Enhancement of hypertrehalosemic hormone biosynthesis by trehalose in isolated cockroach (Periplaneta americana) corpora cardiaca in vitro

J. Insecr Ph.rsiol. Vol. 38. No. 9. pp. 659464. Printed in Great Britain. All rights reserved

0022-1910/92 $5.00 + 0.00 Copyright Q 1992 Pergamon Press Ltd

1992

ENHANCEMENT OF HYPERTREHALOSEMIC HORMONE BIOSYNTHESIS BY TREHALOSE IN ISOLATED COCKROACH (PERIPLANETA AMERICANA) CORPORA CARDIACA IN VITRO M. A. KHAN and J. E. STEELE* Department of Zoology, University of Western Ontario, London,

Ontario,

Canada

MA

5B7

(Received I3 January 1992; revised 10 March 1992)

Abstract-Using [3H]tryptophan as a precursor, the rate of production and release of cardioacceleratory hormone-I and -II by isolated corpora cardiaca of Peripfaneta americana was quantified in vitro. Striking differences in the rate of radiolabelled tryptophan incorporation into these peptides occurred depending on the carbohydrate composition of the incubation media. With medium containing only sucrose, a low rate of hormone accumulation was found. This rate increased more than five-fold when 50 mM trehalose was included in the medium. The effect of trehalose was rather specific and could not be mimicked by 5 mM glucose, although extremely high levels of glucose did stimulate some glands. The enhanced rate of cardioacceleratory hormone-I and -II accumulation appears to be due to an increased rate of peptide synthesis rather than a lowered rate of release. Key Word Index:

Periplaneta americana; corpora

trehalose; P. americana cardioacceleratory acceleratory hormone release

cardiaca; hypertrehalosemic hormones; hormone biosynthesis; P. americana cardio-

lNTRODUCTlON

synthesis/release of the hormones, which originate in the glandular cells of the corpora cardiaca. In the locust, it was suspected that the decrease in haemolymph trehalose shortly after the initiation of flight may exert a positive feedback effect on the release of adipokinetic hormone, because experimental elevation of the trehalose concentrations delayed the flight-induced increase in haemolymph lipid (Van der Horst, 1979; Cheeseman et al., 1976). However, Cheeseman et al. (1976) also noted that the same effect could be obtained by injecting sucrose, a sugar not readily metabolized by flight muscle, indicating that the delayed release of adipokinetic hormone was not specifically related to trehalose utilization. In the cockroach, P. americana, flight caused an increase in haemolymph sugar (King et al., 1986) but, under certain stressful conditions, e.g. exposure to chlorinated insecticides, a rapid and dramatic decrease in haemolymph carbohydrate was observed (Granett and Leeling, 1971; Orr and Downer, 1982). Curiously, these subnormal levels of trehalose were accompanied by increased hypertrehalosemic activity in the haemolymph, and the question as to whether a positive feedback mechanism between substrate

A primary function of the neuropeptides belonging to the adipokinetic hormone-red pigment concentrating hormone family in insects is believed to be the rapid mobilization of substrates required for fuelling high energy activities such as flight. In the locust, Locusta migratoria, which utilizes carbohyrdrate initially and changes to lipid during prolonged flight, the role of adipokinetic hormones-I and -11 in stimulating the release of diacylglycerol from the fat body has been welldocumented (Mayer and Candy, 1969; Goldsworthy, 1983; Gade, 1990). The analogous hypertrehalosemic hormones, designated cardioacceleratory hormone-I and -11, of the cockroach, Periplaneta americana, not only stimulate the release of trehalose from the fat body, but also possess cardioacceleratory and myotropic properties (Steele, 1961, 1984; Scarborough ef al., 1984; O’Shea et al., 1984). While there is ample evidence indicating that these neuropeptides induce the release of lipid or carbohydrate from locust and cockroach fat body respectively, little is known about the effects of fluctuating substrate levels on the *To whom all correspondence

should

be addressed. 659

660

M. A. KHANand J. E. STEELE

levels and hormone release is operative under these circumstances has remained unresolved (Granett and Leeling, 1972). Previously, O’Shea et al. (1984) showed that radioactive tryptophan could readily be taken up by isolated cockroach corpora cardiaca in vitro and incorporated into P. americana cardioacceleratory hormone-I and -II. In the present paper this technique has been adapted for quantifying the rates of synthesis and release of these neuropeptides in vitro. The method was then used to assess the effects of different levels of trehalose directly on these processes. The effects of glucose and sucrose on the rate of hormone synthesis were also investigated. MATERIALSAND

METHODS

Insects

Adult male cockroaches (P. americana), l-4 old were used in this study. They were obtained a colony reared at 28°C and 65-70% r.h. under light-12 h dark photoregimen. Food (dog mixed with refined sugar) and fresh water supplied ad libitum.

weeks from a 12 h chow were

Corpus cardiacum culture in vitro

A sterilized physiological saline, modified from O’Shea et al. (1984), containing 1 mM CaCl,; 5.4 mM

KCI; 0.4mM KH,PO,; 0.5 mM MgCl,. 6H20; 0.4 mM MgSO., .7H,O; 137 mM NaCl; 0.3 mM Na,HPO, .7H,O; 4 mM NaHCO, and 5 mM Hepes, pH 7.2, formed the basic incubation medium for gland culture. Sucrose, trehalose, glucose and certain amino acids were added to this primary medium separately or in various combinations as indicated. Unanaesthetized cockroaches were decapitated and the corpora cardiaca-allata complexes removed under saline. For determining the rate of synthesis of cardioacceleratory hormone-I and -11, individual pairs of glands were incubated in 50 ~1 saline containing 5 PCi L-[5-3H]tryptophan (sp. activity = 1165.5 GBq/mmol; 31.5 Ci/mmol NEN) for 6 h in darkness at 28°C with gentle shaking using a wrist action shaker. After completion of the incubation period, the radioactive saline was discarded. The glands were washed twice with 100 ~1 cold saline before suspension in 100 ~1 of a methanolic mixture (methanol : water: acetic acid, 90: 9: 1) and stored at - 20°C until analysis. In order to quantify the rate of release of cardioacceleratory hormone-I and -11, three pairs of glands were incubated together in 100~1 saline containing 10 PCi [3H]tryptophan. Further details of the procedure for quantifying rate of release are presented under Results and in the legend of Table 1.

Table 1. Rate of cardioacceleratory hormone release from corpora cardiaca under various incubation conditions Hormone synthesized dom/oair CC/h

Release conditions

Hormone released dnm/pair CC/h

Hormone released (%) 16.6 22.4 26.7

4313 4047 2990

No trehalose 90 mM sucrose

720 906 798

4255 3546 2259

50 mM trehalose 90 mM sucrose

949 405 742

2305 1213 2850

50 mM trehalose 90 mM sucrose +45mM NaCl

396 183 410

3499 4057 3261

50 mM trehalose 90 mM sucrose +45 mM KC1

1755 2212 1858

21.9* 2.9

22.3 11.4 32.8 22.2 f 6.2 17.2 15.1 11.8 15.6 k 0.8 50.2 54.5 57.0 53.9 + 2.0”

Each value was obtained by incubating three pairs of glands in 100 ~1 medium containing 10 FCi [3H]tryptophan, in addition to 50 mM trehalose and 90 mM sucrose and normal concentrations of K + and Na + during the first 4 h of incubation. After washing the glands and replacing the labelled tryptophan with cold tryptophan, the incubation was continued for another 2 h in medium with altered carbohydrate and K+ and Na + composition as specified under “release conditions”. Upon completion of the second incubation, both glands and incubation media were extracted and analysed. The amounts of cardioacceleratory hormone in glands and corresponding incubation medium, expressed in dpm/pair CC/h are shown above as “hormone synthesized” and “hormone released” respectively. The per cent hormone released is calculated from these values and the means _+SEM are shown in bold type. *P < 0.01.

Enhancement of hypertrehalosemic hormone biosynthesis Extraction, ident$cation, Ssolation and quantt$cation of radiolabelled cardioacceieratory hormone-I and -II

Synthetic cardioacceleratory hormone-I and -11 were added, 1 pg each, to samples prior to extraction for HPLC. These synthetic peptides were included to act as markers and to enhance recovery of the radiolabelled hormones. The glands were disrupted by sonication (Kontes micro-ultrasound cell disrupter) and the methanol removed under nitrogen. Before loading, samples were centrifuged at 10,OOOgfor 5 min. Peptide separation was performed on a Vydac C- 18 column (dimensions 45 x 250 mm; particle size 10 pm) at ambient temperature with a Waters HPLC system attached to a Waters LC spectrophotometer (LambdaMax Model 481) and a recorder (SE 120, BBC/ Goertz Metrawatt). Two solvents, A =20% ACN (acetonitrile) in IO mM ammonium acetate buffer and B = 50% ACN in water were used to produce the following gradient: 0% B/5 min; O-100% B/40 min; 100% B/S min. The flow rate was set at 1 ml/min and the eluted peptides detected at 219 nm, 0.1 AUFS. One ml fractions were collected in scintillation vials. After addition of 5 ml scintillant (ScintiVerse E, Fisher Scientific) the radioactivity was measured in a Packard Minaxi liquid scintillation counter. Statistical analysis

The results were analysed statistically using ANOVA and Duncan’s new multiple range test.

20

661

1

Lh I71

17)

3

6

9

hours

12

Fig. I. Time course of [‘Hltryptophan incorporation into intraglandular cardioacceleratory hormone-l (open columns) and -II (hatched columns) by individual pairs of corpora cardiaca (CC). The incubation medium contained 90 mM sucrose and 50 mM trehalose. In all figures, columns and bars represent meansf SEM and the number of determinations is shown in parentheses.

experiments we found that in medium containing 90mM sucrose and 5mM trehalose, the rate of radiolabelled hormone accumulation was significantly lower and was constant for about 12 h. For all further experiments, a maximal incubation period of 6 h was used for estimating the rate of hormone synthesis. E’ect of different concentrations of trehalose, added to medium containing 90 mM sucrose, on intraglandular accumulation of cardioacceleratory hormone-I and -iI

REStiLTS

Identtfication of radiolabelled cardioacceleratory hormone-I and -II and time course of [‘Hltryptophan incorporation

Using the HPLC conditions described in Materials and Methods, the peaks of radiolabelled P. americana cardioacceleratory hormone-I and -II were clearly separated with retention times of about 22 and 27 min, respectively, identical to those of the synthetic peptides, detected at 219nm. Cardioacceleratory hormone-I was the predominant peptide synthesized. The ratio of radiolabelled cardioacceleratory hormone-Ijcardioacceleratory hormoneII was reasonably constant (2.88 f 0.31, n = IO) and reflected the ratio of these peptides present in the glands (Scarborough et al., 1984). Figure 1 shows the accumulation of radiolabelled cardioacceleratory hormone-I and -II by individual pairs of glands during 3, 6, 9 and 12 h incubation. These data indicate that the rate of [‘Hltryptophan incorporation into cardioacceleratory hormone-I and -II is constant for at least 9 h. In this medium, which contains 90mM sucrose and 50mM trehalose, the glands were maximally active. In preliminary

In initial experiments the carbohydrate composition of the corpus cardiacum culture medium was similar to that used by O’Shea et al. (1984) namely 90 mM sucrose and 5 mM trehalose. The rate of labelled cardioacceleratory hormone accumulation obtained was not significantly affected when trehalose was omitted from the medium but increased by about three-fold when the concentration of trehalose was elevated to 55 mM. Figure 2 shows the results from a similar experiment using individual pairs of glands. A striking increase in the rate of intraglandular accumulation of both cardioacceleratory hormone-I and -11 was seen when 50mM trehalose was added to medium containing 90 mM sucrose. Increasing the trehalose concentration to 100 mM did not produce an additional increase in radiolabelled hormone accumulation. Efiect of 140mM glucose, sucrose or trehalose on intraglandular accumulation of cardioacceleratory hormone-I and -II

In the previous experiment maximal rates of radiolabelled hormone were observed with 50mM trehalose and 90mM sucrose (total sugar

662

M. A. KHAN and J. E. STEELE

(61

: a

trehalose

concentration

Comb1

(mMl

Fig. 2. Relation between the rate of [‘Hltryptophan incorporation in intraglandular cardioacceleratory hormone-I (open columns) and -II (hatched columns) and the trehalose concentration of the incubation medium which also contains 90 mM sucrose. Individual pairs of glands were incubated for 6 h. Statistics: a/c, P < 0.01; a/e, P < 0.01; c/e, NS (not significant); b/d, P < 0.01; b/f, P < 0.01; d/f, NS.

b

c

d

Comb2

Fig. 4. Effect of 5mM glucose plus 135 mM sucrose (Combl) and 5 mM glucose plus 90 mM sucrose and 45 mM trehalose (Comb2) on the rate of [3H]tryptophan incorporation into intraglandular cardioacceleratory hormone-I (open columns) and -II (hatched columns). Statistics: a/c, P < 0.01; b/d, P < 0.01.

levels of this sugar was devised. When 5 mM glucose was mixed with 135 mM sucrose (combination 1) or with 90 mM sucrose and 45 mM trehalose (combination 2), the maximal rate of radiolabel incorporation into cardioacceleratory hormone-1 and -11 was observed only with the latter combination (Fig. 4). This result suggests strongly that, at physiological concentrations, glucose cannot mimic the enhancing effect of trehalose. Amino acids which make up the building blocks of cardioacceleratory hormone-I and -II, when added to the incubation medium at a final concentration of 1 mM each, did not further enhance the rate of hormone synthesis (results not shown). logical”

medium. When glucose, sucrose and trehalose were tested separately at a final concentration of 140 mM each, the results shown in Fig. 3 were obtained. The maximal rate of labelled cardioacceleratory hormone-1 and -II accumulation occurred in the presence of glucose or trehalose. In contrast, a low rate persisted in medium containing only sucrose. It was noticed that while trehalose invariably enhanced the rate of hormone accumulation by all glands, glucose appeared to have an all-or-none effect with about half the glands showing no stimulation at all. concentration

= 140 nm) in the incubation

Effect of 5mMglucose

on intraglandular accumulation

of cardioacceleratory hormone-I and -II In order to gain further insight into the precise role of glucose,

an experiment

employing

c

glucose

more “physio-

d

sucrose

rrehalose

Fig. 3. Effect of 140 mM glucose, sucrose or trehalose on the rate of intraglandular accumulation of labelled cardioacceleratory hormone-I (open columns) and -11 (hatched columns). Individual pairs of corpora cardiaca were incubated for 6 h. Statistics: a/c, P < 0.01; a/e, NS; c/e, P < 0.01; b/d, P < 0.05; b/f, NS; d/f, P < 0.01.

Effect of trehalose on release of cardioacceleratory hormone-I and -II

The possibility that the difference in the rate of cardioacceleratory hormone accumulation in glands incubated with and without trehalose could be due to a difference in the rate of release of these peptides was investigated. Attempts to quantify the radiolabelled cardioacceleratory hormone-I and -11 released into the original incubation medium were not successful because of the high levels of contaminating radioactivity, probably due to the presence of radiochemical impurities in the [3H]tryptophan sample. We therefore devised the following experimental protocol to study the effect of trehalose on the rate of release of the labelled peptides. All glands were first incubated for 4 h in medium containing 90mM sucrose and 50mM trehalose to ensure optimal accumulation of radiolabelled hormone. The incubation medium was then discarded and the glands washed twice in cold saline. The corpora cardiaca were resuspended in saline containing either 90 mM

Enhancement of hypertrehalosemic

sucrose alone or 90mM sucrose plus 50mM trehalose. The radioactive tryptophan was replaced by the non-radioactive analogue and the incubation continued for another 2 h. Upon completion of incubation, both glands and incubation media were extracted and analysed. The gland extracts give a relative measure of the rate of cardioacceleratory hormone biosynthesized whereas the media extracts provide an estimate of the rate of release of the peptides. From these two values, expressed in dpm/pair corpora cardiacs/h. the percentage cardioacceleratory hormone released during each incubation could be derived. As shown in Table 1, there was no significant difference in the percentage of cardioacceleratory hormone released in medium lacking trehalose and in that containing 50 mM trehalose. To verify that the copora cardiaca are viable under these conditions and respond to release stimuli, incubations with elevated K + or Na+ were included. Replacing the normal medium with high K + medium resulted in a striking increase in the percentage of cardioacceleratory hormone released while the high Na + medium had no effect (Table I). Taken together these results seem to suggest that the enhanced rate of labelled cardioacceleratory hormone accumulation in corpora cardiaca in the presence of 50mM trehalose in the medium is caused by an accelerated rate of hormone synthesis. DISCUSSION

The hypothesis that a decrease in haemolymph trehalose may trigger the release of hypertrehalosemic hormones does not appear to be supported by the in rGtro studies reported here. On the contrary, normal levels of trehalose (ca 50 mM) seem to be necessary for optimal rates of production of cardioacceleratory hormone-I and -II and any drastic decrease in the concentration of this sugar leads to a reduced rate of synthesis (and net release) of these peptides. Oudejans et a/.(1990) commented briefly on a similar enhancing effect of trehalose on adipokinetic hormone biosynthesis by L. migratoria corpus cardiacum in Ctro. The physiological significance of this phenomenon and the mechanism of action of the disaccharide remain unclear. The enhancing effect of trehalose on hormone synthesis could not be reproduced by sucrose or by physiological concentrations of glucose. Since trehalose would have to be converted to glucose before utilization, the possibility that the disaccharide may play a role as energy source during biosynthesis of the neuropeptides and thus enhance their rate of production seems difficult to reconcile with the present findings. Another possibility, that the sugar may facilitate increased uptake

hormone biosynthesis

663

of tryptophan into the corpora cardiaca, does not. appear to be the case either because after 2 and 6 h incubation there was no significant difference in the total amount of radioactivity present in glands incubated in 140 mM sucrose and those incubated in 140 mM trehalose (unpublished findings). The rate of release of the hormones does not seem to be immediately affected by the trehalose concentration in uitro. Enhanced release of these neuropeptides in vitro has been shown to occur in the presence of elevated K+ which is believed to act by depolarizing the glandular cells (Downer et al., 1984; O’Shea et al., 1984). This criterion was used in the present study to check the viability of the glands to release stimuli. Although it remains debatable whether the results obtained here, with isolated corpora cardiaca devoid of nervous connections to the brain, reflect the situation in uivo, the approach described does provide an interesting insight into the direct effects of trehalose on the production and release of cardioacceleratory hormone-I and -II by the corpus cardiacum. The observation that radioactive tryptophan is readily taken up by the corpus cardiacum and incorporated in P. americana cardioacceleratory hormone-I and -11 in vitro (O’Shea et al., 1984) has been useful in identifying the prohormones of L. migratoria adipokinetic hormone-I and -II and the adipokinetic hormone precursor related peptides (Hekimi and O’Shea, 1987) and in isolating and establishing the structure of the adipokinetic hormone-I prohormone (Hekimi et al.. 1989). In its present adaptation, the in Gtro method is sensitive enough to monitor individual pairs of glands and can be employed to investigate factors affecting synthesis and release of cardioacceleratory hormone-I and -II. including the role of factors originating in the brain. It is likely that such factors are directly responsible for enhanced release of these peptides under physiological conditions. For example, it has been proposed that the biogenic amine. octopamine, may be involved in the release of these neuropeptides (Downer et al., 1984; Orchard, 1984). Inclusion of octopamine in incubation media in vitro resulted in enhanced release of hormones from the corpus cardiacum, an effect suggested to be mediated via CAMP (Downer et al., 1984; Cole et al., 1987). The in uitro method presented in this paper is suitable for investigating some of these questions in a more direct fashion, including the effects of biogenic amines on synthesis of the neuropeptides. Acknowledgements-Thiswork was funded by a grant from Insect Biotech Canada to J.E.S. Synthetic cardioacceleratory hormone-I and -II were a generous gift from D. Schooley.

M. A. KHAN amd J. E. STEELE

664 REFERENCES

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