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Adenylate cyclase system and the hyperglycemic factor in the cockroach, Periplaneta americana
Insect Biochem., 1977, Vol. 7, pp. 95 to 99. Pergamon Press. Printed in Great Britain.
ADENYLATE CYCLASE SYSTEM A N D THE HYPERGLYCEMIC FACTOR IN THE COCKROACH, PERIPLANETA AMERICANA KAZUNORI HANAOKA and Susv~crc Y. TAKAHASHI Biological Institute, Faculty of Science, Nagoya University, Nagoya 464, Japan
(Received 22 April 1976) Abstraet--Adenylate cyclase (EC 4.6.1.1) and cyclic AMP in the fat body of the cockroach, Periplaneta
americana, were investigated in relation to the mode of action of the hyperglycemic factor. The adenylate cyclase was activated about 3-fold by the injection of an extract of the corpora cardiaca. Concomitantly with the activation, the cyclic AMP content in fat body was increased nearly twice, preceding the rise of haemolymph trehalose concentration. Injection of theophylline or dibutyryl cyclic AMP induced a sustained hyperglycemia in the cockroach. These results strongly indicate the involvement of adenylate cyclase system in the hyperglycemic action of the corpora cardiaca extract.
INTRODUCTION THE HYPERGLYCEMICfactor raises haemolymph trehalose level by exhausting the glycogen stored in fat body (STEELE, 1963; BOWERS and FRWDMAN, 1963; RALPH and McCARXHV, 1964; HANAOKA and TAr,AHASm, 1976). An enzymic basis for the glycogenolysis has been attributed to the activation of glycogen phosphorylase in fat body (STEELE, 1963; WRENS and GILBERT,1967; GOLDSWORTHY,1970). However, very little has been revealed about the detailed mechanism of the hormonal action. STEELE (1964) reported that adenosine 3':5'-monophosphate (cyclic AMP) was able to mimic the action of the hyperglycemic factor (activation of the glycogen phosphorylase) when added to a fat body preparation incubated in a saline medium, suggesting the mediation of cyclic AMP. In this study, we measured the activity of adenylate cyclase (EC 4.6.1.1) and the level of cyclic A M P in the fat body after injection of the aqueous extract of the corpora cardiaca (CC). The effect of the injection of N6-2'-0-dibutyryl adenosine 3"5'-monophosphate (dibutyryl cyclic AMP) and also of theophylline on the haemolymph trehalose level was analyzed, and the mode of action of the hyperglycemic factor is discussed.
MATERIALS AND METHODS
Experimental animals Cockroaches were reared on mouse food and water at 25 + 1°C. Adult males, 20 to 30 days after adult ecdysis, were used for experiments.
Chemicals Cyclic AMP, adenosine 5'-monophosphate (Y-AMP), adenosine 5'-triphosphate (ATP), crcatine phosphokinase creatine phosphate, dibutyryl cyclic AMP, and bovine heart phosphodiesterase were obtained from Boeringer Mannheim GmbH. [~-32p]-ATP and [8-3H]-adenosine Y:5'-monophosphate were purchased from New England Nuclear Corp. Dowex 1-X8 resin (2(X)-400mesh) was obtained from Dow Chemicals, and neutral aluminium oxide from M. Woelm. Other chemicals were of reagent grade.
Assay of adenylate cyclase Fat body tissues were dissected from 4 males in ice-cold 0.9% NaC1 solution and were homogenized in a glass homogenizer for 5 min. with 2 volumes of cold 10 mM Tris-HC1 buffer, pH 7.5, containing 5 mM 2-mercaptoethanol and 1 mM ethylenediamine tetraacetate (disodium salt). The homogenate was used for the enzyme preparation for incubation. The reaction mixture contained 1.25raM [~t-32p]-ATP (1 × 106c.p.m.). 5.0mM MgCl,, 5.0mM NaF, 1.0mM theophylline, 0.25 mM cyclic AMP, 10 mM creatine phosphate, l0 ~g creatine phosphokinase, 1.0mM 2-mercaptoethanol, 45 mM Tris-HC1 buffer, pH 7.5, and tissue homogenate (ca. 0.1 mg protein): total volume was made up to 200/d with water. The reaction was initiated by adding the tissue homogenate. After incubation at 30°C with shaking for the desired time, the reaction was terminated by immersing the tubes into a boiling water-bath for 3 min and then cooled in an ice-bath. To each tube was added 2.0ml of 50 mM Tris-HC1 buffer, pH 7.5, and the precipitate was centrifuged off. The supernatant was applied to a column (0.5 x 3 cm) of neutral alumina and the fraction not absorbed was collected (WHITEand ZENSER, 1971). The eluates were dried on a rotary evaporator. The dried materials were dissolved in 1 ml of water, and the radioactivity of the solution was counted in a liquid scintillation counter, TEN GSL-163,
96
KAZUNORI HANAOKA AND SUSUMI~ Y. TAKAHASIII
using the dioxane scintillator consisting o1 PPO6g, dimethyl P O P O P 0.27g and naphthalene 112 g in I1 dioxane. Protein concentration in the tissuc homogenate was determined by the method of LOWR~ et al. (1951).
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Determination ¢ff cyclic A M P in the ahdomimd.lia body The abdominal fat body tissues from 5 males were homogenized with a glass homogenizer in 10 vols of cold 5% trichloroacetic acid (TCA). Belore homogenization. [8-3HI-cyclic AMP (20{X)0counts mini was added to the solution to know the recovery of cyclic AMP. After removal of the precipitate by centrifugation, the supernatant was washed with ethylether 5 times. The aqueous phase was taken and neutralized with 1 M Tris. Cyclic AMP in the solution was purified using columns of neutral alumina and Dowex l-X8 (200-400 mesh, C1 form), according to the method of TAKAHASHI et al. 09751. The fraction containing cyclic AMP was diluted with an appropriate volume of water and used for determination of cyclic AMP. Content of cyclic AMP was determined according to the method of Kuo and GREI!N(IARI)(1970) using cyclic AMP-dependent protein kinase. Assay and preparation of cyclic AMP-dependent protein kinase from the developing adults of the silkworm, Bomby.,: mort. were done according to the method reported previously (TARAitASlnet al., 1975).
Thin layer chromatography aml palWr chromatoHraph )' Thin layer chromatography was carried out on a Wako silica layer PA plate (6 × 2(Icm) with a solvent system of isopropanol water 28'~, ammonia water (7:2: 1. v.v). Ascending paper chromatography was carried out on Toyo filter paper No. 51 with three solvent systems: isopropanol water 28"0 ammonia water (7:2:1.x v), n-butanol acetic acid water (4:1: I, vv) and ethanol 0.5 M ammonium acetate (5:2, v/v). Spots of the authentic Y-AMP and cyclic AMP, which wcrc co-chromatographed with the radioactive products, were visualized by' spraying Hanes lsherwood reagent (BANI)t;RSKI alld AXEI.R()I). 1952). The radioactivity of the scraped silica gel or paper strip was measured directly' with a liquid scintillation cotinter using dioxane scintillator.
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Fig. 1. Formation of cyclic AMP b} the homogenate ol fat body tissues. Cockroaches were injected with 10HI of CC extract ( 1/4(I pair of the gland). After 1 hr, the homogenate was prepared, lbllowed by the incubation with [~-3ep]-ATP under the standard assay conditions. a n d Methods. In any case, the radioactive spot corresponding to the authentic cyclic A M P was revealed. W h e n the radioactive product had been pre-treated with bovine heart phosphodiesterase tinder the assay condition as described by BuwctniR (1975). it was convetted to the substance whose Rf value was identical with the authentic 5 ' - A M P on the thin layer chromatogram. Therefore, it is concluded that cyclic A M P is formed by the fat body homogenate from A T P under the asaty conditions. As shown in Fig. 1, production of cyclic A M P is linear with time for ;.it least 15 min, a n d is proportional to the protein concentration of the homogenate (protein content was less than 100Hg).
Preparation o/the corpora cardiaca c.vtract aml deterruination of haemolymph trehalosc
E~i'ct o! the ilijection o! ('C t_,.\lP~.lct otl lilt" acticitv o[ adenylate c3,clase
The methods of the preparation of ('C extract, and of determination of haemolymph trehalose were described in the preceding paper {HANAOKA and TAKaHASHL 1976).
Effect of the injection of CC extract on adenylatc cyclase was investigated by comparing between the
RESULTS
Enzymic .lbrmation o] cyclic A M P by Jar body homogem, te The h o m o g e n a t e of fat body tissues from male adult cockroaches, which had previously been injected with CC extract, was quickly prepared a n d incubated with [~-32p]-ATP under the assay conditions described in Materials a n d Methods. The incubated mixture was applied on a neutral alumina column, a n d the fraction not adsorbed on the column was collected. Identification of the radioactive product in the eluate as cyclic A M P was carried out by thin layer c h r o m a t o g r a p h y a n d paper c h r o m a t o g r a p h y with the solvent systems described under Materials
Table 1. Adenylate cyclase activity in tile homogenate ol fat body tissues Pretreatment None Injected with CC extract* ( 1,'40 gland-pair)
Cyclic AMP formed (pmoles/10 min/mg protein ) 27.5 31.2 92.5 98.S I 01.3 105.4
* The amount of injected CC extract was equivalent lo 140 pair of the gland. Alter I hr at 25 C., the fat body homogenates were prepared and adenylate cyclase acti,&v was determined.
Adenylate cyclase in the fat body
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Fig. 2. Time-course of changes in the level of cyclic AMP in the abdominal fat body and in the level of haemolymph trehalose after the injection of CC extract. The level of cyclic AMP in the abdominal fat body, O; and the level of haemolymph trehalose, A; in the CC extract (1/40 pair of the gland) injected cockroaches. The level of cyclic AMP in the abdominal fat body, O; and the level of haemolymph trehalose, A; in the water injected controls.
CC extract-injected and control insects of the activity in the homogenate of fat body tissues. Results are shown in Table 1. The injection of CC extract (1/40 pair of the gland) caused activation of the enzyme up to about 3 times after 1 hr.
Effect of the injection of CC extract on cyclic AMP level in the fat body Cockroaches were injected with CC extract (1/40 pair of the gland) or water as control. After various intervals, content of cyclic AMP in the abdominal fat body was determined. Results are shown in Fig. 2. The level of cyclic AMP in the abdominal fat body increased very rapidly after the injection of CC extract: the level of cyclic AMP increased from 475pmole/animal to 659pmole/anima1 during the first 5 min after the injection. After l hr, it reached the maximal value of 799 pmole/animal, and this level maintained for at least 5 hr. When distilled water was injected, the cyclic AMP level showed a slight increase after 5 min, but it returned to the original level soon after. The time-course of the initial change in the haemolymph trehalose level after the injection of the CC extract was examined in order to discover the relationship with the change of cyclic AMP level in the fat body. As shown in Fig. 2, the rise in the cyclic AMP level in the fat body is apparently preceding the elevation of haemolymph trehalose level.
Effect of the injection of theophylline and dibutyryl cyclic A M P The effect of the injections of theophylline and of dibutyryl cyclic AMP on the haemolymph trehalose level was examined in order to confirm further the
involvement of cyclic AMP in the hyperglycemic action. The time-course of the changes in haemolymph trehalose level after the injections is shown in Fig. 3. The injection of theophyUine (40 mM, 20 #1) exerted a strong hyperglycemic effect, whereas that of dibutyryl cyclic AMP (10 mM, 20 pl) did only a slight rise. In both eases, the elevated level of haemolymph trehalose maintained for a long period, resembling the effect of injecting the CC extract.
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Hrs.
Fig. 3. Time-course of changes in the level of haemolymph trehalose after the injection of theophylline or dibutyryl cyclic AMP. 40 mM theophylline, O; 10 mM dibutyryl cyclic AMP, El; distilled water (control), &. The vertical bar indicates the standard error of the mean. The volume of the injected solution was 20/~1, and the volume of haemolymph was determined to be 125 #1 by amaranth dilution method (MOROUE, 1969).
98
KAZUNORI HANAOKA AND SUSUMU Y. TAKAHASHI
Table 2. Effect of injection of theophylline and dibutyryl cyclic AMP on the haemolymph trehalose level Haemolymph trehalose* concentration (mg/ml)
Injection Experiment l
distilled water 0.05 mM dibutyryl cAMP 0.25 mM dibutyryl cAMP 1.0 mM dibutyryl cAMP 10 mM dibutyryl cAMP 2.0mM theophylline 20 mM theophylline 40 mM theophylline
17.2 ,+ 0.46 21.2 ,+ 2.35 24.9 _+ 0.21 24.5 _+ 2.33 24.6 + 1.32 21.2 ,+ 2.(X) 26.2 + 0.32 32.1 + 1.32
Experiment 2
distilled water 5.0 mM dibutyryl cAMP 40 mM theophylline CC extract (1/500 pair/20 #1) 5.0mM dibutyryl cAMP plus 40 mM theophylline CC extract plus 40raM theophylline CC extract plus 5.0 mM dibutyryl cAMP
17.2 _+ 0.80 22.3 _+ 0.76 a 27.8 _+ 0.76 b 28.5 +_ 0.54 c 28.1 4- 1.54d 30.3 +_ 1.32 e 32.9 + 1.91 f
Comparison of the means: a-f. P < 0.005: c-f, P < 0.1: b-d, c-e, not significant. * Haemolymph trehalose was determined by the anthrone method 3 hr after the injections. When the dose of these agents was lowered, their effect on haemolymph trehalose level was much reduced or abolished (Table 2). The effect of injecting the mixture (20/d) of any of two, among theophylline (40raM), dibutyryl cyclic AMP (5.0raM) and CC extract (1/500 pair of the gland per 20/d), was also examined. An additional effect was observed when the mixture of dibutyryl cyclic AMP and CC extract was injected (Table 2).
In mammalian liver, cyclic AMP regulates the conversion of an inactive form of phosphorylase to active form by activating protein kinase and phosphorylase kinase. In insects also, an activation of phosphorylase in the fat body by CC extract has aheady been reported (STEELE, 1963; GOLDSWORTHY, 1970). Wlt;NS and GILBeRt (1967) further investigated this phenomenon and found a near maximal stimulation of phosphorylase activity' within minutes of CC extract addition. Recently, cyclic AMP-dependent protein kinase has been partially purified from [hi body of P. americana (Takahashi and Hanaoka, unpublishedJ. From results of above investigations and of this paper, it is proposed that strikingly similar regulatory mechanism to the mammalian liver system is operative in the insect fat body on the hyperglycemic action, it includes the following sequence: (1) the rise of cyclic AMP level in the fat body as the result of adenylatc cyclase activation, (2) activation of protein kinase, (3) activation of phosphorylase through the phosphorylase kinase reaction, (41 increased rate of glycogen degradation, and (5j the rise of haemolymph trehalose level. As previously reported, a high level of haemolymph trehalose induced by the injection of CC extract maintained for a rather long period (STFEUi, 1963; HANAOKA and TAKAHASHL 1976t. In the present study, the elevated level of cyclic AMP in the tht body, caused by the injection of CC extract, was also maintained for at least 5 hr (Fig. 2). Injection of theophylline or dibutyryl cyclic AMP also induced sustained hyperglycemia. The sustained high level of cyclic AMP in the fat body may possibly be responsible for the sustained hyperglycemia, but the details remain to be clarified. Acknowledgements The authors are grateful to Prof. E. OHNISHI and Dr. H. 1StlIZAKI.Nagoya University, for constant guidance and encouragement during the course of this work and for critical reading of the manuscript. This study was partly aided by the grant No. 030404 from the Ministr3 of Education, Japan.
DISCUSSION The present investigation demonstrated the presence of adenylate cyclase in the homogenate of fat body tissues of the cockroach, P. americana. The activation of the adenylate cyclase by the injection of CC extract indicated the involvement of adenylate cyclase system in the hyperglycemic action. Corresponding to the activation of the adenylate cyclase, the content of cyclic A M P in the fat body increased to near maximum within a few minutes after the injection of the CC extract, and this effect preceded the elevation of haemolymph trehalose concentration (Fig. 2). The injection of theophylline caused about 2-fold increase of haemolymph trehalose (Fig. 3). Dibutyryl cyclic A M P also showed hyperglycemic effect, although not quite strong. From these facts, it is concluded that cyclic A M P mediates the insect hyperg!ycemic factor.
REFERENCES BANDURSKI R. S. and AX~HtOD B. (1952) The chromatographic identification of some biological important phosphate esters. J. biol. Chem. 193, 405 410. BOWERS W. S. and FRIEDMAN S. (1963) Mobilization of fat body glycogen by an extract of corpus cardiacum Nature, Lond. 198, 685. BUTCHERR. W. (1975) Cyclic 3',5' nucleotide phosphodiesterase from bovine heart. In Methods ili Enzymolo,qy (Ed. by HARDMAN J. C. and O'MALLEY B. W.) 38, pp. 218 223. Academic Press, New York. GOLDSWORTHYG. J. (1970) The action of hyperglycacmic factors from the corpus cardiacum of Locusta migratoria on glycogen phosphorylase. Gen. comp. Emloe. 14, 78 85. HANAOKAK. and TAKAHASHIS. Y. (1976) Effect of a hyperglycemic factor on haemolymph trehalose and fat body carbohydrates in the American cockroach. Insect Biochem.
Adenylate cyclase in the fat body Kuo J. F. and GREENGARD P. (1970) Cyclic nucleotidedependent protein kinase activated by guanosine 3',5'-monophosphate. J. biol. Chem. 245, 2493-2498. LOWRY O. H., ROSEBROUGHN. J., FARR A. L., and RANDALL R. J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265-275. MORDUEW. (1969) Hormonal control of Malpighian tube and rectal function in the desert locust, Shistcerca gregaria. J. Insect Physiol. 15, 273-285. RALPH C. L. and MCCARTHY C. (1964) Effect of the brain and corpus cardiacum extracts on haemolymph trehalose of the cockroach, Periplaneta americana. Nature, Lond. 203, 1195. STEELEJ. E. (1963)The site of action of insect hyperglycemic hormone. Gen. comp. Endoc. 3. 4(~52.
99
STEELE J. E. (1964) The activation of phosphorylase in an insect by adenosine Y,5' monophosphate and other agent. Am. Zool. 4, 328. TAKAHASHIS. Y., OHOKAT., HANAOKAK., KAGEYAMAT., and OHNISHIE. (1975) Cyclic nucleotide-dependent protein kinases in the eggs of the silkworm, Bombyx mori. Occurrence of two cyclic nucleotide-dependent protein kinases and changes in the activity during development of eggs. Develop. Growth Diff. 13, 97-106. WHITE A. A. and ZENSERT. V. (1971) Separation of cyclic Y,5'-monophosphate from other nucleotides on alumina oxide columns. Applicapion to the assay of adenyl cyclase. Anal. Biochem. 41, 372-396. WIENS A. W. and GILBERTL. I. (1967) Regulation of carbohydrate mobilization and utilization in Leucophaea maderae. J. Insect Physiol. 13, 779 794.
ADENYLATE CYCLASE SYSTEM A N D THE HYPERGLYCEMIC FACTOR IN THE COCKROACH, PERIPLANETA AMERICANA KAZUNORI HANAOKA and Susv~crc Y. TAKAHASHI Biological Institute, Faculty of Science, Nagoya University, Nagoya 464, Japan
(Received 22 April 1976) Abstraet--Adenylate cyclase (EC 4.6.1.1) and cyclic AMP in the fat body of the cockroach, Periplaneta
americana, were investigated in relation to the mode of action of the hyperglycemic factor. The adenylate cyclase was activated about 3-fold by the injection of an extract of the corpora cardiaca. Concomitantly with the activation, the cyclic AMP content in fat body was increased nearly twice, preceding the rise of haemolymph trehalose concentration. Injection of theophylline or dibutyryl cyclic AMP induced a sustained hyperglycemia in the cockroach. These results strongly indicate the involvement of adenylate cyclase system in the hyperglycemic action of the corpora cardiaca extract.
INTRODUCTION THE HYPERGLYCEMICfactor raises haemolymph trehalose level by exhausting the glycogen stored in fat body (STEELE, 1963; BOWERS and FRWDMAN, 1963; RALPH and McCARXHV, 1964; HANAOKA and TAr,AHASm, 1976). An enzymic basis for the glycogenolysis has been attributed to the activation of glycogen phosphorylase in fat body (STEELE, 1963; WRENS and GILBERT,1967; GOLDSWORTHY,1970). However, very little has been revealed about the detailed mechanism of the hormonal action. STEELE (1964) reported that adenosine 3':5'-monophosphate (cyclic AMP) was able to mimic the action of the hyperglycemic factor (activation of the glycogen phosphorylase) when added to a fat body preparation incubated in a saline medium, suggesting the mediation of cyclic AMP. In this study, we measured the activity of adenylate cyclase (EC 4.6.1.1) and the level of cyclic A M P in the fat body after injection of the aqueous extract of the corpora cardiaca (CC). The effect of the injection of N6-2'-0-dibutyryl adenosine 3"5'-monophosphate (dibutyryl cyclic AMP) and also of theophylline on the haemolymph trehalose level was analyzed, and the mode of action of the hyperglycemic factor is discussed.
MATERIALS AND METHODS
Experimental animals Cockroaches were reared on mouse food and water at 25 + 1°C. Adult males, 20 to 30 days after adult ecdysis, were used for experiments.
Chemicals Cyclic AMP, adenosine 5'-monophosphate (Y-AMP), adenosine 5'-triphosphate (ATP), crcatine phosphokinase creatine phosphate, dibutyryl cyclic AMP, and bovine heart phosphodiesterase were obtained from Boeringer Mannheim GmbH. [~-32p]-ATP and [8-3H]-adenosine Y:5'-monophosphate were purchased from New England Nuclear Corp. Dowex 1-X8 resin (2(X)-400mesh) was obtained from Dow Chemicals, and neutral aluminium oxide from M. Woelm. Other chemicals were of reagent grade.
Assay of adenylate cyclase Fat body tissues were dissected from 4 males in ice-cold 0.9% NaC1 solution and were homogenized in a glass homogenizer for 5 min. with 2 volumes of cold 10 mM Tris-HC1 buffer, pH 7.5, containing 5 mM 2-mercaptoethanol and 1 mM ethylenediamine tetraacetate (disodium salt). The homogenate was used for the enzyme preparation for incubation. The reaction mixture contained 1.25raM [~t-32p]-ATP (1 × 106c.p.m.). 5.0mM MgCl,, 5.0mM NaF, 1.0mM theophylline, 0.25 mM cyclic AMP, 10 mM creatine phosphate, l0 ~g creatine phosphokinase, 1.0mM 2-mercaptoethanol, 45 mM Tris-HC1 buffer, pH 7.5, and tissue homogenate (ca. 0.1 mg protein): total volume was made up to 200/d with water. The reaction was initiated by adding the tissue homogenate. After incubation at 30°C with shaking for the desired time, the reaction was terminated by immersing the tubes into a boiling water-bath for 3 min and then cooled in an ice-bath. To each tube was added 2.0ml of 50 mM Tris-HC1 buffer, pH 7.5, and the precipitate was centrifuged off. The supernatant was applied to a column (0.5 x 3 cm) of neutral alumina and the fraction not absorbed was collected (WHITEand ZENSER, 1971). The eluates were dried on a rotary evaporator. The dried materials were dissolved in 1 ml of water, and the radioactivity of the solution was counted in a liquid scintillation counter, TEN GSL-163,
96
KAZUNORI HANAOKA AND SUSUMI~ Y. TAKAHASIII
using the dioxane scintillator consisting o1 PPO6g, dimethyl P O P O P 0.27g and naphthalene 112 g in I1 dioxane. Protein concentration in the tissuc homogenate was determined by the method of LOWR~ et al. (1951).
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Determination ¢ff cyclic A M P in the ahdomimd.lia body The abdominal fat body tissues from 5 males were homogenized with a glass homogenizer in 10 vols of cold 5% trichloroacetic acid (TCA). Belore homogenization. [8-3HI-cyclic AMP (20{X)0counts mini was added to the solution to know the recovery of cyclic AMP. After removal of the precipitate by centrifugation, the supernatant was washed with ethylether 5 times. The aqueous phase was taken and neutralized with 1 M Tris. Cyclic AMP in the solution was purified using columns of neutral alumina and Dowex l-X8 (200-400 mesh, C1 form), according to the method of TAKAHASHI et al. 09751. The fraction containing cyclic AMP was diluted with an appropriate volume of water and used for determination of cyclic AMP. Content of cyclic AMP was determined according to the method of Kuo and GREI!N(IARI)(1970) using cyclic AMP-dependent protein kinase. Assay and preparation of cyclic AMP-dependent protein kinase from the developing adults of the silkworm, Bomby.,: mort. were done according to the method reported previously (TARAitASlnet al., 1975).
Thin layer chromatography aml palWr chromatoHraph )' Thin layer chromatography was carried out on a Wako silica layer PA plate (6 × 2(Icm) with a solvent system of isopropanol water 28'~, ammonia water (7:2: 1. v.v). Ascending paper chromatography was carried out on Toyo filter paper No. 51 with three solvent systems: isopropanol water 28"0 ammonia water (7:2:1.x v), n-butanol acetic acid water (4:1: I, vv) and ethanol 0.5 M ammonium acetate (5:2, v/v). Spots of the authentic Y-AMP and cyclic AMP, which wcrc co-chromatographed with the radioactive products, were visualized by' spraying Hanes lsherwood reagent (BANI)t;RSKI alld AXEI.R()I). 1952). The radioactivity of the scraped silica gel or paper strip was measured directly' with a liquid scintillation cotinter using dioxane scintillator.
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TIME
Fig. 1. Formation of cyclic AMP b} the homogenate ol fat body tissues. Cockroaches were injected with 10HI of CC extract ( 1/4(I pair of the gland). After 1 hr, the homogenate was prepared, lbllowed by the incubation with [~-3ep]-ATP under the standard assay conditions. a n d Methods. In any case, the radioactive spot corresponding to the authentic cyclic A M P was revealed. W h e n the radioactive product had been pre-treated with bovine heart phosphodiesterase tinder the assay condition as described by BuwctniR (1975). it was convetted to the substance whose Rf value was identical with the authentic 5 ' - A M P on the thin layer chromatogram. Therefore, it is concluded that cyclic A M P is formed by the fat body homogenate from A T P under the asaty conditions. As shown in Fig. 1, production of cyclic A M P is linear with time for ;.it least 15 min, a n d is proportional to the protein concentration of the homogenate (protein content was less than 100Hg).
Preparation o/the corpora cardiaca c.vtract aml deterruination of haemolymph trehalosc
E~i'ct o! the ilijection o! ('C t_,.\lP~.lct otl lilt" acticitv o[ adenylate c3,clase
The methods of the preparation of ('C extract, and of determination of haemolymph trehalose were described in the preceding paper {HANAOKA and TAKaHASHL 1976).
Effect of the injection of CC extract on adenylatc cyclase was investigated by comparing between the
RESULTS
Enzymic .lbrmation o] cyclic A M P by Jar body homogem, te The h o m o g e n a t e of fat body tissues from male adult cockroaches, which had previously been injected with CC extract, was quickly prepared a n d incubated with [~-32p]-ATP under the assay conditions described in Materials a n d Methods. The incubated mixture was applied on a neutral alumina column, a n d the fraction not adsorbed on the column was collected. Identification of the radioactive product in the eluate as cyclic A M P was carried out by thin layer c h r o m a t o g r a p h y a n d paper c h r o m a t o g r a p h y with the solvent systems described under Materials
Table 1. Adenylate cyclase activity in tile homogenate ol fat body tissues Pretreatment None Injected with CC extract* ( 1,'40 gland-pair)
Cyclic AMP formed (pmoles/10 min/mg protein ) 27.5 31.2 92.5 98.S I 01.3 105.4
* The amount of injected CC extract was equivalent lo 140 pair of the gland. Alter I hr at 25 C., the fat body homogenates were prepared and adenylate cyclase acti,&v was determined.
Adenylate cyclase in the fat body
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Fig. 2. Time-course of changes in the level of cyclic AMP in the abdominal fat body and in the level of haemolymph trehalose after the injection of CC extract. The level of cyclic AMP in the abdominal fat body, O; and the level of haemolymph trehalose, A; in the CC extract (1/40 pair of the gland) injected cockroaches. The level of cyclic AMP in the abdominal fat body, O; and the level of haemolymph trehalose, A; in the water injected controls.
CC extract-injected and control insects of the activity in the homogenate of fat body tissues. Results are shown in Table 1. The injection of CC extract (1/40 pair of the gland) caused activation of the enzyme up to about 3 times after 1 hr.
Effect of the injection of CC extract on cyclic AMP level in the fat body Cockroaches were injected with CC extract (1/40 pair of the gland) or water as control. After various intervals, content of cyclic AMP in the abdominal fat body was determined. Results are shown in Fig. 2. The level of cyclic AMP in the abdominal fat body increased very rapidly after the injection of CC extract: the level of cyclic AMP increased from 475pmole/animal to 659pmole/anima1 during the first 5 min after the injection. After l hr, it reached the maximal value of 799 pmole/animal, and this level maintained for at least 5 hr. When distilled water was injected, the cyclic AMP level showed a slight increase after 5 min, but it returned to the original level soon after. The time-course of the initial change in the haemolymph trehalose level after the injection of the CC extract was examined in order to discover the relationship with the change of cyclic AMP level in the fat body. As shown in Fig. 2, the rise in the cyclic AMP level in the fat body is apparently preceding the elevation of haemolymph trehalose level.
Effect of the injection of theophylline and dibutyryl cyclic A M P The effect of the injections of theophylline and of dibutyryl cyclic AMP on the haemolymph trehalose level was examined in order to confirm further the
involvement of cyclic AMP in the hyperglycemic action. The time-course of the changes in haemolymph trehalose level after the injections is shown in Fig. 3. The injection of theophyUine (40 mM, 20 #1) exerted a strong hyperglycemic effect, whereas that of dibutyryl cyclic AMP (10 mM, 20 pl) did only a slight rise. In both eases, the elevated level of haemolymph trehalose maintained for a long period, resembling the effect of injecting the CC extract.
laJ tt)
t,0
9
"llad t'r I--
3O
20 laJ "I-
10 0
I
I
I
I
i
I
2
3
4
5
TIME
AFTER
INJECTION
Hrs.
Fig. 3. Time-course of changes in the level of haemolymph trehalose after the injection of theophylline or dibutyryl cyclic AMP. 40 mM theophylline, O; 10 mM dibutyryl cyclic AMP, El; distilled water (control), &. The vertical bar indicates the standard error of the mean. The volume of the injected solution was 20/~1, and the volume of haemolymph was determined to be 125 #1 by amaranth dilution method (MOROUE, 1969).
98
KAZUNORI HANAOKA AND SUSUMU Y. TAKAHASHI
Table 2. Effect of injection of theophylline and dibutyryl cyclic AMP on the haemolymph trehalose level Haemolymph trehalose* concentration (mg/ml)
Injection Experiment l
distilled water 0.05 mM dibutyryl cAMP 0.25 mM dibutyryl cAMP 1.0 mM dibutyryl cAMP 10 mM dibutyryl cAMP 2.0mM theophylline 20 mM theophylline 40 mM theophylline
17.2 ,+ 0.46 21.2 ,+ 2.35 24.9 _+ 0.21 24.5 _+ 2.33 24.6 + 1.32 21.2 ,+ 2.(X) 26.2 + 0.32 32.1 + 1.32
Experiment 2
distilled water 5.0 mM dibutyryl cAMP 40 mM theophylline CC extract (1/500 pair/20 #1) 5.0mM dibutyryl cAMP plus 40 mM theophylline CC extract plus 40raM theophylline CC extract plus 5.0 mM dibutyryl cAMP
17.2 _+ 0.80 22.3 _+ 0.76 a 27.8 _+ 0.76 b 28.5 +_ 0.54 c 28.1 4- 1.54d 30.3 +_ 1.32 e 32.9 + 1.91 f
Comparison of the means: a-f. P < 0.005: c-f, P < 0.1: b-d, c-e, not significant. * Haemolymph trehalose was determined by the anthrone method 3 hr after the injections. When the dose of these agents was lowered, their effect on haemolymph trehalose level was much reduced or abolished (Table 2). The effect of injecting the mixture (20/d) of any of two, among theophylline (40raM), dibutyryl cyclic AMP (5.0raM) and CC extract (1/500 pair of the gland per 20/d), was also examined. An additional effect was observed when the mixture of dibutyryl cyclic AMP and CC extract was injected (Table 2).
In mammalian liver, cyclic AMP regulates the conversion of an inactive form of phosphorylase to active form by activating protein kinase and phosphorylase kinase. In insects also, an activation of phosphorylase in the fat body by CC extract has aheady been reported (STEELE, 1963; GOLDSWORTHY, 1970). Wlt;NS and GILBeRt (1967) further investigated this phenomenon and found a near maximal stimulation of phosphorylase activity' within minutes of CC extract addition. Recently, cyclic AMP-dependent protein kinase has been partially purified from [hi body of P. americana (Takahashi and Hanaoka, unpublishedJ. From results of above investigations and of this paper, it is proposed that strikingly similar regulatory mechanism to the mammalian liver system is operative in the insect fat body on the hyperglycemic action, it includes the following sequence: (1) the rise of cyclic AMP level in the fat body as the result of adenylatc cyclase activation, (2) activation of protein kinase, (3) activation of phosphorylase through the phosphorylase kinase reaction, (41 increased rate of glycogen degradation, and (5j the rise of haemolymph trehalose level. As previously reported, a high level of haemolymph trehalose induced by the injection of CC extract maintained for a rather long period (STFEUi, 1963; HANAOKA and TAKAHASHL 1976t. In the present study, the elevated level of cyclic AMP in the tht body, caused by the injection of CC extract, was also maintained for at least 5 hr (Fig. 2). Injection of theophylline or dibutyryl cyclic AMP also induced sustained hyperglycemia. The sustained high level of cyclic AMP in the fat body may possibly be responsible for the sustained hyperglycemia, but the details remain to be clarified. Acknowledgements The authors are grateful to Prof. E. OHNISHI and Dr. H. 1StlIZAKI.Nagoya University, for constant guidance and encouragement during the course of this work and for critical reading of the manuscript. This study was partly aided by the grant No. 030404 from the Ministr3 of Education, Japan.
DISCUSSION The present investigation demonstrated the presence of adenylate cyclase in the homogenate of fat body tissues of the cockroach, P. americana. The activation of the adenylate cyclase by the injection of CC extract indicated the involvement of adenylate cyclase system in the hyperglycemic action. Corresponding to the activation of the adenylate cyclase, the content of cyclic A M P in the fat body increased to near maximum within a few minutes after the injection of the CC extract, and this effect preceded the elevation of haemolymph trehalose concentration (Fig. 2). The injection of theophylline caused about 2-fold increase of haemolymph trehalose (Fig. 3). Dibutyryl cyclic A M P also showed hyperglycemic effect, although not quite strong. From these facts, it is concluded that cyclic A M P mediates the insect hyperg!ycemic factor.
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