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Release of identified adipokinetic hormones during flight and following neural stimulation in Locusta migratoria
0022-1910/83/050425-05$03.00/O 0 1983 Perganm Prrss Ltd
J. Insect Physiol. Vol. 29. No. 5, pp. 425 to 429. 1983 Printed in Greut Brituin.
RELEASE OF IDENTIFIED ADIPOKINETIC HORMONES DURING FLIGHT AND FOLLOWING NEURAL STIMULATION IN LOCUSTA MZGRATORlA IAN ORCHARD* and ANGELA B. LAiiGEt *Department of Zoology, tDepartment
University of Toronto, Toronto, Ontario, Canada M5S lA1:
of Biology.
York University,
Downsview.
Ontario,
Canada
M3J lP3
(Received 4 October 1982: revised 2 December 1982) Abstract-Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci 11 (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R, value similar to Carlsen’s peptide (adipokinetic hormone II). These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta. and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in
ligated locusts. It is concluded that NCC II controls adipokinetic hormones acts as a hyperglycaemic
the release of adipokinetic hormones during flight and that two are released during flight. One of these hormones adipokinetic hormone II also hormone illustrating that a hyperglycaemic hormone is released, during flight.
Key Word /nde?c: Adipokinetic
hormone,
release, flight, neural
INTRODUCIION THE MOBILIZATIONof diglycerides induced by an adipokinetic hormone during flight in the locust is well established (BEENAKKERS,1969; MAYER and CANDY, 1969: CHEESEMANand GOLDSWORTHY, 1979), and a hormone (adipokinetic hormone I) derived from the glandular lobe has been characterized, sequenced and synthesized (STONE et al., 1976; BROOMFIELD and HARDY, 1977). CHEESEMANet al. (1976) demonstrated the presence of an adipokinetic hormone in the haemolymph of flown locusts, and also measured the kinetics of hormone release during flight (CHEESEMANand GOLDSWORTHY,1979). However, the interpretation of their data has now been complicated by the recent finding of a second peptide with adipokinetic activity in the glandular lobe (CARLSEN et al., 1979). This second peptide (adipokinetic hormone 11) was characterized and isolated by CARLSEN et al. (1979) and shown to be different in amino acid composition to hormone 1. LOUGHTONand ORCHARD (1981) subsequently showed that adipokinetic hormone 11 also possessed hyperglycaemic activity. Since it has been suggested that a hyperglycaemic hormone is also required during flight to mobilise trehalose (VAN DER HORST et al., 1978) it is possible that adipokinetic hormone 11 may be released during flight to serve this purpose and also is responsible in part, for the elevation in haemolymph lipid. The activity attributed to adipokinetic hormone 1 by CHEESEMAN et al. (1976) and CHEESEMAN and GOLDSWORTHY(1979) was eluted from Sephadex LH 20 and may have been contaminated by hormone 11
I P.?9!5
Locusta
(see CARLSEN et al., 1979). Indeed some of the elution profiles shown by CHEESEMANet al. (1976) do suggest
two peaks of hyperlipaemic activity. It is possible, therefore, that both adipokinetic hormones 1 and 11 are released during flight. Similarly, ORCHARD and LQUGHTON (1981a,b) described the release of hyperlipaemic hormone from the glandular lobe of Locusta following stimulation of the nervi corporis cardiaci II (NCC II). However it is known that this nerve controls the release of three hormones associated with lipid levels, namely hypolipaemic hormone (ORCHARD and LOUGHTON. 1980), adipokinetic hormone 11 (LOUGHTON and ORCHARD. 1981) and presumably adipokinetic hormone I (ORCHARD and LQUGHTON, 1981a,b). Thus, the bioassay of perfusates from NCC II-stimulated preparations, in the absence of any purification, is indeed complex and results from the combined effects of these three hormones. In an attempt to resolve the complexities of hormone release we looked for a simple procedure to distinguish between these hormones, and have made use of the separation of adipokinetic hormones I and 11 on thin layer chromatography (TLC) (CARLSEN et al., 1979) and on our own observation of the R, of hypolipaemic hormone. In this report we show that factors co-migrating on TLC with both adipokinetic hormone 1 and with the RF of adipokinetic hormone 11 are released during flight and that NCC 11 release factors co-migrating with adipokinetic hormone I. the RF of hormone 11 and hypolipaemic hormone.
425 -I>
stimulation,
IAN OR~HAKUand ANGELAB. LANGE
426
MATERIALS
AND METHODS
Adults of Locusru ~nigraforiu reared under crowded conditions were used throughout the investigation. All experimental locusts were used 17 hr after being provided with fresh grass.
Corpora cardiaca were dissected from adult male locusts, 10 days post-fledging, and washed for 30 min in locust Ringer (SPENCER and CANDY, 1976). An NCC II was drawn into a suction electrode and stimulated at 5 Hz as previously described (ORCHARD and LOUGHTON, 198la). The compound action potential was recorded from the glandular lobe. The perfusates from control 30-min incubations and from stimulated 30-min incubations were collected, evaporated to dryness and resuspended in 50;tl methanol. The undissolved salts were removed by centrifugation and the supernatant applied to TLC. Flight
Adult virgin females, 13-15 days post-fledging were either rested for 30min (unflown) or flown in groups of four on a flight wheel. 500 ~tl of haemolymph were then collected from groups of ten locusts (SO &locust) and pipetted immediately into 5 ml of methanol. The samples were sonicated and centrifuged. The supernatant was left for 30 min at - 20’ C to precipitate lipid and carbohydrate (CHEESEMANet d., 1976). After centrifugation the supernatant was evaporated to dryness, resuspended in methanol and centrifuged. The supernatant was again evaporated to dryness and then taken up in 50 PI methanol. The supernatant was applied to TLC.
TLC was performed on precoated Silica Gel TLC plates with fluorescent indicator (Eastman 13 I8 I ). Samples were streaked at the origin and parallel lanes run with standards of synthetic adipokinetic hormone I and synthetic red pigment concentrating hormone (RPCH) (Peninsula Laboratories). Chromatograms were visualized by either U.V. absorbance or by spraying with Ehrlich’s reagent (IO?;, (w/v) 4-dimethylamine-benzaldehyde in concentrated HCI, mixed with acetone (I :4 v/v) prior to use). The solvent were S- 1: isopropanol-water-acetic systems used acid (25: IO: I by vol); S-2: ethyl acetate-ethanolwater-pyridine-acetic acid (60:30:8:2: 1 by ~01); S-3: acid acetate-ethanol-water-pyridineacetic ethyl (60:30:8:0.5: I by vol). Areas were scraped off the TLC and extracted in 80 ~(1Ringers. After centrifugation the supernatant was assayed for hyperlipaemic or hyperglycaemic activity. Hyperlipurmic~
(ROE, 1955). The rest of the supernatant
was discarded and the pellet dissolved in 0.2 ml isopropanol. The lipid content of the sample was determined by the method of FLETCHER(1968). Blood volume Blood volume was determined according to the method of LCWGHTON and TOBE (1969) using [’ 4C]-carboxyl inulin. RESULTS
Stimulution
qf NCC
II
An initial test revealed that the perfusate from control corpora cardiaca possessed minimal hyperlipaemic activity. The perfusate from corpora cardiaca in which NCC II had been stimulated possessed a significant amount of hyperlipaemic activity, so confirming previous findings (ORCHARD and LOUGHTON, 198la). No areas of activity were found when the control perfusate was chromatogrammed. Chromatography of perfusates from stimulated corpora cardiaca showed two areas of hyperlipaemic activity and one area of hypolipaemic activity (Fig. 1). In all three solvent systems a major peak of activity co-migrated
c Solvent 2
urd hypergl_vcaemic uchiry
70 /tl
samples were injected into IO-l2-day-old adult males through the membrane at the base of the hind leg, following CO, anaesthesia. For hyperglycaemic activity these males had been ligated at the neck for 3 hr (LOLICHTONand ORCHARD, I98 I ). One hour after injection. 5 jtl of haemolymph were collected through the neck membrane and precipitated in 50 ~1 of 10:;; trichloracetic acid. After centrifugation 5 ~1 of supernatant was removed for carbohydrate assay
Fig. 1
Release of adipokinetic ‘0
with synthetic adipokinetic hormone I. The second peak migrated in solvent I and 2 with the same R, relative to synthetic adipokinetic hormone I and RPCH as reported by CARLSEN et al. (1979) for adipokinetic hormone II. Solvent 3 was devised to further separate these two peaks and revealed that they were indeed discrete. Rechromatography of the hormone II peak from solvent 2 using the solvent 3 system revealed that the second peak co-migrated with an RF value similar to adipokinetic hormone 11. This second peak of hyperlipaemic activity also possessed hyperglycaemic activity (Fig. 2). Examination of the hyperlipaemic activities revealed that similar amounts of adipokinetic hormone I and II were released (assuming equipotency).
sohWlt3 r
427
hormones
T
0
Flight
Fig. 1. Adipokinetic activity recovered following chromatographic separation of perfusates of the NCC II-stimulated preparations. Saline-injected controls also shown. Synthetic adipokinetic hormone (AKH I) and red pigment concentrating hormone (RPCH) were run as standards in parallel lanes. All assays performed on day-10 adult male Locustn. mean k S.E. of four locusts. Solvent I. perfusate from two glands stimulated for 30 min each. Solvent 2 and 3, one gland stimulated for 30min. 0. origin, F. front. RF values for AKH I. RPCH and second peak of adipokinetic activity are. Solvent 1 : 0.70, 0.77, 0.82: Solvent 2: 0.17. 0.26. 0.32: Solvent 3: 0.30. 0.43. 0.56.
Since solvent 3 provided better separation of the two peaks of activity this solvent was used to analyse the haemolymph from flown and unflown locusts. Figure 3 shows that a large amount of hyperlipaemic activity, distributed between two areas on TLC, was present in the haemobmph of flown locusts, but comparatively little (though’ A measurable amount) was found in the unflown locusts. The two peaks of activity again co-migrated with adipokinetic hormones I and II. The area corresponding to hormone II also possessed hyperglycaemic activity (Fig. 2) while that of hormone I did not. These changes in hormone level were due to the release of hormone and not due to any concentrating effect of the haemolymph since blood volume of day-l 7 females did not change significantly during a 30-min flight (resting blood volume 765 + 90,& JZ= 8; flown blood volume 812 * 71 /d, II = 8). The increases in lipid brought about by the bioassay of adipokinetic hormone I were approximately 2.5 LLg/$ for unflown and 16.2 /(g/p1 for flown. Similarly for hormone II they were 1.5 pg/ptl and 6.6 pg/pl. Thus. the adipokinetic hormones I and II released and present at the end of a 30-min flight have adipokinetic activities of 13.7 pg/$ and 5.1 fig//A, respectively. This represents the bioassay of approximately 125 ~1 haemolymph. Day-13-15 female locusts had a blood volume of 376.3 k 40.5 ~1 (n = 8) and so the adipokinetic activities corrected as if hormone from 376 /Alof haemolymph were bioassayed in adult males represent 41.1 pg/ld and 15.3 pg/gl for adipokinetic hormones I and II. The relative contributions of adipokinetic hormones I and II to final lipid elevation are therefore approximately 73 and 27”~. respectively.
DISCUSSION
saline
a
saline 13 C
Fig. 2. Hyperglycaemic activity of fractions from TLC (a) AKH II fraction from perfusate following NCC II stimulation, one gland stimulated for 30 min. (b) AKH II fraction from haemolymph of flown Locusta. (c) AKH I fraction from haemolymph of flown Locusta. Saline-injected controls also shown. Mean k S.E. of three locusts.
The results clearly show that 30min of flight induces the release of two hormones with adipokinetic activity in Locusta. CHEESEMANet al. (1976) have previously shown adipokinetic activity to be present in the haemolymph of flown Locllsta. They found that this activity, obtained from a methanolic extract of haemolymph, was recovered at the same elution volume from Sephadex LH20 as the adipokinetic activity of a purified extract of corpora cardiaca. No other fractions containing adipokinetic activity were found in the haemolymph. In our studies, we have
IAN ORCHARDand ANGELA13.LANC;t:
FLOWN
UNFLOWN
Fig. 3. Adipokinetlc actlvlty recovered following chromatographic separation of haemolymph from unflown and flown Loccc.vtcz.Methanol extract of 5OOh11haemolymph from day-13-15 adult virgin females were fractionated on TLC in solvent 3. Lanes were extracted in 8Olil saline. and 2O-pI samples bioassayed using day-10 adult males. 0, origin. F, front. Synthetic AKH 1 and RPCH run as standards in parallel lanes. except for AKH
Data
represents
mean
f
SE.
of two replicate
I and AKH II which are for three replicates.
AKH I, RPCH and second peak of adipokinetic
been able to show that the adipokinetic activity in the haemolymph can be separated into two regions on TLC which possess the same RF values as synthetic adipokinetic hormone I (STONE et al.. 1976: BROOMFIELDand HARDY, 1977) and adipokinetic hormone II (CARLSEN et a/., 1979). It seems reasonable to conclude that these two active regions are indeed adipokinetic hormones I and 11. Blood volume remained constant during a 30-min flight and so these changes in hormone titre are brought about by release of hormone and not due to any concentrating effect due to lower blood volume. This confirms the previous data of JUTSUM and GOLDSWORTHY (1976) who showed blood volume remained constant during a 60-min flight of day-21 adult male Locusta. CHEESEMANand GOLDSWORTHY( 1979) assessed the release of adipokinetic hormone during flight in Locusta by testing pooled fractions containing possible adipokinetic activity from Sephadex LH20. These fractions may have emerged over a large volume (CHEESEMANer ~1.. 1976) and in the absence of clear data as to which fractions were pooled, could have included hormone II as well as hormone I (see CARLSENut uI., 1979). However. regarding the relative contributions of
activity
experiments assayed on six locusts, assayed on nine locusts. R, values for are: 0.30, 0.43 and 0.56, respectively.
adipokinetic hormones I and II in raising haemolymph lipid in our bioassay, we find hormone I possesses about 737: of the total activity and hormone II 27% (this makes the assumption that both hormones can express themselves fully in the presence of each other). Adipokinetic hormone I is, therefore, the predominant hyperlipaemic hormone and in view of the reported equal potencies of hormones I and II (CARLSEN et al., 1979), adipokinetic hormone I must be present in greater quantities after 30 min of flight. Adipokinetic hormone II also possesses hyperglycaemic activity (LOUGHTONand ORCHARD, 1981, also this study). Thus there is now clear evidence for the release of a hyperglycaemic hormone during locust flight. This extends the observations of VAN DER HORST et al. (1978) who showed that in the initial periods of flight, consumption of haemolymph trehalose was high with little mobilisation of trehalose from body stores. However, after 30min of flight a steady state was reached in which utilisation of trehalose was matched by mobilisation. Adipokinetic hormone II would appear to be responsible for maintenance of this steady condition. Both hormones are present in the haemolymph of unflown locusts. Similar levels of adipokinetic activity
429
Release of adipokinetic hormones were reported by CHEESEMAN and GOLDSWORTHY (1979). Whether this represents a true ‘resting’ level is hard to ascertain. These hormones may be used for maintenance of homeostasis, although CHEESEMAN and GOLDSWORTHY (1979) found no change in adipokinetic titre during starvation in locusts even though lipid levels rise significantly. It would also seem unlikely that adipokinetic hormone II was responsible for this rise during starvation since lipid levels still rise in locusts devoid of their glandular lobes (JUTSUM et al., 1975). NCC II stimulation results in the release of three hormones controlling haemolymph lipid levels; namely hypolipaemic hormone, adipokinetic hormones I and 11. This confirms the more indirect evidence of ORCHARD and LOUGHTON (1980, 1981a, b) and LOUGHTON and ORCHARD (198 1).We have shown previously that the release of hormone following NCC II stimulation is blocked by sodium-free saline and by tetrodotoxin (ORCHARD and LOUGHTON, 198la). Thus, release is due to the presence of a compound action potential and not an artefact of stimulation. Hypolipaemic hormone is stored and released from the storage lobes of the corpora cardiaca, adipokinetic hormones I and II originate within the glandular lobe and the control by NCC II is via aminergic synapses. Both hormones I and II would appear to be controlled by aminergic input since no adipokinetic activity was released following treatment with r-aminergic antagonists or by prior treatment with reserpine (ORCHARD and LOUGHTON. 1981b). However. since equal amounts of adipokinetic hormones I and II appear to be released by stimulating the NCC II, and different amounts appear to be present in the haemolymph after a 30-min flight, it would seem that the pre-synaptic axons in the NCC II are different (assuming equal potencies for adipokinetic hormones I and II and equal clearing rates from haemolymph). Thus hormone I and II may be released independently of one another. The simple procedure for separating these hormones now provides a useful tool for examining in a more quantitative manner the precise titres of both adipokinetic hormones I and II during flight, and the neural control of the release of hormones by the NCC 11.
/Ick,lowledyeme,lto-This work was supported by the Natural Sciences and Engineering Research Council of Canada. We are most grateful to Professor B. G. LOUGHTONfor much help and encouragement, and for supplying .I I me locusts.
REFERENCES BEENAKKERS A. M. Th. (1969) The influence of corpus allatum and corpus cardiacum on lipid metabolism in Locu.sta migratoria. Gen. camp. Endocr. 13, Abstract 12. BROOMFIELD C. E. and HARDYP. M. (1977) The synthesis of locust adipokinetic hormone. Tetruhedron Lett. 25. 2201-2204. CARLSENJ., HERMANW. S., CHRISTENSEN M. and JOSEFSK)N L. f1979) Characterisation of a second peptide with adi. . pokinetic and red pigment-concentrating activity from ihe locust corpora c&iaca. Insect. Biochem. 9,497 501. CHEESEMAN P. and GOLDSWORTHY G. J. (1979) The release of adipokinetic hormone during Right and starvation in Locusta.
Gen. camp. Eudocr.
37. 35-43.
CHEESEMAN P., JUTSUMA. R. and GOLDSWORTHY G. J. (1976) Quantitative studies on the release of locust adipokinetic hormone. Phvsiol. Ent. 1. 115-121. FLETCHERM. J. (1968) A’colorimetric method for estimating serum triglycerides. CICka chim. Acta 22. 393.-397. JUT&M A. R. and GOLDSWORTHY G. J. (1976) Fuels for flight in Locusra. J. Insecr. Pl7,vsiol. 22, 243-249. JUTSUMA. R., ARGARWALH. C. and GOLDSWOIYT~IY (i. J. (1975) Starvation and haemolymph lipids in Loc,u.sf~ migratoria (R & Ft. Acrida 4, 47-56. LOUGHTON B. G. and ORCHARDI. (198 1)The nature of the hyperglycaemic factor from the glandular lobe of the corpus cardiacum of Locusta migrutorru. J. /mast. P/IV Go/.
27, 383-385.
LOUGHTONB. G. and TORES. S. (1969) Blood volume m the African migratory locust. CUJI. ./. Zoo/. 47. 1333-1336. MAYERR. J. and CANDYD. J. (1969) Changes in energy reserves during flight of the desert locust, Sch~.stocerc~lr gregaria. Comp. Biochem. Plzysiol. 31, 409-41X. ORCHARDI. and LOUGHTONB. G. (1980) A hypolipacmic factor from the corpus cardiacum of locusts. Nurwc. Land. 286, 494496.
ORCHARD1. and LOUCHTONB. G. (1981a) The neural control of release of hyperlipaemic hormone from the corpus cardiacum of Locusta migratoria. Cor77p. Bitrc ht7. Pl7KGOl. 68A, 25530. ORCHARDI. and LOUGHTONB. G. (1981b) Is octopamlnc a transmitter mediating hormone release in insect>‘? J. Nrrrrohiol. 12, 143-153. ROF J. H. (1955) The determination of sugar in the blood and
spinal
fluid with anthrone
reagent.
1. hioi.
C~7~w1.
212, 335-343. SPENCERI. M. and CANDYD. J. (1976) Hormonal control of diacyl glycerol mobilisation from fat body of the desert locust. Scllistocrrca gregaria. Ir~setr B~oc~l~m 6.
289-296. STONEJ. V., MORDUEW.. BAILEYK. E. and MORRISH. R. (1976) Structure of locust adipokinetic hormone. a neurohormone that regulates lipid utilisation during flight. Nature. LorId. 263. 207-2 I I. VAN DERHORSTD. J.. VAN D~ORN J. M. and BE~NA~~KI RS A. M. Th. (1978) Dynamics in the haemolymph trehalose pool during flight of the locust. Locusta migrntoria. Insect Biocl7ur17. 8, 4 13-416.
J. Insect Physiol. Vol. 29. No. 5, pp. 425 to 429. 1983 Printed in Greut Brituin.
RELEASE OF IDENTIFIED ADIPOKINETIC HORMONES DURING FLIGHT AND FOLLOWING NEURAL STIMULATION IN LOCUSTA MZGRATORlA IAN ORCHARD* and ANGELA B. LAiiGEt *Department of Zoology, tDepartment
University of Toronto, Toronto, Ontario, Canada M5S lA1:
of Biology.
York University,
Downsview.
Ontario,
Canada
M3J lP3
(Received 4 October 1982: revised 2 December 1982) Abstract-Fractionation of methanol extracts of perfusate and haemolymph on thin-layer chromatography was used to separate hormones associated with haemolymph lipid regulation in Locusta. Electrical stimulation of the nervi corporis cardiaci 11 (NCC II) of isolated corpora cardiaca resulted in the release of three hormones into the perfusate; hypolipaemic hormone and two adipokinetic hormones. The two adipokinetic hormones co-migrated with synthetic adipokinetic hormone (adipokinetic hormone I) and with the R, value similar to Carlsen’s peptide (adipokinetic hormone II). These two adipokinetic hormones were also present in small amounts in the haemolymph of unflown Locusta. and shown to be released during a 30-min flight. The adipokinetic hormone II fraction from the NCC II-stimulated perfusate and haemolymph also possessed hyperglycaemic activity when assayed in
ligated locusts. It is concluded that NCC II controls adipokinetic hormones acts as a hyperglycaemic
the release of adipokinetic hormones during flight and that two are released during flight. One of these hormones adipokinetic hormone II also hormone illustrating that a hyperglycaemic hormone is released, during flight.
Key Word /nde?c: Adipokinetic
hormone,
release, flight, neural
INTRODUCIION THE MOBILIZATIONof diglycerides induced by an adipokinetic hormone during flight in the locust is well established (BEENAKKERS,1969; MAYER and CANDY, 1969: CHEESEMANand GOLDSWORTHY, 1979), and a hormone (adipokinetic hormone I) derived from the glandular lobe has been characterized, sequenced and synthesized (STONE et al., 1976; BROOMFIELD and HARDY, 1977). CHEESEMANet al. (1976) demonstrated the presence of an adipokinetic hormone in the haemolymph of flown locusts, and also measured the kinetics of hormone release during flight (CHEESEMANand GOLDSWORTHY,1979). However, the interpretation of their data has now been complicated by the recent finding of a second peptide with adipokinetic activity in the glandular lobe (CARLSEN et al., 1979). This second peptide (adipokinetic hormone 11) was characterized and isolated by CARLSEN et al. (1979) and shown to be different in amino acid composition to hormone 1. LOUGHTONand ORCHARD (1981) subsequently showed that adipokinetic hormone 11 also possessed hyperglycaemic activity. Since it has been suggested that a hyperglycaemic hormone is also required during flight to mobilise trehalose (VAN DER HORST et al., 1978) it is possible that adipokinetic hormone 11 may be released during flight to serve this purpose and also is responsible in part, for the elevation in haemolymph lipid. The activity attributed to adipokinetic hormone 1 by CHEESEMAN et al. (1976) and CHEESEMAN and GOLDSWORTHY(1979) was eluted from Sephadex LH 20 and may have been contaminated by hormone 11
I P.?9!5
Locusta
(see CARLSEN et al., 1979). Indeed some of the elution profiles shown by CHEESEMANet al. (1976) do suggest
two peaks of hyperlipaemic activity. It is possible, therefore, that both adipokinetic hormones 1 and 11 are released during flight. Similarly, ORCHARD and LQUGHTON (1981a,b) described the release of hyperlipaemic hormone from the glandular lobe of Locusta following stimulation of the nervi corporis cardiaci II (NCC II). However it is known that this nerve controls the release of three hormones associated with lipid levels, namely hypolipaemic hormone (ORCHARD and LOUGHTON. 1980), adipokinetic hormone 11 (LOUGHTON and ORCHARD. 1981) and presumably adipokinetic hormone I (ORCHARD and LQUGHTON, 1981a,b). Thus, the bioassay of perfusates from NCC II-stimulated preparations, in the absence of any purification, is indeed complex and results from the combined effects of these three hormones. In an attempt to resolve the complexities of hormone release we looked for a simple procedure to distinguish between these hormones, and have made use of the separation of adipokinetic hormones I and 11 on thin layer chromatography (TLC) (CARLSEN et al., 1979) and on our own observation of the R, of hypolipaemic hormone. In this report we show that factors co-migrating on TLC with both adipokinetic hormone 1 and with the RF of adipokinetic hormone 11 are released during flight and that NCC 11 release factors co-migrating with adipokinetic hormone I. the RF of hormone 11 and hypolipaemic hormone.
425 -I>
stimulation,
IAN OR~HAKUand ANGELAB. LANGE
426
MATERIALS
AND METHODS
Adults of Locusru ~nigraforiu reared under crowded conditions were used throughout the investigation. All experimental locusts were used 17 hr after being provided with fresh grass.
Corpora cardiaca were dissected from adult male locusts, 10 days post-fledging, and washed for 30 min in locust Ringer (SPENCER and CANDY, 1976). An NCC II was drawn into a suction electrode and stimulated at 5 Hz as previously described (ORCHARD and LOUGHTON, 198la). The compound action potential was recorded from the glandular lobe. The perfusates from control 30-min incubations and from stimulated 30-min incubations were collected, evaporated to dryness and resuspended in 50;tl methanol. The undissolved salts were removed by centrifugation and the supernatant applied to TLC. Flight
Adult virgin females, 13-15 days post-fledging were either rested for 30min (unflown) or flown in groups of four on a flight wheel. 500 ~tl of haemolymph were then collected from groups of ten locusts (SO &locust) and pipetted immediately into 5 ml of methanol. The samples were sonicated and centrifuged. The supernatant was left for 30 min at - 20’ C to precipitate lipid and carbohydrate (CHEESEMANet d., 1976). After centrifugation the supernatant was evaporated to dryness, resuspended in methanol and centrifuged. The supernatant was again evaporated to dryness and then taken up in 50 PI methanol. The supernatant was applied to TLC.
TLC was performed on precoated Silica Gel TLC plates with fluorescent indicator (Eastman 13 I8 I ). Samples were streaked at the origin and parallel lanes run with standards of synthetic adipokinetic hormone I and synthetic red pigment concentrating hormone (RPCH) (Peninsula Laboratories). Chromatograms were visualized by either U.V. absorbance or by spraying with Ehrlich’s reagent (IO?;, (w/v) 4-dimethylamine-benzaldehyde in concentrated HCI, mixed with acetone (I :4 v/v) prior to use). The solvent were S- 1: isopropanol-water-acetic systems used acid (25: IO: I by vol); S-2: ethyl acetate-ethanolwater-pyridine-acetic acid (60:30:8:2: 1 by ~01); S-3: acid acetate-ethanol-water-pyridineacetic ethyl (60:30:8:0.5: I by vol). Areas were scraped off the TLC and extracted in 80 ~(1Ringers. After centrifugation the supernatant was assayed for hyperlipaemic or hyperglycaemic activity. Hyperlipurmic~
(ROE, 1955). The rest of the supernatant
was discarded and the pellet dissolved in 0.2 ml isopropanol. The lipid content of the sample was determined by the method of FLETCHER(1968). Blood volume Blood volume was determined according to the method of LCWGHTON and TOBE (1969) using [’ 4C]-carboxyl inulin. RESULTS
Stimulution
qf NCC
II
An initial test revealed that the perfusate from control corpora cardiaca possessed minimal hyperlipaemic activity. The perfusate from corpora cardiaca in which NCC II had been stimulated possessed a significant amount of hyperlipaemic activity, so confirming previous findings (ORCHARD and LOUGHTON, 198la). No areas of activity were found when the control perfusate was chromatogrammed. Chromatography of perfusates from stimulated corpora cardiaca showed two areas of hyperlipaemic activity and one area of hypolipaemic activity (Fig. 1). In all three solvent systems a major peak of activity co-migrated
c Solvent 2
urd hypergl_vcaemic uchiry
70 /tl
samples were injected into IO-l2-day-old adult males through the membrane at the base of the hind leg, following CO, anaesthesia. For hyperglycaemic activity these males had been ligated at the neck for 3 hr (LOLICHTONand ORCHARD, I98 I ). One hour after injection. 5 jtl of haemolymph were collected through the neck membrane and precipitated in 50 ~1 of 10:;; trichloracetic acid. After centrifugation 5 ~1 of supernatant was removed for carbohydrate assay
Fig. 1
Release of adipokinetic ‘0
with synthetic adipokinetic hormone I. The second peak migrated in solvent I and 2 with the same R, relative to synthetic adipokinetic hormone I and RPCH as reported by CARLSEN et al. (1979) for adipokinetic hormone II. Solvent 3 was devised to further separate these two peaks and revealed that they were indeed discrete. Rechromatography of the hormone II peak from solvent 2 using the solvent 3 system revealed that the second peak co-migrated with an RF value similar to adipokinetic hormone 11. This second peak of hyperlipaemic activity also possessed hyperglycaemic activity (Fig. 2). Examination of the hyperlipaemic activities revealed that similar amounts of adipokinetic hormone I and II were released (assuming equipotency).
sohWlt3 r
427
hormones
T
0
Flight
Fig. 1. Adipokinetic activity recovered following chromatographic separation of perfusates of the NCC II-stimulated preparations. Saline-injected controls also shown. Synthetic adipokinetic hormone (AKH I) and red pigment concentrating hormone (RPCH) were run as standards in parallel lanes. All assays performed on day-10 adult male Locustn. mean k S.E. of four locusts. Solvent I. perfusate from two glands stimulated for 30 min each. Solvent 2 and 3, one gland stimulated for 30min. 0. origin, F. front. RF values for AKH I. RPCH and second peak of adipokinetic activity are. Solvent 1 : 0.70, 0.77, 0.82: Solvent 2: 0.17. 0.26. 0.32: Solvent 3: 0.30. 0.43. 0.56.
Since solvent 3 provided better separation of the two peaks of activity this solvent was used to analyse the haemolymph from flown and unflown locusts. Figure 3 shows that a large amount of hyperlipaemic activity, distributed between two areas on TLC, was present in the haemobmph of flown locusts, but comparatively little (though’ A measurable amount) was found in the unflown locusts. The two peaks of activity again co-migrated with adipokinetic hormones I and II. The area corresponding to hormone II also possessed hyperglycaemic activity (Fig. 2) while that of hormone I did not. These changes in hormone level were due to the release of hormone and not due to any concentrating effect of the haemolymph since blood volume of day-l 7 females did not change significantly during a 30-min flight (resting blood volume 765 + 90,& JZ= 8; flown blood volume 812 * 71 /d, II = 8). The increases in lipid brought about by the bioassay of adipokinetic hormone I were approximately 2.5 LLg/$ for unflown and 16.2 /(g/p1 for flown. Similarly for hormone II they were 1.5 pg/ptl and 6.6 pg/pl. Thus. the adipokinetic hormones I and II released and present at the end of a 30-min flight have adipokinetic activities of 13.7 pg/$ and 5.1 fig//A, respectively. This represents the bioassay of approximately 125 ~1 haemolymph. Day-13-15 female locusts had a blood volume of 376.3 k 40.5 ~1 (n = 8) and so the adipokinetic activities corrected as if hormone from 376 /Alof haemolymph were bioassayed in adult males represent 41.1 pg/ld and 15.3 pg/gl for adipokinetic hormones I and II. The relative contributions of adipokinetic hormones I and II to final lipid elevation are therefore approximately 73 and 27”~. respectively.
DISCUSSION
saline
a
saline 13 C
Fig. 2. Hyperglycaemic activity of fractions from TLC (a) AKH II fraction from perfusate following NCC II stimulation, one gland stimulated for 30 min. (b) AKH II fraction from haemolymph of flown Locusta. (c) AKH I fraction from haemolymph of flown Locusta. Saline-injected controls also shown. Mean k S.E. of three locusts.
The results clearly show that 30min of flight induces the release of two hormones with adipokinetic activity in Locusta. CHEESEMANet al. (1976) have previously shown adipokinetic activity to be present in the haemolymph of flown Locllsta. They found that this activity, obtained from a methanolic extract of haemolymph, was recovered at the same elution volume from Sephadex LH20 as the adipokinetic activity of a purified extract of corpora cardiaca. No other fractions containing adipokinetic activity were found in the haemolymph. In our studies, we have
IAN ORCHARDand ANGELA13.LANC;t:
FLOWN
UNFLOWN
Fig. 3. Adipokinetlc actlvlty recovered following chromatographic separation of haemolymph from unflown and flown Loccc.vtcz.Methanol extract of 5OOh11haemolymph from day-13-15 adult virgin females were fractionated on TLC in solvent 3. Lanes were extracted in 8Olil saline. and 2O-pI samples bioassayed using day-10 adult males. 0, origin. F, front. Synthetic AKH 1 and RPCH run as standards in parallel lanes. except for AKH
Data
represents
mean
f
SE.
of two replicate
I and AKH II which are for three replicates.
AKH I, RPCH and second peak of adipokinetic
been able to show that the adipokinetic activity in the haemolymph can be separated into two regions on TLC which possess the same RF values as synthetic adipokinetic hormone I (STONE et al.. 1976: BROOMFIELDand HARDY, 1977) and adipokinetic hormone II (CARLSEN et a/., 1979). It seems reasonable to conclude that these two active regions are indeed adipokinetic hormones I and 11. Blood volume remained constant during a 30-min flight and so these changes in hormone titre are brought about by release of hormone and not due to any concentrating effect due to lower blood volume. This confirms the previous data of JUTSUM and GOLDSWORTHY (1976) who showed blood volume remained constant during a 60-min flight of day-21 adult male Locusta. CHEESEMANand GOLDSWORTHY( 1979) assessed the release of adipokinetic hormone during flight in Locusta by testing pooled fractions containing possible adipokinetic activity from Sephadex LH20. These fractions may have emerged over a large volume (CHEESEMANer ~1.. 1976) and in the absence of clear data as to which fractions were pooled, could have included hormone II as well as hormone I (see CARLSENut uI., 1979). However. regarding the relative contributions of
activity
experiments assayed on six locusts, assayed on nine locusts. R, values for are: 0.30, 0.43 and 0.56, respectively.
adipokinetic hormones I and II in raising haemolymph lipid in our bioassay, we find hormone I possesses about 737: of the total activity and hormone II 27% (this makes the assumption that both hormones can express themselves fully in the presence of each other). Adipokinetic hormone I is, therefore, the predominant hyperlipaemic hormone and in view of the reported equal potencies of hormones I and II (CARLSEN et al., 1979), adipokinetic hormone I must be present in greater quantities after 30 min of flight. Adipokinetic hormone II also possesses hyperglycaemic activity (LOUGHTONand ORCHARD, 1981, also this study). Thus there is now clear evidence for the release of a hyperglycaemic hormone during locust flight. This extends the observations of VAN DER HORST et al. (1978) who showed that in the initial periods of flight, consumption of haemolymph trehalose was high with little mobilisation of trehalose from body stores. However, after 30min of flight a steady state was reached in which utilisation of trehalose was matched by mobilisation. Adipokinetic hormone II would appear to be responsible for maintenance of this steady condition. Both hormones are present in the haemolymph of unflown locusts. Similar levels of adipokinetic activity
429
Release of adipokinetic hormones were reported by CHEESEMAN and GOLDSWORTHY (1979). Whether this represents a true ‘resting’ level is hard to ascertain. These hormones may be used for maintenance of homeostasis, although CHEESEMAN and GOLDSWORTHY (1979) found no change in adipokinetic titre during starvation in locusts even though lipid levels rise significantly. It would also seem unlikely that adipokinetic hormone II was responsible for this rise during starvation since lipid levels still rise in locusts devoid of their glandular lobes (JUTSUM et al., 1975). NCC II stimulation results in the release of three hormones controlling haemolymph lipid levels; namely hypolipaemic hormone, adipokinetic hormones I and 11. This confirms the more indirect evidence of ORCHARD and LOUGHTON (1980, 1981a, b) and LOUGHTON and ORCHARD (198 1).We have shown previously that the release of hormone following NCC II stimulation is blocked by sodium-free saline and by tetrodotoxin (ORCHARD and LOUGHTON, 198la). Thus, release is due to the presence of a compound action potential and not an artefact of stimulation. Hypolipaemic hormone is stored and released from the storage lobes of the corpora cardiaca, adipokinetic hormones I and II originate within the glandular lobe and the control by NCC II is via aminergic synapses. Both hormones I and II would appear to be controlled by aminergic input since no adipokinetic activity was released following treatment with r-aminergic antagonists or by prior treatment with reserpine (ORCHARD and LOUGHTON. 1981b). However. since equal amounts of adipokinetic hormones I and II appear to be released by stimulating the NCC II, and different amounts appear to be present in the haemolymph after a 30-min flight, it would seem that the pre-synaptic axons in the NCC II are different (assuming equal potencies for adipokinetic hormones I and II and equal clearing rates from haemolymph). Thus hormone I and II may be released independently of one another. The simple procedure for separating these hormones now provides a useful tool for examining in a more quantitative manner the precise titres of both adipokinetic hormones I and II during flight, and the neural control of the release of hormones by the NCC 11.
/Ick,lowledyeme,lto-This work was supported by the Natural Sciences and Engineering Research Council of Canada. We are most grateful to Professor B. G. LOUGHTONfor much help and encouragement, and for supplying .I I me locusts.
REFERENCES BEENAKKERS A. M. Th. (1969) The influence of corpus allatum and corpus cardiacum on lipid metabolism in Locu.sta migratoria. Gen. camp. Endocr. 13, Abstract 12. BROOMFIELD C. E. and HARDYP. M. (1977) The synthesis of locust adipokinetic hormone. Tetruhedron Lett. 25. 2201-2204. CARLSENJ., HERMANW. S., CHRISTENSEN M. and JOSEFSK)N L. f1979) Characterisation of a second peptide with adi. . pokinetic and red pigment-concentrating activity from ihe locust corpora c&iaca. Insect. Biochem. 9,497 501. CHEESEMAN P. and GOLDSWORTHY G. J. (1979) The release of adipokinetic hormone during Right and starvation in Locusta.
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37. 35-43.
CHEESEMAN P., JUTSUMA. R. and GOLDSWORTHY G. J. (1976) Quantitative studies on the release of locust adipokinetic hormone. Phvsiol. Ent. 1. 115-121. FLETCHERM. J. (1968) A’colorimetric method for estimating serum triglycerides. CICka chim. Acta 22. 393.-397. JUT&M A. R. and GOLDSWORTHY G. J. (1976) Fuels for flight in Locusra. J. Insecr. Pl7,vsiol. 22, 243-249. JUTSUMA. R., ARGARWALH. C. and GOLDSWOIYT~IY (i. J. (1975) Starvation and haemolymph lipids in Loc,u.sf~ migratoria (R & Ft. Acrida 4, 47-56. LOUGHTON B. G. and ORCHARDI. (198 1)The nature of the hyperglycaemic factor from the glandular lobe of the corpus cardiacum of Locusta migrutorru. J. /mast. P/IV Go/.
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LOUGHTONB. G. and TORES. S. (1969) Blood volume m the African migratory locust. CUJI. ./. Zoo/. 47. 1333-1336. MAYERR. J. and CANDYD. J. (1969) Changes in energy reserves during flight of the desert locust, Sch~.stocerc~lr gregaria. Comp. Biochem. Plzysiol. 31, 409-41X. ORCHARDI. and LOUGHTONB. G. (1980) A hypolipacmic factor from the corpus cardiacum of locusts. Nurwc. Land. 286, 494496.
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fluid with anthrone
reagent.
1. hioi.
C~7~w1.
212, 335-343. SPENCERI. M. and CANDYD. J. (1976) Hormonal control of diacyl glycerol mobilisation from fat body of the desert locust. Scllistocrrca gregaria. Ir~setr B~oc~l~m 6.
289-296. STONEJ. V., MORDUEW.. BAILEYK. E. and MORRISH. R. (1976) Structure of locust adipokinetic hormone. a neurohormone that regulates lipid utilisation during flight. Nature. LorId. 263. 207-2 I I. VAN DERHORSTD. J.. VAN D~ORN J. M. and BE~NA~~KI RS A. M. Th. (1978) Dynamics in the haemolymph trehalose pool during flight of the locust. Locusta migrntoria. Insect Biocl7ur17. 8, 4 13-416.