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The relative importance of glucose and trehalose in the nutrition of the nervous system of the locust Schistocerca americana gregaria
Insect Biochem., Vol. 10, pp. 155 to 161. 'f;) Pergamon Press Ltd. 1980. Printed in Great Britain.
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THE RELATIVE IMPORTANCE OF GLUCOSE A N D TREHALOSE IN THE NUTRITION OF THE NERVOUS SYSTEM OF THE LOCUST SCHISTOCERCA AMERICANA GREGARIA R. H. C. STRANG~and E. M. CLEMENT* Department of Biochemistry, University of Glasgow, Glasgow, Scotland (Received 17 May 1979)
Abstract--0) The work presented, attempts by a combination of results obtained in vivo and in vitro, to determine the importance of trehalose as an energy substrate for the central nervous system when the locust •is at rest or flying. (2) In agreement with many previous authors, a substantial fall (> 70%) was found in the concentration of trehalose in the haemolymph in the course of a two hr tethered flight. The much lower concentration of glucose was more stable. (3) Oxygen uptake studies showed that trehalose was not an efficient substrate for the nervous system in vitro. Possible reasons for this were, (a) the high K,, (10 mM) of the trehalase of the nervous system for its substrate, and (b) the low concentration of the sugar in the nervous tissue, both in vivo and in vitro. (4) When the concentrations of glucose and trehalose found in the haemolymph were reproduced in vitro, the proportional utilization of the trehalose by the thoracic ganglia was much less than might have been expected on the basis of its high concentration. Key Word Index: Locust, Schistocerca americana gregaria, nervous system, trehalose, energy metabolism.
INTRODUCTION PREVIOUSwork in vitro with the thoracic ganglia of the nervous system of the locust Schistocerca americana gregaria indicated that carbohydrate in the form of glucose was an adequate substrate for oxidation by the tissue (CLEMENT and STRANG, 1978). The minimum concentration of glucose required to produce the maximum rate o f O 2 uptake was found to be 10 mM. Although glucose concentrations in excess of this value has been recorded for locusts (MAYER and CANDY, 1969; HANSEN, 1964), the present authors found concentration in the resting locust which did not exceed 4 mM. Others have recorded glucose concentrations from 0 to 10 m M (TREHERNE, 1958; HOWDEN and KILBY, 1956). Glucose is not the predominant carbohydrate in the haemolymph of the locust. In the resting insect trehalose is present in concentrations of from 40 to 80 m M (HOWDEN and KILBY,1956; HANSEN,1964; MAYER and CANDY,1969; JUTSUM and GOLDSWORTHY, 1976). The disaccharide may be expected to constitute a large part of the oxidizable substrate for the nervous system. This was found to be the case for the cockroach Periplaneta americana by TREHERNE (1960). However, the concentration of trehalose in the locust falls steeply during flight. Although there is some dispute about the extent of this fall, there is agreement that the fall is considerable. The concentration of trehalose available to the nervous system may thus vary quite widely. In contrast, the
concentration of glucose, although much lower, has been shown to be much more stable during flight (MAYER and CANDY, 1969). The experiments reported here were undertaken to investigate the relative importance of glucose and trehalose as energy substrates for the nervous system. It was also hoped to discover if the depleted concentrations of carbohydrate in the haemolymph after extended periods of flight, were still adequate to support the maximum rate of 0 2 consumption of the isolated thoracic ganglia, and whether there was any activation of the trehalase of the nervous system induced by flying.
M A T E R I A L S AND M E T H O D S Locusts
Adult locusts, Schistocerca americana gregaria, of both sexes were used. They were obtained from Larujon Locust Suppliers Ltd., Welsh Mountain Zoo, Colwyn Bay, North Wales. Fed on a diet of bran, they were kept at a temperature of 30-32°C. Reagents
The organic and inorganic chemicals were the product of BDH Ltd., Poole, Dorset, U.K. Purified enzymes and cofactors came from Boehringer Ltd., London. [U-t4C] glucose (sp. act. 230 mCi/mmole) was supplied by the Radiochemical Centre, Amersham, Bucks, U.K. PPO was supplied by International Enzymes ltd., Windsor, Berks, U.K. and POPOP by Koch-Light Ltd., Colnbrook, Bucks, U.K. Estimation of the carbohydrates in the haemolymph and ganglia of the nervous system
*Present address: Department of Human Anatomy, University of Oxford. t Communication to the first author.
After the insects had been cooled in ice for 15 min the head 155
156
R . H . C . STRANG AND E. M. CLEMENT
and tip of the abdomen were carefully cut off, and the bodies eviscerated. Haemolymph was collected from the bodies by centrifugation for 5 min in conical glass centrifuge tubes at the lowest possible speed (5-10g) on a bench centrifuge. Once the protein had been precipitated from the haemolymph with two volumes of 95°~ ethanol, the clear supernatant was lyophilised, and the carbohydrate redissolved in lml of distilled water, samples of which were taken for estimation of glucose and trehalose. The methods for the treatment of the ganglia of the thorax, and for the estimation of trehalose and glucose have already been described (STRANG et al., 1979).
Flight The locusts were flown on a simplified version of the roundabout of KROGH and WEIs-FoGH (1952). The insects were attached to light rods by means of wax yokes around their thoraces. The roundabout was driven both by the flight of the insects, and by a stream of compressed air. Throughout the flight, the locusts were kept warm by a current of air from a hair drier. Samples of the haemolymph and ganglia were taken immediately after the locusts had been removed from the roundabout after periods of flight of up to 2 hr.
Incubation of the ganglia Entire meso- and meta-thoracic ganglia were freed of adhering fatty and tracheal tissue and suspended in a saline based on that of HOYLE (1953) (omitting the MgCI 2 and NaHCO3; pH 6.8, osmolarity about 60~o that of haemolymph), saturated with warm, moist 100~o O z, and containing different concentrations of glucose and trehalose according to the experiment. Oxygen uptake studies were made at 37°C with an 02 electrode (Rank Bros., Bottisham, Cambridge, U.K.), containing 3 ml of medium and up to eight ganglia. Rates of O2 uptake were recorded as initial rates during the first 20 min of incubation. To follow the accumulation of glucose in the medium in the presence of trehalose, two meso- and two meta-thoracic ganglia were suspended in 0.5 ml of saline which was continually aerated with warm, moist 100~o 02, and kept at 37~oC. Samples (100 ,ul) were taken at 0, 10, 20 and 30 min. After each sampling, the volume was restored to 0.5 ml with the original saline. The total production of glucose was estimated making due allowance for the sugar removed in sampling. Blanks contained tissue only with no trehalose in the medium. A n y glucose produced under these conditions was subtracted from the production in the presence of trehalose.
Production and trapping of metabolic CO z In experiments to find the relative oxidative utilisation of trehalose and glucose, the latter was labelled with 14C, and the radioactive CO 2 accumulating in the medium was trapped and counted. U p to five pairs of meso- and metathoracic ganglia were suspended in 3 ml of medium with the appropriate concentrations of trehalose and glucose in an O z electrode. The medium had been previously saturated with 100~00 2. Throughout the experiments the specific activity of the glucose was constant at 0.03 mCi/mmole. After the ganglia had been introduced into the electrode, it was sealed, and the tissue allowed to deplete the oxygen in the electrode to a known extent over a period of 3-4 hr at 37°C. Without opening the electrode, samples (0.1-0.6 ml) of the medium were expelled via a plastic tube and steel needle into Warburg flasks which had been sealed with rubber closures. Three such samples were taken from each incubation. The flasks contained 0.1 ml of 12 M H2SO 4 in the outer compartment, and 0.25 ml of a 10~0 (w/v) methanolic solution of Hyamine 10-X hydroxide solution in the inner well. Trapping of the CO 2 was aided by means of a paper wick. By weighing the flask before and after the addition of the medium, the volume of each sample was calculated. The CO 2 released was trapped overnight at room temperature, and the whole of the contents
of the inner well transferred to 3 ml of a scintillator consisting of 5 g PPO and 0.3 g POPOP/1 of toluene. The trapped radioactivity was estimated on a Phillips liquid scintillation counter programmed for quench correction by the channels ratio method. The efficiency of counting was greater than 90'/,,.
The pH of the incubation media While most of these incubations were carried out at pH 6.8, a few were done at pH 6.2 to see if the lower pH would alter the proportions of trehalose and glucose oxidised by the tissue, in the absence of any definite information as to the pH which obtains in the haemolymph of flying locusts, the pH of 6.2 was chosen on the strength of two pieces of information: one was that this pH had been recorded in the haemolymph of cockroaches after a short period of flight (MATTHEWS el al., 1976); and the other was that while the haemolymph of insects buffers poorly in the normal physiological range, the buffering capacity rises steeply below 6.3 (LEVENBROOK, 1950).
Characteristics of trehalase (c~, ~ "glucoside-l-glucohydrolase; E.C.3.2.1.28) of the thoracic ganglia The m a x i m u m specific activity of the enzyme and its K,, for trehalose were estimated in the supernatants of homogenates of the thoracic ganglia. Adhering tissue was removed from several ganglia which were then blotted gently with a tissue, and weighed on a small piece of aluminium foil. Approximately 10 mg of tissue were homogenised in 50 #1 of ice-cold medium at pH 7.2 (CRABTREE and NEWSHOLME, 1972). The homogenate was centrifuged at 4°C for 5 min at 12,000 g. Preliminary assays had confirmed that no activity was lost in the pellet. Samples of the clear supernatant were added to a total of I ml of an assay medium which had the following composition: 70 m M Tris/HCl buffer (pH 7.2), 1 m M MgClz; 1.5 m M N A D P + ; 1.0 m M ATP: and 4-5U of hexokinase (E.C.2.7.1.1.) and 2-3U glucose 6-phosphate dehydrogenase (E.C.1.1.1.49). Concentrations of trehalose ranged from 2 to 50 mM. To obtain blanks either trehalose or N A D P ÷ was omitted. The rate of glucose production was followed spectrophotometrically at 340 nm. Addition of glucose to some assays confirmed that the activities of the auxiliary enzymes were in adequate excess. The estimates were made at 37°C. In addition to the estimates made at pH 7.2, the m a x i m u m specific activity of the enzyme was also determined at pH 6.2. The ganglia were homogenised in 0.05 M phosphate buffer at this pH. The homogenate was then carefully transferred into a total of 350/~1 of the buffer, and 50/d of 400 m M trehalose added to give a final concentration of 50 m M . At 1, 6 and 11 min after the addition of the trehalose 100 #1 samples were taken and the glucose estimated at pH 7.2 by the previously described assay. (It proved impossible to obtain a continuous record of glucose production as the hexokinase and glucose 6-phosphate dehydrogenase were rather inactive in the phosphate buffer at the lower pH). To ensure that the rate of glucose production was not being underestimated due to the metabolic loss of glucose in the discontinuous procedure, known amounts of glucose, in the concentrations encountered, were added to samples of the homogenates at pH 6.2. No detectable loss of glucose took place in the time required for the estimation.
Statistical treatment of results Estimation of the significance or otherwise of results, where this was in doubt, was made by the application of Student's t-test. RESULTS
Effect o f flight on the concentrations carbohydrates in the haernolymph The
average
concentrations
of
of
trehalose
the and
Trehalose and locust nervous system Table 1. Rates of 0 2 consumption by thoracic ganglia in
vitro Equivalent
in v i v o conditions --
Concentrations of carbohydrate Rate of 02 in medium (mM) uptake (/tmole/g/hr) Glucose Trehalose 10
--
---
Resting
4 4
--
--
1 hr of flight --2 hr of flight
3 3 -1
--
--
1
50 -50 25 -25 15 --
15
268___23 (12) 103_+ 13t ( 1 2 ) 250_+ 20 (8) 220_+ 10" (8) 184_+ 10" ( 8 ) 258 + 20 (6) 200_+ 15" (6) 140_+7" (6) 164-+5t (6) 165 _+8t ( 4 ) 146_+5"f" (4)
Rates are all initial rates. Each average (+ S.D.) is the mean of the number of estimates in the brackets. Figures marked thus * are all significantly different from the maximum rate of 02 uptake; * - p <0.01, t - p <0.001.
glucose ( + the standard deviation) in the resting locusts were found to be 54+ 12 mM and 4 + 1 mM respectively. After 1 hr of flight they had fallen to 26+11 mM and 3+0.7 mM, and after 2 hr the concentrations were 15___9 mM and 1+0.6 mM. Haemolymph samples were taken from six to twelve locusts to obtain these estimates. There was a great deal of variation from one locust to another even among the unflown insects, a fact reflected in the high standard deviation. During flight the inevitable differences in the vigour of individual's performances made the variations in carbohydrate concentrations proportionately greater.
Oxygen uptake by the isolated ganglia in the presence o f different concentrations o f glucose and trehalose The average concentrations o f glucose and trehalose found in resting and exercised locusts were tested both together and separately for their ability to support the oxidative metabolism of isolated thoracic ganglia. The rates of 0 2 uptake are shown in Table 1. Standards of comparison are, as a maximum, the rate found in the presence of 10 mM glucose (CLEMENTand STRANG, 1978) and as a minimum, the rate found in the absence of any exogenous substrate. When the sugars were present in the concentrations found in the haemolymph of the resting locust and after one hours flying, the combination of glucose and trehalose supported the maximum rate of 0 2 uptake. Alone, however, neither sugar was present in sufficient concentration to do this, and despite its very high concentration, trehalose was much less effective as a substrate than was glucose. Even in combination, the concentrations of glucose and trehalose at the end of two hours of flight were insufficient to support the maximum rate o f O 2 uptake recorded in vitro.
The characteristics o f the unpurified trehalase o f the thoracic ganglia The Lineweaver-Burk plot of the results of velocity against substrate concentration gave a straight line. La. 10/2
c
157
Table 2. Concentrations of trehalose in the thoracic ganglia in vivo and in vitro
Conditions
Concentrations of trehalose in the Concentration of medium or haemolymph trehalose in the (raM) ganglia (pmole/g)
Intact ganglia
In vitro In vivo
10 25 50 54-t- 12
2.2 + 0.01 4.9+0.3 6.3+0.2 6.4+_0.4
Tissues were washed by brief immersion in ice-cold medium free of trehalose, before homogenisation. The concentrations are the averages (+ S.D.) of four estimations in vitro, and six in vivo. From the plot the K m of the enzyme with reference to trehalose was found to be 1 x 10 -2 M (10 mM). The Vmax at infinite substrate concentration was found to be 1.53/~mole glucose produced/g/min. An average of four estimates (_+ S.D.) of the specific activity in the presence of 50 mM trehalose gave a figure of 1.45 _-4-0.3 #mole glucose produced/g/rain. When a phosphate buffer pH 6.2 was used, the average of these estimates (_+ S.D.) in the presence of 50 mM trehalose was found to be 2.56+ 0.11 #mole glucose produced/g/min.
Concentrations o f trehalose in the thoracic ganglia in vivo and in vitro In Table 2 are shown the concentrations in vitro after a 30 min incubation in the presence of different concentrations of trehalose, and these are compared with the average concentrations in the haemolymph and tissue in the living insect. It has already been shown (STRANG et al., 1979) that after 30 min of incubation in the absence of trehalose, the concentration in the tissue has fallen to 1.9 #mole/g and thus the concentrations found at this time in the presence of trehalose may be taken to reflect the tissue concentration in equilibrium with the medium. It is clear that at no concentration tested does the tissue concentration approach that of the medium; being a factor of four to five times lower at 10 and 25 mM trehalose and of about eight times lower in the presence of 50 mM trehalose. Also clear is the agreement of the tissue content in vivo with that in vitro, in the presence of similar concentrations of trehalose. This fact gives some confidence that reasonable extrapolations may be made from the results in vitro to the situation in vivo.
Efflux o f glucose from the ganglia in the presence o f trehalose In Table 3 are shown the rates of accumulation of glucose i n the medium in the presence of different concentrations of trehalose. Rates were quite linear during the first 30 min of the incubation, and have been corrected by subtraction of the blank values. There was clearly a brisk efflux of glucose from the intact tissue, which was proportional (at least at 10 and 25 mM trehalose) to the concentration of the disaccharide in the medium. When the total activity of the trehalose is estimated by combining these two rates of glucose production and utilization, the maximum rate found, 1.61 #mol glucose produced/g/min, is quite
158
R. H. C. STRANG AND E. M. CLEMENT Table 3. Estimation of the total activity of trehalase in the intact and homogenised ganglia
Concentration of trehalose in medium (mM)
Conditions Intact ganglia
25 50
0,46 _+0.09 0,97+0.13 1.11 +0.05
50 50
---
10
Hom6genised ganglia pH 7.2 pH 6.2
Rate of glucose accumulation in the medium Rate of glucose -+ S.D. (n = 4) consumption (a) (b) (/~mole/g/min)
Estimated total activity of trehalase ± S.D. (n = 6) (a) + (b)
0.36 0.39 0.50
0.82 1.36 1.6l 1.45_+0.3 2.56_+0.1
--
The rates of glucose consumption by the intact tissue (b) are based on the rates of oxygen uptake in the presence of the appropriate concentrations of trehalose. All incubations were carried out at 37°C ' similar to the figure of 1.45/zmole/g/min, which was the m a x i m u m specific activity of the trehalase at pH 7.2 f o u n d in h o m o g e n a t e s of the ganglia.
Relative utilization of trehalose and glucose by the intact ganglia in vitro The most i m p o r t a n t test of the relative usefulness o f trehalose a n d glucose would be the p r o p o r t i o n s of the C O 2 produced by the tissue originating from each c a r b o h y d r a t e when b o t h were present, as they would be in the insect. T o determine this, [U-14C]-glucose of a c o n s t a n t specific activity was mixed with unlabelled trehalose in the p r o p o r t i o n s which h a d been f o u n d in the h a e m o l y m p h , the ganglia incubated in these media, a n d the radioactivity in the C O 2 produced estimated. In Table 4 are presented the results. T o establish the figure when 100~o of the C O 2 was derived from glucose, the m e d i u m contained 10 m M [14C]-glucose only. W h e n trehalose was present in the medium, the CO 2 produced from glucose was almost always less
than this figure, as would be expected. The results, however, for the suitability of trehalose as a substrate for oxidation by the thoracic ganglia in competition with glucose, do not in any way contradict the impression gained from the Oz uptake studies. Glucose supplied a m u c h higher p r o p o r t i o n of the metabolic C O 2 t h a n its c o n c e n t r a t i o n would seem to warrant. This was especially true if the c o n c e n t r a t i o n s of the two sugars are those e n c o u n t e r e d u n d e r resting conditions, the glucose, a l t h o u g h only 3% of the total c o n c e n t r a t i o n in terms of glucose units, supplied 18~o of the C O r W h e n at the end of an h o u r ' s flight the c o n c e n t r a t i o n of the trehalose had fallen to less t h a n h a l f its original concentration, while that of the glucose h a d remained a b o u t the same, the ~o from glucose rose to 25~o. This increase was statistically significant (p < 0.05). If the calculations are based on the c o n c e n t r a t i o n s f o u n d by MAYER and CANDY (1969) the figures are even more dramatic, with only 3 2 ~ of the metabolic C O 2 coming from trehalose in the resting locust. After 30 min flying, when the
Table 4. Proportions of CO 2 derived from glucose and trehalose by intact ganglia in vitro Concentration of carbohydrate in medium Glucose trehalose (raM) 1.
10
2.
10
60
3.
4
60
4.
3
25
5.
15
40
6.
20
15
Total radioactivity trapped CO 2 _+S.D. (dpm) 10,950 ± 900 (6) 5,190 _+730 (3) 1,940 +40 (6) 2,780 + 190 (3) 7,460 + 510 (3) 10,450 _+580 (3)
°~o CO 2 derived from glucose
Ratio of glucose to trehalose (as glucose) (~)
Equivalent conditions in vivo
100 47
8
18
3
25
6
68
19
95
67
Resting (present authors) After one hour flight (present authors) Resting (MAYER and CANDY, 1969) After 30 min of flight (MAYERand CANDY, 1969)
Incubations were carried out in 3 ml of medium in an oxygen electrode at 37°C, and the consumption of oxygen noted. An equivalence ofO 2 uptake and CO 2 output was assumed, and the output standardised to 900 nmoles/ml. The quoted figures are averages of the number of incubations in brackets. In each incubation three samples were taken for counting. Carbohydrate concentrations do not exactly correspond to the averages found in vivo, but have been rounded off for convenience. The trapped radioactivity has also been rounded off to the nearest 10. The percentages of CO 2 derived from glucose in the presence of trehalose are all significantly different from the counts derived from glucose alone (p < 0.05) with the exception of No. 6.
Trehalose and locust nervous system
159
Table 5. Effect of flying and lowered pH on the utilization of trehalose by the intact thoracic ganglia
pH 6.8 6.8 6.8 6.2 6.2
Concentration of carbohydrate in the medium (mM) Glucose Trehalose 10 20 3 20 3
-15 25 15 25
Total radio-activity in trapped CO2 (dpm) % CO2 derived + S.D. from glucose 1 2 , 0 1 0 + 9 9 0(3) 11,770+ 1300 (3) 3,230+210 (3) 9,200+230 (3) 3,000+220 (3)
100 98 27 77* 25
Incubations were carried out and the C O 2 trapped as in Table 4. The ganglia were taken from locusts which had flown for one hr immediately prior to the experiment. The only conditions which resulted in a CO2 output from trehalose significantlydifferent from the equivalent conditions in Table 5, were those marked thus * (0.05 < p <0.1). concentration of glucose actually exceeds that of trehalose, the latter sugar contributes virtually nothing to the metabolic CO 2. Activation o f trehalase in the course of flight To the experiments in vitro two conditions were applied which were intended to mimic more closely the conditions in vivo. One was that the tissues used were those taken from insects which had flown for an hour. The other was that the pH of the medium was lowered to 6.2. The results of these modifications are shown in Table 5. The only conditions under which there was any evidence for an increased utilization of trehalose was at pH 6.2 in the presence of 20 mM glucose and 15 mM trehalose. Although the reduction of 18% in CO 2 coming from glucose seemed quite substantial, the statistical analysis shows that it has only marginal significance (0.05 < p < 0.1). DISCUSSION The decline in the concentration of total carbohydrate in the course of a two hour flight to 25% of the resting values, is in general agreement with previous findings. Reports range from the almost complete disappearance of carbohydrate as a result of extended flights (WEIs-FOGH, 1964; MAYER and CANDY, 1969; TIETZ, 1967), to a reduction of only 50% (ROBINSON and GOLDSWORTHY, 1976; JUTSUM and GOLDSWORTHY, 1976). The extent of the depletion of the carbohydrate has implications for the nervous system, which by analogy with the cockroach, (TREHERNE, 1960) may be expected to make considerable use of carbohydrate as an energy substrate. In fact ROBINSONand GOLDSWORTaY(1976) suggest that the homeostatic conservation of trehalose may be a mechanism to spare this carbohydrate for the use of tissues other than the flight muscle (e.g. the nervous system) in the course of a prolonged flight. There seems no reason to doubt that the oxidation of diglyceride by the flight muscle favoured by the action of adipokinetic hormone (ROBINSON and GOLDSWORTHV, 1977 a, b) does inhibit the utilization of trehalose by the muscle as has been reported on more than one occasion (ROBINSON and GOLDSWORTHY, 1976; FORD and CANDY, 1972). The findings of the present and other authors, however,
imply that under some circumstances, either the sparing mechanism is rather ineffective (due possibly to low titres of hormone and low concentration of diglyceride), or that it functions to produce a variety of final concentrations of trehalose in the haemolymph. The idea of variability has received recent confirmation in the results of A.M. TH. BEENAKKERS (personal communication) who, like GOLDSWORTHY and his colleagues, found that the depletion of carbohydrate ceases after a 50% decline from the resting concentration, but that there exists between individual locusts a wide variation in initial concenrations ranging from more than 220 mM to about 100 mM (as glucose). Although glucose is usually disregarded in the context of insects, as its concentration is generally much lower than that of the disaccharide, the fact that it is only in combination that the concentrations of glucose and trehalose found in vivo can support the maximum rate of 0 2 uptake in vitro, suggests that glucose is an equally important or even a more important source of energy for the nervous system. Even the total carbohydrate concentration, however, at the end of two hours of flight was found unable to support the maximum rate of 0 2 uptake. In this finding lies the implication that under such conditions some alternative source of energy, exogenous or endogenous, may be required. Glycogen in the nerve cord is a potential source of endogenous energy. Although there is evidence that glycogen in the isolated nerve cord does supply most of the energy in the absence of exogenous substrates (STRANGet al., 1979) it has yet to be confirmed that the same applies in vivo, when the ambient carbohydrate concentration is low. The high Ks of the trehalase means that the enzyme will function at rates less than the maximum when the concentration of substrate falls below 50 mM. In addition, the concentration of the disaceharide in the nervous tissue is much lower than in the surrouding medium, [a situation similar to that in the cockroach (TREHERNE, 1974)]. These two aspects, high Km and low tissue concentration, would seem sufficient to account for the inadequacy oftrehalose as a substrate, were it not for the fact that the efflux of glucose from the tissue is about twice the estimated rate of glucose oxidation. One explanation for these rather paradoxical results is that both enzyme and
160
R . H . C . STRANGAND E.M. CLEMENT
disaccharide are confined to some (presumably outer) compartment of the ganglia, where the concentration of the substrate approaches that of the surrounding fluid. Here also the maximum specific activity of the trehalase would be greater than that of the hexokinase, which would be more homogenous in its distribution. This would result in a greater production of glucose from trehalose than could be further metabolised, causing the glucose efflux found. Under the artificial conditions in vitro in the absence of glucose from the medium, there would be a concentration gradient down which the glucose could diffuse to the outside. In view of this, it was thought that the experiments involving trehalose alone gave a false impression of its utilization by the tissue. On the contrary, however, when intact ganglia were incubated in the presence of both sugars, the proportions of the CO 2 originating from trehalose were still low. It is interesting to note that the results reported here for the locust nervous system, are similar to those for the cockroach under the same sort of conditions (TREHERNE, 1960). TREHERNE found that with a ratio of trehalose to glucose of seventeen, the glucose was taken up 2.5 times as rapidly as the trehalose. The corresponding ratio in the case of the locust ranged from two to three for similar ratios of trehalose to glucose. Without exception, the insect trehalases so far investigated have optimum pH values between 5.5 and 6.5 (DuvE, 1972) and most have a K,, for trehalose of about l m M (GussIN and WYATT, 1965; GILBY et al., 1967). The trehalase of the flight muscle of the desert locust has a maximum specific activity of 4-10 #mole glucose produced/g/min at 37°C (depending on the method of activation) and a K mof 3mM (CANDY,1974; FORD and CANDY, 1972). Both these parameters indicate that the flight muscle is likely to be a more efficient user of trehalose as the concentration of the sugar falls. This would be perfectly consistent with the suggestion that the main function of the trehalose is to provide an immediately available reserve of carbohydrate for the almost exclusive use of flight muscle (BEDFORD, 1977). Although the deductions regarding the proportional utilization of trehalose were made on the basis of results obtained with tissue from resting locusts, there did not seem to be any proportional increase in the use of trehalose when the tissue from flown locusts was used in vitro. This would argue against any persisent activation of the trehalase. There is however some evidence, of a rather low statistical significance, that a lower pH does alter the proportional use of trehalose and glucose in favour of the former. Due to the lack of information about the pH values encountered in flight it is difficult to estimate the importance of this effect in vivo. The factors which control the activity of trehalase have so far eluded detection. In the intact ganglia the enzyme seems to be fully active, if the maximum specific activity found in homogenates is correct. If this is a true representation of the conditions in vivo, then the trehalose is likely to contribute glucose to the pool of that sugar in the haemolymph, and thus help to maintain the equilibrium between the two circulating sugars. To conclude, the importance of trehalose as an energy substrate to the nervous system depends very
much upon its concentration and upon that of its hydrolytic product, glucose. Of the two, glucose is far more effective in low concentrations, and in view of its greater consistency in the haemolymph under flying conditions, the monosaccharide may be relatively more important to the tissue. All the evidence indicates that the thoracic ganglia are not well adapted to the use of trehalose. Although the evidence is at present scanty, it may be, in any case, that the nervous tissue can utilise more than carbohydrate for its energy requirements. Acknowledgements--The authors gratefully acknowledge the valuable advice and information of Professor R. M. S. SMELUE,Dr. C. A. FEWSON,Dr. D. J. CANDYand Dr. A. M. TH. BEENAKKERS.
REFERENCES BEDFORD J. J. (1977) The carbohydrate levels of insect haemolymph. Comp. Biochem. Physiol. 57, 83-86. CANDY D. (1974) The control of muscle trehalase activity during locust flight. Biochem. Soc. Trans. 2, 1107-1109. CLEMENTE. M. and STRANGR. H. C. (1978) A comparison of some aspects of the physiology and metabolism of the nervous system of the locust Schistocerca gregaria in vitro with those in vivo. J. Neurochem. 31, 133-145. CRABTREEB. and NEWSHOLMEE. A. (1972) The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and glycerol 3-phosphate dehydrogenase in muscles from vertebrates and invertebrates. Biochem. J. 126, 49-58. DUVE H. (1972) Purification and properties of trehalase isolated from the blowfly Calliphora erythrocephala. Insect Biochem. 2, 445-450. FORD W. C. L. and CANDY D. J. (1972) The regulation of glycolysis in perfused locust flight muscle. Biochem. J. 130, 1101-1112. GILBY A. R., WYATT S. S. and WYATT G. R. (1967) Trehalases from the cockroach Blaberus discoidalis: activation, solubilization and properties of the muscle enzymes, and some properties of the intestinal enzyme. Acta biochim, pol. 14, 83-100. GusslN A. E. S. and WYATTG. R. (1965) Membrane-bound trehalase from Cecropia silkmoth muscle. Archs. Biochem. Biophys. 112, 626-634. HANSEN O. (1964) Effect of diet on the amount and composition of locust blood carbohydrates. Biochem. J. 92, 333-337. HOWDEN G. F. and KILBY B. A. 0956) Trehalose and trehalase in the locust. Chemy Ind. t453-1454. HOYLEG. (1953) Potassium ions and insect nerve muscle J. exp. Biol. 30, 121-133. JUTSUMA. R. and GOLDSWORTHYG. J. (1976) Fuels for flight in Locusta. J. Insect Physiol. 22, 243-249. KROGH A. and WEIS-FOGH T. (1952) A roundabout for studying sustained flight of locusts. J. exp. Biol. 29, 211-219. LEVENBROOKL. (1950) The physiology of carbon dioxide transport in insect blood. J. exp. Biol. 27, 184-191. MATTHEWSJ. R., DOWNER R. G. A. and MORRISONP. E. (1976) ct-glucosidase activity in haemolymph of the American cockroach, Periplaneta americana. J. Insect Physiol. 22, 157-163. MAYER R. J. and CANDY D. J. (1969) Changes in energy reserves during flight of the desert locust, Schistocerca gregaria. Comp. Biochem. Physiol. 31, 409-418. ROBINSON N. L. and GOLDSWORTHY G. J. (1976) Adipokinetic hormone and flight metabolism in the locust. J. Insect Physiol. 22, 1559-1564.
Trehalose and locust nervous system ROBINSON N. L. and GOLDSWORTHY G. J. (1977a) Adipokinetic hormone and the regulation of carbohydrate and lipid metabolism in a working flight-muscle preparation. J. Insect Physiol. 23, 9-16. ROB1NSONN. L. and GOLDSWORTHYG. J. (1977b) A possible site of action of adipokinetic hormone on the flight muscle of locusts. J. Insect Physiol. 23, 152-158. STRANG R. H. C., CLEMENTE. M. and RAE R. C. (1979) Some aspects of the carbohydrate metabolism of the thoracic ganglia of the locust Schistocerca gregaria. Comp. Biochem. Physiol. B62, 217-224. TIETZ A. (1967) Fat transport in the locust: the role of diglyceride. Eur. J. Biochem. 2, 236-232.
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TREHERNE J. E. (1958) Facilitated diffusion and exchange in absorption of glucose by the locust, Schistocerca gregaria. Nature, Lond. 181, 1280-1281. TREHERNE J. E. (1960) The nutrition of the central nervous system of the cockroach, Periplaneta americana. J. exp. Biol. 37, 513-533. TREHERNE J. E. (1974) In Insect Neurobiology (Ed. by TREHERNEJ. E.), p. 229, North Holland Press, Amsterdam. WEIS-FO6H T. (1964) Diffusion in insect wing muscle, the most active tissue known. J. exp. Biol. 41, 229-256.
0020-1700/80/0401-0443
$02.00/0
THE RELATIVE IMPORTANCE OF GLUCOSE A N D TREHALOSE IN THE NUTRITION OF THE NERVOUS SYSTEM OF THE LOCUST SCHISTOCERCA AMERICANA GREGARIA R. H. C. STRANG~and E. M. CLEMENT* Department of Biochemistry, University of Glasgow, Glasgow, Scotland (Received 17 May 1979)
Abstract--0) The work presented, attempts by a combination of results obtained in vivo and in vitro, to determine the importance of trehalose as an energy substrate for the central nervous system when the locust •is at rest or flying. (2) In agreement with many previous authors, a substantial fall (> 70%) was found in the concentration of trehalose in the haemolymph in the course of a two hr tethered flight. The much lower concentration of glucose was more stable. (3) Oxygen uptake studies showed that trehalose was not an efficient substrate for the nervous system in vitro. Possible reasons for this were, (a) the high K,, (10 mM) of the trehalase of the nervous system for its substrate, and (b) the low concentration of the sugar in the nervous tissue, both in vivo and in vitro. (4) When the concentrations of glucose and trehalose found in the haemolymph were reproduced in vitro, the proportional utilization of the trehalose by the thoracic ganglia was much less than might have been expected on the basis of its high concentration. Key Word Index: Locust, Schistocerca americana gregaria, nervous system, trehalose, energy metabolism.
INTRODUCTION PREVIOUSwork in vitro with the thoracic ganglia of the nervous system of the locust Schistocerca americana gregaria indicated that carbohydrate in the form of glucose was an adequate substrate for oxidation by the tissue (CLEMENT and STRANG, 1978). The minimum concentration of glucose required to produce the maximum rate o f O 2 uptake was found to be 10 mM. Although glucose concentrations in excess of this value has been recorded for locusts (MAYER and CANDY, 1969; HANSEN, 1964), the present authors found concentration in the resting locust which did not exceed 4 mM. Others have recorded glucose concentrations from 0 to 10 m M (TREHERNE, 1958; HOWDEN and KILBY, 1956). Glucose is not the predominant carbohydrate in the haemolymph of the locust. In the resting insect trehalose is present in concentrations of from 40 to 80 m M (HOWDEN and KILBY,1956; HANSEN,1964; MAYER and CANDY,1969; JUTSUM and GOLDSWORTHY, 1976). The disaccharide may be expected to constitute a large part of the oxidizable substrate for the nervous system. This was found to be the case for the cockroach Periplaneta americana by TREHERNE (1960). However, the concentration of trehalose in the locust falls steeply during flight. Although there is some dispute about the extent of this fall, there is agreement that the fall is considerable. The concentration of trehalose available to the nervous system may thus vary quite widely. In contrast, the
concentration of glucose, although much lower, has been shown to be much more stable during flight (MAYER and CANDY, 1969). The experiments reported here were undertaken to investigate the relative importance of glucose and trehalose as energy substrates for the nervous system. It was also hoped to discover if the depleted concentrations of carbohydrate in the haemolymph after extended periods of flight, were still adequate to support the maximum rate of 0 2 consumption of the isolated thoracic ganglia, and whether there was any activation of the trehalase of the nervous system induced by flying.
M A T E R I A L S AND M E T H O D S Locusts
Adult locusts, Schistocerca americana gregaria, of both sexes were used. They were obtained from Larujon Locust Suppliers Ltd., Welsh Mountain Zoo, Colwyn Bay, North Wales. Fed on a diet of bran, they were kept at a temperature of 30-32°C. Reagents
The organic and inorganic chemicals were the product of BDH Ltd., Poole, Dorset, U.K. Purified enzymes and cofactors came from Boehringer Ltd., London. [U-t4C] glucose (sp. act. 230 mCi/mmole) was supplied by the Radiochemical Centre, Amersham, Bucks, U.K. PPO was supplied by International Enzymes ltd., Windsor, Berks, U.K. and POPOP by Koch-Light Ltd., Colnbrook, Bucks, U.K. Estimation of the carbohydrates in the haemolymph and ganglia of the nervous system
*Present address: Department of Human Anatomy, University of Oxford. t Communication to the first author.
After the insects had been cooled in ice for 15 min the head 155
156
R . H . C . STRANG AND E. M. CLEMENT
and tip of the abdomen were carefully cut off, and the bodies eviscerated. Haemolymph was collected from the bodies by centrifugation for 5 min in conical glass centrifuge tubes at the lowest possible speed (5-10g) on a bench centrifuge. Once the protein had been precipitated from the haemolymph with two volumes of 95°~ ethanol, the clear supernatant was lyophilised, and the carbohydrate redissolved in lml of distilled water, samples of which were taken for estimation of glucose and trehalose. The methods for the treatment of the ganglia of the thorax, and for the estimation of trehalose and glucose have already been described (STRANG et al., 1979).
Flight The locusts were flown on a simplified version of the roundabout of KROGH and WEIs-FoGH (1952). The insects were attached to light rods by means of wax yokes around their thoraces. The roundabout was driven both by the flight of the insects, and by a stream of compressed air. Throughout the flight, the locusts were kept warm by a current of air from a hair drier. Samples of the haemolymph and ganglia were taken immediately after the locusts had been removed from the roundabout after periods of flight of up to 2 hr.
Incubation of the ganglia Entire meso- and meta-thoracic ganglia were freed of adhering fatty and tracheal tissue and suspended in a saline based on that of HOYLE (1953) (omitting the MgCI 2 and NaHCO3; pH 6.8, osmolarity about 60~o that of haemolymph), saturated with warm, moist 100~o O z, and containing different concentrations of glucose and trehalose according to the experiment. Oxygen uptake studies were made at 37°C with an 02 electrode (Rank Bros., Bottisham, Cambridge, U.K.), containing 3 ml of medium and up to eight ganglia. Rates of O2 uptake were recorded as initial rates during the first 20 min of incubation. To follow the accumulation of glucose in the medium in the presence of trehalose, two meso- and two meta-thoracic ganglia were suspended in 0.5 ml of saline which was continually aerated with warm, moist 100~o 02, and kept at 37~oC. Samples (100 ,ul) were taken at 0, 10, 20 and 30 min. After each sampling, the volume was restored to 0.5 ml with the original saline. The total production of glucose was estimated making due allowance for the sugar removed in sampling. Blanks contained tissue only with no trehalose in the medium. A n y glucose produced under these conditions was subtracted from the production in the presence of trehalose.
Production and trapping of metabolic CO z In experiments to find the relative oxidative utilisation of trehalose and glucose, the latter was labelled with 14C, and the radioactive CO 2 accumulating in the medium was trapped and counted. U p to five pairs of meso- and metathoracic ganglia were suspended in 3 ml of medium with the appropriate concentrations of trehalose and glucose in an O z electrode. The medium had been previously saturated with 100~00 2. Throughout the experiments the specific activity of the glucose was constant at 0.03 mCi/mmole. After the ganglia had been introduced into the electrode, it was sealed, and the tissue allowed to deplete the oxygen in the electrode to a known extent over a period of 3-4 hr at 37°C. Without opening the electrode, samples (0.1-0.6 ml) of the medium were expelled via a plastic tube and steel needle into Warburg flasks which had been sealed with rubber closures. Three such samples were taken from each incubation. The flasks contained 0.1 ml of 12 M H2SO 4 in the outer compartment, and 0.25 ml of a 10~0 (w/v) methanolic solution of Hyamine 10-X hydroxide solution in the inner well. Trapping of the CO 2 was aided by means of a paper wick. By weighing the flask before and after the addition of the medium, the volume of each sample was calculated. The CO 2 released was trapped overnight at room temperature, and the whole of the contents
of the inner well transferred to 3 ml of a scintillator consisting of 5 g PPO and 0.3 g POPOP/1 of toluene. The trapped radioactivity was estimated on a Phillips liquid scintillation counter programmed for quench correction by the channels ratio method. The efficiency of counting was greater than 90'/,,.
The pH of the incubation media While most of these incubations were carried out at pH 6.8, a few were done at pH 6.2 to see if the lower pH would alter the proportions of trehalose and glucose oxidised by the tissue, in the absence of any definite information as to the pH which obtains in the haemolymph of flying locusts, the pH of 6.2 was chosen on the strength of two pieces of information: one was that this pH had been recorded in the haemolymph of cockroaches after a short period of flight (MATTHEWS el al., 1976); and the other was that while the haemolymph of insects buffers poorly in the normal physiological range, the buffering capacity rises steeply below 6.3 (LEVENBROOK, 1950).
Characteristics of trehalase (c~, ~ "glucoside-l-glucohydrolase; E.C.3.2.1.28) of the thoracic ganglia The m a x i m u m specific activity of the enzyme and its K,, for trehalose were estimated in the supernatants of homogenates of the thoracic ganglia. Adhering tissue was removed from several ganglia which were then blotted gently with a tissue, and weighed on a small piece of aluminium foil. Approximately 10 mg of tissue were homogenised in 50 #1 of ice-cold medium at pH 7.2 (CRABTREE and NEWSHOLME, 1972). The homogenate was centrifuged at 4°C for 5 min at 12,000 g. Preliminary assays had confirmed that no activity was lost in the pellet. Samples of the clear supernatant were added to a total of I ml of an assay medium which had the following composition: 70 m M Tris/HCl buffer (pH 7.2), 1 m M MgClz; 1.5 m M N A D P + ; 1.0 m M ATP: and 4-5U of hexokinase (E.C.2.7.1.1.) and 2-3U glucose 6-phosphate dehydrogenase (E.C.1.1.1.49). Concentrations of trehalose ranged from 2 to 50 mM. To obtain blanks either trehalose or N A D P ÷ was omitted. The rate of glucose production was followed spectrophotometrically at 340 nm. Addition of glucose to some assays confirmed that the activities of the auxiliary enzymes were in adequate excess. The estimates were made at 37°C. In addition to the estimates made at pH 7.2, the m a x i m u m specific activity of the enzyme was also determined at pH 6.2. The ganglia were homogenised in 0.05 M phosphate buffer at this pH. The homogenate was then carefully transferred into a total of 350/~1 of the buffer, and 50/d of 400 m M trehalose added to give a final concentration of 50 m M . At 1, 6 and 11 min after the addition of the trehalose 100 #1 samples were taken and the glucose estimated at pH 7.2 by the previously described assay. (It proved impossible to obtain a continuous record of glucose production as the hexokinase and glucose 6-phosphate dehydrogenase were rather inactive in the phosphate buffer at the lower pH). To ensure that the rate of glucose production was not being underestimated due to the metabolic loss of glucose in the discontinuous procedure, known amounts of glucose, in the concentrations encountered, were added to samples of the homogenates at pH 6.2. No detectable loss of glucose took place in the time required for the estimation.
Statistical treatment of results Estimation of the significance or otherwise of results, where this was in doubt, was made by the application of Student's t-test. RESULTS
Effect o f flight on the concentrations carbohydrates in the haernolymph The
average
concentrations
of
of
trehalose
the and
Trehalose and locust nervous system Table 1. Rates of 0 2 consumption by thoracic ganglia in
vitro Equivalent
in v i v o conditions --
Concentrations of carbohydrate Rate of 02 in medium (mM) uptake (/tmole/g/hr) Glucose Trehalose 10
--
---
Resting
4 4
--
--
1 hr of flight --2 hr of flight
3 3 -1
--
--
1
50 -50 25 -25 15 --
15
268___23 (12) 103_+ 13t ( 1 2 ) 250_+ 20 (8) 220_+ 10" (8) 184_+ 10" ( 8 ) 258 + 20 (6) 200_+ 15" (6) 140_+7" (6) 164-+5t (6) 165 _+8t ( 4 ) 146_+5"f" (4)
Rates are all initial rates. Each average (+ S.D.) is the mean of the number of estimates in the brackets. Figures marked thus * are all significantly different from the maximum rate of 02 uptake; * - p <0.01, t - p <0.001.
glucose ( + the standard deviation) in the resting locusts were found to be 54+ 12 mM and 4 + 1 mM respectively. After 1 hr of flight they had fallen to 26+11 mM and 3+0.7 mM, and after 2 hr the concentrations were 15___9 mM and 1+0.6 mM. Haemolymph samples were taken from six to twelve locusts to obtain these estimates. There was a great deal of variation from one locust to another even among the unflown insects, a fact reflected in the high standard deviation. During flight the inevitable differences in the vigour of individual's performances made the variations in carbohydrate concentrations proportionately greater.
Oxygen uptake by the isolated ganglia in the presence o f different concentrations o f glucose and trehalose The average concentrations o f glucose and trehalose found in resting and exercised locusts were tested both together and separately for their ability to support the oxidative metabolism of isolated thoracic ganglia. The rates of 0 2 uptake are shown in Table 1. Standards of comparison are, as a maximum, the rate found in the presence of 10 mM glucose (CLEMENTand STRANG, 1978) and as a minimum, the rate found in the absence of any exogenous substrate. When the sugars were present in the concentrations found in the haemolymph of the resting locust and after one hours flying, the combination of glucose and trehalose supported the maximum rate of 0 2 uptake. Alone, however, neither sugar was present in sufficient concentration to do this, and despite its very high concentration, trehalose was much less effective as a substrate than was glucose. Even in combination, the concentrations of glucose and trehalose at the end of two hours of flight were insufficient to support the maximum rate o f O 2 uptake recorded in vitro.
The characteristics o f the unpurified trehalase o f the thoracic ganglia The Lineweaver-Burk plot of the results of velocity against substrate concentration gave a straight line. La. 10/2
c
157
Table 2. Concentrations of trehalose in the thoracic ganglia in vivo and in vitro
Conditions
Concentrations of trehalose in the Concentration of medium or haemolymph trehalose in the (raM) ganglia (pmole/g)
Intact ganglia
In vitro In vivo
10 25 50 54-t- 12
2.2 + 0.01 4.9+0.3 6.3+0.2 6.4+_0.4
Tissues were washed by brief immersion in ice-cold medium free of trehalose, before homogenisation. The concentrations are the averages (+ S.D.) of four estimations in vitro, and six in vivo. From the plot the K m of the enzyme with reference to trehalose was found to be 1 x 10 -2 M (10 mM). The Vmax at infinite substrate concentration was found to be 1.53/~mole glucose produced/g/min. An average of four estimates (_+ S.D.) of the specific activity in the presence of 50 mM trehalose gave a figure of 1.45 _-4-0.3 #mole glucose produced/g/rain. When a phosphate buffer pH 6.2 was used, the average of these estimates (_+ S.D.) in the presence of 50 mM trehalose was found to be 2.56+ 0.11 #mole glucose produced/g/min.
Concentrations o f trehalose in the thoracic ganglia in vivo and in vitro In Table 2 are shown the concentrations in vitro after a 30 min incubation in the presence of different concentrations of trehalose, and these are compared with the average concentrations in the haemolymph and tissue in the living insect. It has already been shown (STRANG et al., 1979) that after 30 min of incubation in the absence of trehalose, the concentration in the tissue has fallen to 1.9 #mole/g and thus the concentrations found at this time in the presence of trehalose may be taken to reflect the tissue concentration in equilibrium with the medium. It is clear that at no concentration tested does the tissue concentration approach that of the medium; being a factor of four to five times lower at 10 and 25 mM trehalose and of about eight times lower in the presence of 50 mM trehalose. Also clear is the agreement of the tissue content in vivo with that in vitro, in the presence of similar concentrations of trehalose. This fact gives some confidence that reasonable extrapolations may be made from the results in vitro to the situation in vivo.
Efflux o f glucose from the ganglia in the presence o f trehalose In Table 3 are shown the rates of accumulation of glucose i n the medium in the presence of different concentrations of trehalose. Rates were quite linear during the first 30 min of the incubation, and have been corrected by subtraction of the blank values. There was clearly a brisk efflux of glucose from the intact tissue, which was proportional (at least at 10 and 25 mM trehalose) to the concentration of the disaccharide in the medium. When the total activity of the trehalose is estimated by combining these two rates of glucose production and utilization, the maximum rate found, 1.61 #mol glucose produced/g/min, is quite
158
R. H. C. STRANG AND E. M. CLEMENT Table 3. Estimation of the total activity of trehalase in the intact and homogenised ganglia
Concentration of trehalose in medium (mM)
Conditions Intact ganglia
25 50
0,46 _+0.09 0,97+0.13 1.11 +0.05
50 50
---
10
Hom6genised ganglia pH 7.2 pH 6.2
Rate of glucose accumulation in the medium Rate of glucose -+ S.D. (n = 4) consumption (a) (b) (/~mole/g/min)
Estimated total activity of trehalase ± S.D. (n = 6) (a) + (b)
0.36 0.39 0.50
0.82 1.36 1.6l 1.45_+0.3 2.56_+0.1
--
The rates of glucose consumption by the intact tissue (b) are based on the rates of oxygen uptake in the presence of the appropriate concentrations of trehalose. All incubations were carried out at 37°C ' similar to the figure of 1.45/zmole/g/min, which was the m a x i m u m specific activity of the trehalase at pH 7.2 f o u n d in h o m o g e n a t e s of the ganglia.
Relative utilization of trehalose and glucose by the intact ganglia in vitro The most i m p o r t a n t test of the relative usefulness o f trehalose a n d glucose would be the p r o p o r t i o n s of the C O 2 produced by the tissue originating from each c a r b o h y d r a t e when b o t h were present, as they would be in the insect. T o determine this, [U-14C]-glucose of a c o n s t a n t specific activity was mixed with unlabelled trehalose in the p r o p o r t i o n s which h a d been f o u n d in the h a e m o l y m p h , the ganglia incubated in these media, a n d the radioactivity in the C O 2 produced estimated. In Table 4 are presented the results. T o establish the figure when 100~o of the C O 2 was derived from glucose, the m e d i u m contained 10 m M [14C]-glucose only. W h e n trehalose was present in the medium, the CO 2 produced from glucose was almost always less
than this figure, as would be expected. The results, however, for the suitability of trehalose as a substrate for oxidation by the thoracic ganglia in competition with glucose, do not in any way contradict the impression gained from the Oz uptake studies. Glucose supplied a m u c h higher p r o p o r t i o n of the metabolic C O 2 t h a n its c o n c e n t r a t i o n would seem to warrant. This was especially true if the c o n c e n t r a t i o n s of the two sugars are those e n c o u n t e r e d u n d e r resting conditions, the glucose, a l t h o u g h only 3% of the total c o n c e n t r a t i o n in terms of glucose units, supplied 18~o of the C O r W h e n at the end of an h o u r ' s flight the c o n c e n t r a t i o n of the trehalose had fallen to less t h a n h a l f its original concentration, while that of the glucose h a d remained a b o u t the same, the ~o from glucose rose to 25~o. This increase was statistically significant (p < 0.05). If the calculations are based on the c o n c e n t r a t i o n s f o u n d by MAYER and CANDY (1969) the figures are even more dramatic, with only 3 2 ~ of the metabolic C O 2 coming from trehalose in the resting locust. After 30 min flying, when the
Table 4. Proportions of CO 2 derived from glucose and trehalose by intact ganglia in vitro Concentration of carbohydrate in medium Glucose trehalose (raM) 1.
10
2.
10
60
3.
4
60
4.
3
25
5.
15
40
6.
20
15
Total radioactivity trapped CO 2 _+S.D. (dpm) 10,950 ± 900 (6) 5,190 _+730 (3) 1,940 +40 (6) 2,780 + 190 (3) 7,460 + 510 (3) 10,450 _+580 (3)
°~o CO 2 derived from glucose
Ratio of glucose to trehalose (as glucose) (~)
Equivalent conditions in vivo
100 47
8
18
3
25
6
68
19
95
67
Resting (present authors) After one hour flight (present authors) Resting (MAYER and CANDY, 1969) After 30 min of flight (MAYERand CANDY, 1969)
Incubations were carried out in 3 ml of medium in an oxygen electrode at 37°C, and the consumption of oxygen noted. An equivalence ofO 2 uptake and CO 2 output was assumed, and the output standardised to 900 nmoles/ml. The quoted figures are averages of the number of incubations in brackets. In each incubation three samples were taken for counting. Carbohydrate concentrations do not exactly correspond to the averages found in vivo, but have been rounded off for convenience. The trapped radioactivity has also been rounded off to the nearest 10. The percentages of CO 2 derived from glucose in the presence of trehalose are all significantly different from the counts derived from glucose alone (p < 0.05) with the exception of No. 6.
Trehalose and locust nervous system
159
Table 5. Effect of flying and lowered pH on the utilization of trehalose by the intact thoracic ganglia
pH 6.8 6.8 6.8 6.2 6.2
Concentration of carbohydrate in the medium (mM) Glucose Trehalose 10 20 3 20 3
-15 25 15 25
Total radio-activity in trapped CO2 (dpm) % CO2 derived + S.D. from glucose 1 2 , 0 1 0 + 9 9 0(3) 11,770+ 1300 (3) 3,230+210 (3) 9,200+230 (3) 3,000+220 (3)
100 98 27 77* 25
Incubations were carried out and the C O 2 trapped as in Table 4. The ganglia were taken from locusts which had flown for one hr immediately prior to the experiment. The only conditions which resulted in a CO2 output from trehalose significantlydifferent from the equivalent conditions in Table 5, were those marked thus * (0.05 < p <0.1). concentration of glucose actually exceeds that of trehalose, the latter sugar contributes virtually nothing to the metabolic CO 2. Activation o f trehalase in the course of flight To the experiments in vitro two conditions were applied which were intended to mimic more closely the conditions in vivo. One was that the tissues used were those taken from insects which had flown for an hour. The other was that the pH of the medium was lowered to 6.2. The results of these modifications are shown in Table 5. The only conditions under which there was any evidence for an increased utilization of trehalose was at pH 6.2 in the presence of 20 mM glucose and 15 mM trehalose. Although the reduction of 18% in CO 2 coming from glucose seemed quite substantial, the statistical analysis shows that it has only marginal significance (0.05 < p < 0.1). DISCUSSION The decline in the concentration of total carbohydrate in the course of a two hour flight to 25% of the resting values, is in general agreement with previous findings. Reports range from the almost complete disappearance of carbohydrate as a result of extended flights (WEIs-FOGH, 1964; MAYER and CANDY, 1969; TIETZ, 1967), to a reduction of only 50% (ROBINSON and GOLDSWORTHY, 1976; JUTSUM and GOLDSWORTHY, 1976). The extent of the depletion of the carbohydrate has implications for the nervous system, which by analogy with the cockroach, (TREHERNE, 1960) may be expected to make considerable use of carbohydrate as an energy substrate. In fact ROBINSONand GOLDSWORTaY(1976) suggest that the homeostatic conservation of trehalose may be a mechanism to spare this carbohydrate for the use of tissues other than the flight muscle (e.g. the nervous system) in the course of a prolonged flight. There seems no reason to doubt that the oxidation of diglyceride by the flight muscle favoured by the action of adipokinetic hormone (ROBINSON and GOLDSWORTHV, 1977 a, b) does inhibit the utilization of trehalose by the muscle as has been reported on more than one occasion (ROBINSON and GOLDSWORTHY, 1976; FORD and CANDY, 1972). The findings of the present and other authors, however,
imply that under some circumstances, either the sparing mechanism is rather ineffective (due possibly to low titres of hormone and low concentration of diglyceride), or that it functions to produce a variety of final concentrations of trehalose in the haemolymph. The idea of variability has received recent confirmation in the results of A.M. TH. BEENAKKERS (personal communication) who, like GOLDSWORTHY and his colleagues, found that the depletion of carbohydrate ceases after a 50% decline from the resting concentration, but that there exists between individual locusts a wide variation in initial concenrations ranging from more than 220 mM to about 100 mM (as glucose). Although glucose is usually disregarded in the context of insects, as its concentration is generally much lower than that of the disaccharide, the fact that it is only in combination that the concentrations of glucose and trehalose found in vivo can support the maximum rate of 0 2 uptake in vitro, suggests that glucose is an equally important or even a more important source of energy for the nervous system. Even the total carbohydrate concentration, however, at the end of two hours of flight was found unable to support the maximum rate of 0 2 uptake. In this finding lies the implication that under such conditions some alternative source of energy, exogenous or endogenous, may be required. Glycogen in the nerve cord is a potential source of endogenous energy. Although there is evidence that glycogen in the isolated nerve cord does supply most of the energy in the absence of exogenous substrates (STRANGet al., 1979) it has yet to be confirmed that the same applies in vivo, when the ambient carbohydrate concentration is low. The high Ks of the trehalase means that the enzyme will function at rates less than the maximum when the concentration of substrate falls below 50 mM. In addition, the concentration of the disaceharide in the nervous tissue is much lower than in the surrouding medium, [a situation similar to that in the cockroach (TREHERNE, 1974)]. These two aspects, high Km and low tissue concentration, would seem sufficient to account for the inadequacy oftrehalose as a substrate, were it not for the fact that the efflux of glucose from the tissue is about twice the estimated rate of glucose oxidation. One explanation for these rather paradoxical results is that both enzyme and
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disaccharide are confined to some (presumably outer) compartment of the ganglia, where the concentration of the substrate approaches that of the surrounding fluid. Here also the maximum specific activity of the trehalase would be greater than that of the hexokinase, which would be more homogenous in its distribution. This would result in a greater production of glucose from trehalose than could be further metabolised, causing the glucose efflux found. Under the artificial conditions in vitro in the absence of glucose from the medium, there would be a concentration gradient down which the glucose could diffuse to the outside. In view of this, it was thought that the experiments involving trehalose alone gave a false impression of its utilization by the tissue. On the contrary, however, when intact ganglia were incubated in the presence of both sugars, the proportions of the CO 2 originating from trehalose were still low. It is interesting to note that the results reported here for the locust nervous system, are similar to those for the cockroach under the same sort of conditions (TREHERNE, 1960). TREHERNE found that with a ratio of trehalose to glucose of seventeen, the glucose was taken up 2.5 times as rapidly as the trehalose. The corresponding ratio in the case of the locust ranged from two to three for similar ratios of trehalose to glucose. Without exception, the insect trehalases so far investigated have optimum pH values between 5.5 and 6.5 (DuvE, 1972) and most have a K,, for trehalose of about l m M (GussIN and WYATT, 1965; GILBY et al., 1967). The trehalase of the flight muscle of the desert locust has a maximum specific activity of 4-10 #mole glucose produced/g/min at 37°C (depending on the method of activation) and a K mof 3mM (CANDY,1974; FORD and CANDY, 1972). Both these parameters indicate that the flight muscle is likely to be a more efficient user of trehalose as the concentration of the sugar falls. This would be perfectly consistent with the suggestion that the main function of the trehalose is to provide an immediately available reserve of carbohydrate for the almost exclusive use of flight muscle (BEDFORD, 1977). Although the deductions regarding the proportional utilization of trehalose were made on the basis of results obtained with tissue from resting locusts, there did not seem to be any proportional increase in the use of trehalose when the tissue from flown locusts was used in vitro. This would argue against any persisent activation of the trehalase. There is however some evidence, of a rather low statistical significance, that a lower pH does alter the proportional use of trehalose and glucose in favour of the former. Due to the lack of information about the pH values encountered in flight it is difficult to estimate the importance of this effect in vivo. The factors which control the activity of trehalase have so far eluded detection. In the intact ganglia the enzyme seems to be fully active, if the maximum specific activity found in homogenates is correct. If this is a true representation of the conditions in vivo, then the trehalose is likely to contribute glucose to the pool of that sugar in the haemolymph, and thus help to maintain the equilibrium between the two circulating sugars. To conclude, the importance of trehalose as an energy substrate to the nervous system depends very
much upon its concentration and upon that of its hydrolytic product, glucose. Of the two, glucose is far more effective in low concentrations, and in view of its greater consistency in the haemolymph under flying conditions, the monosaccharide may be relatively more important to the tissue. All the evidence indicates that the thoracic ganglia are not well adapted to the use of trehalose. Although the evidence is at present scanty, it may be, in any case, that the nervous tissue can utilise more than carbohydrate for its energy requirements. Acknowledgements--The authors gratefully acknowledge the valuable advice and information of Professor R. M. S. SMELUE,Dr. C. A. FEWSON,Dr. D. J. CANDYand Dr. A. M. TH. BEENAKKERS.
REFERENCES BEDFORD J. J. (1977) The carbohydrate levels of insect haemolymph. Comp. Biochem. Physiol. 57, 83-86. CANDY D. (1974) The control of muscle trehalase activity during locust flight. Biochem. Soc. Trans. 2, 1107-1109. CLEMENTE. M. and STRANGR. H. C. (1978) A comparison of some aspects of the physiology and metabolism of the nervous system of the locust Schistocerca gregaria in vitro with those in vivo. J. Neurochem. 31, 133-145. CRABTREEB. and NEWSHOLMEE. A. (1972) The activities of phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase and glycerol 3-phosphate dehydrogenase in muscles from vertebrates and invertebrates. Biochem. J. 126, 49-58. DUVE H. (1972) Purification and properties of trehalase isolated from the blowfly Calliphora erythrocephala. Insect Biochem. 2, 445-450. FORD W. C. L. and CANDY D. J. (1972) The regulation of glycolysis in perfused locust flight muscle. Biochem. J. 130, 1101-1112. GILBY A. R., WYATT S. S. and WYATT G. R. (1967) Trehalases from the cockroach Blaberus discoidalis: activation, solubilization and properties of the muscle enzymes, and some properties of the intestinal enzyme. Acta biochim, pol. 14, 83-100. GusslN A. E. S. and WYATTG. R. (1965) Membrane-bound trehalase from Cecropia silkmoth muscle. Archs. Biochem. Biophys. 112, 626-634. HANSEN O. (1964) Effect of diet on the amount and composition of locust blood carbohydrates. Biochem. J. 92, 333-337. HOWDEN G. F. and KILBY B. A. 0956) Trehalose and trehalase in the locust. Chemy Ind. t453-1454. HOYLEG. (1953) Potassium ions and insect nerve muscle J. exp. Biol. 30, 121-133. JUTSUMA. R. and GOLDSWORTHYG. J. (1976) Fuels for flight in Locusta. J. Insect Physiol. 22, 243-249. KROGH A. and WEIS-FOGH T. (1952) A roundabout for studying sustained flight of locusts. J. exp. Biol. 29, 211-219. LEVENBROOKL. (1950) The physiology of carbon dioxide transport in insect blood. J. exp. Biol. 27, 184-191. MATTHEWSJ. R., DOWNER R. G. A. and MORRISONP. E. (1976) ct-glucosidase activity in haemolymph of the American cockroach, Periplaneta americana. J. Insect Physiol. 22, 157-163. MAYER R. J. and CANDY D. J. (1969) Changes in energy reserves during flight of the desert locust, Schistocerca gregaria. Comp. Biochem. Physiol. 31, 409-418. ROBINSON N. L. and GOLDSWORTHY G. J. (1976) Adipokinetic hormone and flight metabolism in the locust. J. Insect Physiol. 22, 1559-1564.
Trehalose and locust nervous system ROBINSON N. L. and GOLDSWORTHY G. J. (1977a) Adipokinetic hormone and the regulation of carbohydrate and lipid metabolism in a working flight-muscle preparation. J. Insect Physiol. 23, 9-16. ROB1NSONN. L. and GOLDSWORTHYG. J. (1977b) A possible site of action of adipokinetic hormone on the flight muscle of locusts. J. Insect Physiol. 23, 152-158. STRANG R. H. C., CLEMENTE. M. and RAE R. C. (1979) Some aspects of the carbohydrate metabolism of the thoracic ganglia of the locust Schistocerca gregaria. Comp. Biochem. Physiol. B62, 217-224. TIETZ A. (1967) Fat transport in the locust: the role of diglyceride. Eur. J. Biochem. 2, 236-232.
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TREHERNE J. E. (1958) Facilitated diffusion and exchange in absorption of glucose by the locust, Schistocerca gregaria. Nature, Lond. 181, 1280-1281. TREHERNE J. E. (1960) The nutrition of the central nervous system of the cockroach, Periplaneta americana. J. exp. Biol. 37, 513-533. TREHERNE J. E. (1974) In Insect Neurobiology (Ed. by TREHERNEJ. E.), p. 229, North Holland Press, Amsterdam. WEIS-FO6H T. (1964) Diffusion in insect wing muscle, the most active tissue known. J. exp. Biol. 41, 229-256.