Studies on the Metabolism of Locust Fat Body

The fat body of S. gregaria becomes packed with fat droplets, the predominant reserve. In addition it contains abundant glycogen and protein, and ribonucleic acid can be readily demonstrated in the cytoplasm; the tissue is free from symbiotic organisms (Coupland, 1957). Schistocerca fat body has been shown to contain a number of enzymes including transaminases and the glutamic dehydrogenase system (Kilby & Neville, 1957) and an enzyme system which synthesizes trehalose from glucose (Candy & Kilby, 1959).

S. gregaria Forskal was chosen because its fat body could be removed in a sheet which remained intact throughout experiments. The fat body was taken from sexually immature males of the gregarious phase approximately 5 days after the moult to the adult stage when it was actively laying down reserves. In the few experiments where locusts of a different age were used this is mentioned in the text. The locusts were obtained from the Anti-Locust Research Centre a day or two before use and were fed.

Locusts were anaesthetized and opened in a saline devised for Schistocerca (Weis-Fogh, 1956; note erratum). The perivisceral fat body, which contains fewer oenocytes than the peripheral fat body (Coupland, 1957), was removed from the insect and freed from testes and air sacs. The sheets of fat body were incubated separately in manometer flasks containing locust saline and trace amounts (approximately 0·5 μc.) of a metabolite labelled with carbon-14. Incubation lasted for 4 hr., the flasks being kept at 30° C. and shaken, and the carbon dioxide produced was collected on slips of filter paper moistened with potassium hydroxide solution which had been placed in the centre wells.

After incubation the fat body was carefully rinsed and then divided into the following fractions: acid-soluble, glycogen, fat, carbonate-soluble and protein. After addition of carrier glycogen, the tissue was ground with extra-fine carborundum powder in the presence of freshly-made 8% metaphosphoric acid at 0° C. A flow diagram of the fractionation procedure is shown below. The procedure was designed to remove both water-soluble and protein-bound glycogen. Extraction of lipids was carried out at 45-50° C. and the acetone-soluble protein was separated from the lipids by its insolubility in light petroleum.

The extraction with sodium carbonate solution was included to remove uric acid which is known to be present in fat body. Nucleic acids also dissolve in this solution. It was subsequently found that the small amount of radioactivity in the carbonate-soluble fraction (1–4%) was not in uric acid or nucleic acids. A tentative identification of labelled glycine in a hydrolysate of the fraction, after incubation with glycine-¹⁴C, suggested that some protein had been taken up in solution.

Sheets of perivisceral fat body were dissected from locusts as described above, rinsed in 0·15 M potassium chloride and homogenized by hand in a glass homogenizer at 0° C. The homogenate was suspended in the potassium chloride solution and centrifuged twice at 500 g. to remove cellular debris, nuclei and fat. The contents of each manometer flask consisted of a cell-free suspension of fat body, equivalent to the sheet normally removed from one locust, in 0·15 m potassium chloride, pH 7·4, and 0·5 μc. succinic acid-1,4-¹⁴C. Half of the flasks also contained 0·001m sodium malonate and in one experiment 0·001m ethylene-diamine-tetra-acetic acid was added to each flask. Incubation proceeded for 1 hr. at 30° C. in a gas phase of air, the flasks being shaken. Carbon dioxide produced during the experiment was released from solution by addition of acid and collected on filter paper moistened with potassium hydroxide solution previously placed in the centre well.

A sheep heart succinic oxidase system was prepared as described by Umbreit, Burris & Stauffer (1957). A second preparation was made in which the sheep heart muscle was homogenized with half its weight of locust fat body and the homogenate allowed to stand for an hour at 0° C. before removal of the fat by centrifuging. The final incubation mixture consisted of the homogenate in 0·1m phosphate buffer, pH 7·4, and 0·05 m sodium succinate. The homogenates were incubated with shaking at 37° C. in an atmosphere of air. Oxygen uptake was expressed as $QO2$⁠, i-e-μ1 oxygen/mg. fat free dry wt./hr.

The radioactivity of the fat-body fractions was measured by gas analysis after oxidation of tissue carbon to carbon dioxide (Glascock, 1954). The radioactive compounds of the acid-soluble fraction were separated on paper chromatograms and located by the automatic scanning technique of Winteringham, Harrison & Bridges (1952). The radioactive spots were eluted and run again (Winteringham, 1953) and were identified by co-chromatography with known compounds in at least three solvent systems, those principally used being (i) water-saturated phenol; (ii) butanol:ethanol:water (40:10:50), and (iii) formic acid: acetone:water (14:60:26). The radioactive amino acids in the protein fraction were identified in the same way after hydrolysis of the protein. Lipids were analysed on silicic acid columns (Borgstrom, 1952a, b).

Sheets of perivisceral fat body from immature adult male locusts were incubated for 4 hr. at 30° C. in locust saline with trace amounts of metabolites labelled with carbon-14. The carbon dioxide produced during incubation was collected in the usual way. After incubation the fat body was rinsed and fractionated. The distribution of radioactivity in the various fractions and the radioactivity of the carbon dioxide, after incubation with various metabolites, is shown in Table 1.

In the fat body of immature adults one-third of the radioactivity was in the acid-soluble fraction. The radioactivity of the glycogen and of the carbonate-soluble fraction was very low. Fat radioactivity was fairly high (23 %) as was that of the protein (35 %). The radioactivity of the carbon dioxide was higher than that of all the fat body fractions combined, showing that the tissue oxidized more glycine than it incorporated. To illustrate this point, carbon dioxide radioactivity is expressed, in Table 1, as percentage of the total uptake of carbon-14, i.e. as percentage of fat body plus carbon dioxide radioactivity.

The results obtained with fat body taken from male larvae 5 days after entering the 5th instar were almost identical with those from immature adults (Table 1) and preliminary results with sexually mature adult males showed that the pattern of incorporation had not changed.

The various fractions of fat body from the immature adult were analysed further. Radiochromatography of the acid-soluble fraction showed that 76 % of the radioactivity was in glycine and serine. The remainder of the activity was in two spots which were not identified. Hydrolysis of the protein fraction showed that the radioactivity was again in glycine and serine. Analysis of the fat fraction showed that 95 % of the radioactivity was in neutral fat. The remainder was distributed between free fatty acids, phospholipids, cholesterol and cholesterol esters. Hydrolysis of the neutral fat showed that 55 % of its activity was in fatty acid. Hydrolysis of the carbonate-soluble fraction yielded a radioactive compound tentatively identified as glycine.

Incubation with generally-labelled leucine yielded a pattern of incorporation somewhat similar to that obtained with glycine. The principal differences were in lower fat and higher protein radioactivity. Once again the carbon dioxide was highly radioactive, containing more than half of the carbon-14 taken up by the fat body.

The radioactivity of the acid-soluble fraction was lower and that of the fat fraction higher than after incubation with the other metabolites. Glycogen radioactivity was again negligible and the protein showed moderate radioactivity. Only one-third of the carbon-14 taken up by the fat body was converted to carbon dioxide. The radioactivity of the acid-soluble fraction was principally in proline and glutamate, the proline usually being rather more radioactive than the glutamate. Aspartate was found to be radioactive on one occasion and radioactive trehalose was also found once. There was no evidence of labelled glutamine. The acid-soluble fraction gave clearer radiochromatograms after hydrolysis suggesting that labelled peptides were present. After hydrolysis of the protein fraction radioactivity was found in proline, glutamate, aspartate and alanine, the glutamate and aspartate being the most heavily labelled.

The most striking feature of the incorporation of generally-labelled glucose into fat body was the high radioactivity of the acid-soluble fraction which comprised nearly 60% of the fat-body activity. Radiochromatography showed that the activity was almost entirely confined to trehalose. Only 3 % of the glucose was incorporated into glycogen; 22% was incorporated into fat correspending to the result obtained with glycine; incorporation into protein was low. Hydrolysis of the carbonate-soluble fraction yielded a single radioactive compound. This was not identified but it was found not to be glucose, ribose, alanine or glutamate. Less than one-third of the carbon-14 taken up by the fat body was converted to carbon dioxide.

Hearfield & Kilby (1958) made an intensive study of oxidative enzymes in the fat body of Schistocerca gregaria. They showed that a number of enzymes associated with the tricarboxylic acid cycle were present, namely, aconitase, wo-citric dehydrogenase, fumarase, malic dehydrogenase and cytochrome oxidase; but they were unable to show the presence of the condensing enzyme, a-ketoglutarate oxidase or succinic dehydrogenase and for this reason suggested that the tricarboxylic acid cycle might not play an important part in the intermediary metabolism of locust fat body.

Succinic dehydrogenase has been demonstrated within isolated mitochondria of the fat body of Periplaneta americana (L.) by the reduction of tétrazolium salts to formazan dyes (Pearse & Scarpelli, 1958). Using a similar technique, β-hydroxybutyric dehydrogenase was demonstrated in mitochondria isolated from the fat body of Locusta mgratoria L. (Hess, Scarpelli & Pearse, 1958), but attempts to obtain a reaction for succinic dehydrogenase in these mitochondria were unsuccessful (A. G. E. Pearse, personal communication). Bellamy (1958), obtained a very small uptake of oxygen on incubating homogenates of Schistocerca fat body with a-ketoglutarate and succinate and he suggested that a-ketoglutarate oxidase and succinic dehydrogenase were present but were labile on homogenizing.

When Schistocerca fat body was incubated with sodium acetate-2-¹⁴C the radioactivity of the acid-soluble fraction was found to be in glutamate,. proline and aspartate, as described above. This suggested that the tricarboxylic acid cycle was functioning normally since glutamate is formed from α-ketoglutarate by transamination and aspartate is formed from oxaloacetate in the same manner. Proline can be formed from glutamate. These experiments were therefore extended to study the respiratory metabolism of the fat body.

In a series of experiments, sheets of fat body and cell-free suspensions of fat body were incubated with sodium acetate-2-¹⁴C or succinic acid-1,4-¹⁴C and the radioactivity of the carbon dioxide produced was taken as an indication of the oxidation of the substrate. The results are given in Table 2.

Incubation of whole fat body with sodium acetate-2-¹⁴C in the presence of 0-015 M sodium fluoroacetate inhibited the production of ¹⁴CO₂ by 95 %. The inhibitory effect of fluoroacetate has been described by Peters (1955). Fluoroacetate itself has no known action upon isolated enzymes in vitro, but in vivo fluoroacetate is synthesized by the condensing system to monofluorocitric acid and this competitively inhibits aconitase, blocking the tricarboxylic acid cycle at the citric acid stage. The inhibitory effect of fluoroacetate on ¹⁴CO₂ production by whole fat body thus provides support for Hearfield & Kilby’s claim to have found aconitase and provides indirect evidence for the presence of the condensing system of enzymes in locust fat body (Table 2).

Incubation of whole fat body with succinic acid-1,4-¹⁴C yielded highly radioactive carbon dioxide, showing that an active succinic dehydrogenase was present. To confirm this finding, the effect of malonate on ¹⁴CO₂ production was examined. O’OiM malonate had no inhibitory effect on ¹⁴CO₂ production during incubation of whole fat body with trace amounts of acetate-2-¹⁴C or succinic acid-1,4-¹⁴C; in fact, in all experiments it appeared to have a slightly stimulating effect. Malonate is known to stimulate endogenous respiration in Chlorella (M. Merritt, personal communication). The presence of 0·02m malonate during incubation of whole fat body with succinic acid-1,4-¹⁴C caused 20% inhibition in the production of ¹⁴CO₂ and the presence of O-OIM malonate during incubation of a cell-free fat-body suspension with this metabolite caused inhibition of 59 and 79% in two experiments.

Homogenization of fat body greatly reduced formation of ¹⁴CO₂ on subsequent incubation with succinic acid-1,4-¹⁴C. Addition of 0·001m ethylene-diaminetetra-acetic acid to the preparation had no marked effect. The reduction was greater than the figures in Table 2 suggest, for there the radioactivity of the carbon dioxide is expressed in terms of the dry weight of the final preparations. In whole fat body the dry weight included the inactive reserves, but in the cell-free suspensions the fat reserves were removed after centrifuging. Homogenization in fact reduced ¹⁴CO₂ production by about 95 %.

Sodium oleate is known to inhibit succinic oxidase (Edwards & Ball, 1954) and rat intestinal mucosa has been shown to contain a factor, identified as fatty acid, which inhibits succinic oxidase after homogenization (Nakamura, Pichette, Broitman & Bezman, 1959). It seemed likely that such an inhibitor must be released during homogenization of fat body so the effect of fat body homogenate on a succinic oxidase system known to be active was studied. Sheep heart muscle homogenized with half its weight of locust fat body gave a $QO2$ of 257 on incubation with 0-05 M succinate compared with the $QO2$ of 241 of the control preparation. The fat-body homogenate thus had no inhibitory effect on the sheep heart muscle succinic oxidase system.

When whole fat body was incubated with glycine-¹⁴C (G) or leucine-¹⁴C (G) well over half of the carbon-14 taken up was converted to carbon dioxide yet incubation with acetate-2-¹⁴C or glucose-¹⁴C (G) resulted in much less than half of the carbon-14 taken up appearing in this form (Table 1). This suggested that amino acids might form an important substrate for respiration so a comparison was made of the oxidation of amino acids by the fat body and by the indirect flight muscles. These tissues were incubated in locust saline with trace amounts of glycine-¹⁴C (G) and leucine-¹⁴C (G) and the radioactivity of the carbon dioxide produced was measured and expressed in terms of the dry weight of the tissue. Bellamy (1958) has shown that Schistocerca fat body and flight muscle have similar rates of respiration when incubated in 0·25 m sucrose. The results are given in Table 3.

The fat body produced seven times as much radioactive carbon dioxide from glycine-¹⁴C (G) as the flight muscle and it produced twenty-six times as much radioactive carbon dioxide from leucine-¹⁴C (G) as the flight muscle.

Studies of isotope incorporation can provide very useful data on the pathways of metabolism but the recycling of isotopes in living tissues is liable to give an impression of synthesis where in fact there is no net gain. Thus the incorporation of radioactive glycine into protein may not represent synthesis of protein, but may simply reflect the rate of turnover of protein in the tissue. However, turnover rates are much lower in growing cells. In yeast the protein breakdown in growing cells is only 4% of that in resting cells (Halvorson, 1958), and in rapidly growing cells of Escherichia coli there is no detectable turnover of protein (Mandelstam, 1958). As the fat body of Schistocerca was studied at a time when it was rapidly laying down reserves it may well be that the pattern of incorporation reflected synthesis rather than turnover but this cannot be taken for granted.

The pattern of incorporation obtained with the various metabolites shows that fat body is able to use a wide range of substrates for the formation of fat and protein although, as would be expected, glycine and leucine were more readily incorporated into protein than the other metabolites and acetate was more readily incorporated into fat. Sugars seem to be the only important source of trehalose. The high level of incorporation of various metabolites into protein suggests that fat body may synthesize much protein. In contrast, glycogen appears to be a minor product of fat-body synthesis and the high incorporation of glucose into trehalose may reflect the importance of trehalose as a fat-body reserve. It is known to be used during flight in Locusta (Bücher & Klingenberg, 1958).

Incubation with acetate-2-¹⁴C led to the appearance of radioactivity in glutamate, aspartate and alanine. It is well known that these amino acids can become labelled with acetate via the tricarboxylic acid cycle and the result shows that in insects, as in other organisms, the intermediates of the tricarboxylic acid cycle can provide the carbon skeletons of the non-essential amino acids. It is surprising that no radioactivity was found in glutamine as Bellamy (1958) found glutamine to be twice as abundant as glutamate in Schistocerca fat body.

The inhibitory effect of fluoroacetate on fat body respiration confirms the presence of aconitase, found by Hearfield & Kilby (1958), and provides indirect evidence for the presence of the condensing enzyme system, not found by these authors.

The production of ¹⁴CO₂ from succinic acid-1,4-¹⁴C shows that whole fat body contains succinic dehydrogenase, and this enzyme could be inhibited with 0·02 m malonate. The succinic oxidase system appears to be labile on homogenization, as was suggested by Bellamy (1958). The failure of Hearfield & Kilby (1958) to find the condensing system suggests that it also is labile on homogenization and it is of interest that inhibition at this point in the tricarboxylic acid cycle would lead to accumulation of oxaloacetate which would competitively inhibit succinic dehydrogenase. That the inhibition of succinic dehydrogenase is not due only to oxaloacetate accumulation is shown by Pearse’s failure to demonstrate the enzyme in isolated mitochondria using a tetrazolium salt technique (see p. 670). It may be concluded that some unknown factor of the homogenate inhibits the fat body condensing system and succinic dehydrogenase and, probably, α-ketoglutarate oxidase also. This factor has no effect on sheep heart muscle succinic dehydrogenase.

The much higher rate of oxidation of amino acids than of glucose to carbon dioxide is surprising as glucose is the most generally usable fuel, but it may be related to the functioning of the fat body in the intermediary metabolism of the insect. The demonstration in Schistocerca fat body of active transaminases and of a glutamic dehydrogenase system which links nitrogen and carbohydrate metabolism (Kilby & Neville, 1957) lends support to this idea. The finding that fat body oxidizes glycine and leucine many times faster than do the flight muscles parallels observations on the rat which showed that liver and kidney were able to oxidize glycine at a high rate while skeletal muscle had no such ability (Nakada & Weinhouse, 1953). It is possible that the fat body performs one of the functions of the vertebrate liver, transdeaminating amino acids and making the residues available for metabolism by other tissues.

1. The incorporation of glycine-¹⁴C (G), leucine-¹⁴C (G), sodium acetate-2-¹⁴C and glucose-¹⁴C (G) into Schistocerca fat body was studied under in vitro conditions, and the distribution of radioactivity in the various fat body fractions and the labelling of compounds within the fractions is described.

2. The overall picture was of high incorporation into fat and protein and of very low incorporation into glycogen.

3. Incubation with glycine-¹⁴C led to radioactivity appearing in the glycine and serine of the protein and of the amino acid pool. Incubation with sodium acetate-2-¹⁴C led to radioactivity appearing in glutamate, proline, aspartate and alanine, showing that the intermediates of the tricarboxylic acid cycle provide the carbon skeletons of certain amino acids. Glucose-¹⁴C was largely converted to trehalose.

4. Succinic dehydrogenase and the condensing enzyme system were shown to be present in fat body, contrary to previous reports. The succinic oxidase system was highly labile on homogenizing the tissue.

5. Fat body, unlike flight muscle, used glycine-¹⁴C and leucine-¹⁴C as respiratory substrates, and it is suggested that fat body acts like the vertebrate liver by transdeaminating amino acids and making them available for further metabolism by other tissues.

The radiochromatography was carried out in the Biochemistry Section of the Pest Infestation Laboratory, Slough, and I am most grateful to Mr F. P. W. Winteringham for putting the equipment at my disposal. It is a pleasure to thank Dr S. E. Lewis, Dr J. H. Ottaway and Dr A. L. Greenbaum for their advice. A grant was received from the Central Research Funds of London University and the locusts were provided by the Anti-Locust Research Centre.