RECENT ADVANCES INARTHROPOD ENDOCRINOLOGY

Edited by

i Geoffrev M. Coast

\ O"partmmt of Biology, Bírkbeck College,

) University of London, London, UK

Simon G. Webster

, r School of Biological Sciences, University of'!,/ Wales Bangor, Bangor Gwynedd, UK

CaunmDGEUN TERSITY PRESS 1998

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WIL J. A. VAN MARREWIJKANd DICK J. VAN DER HORST

Signal transduction of adipokinetichormone

Introduction

Flight activity of insects provides an attractive model system to study regula-tion of metabolic processes during physical exercise. Several long-distanceflying insects mobilise both carbohydrate and lipid reserves as fuels formigratory flight from the fat body, which combines many of the propertiesand functions of vertebrate liver and adipose tissue, and plays a fundamentalrole in energy metabolism. This chapter focuses on current knowledge on afew aspects of metabolic processes in the fat body, employing particularly themigratory locust, Locusta migratorin, as an insect model. The mobilisationof energy substrates from the fat body is controlled by adipokinetic hor-mones (AKHs), peptide neurohormones which upon flight are released bythe corpus cardiacum, a neuroendocrine gland connected with the brain.Recent data on the involvement of the distinct AKHs identified in locustcorpora cardiaca in flight-related processes is discussed, with special empha-sis on their role in substrate mobilisation. However, the major focus of thischapter is on the rnechanism through which the AKH signal is transducedintracellularly. The structural features of the fat body, which is capable offunctioning in vitro, render this metabolically highly active tissue a suitablemodel for studies on signal perception and transduction in an intact animalsystem. Therefore data on AKH signal transduction presented in thischapter result primarily from studies performed on fat body in vitro.

Adipokinetic hormones and substrate mobilisation

Insect flight activity stimulates the adipokinetic cells in the glandular lobes ofthe corpus cardiacum to release the adipokinetic hormones. These are N-and C-terminal blocked small peptides which are structurally related; inglandular lobes of the migratory locust, a decapeptide AKH-I is present inaddition to two octapeptides (AKH-II and III), the molar proportions ofAKH-I:AKH-II:AKH-III being approximately 14:2:1 (Oudejans er a/.,1993). A1l three AKHs are synthesised as preprohormones; recent data using

AKH signal transductíon 173

in situhybridisation showed the mRNA signals encoding the three differentAKH precursors to be colocalised in cell bodies of the glandular lobes ofthe corpus cardiacum (Bogerd et a1.,1995). Additionally, expression of thegenes for all three AKH precursors is stimulated byflight activity. These datasuggest that all three AKHs are involved in flight-related processes. A physio-logically relevant question therefore is why these three related hormonesoccur simultaneously.

The action of the hormones on the insect fat body results ultimately in themobilisation of stored reserves as fuels for flight. Carbohydrate (trehalose) ismobilised from glycogen reserves, implying hormonal activation, byphosphorylation, of the key enzyme glycogen phosphorylase. Similarly, lipid(sn-l,2-diacylglycerol, DAG) is mobilised from stored triacylglycerol, whichimplies hormonal activation of the fat body triacylglycerol lipase (for areview, see Beenakkers, Van der Horst & Van Marrewijk, 1985). The carbo-hydrate and lipid substrates are transported in the haemolymph to theworking flight muscles. Carbohydrate provides most of the energy for theinitial period of flight; thereafter, in long-term flying insects like locusts, theconcentration of lipid substrate in the blood is increased and gradually takesove! so the principal substrate for long-term flight is lipid. Although glyco-gen phosphorylase in the locust fat body is converted into its phosphorylatedactive form within a few minutes of flight activity, it takes almost half anhour before newly synthesised trehalose is released from the fat body(Beenakkers et a1.,1985). A possible explanation for this lag period betweenthe phosphorylation of glycogen phosphorylase and the release of newlysynthesised trehalose may be that the allosteric enzyme is kept in its inactiveT (tense) state as long as the concentration of trehalose is high, and willswitch to its active R (relaxed) state only after a substantial reduction of tre-halose levels in the fat body, as may occur during flight activity. A similarmechanism has been described for the inhibition of phosphorylase a byglucose in vertebrate liver (Johnson,1992).

In a bioassay, all three AKHs are able to activate both glycogen phospho-rylase and triacylglycerol lipase, although there may be differences inpotency, as shown in Fig. 1. Recent preliminary results obtained withcombinations of two or three AKHs, which are likely to occur together ininsect haemolymph under physiological conditions in vivo, revealed that themaximal responses for both the carbohydrate- and the lipid-mobilisingeffects were much lower than the theoretically calculated responses that werebased on dose-response curves for the individual hormones. In the lower(probably physiological) range, however, combinations of the AKHs weremore effective or at least as effective as the theoretical values calculated fromthe responses to the individual honnones (Oudejans et a1.,1992).In additionto their different potencies in eliciting substrate mobilisation, all three AKHs

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174 W. J. A. VAN MARREWIJK AND D. J. VAN DER HORST

Dose injected (Pmol)

Fig. l. Dose-response curves for the lipid mobilising effects of AKH-I, IIand III in L. migraÍorua. Adult male locusts were injected with differentdoses of AKH or, in the controls, with saline, and after 120 min the lipidcontent in haemolymph was determined. Responses represent increase inhaemolymph lipid in AKH-injected locusts, expressed as g l-r. For clarity,data points and standard error bars have been omitted. EDro, effective doseeliciting 50% of the maximum response (after Oudej ans et a1.,7992).

inhibit RNA synthesis in the locust fat body in rilro (Kodrík & Goldsworthy,1995). In males, the responses were dose-dependent, with their potenciesdecreasing in the order AKH-III>AKH-II>AKH-I. All three AKHs wereequally efficacious. These data may point to the occurrence of differentreceptors for the AKHs or, alternatively, the observed differences ineffectiveness of the three AKHs may be due to differences in the mechanismof hormonal signal transduction. However, a decisive factor for the ability toelicit physiological responses is the relative abundance of each of the hor-mones, which is determined by their rates of synthesis and release, and bytheir rate of inactivation. Studies of hormone dynamics in intact locustsinjected with physiological amounts of tritiated AKH (l pmol) have demon-strated for all three AKHs a decrease in total radioactivity (non-degradedhormone plus radioactive degradation products) with time, both at rest andduring flight (Fig. 2A). The rate of decrease was quite different for each ofthe hormones (AKH-III>>>AKH-II>AKH-I), and was faster duringflight than at rest (Oudejans et al., 1996). Although the first step in the

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176 w. J. A. vAN MARREwTJK AND D. J. vAN DER HoRsr

inactivation of the hormones occurs by an endopeptidase cleaving the N-terminal tripeptide pGlu-Leu-Asn which they have in common, their half-lives were diferent and were changed by flight activity, as estimated fromsemiJogarithmic plots of remaining AKH in haemolymph versus time (Fig.2B): in resting locusts, halflives were 51, 40 and 5 min for AKH-I, II and III,respectively, whereas during flight values of 35,37 and 3 min were obtained.This rapid and differential degradation of the three AKHs will lead tochanges from the proportions in which they were released, and therefore hasimportant consequences for concerted hormone action at the level of thetarget organ receptor(s), suggesting that each of the AKHs may play its ownbiological role in the overall syndrome of locust flight.

Hormonal signal transduction in insect fat body

Role of extracellular calcium in phosphorylase stimulationby AKH

As to the mechanism of transduction of the three AKH signals - which addi-tionally might provide some possible clues as to why in L. migratoria threedifferent AKHs occur - the activation of glycogen phosphorylase was moni-tored in fat body in vitro, which provides a convenient and very sensitive testsystem. Stimulation of fat body phosphorylase by all three AKHs is depen-dent on the presence of extracellular Caz+ (Vroemen et al., 1995a). At lowconcentrations of calcium ions in the medium (<0.2 mmol l-1) no significantphosphorylase activation by AKH occurs. A concentration of >1.5 mmolCaz+ L-t is required for maximal activation by each of the three hormones,which is consistent with the physiological levels of Ca2* normally present inthe extracellular fluid of eukaryotes (l-2 mmol t-r;. In the absence of AKH,Ca2+ in the medium does not affect the enzyme activity. The presence ofCa2+ was also shown to be indispensable for the induction of both fat bodyphosphorylase activation and the synthesis and release of trehaiose in thecockroach Periplaneta amerícana by corpus cardiacum extract or hyper-trehalosaemic peptides (Steele & Paul, 1985; Orr et a1.,1985), and recentlythe presence of extracellular Ca2+ was found to be necessary for a fullstimulation of fat body phosphorylase by hypertrehalosaemic hormone inthe cockroach Blaberus discoidalis (Park & Keeley, 1995, 1996). An obviousway for Caz* to exert its role in phosphorylase activation is by direct stimula-tion of phosphorylase kinase, which converts inactive phosphorylase intothe active form. Ca2+-calmodulin-dependent phosphorylase kinase activityhas been demonstrated in cell-free preparations ftom Locusta fat body (VanMarrewijk, Van den Broek & Beenakkers, 1991), and stimulation of phos-phorylase kinase by Caz+ has also been shown in several other insects.

AKH signal transduction 177

including the fruitfly Drosophila melanogaster (Dombrádi et a1.,1987) and P.

am e r i c ana (Pallen & Steele, I 9 8 8). Howev er, a Ca2 + -independent phosphory-lase kinase also occurs in insect fat body, as has been demonstrated forPhylosamia cynthia (Hayakawa & Chino, 1983).

A Caz+ dependence similar to that demonstrated for hormone-inducedcarbohydrate mobilisation appears to exist for the effect of AKH on themobilisation of lipids, as both Lum & Chino (1990) and Wang, Hayakawa &Downer (1990) have demonstrated that AKH, which is known to stimulatethe production of DAG from triacylglycerol stores in the locust fat body,requires the presence of extracellular Caz+ to evoke an increase in intra-cellular levels of DAG. Again, Ca2+ alone in the absence of AKH did not sig-nificantly change the fat body DAG content. These studies on the Ca2+dependence of hormonally controlled lipid mobilisation were conductedexclusively with AKH-I. However, an assessment of the Ca2+ dependence ofthe DAG production induced by AKH-II and AKH-III will also be impor-tant in providing some insight into why three dift'erent AKHs occur in locusts.

AKH-induced cyclic AMP production and calciumdependence

All three AKHs of L. migratoria are capable of elevating intracellularlevels of cyclic AMP in the fat body in vitro (Yroemen, Van Marrewijk &Van der Horst, 1995b). This elevation appeared to be very rapid; a signifi-cant increase was measured within 15 s of adding AKH to the medium,whereas maximal levels of cyclic AMP (200% increase compared to thecontrol level) were reached after 1 min (Fig. 3). Dose-response studies haveshown that at 40 nmol l-r the three AKHs are equally potent in enhancingintracellular cyclic AMP, this massive dose probably eliciting a maximalresponse. However, at a more physiological level (4 nmol l-1), their poten-cies in elevating cyclic AMP levels were different, decreasing in the orderAKH-III>AKH-II>AKH-I. Correspondingly, at this concentrationAKH-III was also the most potent in inducing activation of glycogen phos-phorylase, aithough results obtained with AKH-I and AKH-II did notdiffer significantly.

While the hormonally induced activation of phosphorylase describedabove was dependent on the presence of extracellular calcium ions, theability of the AKHs to raise levels of cyclic AMP in the fat body appeared tobe equally Ca2*-dependent: when Ca2+ (normally present at 2 mmol 1-1) waseliminated from the incubation medium, none of the peptides was able toelevate cyclic AMP levels (Vroemen et al.,l995b). For AKH-I, the require-ment of extracellular Ca2* to increase fat body cyclic AMP was shown byWang et al-, (1990). Altogether, the above data on DAG levels, activation of

178 W. J. À. VAN MARREWIJK AND D. J. VAN DER HORST

incubation time (min)

Fig. 3. Time course for the effect of a saturating dose of AKH on intra-celluiar cyclic AMP levels in fat body of L. migratona. Fat body was incu-bated for the times given with 40 nmol AKH-I (A), II (B) or III (C) 1-1, andsubsequently cyclic AMP and protein contents were determined inhomogenates. For each time point, n:6 (after Vroemen et a1.,1995b).

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glycogen phosphorylase and increase in cyclic AMP levels provide convinc-ing evidence that extracellular calcium ions are necessary for the AKHs toexert their effects on the fat body.

Regulation of the influx and eflux of calcium

The activating effect of AKH on phosphorylase can be mimicked by thecalcium ionophore A23187 (Van Marrewijk et al.,l99l). Like AKH, thiscompound also increases the levels of cyclic AMP and DAG in locust fatbody tissue incubated in vitro (Wang et a1.,1990). Although responses to theionophore were smaller than those elicited by AKH, these results suggestthat at least part of the action of AKH consists of enabling extracellularcalcium ions to enter fat body cells, leading to enhanced concentrations ofintracellular Ca2+. Support for this suggestion was obtainedby Wang et al.(1990), who measured a 2.5-fold increase in the uptake of 45Ca2+ from themedium into the fat body upon incubation in the presence of 100 nmolAKH-I l-1 for I h. However, results from this study are not conclusive,because the hormone may also stimulate Ca2* effiux (see below). The effectsof 40 nmol AKH-I, II and III l-r on the uptake of 4sca2+ from the mediumwere examined during the first 5 min after peptide addition because, in thecase of a role for extracellular calcium in signal transduction, effects on Ca2*fluxes may be expected to occur shortly after applying the hormone (VanMarrewijk, Van den Broek & Van der Horst, 1993; Vroemen et al.,l995a).All three AKHs significantly increased the uptake of Ca2* from the medium,the effect being evident within I min. No significant differences between theeffects of the three AKHs were observed, even when AKH concentrationswere reduced to 4 nmol l-1.

In addition to stimulating the influx of extracellular Caz+ into the fatbody, all three hormones enhance the effiux of Caz+ from the fat body, pos-sibly as a result of the increased levels of intracellular Ca2+ (Van Marrewijket a1.,1993; Vroemen et al.,l995a). Fat body tissue was pre-loaded to equi-librium with a5Ca2+ and the radioiabel released into the medium in theabsence or presence of AKH-I, II or III was measured. At a saturating doseof 40 nmol l-1, and assuming that during the first 15 s of incubation're-influx' of 45Ca2+ released into the medium was negligible, 50oÁ of the pre-loaded 4sca2+ was estimated to have been extruded from the fat body withinabout I min of incubation in the presence of an AKH. This indicates aneflux rate that seems to be appropriate for a rapid second-messenger system.The maximal Ca2+ effiux evoked by saturating doses of AKH-I, II and IIIdid not differ. However, at a more physiological level (4 nmol l-1), a small butinteresting difference in potency was observed; the AKH-induced increase inCa2* eflux decreased in the order AKH-III>AKH-II>AKH-I (Fig. 4), as

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Fig. 4. Effect of a physiological dose of AKH on the effux of radioactivecalcium from L. migratoria fat body in vitro. Fat body pre-loaded witha5Ca2* was incubated with 4 nmol AKH-I, II or III 1-r, and radioactivityreleased into the medium was determined at several time intervals. Thefigure shows the percentage of preJoaded radioactivity that has beenreleased after 5 min of incubation. For each bar n=12. The effect inducedby AKH-III was significantly higher than that by AKH-I (P<0.05) (afterVroemen et al.,1995a).

was the case for stimulation of cyclic AMP production and phosphorylaseactivation. From the time courses of both the influx and the effiux of Ca2* itcould be calculated that the influx exceeds the effiux, resulting in an increasein the intracellular concentration of Ca2+. This fits the above observationsthat the AKH-induced formation of cyclic AMP and DAG in the fat body,and the activation of glycogen phosphorylase are dependent on extracellularCa2+. Furthermore, recently Lee & Goldsworthy (1995) described a methodfor the preparation of metabolically active and AKH-sensitive dispersedcells from I,o custa fat body, and preliminary data from fluorimetric measure-ments in these cells indicate that the AKHs cause rapid increases (<10 s) inintracellular Ca2* (Lee, Hyde & Goldsworthy, 1995). Enhancement of intra-cellular Ca2+ levels is not necessarily the sole function of extracellular Caz+,as they may also be required for the binding of the AKHs to their receptor(s)on fat body membranes as recently demonstrated for Manduca sexta AK}l(Ziegler, Jasensky & Morimoto, 1995). However, results from recent bindingstudies with Locusta AKHs have not yet revealed evidence of Ca2+-depen-dent binding.

AKH signal transduction 181

Mobilisation of calcium from intracellular stores:involvement of inositol phosphates

In addition to the influx of extracellular C**, the mobilisation of Ca2* fromintracellular stores appears to be important for the effects of AKH on the fatbody. For instance, the addition of TMB-8, an inhibitor of intracellular Caz+release, to a Ca2+-containing medium led to a significant dose-dependentreduction of the phosphorylase activating action of AKH-I (Van Marrewijket al., L993). From this it can be concluded that for a maximal effect of AKHon phosphorylase activity in locust fat body, release of intracellular Ca2* isrequired in addition to the availability of extracellular Caz+.lntracellularCa2+ release is also a significant step in the hypertrehalosaemic hormonesignal transduction cascade for trehalose biosynthesis in the fat body of ,8.

discoidalís (Keeley & Hesson, 1995). Inclusion in the incubation medium ofdantrolene, which inhibits the release of intracellular Ca2+, partially sup-pressed the stimulating effect of the hypertrehalosaemic hormone on tre-halose synthesis in the fat body in vitro.

In the regulation of Caz+ mobilisation from intracellular stores, inositolphosphates have been shown to play a pivotal role in a variety of cell types(Berridge, 1989,1994; Berridge & Irvine, 1989). In the hormone-stimulatedinositol lipid cycle, binding of the agonist to its cell surface receptor leads tothe activation of G-protein-coupled phospholipase C and, as a result, theconversion of the membrane phospholipid phosphatidylinositol 4,5-bispho-sphate into two potential second messengers: sn-1,2-DAG, an activator ofprotein kinase C, and myo-inositol 1,4,5-trisphosphate (IPr), whose role inthe mobilisation of Ca2+ from intracellular stores has been well established(Berridge & Irvine, 1989). IP3 might be expected to have a role in the AKHsignal transduction mechanism because, in locusts, AKH has been shown toenhance intracellular Ca2* levels in the fat body. In Locusta fat body in vitro,the formation of [3]Ilinositol phosphates from phosphoinositides pre-labelled wÍh myo-l3Hlinositol indicates the functioning of the inositol lipidcycle. Phosphoinositide turnover was stimulated by AKH-I, as evidenced bythe increased production of [3H]inositol phosphates within I min of addingthe hormone to the incubation medium (Van Marrewljk et al., 1992).Preliminary data have demonstrated that, in LocusÍa fat body, IP, is amongthe inositol phosphates that respond to AKH-I with a strong increase inconcentration. Possible effects of AKH-II and III on fat body IP, levelsremain to be established, and may provide clues as to whether the threeAKHs share the same signalling pathways and/or receptor(s). Furthermore,in Schistocerca gregaria, both AKH-I and II were shown to significantlystimulate IP, formation in the fat body (Pancholiet a1.,1991; Stagg & Candy,1995). These data indicate that the action of AKH on the insect fat body may

I82 w. J. A. vAN MARREwIJK AND D. J. vAN DER HoRsr

involve mobilisation of intracellular Ca2+ by IPr. This role for IP, inhormone-induced mobilisation of Ca2* from intracellular stores may be

common among insects, as in ^8. discoidalis fat body hypertrehalosaemichormone has been demonstrated to induce intracellular Ca2* release as wellas to greatly increase IP, levels (Park & Keeley, 1996).

Capacitative calcium entry

Agonist-induced Ca2+ influx into cells has been proposed to depend on adirect activation of plasma membrane Ca2* channels by IP, or inositol1,3,4,5-phosphate (IP) (Kuno & Gardner, i987; Irvine 1991). However,several recent studies (Takemura et al., 1989; Bird et al., 1992; Bird &Putney, 1993) using the sesquiterpene lactone tumour promoter thapsigar-gin, which depletes Ca2+ from IPr-sensitive pools by inhibition of micro-somal Ca2*-ATPase activity without affecting inositol phosphate levels,

have lent considerable support in favour of a mechanism termed capacita-tive calcium entry. According to this concept, the state of filling of the intra-cellular Ca2+ stores is considered as the decisive factor that regulates theinflux of extracellular Ca2*. Thus, depletion of the intracellular Ca2+ stores

would, in some way, activate the mechanism of Ca2+ influx across theplasma membrane (for a recent review, see Berridge,1995). To test whetherthis mechanism applies also to insects, thapsigargin was added to fat bodyincubated in vitro, and its effect was assessed by measuring the influx of4sca2+ (Van Marrewljk et a1.,1993). The presence of thapsigargin led to asignificant increase in 4scaz+ uptake, implying that in the locust fat bodydepletion of intracellular Caz+ stores leads to an enhanced influx of extra-celluiar Ca2+. Furthermore, thapsigargin induced a marked activation ofglycogen phosphorylase only in the presence of extracellular Ca2+, which isconsistent with the view that depletion of intracellular Caz+ stores leads to astimulation of the Ca2+-entry mechanism across the plasma membrane ofinsect fat body cells. However, capacitative calcium entry does not excludethe possibility that other mechanisms are involved in regulating the influx ofextracellular Ca2+, such as a direct activation of Ca2+ channels by AKH.Moreover, the relative importance of extracellular versus intracellular Ca2+

for the signal transduction process may vary among insects. In studies ontrehalose synthesis by the fat body of B. discoidalzi, both thimerosal, whichsensitises IP, receptors of the endoplasmic reticulum for Ca2+ release(Bootman, Taylor & Berridge, 1992), and thapsigargin were shown toproduce signiflcant hypertrehalosaemic responses, whereas the ionophoresA23187 and ionomycin did not significantly increase trehalose synthesis(Keeley & Hesson, 1995). Omission of Ca2* from the incubation mediumdid not lower the stimulation of trehalose synthesis by thimerosal, nor did

AKH sígnal transduction 183

A23187-induced Ca2* influx increase the thimerosal response. For thehypertrehalosaemic responsg the presence of extracellular Caz+ was impor-tant only if intracellular Ca?+ was depleted. It was suggested that for trans-ducing the message of the hypertrehalosaemic hormone in Blaberus fatbody, intracellular Ca2+ release is more important than extracellular Ca2+

entry. This relatively low effectiveness of extracellular Ca2* influx in B. dis-coidalis contrasts with the situation in locust fat body where, as describedabove, extracellular Ca2* is essential for the AKH-induced mobilisation ofboth carbohydrate and lipid.

Interactions between the second-messenger systems

At least a major part of the action of AKH may involve depletion of intra-cellular Ca2+ stores, because in locust fat body the three AKHs produce anincreased uptake of radioactive Caz+ from the medium that is similar to theCa2+ uptake caused by thapsigargin. A likely mechanism would be thatAKH increases the concentration of IP' which induces the emptying ofintracellular Ca2+ stores and this, in turn, in some as yet unknown way,stimulates the influx of extracellular Caz*. This extracellular Ca2+ influx isapparently required for the AKH-induced production of cyciic AMP, pos-sibly by activating a Ca2+-dependent adenylyl cyclase; the cyclic nucleotidesubsequently causes actjvation of glycogen phosphorylase. However, experiments in which cyclic AMP levels in the fat body were increased either byactivation of the catalytic subunit of the adenylyl cyclase complex withforskolin or by inhibition of cyclic nucleotide phosphodiesterase with theo-phylline, both in the presence and in the absence of Caz* in the incubationmedium, have demonstrated that the activating effect on phosphorylase bycyclic AMP itself is independent of extracellular Caz* ffan Marrewljk et al.,1993). Compared with the phosphorylase activation induced by AKH, theeffects caused by the Ca2+ ionophore A23I87, as well as by enhanced intra-cellular levels of cyclic AMP produced by forskolin or theophylline are onlypartial. Therefore it still remains to be established whether the AKH-inducedactivation of phosphorylase occurs exclusively via the serial effects of C**and cyclic AMP, respectively, or whether there is additionally a parallel effectof Ca2+, leading directly to phosphorylase activation independent of cyclicAMP.

Orr et al. (1985) showed that cyclic AMP is not always necessarilyinvolved in Ca2+-dependent hormonal activation of insect fat body phos-phorylase. In fat body of P. qmericana incubated in a Ca2+-containingmedium, two potent hypertrehalosaemic factors from the corpus cardiacumstimulated activation of phosphorylase and increased production of tre-halose without any associated elevation of cyclic AMP leveis, indicating that

184 w. J. A. vAN MARREwIJK AND D. J. vAN DER HoRsr

the effects of these factors were not mediated by the cyclic nucleotide. In theabsence of external Ca2*, neither phosphorylase activation nor stimulationof trehalose production by the hlpertrehalosaemic factors occurred.

Involvement of G-protein-coupled receptors in AKHsignalling

The AKH-induced increases in the levels of cyclic AMP and IP, in the locustfat body described above point to a hormonal activation of both adenylylcyclase and phospholipase C. The activation of these enzymes by many hor-mones that bind to cell surface receptors has been shown to occur via G-protein-coupled receptors, which may also mediate the action of AKHs.Within the group of G-proteins, G, and G, have been the most extensivelystudied; the G, a-subunit stimulates adenylyl cyclase (and activates Ca2*channels), whereas the Q a-subunit inhibits adenylyl cyclase (and activatesphospholipase C, phospholipase A, and K+ channels) (for reviews, see

Hepler & Gilman, 1992; Gilman, 1995). Several bacterial toxins are generallyused to study the role of these G-proteins in signal transduction: choleratoxin (CTX) which irreversibly activates G, by preventing GTPase activity ofthe a-subunit via ADP-ribosylation of an arginine residue, and pertussistoxin (PTX), which irreversibly inhibits Q by ADP-ribosylation of the a-subunit at a cysteine residue, thus stabilising the GDP form of the G-protein.

To examine a possible involvement of G,- and G,-proteins in the AKHsignal transduction mechanism, the effects of CTX and PTX on the produc-tion of cyclic AMP and the activation of glycogen phosphorylase were deter-mined in fat body in vitro upon addition of the toxins to the incubationmedium (Vroemen et al., 1995b). PTX had no effect on either process,whereas CTX caused a significant elevation of intracellular cyclic AMPlevels and also stimulated the activation of phosphorylase. These resultssuggest that for AKH, transduction of the signal in the fat body occursthrough G,-protein-coupled receptor(s). CTX, when applied together withAKH-I, II or III, increased the stimulatory effects of all three AKHs on bothcyclic AMP levels and phosphorylase activation, but the individual effects ofCTX and AKH were not additive. This suggests that CTX and AKH act viaa common pathway involving the G.-protein. Again, PTX had no effect onAKH activity. Additional evidence for the involvement of G-proteins in thesignal transduction of AHK-I, II and III was obtained from experimentswith GDPBS, which is a competitive inhibitor of G-protein activation byGTP. When fat body cells, permeabilised with streptolysin O and pre-incubated in the presence of GDPBS, were incubated with AKH-I, II or III,phosphorylase activation by all three hormones appeared to be stronglyreduced. The involvement of G-proteins (such as Go-o or G,-Fy) in the

AKH signal transduction

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185

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Fig. 5. Proposed modei for the coupiing of AKH signalling pathways in alocust fat body cell. R, receptor; G, G protein; PLC, phospholipase C; IP'inositol 1,4,5-trisphosphate; AC, adenylyl cyclase; Gph, glycogen phospho-

rylase.

AKH-induced activation of phospholipase C in locust fat body is currentlyunder investigation. Figure 5 shows a proposed model for the AKH signaltransduction pathways in locust fat body, based on the various data dis-cussed in this chapter.

References

Beenakkers, A. M. Th., Van der Horst, D. J. & Van Marrewijk, W: J. A.(1985). Biochemical processes directed to flight muscle metabolism. InComprehensive Insect Physíology, Biochemístry and Pharmacology, ed.G. A. Kerkut & L. J. Gilbert, Vol. 10, pp. 451-486. Oxford: PergamonPress.

Berridge, M. J. (1989). Inositol trisphosphate and diacylglycerol: two inter-acting second messengers. Annual Review of Bíochemistry 56,159-193.

Berridge, M. J. (1994). The biology and medicine of calcium signaliing.M o le cular and C e llul ar Endo cr ino I o gy 98, 1 19 -124.

Berridge, M" i. (1995). Capacitative calcium entry. Biochemical Journal3l2,l-11.

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