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Insect acetyl-CoA carboxylase: enzyme activity during adult development and after feeding in the tsetse fly, Glossina morsitans
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Comp. Biochem. Physiol. Vol. 108B, No. 1, pp. 27-33, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
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0305-0491(93)E0003-X
Insect acetyl-CoA carboxylase: enzyme activity during adult development and after feeding in the tsetse fly, Glossina morsitans J. P. Dean Goldring* and John S. Read Department of Biochemistry, University of Zimbabwe, P.O. Box M.P. 167, Harare, Zimbabwe; and *Department of Biochemistry, University of the Witwatersrand, P.O. 2050 Wits, Johannesburg, South Africa Acetyl-CoA carboxylase (EC 6.1.4.2) activity in the adult tsetse fly (Glossina morsitans) increased 2-3 days after pupation to reach a plateau of between 0.4 and 0.6 pmol/min[mg after 7 days, and between 0.6 and 0.8 pmol[min/mg after 6 days in the abdomens of male and female flies, respectively. The enzyme showed a 50-70% increase in specific activity within 20 hr after a blood meal in previously starved flies. Lipogenesis and acetyl-CoA carboxylase activity were detected in the thorax, the abdominal cuticle and, in greatest quantity, in the fat body. Key words: Acetyl-CoA carboxylase; Glossina morsitans; Tsetse fly; Fat body; Lipogenesis; Fatty acid synthesis. Comp. Biochem. Physiol. 108B, 27-33, 1994.
Introduction The fat body is the organ of storage and general mammals (Numa and Tanabe, 1984). The enbiosynthesis in insects. In the tsetse fly zyme fatty acid synthetase has been purified and (Glossina) this organ has relatively little stored characterized from the pea aphid, Acyrthosiphon glycogen and low glycolytic enzyme activity pisum (Ryan et al., 1983) and partially purified (Norden and Patterson, 1969; Konji et al., 1984) from Drosophila melanogaster (de Renobles whereas lipids, and therefore fatty acids, are a et al., 1986). The activity of fatty acid synthetase major energy storage compound for the insect in the Mediterranean fruitfly, Ceratatis capitata, (Jack, 1939; Langley, 1977). Lipid catabolism has been followed during its development by has been shown to contribute to the production Municio et al. (1977). This group subsequently of proline, the major substrate for tsetse fly measured both acetyl-CoA carboxylase and flight muscle metabolism (Bursell and Slack, fatty acid synthetase activity in response to 1976; Bursell, 1963). The synthesis of neutral dietary unsaturated fatty acids in the fruitfly lipids has been followed in the tsetse fly using larvae (Lizarbe et al., 1980). In our laboratory [14C] acetate (McCabe, 1973), [14C] glucose we have determined the activity of acetyl-CoA (Moloo, 1977) and [lac] leucine (Langley and carboxylase during the development of four Bursell, 1980) as substrates. An alternative insects, including the tsetse fly. We found a method, and possibly a more accurate one relationship between total lipid mass and because of the ability to measure peaks of acetyl-CoA carboxylase activity at the larval, lipogenic activity, is to determine the activity of pupal and adult stages of insect development relevant lipogenic enzymes as has been done in (Goldring and Read, 1993). Unlike mammalian lipogenesis, lipogenic enzymes in insects have received little attention (Stanley-Samuelson et al., 1988). In mammals, it has been estabCorrespondence to: J. P. Dean Goldring, Department of lished that acetyl-CoA carboxylase is the rateBiochemistry, University of the Witwatersrand, P.O. limiting enzyme in the fatty acid biosynthetic 2050 Wits, Johannesburg, South Africa. Tel: (27) 11 pathway (Numa and Tanabe, 1984). 716-2211; Fax: (27) 11 403-1733. 27
28
J. P. Dean Goldring and John S. Read
Due to the economic importance of the African tsetse fly (Glossina spp.) as a vector of Trypanosomiasis, and because of the insect's fairly unique life-cycle, involving the nutrition of a single larva in utero, (Moloo, 1976) we selected the tsetse fly for further study of insect acetyl-CoA carboxylase. We wished to examine the changes in enzyme activity in the newly emerged adult, and the enzyme activity after a blood meal in mature adults. It was also important to determine the sites of fatty acid synthesis, and to ascertain if de novo fatty acid synthesis is associated with the cuticle as a precursor to the cuticular hydrocarbons (Blomquist et al., 1987; Juarez et al., 1992). We compared our measurements of enzyme activity with a second radioactive method which measures total lipogenic activity.
Materials and Methods Tsetse flies Tsetse fly pupae (Glossina morsitans) were obtained from the Tsetse Research Laboratory, University of Bristol, Langford, U.K. and were fed on guinea-pigs at the tsetse insectary, Department of Zoology, University of Zimbabwe (Dame and Ford, 1966). Preparation of tsetse tissue for determining enzyme activity during earl), adult development Flies of each sex were separated upon emergence from the pupae and presented daily with guinea-pigs for an hour. Ten flies were removed daily after feeding and immobilized by cooling ( - 2 0 ° C ) for 5min. The abdomen and the thorax of each fly were separated and the residual wet blood meal was squeezed out of the abdomen and weighed on filter paper. Flies without a blood meal were discarded. The abdomens and thoraxes of five flies were pooled and homogenized in 0.25 M sucrose buffer solution (1 ml) containing, 1.0 mM EDTA, 10 mM potassium citrate, and 15 mM Na HPO 4 at pH 7.0 and 4°C. The homogenates were immediately assayed in duplicate for acetyl-CoA carboxylase activity. The entire experiment ((~13 days) was run twice. Preparation of tsetse fly tissue .[or 24hr postfeeding measurements Flies over 10 days old were starved for 2 days and then presented with guinea-pig blood for 1 hr. Every 2-3 hr after feeding, 10 flies were removed from the colony, immobilized by cooling ( - 20°C) for 5 min and dissected into thorax and abdomen. The residual blood meal was removed on to filter paper, and the thorax and abdomen were each homogenized in the 0.25 M sucrose buffer (as outlined above). Flies without
a blood meal were discarded. Acetyl CoA carboxylase activity was determined and protein content measured. Over the 24-hr period enzyme activity was determined within 30 min of the flies being removed from the colony at each time point.
Dissection of tissue To determine the major site of lipogenic activity in these insects, five flies of each sex over 14 days old, were dissected under a binocular microscope in 0.25 M sucrose buffer solution (see above) into head and thorax, fat body, abdominal cuticle, digestive tract, and blood meal. Flies were collectively homogenized in 0.25 M sucrose buffer as before. A 20/~1 sample was assayed in duplicate for carboxylase activity, and the remaining homogenate was incubated with tritiated water. Acetyl-CoA carboxylase assay Acetyl-CoA carboxylase activity was determined as described previously (Goldring and Read, 1993; Rainwater and Kolattukudy, 1982). Briefly, 20/~1 homogenate was added to an assay mixture of 50raM Tris-HC1 buffer, pH 7.0, and 50 mM NaH~4CO3 (specific activity 0.1 mCi/mmol, Amersham, International, Amersham, U.K.), 15mM potassium citrate, 15mM magnesium chloride, 2.5mM ATP, 100/~g bovine serum albumin and l mM acetyl-CoA (Sigma, St Louis, MO) to give a total volume of 0.1 ml. This assay was carried out in duplicate in BEEM capsules held in a water bath at 3T'C. The reaction was stopped after 15 min by the addition of 0.1 ml 6 M HC1 and the mixture transferred to scintillation vials and dried at 100°C on a heating block under a stream of air to drive off unfixed radioactive bicarbonate. The residue was dissolved in 1 ml water and 4ml scintillation fluid ("Lumax", Lumac B.V., Holland) and counted on a Packard tricarb scintillation counter. The counts per min (cpm) were converted to disintegrations per min (dpm) by the channels ratio method using a quench curve produced by standards (Amersham). Tritiated water assay for lipogenisis The incorporation of 3H20 (Herzberg, 1979) was measured on each separate tissue dissected, homogenized as above and incubated at 37°C in 0.25 M sucrose buffer (see above) with 1.0/~1 3H20 (Amersham, 5 Ci/ml). The assay was terminated after 2 hr by the addition of hot 5 M K O H and boiled for l hr. The total radioactivity (dpm) in a petroleum ether fraction (B.pt 60°C) was determined after concentration of the solvent (3 ml) and addition of 5 ml Lumax scintillation fluid (Leyack, B.V., Holland).
Acetyl-CoA carboxylase in the tsetse fly
29
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Fig. 1. Activity of a c e t y l ~ o A carboxylase during the first 13 days of adult life in abdomen and thorax of the female tsetse fly. The points represent the mean of eight readings (two groups of five flies assayed in duplicate, the experiment run twice). Bars represent the spread of readings.
Protein determination Protein content was determined by the modified Coomassie Blue dye binding method (Spector, 1978), using bovine serum albumin as standard. Chemicals All bichemicals were purchased from Sigma, and the solvents and salts were of analytical grade. Results
Acetyl-CoA carboxylase activity in the abdomen and thorax of male and female tsetse flies during the first 13 days of adult life The results of the first experiment to measure lipogenic activity in newly emerged adults showed a noticeable change in the specific activity of the enzyme acetyl-CoA carboxylase
after 2-3 days in both male and female flies. This activity in the abdomen of males rose rapidly and peaked at day 6 and then decreased to a value between 0.4 and 0.6 #mol/min/mg for the next 7 days (Fig. 2). The female tsetse fly showed a gradual increase of abdominal enzyme activity to reach a plateau of 0.64).8 pmol/min/ mg specific activity on day 6 which lasted for the next 7 days (Fig. 1). The activity was noticeably higher in the females when compared to agematched males, and in both sexes the abdomen had higher enzyme activity than the thorax. Thoracic enzyme activity remained between 0.05 and 0.175/zmol/min/mg in males and 0.1 and 0.3 pmol/min/mg in females for the duration of the experiment (Figs 1 and 2). The actual mass of the blood meal reached a plateau at about the same time as enzyme activity, days 6-8, in both sexes, but the females had ingested larger blood meals (Fig. 3).
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Fig. 2. Activity of acetyl~CoA carboxylase during the first 13 days of adult life in the male tsetse fly. The points represent the mean of eight readings (two groups of five flies assayed in duplicate, the experiment run twice). Bars represent the spread of readings.
30
J.P. Dean Goldring and John S. Read
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Fig. 3. Blood meal wet weight for the first 13 days of adult life in male and female tsetse flies. The points represent the blood meal from one fly (10 flies/10).
Acetyl-CoA carboxylase activity in the abdomen and thorax of adult male tsetse flies for 24hr after feeding The change in acetyl-CoA carboxylase activity after the blood meal, over 24 hr (Fig. 4) was approximately 0.10-0.15/~mol/min/mg specific activity in the abdomen, and the thorax showed changes of a similar order. The broad peak of activity, however, could be seen at about 20 hr after feeding.
Lipogenesis and acetyl-CoA carboxylase activity in different tissues in the tsetse fly In Table 1 the results of enzyme determinations on the dissected flies are shown together with the incorporation of tritium into lipid as a measure of lipogenesis. The enzyme activites are expressed as specific activities, i.e. the rate of conversion of the substrate per mg of protein, whereas the other assay measures the total
tritium incorporated into saponifiable lipids in each tissue. The fat body had the highest level of lipogenic activity in male and female flies as determined by both assays, and there was a high level of activity in the thorax and the cuticle. Similar results were obtained for tritium incorporated into intact tissue (results not shown).
Discussion The initial low acetyl-CoA carboxylase activity, lasting 2 3 days, is probably due to both the domination of the insect's metabolism by the need to develop its flight musculature (Bursell, 1961), and the possibility that the insect is still relying on lipid stores laid down by the larva before pupation (Jack, 1939; Goldring and Read, 1993). This suggests that the lipid synthetic activity in the pregnant female is sufficient to sustain its larva through pupation to the
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Fig. 4. Acetyl-CoA carboxylaseactivity measured for a 24-hr period after feeding in male tsetse flies. The points represent the mean of duplicate readings.
31
Acetyl-CoA carboxylase in the tsetse fly Table 1. The activity of acetyl-CoA carboxylase and the incorporation of 3H20 into a saponifiable lipid fraction in tissues dissected from male and female tsetse flies
Tissue Thorax and head Blood meal Digestive tract Fat body Cuticle
Acetyl-CoA carboxylase activity Incorporation of 3H20 (# mol/min/mg protein) (dpm/tissue) Female Male Female Male 0.6 0.0 0.1 4.44 0.7
adult stage. Female tsetse flies produce lipid in the fat body for their own lipid requirements as well for larval nutrition (Moloo, 1976; Langley and Bursell, 1980) and therefore we would expect to see higher acetyl-CoA carboxylase activity in the female compared to the male. Female flies do have more lipid by weight and ingest a larger blood meal (Fig. 3), after 12 days of life, than male flies fed on the same diet (Langley and Pimley, 1979). The female tsetse fly showed a gradual increase of carboxylase activity to reach a plateau on day 6 which lasted for the next 7 days, while males showed a more pronounced rise in enzyme activity which peaked on day 6 and then remained constant for the next 7 days. It is possible that if these were wild flies, with the further energy demands made when they search for food and mates (Langley, 1966b), they would show more peaks of lipogenic activity. In both sexes, the peak in lipogenic activity, as measured by acetyl-CoA carboxylase activity, would appear to occur 1 or 2 days before the peak of total lipid product determined by Langley and Pimley (1979) and Quinlan and Gatehouse (1981). This is not altogether surprising, as this could be the difference between measuring the activity of one enzyme and measuring the total product of a biosynthetic pathway. It is possible that the latter is a period when esterification and accumulation of the fatty acid products as neutral lipids occurs before breakdown begins to predominate. The rise in the weight of the blood meal during insect development follows the same trend as enzyme activity (Fig. 3), suggesting a direct relationship with dietary intake. Such a relationship has been established for mammalian acetyl-CoA carboxylase where carboxylase mRNA levels change in response to fasting and refeeding (Pape et al., 1988). Bursell (1963) showed that as the residual dry weight of the abdomen decreased by over a half, the lipid content in the abdomen rose to a peak in 24hr. McCabe and Bursell (1975a,b) followed the incorporation of radioactive leucine into neutral lipids, palmitic acid and palmitoleic acid and found peak incorporation to be be-
0.585 0.0 0.2 3.3 1.0
243 67 148 3084 2048
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tween 24 and 36 hr. The percent digestion taking place in the abdomen was shown to reach a peak at the same time by Langley (1966a). Our results show that there is a similar increase in acetyl-CoA carboxylase activity, but the peak occurs slightly before 24 hr, at approximately 20 hr. The percentage change is between 50 and 75% over this time period in both the abdomen and thorax. Bursell et al. (1974) discussed this rise and fall in lipid content in relation to a change from a lipogenic to lipolytic phase. To describe this fully would require the simultaneous determination of the activity of enzymes involved in both pathways. Bursell (1963) showed a gradual utilization of lipid stores by conversion to proline in the fat body starting 12 hr after a blood meal. The pathway from lipid to proline may be via acetate but it is unlikely to be the only metabolic route. Therefore the activity of lipolytic enzymes and the lipase, which possibly releases diglycerides from the fat body for lipid transport by lipoproteins (Beenakkers et al., 1984), would also have to be determined to fully describe the lipolytic phase proposed by Bursell. The results obtained in these experiments all show that the abdomen has higher acetyl-CoA carboxylase activity than the thorax, and as we expected, the prime site of activity was indeed the fat body (Table 1). When studied in isolation, the specific enzyme activity is proportionately higher in the abdomen than in the thorax, and this activity is 25% higher in the female fat body compared to the male. The difference in enzyme activity in the thorax of both sexes is less pronounced. The uterine gland could be thought of as analogous to the mammalian mammary gland and Langley and Kaboyo (1981) published a method for the separation of the gland from the fat body, but the technique does not give viable tissue for estimating enzyme activity. Since the uterine gland synthesizes proteins (Riddiford and Dhadialla, 1990) it would be interesting to see if the gland is able to synthesize fatty acids de novo to account for the differences in enzyme activity between the sexes. It is more likely, however, that fat body diglycerides are the source of
32
J. P. Dean Goldring and John S. Read
uterine lipids (Langley and Bursell, 1980). If the uterine gland produced fatty acids, such a pathway would also be unlikely to be active before sufficient maturation had been reached for the female to achieve pregnancy (Ma et al., 1975). These arguments favour the suggestion that the extra enzyme activity in female flies observed in this study is a true measurement of lipogenesis. It would be interesting to measure carboxylase m R N A levels in the fat body during pregnancy to see if a demand for uterine milk by the developing larvae induced increased expression of the carboxylase gene as has been reported for the mammalian mammary gland during lactation (Pape e t aL, 1988). Female tsetse flies produce a hydrocarbon contact sex pheromone and injected succinate can be incorporated into this pheromone (Langley and Carlson, 1983), suggesting that fatty acid synthesis may be the first step to synthesis of these branched-chain hydrocarbons. The incorporation of radioactivity from 3H20 into saponifiable lipid fraction (Table 1) by the cuticle fraction indicates that there was de n o v o fatty acid synthesis associated with the cuticle. Recently, fatty acid synthetase, which produces methyl-branched fatty acids, has been reported in integument-enriched tissue from the German cockroach, B l a t t e l l a g e r m a n i c a (Juarez et al., 1992; Gu et al., 1993). Fatty acid synthetase was found to be active in both a microsomal and soluble form and utilized m a l o n y l - C o A as a substrate. Our study showed acetyl-CoA carboxylase activity in the cuticle fraction. Since tritium was incorporated into saponifiable lipid and the enzyme produces malonyl-CoA, we expect fatty acid synthetase to be present in this tissue in tsetse flies. Acetyl-CoA carboxylase is also a likely producer of integument malonyl~CoA used by fatty acid synthetase in the German cockroach (Gu et al., 1993). Enzyme activity in the cuticle was similar to that in the thorax but lower than that suggested by the incorporated tritium. It is possible that some of the tritium incorporated can be accounted for by post-transport modification of fatty acids. An alternative explanation may be due to residual fat body tissue fragments, or to the presence of oenocytes (Tobe et al., 1973), which have been considered to be the site of hydrocarbon synthesis in insects (Diehl, 1975). The level of acetyl-CoA carboxylase activity in the thorax would be expected to provide for local metabolic requirements. The control of lipid synthesis in the tsetse fly and a role for acetyl-CoA carboxylase remain to be investigated. We did note, however (results not shown) that including thorax extracts into abdominal preparations lowered enzyme activity, suggesting the presence of regulatory
factors in the thorax. Further studies of acetyl-CoA carboxylase from tissues which have been incubated with the peptide hormone, lipid synthesis inhibitory factor (Pimley and Langley, 1981), octopamine (Pimley, 1985) and the adipokinetic hormone (Beenakkers et al., 1984; Strobel et al., 1990) would give insight into the role of acetyl-CoA carboxylase in regulating lipid synthesis, and the supply of lipids for uterine milk in the tsetse fly. Acknowledgements--The authors would like to thank the
University of Zimbabwe for a Scholarship (JPDG), and the Research Board of the Universityof Zimbabwe for financial support. We thank the Tsetse Research Laboratory, Langford, Bristol, U.K. for a supply of tsetse fly pupae, and Dr P. Phelps, Department of Zoology, University of Zimbabwe, for use of the tsetse insectary.
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Juarez P., Chase J. L. and Blomquist G. J. (1992) A microsomal fatty acid synthetase from the integument of Blattella germanica synthesises methyl-branched chain fatty acids, precursors to hydrocarbon and contact sex pheromone. Archs Biochem. Biophys. 239, 333-341. Konji V. N., Olembo N. J. and Pearson D. J. (1984) Enzyme activities in the fat body of the tsetse fly, Glossina morsitans, and the fleshfly, Sarcophaga tibialis in relation to proline metabolism. Insect Biochem. 14, 6854590. Langley P. A. (1966a) The control of digestion in the tsetse
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and the blowfly (Sarcophaga). Comp. Biochem. Physiol. 31, 819-827. Numa S. and Tanabe T. (1984) Acetyl-CoA carboxylase and its regulation. In Fatty Acid Metabolism and Its Regulation (Edited by Numa S.), pp. 1-23. Elsevier, Amsterdam. Pape M. E., Lopez-Casillas F. and Kim K.-H. (1988) Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl~CoA carboxylase mRNA. Archs Biochem. Biophys. 267, 104-109. Pimley R. W. (1985) Cyclic AMP and calcium mediate the regulation of fat cell activity octopamine and peptide hormones in Glossina morsitans. Insect Biochem. 15, 293-298. Pimley R. W. and Langley P. A. (1981) Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans. J. Insect Physiol. 27, 839-847. Quinlan R. J. and Gatehouse A. J. (1981) The effect of low doses of Endosulphan on lipid reserves in the tsetse fly, Glossina morsitans. Ent. exp. appl. 29, 29-38. Rainwater D. L. and Kolattukudy P. E. (1982) Purification and characterisation of acetyl-CoA carboxylase from goose uropygial gland which produces multimethyl branched acids and evidence for its identity with avian acetyl-CoA carboxylase. Archs Biochem. Biophys. 213, 372-383. de Renobles M., Woodin T. S. and Blomquist G. J. (1986) Drosophila melanogaster fatty acid synthetase: characteristics and effect of protease inhibitors. Insect Biochem. 16, 887-894. Riddiford L. M. and Dhadialla T. S. (1990) Protein synthesis by the milk gland and fat body of the tsetse fly, Glossina pallidipes. Insect Biochem. 20, 493-500. Spector T. (1978) Refinement of the Coomassie Blue method for protein quantitation. Analyt. Biochem. 86, 142-146. Stanley-Samuelson D. W., Jurenka R. A., Cripps C., Blomquist G. J. and de Renobles M. (1988) Fatty acids in insects: composition, metabolism and biological significance. Arch. Insect Biochem. Physiol. 9, 1-33. Strobel L. M., Kanost M. R., Zeigler R. and Wells M. A. (1990) Adipokinetic hormone causes formation of a low density lipophorin in the house cricket Acheta domesticus. Insect Biochem. 20, 859-863. Tobe S. S., Davey K. S. and Huebner E. (1973) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.: histology and histochemistry of the milk gland, fat body and oenocytes. Tissue Cell. 5, 633~550.
Comp. Biochem. Physiol. Vol. 108B, No. 1, pp. 27-33, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
0305-0491(93)E0003-X
Insect acetyl-CoA carboxylase: enzyme activity during adult development and after feeding in the tsetse fly, Glossina morsitans J. P. Dean Goldring* and John S. Read Department of Biochemistry, University of Zimbabwe, P.O. Box M.P. 167, Harare, Zimbabwe; and *Department of Biochemistry, University of the Witwatersrand, P.O. 2050 Wits, Johannesburg, South Africa Acetyl-CoA carboxylase (EC 6.1.4.2) activity in the adult tsetse fly (Glossina morsitans) increased 2-3 days after pupation to reach a plateau of between 0.4 and 0.6 pmol/min[mg after 7 days, and between 0.6 and 0.8 pmol[min/mg after 6 days in the abdomens of male and female flies, respectively. The enzyme showed a 50-70% increase in specific activity within 20 hr after a blood meal in previously starved flies. Lipogenesis and acetyl-CoA carboxylase activity were detected in the thorax, the abdominal cuticle and, in greatest quantity, in the fat body. Key words: Acetyl-CoA carboxylase; Glossina morsitans; Tsetse fly; Fat body; Lipogenesis; Fatty acid synthesis. Comp. Biochem. Physiol. 108B, 27-33, 1994.
Introduction The fat body is the organ of storage and general mammals (Numa and Tanabe, 1984). The enbiosynthesis in insects. In the tsetse fly zyme fatty acid synthetase has been purified and (Glossina) this organ has relatively little stored characterized from the pea aphid, Acyrthosiphon glycogen and low glycolytic enzyme activity pisum (Ryan et al., 1983) and partially purified (Norden and Patterson, 1969; Konji et al., 1984) from Drosophila melanogaster (de Renobles whereas lipids, and therefore fatty acids, are a et al., 1986). The activity of fatty acid synthetase major energy storage compound for the insect in the Mediterranean fruitfly, Ceratatis capitata, (Jack, 1939; Langley, 1977). Lipid catabolism has been followed during its development by has been shown to contribute to the production Municio et al. (1977). This group subsequently of proline, the major substrate for tsetse fly measured both acetyl-CoA carboxylase and flight muscle metabolism (Bursell and Slack, fatty acid synthetase activity in response to 1976; Bursell, 1963). The synthesis of neutral dietary unsaturated fatty acids in the fruitfly lipids has been followed in the tsetse fly using larvae (Lizarbe et al., 1980). In our laboratory [14C] acetate (McCabe, 1973), [14C] glucose we have determined the activity of acetyl-CoA (Moloo, 1977) and [lac] leucine (Langley and carboxylase during the development of four Bursell, 1980) as substrates. An alternative insects, including the tsetse fly. We found a method, and possibly a more accurate one relationship between total lipid mass and because of the ability to measure peaks of acetyl-CoA carboxylase activity at the larval, lipogenic activity, is to determine the activity of pupal and adult stages of insect development relevant lipogenic enzymes as has been done in (Goldring and Read, 1993). Unlike mammalian lipogenesis, lipogenic enzymes in insects have received little attention (Stanley-Samuelson et al., 1988). In mammals, it has been estabCorrespondence to: J. P. Dean Goldring, Department of lished that acetyl-CoA carboxylase is the rateBiochemistry, University of the Witwatersrand, P.O. limiting enzyme in the fatty acid biosynthetic 2050 Wits, Johannesburg, South Africa. Tel: (27) 11 pathway (Numa and Tanabe, 1984). 716-2211; Fax: (27) 11 403-1733. 27
28
J. P. Dean Goldring and John S. Read
Due to the economic importance of the African tsetse fly (Glossina spp.) as a vector of Trypanosomiasis, and because of the insect's fairly unique life-cycle, involving the nutrition of a single larva in utero, (Moloo, 1976) we selected the tsetse fly for further study of insect acetyl-CoA carboxylase. We wished to examine the changes in enzyme activity in the newly emerged adult, and the enzyme activity after a blood meal in mature adults. It was also important to determine the sites of fatty acid synthesis, and to ascertain if de novo fatty acid synthesis is associated with the cuticle as a precursor to the cuticular hydrocarbons (Blomquist et al., 1987; Juarez et al., 1992). We compared our measurements of enzyme activity with a second radioactive method which measures total lipogenic activity.
Materials and Methods Tsetse flies Tsetse fly pupae (Glossina morsitans) were obtained from the Tsetse Research Laboratory, University of Bristol, Langford, U.K. and were fed on guinea-pigs at the tsetse insectary, Department of Zoology, University of Zimbabwe (Dame and Ford, 1966). Preparation of tsetse tissue for determining enzyme activity during earl), adult development Flies of each sex were separated upon emergence from the pupae and presented daily with guinea-pigs for an hour. Ten flies were removed daily after feeding and immobilized by cooling ( - 2 0 ° C ) for 5min. The abdomen and the thorax of each fly were separated and the residual wet blood meal was squeezed out of the abdomen and weighed on filter paper. Flies without a blood meal were discarded. The abdomens and thoraxes of five flies were pooled and homogenized in 0.25 M sucrose buffer solution (1 ml) containing, 1.0 mM EDTA, 10 mM potassium citrate, and 15 mM Na HPO 4 at pH 7.0 and 4°C. The homogenates were immediately assayed in duplicate for acetyl-CoA carboxylase activity. The entire experiment ((~13 days) was run twice. Preparation of tsetse fly tissue .[or 24hr postfeeding measurements Flies over 10 days old were starved for 2 days and then presented with guinea-pig blood for 1 hr. Every 2-3 hr after feeding, 10 flies were removed from the colony, immobilized by cooling ( - 20°C) for 5 min and dissected into thorax and abdomen. The residual blood meal was removed on to filter paper, and the thorax and abdomen were each homogenized in the 0.25 M sucrose buffer (as outlined above). Flies without
a blood meal were discarded. Acetyl CoA carboxylase activity was determined and protein content measured. Over the 24-hr period enzyme activity was determined within 30 min of the flies being removed from the colony at each time point.
Dissection of tissue To determine the major site of lipogenic activity in these insects, five flies of each sex over 14 days old, were dissected under a binocular microscope in 0.25 M sucrose buffer solution (see above) into head and thorax, fat body, abdominal cuticle, digestive tract, and blood meal. Flies were collectively homogenized in 0.25 M sucrose buffer as before. A 20/~1 sample was assayed in duplicate for carboxylase activity, and the remaining homogenate was incubated with tritiated water. Acetyl-CoA carboxylase assay Acetyl-CoA carboxylase activity was determined as described previously (Goldring and Read, 1993; Rainwater and Kolattukudy, 1982). Briefly, 20/~1 homogenate was added to an assay mixture of 50raM Tris-HC1 buffer, pH 7.0, and 50 mM NaH~4CO3 (specific activity 0.1 mCi/mmol, Amersham, International, Amersham, U.K.), 15mM potassium citrate, 15mM magnesium chloride, 2.5mM ATP, 100/~g bovine serum albumin and l mM acetyl-CoA (Sigma, St Louis, MO) to give a total volume of 0.1 ml. This assay was carried out in duplicate in BEEM capsules held in a water bath at 3T'C. The reaction was stopped after 15 min by the addition of 0.1 ml 6 M HC1 and the mixture transferred to scintillation vials and dried at 100°C on a heating block under a stream of air to drive off unfixed radioactive bicarbonate. The residue was dissolved in 1 ml water and 4ml scintillation fluid ("Lumax", Lumac B.V., Holland) and counted on a Packard tricarb scintillation counter. The counts per min (cpm) were converted to disintegrations per min (dpm) by the channels ratio method using a quench curve produced by standards (Amersham). Tritiated water assay for lipogenisis The incorporation of 3H20 (Herzberg, 1979) was measured on each separate tissue dissected, homogenized as above and incubated at 37°C in 0.25 M sucrose buffer (see above) with 1.0/~1 3H20 (Amersham, 5 Ci/ml). The assay was terminated after 2 hr by the addition of hot 5 M K O H and boiled for l hr. The total radioactivity (dpm) in a petroleum ether fraction (B.pt 60°C) was determined after concentration of the solvent (3 ml) and addition of 5 ml Lumax scintillation fluid (Leyack, B.V., Holland).
Acetyl-CoA carboxylase in the tsetse fly
29
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Fig. 1. Activity of a c e t y l ~ o A carboxylase during the first 13 days of adult life in abdomen and thorax of the female tsetse fly. The points represent the mean of eight readings (two groups of five flies assayed in duplicate, the experiment run twice). Bars represent the spread of readings.
Protein determination Protein content was determined by the modified Coomassie Blue dye binding method (Spector, 1978), using bovine serum albumin as standard. Chemicals All bichemicals were purchased from Sigma, and the solvents and salts were of analytical grade. Results
Acetyl-CoA carboxylase activity in the abdomen and thorax of male and female tsetse flies during the first 13 days of adult life The results of the first experiment to measure lipogenic activity in newly emerged adults showed a noticeable change in the specific activity of the enzyme acetyl-CoA carboxylase
after 2-3 days in both male and female flies. This activity in the abdomen of males rose rapidly and peaked at day 6 and then decreased to a value between 0.4 and 0.6 #mol/min/mg for the next 7 days (Fig. 2). The female tsetse fly showed a gradual increase of abdominal enzyme activity to reach a plateau of 0.64).8 pmol/min/ mg specific activity on day 6 which lasted for the next 7 days (Fig. 1). The activity was noticeably higher in the females when compared to agematched males, and in both sexes the abdomen had higher enzyme activity than the thorax. Thoracic enzyme activity remained between 0.05 and 0.175/zmol/min/mg in males and 0.1 and 0.3 pmol/min/mg in females for the duration of the experiment (Figs 1 and 2). The actual mass of the blood meal reached a plateau at about the same time as enzyme activity, days 6-8, in both sexes, but the females had ingested larger blood meals (Fig. 3).
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Fig. 2. Activity of acetyl~CoA carboxylase during the first 13 days of adult life in the male tsetse fly. The points represent the mean of eight readings (two groups of five flies assayed in duplicate, the experiment run twice). Bars represent the spread of readings.
30
J.P. Dean Goldring and John S. Read
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Fig. 3. Blood meal wet weight for the first 13 days of adult life in male and female tsetse flies. The points represent the blood meal from one fly (10 flies/10).
Acetyl-CoA carboxylase activity in the abdomen and thorax of adult male tsetse flies for 24hr after feeding The change in acetyl-CoA carboxylase activity after the blood meal, over 24 hr (Fig. 4) was approximately 0.10-0.15/~mol/min/mg specific activity in the abdomen, and the thorax showed changes of a similar order. The broad peak of activity, however, could be seen at about 20 hr after feeding.
Lipogenesis and acetyl-CoA carboxylase activity in different tissues in the tsetse fly In Table 1 the results of enzyme determinations on the dissected flies are shown together with the incorporation of tritium into lipid as a measure of lipogenesis. The enzyme activites are expressed as specific activities, i.e. the rate of conversion of the substrate per mg of protein, whereas the other assay measures the total
tritium incorporated into saponifiable lipids in each tissue. The fat body had the highest level of lipogenic activity in male and female flies as determined by both assays, and there was a high level of activity in the thorax and the cuticle. Similar results were obtained for tritium incorporated into intact tissue (results not shown).
Discussion The initial low acetyl-CoA carboxylase activity, lasting 2 3 days, is probably due to both the domination of the insect's metabolism by the need to develop its flight musculature (Bursell, 1961), and the possibility that the insect is still relying on lipid stores laid down by the larva before pupation (Jack, 1939; Goldring and Read, 1993). This suggests that the lipid synthetic activity in the pregnant female is sufficient to sustain its larva through pupation to the
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Fig. 4. Acetyl-CoA carboxylaseactivity measured for a 24-hr period after feeding in male tsetse flies. The points represent the mean of duplicate readings.
31
Acetyl-CoA carboxylase in the tsetse fly Table 1. The activity of acetyl-CoA carboxylase and the incorporation of 3H20 into a saponifiable lipid fraction in tissues dissected from male and female tsetse flies
Tissue Thorax and head Blood meal Digestive tract Fat body Cuticle
Acetyl-CoA carboxylase activity Incorporation of 3H20 (# mol/min/mg protein) (dpm/tissue) Female Male Female Male 0.6 0.0 0.1 4.44 0.7
adult stage. Female tsetse flies produce lipid in the fat body for their own lipid requirements as well for larval nutrition (Moloo, 1976; Langley and Bursell, 1980) and therefore we would expect to see higher acetyl-CoA carboxylase activity in the female compared to the male. Female flies do have more lipid by weight and ingest a larger blood meal (Fig. 3), after 12 days of life, than male flies fed on the same diet (Langley and Pimley, 1979). The female tsetse fly showed a gradual increase of carboxylase activity to reach a plateau on day 6 which lasted for the next 7 days, while males showed a more pronounced rise in enzyme activity which peaked on day 6 and then remained constant for the next 7 days. It is possible that if these were wild flies, with the further energy demands made when they search for food and mates (Langley, 1966b), they would show more peaks of lipogenic activity. In both sexes, the peak in lipogenic activity, as measured by acetyl-CoA carboxylase activity, would appear to occur 1 or 2 days before the peak of total lipid product determined by Langley and Pimley (1979) and Quinlan and Gatehouse (1981). This is not altogether surprising, as this could be the difference between measuring the activity of one enzyme and measuring the total product of a biosynthetic pathway. It is possible that the latter is a period when esterification and accumulation of the fatty acid products as neutral lipids occurs before breakdown begins to predominate. The rise in the weight of the blood meal during insect development follows the same trend as enzyme activity (Fig. 3), suggesting a direct relationship with dietary intake. Such a relationship has been established for mammalian acetyl-CoA carboxylase where carboxylase mRNA levels change in response to fasting and refeeding (Pape et al., 1988). Bursell (1963) showed that as the residual dry weight of the abdomen decreased by over a half, the lipid content in the abdomen rose to a peak in 24hr. McCabe and Bursell (1975a,b) followed the incorporation of radioactive leucine into neutral lipids, palmitic acid and palmitoleic acid and found peak incorporation to be be-
0.585 0.0 0.2 3.3 1.0
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tween 24 and 36 hr. The percent digestion taking place in the abdomen was shown to reach a peak at the same time by Langley (1966a). Our results show that there is a similar increase in acetyl-CoA carboxylase activity, but the peak occurs slightly before 24 hr, at approximately 20 hr. The percentage change is between 50 and 75% over this time period in both the abdomen and thorax. Bursell et al. (1974) discussed this rise and fall in lipid content in relation to a change from a lipogenic to lipolytic phase. To describe this fully would require the simultaneous determination of the activity of enzymes involved in both pathways. Bursell (1963) showed a gradual utilization of lipid stores by conversion to proline in the fat body starting 12 hr after a blood meal. The pathway from lipid to proline may be via acetate but it is unlikely to be the only metabolic route. Therefore the activity of lipolytic enzymes and the lipase, which possibly releases diglycerides from the fat body for lipid transport by lipoproteins (Beenakkers et al., 1984), would also have to be determined to fully describe the lipolytic phase proposed by Bursell. The results obtained in these experiments all show that the abdomen has higher acetyl-CoA carboxylase activity than the thorax, and as we expected, the prime site of activity was indeed the fat body (Table 1). When studied in isolation, the specific enzyme activity is proportionately higher in the abdomen than in the thorax, and this activity is 25% higher in the female fat body compared to the male. The difference in enzyme activity in the thorax of both sexes is less pronounced. The uterine gland could be thought of as analogous to the mammalian mammary gland and Langley and Kaboyo (1981) published a method for the separation of the gland from the fat body, but the technique does not give viable tissue for estimating enzyme activity. Since the uterine gland synthesizes proteins (Riddiford and Dhadialla, 1990) it would be interesting to see if the gland is able to synthesize fatty acids de novo to account for the differences in enzyme activity between the sexes. It is more likely, however, that fat body diglycerides are the source of
32
J. P. Dean Goldring and John S. Read
uterine lipids (Langley and Bursell, 1980). If the uterine gland produced fatty acids, such a pathway would also be unlikely to be active before sufficient maturation had been reached for the female to achieve pregnancy (Ma et al., 1975). These arguments favour the suggestion that the extra enzyme activity in female flies observed in this study is a true measurement of lipogenesis. It would be interesting to measure carboxylase m R N A levels in the fat body during pregnancy to see if a demand for uterine milk by the developing larvae induced increased expression of the carboxylase gene as has been reported for the mammalian mammary gland during lactation (Pape e t aL, 1988). Female tsetse flies produce a hydrocarbon contact sex pheromone and injected succinate can be incorporated into this pheromone (Langley and Carlson, 1983), suggesting that fatty acid synthesis may be the first step to synthesis of these branched-chain hydrocarbons. The incorporation of radioactivity from 3H20 into saponifiable lipid fraction (Table 1) by the cuticle fraction indicates that there was de n o v o fatty acid synthesis associated with the cuticle. Recently, fatty acid synthetase, which produces methyl-branched fatty acids, has been reported in integument-enriched tissue from the German cockroach, B l a t t e l l a g e r m a n i c a (Juarez et al., 1992; Gu et al., 1993). Fatty acid synthetase was found to be active in both a microsomal and soluble form and utilized m a l o n y l - C o A as a substrate. Our study showed acetyl-CoA carboxylase activity in the cuticle fraction. Since tritium was incorporated into saponifiable lipid and the enzyme produces malonyl-CoA, we expect fatty acid synthetase to be present in this tissue in tsetse flies. Acetyl-CoA carboxylase is also a likely producer of integument malonyl~CoA used by fatty acid synthetase in the German cockroach (Gu et al., 1993). Enzyme activity in the cuticle was similar to that in the thorax but lower than that suggested by the incorporated tritium. It is possible that some of the tritium incorporated can be accounted for by post-transport modification of fatty acids. An alternative explanation may be due to residual fat body tissue fragments, or to the presence of oenocytes (Tobe et al., 1973), which have been considered to be the site of hydrocarbon synthesis in insects (Diehl, 1975). The level of acetyl-CoA carboxylase activity in the thorax would be expected to provide for local metabolic requirements. The control of lipid synthesis in the tsetse fly and a role for acetyl-CoA carboxylase remain to be investigated. We did note, however (results not shown) that including thorax extracts into abdominal preparations lowered enzyme activity, suggesting the presence of regulatory
factors in the thorax. Further studies of acetyl-CoA carboxylase from tissues which have been incubated with the peptide hormone, lipid synthesis inhibitory factor (Pimley and Langley, 1981), octopamine (Pimley, 1985) and the adipokinetic hormone (Beenakkers et al., 1984; Strobel et al., 1990) would give insight into the role of acetyl-CoA carboxylase in regulating lipid synthesis, and the supply of lipids for uterine milk in the tsetse fly. Acknowledgements--The authors would like to thank the
University of Zimbabwe for a Scholarship (JPDG), and the Research Board of the Universityof Zimbabwe for financial support. We thank the Tsetse Research Laboratory, Langford, Bristol, U.K. for a supply of tsetse fly pupae, and Dr P. Phelps, Department of Zoology, University of Zimbabwe, for use of the tsetse insectary.
References Beenakkers A. M. Th., van der Horst D. J. and van Marrewijk W. J. A. (1984) Insect flight muscle metabolism. Insect Biochem. 14, 243-260. Blomquist G. J., Nelson D. R. and de Renobles M. (1987) Chemistry, biochemistryand physiologyof insect cuticular lipids. Arch. Insect Biochem. Physiol. 6, 227-285. Bursell E. (1961) Post-teneral development of the thoracic musculature in tsetse flies. Proc. R. Ent. Soc. Lond. A 36, 69-79. Bursell E. (1963) Aspects of the metabolism of amino acids in the tsetse fly Glossina (Diptera). J. Insect Physiol. 9, 439-452. Bursell E., Billing K. C., HargroveJ. W., McCabe C. T. and Slack E. (1974) Metabolism of the blood meal in tsetse flies (a review). Acta trop. 31, 297-320. Bursell E. and Slack E. (1976) Oxidation of proline by sarcosomes of the tsetse fly, Glossina morsitans. Insect Biochem. 6, 159-167. Dame D. A. and Ford H. R. (1966) The effect of the chemosterilant tepa on Glossina morsitans Westwood. Bull. Ent. Res. 56, 649-658. Diehl P. A. (1975) Synthesis and release of hydrocarbonsby oenocytes of the desert locust. Schistocerca gregaria. J. Insect. Physiol. 21, 1237-1246. Goldring J. P. D. and Read J. S. (1993) Insect acetyl~CoA carboxylase: activity during the larval, pupal and adult stages of insect development. Comp. Biochem. Physiol. 106B, 855458. Gu P., Welch W. H. and Blomquist G. J. (1993) Methylbranched fatty acid biosynthesis in the German cockroach, Blattella germanica: kinetic studies comparing a microsomal and soluble fatty acid synthetase. Insect Biochem. molec. Biol. 23, 263-271. Herzberg G. R. (1979)The effectof diet on in vitro fatty acid synthesis in rat liver. Biochem. Educ. 7, 72-73. Jack (1939) Studies in the physiology and behaviour of Glossina morsitans, Westwood. Mere. Dept. Agric. S. Rhod. Govt. No. 1.
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and the blowfly (Sarcophaga). Comp. Biochem. Physiol. 31, 819-827. Numa S. and Tanabe T. (1984) Acetyl-CoA carboxylase and its regulation. In Fatty Acid Metabolism and Its Regulation (Edited by Numa S.), pp. 1-23. Elsevier, Amsterdam. Pape M. E., Lopez-Casillas F. and Kim K.-H. (1988) Physiological regulation of acetyl-CoA carboxylase gene expression: effects of diet, diabetes, and lactation on acetyl~CoA carboxylase mRNA. Archs Biochem. Biophys. 267, 104-109. Pimley R. W. (1985) Cyclic AMP and calcium mediate the regulation of fat cell activity octopamine and peptide hormones in Glossina morsitans. Insect Biochem. 15, 293-298. Pimley R. W. and Langley P. A. (1981) Hormonal control of lipid synthesis in the fat body of the adult female tsetse fly, Glossina morsitans. J. Insect Physiol. 27, 839-847. Quinlan R. J. and Gatehouse A. J. (1981) The effect of low doses of Endosulphan on lipid reserves in the tsetse fly, Glossina morsitans. Ent. exp. appl. 29, 29-38. Rainwater D. L. and Kolattukudy P. E. (1982) Purification and characterisation of acetyl-CoA carboxylase from goose uropygial gland which produces multimethyl branched acids and evidence for its identity with avian acetyl-CoA carboxylase. Archs Biochem. Biophys. 213, 372-383. de Renobles M., Woodin T. S. and Blomquist G. J. (1986) Drosophila melanogaster fatty acid synthetase: characteristics and effect of protease inhibitors. Insect Biochem. 16, 887-894. Riddiford L. M. and Dhadialla T. S. (1990) Protein synthesis by the milk gland and fat body of the tsetse fly, Glossina pallidipes. Insect Biochem. 20, 493-500. Spector T. (1978) Refinement of the Coomassie Blue method for protein quantitation. Analyt. Biochem. 86, 142-146. Stanley-Samuelson D. W., Jurenka R. A., Cripps C., Blomquist G. J. and de Renobles M. (1988) Fatty acids in insects: composition, metabolism and biological significance. Arch. Insect Biochem. Physiol. 9, 1-33. Strobel L. M., Kanost M. R., Zeigler R. and Wells M. A. (1990) Adipokinetic hormone causes formation of a low density lipophorin in the house cricket Acheta domesticus. Insect Biochem. 20, 859-863. Tobe S. S., Davey K. S. and Huebner E. (1973) Nutrient transfer during the reproductive cycle in Glossina austeni Newst.: histology and histochemistry of the milk gland, fat body and oenocytes. Tissue Cell. 5, 633~550.