www.elsevier.com/locate/jinsphys
Sulfakinins reduce food intake in the desert locust,
Schistocerca
gregaria
Zhu Wei
a, Geert Baggerman
a, Ronald J. Nachman
b, Graham Goldsworthy
c,
Peter Verhaert
a, Arnold De Loof
a, Liliane Schoofs
a,*aLaboratory of Developmental Physiology and Molecular Biology, K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium bVeterinary Entomology Research Unit, FAPRL, US Department of Agriculture, 2881 F&B Rd, College Station, TX 77845, USA
cBiology Department, Birkbeck College, Malet Street, London WC1E 7HX, UK
Received 16 August 1999; accepted 20 January 2000
Abstract
In vertebrates, the peptides cholecystokinin (CCK), neuropeptide Y, galanin, and bombesin are known to be involved in the control of food intake. We report here that insect sulfakinins, peptides which display substantial sequence similarities with the vertebrate gastrin/CCK peptide family, significantly inhibit food uptake in fifth instar nymphs of the locust,Schistocerca gregaria. Upon injection of Lom-sulfakinin, a neuropeptide present in the corpus cardiacum of locusts, food intake was significantly reduced in a dose-dependent manner within a fixed 20 min time period. The induced effect ranged from 13% inhibition (10 pmol of injected peptide) to over 50% inhibition at 1 nmol. Other naturally occurring sulfakinins from different insect species also elicited this satiety effect. Analogous to the satiety effect of CCK in vertebrates, the sulfate group is required for activity. No effect on the palptip resistance was found after injection with sulfakinin. Therefore it seems unlikly that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors.2000 Elsevier Science Ltd. All rights reserved.
Keywords: Schistocerca gregaria; Sulfakinin; CCK; Feeding; Neuropeptide
1. Introduction
Recent developments in insect physiology and endo-crinology reveal that the hormonal control of physiologi-cal and neuro-endocrinologiphysiologi-cal processes are as complex in insects as they are in higher vertebrates. As in ver-tebrates, neuropeptides appear to play pivotal roles in these processes. The control of food intake is a physio-logically complex behavioral system (Geiselman, 1996). During the past four decades, considerable progress has been made in understanding putative signals for hunger, satiation and satiety. These putative physiological con-trols of food intake include positive and negative sensory feedback, gastric and intestinal distension, effects of nutrients, nutrient reserves, and the release of peptides and hormones in the gastrointestinal tract or in the brain. The role of peptides in the regulation of food uptake
* Corresponding author. Fax:+32-16-32-39-02.
E-mail address:[email protected] (L. Schoofs).
0022-1910/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 0 ) 0 0 0 4 6 - 9
has been intensively studied in mammalian vertebrates. Neuropeptide Y and galanin are stimulators of appetite whereas bombesin and cholecystokinin (CCK) are sat-iety inducing peptides (Palmiter et al., 1998; Lee et al., 1994).
consequence of food intake is to stretch the foregut fol-lowed by contraction and relaxation of the intestinal muscles, it can be hypothesized that some myotropins may have an effect on the regulation of food intake.
One of the myotropic peptide families, the sulfakinins, displays sequence homology with the hormonally active portion of the vertebrate gastrin/CCK peptide family (Nachman et al., 1986). The intestinal myotropic action of the sulfakinins is also analogous to that of gastrin. In addition, sulfakinin peptides have been shown to stimu-late release of the digestive enzyme α-amylase in the red palm weevil,Rhynchophorus ferrugineus(Nachman et al., 1997a), analogous to the secretagogue effect of CCK in mammals. Sulfakinins have been identified in various insects including the cockroaches, Leucophaea maderae (Nachman et al. 1986a,b, 1988; Nachman et al., 1989a,b) and Periplaneta americana (Veenstra, 1989), the locust, Locusta migratoria (Schoofs et al., 1990) and the flies, Neobellieria bullata (Fonagy et al., 1992) andCalliphora vomitoria(Duve et al., 1995). The precursor gene encoding sulfakinin was sequenced in the fruit fly,Drosophila melanogaster(Nichols et al., 1988) and the blow fly, Calliphora vomitoria (Duve et al., 1995). In addition to the two major sulfakinins, i.e. Pea-SK, which is unblocked at the N-termius and Lem-SK-2, the corpora cardiaca/corpora allata complex of the american cockroch, P. americanaalso contains a modi-fied Pea-SK with O-methylated glutamic acid and rela-tively high amounts of the nonsulfated forms of these peptides (Predel et al., 1999)
We report here that, comparable to the satiety effect of CCK observed in mammals, insect sulfakinins sig-nificantly inhibit food intake in the locust, S. gregaria.
2. Materials and methods
2.1. Animals
S. gregaria andL. migratoria were raised under lab-oratory conditions. The animals wee kept crowded in cages containing a few hundred specimens. The tempera-ture was kept at 32°C and a light/dark cycle of 16/8 h was used. S. gregaria were fed once a day, in the morning, with fresh cabbage leafs and oatmeal (Veelaert et al., 1997). L. migratoria were reared on grass or cornleafs, depending on the availability, and oatmeal (Ashby, 1972).
2.2. Chemicals
Non-sulfated locustasulfakinin (non-S-Lom-SK or pQLASDDYGHMRFamide) (Schoofs et al., 1990), Lom-SK (pQLASDDY(SO3H)GHMRFamide), [Ser (SO3H)7]Lom-SK (pQLAS(SO3H)DDYGHMRFamide),
Perisulfakinin I (Pea-SK I or EQFDDY(SO3H)
GHMRFamide) (Nachman et al., 1986b), Leucosulfaki-nin (LSK or EQFEDY(SO3H)GHMRFamide) (Nachman et al., 1986b),Leptinotarsa Neuropeptide F (Led-NPF 1 or ARGPQLRLRFamide) (Spittaels et al., 1996) and Lom-AKH-I (pQLNFTPNWGTamide) (Stone et al., 1976) were synthesized via FMOC chemistry on a Milligen/BioSearch 9600 Peptide Synthesizer according to previously described procedures (Nachman et al., 1989a). The sulfated analogs were synthesized by dis-solving the pure non-sulfated sequence (100 nmol) in 100µl of sulfuric acid at 0°C for 25 min, followed by careful neutralization with ammonium hydroxide at 0°C, lyophilization of the neutral solution. Then, they were purified via reverse phase HPLC as previously described (Nachman et al., 1997b).
CCK-8 (DY(SO3H)MGWMDFamide),) and crus-tacean cardioactive peptide (CCAP or PFCNAFTGC amide) (Stangier et al., 1987) were purchased from Bachem (Switzerland).
2.3. Feeding assay
The antifeedant potency of insect peptides was meas-ured in 1-day-old male and female fifth instar nymphs of S. gregaria. The principle of the test consists of presenting to the animals a standardized amount of food during a fixed period of time. The group of animals (n=10 for each peptide) treated with a neuropeptide was then compared to a new control group (n=10) for each experiment. One-day-old fifth instar nymphs, weighing 0.6–0.9 g were kept separately in small plastic containers to standardize their state of hunger prior to the assay. This was achieved by depriving them of food overnight for 16 h. This was based on several periods of starvation (5, 12 and 16 h) we tested, from which a starvation over-night for 16 h gave the best results in terms of intra-experiment variation. The intra-experiments were done the next morning.
Prior to injection, the peptides were dissolved in locust saline (composition: 9.82 g/l NaCl; 0.48 g/l KCl; 0.19 g/l NaH2PO4; 0.25 g/l NaHCO3; 0.73 g/l Mg Cl2
and 0.32 g/l CaCl2). Individual locusts were then injected
calculated as the percentage of inhibition of food intake of each insect in comparison to the average eaten by the control group.
In the experiments with injection of corpora cardiaca extracts, the insects were allowed 40 min to eat. During these experiments the latency prior to food uptake was measured by recording the time from the moment the food was provided until the onset of food intake. Insects not eating during this period, from the test as well as from the control group, were not taken into account when analysing these data.
Since the experiments never took more than 40 min, any weight loss due to evaporation from the leafs proved to be negligible. The Student’st-test was used to analyze the difference between the control and the test groups.
2.4. Palptip assay
The measurement of electrical resistance across the cuticle was based on the technique of Scheie and Smyth (1968). Three-day-old fifth instar nymphs of L. migratoriawere used. Prior to injection the animals were starved for 5 h. Animals of the control group were injected with locust saline (NaCl 9.82 g/l, KCl 0.48 g/l, MgCl2·6H2O 0.73g/l, CaCl2·2H2O 0.32 g/l,
NaH2PO4·6H2O 0.19 g/l, NaHCO30.25 g/l) whereas the
experimental group received a peptide sample dissolved in saline. Each nymph was placed with its ventral surface up on a plasticene block, with the legs immobilized. The maxillary palps were fixed on the block alongside their heads with two small steel hooks. Measurements of the electrical resistance across the head and maxillary palps were accomplished by putting a capillary glass-electrode filled with electrolyte solution over each palp so that the distal one-third of the terminal segment of the palps was immersed in electrolyte. After measuring the resistance across the intact palps, the electrodes were temporarily raised, the domes of the palps were cut off and the resist-ance was measured again. Subtraction of the second value from the first gave the electrical resistance over both palp tips.
2.5. Corpora cardiaca extracts and HPLC separations
One hundred and fifty pairs of corpora cardiaca com-plexes from fifth instarS. gregaria nymphs were homo-genized in 5 ml of extraction methanol/water/acetic acid (90/9/1) solution. After sonication and centrifugation (15 min, 10,000 rpm, 5°C) the methanol was evaporated. The remaining aqueous solution was extracted with ethyl acetate and n-hexane to remove the lipids. The aqueous solution was dried and applied to Megabond Elute C18 cartridges (Varian). The peptides were eluted with 50% acetonitrile, containing 0.1% trifluoroacetic acid (TFA). After drying, the sample was ready to be redissolved in saline and to be injected.
Further fractionation was done on a Microsorb-MV C18 column (5 µm, 100 A˚ , 250×4.6 mm) in a GILSON HPLC system with a variable wavelength detector (214 nm). The conditions were: solvent A: 0.1% TFA in milli-Q water; solvent B: 50% CH3CN in 0.1% aqueous TFA.
Column conditions were: 100% A for 10 min, followed by a linear gradient to 100% B in 30 min; flow rate: 1 ml/min; detector range: 2.0 AUFS (absorbance units full scale). The fractions were screened for in vitro myotropic activity (5 equivalents) on an isolated cock-roach hindgut as described by Holman et al. (1991) and for antifeeding activity (injection of 10 eq/nymph). We ran synthetic Lom-SK (0.5 µg) using the same con-ditions.
3. Results
Fig. 1 shows that 0.1 equivalent of crude extracts of corpara cardiaca (CC) caused 38% inhibition of food intake in locust fifth instar nymphs. In addition, a pro-nounced increase in the latency to feed was observed after injection of the CC-extract (Fig. 2). Among the set of peptides investigated (CCAP, Led-NPF 1, Lom-AKH 1, LSK, Lom-SK and Pea-SK), all of them originating from insect brain–corpus cardiacum complexes (Veelaert et al., 1998), only the sulfakinins reduced food uptake significantly in fifth instar nymphs (Fig. 1). The response of the sulfakinins was dose-dependent, with a threshold concentration of about 1 pmol/locust, coresponding to a concentration of ±1028 M in the hemolymph (Fig. 3).
The most effective dose (1 nmol, ±1025 M in the
hemlymph) decreased the amount of food intake by 55%. The Student’st-test analysis yielded a significance level of 1024%. All the other insect sulfakinins tested
(all having the C-terminal DY(SO3H)GHMRFamide
sequence in common) showed similar dose–response curves. However, the sulfakinins did not elicit an increased latency to feed, as seen in nymphs injected with extracts of CC.
Non-sulfated Lom-SK elicited no significant effect on food uptake even when injected at high doses (up to 10 nmol/insect). Injection of 1 nmol of [Ser(SO3H)7
]Lom-SK, with the sulfate group on the serine residue instead of on the tyrosine residue, was, however, less effective than injection of the same amount of the naturally occur-ring Lom-SK containing a sulfated tyrosine residue.
CCK, a vertebrate peptide that displays a high degree of sequence similarities to peptides of the insect sulfaki-nin family, did not reduce food intake. Sulfakisulfaki-nin had no effect on palptip resistance of fifth instar L. migratoria
nymphs (data not shown).
Fig. 1. Effect of different injections on food intake in locusts. Peptides were injected at 1nmol/insect. Each test group consists of at least 10 animals. Vertical bars indicate standard deviation; *, low significance level (α=5–1%); **, highly significant (α,1%).
Fig. 2. Delay before the onset of food intake in locusts after different injections. Each test group consists of at least 10 locusts. Vertical bars indicate standard deviation; *, low significance level (α=5–1%); **, highly significant (α,1%).
data indicate that the corpora cardiaca of fifth instar nymphs ofS. gregariacontain a peptide identical or very similar to Lom-SK, which was so far only identified in brain–CC complexes of L. migratoria.
4. Discussion
The present data demonstrate clearly for the first time that food uptake in locust fifth instar nymphs is influ-enced by neuropeptides of the sulfakinin family. Fifth instar L. miratoria nymphs have a typical feeding pat-tern, which consists of 5–15 min meals, interrupted by periods of approx. 1 h, in which the animals do not eat (Blaney et al., 1973). The meal size is approximately constant during the whole nymphal stage, although the periods in between feeding behavior become shorter in the middle of the nymphal stage. Crude extracts of corpora cardiaca fromS. gregarianymphs elicit a potent inhibition of food-intake in starved locusts of the same species. As the inhibitory activity co-eluted with syn-thetic Lom-SK, the active peptide in the corpora cardiaca ofS. gregarianymphs is likely to be identical or at least very similar to Lom-SK.
The fact that non-sulfated Lom-SK has no effect on food uptake suggests that the feeding inhibition effect of the sulfakinins is a specific response. The sulfated tyro-sine is also essential for the myotropic activity of the sulfakinins (Nachman et al., 1986b). Injection of [Ser(SO3H)7]Lom-SK is active but less effective than
naturally occurring tyrosine sulfated Lom-SK. The dis-tance between the acidic sulfate group and the peptide backbone is shorter for Ser(SO3H) than for Tyr(SO3H)
Fig. 3. Effect of different insect sulfakinins on food intake. Each test group consists of at least 10 animals. Vertical bars indicate standard deviations; *, low significance level (α=5–1%); **, highly significant (α,1%).
the sulfakinins requires a Tyr(SO3H) moiety.
Replace-ment of Tyr(SO3H) with Ser(SO3H) leads to an even
larger drop in the myotropic activity of the sulfakinins on the cockroach hindgut bioassay (Nachman et al., 1989a).
Relatively high amounts of peptide are needed to get a substantial decrease in food intake. This could be due to enzymatic breakdown of the peptide in the hemo-lymph. Degradation in the hemolymph has, so far, only been studied for a very limited number of neuropeptides. Schoofs et al. (1998) have studied the degradation of 10 schistostatins in the hemolymph of S. gregaria. The avarage half life of these peptides is about 60 min. Neb-TOMF, a peptide with free N- and C-terminus has an even shorter halflife (3 min) (unpublished data). One of the proteases involved in peptide degradation in insects is angiotensin-converting enzyme as demonstrated by Isaac et al. (1998).
The mode of action of sulfakinins in inhibiting food intake is not yet known. As insects have an open circu-latory system, hormones can be transported directly to all of the organs. Therefore, several sites of sulfakinin action are possible. It remains to be determined whether endogenous sulfakinin is active and whether it has a cen-tral action on the brain feeding system or a peripheral hormonal action or both. Lom-SK may activate the stretch receptors in the foregut, which inhibit food uptake through the posterior pharyngeal nerves (Bernays and Chapman, 1973). It is also possible that Lom-SK is
involved in a similar feed-back mechanism that is induced by signaling molecules from the abdominal gan-glion acting on the hindgut (Simpson, 1983).
It seems unlikely that sulfakinins reduce food intake by decreasing the sensitivity of the taste receptors on the maxillae as we found no effect on palptip resistance by injection with sulfakinin in L. migratoria. The chemo-sensory sensillae on the domes of the maxillary palps are predominantly contact chemoreceptors and are thought to mediate taste perception. Each of these gusta-tory sensillae comprises a short peg bearing an apical pore that opens and closes according to the nutritional status of the locust: the pores are open in starved locusts but closed after feeding (see Blaney and Simmonds, 1990). Bernays et al. (1972) showed that changes in the electrical resistance across the tips of the maxillary palps correlate with these opening and closing movements. The electrical resistance across the tips of the maxillary palps is higher after feeding (Bernays et al., 1972) and after injection of extracts of CC (Bernays and Mordue, 1973) or Locusta-diuretic peptide (Coast and Goldswor-thy, 1997). Therefore, other factors, such as the diuretic peptide, are likely to be involved in the control of food uptake. This is also in agreement with our findings that corpora cardiaca extracts not only reduced the meal size, as the sulfakinin peptides do, but also significantly increased the latency to feed.
Cholecystokinin-8, which differs from Lom-SK in its C-terminal pentamer sequence by Asp instead of Arg in the 2nd position from the C-terminus and by Trp instead of His in the 4th position from the C-terminus, does not reduce food uptake in the locust nymphs. Similarly, while CCK is active on intestinal smooth muscle of ver-tebrates, it is unable to alter the spontaneous contractions of the insect hindgut. However, replacement of a single residue, Arg for Asp, transforms the inactive mammalian hormones gastrin II and CCK into active analogs on the cockroach hindgut bioassay (Nachman et al., 1988).
Analogous to the physiological responses observed with the gastrin/CCK family in mammals, the sulfakin-ins induce satiety, stimulate contractions of gut muscles and stimulateα-amylase release in insects. This provides further evidence for the hypothesis that the structurally similar gastrin/CCK and sulfakinin peptides have a long evolutionary origin, and hence an important physiologi-cal function. Prior to the discovery of CCK-analogous biological activities for the sulfakinins in insects, other authors have suggested that this might be an example of convergent rather than divergent evolution (Dockray, 1989; Duve et al., 1995). Our results indicate that sulfa-kinin neuropeptides elicit satiety in locusts, like their vertebrate CCK homologues in mammals. It therefore appears that if gastrin/CCK-like peptides and insect sul-fakinins have the same peptide ancestor, which is likely in view of their homologous structures, they have main-tained very similar physiological roles throughout evol-ution.
Acknowledgements
The authors thank the European Community (FAIR-CT-96-1648) and the F.W.O. (G.0264.96) for financial support.
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