Fate of glucose in haemolymph of the American cockroach, Periplaneta americana

J. Insect Physiol.. 1977. Vol. 23. pp. 525 to 529. Pergamon Press. Printed in Great Britain.

FATE OF GLUCOSE IN H A E M O L Y M P H OF THE AMERICAN COCKROACH, P E R I P L A N E T A AMERICANA J. H. SPRING,* J. R. MATTHEWS,'t"and R. G. H. DOWNER+ Department of Biology, University of Waterloo, Waterloo. Ontario, Canada (Receired 29 October 1976)

Abstract--The rate of removal of high concentrations of glucose (10/~g/gl haemolymph) from haemolymph of adult male cockroaches, Periplaneta america~m, was studied in normal and ligated insects. More than 50°0 of the injected glucose is removed from the haemolymph of normal insects within 20 min of injection. A period of rapid trehalose synthesis occurs during the initial 10 min following injection of glucose into the haemocoele, and this is succeeded by a period of glycogen synthesis. The results are discussed in terms of earlier observations on "stress-induced hypertrehalosemia' and the possible involvement of a glycogenic agent.

INTRODUCTION TI-~ mGr~ levels of glycogen which occur in the fat body of many insect species testify to the existence of an active pathway for glycogen synthesis, and the biochemical properties of the glycogen-synthetasecomplex have been investigated in several species under in vitro conditions (VARDANIS, 1963; MtrgpI-IV and WYATT, 1965). However little information is available to describe the process of glycogen synthesis in the intact insect. When trace amounts of 1*C-glucose are injected into the insect haemocoele, most of the label rapidly appears in the haemolymph trehalose fraction (TREHERNE, 1958; BRICTEAUX-GREGOIRE et al., 1964, 1965; LIPKE et al., 1965; ELA et al., 1970) with the primary site of conversion being the fat body (CLEMENTS, 1959; CANDY and KILBY, 1959; CLEGG and EVANS,1961: SAITO, 1963; MURPHY and WYATT, 1965). Very little label is recovered from the fat-body glycogen fraction under these conditions. In the american cockroach, Periplaneta americana a period of rapid trehalose synthesis results from experimental handling of the insect and, although most of the additional trehalose which appears in the haemolymph is derived from glycogenolysis within the fat body, (MATTHEWS and DOWNER, 1974) it is probable that the glucose---, trehalose pathway (WYATT, 1967) is also activated. Thus when small amounts of 14C-glucose are injected into the haemocoele the rapid appearance of label in haemolymph trehalose may be a consequence of 'stress-induced hypenreha* Present address: Department of Zoology, University of British Columbia. Vancouver, B.C. * Deceased--29th May, 1976. +,To whom all correspondence and reprint requests should be addressed.

losemia" rather than a reflection of metabolic pathways in resting insects. The present report describes the rate at which large concentrations of glucose are removed from the haemolymph of intact and ligated cockroaches, and the respective rates at which the excess glucose is converted into trehalose and glycogen during and following the hypertrehalosemic response. MATERIALS AND METHODS Adult male insects were taken at 1 to 3 months after the adult ecdysis from a colony of Periplaneta americana maintained under standard conditions in this laboratory. Rearing conditions and experimental procedures for injection, collection of haemolymph and for the estimation of trehaiose have been described previously (MATTHEWS and DOWNER. 1973). Haemolymph glucose levels were determined by the glucose oxidase method as modified by MATTHEWS et al. (1976), and glycogen was extracted according to the method of VAN HAhl)EL (1965). The metabolic fate of injected radiolabelled glucose was determined by measuring the radioactivity associated with the total glucose, trehalose, and glycogen fractions from a single insect. The animal was homogenised for 30 sec in 40 ml 95~ ethanol in a Sorvall Omnimixer fitted with a microattachment. The homogenate was centrifuged at 2.33 x l0 s g for 15 min and the resulting pellet twice suspended in ethanol and recentrifuged. The pooled supernatants were evaporated to dryness and the residue dissolved in 0.5 ml distilled water before removing the lipid content by double extraction with 3.0ml diethyl ether. The aqueous layer was applied to a bilayered ionexchange column (WYATT and KAI_~, 1957) from which neutral lipids were eluted with 5.5 ml distilled

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J.H. SPRING,J. R. MATTSEws AND R. G. H. DOWNER RESULTS

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Fig. t. Rate of removal of glucose from haemolymph of adult male Periplaneta americana following injection of 1.8 mg glucose into the haernocoele. (The number of determinations is shown in parentheses).

water. Trehalose and glucose were separated by thin layer chromatography (MArrr~ws and Dowr~a, 1973). Duplicate eluent samples were run on adjacent lanes and the sugars in one lane of each pair visualised with sulphuric acid: ethanol (1:9, v:v). Corresponding areas of the adjacent lane were cut out and placed, gel side up, in a scintillation vial to which was added 200 ltl distilled water, 3.0 ml 2-methoxyethanol and 10.0 ml tluor (2.0g dimethyl POPOP, 6.0g PPO, 1.01 toluene). Glycogen was estimated from the pellet remaining after ethanol extraction by adding 3.0 ml 30°.o potassium hydroxide and heating at 100°C for 10 min. To this solution was added 0.1 ml saturated sodium sulphate solution and 7.0 ml 95°~, ethanol. The mixture was centrifuged, and the dried pellet resuspended in 1.0 ml distilled water. Protein was precipitated by the addition of 2.0 ml 0.6 M perchloric acid (WYATT and KALr, 1957). Glycogen was precipitated from the supernatant by the addition of 0.1 ml saturated sodium sulphate solution and 10.0ml 95°o ethanol. The dried pellet was resuspended in 1.0 ml distilled water, and a 200/zl aliquot transferred to a scintillation vial containing 3.0 ml 2-methoxy ethanol and 10 ml fluor. Insects were ligated by means of a cotton thread tied tightly around the neck. Following ligation the head was removed anterior to the ligation. Ligated animals were maintained for 12 hr in moist petri dishes prior to experimentation.

The capacity of the insect to remove high concentrations of glucose from the haemolymph was determined by injecting insects with 1.8 mg glucose and estimating the glucose content of 5-#1 haemolymph aliquots at varying times following injection. The results which are summarised in Fig. 1 indicate that over half the injected amount (assuming a blood volume of approximately 175#I--MATTHEWS and DOWNER, 1974) is removed within 20 rain of injection. and within 1 hr the haemolymph glucose concentration is reduced to 0.50 + 0.12 #g/#l. Some of the glucose may be converted to either trehalose or glycogen, and, in order to determine the biochemical fate of the substrate, radiolabelled glucose was injected into the haemocoele and the distribution of label in the three major carbohydrate fractions analysed at varying times following injection. Glucose was prepared so that a single 10-/~1 injection contained 1.50mg glucose and 3.72 x 105dpm (t4C)-glucose. The disappearance of (l~C)-glucose from haemolymph is shown in Fig. 2A and closely parallels the earlier observations on removal of cold glucose from haemolymph (Fig. 1). Of particular interest in this regard is the apparent discontinuity in the rate of removal of glucose which occurs between l0 and 20 min following injection. In both curves a statistically significant reduction in the rate of removal is observed during this period. Fig. 2 also demonstrates an initial period of trehalose synthesis during

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Fig. 2. Incorporation of ~'~C-glucose into trehalose and glycogen of adult male Periplaneta americana. (Insects were injected with a solution containing 3.72 x 10~dpm t'C-glucose and 1.5 mg cold glucoset.

Fate of glucose in haemolymph of P. americana 22

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Fig. 3. Elevation of trehalose concentration in haemolymph of adult male Periplaneta americana following injection of 1.8 mg glucose into the haemocoele (The number of determinations is shown in parentheses). the first 10 to 20min following injection, but little or no trehalogenesis after this time. The rate of glycogen synthesis, by contrast, appears to increase at about 20 min after the injection (Fig. 2). The initial burst of trehalose synthesis following the injection of large concentrations of glucose was confirmed by measuring the absolute concentrations of haemolymph trehalose at varying times after injecting 1.8 mg glucose into the insect haemocoele. The results

527

are presented in Fig. 3 and demonstrate that haemolymph trehalose levels increase during the initial 20min following injection, but no appreciable increase is observed after this time. The major site of glycogen synthesis in the insect is the fat body. and the rate of glycogenesis in this tissue was investigated following the injection of 1.5 mg glucose and 1.68 × 1 0 6 dpm (14C)-glucose. Fat bodies were rapidly dissected at varying times following injection and were placed in potassium hydroxide solution for extraction of glycogen. The general shape of the glycogen synthesis curve for fat body (Fig. 4) resembles that presented previously for the whole insect (Fig. 2). However the onset of rapid glycogenesis is evident at 30min following the injection rather than at 20 min as before. Carbohydrate metabolism is known to be influenced by a number of humoral factors, some of which are contained within the head. In order to obtain preliminary information on the possible contribution of such factors to the disappearance of excess glucose from haemolymph, the removal of radiolabelled glucose from haemolymph was studied in insects from which the head was removed 12hr before injection. The distribution of label between glycogen, glucose, and trehalose was measured at various times following the injection of a solution containing 1.5 mg glucose and 1.31 × 106 dpm (l'*C)-glucose. The results which are presented in Fig. 5 show two important differences from those obtained with intact insects (Fig. 2J. The initial rapid burst of trehalose synthesis does not terminate after 20min. and trehalose I00 8O

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Fig. 4. Incorporation of ~4C-glucose into fat body glycogen of adult male Periplaneta americana. (Insects were injected with a solution containing 1.68 x 106dpm 14C-glucose and 1.5 mg cold glucose).

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Fig. 5. Incorporation of 14C-glucose into trehalose and glycogen of adult male Periplanera americana, ligated at the neck. (Insects were injected with a solution containing 1.31 × l0 s dpm J'*C-glucose and 1.5 mg cold glucose).

528

J.H. SPRING.J. R. MATTHEWSAND R. G. H. DOWNER

Table 1. Effect of head-ligation on haemolymph trehalose levels of adult male Periplaneta americana Treatment Ligated Non-ligated

Trehalose concentration (#g/#l) 15.2 -r 0.9* 17.1 + 0.6

* Mean + standard error of the mean for 8 determinations. levels continue to rise after this time. Secondly almost no glycogen synthesis is observed in the first 20 min. although glycogenesis proceeds rapidly after this time. In evaluating the results of experiments with ligated insects the effect of ligation on haemolymph trehalose levels is required. Accordingly the concentration of trehalose in the haemolymph of tigated insects was determined (Table II. It is apparent that ligated insects show a decrease in trehalose levels which are 11°,', below those of normal insects. DISCUSSION Glucose levels in the resting cockroach are low. and even during periods of exercise and/or 'stress' the glucose concentration of haemolymph remains at less than 1/tg/#1 (MATTh'EWSet al.. 1976). Thus the ability of the american cockroach to rapidly remove, from the haemolymph, glucose at concentrations as high as 10/.~3/#1 (assuming haemolymph volume to be 175 #l--MArrm:~ws and DOWNER, 1974) indicates a considerable capacity within the insect to regulate haemolymph glucose levels. Experimental handling of cockroaches causes rapid activation of glycogenolytic processes in the fat body and subsequent release of trehalose into haemolymph tMArrm='WS and DOWNER, 19741. Trehalose may also be synthesised directly from glucose through the intermediates glucose-6-phosphate and trehalose-6-phosphate (WYATT, 1967) and it is probable that this pathway is similarly activated by handling. The maximum hypertrehalosemic response is evident between 10 and 15min after 'stressing' the insect (MArTHEWS and DOWNER. 1974), and it is reasonable to expect that during this time any excess glucose present in the haemolymph will be converted to trehalose. This expectation is confirmed by the results presented in Fig. 2A and 2B which show that radiolabelled glucose becomes rapidly incorporated into trehalose during the ten minute period following injection of the radiolabelled substrate into the insect haemocoele. Furthermore the results presented in Fig. 3 indicate that the rate of trehalose synthesis in insects which have been injected with large concentrations of glucose is similar to that induced by handling. In the present study, haemolymph trehalose levels rose from 15.8#g/#1 to 20.0#g/#l during the twenty minute period following injection. This represents a rate of trehalose synthesis equivalent to 37 :zg/min/insect and compares with the rate of 43 #g/rain/insect reported

for 'stressed" insects during a similar period (MATTI.-IEWS and DOWER, 1974). Therefore, the observed hypertrehalosemic response may result from the trauma of injection rather than from the presence of abnormally high concentrations of glucose. When tracer amounts of ~'~C-glucose were injected into locusts (TRt~Iq~R~, 1958) and blowflies (Ct~GG and EVANS, 1961) less than 10% of the radiolabelled glucose remained in the haemolymph after l0 min and thus only the immediate routes of glucose metabolism were detected. Fig. 2A shows that in the present investigation 36.8 + 4.8°0 of the injected t*C-glucose remains in the haemolymph alter 20 min and is available to participate in the metabolic activities that occur following the initial 'stress-induced hypertrehalosemic' response. Consequently it has been possible to detect a period of pronounced glycogen synthesis that begins at about 20 min after injection. Some glycogen synthesis is also observed during the initial 10 rain period, and as the experimental procedure involved extraction of glycogen from the whole insect it is probable that at least some of this glycogen is synthesised in the musculature. Glycogen synthetase has been demonstrated in the thoracic musculature of locusts (TRIVELLONI, 1960~ and muscle glycogen is an important source of energy during flight in cockroaches (POLACEK and KUBISTA, 1960; DOWNER and MA'rTH~WS, 1976). A depressed rate of glycogen synthesis in the muscles of ligated insects may be advanced to account for the reduced rate of removal of glucose from the haemolymph of headless insects (Fig. 5A), and the virtual lack of glycogenesis during the initial 20 min period lFig. 5C). Ligated insects are sluggish and less responsive to environmental stimulation than normal insects, and it is probable that the reduced muscular activity is reflected in reduced rates of glycogen synthesis in the musculature. However the major site of the increased rate of glycogen synthesis that follows the hypertrehalosemic response, is the fat body IFig. 4). The apparent retardation in the onset of glycogen synthesis that is observed in isolated fat body !Fig. 4) compared with the whole insect (Fig. 2C) may be an artifact resulting from the extra time required for dissection of fat body tissue. MATTHEWS and DOWNER (1974) demonstrated rapid glycogenolysis in fat body during the time taken for dissection, and under these conditions the most recent glucose molecules to be incorporated into the polysaccharide would be the first to be hydrolysed. Thus the appearance of radioactivity in the glycogen fraction may be delayed. The results of Figs. 1 to 4 are strongly suggestive of a 'metabolic switch' operating at between 10 and 20 rain following injection to arrest the incorporation of glucose into trehalose in favour of glycogen synthesis. Such a switch would explain the observed discontinuity in the glucose disappearance curve, but the mechanism awaits elucidation. MtTRPHY and WYATT (1965) demonstrated the inhibition of trehalose-6phosphate synthetase by trehalose and suggested that

Fate of glucose in haemolymph of P. americana this inhibition may be of physiological significance in the homeostatic regulation of haemolymph trehalose levels. Thus when haemolymph trehalose levels exceed a certain concentration, trehalose-6-phosphate synthetase may be inhibited and glucose-6-phosphate made available for glycogen synthesis. However, the results obtained with ligated insects (Fig. 5) indicate that the antagonistic processes of glycogenesis and trehalogenesis can proceed simultaneously and may also be under nervous or hormonal control. The onset of trehalose synthesis in response to handling is thought to be effected through a chemical activator of glycogenolysis similar to the 'hyperglycaemic factor' described by STEELE (1961, 1963) although the demonstration of this response in ligated insects suggests that the effector may not be released from the head. The continued trehalose synthesis after 10 min in ligated insects does not negate the proposal that trehalose inhibition of trehalose-6-phosphate synthetase is involved in the termination of stressinduced hypertrehalosemia, because the trehalose levels of ligated insects are reduced (Table 1) and inhibiting concentrations may not be achieved under these conditions. However the observation that glycogen synthesis is activated after 20 min in ligated insects, although trehalose synthesis is occurring concurrently, indicates the presence of an additional mechanism for controlling glycogen synthesis. Glycogenic factors have previously been proposed for several species of Diptera. LIE (1974) reported such a factor in the corpora allata of houseflies, NORM.ANN ~19751 indicated the release of a hypoglycaemic factor from the medial neurosecretory cells of Calliphora and SEECOI:Fand DEWHURST (1974) have claimed that insulin is an active hormone in Drosophila. The results of the present study suggest that either a glycogenic factor is present in the cockroach, or a substance which inhibits glycogen synthesis occurs in the haemolymph during the initial 20-min period following injection of large concentrations of glucose into the haemocoele. The enhanced glycogenic effect that is observed in ligated insects indicates that the proposed substance is released or generated from an abdominal or thoracic location, and studies are presently in progress to elucidate the existence, source and nature of the factor. Acknowledgements--The study was supported by operating grant No. A 6084 from the National Research Council of Canada to R. G. H. Downer.

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