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Synthesis of proline by fat body of the tsetse fly (Glossina morsitans): metabolic pathways
Insect Biochem., 1977, Vol. 7, pp. 427 to 434. Pergamon Press. Printed in Great Britain.
SYNTHESIS OF P R O L I N E BY FAT BODY OF THE TSETSE FLY (GLOSSINA MORSITANS): METABOLIC PATHWAYS E. BURSELL Department of Zoology, University of Rhodesia, Salisbury, Rhodesia (Received 22 April 1977)
Abstract--Fat body from the tsetse fly is capable of synthesising proline from lipid and amino acid raw materials, and the metabolic pathways involved have been identified. Maximum rates of proline synthesis, at levels of about 1.1 #moles proline/hr/mg non-fatty dry weight, are achieved with alanine and lipid as substrates. The use of alanine as a dominant raw material establishes a close relationship between flight muscle and fat body metabolism, and the system as a whole is seen to constitute a special mechanism for the oxidation of lipid reserves. INTRODUCTION INDICATIONShave been obtained that fat body of the tsetse fly is capable of synthesising proline from a variety of raw materials, of which alanine and lipid carbon may be the most important (BuRSELLe t al., 1974; HARGROVE, 1976), and a scheme for the synthesis of proline has been proposed (McCABE and BURSELL, 1975b). As shown in Fig. l, it involves a transamination between alanine and oxoglutarate, carboxylation of the pyruvate so formed to give oxaloacetate, the condensation of this with acetyl-CoA (from fl-oxidation of fatty acids) to form citrate, isocitrate and oxoglutarate. The latter replaces substrate used in the initial transamination, and the glutamate formed is reduced to proline in the overall reaction: alanine + acetyl-CoA + N A D H + H ÷ --. proline + 2 H 2 0 + N A D + + CoA. In what follows this will be denoted Scheme 1, and as can be seen from Fig. 1 it would involve the incorporation of two of the three carbon atoms of alanine into the proline molecule (at positions 2 and 3) with the third appearing as carbon dioxide. Results obtained with in oivo systems have indicated, however, that a substantial reincorporation of carbon dioxide may take place (McCABE and BURSELL, 1975b) and it seems possible that, as illustrated, some of the released carbon dioxide may be used for carboxylation of pyruvate. Since alanine may itself serve as a source of acetylCoA, it could be envisaged that alanine might constitute the sole raw material for proline synthesis, one molecule being carboxylated to give the four-carbon oxaloacetate as before, while a second molecule (see dotted line in Fig. 1) could be decarboxylated to provide the two-carbon fragment required for the condensation reaction. The overall reaction would then be: 2 alanine + NAD ---~proline + NHs + CO2 + N A D H + H + t.B. 7--5/6--B
which in what follows will be referred to as Scheme 2. Under these circumstances a minimum of four alanine carbons would be expected to become incorporated into the proline molecule, with partial labelling of the carboxyl-carbon according to the extent of carbon dioxide incorporation. The overall reaction of Scheme 1 is seen to involve an input of reducing power to the system, but the fl-oxidation which gives rise to acetyl-CoA includes two NAD-linked oxidations thus providing a net surplus of reducing power for Scheme 1 as well as for Scheme 2. Other amino acids could feed into these pathways to the extent that they give rise to one or other of the intermediaries, as shown for leucine, aspartate and glutamate in the figure. The present investigation was undertaken to provide direct evidence of the capacity of the tsetse fat body to synthesise proline from amino acids and lipid substrates, and to determine whether the proposed pathways are in fact operative during synthesis. MATERIALS A N D M E T H O D S Material was isolated from virgin females of Glossina morsitans morsitans Westwood, maintained for 2 to 4 weeks on the blood of guinea pigs. Unmated females show hypertrophy of the fat body, and adequate amounts of tissue can be conveniently isolated from a single female. The postero-lateral margins of the abdomen were slit to allow extrusion of fat body, which was then cut off with fine scissors for transfer to a droplet of insect saline (the formulation of GEE (1976) was used with omission of organic constituents). Contaminating segments of Malpighian tubules, which in such females are generally full of uric acid crystals, can readily be identified and removed, but interwoven tubules of the milk gland remain. Since the milk gland is metabolically quiescent in unmated females its presence as a contaminant is unlikely to be of major importance, and in fact, the occasional use of fat body from male flies has given identical results as far as metabolic pathways are concerned. In what follows the term "fat body" will be loosely used to denote tissue isolated
427
428
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Fig. 1. Metabolic pathways presumed to be involved in the synthesis of proline from various amino acids and lipid. The.expected pattern of radioactive labelling with alanine and acetyl-CoA as substrates is indicated. For further explanation see text. from virgin females in the way described. A few experiments were carried out with faty body isolated from males of Sarcophaga nodosa (Engels). In addition to inorganic constituents, the media used for isolation and assay contained 10~ bovine serum albumin ("essentially fatty-acid free") serving as a trap for fatty acids released by lipase activity. After isolation the tissue was incubated for 5 min in each of two droplets of fresh medium to reduce the content of endogenous amino acids. It was then washed briefly in a third droplet and transferred to an incubation chamber of 0.5 ml capacity containing 20/d of medium. Radioactive substrates were added (usually l ld of an 0.5 M solution) and the chamber was then closed by a coverslip sealed to the rim with silicone grease, the inner surface of the coverslip bearing a small glass-fibre disc (3 mm diameter) wetted with 3.5/.d of 10~ NaOH for carbon dioxide absorption. The incubation chamber was mounted on a vibrator oscillating at 50 Hz with an amplitude of 0.3 mm, to ensure bulk movement of fluid and thereby facilitate exchange of material between fat body and medium. After 30 min the coverslip was removed and the medium was transferred by Hamilton syringe into a small centrifuge tube containing 160/~1 of absolute alcohol. Then 20 #1 of washing medium without substrate was added to the chamber, the coverslip was replaced, and incubation continued for 3 min to recover the bulk of endogenous substrates. The coverslip was again removed and the glassfibre disc transferred to a scintillation vial for in vacuo drying and subsequent counting (see below). The washing medium was syringed into the centrifuge tube to giye a final concentration of 80% alcohol, ensuring precipitation of the serum albumin. Fresh medium and substrate was now added to the incubation chamber, a new glass-fibre disc was prepared and
mounted on the coverslip, the chamber was closed and incubation continued for another 30 min. The whole process was repeated twice more to give a total of four halfhour periods of incubation, except in experiments involving labelled triglyceride of low specific activity, where a single 2 hr incubation was used. Rates of metabolism were found to be sustained at a constant level throughout the period of incubation. The protein precipitate of the extract was finely dispersed with a glass rod, and the extract was centrifuged at 4000 O for 5 min. The resulting supernatant was used for assay of proline and of amino acid radioactivity. For the separation of amino acids a 100/11 aliquot or the supernatant was spotted on the baseline of a 40 x 6 cm strip of Whatman No. 1 chromatography paper, side by side with an aliquot of a solution containing selected amino acid standards, for descending development in the phenol:ethanol:ammonia or the butanol:acetic acid solvents of S~TH (1960). Following development the strip was scanned for radioactivity as previously described (McCABE and BURSELL, 1975a), and radioactive spots were identified with reference to the standard amino acids made visible by treatment with ninhydrin reagent. Virtually all of the radioactivity was found to be associated with the radioactive substrates and with proline, and the corresponding strips were cut out and inserted into scintillation vials to each of which was added 20 ml of scintillation fluid (5.0 g POP, 0.3g dimethyI-POPOP in 1000ml toluene) for counting on a Packard Scintillation Spectrometer (Model 3330). Corrections for radioactive impurities running in the proline position were made on the basis of a blank run using an appropriate quantity of radioactive substrate. The carbon dioxide absorbed on the glass-fibre disc was assayed for radioactivity by the same procedure, except that the volume of scintillation fluid was reduced to 10 ml.
Proline synthesis by tsetse fat body Vials were counted soon after addition of the scintillation fluid to minimise effects of a slow loss of radioactivity to the gas phase which was found to occur. The proline content of extracts was determined according to the method developed by H~GROVE (1973). Six evenly spaced 10/zl aliquots were applied to a 16.5 x 3.5 cm strip of chromatography paper. After drying at room temperature the strips were dipped through a solution of 0.2% isatin in n-butanol containing 4% glacial acetic acid. The strips were air-dried for 2hr and kept overnigl~t in vacuo at room temperature to allow full development of colour. They were then scanned on the chromatographic attachment of a. Zeiss PMQII spectrophotometer, and the amount of proline estimated (by comparison with standard strips) on the basis of average peak height. At high concentration other amino acids may interfere with colour development, but such effects were minimised by adding substrate amino acids to the proline standards at relative concentrations equal to those of the incubation medium. With careful standardisation this simple and senstive technique proved capable of giving reliable estimates provided the proline content of aliquots was kept between 0.15 and 0.75 #g. To determine the amount of tissue used for incubation, and to estimate lipid radioactivity, the fat body was transferred, at the end of experiments, to a weighed coverslip (Cahn Model 4700 Electrobalance) after washing in saline without serum albumin. The tissue was dried at 80°C, extracted with chloroform, dried again and re-weighed to provide an estimate of fat-less dry weight. The lipid extract was evaporated to dryness in a scintillation vial and l0 ml of scintillation fluid was added for counting. All radioactive materials were from the Radiochemical Centre, Amersham and substrates (from Sigma Chemical Co.) were made up to a specific activity ranging from 0.3 to 0.9 mCi/mmole. To effect substantial labelling of endogenous triglycerides a small pellet of radioactive leucine mixed with oleic acid was implanted under the dorsal cuticle of the second thoracic segment of newly-fed females a week after emergence. In the course of the next 24hr tbe leucine was metabolised and high levels of radioactivity could be recovered in fat body glycerides, with specific activities reaching 0.002 mCi/matom of carbon. To determine the distribution of radioactivity in triglycerides, lipids were extracted as described above, and components were separated on a silica gel column. The triglyceride fraction, eluted with hexane, was saponified by refluxing in methanolic potassium hydroxide for 2 hr, and following acidification the fatty acids were extracted from the aqueous phase with several changes of ether. Aliquots of the aqueous phase were mixed with "Instagel" (Packard Instrument Co.) for scintillation counting, and the fatty acids were taken up in a hexane/ether mixture for counting in the toluene scintillation fluid. Taking tripalmitate as the dominant triglyceride (McCABE, 1973) and assuming uniformly-labelled material, the distribution of label between lipid and aqueous fractions should be 95.0:5.0 which does not differ substantially from the observed distribution of 94.1:5.9. In calculating the extent of incorporation of lipid carbon into proline it has accordingly been assumed that the triglycerides have become uniformly labelled. RESULTS
Metabolic pathways The procedures, described provide estimates of the
429
specific activity of the substrates used, the distribution of radioactivity between substrate and products (i.e. proline, carbon dioxide, and triglyceride) and the amount of proline synthesised during incubation. On the basis of these estimates it is possible to calculate the amount of substrate used per mole of proline synthesised and also the number of substrate carbons incorporated in proline and carbon diox!de per mole of proline synthesised. In Fig. 2 the results of all experiments have been plotted to show the close relation between substrate utilisation and carbon incorporation. F r o m the corresponding regression equations the number of carbon atoms incorporated for every molecule of substrate used can be calculated for comparison with values to be expected on the basis of the proposed metabolic pathways. This comparison has been set out in Table 1, and results for each of the substrates will be briefly discussed. (1) L-Alanme
Universally labelled (u.l.). Figure 1 indicates that two alanine carbons should become incorporated inthe proline molecule per molecule of substrate used for the synthesis, irrespective of whether synthesis is based on Scheme 1 or Scheme 2, since both branches of the pathway involve a loss of one carbon as carbon dioxide. Figure 2a and Table 1 show that the amount of carbon incorporated in proline is significantly in excess of expectation, whereas the carbon dioxide recovery shows a corresponding deficit. In the light of earlier observations the discrepancy could best be accounted for on the basis of a re-incorporation of carbon dioxide, and this interpretation has been verified by the use of specifically labelled alanine. Carboxyl-labelled. In the absence of carbon dioxide incorporation, proline synthesised from carboxyllabelled alanine should be unlabelled because carboxyl-carbons would be lost by decarboxylafions of citrate and pyruvate. As shown in Fig. 2b and Table 1, however, more radioactivity can be recovered in proline.than in carbon dioxide. The results confirm those obtained with universally-labelled alanine in showing that more than half of the carbon dioxide released in decarboxylations is reincorporated during the conversion of pyruvate to oxaloacetate. Figures 2a and b are of further interest in showing that the number of alanine molecules that participate in the synthesis of one molecule of proline varies widely, from 0.5 to 1.6. Values lower than the theoretical minimum of 1,0 were generally obtained during the first 30 rain incubation. It seems probable that the apparent discrepancy is associated with a failure to remove all endogenous substrates by the standard washing procedure. The use of residual substrates for proline synthesis would reduce the requirement for alanine during initial phases of incubation. When consideration is limited to later stages of incubation it was found that fat body isolated during the first two days of the hunger cycle gave high values for the alanine/proline ratio (1.352 _ 0.035) whereas
430
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Fig. 2. The incorporation of substrate carbon into proline by fat body of the tsetse fly. The number of substrate molecules used for tl~e synthesis of one molecule of proline is plotted on the abscissa, while ordinates show the number of substrate carbons incorporated in the proline molecule and in carbon dioxide respectively. Amino acids are denoted by the first three letters of their name; Triglyceride (TGL) and leucine were always tested with alanine as an accessory substrate, and in Fig. 2f (lower curve) results obtained with alanine as an accessory substrate are denoted with a + . For further explanation see text. Table 1. The incorporation of substrate radioactivity in proline and in carbon dioxide by fat body of the tsetse fly Substrate I. L-alanine (i) u.1. observed expected (ii) 1-14C observed expected 2. L-apsartate (u.l.) observed expected 3. Triglyceride (u.l.) observed expected 4. L-leucine (u.l.) observed expected 5. L-glutamate (u.l.) observed expected
Atoms of ]4C/mole of substrate In CO_,
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In proline
Total
26
2.651 -I- 0.014 2.000"I"
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0.920 -t- 0.043 0.909
4.871 -I- 0.087 5.000
Observed values represent the mean _ the standard error of the mean. Where expectations differ significantly from observed values they have been marked with astenslcs, * for the 5 ~ and 1 for the 1~ levels of probability. For further explanation see text.
431
Profine synthesis by tsetse fat body later stages of the hunger cycle were characterised by values near to unity (1.013 _ 0.035). This would be in accord with earlier work (Mc CAaE and BuRSELL, 1975b) which has shown that during the early, so-called lipogenic, phases of the hunger cycle fat body metabolism is geared primarily to the synthesis, rather than the breakdown, of lipids. Under these circumstances lipid carbon would not be readily available for proline synthesis by Scheme 1, and synthesis would have to be accomplished in part by Scheme 2 thus involving the use of more than one molecule of alanine per molecule of proline. Conversely, during the later, lipolytic, phases of the hunger cycle, r-oxidation of lipid becomes a dominant feature of fat body metabolism, providing a ready supply of lipid carbon for synthesis by Scheme 1. (2) L-aspartate (u.l.) As a precursor of oxaloacetate, aspartate should be able to participate in the synthesis of proline by Scheme 1 (Fig. 1); this would involve the incorporation of three aspartate carbons into the proline molecule with the appearance of the fourth as carbon dioxide. In fact the results shown in Fig. 2c and Table 1 indicate that only 2.5 carbons appear in proline whereas 1.5 are recovered as carbon dioxide. The discrepancy could be accounted for on the assumption that oxaloacetate may give rise not only to citrate by condensation with acetyl-CoA, but also to pyruvate by decarboxylation, and hence to acetyl-CoA. The operation of this second pathway is confirmed by the ability of aspartate to contribute substantially to the synthesis of triglyceride, as will be shown below. On this basis aspartate, like alanine, could serve as the sole substrate of proline synthesis according to the overall reaction: 2 aspartate + NADH + H + proline + 3CO2 + NH3 + NAD +. Five carbon atoms would be incorporated in proline and three would appear as carbon dioxide, or 2.5 and 1.5 respectively per molecule of aspartate, in accord with the results shown in Table 1. Confirmation of the partial operation of this scheme is provided by Fig. 2c which shows that the synthesis of one molecule of proline can involve the participation of more than one molecule of aspartate. (3) Triglyceride (u./:) Triglycerides cannot serve as sole substrate for the synthesis of proline since they are incapable of giving rise to four-carbon oxaloacetate required for the condensation reaction (Fig. 1). To ensure net synthesis of proline it is therefore necessary to include an accessory substrate, and Fig. 2d and Table 1 show the results obtained with alanine as the accessory substrate. They are fully in accord with expectation in showing that both carbons of the two-carbon lipid fragments that arise in the course of r-oxidation are incorporated into the proline molecule, while recov-
ery of radioactivity in carbon dioxide is negligible. The amount of two-carbon material used in the synthesis of one molecule of proline ranged from about 1.0 during the lipolytic phase, where synthesis is based on the operation of Scheme 1, to 0.6 during the lipogenic phase when Scheme 2 is partially operative and alanine itself provides a secondary source of acetylCoA. (4) L-Leucine (u.l.) Leucine serves as a raw material for the produc~tion of acetyl-CoA, and as with triglyceride, the synthesis of proline is dependent on the provision of an accessory substrate, such as alanine. Early stages in the metabolism of leucine involves the loss of one carbon atom as carbon dioxide, and incorporation of the remaining 5 atoms of carbon into 3 molecules of acetyl-CoA. All five carbons should therefore become incorporated into the proline molecule, whereas the sixth should appear as carbon dioxide, which is in broad accord with the results shown in Fig. 2e and Table 1 although there appeared to be a slight deficit of carbon dioxide. Only one third of each leucine molecule can be incorporated in any one molecule of proline, which conforms with values plotted in Fig. 2e, where the leucine/proline ratio ranges from 0.01 to 0.35. As with triglyceride, the lower values presumably result from the participation of Scheme 2 in the total synthesis. (5) L-Glutamate (u.l.) The results obtained with glutamate (Fig. 2f and Table 1) were unexpected in showing substantial recovery of carbon dioxide radioactivity. On the basis of Fig. 1, all five carbons of the glutamate molecule should be recoverable in proline. However, the conversion of glutamate to proline involves a double reduction, and it is possible that the appearance of carbon dioxide indicates the involvement of an oxidative process which could serve to generate the requisite reducing power. Complete oxidation of glutamate (BURSELLand SLACK,1976 for details of the pathway) would provide a total of 9 units of reducing power, adequate for the reduction of 4.5 molecules of glutamate to proline. On this basis the distribution of radioactivity between proline and carbon dioxide should be 4.09:0.91 which is in good accord with observed values of 3.95:0.92. The interpretation is strengthened by the observation that when alanine is added as an accessory substrate, thereby providing a surplus of reducing power, the carbon dioxide production is reduced to negligible levels, as shown in Fig. 2e (lower graph, +).
Rates of synthesis From the results obtained estimates have been made of the rates at which different substrates are utilised, and also of the rates at which substrate carbons are incorporated into metabolic end products. A few tests were carried out with glucose as a sub~L
432
E. BURSELL
Table 2. The rate of substrate utilisation and the rate of incorporation of substrate carbon into metabolic end products
Species I. GIossina
2. Sarcophaga
Substrate Alanine TGL Aspartate Leucine Glutamate Proline Glucose Alanine Glucose
n
Substrate utilisation pmoles/mg/hr
26 4 10 9 9 3 l 1 1
1.083 + 0.048 1.109 4- 0.196 0.247 4- 0.014 0.114 4- 0.028 0.365 + 0.033 0.003 0.051 0.537 0.470
Carbon incorporation taatoms substrate-~*C/mg/hr proline TGL trehalose 2.84 2.16 0.60 0.56 1.47 -0.00 0.69 0.17
0.04 -0.02 0.04 0.02 0.01 0.04 0.15 0.05
------0.18 -2.28
CO2 0.37 0.09 0.36 0.08 0.34 0.01 0.10 0.55 0.31
Substrate utilisation by tsetse fat body has been partitioned between proline and carbon dioxide in accord with the values given in Table 1. Triglyceride (TGL) incorporation is based on a separate set of experiments involving fat body isolated from females on the first day of the hunger cycle, and values represent the mean of 5 determinations. strate, and with fat body isolated from the blowfly, and the results of all experiments are set out in Table 2. Proline concentrations were never allowed to exceed 10mM since the synthesis of proline was found to be strongly inhibited by high concentrations. The first part of the table shows that alanine and lipid constitute by far the most important raw materials for proline synthesis, with amino acids other than alanine making a smaller, though by no means negligible, contribution. The rate of incorporation into triglyceride is very small by comparison, while values for the release of carbon dioxide are of interest in showing that proline itself is only oxidised at very slow rates. Glucose is also metabolised comparatively slowly, in accord with the work of NORDEN and PATERSON (1969, 1970) which shows that the capacity for carbohydrate metabolism is poorly developed in Glossina. The pattern of glucose metabolism is normal in the sense that trehalose appears as the main reaction product. Previous work on insect fat body has shown that radioactive carbon from acetate and glucose is readily incorporated into proline and other amino acids ('~¢'INTERINGHAM and HARRISON, 1956; CLEMEqqTS, 1959; PRICE, 1961), which suggests that the capacity to synthesise proline may be a general feature of fat body metabolism in insects. This view is confirmed by observations on the fat body of the blowfly, as shown in the second section of Table 2. The synthesis of proline, however, is substantially slower than in the tsetse fly, and it accounts for less than half of the alanine carbon utilised. Much of the balance would presumably be accounted for by gluconeogenesis, but this possibility has not been checked. Glucose itself is also seen to be rapidly metabolised by blowfly fat body, with the bulk of radioactivity appearing in trehalose, as would be expected on the basis of earlier work (CLEGG and EVANS, 1961). Investigations of the composition of tsetse fat body have shown that the lipid content is about 75~ of total dry weight, and since the i'at content of an adult male of G. morsitans is in the region of 3.0 mg (JACK-
SON, 1953) non-lipid components would make up about 1.0 mg. With alanine and lipid carbon as raw material, the corresponding rate of proline synthesis would then be about 1.1 pmoles/fly/hr or 125 Flg/fly/hr sufficient to account for the observed rate of synthesis in the resting fly following flight (BURSELL, 1963). With the average level of triglyceride synthesis at about 0.03 patoms carbon/mg/hr the corresponding value for triglyceride synthesis would be about 1.1 mg/fty per day, which again is ample to account for observed in viuo rates (McCABE and BURSELL, 1975b). DISCUSSION The present work has confirmed the ability of tsetse fat body to synthesise proline from lipid and amino acid carbon, and has served to identify the metabolic pathways involved. Alanine constitutes the main amino acid substrate, and this is of particular interest in view of the fact that it is the principal end-product of oxidative metabolism in the flight muscle (BuRSELL and SLACK, 1976). A very close relation is thus established between flight muscle and fat body metabolism, as illustrated in Fig. 3. Energy for the contraction of flight muscle is shown to arise from the partial oxidation of proline on the left of the diagram. The alanine so produced passes from flight muscle into the haemolymph and is transported to the abdominal fat body, on the right, where it couples with two-carbon lipid fragments to reconstitute proline; and proline passes back into the haemolymph for transport to the flight muscle, thus completing the cycle. During steady-state operation the sole input to the system as a whole would be lipid carbon, whereas the output would be energy for the contraction and maintenance of the flight muscles. The system could thus be seen as constituting a special mechanism for the oxidation of lipid reserves, geared to the special profine-centred metabolism of the tsetse fly. An interesting parallel is provided by the situation in locusts, where glycerol has been shown to play a rfle similar to that of
433
Proline synthesis by tsetse fat body
Myofibrils Sorcoplosm
Sorcosome
Hoemolymph
Foi" body
Fig. 3. Diagrammatic representation of the relationship between flight muscle and fat body metabolism during lipolytic phases of the hunger cycle. Substrates are denoted by the first three letters of their name. EN = energy, ACoA = acetyl-CoA. In the flight muscle pyruvate is shown to arise by oxidative decarboxylation of malate in accord with the findings of HOEK et al. (1976). For further explanation see text. alanine as a carrier of lipid from fat body to thoracic musculature (CANDY et al., 1976). It must be emphasised that in the tsetse fly the cyclic system will be strongly displaced from steadystate operation during flight and during the restorative phase of rest following flight. At initiation of flight in male G. morsitans proline is oxidised at a rate of about 40/amoles/hr (BURSELL and SLACK, 1976) which greatly exceeds the capacity of the fat body for proline synthesis, at about 1.1 #moles/hr. During flight, therefore, the oxidative part of the cycle will dominate to give the observed decrease in the level of proline and increase in the level of alanine (BURSELL, 1963). Conversely, during rest following flight the metabolic rate, and hence the rate of proline oxidation, decreases by a factor of about 100 (BURSELL et al., 1974; HARGROVE, 1976) to levels which are exceeded by the synthetic powers of the fat body, thus giving the observed slow reconstitution of the proline reserve associated with a corresponding decrease in the concentration of alanine. As the proline concentration increases, proline synthesis will be progressively inhibited until its rate exactly matches the rate of proline utilisation by the metabolic system as a whole, and at this point the cycle will resume steady-state operation. It has been suggested by HARGROVE(1976) that the synthesis of proline may contribute substantially to the maintenance of proline levels during flight, and so prolong the duration of flight. This suggestion is not borne out by present results which indicate that the capacity of the fat body for proline synthesis is negligible by comparison with the capacity of flight muscle for proline oxidation. The possibifity should not be ignored, however, that the mobilisation of reserves from the fat body of tsetse flies might be under neuroendocrine control, as it is in other insects (review by BArnEY, 1975). In that case the synthesis
of proline might be increased during flight to levels where it could make an effective contribution to flight metabolism, though preliminary attempts to demonstrate such an effect by incubation of fat body with haemolymph from flown flies, or with extracts of corpora cardiaca, have so far proved unsuccessful. Acknowledgements--My thanks are due to Dr D. A. NORDEN and Dr J. W. HARGROVEfor helpful comments on the manuscript.
REFERENCES BAILEYE. (1975) Biochemistry of insect flight. Part 2: Fuel supply. In Insect Biochemistry and Function (Ed. by CANDY D. J. and K.ILBYB. A.) pp. 95-176. Chapman & Hall, London. BURSELL E. (1963) Aspects of the metabolism of amino acids in the tsetse fly GIossina (Diptera). J. Insect Physiol. 9, 439-452. BURSELLE. and SLACKE. (1976) Oxidation of proline by sarcosomes of the tsetse fly Glossina morsitans. Insect Biochem. 6, 159-167. BURSar E., BmLING K. C., HARGROVEJ. W., MCCABE C. T., and SLACKE. (1974) Metabolism of the bloodmeal in tsetse flies (a review). Acta trop. 31, 297-320. CANDY D. J., HALL L. J., and SPENCERF. M. (1976) The metabolism of glycerol in the locust Schistocerca oregaria during flight. J. Insect Physiol. 22, 583-587. CLEOG J. S. and EvANs D. R. (1961) The physiology of blood trehalose and its function during flight in the blowfly. J. exp. Biol. 38, 771-792. CLE~-~TS A. N. (1959) Studies on the metabolism of locust fat body. J. exp. Biol. 36, 665-675. GEE J. D. (1976) Active transport of sodium by the Malpighian tubules of the tsetse fly Glossina morsitans. J. exp. Biol. 64, 357-368. HARGROVEJ. W. (1973) The Physiology of Flight in Tsetse. Ph.D. thesis, University of London. HARGROW J. W. (1976) Amino acid metabolism during flight in tsetse flies. J. Insect Physiol. 22, 309-313.
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E. BURSELL
HOEK J. B., PEARSOND. J., and OLEMBON. K. (1976) Nicotinamideadenine dinucleotide-linked "malic" enzyme in flight muscle of the tsetse fly (Glossina) and other insects. Biochem. d. 160, 253-262. JACKSONC. H. N. (1953) An artificially isolated generation of tsetse flies (Diptera). Bull. ent. Res. 37, 291-299. MCCABE C. T. (1973) The Metabolic lnterrelationsbips of Amino Acids and Lipids in the Tsetse Fly, GIossina morsitans Westwood. Ph.D. thesis, University of London. MCCAaE C. T. and BURSELL E. (1975a) Metabolism of digestive products in the tsetse fly, GIossina morsitans. Insect Biochem. 5, 769-779. McCABE C. T. and BURSELLE. (1975b) Interrelationships between amino acid and lipid metabolism in the tsetse fly, GIossina morsitans. Insect Biochem. 5, 781-789.
NORDEN D. A. and PATERSOND. J. (1969) Carbohydrate metabolism in flight muscle of the tsetse fly (Glossina) and the blowfly (Sarcophaga). Comp. Biochem. Physiol. 31, 819-827. NORDEN D. A. and PATERSOND. J. (1970) Carbohydrate metabolism in flight muscle of the tsetse fly (GIossina) and the blowfly (Sarcophaga). Part II. Int. J. Biochem. 1, 81-84. I:~ICE G. M. (1961) Some aspects of amino acid metabolism in the adult housefly, Musca domestica. Biochem. J. 80, 420-428. SMITH I. (1960) Chromatographic and Electrophoretic Techniques, 1, 82. Heineman, London. WIN'rERINGHAMF. P. W. and HARRISONA. H. (1956) Study of anticholinesterase action in insects by a labelled pool technique. Nature, Lond. 178, 81-83.
SYNTHESIS OF P R O L I N E BY FAT BODY OF THE TSETSE FLY (GLOSSINA MORSITANS): METABOLIC PATHWAYS E. BURSELL Department of Zoology, University of Rhodesia, Salisbury, Rhodesia (Received 22 April 1977)
Abstract--Fat body from the tsetse fly is capable of synthesising proline from lipid and amino acid raw materials, and the metabolic pathways involved have been identified. Maximum rates of proline synthesis, at levels of about 1.1 #moles proline/hr/mg non-fatty dry weight, are achieved with alanine and lipid as substrates. The use of alanine as a dominant raw material establishes a close relationship between flight muscle and fat body metabolism, and the system as a whole is seen to constitute a special mechanism for the oxidation of lipid reserves. INTRODUCTION INDICATIONShave been obtained that fat body of the tsetse fly is capable of synthesising proline from a variety of raw materials, of which alanine and lipid carbon may be the most important (BuRSELLe t al., 1974; HARGROVE, 1976), and a scheme for the synthesis of proline has been proposed (McCABE and BURSELL, 1975b). As shown in Fig. l, it involves a transamination between alanine and oxoglutarate, carboxylation of the pyruvate so formed to give oxaloacetate, the condensation of this with acetyl-CoA (from fl-oxidation of fatty acids) to form citrate, isocitrate and oxoglutarate. The latter replaces substrate used in the initial transamination, and the glutamate formed is reduced to proline in the overall reaction: alanine + acetyl-CoA + N A D H + H ÷ --. proline + 2 H 2 0 + N A D + + CoA. In what follows this will be denoted Scheme 1, and as can be seen from Fig. 1 it would involve the incorporation of two of the three carbon atoms of alanine into the proline molecule (at positions 2 and 3) with the third appearing as carbon dioxide. Results obtained with in oivo systems have indicated, however, that a substantial reincorporation of carbon dioxide may take place (McCABE and BURSELL, 1975b) and it seems possible that, as illustrated, some of the released carbon dioxide may be used for carboxylation of pyruvate. Since alanine may itself serve as a source of acetylCoA, it could be envisaged that alanine might constitute the sole raw material for proline synthesis, one molecule being carboxylated to give the four-carbon oxaloacetate as before, while a second molecule (see dotted line in Fig. 1) could be decarboxylated to provide the two-carbon fragment required for the condensation reaction. The overall reaction would then be: 2 alanine + NAD ---~proline + NHs + CO2 + N A D H + H + t.B. 7--5/6--B
which in what follows will be referred to as Scheme 2. Under these circumstances a minimum of four alanine carbons would be expected to become incorporated into the proline molecule, with partial labelling of the carboxyl-carbon according to the extent of carbon dioxide incorporation. The overall reaction of Scheme 1 is seen to involve an input of reducing power to the system, but the fl-oxidation which gives rise to acetyl-CoA includes two NAD-linked oxidations thus providing a net surplus of reducing power for Scheme 1 as well as for Scheme 2. Other amino acids could feed into these pathways to the extent that they give rise to one or other of the intermediaries, as shown for leucine, aspartate and glutamate in the figure. The present investigation was undertaken to provide direct evidence of the capacity of the tsetse fat body to synthesise proline from amino acids and lipid substrates, and to determine whether the proposed pathways are in fact operative during synthesis. MATERIALS A N D M E T H O D S Material was isolated from virgin females of Glossina morsitans morsitans Westwood, maintained for 2 to 4 weeks on the blood of guinea pigs. Unmated females show hypertrophy of the fat body, and adequate amounts of tissue can be conveniently isolated from a single female. The postero-lateral margins of the abdomen were slit to allow extrusion of fat body, which was then cut off with fine scissors for transfer to a droplet of insect saline (the formulation of GEE (1976) was used with omission of organic constituents). Contaminating segments of Malpighian tubules, which in such females are generally full of uric acid crystals, can readily be identified and removed, but interwoven tubules of the milk gland remain. Since the milk gland is metabolically quiescent in unmated females its presence as a contaminant is unlikely to be of major importance, and in fact, the occasional use of fat body from male flies has given identical results as far as metabolic pathways are concerned. In what follows the term "fat body" will be loosely used to denote tissue isolated
427
428
E
BURSELL
+CH~ ,
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.
COOH COOH "COOH I I .I " ~ H N H 2 - [ ~ ' C : O ~ C:O I / .I / ol "CH3 / CH3/ ~H2
I
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+CHz .I . ~COHCOOH .I ~H2 COOH
+COOHI +CHz "CIH2
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.............................."CO~, ..........................
Pyruvate Oxaloocetate
Citrate
OxocjIutarace
Fig. 1. Metabolic pathways presumed to be involved in the synthesis of proline from various amino acids and lipid. The.expected pattern of radioactive labelling with alanine and acetyl-CoA as substrates is indicated. For further explanation see text. from virgin females in the way described. A few experiments were carried out with faty body isolated from males of Sarcophaga nodosa (Engels). In addition to inorganic constituents, the media used for isolation and assay contained 10~ bovine serum albumin ("essentially fatty-acid free") serving as a trap for fatty acids released by lipase activity. After isolation the tissue was incubated for 5 min in each of two droplets of fresh medium to reduce the content of endogenous amino acids. It was then washed briefly in a third droplet and transferred to an incubation chamber of 0.5 ml capacity containing 20/d of medium. Radioactive substrates were added (usually l ld of an 0.5 M solution) and the chamber was then closed by a coverslip sealed to the rim with silicone grease, the inner surface of the coverslip bearing a small glass-fibre disc (3 mm diameter) wetted with 3.5/.d of 10~ NaOH for carbon dioxide absorption. The incubation chamber was mounted on a vibrator oscillating at 50 Hz with an amplitude of 0.3 mm, to ensure bulk movement of fluid and thereby facilitate exchange of material between fat body and medium. After 30 min the coverslip was removed and the medium was transferred by Hamilton syringe into a small centrifuge tube containing 160/~1 of absolute alcohol. Then 20 #1 of washing medium without substrate was added to the chamber, the coverslip was replaced, and incubation continued for 3 min to recover the bulk of endogenous substrates. The coverslip was again removed and the glassfibre disc transferred to a scintillation vial for in vacuo drying and subsequent counting (see below). The washing medium was syringed into the centrifuge tube to giye a final concentration of 80% alcohol, ensuring precipitation of the serum albumin. Fresh medium and substrate was now added to the incubation chamber, a new glass-fibre disc was prepared and
mounted on the coverslip, the chamber was closed and incubation continued for another 30 min. The whole process was repeated twice more to give a total of four halfhour periods of incubation, except in experiments involving labelled triglyceride of low specific activity, where a single 2 hr incubation was used. Rates of metabolism were found to be sustained at a constant level throughout the period of incubation. The protein precipitate of the extract was finely dispersed with a glass rod, and the extract was centrifuged at 4000 O for 5 min. The resulting supernatant was used for assay of proline and of amino acid radioactivity. For the separation of amino acids a 100/11 aliquot or the supernatant was spotted on the baseline of a 40 x 6 cm strip of Whatman No. 1 chromatography paper, side by side with an aliquot of a solution containing selected amino acid standards, for descending development in the phenol:ethanol:ammonia or the butanol:acetic acid solvents of S~TH (1960). Following development the strip was scanned for radioactivity as previously described (McCABE and BURSELL, 1975a), and radioactive spots were identified with reference to the standard amino acids made visible by treatment with ninhydrin reagent. Virtually all of the radioactivity was found to be associated with the radioactive substrates and with proline, and the corresponding strips were cut out and inserted into scintillation vials to each of which was added 20 ml of scintillation fluid (5.0 g POP, 0.3g dimethyI-POPOP in 1000ml toluene) for counting on a Packard Scintillation Spectrometer (Model 3330). Corrections for radioactive impurities running in the proline position were made on the basis of a blank run using an appropriate quantity of radioactive substrate. The carbon dioxide absorbed on the glass-fibre disc was assayed for radioactivity by the same procedure, except that the volume of scintillation fluid was reduced to 10 ml.
Proline synthesis by tsetse fat body Vials were counted soon after addition of the scintillation fluid to minimise effects of a slow loss of radioactivity to the gas phase which was found to occur. The proline content of extracts was determined according to the method developed by H~GROVE (1973). Six evenly spaced 10/zl aliquots were applied to a 16.5 x 3.5 cm strip of chromatography paper. After drying at room temperature the strips were dipped through a solution of 0.2% isatin in n-butanol containing 4% glacial acetic acid. The strips were air-dried for 2hr and kept overnigl~t in vacuo at room temperature to allow full development of colour. They were then scanned on the chromatographic attachment of a. Zeiss PMQII spectrophotometer, and the amount of proline estimated (by comparison with standard strips) on the basis of average peak height. At high concentration other amino acids may interfere with colour development, but such effects were minimised by adding substrate amino acids to the proline standards at relative concentrations equal to those of the incubation medium. With careful standardisation this simple and senstive technique proved capable of giving reliable estimates provided the proline content of aliquots was kept between 0.15 and 0.75 #g. To determine the amount of tissue used for incubation, and to estimate lipid radioactivity, the fat body was transferred, at the end of experiments, to a weighed coverslip (Cahn Model 4700 Electrobalance) after washing in saline without serum albumin. The tissue was dried at 80°C, extracted with chloroform, dried again and re-weighed to provide an estimate of fat-less dry weight. The lipid extract was evaporated to dryness in a scintillation vial and l0 ml of scintillation fluid was added for counting. All radioactive materials were from the Radiochemical Centre, Amersham and substrates (from Sigma Chemical Co.) were made up to a specific activity ranging from 0.3 to 0.9 mCi/mmole. To effect substantial labelling of endogenous triglycerides a small pellet of radioactive leucine mixed with oleic acid was implanted under the dorsal cuticle of the second thoracic segment of newly-fed females a week after emergence. In the course of the next 24hr tbe leucine was metabolised and high levels of radioactivity could be recovered in fat body glycerides, with specific activities reaching 0.002 mCi/matom of carbon. To determine the distribution of radioactivity in triglycerides, lipids were extracted as described above, and components were separated on a silica gel column. The triglyceride fraction, eluted with hexane, was saponified by refluxing in methanolic potassium hydroxide for 2 hr, and following acidification the fatty acids were extracted from the aqueous phase with several changes of ether. Aliquots of the aqueous phase were mixed with "Instagel" (Packard Instrument Co.) for scintillation counting, and the fatty acids were taken up in a hexane/ether mixture for counting in the toluene scintillation fluid. Taking tripalmitate as the dominant triglyceride (McCABE, 1973) and assuming uniformly-labelled material, the distribution of label between lipid and aqueous fractions should be 95.0:5.0 which does not differ substantially from the observed distribution of 94.1:5.9. In calculating the extent of incorporation of lipid carbon into proline it has accordingly been assumed that the triglycerides have become uniformly labelled. RESULTS
Metabolic pathways The procedures, described provide estimates of the
429
specific activity of the substrates used, the distribution of radioactivity between substrate and products (i.e. proline, carbon dioxide, and triglyceride) and the amount of proline synthesised during incubation. On the basis of these estimates it is possible to calculate the amount of substrate used per mole of proline synthesised and also the number of substrate carbons incorporated in proline and carbon diox!de per mole of proline synthesised. In Fig. 2 the results of all experiments have been plotted to show the close relation between substrate utilisation and carbon incorporation. F r o m the corresponding regression equations the number of carbon atoms incorporated for every molecule of substrate used can be calculated for comparison with values to be expected on the basis of the proposed metabolic pathways. This comparison has been set out in Table 1, and results for each of the substrates will be briefly discussed. (1) L-Alanme
Universally labelled (u.l.). Figure 1 indicates that two alanine carbons should become incorporated inthe proline molecule per molecule of substrate used for the synthesis, irrespective of whether synthesis is based on Scheme 1 or Scheme 2, since both branches of the pathway involve a loss of one carbon as carbon dioxide. Figure 2a and Table 1 show that the amount of carbon incorporated in proline is significantly in excess of expectation, whereas the carbon dioxide recovery shows a corresponding deficit. In the light of earlier observations the discrepancy could best be accounted for on the basis of a re-incorporation of carbon dioxide, and this interpretation has been verified by the use of specifically labelled alanine. Carboxyl-labelled. In the absence of carbon dioxide incorporation, proline synthesised from carboxyllabelled alanine should be unlabelled because carboxyl-carbons would be lost by decarboxylafions of citrate and pyruvate. As shown in Fig. 2b and Table 1, however, more radioactivity can be recovered in proline.than in carbon dioxide. The results confirm those obtained with universally-labelled alanine in showing that more than half of the carbon dioxide released in decarboxylations is reincorporated during the conversion of pyruvate to oxaloacetate. Figures 2a and b are of further interest in showing that the number of alanine molecules that participate in the synthesis of one molecule of proline varies widely, from 0.5 to 1.6. Values lower than the theoretical minimum of 1,0 were generally obtained during the first 30 rain incubation. It seems probable that the apparent discrepancy is associated with a failure to remove all endogenous substrates by the standard washing procedure. The use of residual substrates for proline synthesis would reduce the requirement for alanine during initial phases of incubation. When consideration is limited to later stages of incubation it was found that fat body isolated during the first two days of the hunger cycle gave high values for the alanine/proline ratio (1.352 _ 0.035) whereas
430
E. BURSELL 5
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Fig. 2. The incorporation of substrate carbon into proline by fat body of the tsetse fly. The number of substrate molecules used for tl~e synthesis of one molecule of proline is plotted on the abscissa, while ordinates show the number of substrate carbons incorporated in the proline molecule and in carbon dioxide respectively. Amino acids are denoted by the first three letters of their name; Triglyceride (TGL) and leucine were always tested with alanine as an accessory substrate, and in Fig. 2f (lower curve) results obtained with alanine as an accessory substrate are denoted with a + . For further explanation see text. Table 1. The incorporation of substrate radioactivity in proline and in carbon dioxide by fat body of the tsetse fly Substrate I. L-alanine (i) u.1. observed expected (ii) 1-14C observed expected 2. L-apsartate (u.l.) observed expected 3. Triglyceride (u.l.) observed expected 4. L-leucine (u.l.) observed expected 5. L-glutamate (u.l.) observed expected
Atoms of ]4C/mole of substrate In CO_,
n
In proline
Total
26
2.651 -I- 0.014 2.000"I"
0.344 ___0.016 1.0001
2.995 __+0.022 3.000
4
0.523 -I- 0.034 0.000I"
0.471 -I- 0.031 1.000 t
0.994 + 0.046 1.000
10
2.457 _ 0.062 2.500
1.505 -I- 0.054 1.500
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4
2.033 -I- 0.027 2.000
0.081 _ 0.034 0.000
2.114 _+ 0.044 2.000
9
4.972 ___0.094 5.000
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5.688 _ 0.110 6.000*
9
3.951 -t- 0.076 4.091
0.920 -t- 0.043 0.909
4.871 -I- 0.087 5.000
Observed values represent the mean _ the standard error of the mean. Where expectations differ significantly from observed values they have been marked with astenslcs, * for the 5 ~ and 1 for the 1~ levels of probability. For further explanation see text.
431
Profine synthesis by tsetse fat body later stages of the hunger cycle were characterised by values near to unity (1.013 _ 0.035). This would be in accord with earlier work (Mc CAaE and BuRSELL, 1975b) which has shown that during the early, so-called lipogenic, phases of the hunger cycle fat body metabolism is geared primarily to the synthesis, rather than the breakdown, of lipids. Under these circumstances lipid carbon would not be readily available for proline synthesis by Scheme 1, and synthesis would have to be accomplished in part by Scheme 2 thus involving the use of more than one molecule of alanine per molecule of proline. Conversely, during the later, lipolytic, phases of the hunger cycle, r-oxidation of lipid becomes a dominant feature of fat body metabolism, providing a ready supply of lipid carbon for synthesis by Scheme 1. (2) L-aspartate (u.l.) As a precursor of oxaloacetate, aspartate should be able to participate in the synthesis of proline by Scheme 1 (Fig. 1); this would involve the incorporation of three aspartate carbons into the proline molecule with the appearance of the fourth as carbon dioxide. In fact the results shown in Fig. 2c and Table 1 indicate that only 2.5 carbons appear in proline whereas 1.5 are recovered as carbon dioxide. The discrepancy could be accounted for on the assumption that oxaloacetate may give rise not only to citrate by condensation with acetyl-CoA, but also to pyruvate by decarboxylation, and hence to acetyl-CoA. The operation of this second pathway is confirmed by the ability of aspartate to contribute substantially to the synthesis of triglyceride, as will be shown below. On this basis aspartate, like alanine, could serve as the sole substrate of proline synthesis according to the overall reaction: 2 aspartate + NADH + H + proline + 3CO2 + NH3 + NAD +. Five carbon atoms would be incorporated in proline and three would appear as carbon dioxide, or 2.5 and 1.5 respectively per molecule of aspartate, in accord with the results shown in Table 1. Confirmation of the partial operation of this scheme is provided by Fig. 2c which shows that the synthesis of one molecule of proline can involve the participation of more than one molecule of aspartate. (3) Triglyceride (u./:) Triglycerides cannot serve as sole substrate for the synthesis of proline since they are incapable of giving rise to four-carbon oxaloacetate required for the condensation reaction (Fig. 1). To ensure net synthesis of proline it is therefore necessary to include an accessory substrate, and Fig. 2d and Table 1 show the results obtained with alanine as the accessory substrate. They are fully in accord with expectation in showing that both carbons of the two-carbon lipid fragments that arise in the course of r-oxidation are incorporated into the proline molecule, while recov-
ery of radioactivity in carbon dioxide is negligible. The amount of two-carbon material used in the synthesis of one molecule of proline ranged from about 1.0 during the lipolytic phase, where synthesis is based on the operation of Scheme 1, to 0.6 during the lipogenic phase when Scheme 2 is partially operative and alanine itself provides a secondary source of acetylCoA. (4) L-Leucine (u.l.) Leucine serves as a raw material for the produc~tion of acetyl-CoA, and as with triglyceride, the synthesis of proline is dependent on the provision of an accessory substrate, such as alanine. Early stages in the metabolism of leucine involves the loss of one carbon atom as carbon dioxide, and incorporation of the remaining 5 atoms of carbon into 3 molecules of acetyl-CoA. All five carbons should therefore become incorporated into the proline molecule, whereas the sixth should appear as carbon dioxide, which is in broad accord with the results shown in Fig. 2e and Table 1 although there appeared to be a slight deficit of carbon dioxide. Only one third of each leucine molecule can be incorporated in any one molecule of proline, which conforms with values plotted in Fig. 2e, where the leucine/proline ratio ranges from 0.01 to 0.35. As with triglyceride, the lower values presumably result from the participation of Scheme 2 in the total synthesis. (5) L-Glutamate (u.l.) The results obtained with glutamate (Fig. 2f and Table 1) were unexpected in showing substantial recovery of carbon dioxide radioactivity. On the basis of Fig. 1, all five carbons of the glutamate molecule should be recoverable in proline. However, the conversion of glutamate to proline involves a double reduction, and it is possible that the appearance of carbon dioxide indicates the involvement of an oxidative process which could serve to generate the requisite reducing power. Complete oxidation of glutamate (BURSELLand SLACK,1976 for details of the pathway) would provide a total of 9 units of reducing power, adequate for the reduction of 4.5 molecules of glutamate to proline. On this basis the distribution of radioactivity between proline and carbon dioxide should be 4.09:0.91 which is in good accord with observed values of 3.95:0.92. The interpretation is strengthened by the observation that when alanine is added as an accessory substrate, thereby providing a surplus of reducing power, the carbon dioxide production is reduced to negligible levels, as shown in Fig. 2e (lower graph, +).
Rates of synthesis From the results obtained estimates have been made of the rates at which different substrates are utilised, and also of the rates at which substrate carbons are incorporated into metabolic end products. A few tests were carried out with glucose as a sub~L
432
E. BURSELL
Table 2. The rate of substrate utilisation and the rate of incorporation of substrate carbon into metabolic end products
Species I. GIossina
2. Sarcophaga
Substrate Alanine TGL Aspartate Leucine Glutamate Proline Glucose Alanine Glucose
n
Substrate utilisation pmoles/mg/hr
26 4 10 9 9 3 l 1 1
1.083 + 0.048 1.109 4- 0.196 0.247 4- 0.014 0.114 4- 0.028 0.365 + 0.033 0.003 0.051 0.537 0.470
Carbon incorporation taatoms substrate-~*C/mg/hr proline TGL trehalose 2.84 2.16 0.60 0.56 1.47 -0.00 0.69 0.17
0.04 -0.02 0.04 0.02 0.01 0.04 0.15 0.05
------0.18 -2.28
CO2 0.37 0.09 0.36 0.08 0.34 0.01 0.10 0.55 0.31
Substrate utilisation by tsetse fat body has been partitioned between proline and carbon dioxide in accord with the values given in Table 1. Triglyceride (TGL) incorporation is based on a separate set of experiments involving fat body isolated from females on the first day of the hunger cycle, and values represent the mean of 5 determinations. strate, and with fat body isolated from the blowfly, and the results of all experiments are set out in Table 2. Proline concentrations were never allowed to exceed 10mM since the synthesis of proline was found to be strongly inhibited by high concentrations. The first part of the table shows that alanine and lipid constitute by far the most important raw materials for proline synthesis, with amino acids other than alanine making a smaller, though by no means negligible, contribution. The rate of incorporation into triglyceride is very small by comparison, while values for the release of carbon dioxide are of interest in showing that proline itself is only oxidised at very slow rates. Glucose is also metabolised comparatively slowly, in accord with the work of NORDEN and PATERSON (1969, 1970) which shows that the capacity for carbohydrate metabolism is poorly developed in Glossina. The pattern of glucose metabolism is normal in the sense that trehalose appears as the main reaction product. Previous work on insect fat body has shown that radioactive carbon from acetate and glucose is readily incorporated into proline and other amino acids ('~¢'INTERINGHAM and HARRISON, 1956; CLEMEqqTS, 1959; PRICE, 1961), which suggests that the capacity to synthesise proline may be a general feature of fat body metabolism in insects. This view is confirmed by observations on the fat body of the blowfly, as shown in the second section of Table 2. The synthesis of proline, however, is substantially slower than in the tsetse fly, and it accounts for less than half of the alanine carbon utilised. Much of the balance would presumably be accounted for by gluconeogenesis, but this possibility has not been checked. Glucose itself is also seen to be rapidly metabolised by blowfly fat body, with the bulk of radioactivity appearing in trehalose, as would be expected on the basis of earlier work (CLEGG and EVANS, 1961). Investigations of the composition of tsetse fat body have shown that the lipid content is about 75~ of total dry weight, and since the i'at content of an adult male of G. morsitans is in the region of 3.0 mg (JACK-
SON, 1953) non-lipid components would make up about 1.0 mg. With alanine and lipid carbon as raw material, the corresponding rate of proline synthesis would then be about 1.1 pmoles/fly/hr or 125 Flg/fly/hr sufficient to account for the observed rate of synthesis in the resting fly following flight (BURSELL, 1963). With the average level of triglyceride synthesis at about 0.03 patoms carbon/mg/hr the corresponding value for triglyceride synthesis would be about 1.1 mg/fty per day, which again is ample to account for observed in viuo rates (McCABE and BURSELL, 1975b). DISCUSSION The present work has confirmed the ability of tsetse fat body to synthesise proline from lipid and amino acid carbon, and has served to identify the metabolic pathways involved. Alanine constitutes the main amino acid substrate, and this is of particular interest in view of the fact that it is the principal end-product of oxidative metabolism in the flight muscle (BuRSELL and SLACK, 1976). A very close relation is thus established between flight muscle and fat body metabolism, as illustrated in Fig. 3. Energy for the contraction of flight muscle is shown to arise from the partial oxidation of proline on the left of the diagram. The alanine so produced passes from flight muscle into the haemolymph and is transported to the abdominal fat body, on the right, where it couples with two-carbon lipid fragments to reconstitute proline; and proline passes back into the haemolymph for transport to the flight muscle, thus completing the cycle. During steady-state operation the sole input to the system as a whole would be lipid carbon, whereas the output would be energy for the contraction and maintenance of the flight muscles. The system could thus be seen as constituting a special mechanism for the oxidation of lipid reserves, geared to the special profine-centred metabolism of the tsetse fly. An interesting parallel is provided by the situation in locusts, where glycerol has been shown to play a rfle similar to that of
433
Proline synthesis by tsetse fat body
Myofibrils Sorcoplosm
Sorcosome
Hoemolymph
Foi" body
Fig. 3. Diagrammatic representation of the relationship between flight muscle and fat body metabolism during lipolytic phases of the hunger cycle. Substrates are denoted by the first three letters of their name. EN = energy, ACoA = acetyl-CoA. In the flight muscle pyruvate is shown to arise by oxidative decarboxylation of malate in accord with the findings of HOEK et al. (1976). For further explanation see text. alanine as a carrier of lipid from fat body to thoracic musculature (CANDY et al., 1976). It must be emphasised that in the tsetse fly the cyclic system will be strongly displaced from steadystate operation during flight and during the restorative phase of rest following flight. At initiation of flight in male G. morsitans proline is oxidised at a rate of about 40/amoles/hr (BURSELL and SLACK, 1976) which greatly exceeds the capacity of the fat body for proline synthesis, at about 1.1 #moles/hr. During flight, therefore, the oxidative part of the cycle will dominate to give the observed decrease in the level of proline and increase in the level of alanine (BURSELL, 1963). Conversely, during rest following flight the metabolic rate, and hence the rate of proline oxidation, decreases by a factor of about 100 (BURSELL et al., 1974; HARGROVE, 1976) to levels which are exceeded by the synthetic powers of the fat body, thus giving the observed slow reconstitution of the proline reserve associated with a corresponding decrease in the concentration of alanine. As the proline concentration increases, proline synthesis will be progressively inhibited until its rate exactly matches the rate of proline utilisation by the metabolic system as a whole, and at this point the cycle will resume steady-state operation. It has been suggested by HARGROVE(1976) that the synthesis of proline may contribute substantially to the maintenance of proline levels during flight, and so prolong the duration of flight. This suggestion is not borne out by present results which indicate that the capacity of the fat body for proline synthesis is negligible by comparison with the capacity of flight muscle for proline oxidation. The possibifity should not be ignored, however, that the mobilisation of reserves from the fat body of tsetse flies might be under neuroendocrine control, as it is in other insects (review by BArnEY, 1975). In that case the synthesis
of proline might be increased during flight to levels where it could make an effective contribution to flight metabolism, though preliminary attempts to demonstrate such an effect by incubation of fat body with haemolymph from flown flies, or with extracts of corpora cardiaca, have so far proved unsuccessful. Acknowledgements--My thanks are due to Dr D. A. NORDEN and Dr J. W. HARGROVEfor helpful comments on the manuscript.
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