

t

,L

Lyso-PL .I

Hemolyrnph

Mucosa

(DG: PL:Sterol)

Fatty acyl- CoA

FFA

----"

Other derivatives

31

S!

ynt h esis of PL --=-

Water-soluble metabolites

,,-

PL

FIG. 8. Proposed scheme for absorption of neutral lipids and phospholipids in insects. MG, monoacylglycerol;DG, diacylglycerol;TG, triacylglycerol;PL, phospholipid;FFA, freefattyacid. After Turunen)~7 vertebrates, the surface-active lysophosphatidylcholine may function in the emulsification of less polar lipids in the intestinal lumen. Lysophosphatidylcholine itself readily enters the midgut cells, where it is reacylated to phosphatidylcholine. Turunen 31s also suggested that PL are directly concerned in transcellular lipid movement by acting as carriers in the mucosal cells and releasing DG onto a protein acceptor in the haemolymph. Data mentioned above indicating a positive correlation between PL synthesis and DG release and a similar 3H/14C-ratio in intestinal PL and DG and haemolymph DG in locusts fed labeled TG TM lend support to this suggestion. A proposed scheme for the absorption of neutral and phospholipids in insects is illustrated in Fig. 8. Available data indicate DG as the major lipid formed and released during the process of lipid absorption in insects. In haemolymph, these DG are present associated with lipoproteins TM which, in P. americana, has been shown to be the same lipoprotein that serves to transport DG from the site of storage in the fat body to utilization sites. 57,63 Observations of Thomas 295 may suggest that the midgut mucosa of S. gregaria is capable of conjugating the DG with proteins as lipoproteins prior to their release into the haemolymph. Incubation of isolated gut preparations from locusts fed radioactive lipids resulted in similar patterns of DG release into medium containing haemolymph and medium void of haemolymph. Latter medium also contained a labeled lipoprotein which had the same electrophoretic mobility as the haemolymph lipoprotein. The major site of absorption of dietary acylglycerols is the midgut, especially the anterior region and the associated caeca, 313'318although the crop of P. americana J53and the hindgut of the crickets Gryllus rubens and Scapteriscus acletus 298'299 have been demonstrated to be capable of significant fatty acid absorption. The midgut is also the major site of cholesterol absorption in most of the insects studied; however, in some species including carnivorous and omnivorous insects, absorption of cholesterol has been reported to take place mainly in the foregut, and particularly in the crop. 17° Cholesterol may be absorbed as free sterol, although temporary intracellular esterification may occur in the gut tissue. 315'317Release of cholesterol into the haemolymph probably occurs mainly in the form of free sterol, as reflected in the haemolymph levels of free and esterified cholesterol (Section II.B). The released cholesterol is incorporated into haemolymph lipoproteins for transport to sites of utilization. 57'62"64Phytophagous insects are adapted to utilize phytosterols from the diet. Usually these phytosterols are in part converted to cholesterol in the midgut cells before release into the haemolymph (Section III.A). C. Lipid Storage in the Fat Body The majority of insect lipids is usually found in the fat body, and in most of the species examined over 90% of total fat body lipid is present as TG (Section II.A). These lipid stores are derived from absorbed dietary lipids transported to the fat body by JPLR 2 4 / 1 ~

32


haemolymph, but they may also be the result of de novo lipogenesis in the fat body itself. The uptake of DG as well as FFA into fat body lipids has been demonstrated in several species of insects both in vivo from haemolymph and in vitro from incubation media, and has been reviewed by Bailey. 9 In P. americana, the incorporation of haemolymph acylglycerols into fat body lipids was reported to be stimulated by a hypolipaemic factor from the corpus cardiacum, which thus may favor the deposition of dietary lipid in the fat body. 98 A similar hypolipaemic factor has been described in the storage lobe of locust corpora cardiaca. 236 Important aspects of de novo lipogenesis include: the conversion of non-lipoidal substrates, largely carbohydrate, to C2 units; the synthesis of long-chain saturated fatty acids from C2 units; desaturation and chain elongation of saturated fatty acids; esterification of fatty acids to yield acylglycerols. The ability of insects to synthesize lipids from non-lipid precursors has been demonstrated in a number of studies carried out mostly on whole animals, as illustrated by several reviews. 9'94"95'122This section will focus on aspects of lipogenesis in the fat body, as this organ not only contains the main storage depots of lipid, but also functions as the primary site of lipid biosynthesis in insects. 1. Biosynthesis o f Fatty Acids

Clements73 has demonstrated that acetate, glucose and some amino acids can be converted to fatty acids by isolated fat body tissue of S. gregaria. Fatty acid synthesis from acetate and glucose was also obtained in homogenates of fat body tissue of the moth, Prodenia eridania. 367The major fatty acid produced was palmitic acid. Tietz 3°2showed that cell-free extracts of the fat body of L. migratoria incorporated J4C-acetate into fatty acids, factors required including ATP, CoA, KHCO3, NADP and malonate. Again, palmitic acid was the major fatty acid synthesized. Fixation of ~4CO: into malonate has also been demonstrated in the locust fat body. 3~°Acetyl-CoA carboxylase activity has been detected in fat body of P. americana and was located in the cytosol.283 Fat body cytosol also contained acetyl-CoA synthetase activity, which was suggested to be concerned predominantly with the conversion to acetyl-CoA of acetate arising in the diet or by the action of the gut flora. The optimum conditions for fatty acid synthesis by insect fat body appear to be similar to those for mammalian and avian systems, and they have palmitic acid as the major end product in common. 9 These observations indicate that fatty acid biosynthesis in the fat body occurs by a pathway similar to that occurring in mammals and other animal groups. It has been shown in a number of studies on whole insects that long-chain saturated fatty acids may be directly desaturated to the monoenic equivalents.9,94"95Tietz and Stern 3°6 have shown that, in the fat body of L. migratoria, being able to convert stearic to oleic acid and palmitic to palmitoleic acid, the desaturase activity resides within the microsomal fraction and has an absolute requirement for oxygen in common with desaturases from yeasts and higher animals. In addition to the direct desaturation pathway, evidence has been obtained for an alternative route for the biosynthesis of palmitoleic and oleic acids in mitochondria of Drosophila melanogaster, 128a86 which was similar to the monoene synthetic pathway reported for microorganisms. ~° Insects are considered to be unable to synthesize polyunsaturated fatty acids de novo (see, however, Section III.A.). It has already been mentioned before (Section III.A.) that phytophagous insects recently examined contained C20 and/or C22 polyunsaturated fatty acids in their tissues, which would suggest that these species are able to elongate and further desaturate essential dietary Cj8 fatty acids. 81,8sInsects can also shorten existing fatty acids. Keith 18~demonstrated that in D. melanogaster dietary stearic acid is shortened from the carboxyl end to myristic acid and its monoene and that linoleic acid is similarly shortened to Ct4 and C~6 dienes. In the boll weevil, Anthonomus grandis, dietary stearic acid was shown to be converted to palmitic acid, which in part was desaturatedJ 98 Another factor that plays an important role in determining fatty acyl chain length was suggested to be the malonyl-CoA:acetyl-CoA ratio (which is dependent upon the activity


33

Glucose .,~ = C-acose=6-p

Fatty acids

1

Fruct~se-6-P ~ ....

ICYTOPLASM I

V T~- . . . . . . . . . . .

Pyruvate 9

PEP =

U

= Oxaloacetate 4

= AcetyI-CoA

[] fruv

Idalate/ =, AcetyI-CoA

ate I

I

f

,~ Citrate

1

I~c~'ate

,

[MrrOcHOM:)RJON I I.

1

2-Oxoglutarate-

- 2-Oxoglutarate

FIG. 9. Metabolic pathway involved in the conversion of carbohydrate to fatty acids. Numbers refer to the following enzymes: 1. citrate lyase; 2. isocitrate dehydrogenase; 3. 'malic enzyme'.

of acetyl-CoA carboxylase), a high ratio favoring, like in vertebrates, the formation of longer chains. 95 2. Conversion of Carbohydrate to Fatty Acids

In a number of studies, Walker and Bailey343-347have demonstrated the conversion of carbohydrate to lipid in the fat body of S. gregaria and obtained evidence for the underlying mechanism. The proposed pathway is essentially the same as that proposed for mammalian tissues) 89 Glucose is converted to pyruvate via the glycolytic and pentose phosphate pathways. The pyruvate is transported into mitochondria and oxidized to acetyl-CoA, which is converted to citrate. The citrate is transported into the cytosol and acetyl-CoA for fatty acid synthesis is regenerated by the action of citrate lyase (Fig. 9). Further support for such a pathway in the insect fat body has been presented by Storey and Bailey, 2s2'283 who determined maximal activities and intracellular distribution of enzymes associated with lipogenesis in fat body of P. americana. N A D P H required for fatty acid synthesis in insect fat body was found to be generated by the pentose phosphate pathway and by the action of cytosolic NADP-dependent isocitrate dehydrogenase (Fig. 9). 282'283'344'346 Like in mammalian tissues, 189 part of the N A D P H required may also be provided by 'malic enzyme', as has been demonstrated in larvae of D. melanogaster.lt8:19 3. Biosynthesis of Triacylglyeerols

Fatty acids synthesized de novo or produced by lipolysis from dietary lipids are stored in the fat body mainly in esterified form as TG. The incorporation of fatty acids into fat body acylglycerols has been demonstrated in a number of insect species both in vivo and in vitro (cf. Bailey9). Municio et al. 2~7"2~8using the fruit fly, Ceratitis capitata, have shown a stereospecific incorporation of fatty acids into TG resulting in a positional distribution similar to the general positional tendencies described for animal depot fats: 37 sn-1 and sn-2 are mainly acylated by saturated and unsaturated fatty acids, respectively, while position sn-3 is randomly occupied by the long-chain fatty acids. The stereospecific distribution of fatty acids changed during development of the insect (Fig. 10), leading to a reduction of palmitoleic acid in each of the three positions which was counterbalanced by an increase of palmitic acid in sn-1 and sn-3 positions and of oleic acid in the position sn-2. n7 These changes were interpreted as suggesting the existence of differences in the fatty acid specificity of the acyltransferase system at different stages of development.

34

Ad M. Th. Beenakkers et al. % sn- 1

80

o

o 16:0 ~x 18:0 I,-----Q 16:1[v 9) A-----~ 18:1(" 9}

sn-2

70 60

m - - - - - m 18;2

50

,, . . . . . . . -",_,-"

40 30 .

0.,

",.

/:

///

"'0

20 10 0 L %

P-A

A

E

L

P-A

A

P-A

A

TG

sn-3

50 40 30 2C 10 0 E

L

P-A

A

E

L

FIG. 10. Quantitative variations of fatty acids in the triacylglycerols (TG) and in the positions sn-1, sn-2 and sn-3 of TG during development of C. capitata. E, eggs; L, larvae; P-A, pharate adults; A, adults. After Garcia and Municio):

The two pathways known for the synthesis of TG in mammals, i.e. the c(-glycerophosphate and the monoacylglycerol pathways (Fig. 7), also occur in insects, as described for the intestine in Section III.B., while evidence has been obtained for their functioning in the fat body as well. Fat body of Hyalophora cecropia was shown to contain phosphatidate phosphohydrolase activity which, like the vertebrate enzyme, was associated with the microsomes.~52 In fat body of P. americana, the presence of monoacylglycerol acyltransferase was demonstrated by the capacity of tissue homogenates to acylate 2-monooleoyl glycerol with palmitic acid) 55 Both pathways have been shown in fat body of L. migratoria. The microsomal fraction incorporated palmitate into phosphatidate, and addition of supernatant obtained from centrifugation at 140,000 g led to conversion of phosphatidate to DG and TG. 3°4'3°5 These results indicate that in the locust fat body glycerol phosphate and diacylglycerol acyltransferase activities are associated with the microsomes whereas, differently from H. cecropia and vertebrates, the supernatant fraction contains phosphatidate phosphohydrolase. Glycerol 3-phosphate as well as glycerol could be utilized by fat body homogenates for acylglycerol synthesis, the presence of glycerol kinase in the mitochondrial fraction enabling the conversion of glycerol to glycerol 3-phosphate. The monoacylglycerol pathway in locust fat body is also associated with the microsomes, as indicated by the monoacylglycerol acyltransferase activity in this fraction. TM Tietz et al. 3°9 have shown that the enzyme specifically acylates 2-acyl-sn-glycerol, resulting in the formation of 1,2-DG (see also Section IV.B). It has been proposed by Peled and Tietz z4~ that the two pathways for acylglycerol synthesis serve different physiological functions, and that their relative contributions largely depend upon the availability of glycerol 3-phosphate. In feeding locusts, there will be a continuous formation of glycerol 3-phosphate from glucose, which permits synthesis of storage TG via the c(-glycerophosphate pathway. During flight, the concentration of glycerol 3-phosphate would be too low to serve as a substrate for acylglycerol synthesis, and the monoacylglycerol pathway might predominate: 2-MG formed by lipolysis from storage TG are reacylated to 1,2-DG, which are released into the haemolymph to provide energy to the flight muscles. However, the availability of glycerol that is produced during flight by DG hydrolysis in the flight muscles and transported to the fat body to be used for the re-esterification of fatty acids (see also Section IV.B) is neglected in this model, and


35

this may lead to an underestimation of the role of the ~t-glycerophosphate pathway during flight. IV. LIPID METABOLISM AND FLIGHT A. Lipid and Flight Economy

Since in the early fifties first evidence for participation or exclusive utilization of lipid during insect flight was furnished by measurements of the respiratory quotient during flight of the desert locust 19° and some lepidopteran species, 365 the role of lipids in energy metabolism and the biochemical mechanism underlying lipid utilization have been studied extensively, as will be discussed below. In earlier studies, the exclusive importance of carbohydrate for flight of the honey-bee ~69 and the fruit fly49 had been discovered. These data raised the question of possible species-specificity in substrate utilization during flight and an eventual relation between the biology of a given species and a particular substrate utilized. In a discussion on the relationship between the endurance of flight and the kind of substrate used, Weis-Fogh 352 indicated that insects using lipid as a fuel can make long-range flights, whereas those depending on only carbohydrate are capable of relatively short periods of continuous flight. Reasons for this disparity are the higher energetic value of lipid compared to carbohydrate and the fact that glycogen, the main carbohydrate reserve in animals, is always stored with an appreciable amount of water. It can be calculated that 1 mg of lipid supplies as much energy as 8 mg of stored glycogen; so, for reasons of weight economy, lipid is a more desirable substrate than carbohydrate (cf. Beenakkers et al.2°). If a locust for an uninterrupted flight period of 10 hr (as observed indeed) would rely exclusively on carbohydrate, pre-flight loading with glycogen to more than 50% of its normal weight would be required, whereas in using lipid only 7% is needed. Therefore, insects that remain airborn for many hours may be expected to utilize lipid during continuous flight. This has been demonstrated for a number of insects. 4°'~74 Storage of energy in the more compact form of lipid is advantageous also for insects that do not feed as adults. Stores of lipid built up during larval stages can be utilized for adult physiological processes, for instance mating flights. Comparison of the activities of enzymes of the main catabolic pathways in flight muscles of various insect species reveals high activities of enzymes responsible for fatty acid oxidation and very low activities of the enzymes regulating carbohydrate metabolism in two butterfly species not feeding as adults (cf. Beenakkers et al.22). Species, both feeding in the adult stage and capable of prolonged flight, were found to have the capacity of oxidizing both carbohydrate and lipid in the flight muscles; as evidenced for particularly the migratory locust, carbohydrate is the fuel used in the initial flight period, lipid becoming the predominant substrate during prolonged flight (cf. Beenakkers et al.2°). It must be emphasized that metabolic activity generally is very high during flight; in many insect species the metabolic rate increases 50to 100-fold during the transition from rest to flight (Table 1). Consequently, substrate mobilization from fat body stores and utilization in the flight muscles, whether carbohydrate or lipid, must reach high values as well. In comparison, small mammals running at maximal speed and flying birds achieve metabolic rates exceeding resting levels by only about 10-fold. Therefore, flight metabolism in insects has a profound impact on various TABLE1. OxygenConsumption Rates of Insects at Rest and during Flighta pl O2g body wt-t min 1 Species Rest Flight Periplaneta americana 6 600 Schistocerca gregaria 10 500 Metopsilus procellus 12 1700 Tabanus affinis 18 930 Apis mellifera 53 1000 ~After Beenakkers et aL 22

36


organs, including fat body, haemolymph and flight muscles, as will be discussed with regard to the metabolism of lipids. B. Lipid Release from Fat Body As indicated before (see Section II), the insect fat body is the metabolically most important organ in both synthesis and storage of lipid and the supply of lipid to the haemolymph, for instance during flight. The dominant lipid class for storage is TG which is mobilized in response to metabolic demands. Tietz 3°2'3°4demonstrated the release of lipid from adult L. migratoria fat body in vitro. In a medium containing 14C-labeled fatty acids, fat body acylglycerols incorporated the radiolabel and upon transfer of the fat body to a medium obligatory containing haemolymph components (which afterwards turned out to be the acceptor lipoproteins in order to transport lipids in the aqueous haemolymph, see Section IV.D), acylglycerols were released. Chino and Gilbert, 6~ using prelabeled fat bodies of the silkmoth, H. cecropia and the grasshopper, Melanoplus differentialis, noticed that the amount of labeled TG released into the medium was but very small, and were the first to demonstrate the specific release of DG which is compellingly confirmed in a great many insect species (for reviews, see Bailey; 9 Downer; 94,95 Beenakkers et al.; 2°'22 Chin057), although little is known about the biochemical and physiological attributes of DG esters. 1. Stereospecificity of Released Diacylglycerols Concerning the structure of the haemolymph DG, Tietz et al. 3°9 showed that 1,2-DG is released from locust fat body. These 1,2-DG are stereospecific revealing the sn-l,2 configuration with a very high optical purity, z°2'3°8Tietz and Weintraub 3°8 analyzed both fat body and haemolymph 1,2-DG by enantioselective enzymatic degradations using phospholipase A 2 after phosphenylation of the DG and obtained an optical purity of 80-90%. Lok and Van der Horst 2°2 applied a ~H-NMR method using chiral shift reagents after conversion of the haemolymph 1,2-DG into 1,2-diacetyl-3-tritylglycerols and found the optical purity of the sn-l,2-DG to be over 96% (Fig. 11). These data demonstrate stereospecific conversions to be involved in the production of DG, which will be discussed later on. 2. Diacylglycerol Production in the Fat Body The initial event in the lipolysis of the fat body TG involves hydrolysis of the long-chain fatty acylglycerol esters by the action of lipases (cf. Van der Horst3:2). Compared to mammalian adipose tissue TG lipase (for a review, see Steinberg277), only little is known from insect fat body TG lipase (for reviews, see Beenakkers et al.2°; Van der Horst; 323

o

CH,C,H

u

9

~H20-~)-RI 3

c.,o-o. . . .

haemolym~ 1,2-dlacyl- sn-~ycerol s n ~23

42

4O

3~

FIG. 11. ~H-NMR spectrum (200MHz, C2HC13 solution) of the acety] methyl protons of 1,2-diacetyl-3-tritylglycerol originatingfrom the haemolymph1,2-diacylglycerolsof Locusta after a 2 hr flight. From Lok and Van der Horstfl°2


37

Downer95). Lipase activity has been reported in the fat body of H. cecropia ~24 with trioleoylglycerol as a substrate. In the locust fat body, an alkaline lipase preferentially hydrolyzed MG, whereas an acid lipase was also active against DG. TG hydrolysis, however, occurred but very slowly, yielding fatty acids as major end product. 3°7 Since it proved difficult to obtain stable micellar TG emulsions, these in vitro results may not reflect the in vivo situation. Significant TG lipase activity was observed in the fat body of the cockroach, P. americana, ~54 an insect species which, in contrast to the locust, is not depending on a supply of lipid during flight activity. 97 DG was the primary product of lipolysis. For the conversion of TG to DG in the insect fat body during flight, two alternatives have been proposed. From in vitro experiments in which fat body extracts of the desert locust, S. gregaria, were incubated with J4C-labeled TG, yielding radiolabeled DG and free fatty acids, Spencer and Candy 27~ proposed monoacyl cleavage from the stored TG to be the primary route of 1,2-DG production. This pathway may also prevail in the cockroach fat body. t54 A second pathway is degradation of TG to 2-MG, followed by reacylation to 1,2-DG. 3°7'3°8On the basis of differences in the fatty acid composition of fat body TG and haemolymph DG in H. cecropia and L. migratoria, Beenakkers and Gilbert t7 and Beenakkers ~5 postulated the existence of two different acylglycerol pools in the fat body. TG in the storage compartment is hydrolyzed mainly to MG and free fatty acids, which are translocated to an active compartment with subsequent synthesis of DG with a specific fatty acid pattern for release into the haemolymph. Evidence for either pathway is inconclusive; 2°2 however, the in vitro studies by Tietz et al. 3°9 showing that locust fat body microsomal acyltransferase specifically used 2-MG and, in addition, that the enzyme is stereospecific and preferentially synthesizes sn-1,2-DG, 3°8 are clearly in favor of the latter pathway, although direct production of sn-l,2-DG by a stereospecific lipase cannot be discounted. Incubations with the fat body lipase system and optically active radiolabeled TG could possibly distinguish more conclusively between the alternatives. 22 By an approach in which toxic enantiomeric og-fluorinated alkyldiacylglycerols were fed to the termite, R. flavipes, Carvalho and Prestwich 48 propose evidence for stereospecific lipolysis of TG. However, this applies to the metabolism of dietary TG in the gut rather than to mobilization of fat body reserves. In the locust, it has been recognized that irrespective of the pathway involved, conversion of TG to DG in the fat body leads to the production of free fatty acids as well. Candy et al. 4~ showed that injection of ~4C-glycerol resulted in the appearance of both labeled DG and trehalose in the haemolymph, and proposed that when acylglycerol is used for flight muscle energy generation, the fatty acids are oxidized but most of the glycerol is transported to fat body and there converted to glycerol 3-phosphate, thus providing a mechanism for the re-esterification of the free fatty acids secondarily produced during DG formation from TG stores. Using pulse-labeling of the haemolymph glycerol pool, Van der Horst et al. 3z4 substantiated this glycerol shuttle and demonstrated a rapid transport of glycerol from the flight muscles to the fat body during flight (see Section IV.F). The re-esterification of free fatty acids in the fat body apparently also is a stereospecific process in view of the persisting exclusive stereospecificity of the DG in the haemolymph during flight. 2°2 The rationale for the stereospecificity of these lipid conversions remains to be explained, but has been proposed to be related to the possible specificity of the flight muscle lipoprotein lipase 323by analogy to the stereospecificity observed in mammalian lipoprotein lipases (for a review, see Jensen et al.168), in the cockroach, the single other insect in which stereospecificity of DG has been studied, only a slight preference for the sn- 1,2 enantiomer over the sn-2,3 compound was found 154 but, in this insect, DG is not used as a fuel for flight. 3. Diacylglycerol Dynamics during Flight

Flight activity in the locust is accompanied by a considerable elevation of the DG level in the haemolymph, reaching a plateau phase at about 3-fold its resting concen-

38


mglml

~

=t~a6 101 .~

,ol/ "

0

,¢~,

3'o 60 ~35 ~s l~S 2~s 2~5 2~s 3~s 3~s flight time (min)

FIG. 12. Increase in locust haemolymph diacylglycerol concentration during flight, and decrease in specific radioactivity of the haemolymph diacylglycerol pool after pulse-labeling with ~4C-oleic acid at the steady state flight level. From Van der Horst. 322 tration 15'173'2~1 (Fig. 12) indicative o f the attainment o f an equilibrium between the mobilization o f these lipids by the fat b o d y and their utilization as substrate for flight. The mobilized D G p r e d o m i n a n t l y are C34 and C36 acylglycerols (16:18 and 18:18), 173 their constituent fatty acids being mainly oleic (18:1) and palmitic (16:0) acid, the relative composition remaining fairly constant during flight periods up to 6 hr j3'325(Table 2). Only m i n o r changes in other neutral lipid classes are noticed although the low levels o f F F A show a relatively large increase (Table 3). Evidence for the actual use o f D G for flight muscle energy generation was obtained in the moth, S p o d o p t e r a f r u g i p e r d a , as the turnover o f the total pool o f D G during flight appeared to m a t c h the metabolic activity o f the insects. TMT u r n o v e r studies o f the h a e m o l y m p h D G pool o f the locust u p o n pulse-labeling with ]4C-fatty acids revealed that the plateau phase in D G level maintained during prolonged flight is a steady state in which the utilization o f D G for energy production a m o u n t s to 3.4 mg D G locust-~ hr-J, a value m o r e than 8-fold the utilization rate o f D G at rest 325 (Fig. 12). T u r n o v e r rate o f D G appeared to be independent o f the fatty acid used for the pulse-labeling, indicating r a n d o m utilization o f the D G fatty acids. Oxidation o f D G in the first period o f flight period o f flight increases with the rise in h a e m o l y m p h D G level. 326 C. H o r m o n a l C o n t r o l o f Lipid Release In m a m m a l i a n adipose tissue, mobilization o f T G is mediated by the intracellular 'hormone-sensitive lipase', which m a y encompass a n u m b e r o f enzymes specific for T G , TABLE2. Proportional Composition of the Quantitatively Most Important Fatty Acids of the Haemolymph Diacylglycerols from Locusts at Rest and after Flights of 2, 4 and 6 hr Flight (hr) Fatty acid 0 2 4 6 14:0 0.9 1.1 1.1 1.1 16:0 15.4 22.6 22.8 23.0 16:1 2.6 2.7 2.6 2.8 18:0 5.1 4.6 4.6 4.5 18:1 31.0 47.2 47.6 47.2 18:2co6 9.1 10.5 10.8 10.5 18:3(o3 3.0 8.0 8.6 8.3 Total 67.1 96.7 98.1 97.4 *All figures expressed als mol.%. Average values of groups of 12 male locusts are given. From Van der Horst et al. 32s

TABLE 3. Neutral Lipids and Free Fatty Acids in the Haemolymph of Male Locusts At rest After flight Lipid class % mg ml ~ % mg ml- i TG 7.1 0.33 3.7 0.52 DG 84.1 3.89 86.1 12.0l MG 3.9 0.18 1.6 0.22 Free Fatty acids 4.9 0.23 8.6 1.20 Total 4.63 13.95 *Haemolymph lipids were analyzed after a 3 hr flight period. From Beenakkers/5


39

DG and MG. The enzyme complex is activated by several lipolytic hormones such as catecholamines, ACTH and glucagon. The activation pathway is supposed to include adenylate cyclase stimulation, intracellular cAMP accumulation, activation of a cAMPdependent protein kinase, and phosphorylation of the lipase (for a review, see Steinberg277). In insects, particularly, adipokinetic hormone and octopamine may be related to hormonal control of fat body lipid release during flight. 1. Adipokinetic Hormone

Since nearly two decades ago Beenakkers ~4 and Mayer and Candy2t° independently reported the presence of a lipid mobilizing factor in the corpora cardiaca of the migratory locust, L. migratoria, and the desert locust, S. gregaria, respectively, hormonal control of lipid release has become an important topic in insect biochemistry and physiology which has been amply discussed in a number of recent reviews (Goldsworthy;129 Goldsworthy and Wheeler; 132 Beenakkers et al.16'23'24). The peptidergic factor, which was named adipokinetic hormone, is synthesized in the corpora cardiaca, endocrine organs located in the head region, and more accurately in their constituent glandular lobes. TM Upon injection, this factor elicites elevation of DG in locust haemolymph at the expense of fat body TG. The factor had been isolated and identified as an N- and C-terminally blocked decapeptide, the structure of which has been sequenced 28° and confirmed by synthesis,39'363 and is commercially available. This adipokinetic hormone (I~iu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), which will be referred to as AKH I, is released during flight and is proposed to activate fat body TG lipase. Flight activity as well as injection of the hormone induce an increase in cAMP in the locust fat body. ~4'~~6In addition, the stimulatory action of the hormone on fat body DG release is mimicked by dibutyryl-cAMP.TM Protein kinase activity in locust fat body was demonstrated, the enzyme was activated in vitro both by cAMP and cGMP, 19"339suggesting mobilization of DG to be mediated by cAMP or cGMP-dependent protein kinases. Whereas Pines et al. z45 showed in vitro activation of locust TG lipase by cyclic nucleotides, neither protein kinase activation nor lipase stimulation was accomplished in vivo after hormone injection. Thus, the ultimate mechanism for hormonal control of TG lipase is still uncertain, although the data obtained suggest TG mobilization to proceed in the same sequence as that in adipose tissue of vertebrates indicated above. Lipid mobilization in the monarch butterfly, Danaus plexippus, and M. sexta was shown to be dependent on factors in the corpora cardiaca as well; in M. sexta, a nonapeptide adipokinetic hormone was recently reported 37° although lipid mobilization was also achieved after injection of locust AKH. ~9Insects like the mealworm, T. molitor, the cockroach, P. americana, and the stick insect, Carausius morosus, on the other hand, reveal no enhanced lipid release after flight or injection of the hormone extract of their corpora cardiaca but, when injected into locusts, these extracts have an adipokinetic effect (for reviews, see Goldsworthy; ~29 Beenakkers et al. 16 This cross-reactivity leading to species-specific responses may indicate partial similarity between the hormones involved, and also shows some correspondence in fat body hormone receptors. ~6 In addition to AKH I, in locusts, a second lipid mobilizing peptide (AKH II) has been found in the corpora cardiaca of S. gregaria 47 and L. migratoria. ~5 This AKH II, the primary structure of which has recently been elucidated (intriguingly, differing for Schistocerca and Locusta by one amino acid), 267 is a blocked octapeptide; like AKH I, compound II is primarily confined to the glandular lobes of the corpus cardiacum, but is present in smaller amounts accounting for some 20% of the total adipokinetic activity.47 It should be remarked, however, that during flight only very little quantities of hormones are released. Recent studies by Orchard and Lange234,235suggest that, in addition to AKH I, AKH II is also released during flight of Locusta and may be responsible for some lipid mobilization, the titer of AKH I being elevated earlier than that of AKH II. Goldsworthy and Wheeler ~32have assayed the relative potencies of natural AKH I and II in stimulating lipid mobilization, and conclude that considerably higher doses of AKH II are required

40


tO give a lipid increase of significantly less than the maximum response obtained at the optimum dose of AKH I. 2. Octopamine

Besides adipokinetic hormones I and II, a further complication in the release of lipid from the fat body arises since the report of elevated levels of the biogenic amine, octopamine, the phenolic analogue of noradrenaline, in the haemolymph of locusts during the first minutes of flight. ~37As flight continues, however, there is a rapid decline towards resting levels. Octopamine was shown to act upon the locust fat body to stimulate lipid release in v i t r o . 232"233"335The site of release or origin of the hormone is not clear, although release from nerves innervating the flight muscles has been suggested. ~38Octopamine may be involved in rapid mobilization of DG during the first few minutes of flight in view that haemolymph lipid is elevated 52'3z6in spite of the apparent absence of a rise in the titer of AKH in this period. 52 There is, however, evidence for a very rapid release of AKH as well, 248'34° thus, as proposed by Goldsworthy, 129 octopamine and adipokinetic hormone may act together on the fat body during the early minutes of flight activity. D. Haemolymph Proteins and Lipid Transport 1. Diacylglycerol- Transporting Lipoproteins

As indicated above, release of DG from insect fat body requires the presence of specific lipoproteins in the haemolymph which function in taking up and carrying the lipid in the aqueous medium. Two classes of lipoproteins have been characterized as common constituents of insect haemolymph plasma: vitellogenins, which are generally femalespecific and will be dealt with later (Section V), and the sex-unspecific lipoproteins, which are specifically involved in the transport of DG from the fat body to sites of utilization. Latter lipoproteins have been identified in several insect species including moths, locusts and cockroaches (for reviews, see Wyatt and Pan; 361 Beenakkers e t al.; 2°'22"23 Van der Horst323). Methods employed for isolation and characterization of these lipoproteins have led to the adoption of rather confusing systems of nomenclature. Thus, Thomas and Gilbert 296'297 used the term high density lipoproteins (HDL) by analogy to mammalian serum proteins after preparative ultracentrifugation of these lipoproteins from H. cecropia and H. gloveri, whereas Chino et al. 65 used DG-carrying lipoprotein I (DGLP-I) for the equivalent lipoprotein in P. cynthia after isolation by DEAE cellulose column chromatography. Later, the designation of these lipoproteins was changed to lipoprotein I (LP-I), 123 the indications DGLP and HDL were, however, not abandoned. 63'z39 In the locust, the yellow colored DG-transporting lipoprotein has been referred to as Ayellow, a classification based on the sequence of elution of protein fractions separated by gel permeation chromatography, 222'33° although LP-I and DGLP have been used as we11.64.120 Since the sex-unspecific DG-carrying lipoprotein serves to transport cholesterol and hydrocarbons in addition to D G , 57'63'64Chino et al. 6° proposed the generic term 'lipophorin' (Lp) for these insect lipoproteins; however, as will be perceived later on, this does not abolish difficulties encountered in the indication of the lipoproteins on changes in density, lipid loading, or molecular weight which are occurring, for instance, during flight or development of insects. 2. General Characteristics of Lipophorins

The lipophorins are synthesized in the fat body, j2j'~45lipid loading of lipophorins in the haemolymph apparently occurs independently of de novo synthesis since inhibition of protein synthesis in vitro does not affect the release of DG nor the uptake of lipid by the lipophorins. 24°These lipophorins are considered to be a lipid shuttle, reutilized many times


41

TABLE4. Relative PercentageComposition of Lipid Classes in Lipophorins from Insects of Four Orders Cockroachb Silkworm b Honey-beea Locustb (Periplaneta (Phylosamia Lipid class (Apis mellifera) (Locusta migratoria) americana) cynthia) PL 31.3 36.1 42.8 25.8 MG 1.7 0.0 0.0 0.0 DG 32.4 32.8 15.2 56.3 TG 9.5 1.7 2.0 1.2 Cholesterol 14.7 7.8 5.0 13.2 Free fatty acids 5.4 Cholesterol esters trace 0.1 0.0 0.0 Hydrocarbons 4.8 21.1 28.3 1.4 Total 99.8 99.6 93.4 97.9 afrom Robbs et al. 252 bfrom Chino and Kitazawa.64 without synthesis of degradation 2°-22'96'123 and, therefore, lipophorins are unique lipoproteins different from mammalian plasma lipoproteins that appear to function only once. Compared to mammalian lipoproteins, the composition of lipophorins, although as yet limited in scope, is unusual in both lipid and apoprotein content. Lipids, which constitute 39-48% of the lipophorin mass, consist predominantly of D G and PL with some 5q5% of unesterified cholesterol, while amounts of T G are very low and cholesteryl esters are virtually absent (for reviews, see Gilbert et al.; t25 ChapmanS]). A few more recent data on the lipid composition of lipophorins from insects of four different orders, summarized in Table 4, show the same tendency although the high contents of hydrocarbons reported for locust and cockroach are remarkable. In addition, lipophorins generally contain two nonidentical and relatively large apoproteins, apoLP-I (molecular weight ~250,000 daltons) and apoLp-II (molecular weight ~ 80,000 daltons), as will be discussed below; the high protein content of these lipoproteins destines them to the class of high density lipoproteins (HDLp). In contrast, mammalian lipoproteins contain little D G and large amounts of T G and cholesteryl esters, and with the exception of apoB, molecular weight of which may be as high as 400,000 daltons, 2°3 their various apoproteins are relatively small polypeptides (molecular weight < 40,000). Insect lipophorins appear to be a high molecular weight macromolecules; molecular weights reported range from about 450,000 to 850,000, depending on species investigated but also on methods employed for isolation. Thus, by gel permeation chromatography, the molecular weight of locust lipophorin was found to be c a . 450,000130'330 or about 700,000, t2° whereas by sedimentation diffusion equilibrium analyses about 850,000 ~2° and ca. 580,00064 were determined. In the electron microscope, high density lipophorins examined thus far appear as globular shaped structures in the range 12-16 rim. 63~5'356 A recent electron micrograph of pure locust lipophorin isolated by gel permeation chromatography and visualized by modified negative staining techniques is shown in Fig. 13; the mean diameter of the well-defined lipophorin particles is ca. 17 nm. 321 The lipophorin apoproteins of two insect species depending on lipid as a fuel for flight have been studied extensively and will be considered in more detail. Lipophorin isolated from the larval tobacco hornworm, M . sexta, was shown to be a high density lipoprotein (d ,-~ 1.13 g ml 1)239,266of which the two apolipoproteins (molecular weights ~ 245,000 and ~78,000, respectively) were isolated and examined using immunological probes. 265 Although the marked similarity in amino acid composition of the apoproteins suggested possible homology, apoLp-I and apoLp-ll showed no immunological cross-reactivity. ApoLp-I and apoLp-II from the adult moth lipophorin were immunologically indistinguishable from their larval counterparts. Both apoproteins bind fluorescein-labeled concanavalin A (FITC-conA), indicating that they are both high mannose glycoproteins. 26~ For lipophorin (or Ayellow) of L. migratoria, some authors reported a single subunit of about molecular weight 85,000, whereas a larger component of approximately molecular weight 240,000 was considered to be an artificially aggregated product of the 85,000

42


FIG. 13. Electronmicrographsof lipophorins isolated from haemolymphof resting or AKH-injected locusts by gel permeation chromatography on Ultrogel AcA 22. Negativelystained preparations are shown of (A) O-lipoproteins, (B) O+-lipoproteins, (C) lipophorin (or Ayeuow),(D) A +-Iipoproteins. Scale bars represent 100 nm. From Van Antwerpen et al.32~ subunit. ~2°'~21 Indeed, after pulse-labeling of locust fat body & vitro using radioactive leucine, the early translation product of lipophorin appeared to be the 85,000 subunit, j2~ However, Chino and Kitazawa 64 also identified both a light-chain (85,000) and a heavy-chain (250,000) subunit in locust lipophorin, but demonstrated in electropherograms stained with the periodate-Schiff technique that, in contrast to the light-chain, heavy-chain subunit was associated with carbohydrates, rendering an unphysiological aggregation of light-chain subunits less likely. Studies in our laboratory on locust lipophorin show that both apoproteins separated in SDS-polyacrylamide gels bind FITC-conA, indicating that, like in Manduca, both are mannose-containing glycoproteins (T.K.F. Schulz, J. M. Van Doorn and D. J. Van der Horst, unpublished results). Immunological probes using monoclonal antibodies directed against apoLp-I and apoLpI1263 clearly demonstrate that the two apoproteins are not homologous. 262"327"332 3. Structural Organization o f Lipophor&s

As to the structural organization of lipophorin apoproteins, limited proteolytic degradation of larval M . sexta lipophorin by bovine trypsin showed that apoLP-I was extensively cleaved into large fragments while apoLp-II remained unaffected, suggesting that a large portion of apoLp-II may lie inside of the particle. 239 Similar results were


43

obtained for the lipophorin of the larval honey-bee, Apis mellifera, 252and for the lipophorin of both locust and cockroach. 177 Additionally, the lipophorin of the latter two species showed ready cross-linking of the apoproteins with dimethylsuberimidate, indicating that the apoproteins lie close to each other. 177Also, by immunological tests in which antiserum against apoLp-I or whole M. sexta lipophorin strongly precipitated lipophorin whereas antiserum against apoLp-II caused only minor precipitation, relatively greater exposure of apoLp-I to the aqueous environment was demonstrated. 265 However, enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies specific for locust lipophorin apoLp-I and apoLp-II, respectively, revealed that both apoLp-I and apoLp-II are exposed and, therefore, may reflect distinct recognition sites for target cells. 262'327'332Ryan et al. 26° showed cross-reactivity between Manduca anti-apoLp-II and apoLp-II of lipophorins from seven insect orders, whereas anti-Manduca sexta-apoLp-I failed to show antigenic cross-activity with the apolipophorins I of any species other than Manduca, suggesting that important structural features of apoLp-II must be conserved to maintain structural integrity of the lipophorin particle, which apparently does not apply to apoLp-I. Concerning the localization of the lipophorin lipids, it has been suggested that, due to the polar nature of the phospholipids and the 1,2-DG, they should occupy the surface of the particle, 239 which was substantiated by calculations based upon size and composition of the lipophorin from larval M. sexta. The presence of DG in the easily accessible shell of the lipophorins may allow their exchange without degradation of the rest of the particle, enabling the lipoprotein to act as a reusable lipid shuttle (see Gilbert and Chino; 123Van der Horst; 323 Downer and Chin096). Evidence for the localization of phospholipids at the surface of locust iipophorin was recently obtained by 3Jp N M R spectroscopy, ~79which was further supported by enzymatic studies using phospholipase A2. Localization of the other lipid constituents, particularly the sn-l,2-DG, awaits to be established. 4. Lipophorin Complexes

In the locust, in addition to lipophorin (or AyeHow),large complexes of lipoproteins (O-lipoproteins) have been found in chromatographic isolations of haemolymph proteins, 33° the function of which is undefined. Immunologically these O-lipoproteins are identical to lipophorin 329 and are not containing other haemolymph protein components, which is also reflected in their subunit composition obtained by SDS-polyacrylamide gel electrophoresis. 33~ In the electron microscope, O-lipoproteins also appear as complexes composed of discrete lipophorin particles 32~ (Fig. 13). The existence of the O-lipoproteins in vivo has been questioned 129,356and suggested to be an artifact of the isolation procedures. However, fresh haemolymph visualized in the electron microscope contains identical complexes. 321 Besides, the appearance of the voided amorphous material Wheeler et al. 356 obtained by°chromatographic fractionation of haemolymph after ammonium sulphate treatment is completely different from the O-lipoprotein complexes described above and indeed may represent denatured haemolymph protein components. Nevertheless, the physiological significance of the O-lipoproteins at present remains an open question. E. Lipoprotein Dynamics during Flight 1. Hormone-induced Lipophorin Rearrangements

Our present knowledge of changes in lipophorins resulting from DG loading during flight appears to be confined to the locust, although the lipophorin transformations concomitant with flight have recently been confirmed in a few other insect species. In the locust, it was shown that AKH-stimulated lipid mobilization induced by flight activity, or injection of the hormone (AKH I) into resting insects, results in significant shifts in the haemolymph protein pattern involving lipoprotein-protein interactions; a model for these lipoprotein rearrangements during AKH action has emerged particularly from the work of our laboratory and that of Goldsworthy and co-workers in Hull and has been reviewed

44


/AKH

/ 1 / FFA~ fat body

enemy glycerol: haemolymph

I flight muscle

FIG. [4. AKH-induced ]ipophorin transformations in the locust during flight. recently (Beenakkers et al.; 16"2e~23 Van der Horst; 323 Goldsworthy; 129 Goldsworthy and Wheeler132). A schematic representation is given in Fig. 14. Thus, the lipid mobilizing effect of AKH on the fat body is attended by the association of the lipophorin in the haemolymph with a nonlipid containing protein fraction (C-proteins) and the increased amount of DG released from the fat body to form a new, higher molecular weight lipoprotein particle with a lower density, A ÷ (cf. Mwangi and Goldsworthy;22z223 Van der Horst e t a1.329'33°'333). The locust A ÷ lipophorin is a low density lipoprotein (d ~ 1.01 g ml-J), ~29 molecular weight of which was estimated by gel permeation chromatography to be ca. 3,500,000330 although lower values (1.65-2.12 x 106) have been calculated as well. ~3° In the light of the nomenclature of lipoproteins discussed in Section IV.D, the high density lipophorin (or HDLp) is converted into a low density form (or LDLp). The model for lipophorin A ÷ formation has obtained convincing evidence by immunological data 329 and radiolabeling experiments. 333,354,35sIn vitro studies using fat body tissue and isolated haemolymph protein fractions have demonstrated that the lipid mobilizing effect of AKH and the subsequent formation of A ÷ only occurs when the incubation medium contains both lipophorin and the C-protein fraction. 335 Interestingly, the shortterm lipid mobilizing effect of octopamine did not induce lipophorin conversions in these in vitro studies. Recently, only one specific component of the C-protein fraction was shown to be actually involved in A + formation and is recovered in the subunit pattern of A ÷ in SDS-polyacrylamide gelsTM (Fig. 15). This C2-protein (molecular weight ~20,000) is a glycoprotein containing relatively large amounts (12.5%) of carbohydrate. The complex biantennary sugar chain has been analyzed; j in contrast to the two other apolipophorins in A + (originating from lipophorin), however, the C2-protein does not bind to concanavalin A (see Section IV.D). Goldsworthy e t a / . 13° have reported that, in addition to the major (molecular weight ~ 20,000) glycoprotein, a minor (molecular weight ~ 16,000) glycoprotein may contribute to A ÷ formation; both glycoproteins do not bind to concanavalin A. However, their data seem to be based on polyacrylamide gel electrophoresis and the detection of a minor lower molecular weight component might be explained by the microheterogeneity in the carbohydrate chain we have observed in our analyses. Recently, Haunerland et al. 146 demonstrated a glycosylated apoprotein (molecular weight ~20,000) containing mannose (2.1%) and glucosamine (1.2%) in the low density lipophorin from the grasshopper, Gastrimargus africanus, resulting from the administration of synthetic locust AKH I, which may be taken to indicate a similar system of lipophorin conversions in Orthoptera other than locusts. In contrast to the locust protein Cz (or apoLp-III), however, this glycoprotein stained with FITC-conA. In recent electronmicrographs of pure locust lipophorin A ÷ isolated by gel permeation chromatography, the A + lipoprotein appears as a homogenous fraction containing spherical particles with a diameter of 25-32 nm 32~ (Fig. 13), clearly lacking the possession of short projections or the aggregation into short chains by such processes as described by Wheeler et al. 356


45

94.000 67.000 ¸,

i

!

43.000

i

30.000 20.100 !

14.400

Ay

A+

C2

FIG. 15. Subunit patterns of lipophorin (or Ayellow), lipophorin A +, and C2-protein (or apoLp-llI) obtained by SDS-polyacrylamide gradient gel (5-20%) electrophoresis, electrophoretically transferred to nitrocellulose paper and stained for protein. Haemolymph protein fractions were isolated from resting locusts (Aye.ow,C2) or AKH-injected insects (A +) by gel permeation chromatography on Ultrogel AcA 22. From Van der Horst et al. 32s

The hormone-induced formation of lipophorin A + during flight activity resulting in a higher capacity for DG uptake and transport in the haemolymph is essential for the progressive delivery of lipid to the working flight muscles which is reflected in the increased turnover rate of the DG (cf. Van der Horst et al.; 33° Beenakkers et al.22'23). Particularly, the lipid-rich A ÷ lipoprotein is thought to be unloaded by the flight muscles (see Section IV.F); both lipophorin and protein C2 are recovered and may reload DG at the fat body site (see Fig. 14). Recently, in the adult sphinx moth, M. sexta, a similar model for AKH-induced lipophorin rearrangement has been reported by Law and his colleagues in Tucson, Arizona. Injection of locust synthetic AKH I into moths stimulates lipid loading of the lipophorin coincident with the association of an additional third apoprotein which was named apolipophorin-III (apoLp-III). 266The lipophorin decreases in density from 1.11 to 1.06 g ml -l, the high density resting form (HDLp) being converted to a low density lipophorin (LDLp), while no appreciable amounts of intermediate density particles are formed on administration of lower doses of the hormone. Due to the ultracentrifugal isolation technique used, no data on the molecular weight of the LDLp are available. Molecular weight of the apoLp-III (,-, 17,000) is comparable to that of locust C2-protein; however, in contrast to the glycoprotein of the locust, apoLp-III contains no carbohydrateJ s° The amino acid composition of Manduca apoLp-III is given in Table 5 along with that of locust C2-protein and that of a bug, and will be discussed below.

46

Ad M. Th. Beenakkers el al. TABLE5. Amino Acid Composition of ApoLp-III from Three Insect Species Representing Three Different Orders Locusta migra-

Munduca

Thasus

Amino acid toria a sexta b acutangulusc Asp/Asn 22 19 24 Thr 12 8 7 Ser 11 13 10 Glu/Gln 35 31 30 Pro 6 2 4 Gly 6 6 9 Ala 31 24 17 Val 8 10 15 Met 0 2 2 Ile 7 2 9 Leu 19 11 16 Tyr 1 1 2 Phe 2 8 9 His 7 4 4 Lys 6 22 29 Arg 2 2 4 Trp ND 0 0 Cys 0 0 0 Data derived from analyses of duplicate samples hydrolyzedfor 24, 48 and 72 hr in 6N HC1 in vacuo at ll0°C. Compositions are given in residues per molecular weight = 20,000 (Locusta), 17,000 (Manduca) and 20,000 (Thasus) apoLp-III; molecular weight of Locusta apoLp-III was corrected for 12.5% carbohydrate. ~After Van der Horst et al. TM bFrom Kawooya et al) 8° CFrom Wells eta/. 353

The interspecies activity of locust A K H I but also the apparent differences in composition of the decapeptide and the nonapeptide adipokinetic hormone isolated from M a n d u c a have been discussed above (see Section IV.C), and may explain why high doses of locust A K H (100-200 pmol) were required to elicit the lipoprotein conversions in the sphinx moth, 266 while in Locusta response to injection of A K H I is maximal at a dosage of only 2-4 pmol. m

2. Occurrence and A m i n o A c i d Composition o f Apolipophorin-III

Interestingly, in a study of lipophorins from adult insects representing seven orders, evidence for an apolipophorin-III was only found in M . sexta and in a hemipteran species, the leaffoot bug Leptoglossus zonatus, z6° suggesting that in addition to Orthoptera and Lepidoptera, hemipteran lipophorin may also undergo transitions similar to those described for L. migratoria and M . sexta, as much as suggesting the model of hormoneinduced lipophorin rearrangements to be a general feature for insects mobilizing lipid as an energy substrate for flight. However, the hemipteran, Thasus acutangulus, appeared to contain large amounts of a p o L p - I I I which, in contrast to both locust and sphinx moth, were associated with the lipophorin already in the resting situation, whereas injection of locust A K H had no effect on lipophorin density or apolipophorin content. 353 The amino acid composition of the pure isolated a p o L p - I I I which, like that of Manduca, was not glycosylated (molecular weight ~ 20,000) is listed in Table 5 along with that of M . sexta and pure L. migratoria protein C2 (or apoLp-III). There are obvious similarities in amino acid composition; all species lack cysteine whereas in addition M . sexta and T. acutangulus lack tryptophane which was not determined for the locust. Striking differences between the locust a p o L p - I I I and the other two are the relatively low levels of lysine and


47

phenylalanine residues and the large number of alanine residues in the locust. Possibly these differences relate to the glycosylation which is only found in the locust apoLp-III. 3. Function and Nature o f Lipophorin-Protein Interactions

In spite of the rapid advances in the field of insect lipophorin biochemistry and physiology, many problems concerning the A ÷ lipophorin (or LDLp) remain to be elucidated. For instance, it is not clear why protein C2 (or apoLp-III) associates with locust lipophorin during flight, although a few explanations have been advanced (see Van der Horst; 323 Beenakkers et al.Z2'23), including a role in the maintenance of the structural integrity of the lipophorin on DG loading, and a specific interaction of the incorporated apoLp-III with receptors on the flight muscles, possibly resulting in activation of the flight muscle lipoprotein lipase by analogy to activation of mammalian lipoprotein lipase requiring the presence of apoC-II (for reviews see Cryer; TM Sparrow and Gotto; 27° Quinn et al.; 247 Mahley et al.2°3). Immunological probes using native lipophorin A ÷ and monoclonal antibodies specific for its three apoprotein constituents indicated major localization of apoLp-III at the lipoprotein surface, which is compatible with an involvement in flight muscle cell membrane recognition and/or enzyme activation; 262'263'327'328however, ELISA with anti-apoLp-I and anti-apoLp-II showed that, despite the dramatic increase in lipid and apoLp-III contents, both apoLp-I and apoLp-II are still exposed in the A ÷ particle and thus may reflect recognition sites as well. Another intriguing problem is the nature of the association of the apoLp-III (or Cz) and lipophorin. In a study on the time course in lipophorin A ÷ formation in locust in vivo, Wheeler and Goldsworthy TM reported that lipophorin (or Ayeuow) does not contribute to A + until some 15-20 min after injection of AKH, whereas A ÷ is formed without delay as is the association of injected 3H-labeled protein fraction C with A +. This conclusion was confirmed recently) 3° However, from in vitro incubation studies of fat body tissue carried out in our laboratory 335 already discussed above, it appeared that no lipophorin A + was formed when the incubation medium contained either lipophorin or the C-protein fraction. Clearly, therefore, additional investigations will be needed to gain an understanding of the process of DG loading and apoLp-III association. Interestingly, the phenomenon of AKH-induced lipophorin transformations appears to be virtually restricted to the adult form of the insect, which, in view of the proposed function of 'loaded' lipophorin in flight metabolism is however not unexpected. In the last larval stage of the locust, the magnitude of the hyperlipaemic response to injected A K H remains substantially below the level reached in adults although also in larvae the mobilized lipid consists mainly of DG. 341The amount of lipophorin A ÷ formed is but very low. This incomplete response has been related to the inability of larvae to form lipophorin A ÷, which may be explained by both the limited ability of the larval fat body to respond to AKH and the very low levels of the C2-protein (or apoLp-III) in larval haemolymph.222,332.354 Also, in M. sexta, it was shown that the larval fat body does not respond to AKH although apoLp-III level which, in the last larval stage, is present in concentrations