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In vitro and in vivo fate of saturated and unsaturated fatty acids during development of insects
289
Biochimica et Biophysics Acta, 360 (1974) 289-297 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56485
IN VITRO AND IN VIVO FATE OF SATURATED AND UNSATURATED FATTY ACIDS DURING DEVELOPMENT OF INSECTS
A.M. MUNICIO,
J.M. ODRIOZOLA,
Department of Biochemistry, (Received
March 14th,
M.A. PiREZ-ALBARSANZ
Faculty of Sciences,
and J.A. RAMOS
University of Madrid, Madrid (Spain)
1974)
Summary 1. In vitro elongation, saturation and desaturation reactions of frtty acids using labelled 10 : 0, 12 ?*O, 14 : 0, 18 : 0, 18 : 1 and 18 : 2 have been investigated with homogenates of larvae and pharste adults of the Dipterous Ceratitis capitata.
2. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. Direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. Unsaturated fatty acids are scarcely hydrogenated to stearic acid by larval homogenates; pharate adult homogenates does not exhibit any saturating activity. 3. Larval and pharate adult homogenates show a different pattern of incorporation of labelled fatty acids into the main classes of lipids. As a general rule, labelled fatty acids are preferentially incorporated into triacylglycerols by larval homogenates; C, 8 fatty acids exhibit a different incorporating pattern according to the degree of unsaturation. Labelled fatty acids remain mainly as free fatty acids in the presence of pharate adult homogenates. This different behaviour of larval and pharate adult homogenates can not be explained through differences in the fatty acid activating capacity of both stages of development. 4. In vivo experiments of feeding larvae with 16 : 0, 18 : 0, 18 : 1 and 18 : 2 have been also carried out. Transformations of these fatty acids are followed through the different stages of development of the insect. The observed elongation activity is in general agreement with the in vitro results. Palmitic and stearic acids are increasingly desaturated during the larval stage of development, that is also in agreement with the in vitro results. A de novo synthesis from labelled acetate, obtained from oxidative degradation of labelled fatty acids, may explain the presence in the lipid fraction of shorter-chain labelled fatty acids than the substrates. 5. Incorporation of 18 : 0 and 18 : 2 into the main classes of lipids was also determined in the ih vivo experiments. An incorporation of free fatty acids into triacylglycerols during larval and pharate adult stages of development is
290
noticed. Incorporation of 18 : 2 into phospholipids was 18 : 0. In the main, the in vivo results of incorporation do vitro experiments, as far as the triacylglycerols-free fatty concerned. ___ ~-- ._..._~ ~_.__
higher than that of not reproduce the in acids relationship is ___-_
.___
Introduction The fatty acid synthetic capabilities of the insects as a group and their changes during different stages of development constitute important aspects of the metabolic activity of lipids in insects. Experiments involving the fate of saturated fatty acids in the presence of homogenates of different stages of development of the insect Ceratitis capitatu have been previously undertaken [ 1,2] . The results obtained show clear differences in the behaviour of larval and pharate adult stages of development. Larval homogenates desaturate and elongate the labelled substrates according to their chain length, whereas direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. Microsomes isolated from the fat body of the migratory locust readily desaturated stearic to oleic acid when incubated aerobically in the presence of reduced pyridine nucleotides [ 31. The presence of a reversible desaturation reaction catalyzed by the microsomes of both hepatopancreas and muscle of some Crustacea has been suggested [ 41. The fatty acid metabolism in insects requires more investigation and to gather further information on this problem, the in vitro and in vivo behaviour of saturated, monounsaturated and polyunsaturated fatty acids was studied in the Dipterous C. capitata. Materials and Methods Materials ATP, CoA and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. [l-l 4 C] -1abelled fatty acids were obtained from The Radiochemical Centre, Amersham, Bucks. Rearing of inset ts C. capitata (Wiedemann) was used during the larval, pharate adult and adult stages of development. Diet, temperature and humidity conditions of culturing were carefully controlled, as reported previously [l] . For the in vitro experiments, larvae, pharate adults and adults were reared at different development times according to the particular experiments. Larvae were starved for 4-5 h before homogenization. Adults were collected during the first 24 h after emergence. For the in vivo experiments, larvae were reared at the corresponding times of development and fasted for 4-5 h before feeding [l-’ 4 C] -1abelled fatty acids.
291
Incubation mhture Insects were directly homogenized with 3 vols of cold homogenizing buffer (0.35 M sucrose-O.05 M Tris, pH 7.4) according to the method previously described [ 21. The assay mixture for incorporating [l-’ 4 C] -labelled fatty acids contained ATP (6 pmoles/ml), CoA (2 pmoles/ml), NADPH (1 E.tmole/ml), MgCl* (2 pmoles/ml) in 0.05 M potassium phosphate buffer, pH 7.4 (22 pmoles/ml), streptomycin (1 mg/ml) and 2 /.Si of the labelled fatty acid and 0.5 ml of the total homogenate. Labelled fatty acids (10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2) having a range of specific activity 20-40 Ci/mole, were suspended in potassium phosphate buffer and the mixture was sonicated for 2 min prior to the addition to the incubation mixture at 0.20 ymole/ml. Incubations were made in a shaker at 37°C for 180 min. Final volume 1 ml containing 10 mg/ml of proteins. ‘In vivo experiments Larvae reared at the corresponding times of development were fasted during 4-5 h and fed a single dose of the [l- ’ 4 C] -1abelled fatty acid (10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2) (1 mCi/2 g of larvae) for 5 h. After this time, larvae were allowed to follow the development feeding the normal diet; aliquots of the insect pool were taken for lipid analysis according to the experiment. Extraction and separation of lipids At the end of the incubation time, the reaction was stopped by the addition of 1.0 ml of chloroform and 2.1 ml of methanol. The crude lipid extract was obtained and purified by the Bligh and Dyer procedure [5]. Insects from the in vivo experiments were homogenized and extracted by the same method. Purified lipids were fractionated into lipid classes by thin-layer chromatography on silica gel G by the method described [l] . Thin-layer bands were scraped off the plate and either directly transferred to scintillation vials for counting or submitted to the methanolysis procedure. Fatty acid methyl esters Fatty acid methyl esters were obtained either from total lipids or silica gel bands after separating into lipid classes. Methanolysis was carried out with boron trifluoride-methanol reagent as described [ 11. Incorporation of radioactivity into lipid classes and Details of the experimental procedures for the activity in both cases were previously described quoted represent an average of three determinations
individual fatty acids determination of the radio[ 11. The analytical figures (P < 0.05).
Results and Discussion In vitro experiments In a series of in vitro experiments the fatty acids 10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2 have been used. The fatty acid-incorporating
Fig. 1. Incorporation of [l- * 4C]-labelled fatty acids by &lay-old larval homogenates and B-day-old pharate adult homogenates into triacylglycerols (first bars from the left). free fatty acids (second bars), diacylglycerols (third bars) and phopholipids (fourth bars).
capacity of the larval homogenates (Fig. 1) appears to be unequally shown in the different classes of lipids, triacylglycerols, free fatty acids, diacylglycerols and phopholipids. Triacylglycerols exhibited the highest levels of incorporation of 10 : 0, 12 : 0, 14 : 0 and 16 :0 by larval homogenates whereas very low levels of these fatty acids remained as free acids. On the other hand, very sharp differences were observed in the fate of the C , B fatty acids. Stearic acid showed a sharp drop in the incorporation into triacylglycerols remaining mainly as free fatty acids. The incorporation ability of the Cl 8 fatty acids into triacylglycerols by larval homogenates depended on the unsaturation degree; the more unsaturated the fatty acid, the more the incorporating capacity into triacylglycerols and the lower the levels of free fatty acids. The incorporating capacity of labelled fatty acids into phospholipids by larval homogenates exhibited rather low values, oleic and linoleic acids showing the highest incorporation. It has been clearly established by earlier studies [2] that the incorporation of labelled acetate takes place mainly into phopholipids and at lower levels into triacylglycerols and as free fatty acids. From these facts, therefore, the existence of an inverse correspondence between the fatty acid incorporation into triacylglycerols and its presence as free fatty acids is clear.
293
Pharate
adult
homogenates show a completely different pattern of fatty activity. Fatty acids remained mainly present as free fatty acids whereas their incorporation into triacylglycerols was rather low; steak acid showed the lowest incorporating capacity into triacylglycerols by pharate adult homogenates, remaining present mainly as free fatty acid. Thus, the very high percentage of labelled fatty acids present in the lipids as free fatty acids is one of the most characteristic facts in the biochemical behaviour of the pharate adult homogenates. The incorporating capacity of fatty acids into phospholipids by pharate adult homogenates was very low, oleic and linoleic also being the acids exhibiting the highest values of incorporation. From these data, the existence of a different in vitro behaviour of larval and pharate adult homogenates, where the fatty acid incorporation into triacylglycerols is concerned, is again clear. This different behaviour can not be explained through the differences in the fatty acid activating capacity exhibited by either larval or pharate adult stages of development [6]. The changes that each labelled fatty acid undergoes during the in vitro incubation in the presence of homogenates of either larvae or pharate adult are given in Table I. These results are in general agreement with previous results on the fate of saturated fatty acids [2]. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. The longer the length of the fatty acids used as substrates, the more pronounced the abundance of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Direct unsaturation and elongation of fatty acids by pharate adult homogenates were insignificant. The unsaturated fatty acids, 18 : 1 and 18 : 2, were scarcely hydrogenated to stearic acid by larval homogenates whereas pharate adult homogenates did not show any saturating activity. Oleic acid was elongated to 20 : 1 in the presence of larval homogenates.
acid-incorporating
TABLE I SATURATION, DESATURATION AND ELONGATION NATES OF THE INSECT C. CAPITATA Values
ACIDS
BY LARVAL
HOMOGE-
are given as percentages of the fatty acids used as substrates.
Recovered
Substrate lo:o
lo:o 1O:l 12:o 12:l 14:o 14:l 16:0 16:l 18:0 18:l 18:2 2O:l
OF FATTY
12:o
14:o
16:0
18:O
18:l
18:2
0.8
0.2
2.0 12.0 0.8
3.0 5.0 1.0
1.5 1.5 40.0 50.0 3.7
294
In vivo experiments In vivo experiments of feeding palmitic, stearic, oleic and linoleic acids to larve of C. capitata have been also carried out. Individual changes have been examined by radio gas-liquid chromatography in the fatty acids from total lipids of larvae (5- and 6-day old), pharate adults (l- and g-day old) and l-day-old adults. Results are given in Fig. 2. Palmitic and stearic acids were increasingly desaturated during the larval stage of development of the insect until the first day of the pharate adult stage; the levels of palmitoleic and oleic acids thus formed represent a rather high desaturation activity that, on the other side, is in agreement with the in vitro results. Palmitic acid was elongated to 18 : 0 and 20 : 0 and the elongation product, 18 : 0, was simultaneously desaturated to oleic acid. Stearic acid was elongated to 20 : 0. This elongation activity is also in general agreement with the in vitro results since the percentages of elongation products from 18 : 0 were smaller than those obtained from 16 : 0. It is interesting to observe that in vivo admimstration of labelled stearic acid yielded estimable amounts of both 16 : 0 and 16 : 1. Since fatty acids used in these experiments were labelled at the C, atom, the presence of labelled shorter fatty acids involves several possibilities that exclude, therefore, chain shortening by the direct reversal of chain elongation. Palmitate and stearate could be oxidized to acetate in the mitochondria and the radioactive acetate can be used for many synthetic reactions. Among these reactions acetate can be utilized for chain elongation of saturated and monounsaturated fatty acids in the mitochondria themselves [7] ; it can be utilized by the microsomes, previously transported out of the mitochondria under the form of citrate, to form polyunsaturated fatty acids by chain elongation of 18 : 2 and 18 : 3 [8] ; it can be used for de novo synthesis of fatty acids in the cytosol, palmitate being the major end product of this reaction [9,10]. Thus, direct desaturation, mitochondrial elongation and de novo synthesis from labelled acetate obtained from oxidative degradation might explain the origin of the fatty acids formed after feeding larve with either palmitic or
V c, 18 1
Fig. 2. Percentages of relative abundance of labelled fatty acids (calculated from used as substrates) at different stages of the insect development after feeding 16 : 0, 18 : 0, 18 : 1 or 18 : 2 (1.. larvae: ph.ad., pharate adults: ad., adults).
182
7
the labelled fatty acids B-day-old larvae either
295
stearic acids. All these fatty acids remained at practically constant levels during the pharate adult stage of development. Administration of labelled oleic acid yielded the elongation product 20 : 1 and the shorter fatty acids palmitic and palmitoleic acids. Both these labelled fatty acids had to be formed by de novo synthesis. However, stearate could be synthesised by either a direct hydrogenation or elongation of a previously de novo synthesised palmitate. Administration of labelled polyunsaturated fatty acid, 18 : 2, yielded several labelled fatty acids, the polyunsaturated 20 : 4 being the fatty acid obtained in the highest proportion; this fatty acid could be formed through microsomal chain elongation. Palmitate and palmitoleate were also formed from linoleate although in lower proportions than they were from oleate. The presence of oleate would be explained by a partial hydrogenation of linoleate. The absence of labelled stearic acid after feeding linoleic acid is consistent with the hydrogenation reaction 18 : 2 -+ 18 : 1 since the formation of oleate from linoleate through an elongation mechanism of palmitate seemed to require the simultaneous presence of stearate. Direct hydrogenation would be, therefore, the simplest explanation although there are not many experimental results on enoyl-CoA reductases. A pans-2,3-enoyECoA reductase activity in microsomal fractions of lactating rabbit mammary gland and of rat liver [11,12] has been described. Nugteren [13] observed the conversion of 3,6,9,12-Cl s : 4 to 8,11,14-C2,, : 3 in rat liver microsomes and proposed a mechanism consisting of reduction of the first double bound and subsequent chain elongation. The saturation of oleic acid by microsomal preparations from muscle of Eriocheir sinensis [4] have been also described. In the mitochondrial fraction of rat liver, the presence of enzyme activities that carry out chain shortening and partial hydrogenation of double bonds has been shown [14]. It may be pointed here that the diet administration of [l-l 4 C] acetate gave rise to the saturated fatty acids 12 : 0, 14 : 0, 16 : 0 and 18 : 0, palmitic acid showing the highest levels. Nevertheless, the presence of C 16 fatty acids formed after feeding either 18 : 0 or 18 : 1 or 18 : 2 was not followed by both shorter fatty acids 12 : 0 and 14 : 0. This apparent lack of coincidence might be explained because’of a large dilution of the radioactive acetate pool by the acetate from carbohydrates and other metabolic sources. Under these circumstances the de novo synthesis of fatty acids could scarcely manifest itself. Nevertheless, the results obtained in the [l- ’ 4 C] acetate feeding experiments did not agree with this assumption. Another hypothesis would be the existence of a compartmentalized pool of acetyl-CoA, as suggested by Yates and Garland [15] , that could make the radioactivity acetate obtained from oxidative degradation of C, H fatty acids more utilizable for chain elongation than for de novo synthesis of palmitate. Fig. 3 shows the percentages of incorporation of 18 : 0 and 18 : 2 into the most abundant classes of lipids after feeding larvae with the labelled fatty acids. From these results is is possible to outline several features as to the in vitro and in vivo differences of fatty acid incorporation during the development of the insect.
296
In the in vitro experiments of fatty acid incorporation by larval homogenates, 18 : 2 showed a very high incorporation into triacylglycerols whereas 18 : 0 remained mainly as free fatty acid. This different behaviour was also observed in the in vivo experiments from a qualitative point of view; thus, 18 : 0 from the lipids of 5-day-old larvae was present as free fatty acid at a small higher level than it was incorporated into triacylglycerols whereas 18 : 2 was mainly incorporated into triacylglycerols. Nevertheless, the high percentages that 18 : 0 and 18 : 2 exhibited as free fatty acids after incubating with pharate adult homogenates were not present in this stage of development after feeding larvae with the labelled fatty acids. It is interesting to remark that, on the contrary, the in vivo fate of either 18 : 0 or 18 : 2 followed a similar pattern, the most outstanding fact being the incorporation of free fatty acids into triacylglycerols during larval and pharate adult stages of development. Incorporation of 18 : 2 into phospholipids was rather higher than of 18 : 0, in agreement with the in vitro results using either larval or pharate adult homogenates. The emergence of the adults was followed by a decrease in the levels of triacylglycerols that were counterbalanced by an increase of free fatty acids and phospholipids. These facts confirmed directly the use of reserves of triacylglycerols for the synthesis of phospholipids that require the rearrangement of tissues during metamorphosis; these biochemical changes take place, therefore, in the last period before adult emergence. It is clear, then, that in the main the 180
,
182
7 6 5 4 3 2 1 70 60
FFA
50 40 30 20 10
70 60 53 I
PL
Incorporation of 18 : 0 and 18 : 2 in the viva experiments into the main classes of lipids (triacylfree fatty acids and phospholipids) at different stages of insect development Cl., larvae: ph.ad., pharate adults: ad., adults). Fig. 3.
glycerols,
297
in vitro experiments using pharate adult homogenates did not reproduce the in vivo results, as far as triacylglycerols-free fatty acids relationship is concerned. References 1 Municm, A.M., Odriozola. J.M., Pitieiro, A. and Ribera, A. (1971) Biochim. Biophys. 212-225 2 Municio, A.M., Odriozola. J.M., Pifieiro. A. and Ribera. A. (1972) Biochnn. Biophys. 248-257 3 Tietz, A. and Stern, N. (1969) FEBS L&t. 2. 286-288 4 Heinen, E. and Dandrifosse, G. (1973) Arch. Int. Physiol. Biochem. 81,9-15 5 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 39. 911-917 6 P&z-Albarsanz, M.A. (1974) Doctoral Thesis, Unwersity of Madrid 7 Boone. S.C. and Wakil. S.J. (1970) Biochemistry 9. 1470-1479 8 Watson, J.A. and Lowenstein. J.M. (1970) J. Biol. Chem. 245, 5993-6002 9 Lynen, F. (1961) Fed. Proc. 20.941-951 10 Bressler, R. and Wakil, S.J. (1962) J. Biol. Chem. 237. 1441-1448 11 Lachance, J.P., Popjak, G. and de Waard, A. (1958) Biochem. J. 68. 7p 12 Matthes, K.J., Abraham, S. and Chaikoff, I.L. (1960) Biochnn. Biophys. Acta 37.180-181 13 Nugteren, D.H. (1965) Biochim. Biophys. Acta 106. 280-290 14 Kunau, W.H. and Couzens, B. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 352.1297-1305 15 Yates, D.W. and Garland, P.P. (1966) Biochem. Biophys. Res. Commun. 23. 460465
Acta 248, Acta 280,
Biochimica et Biophysics Acta, 360 (1974) 289-297 0 Elsevier Scientific Publishing Company, Amsterdam
- Printed
in The Netherlands
BBA 56485
IN VITRO AND IN VIVO FATE OF SATURATED AND UNSATURATED FATTY ACIDS DURING DEVELOPMENT OF INSECTS
A.M. MUNICIO,
J.M. ODRIOZOLA,
Department of Biochemistry, (Received
March 14th,
M.A. PiREZ-ALBARSANZ
Faculty of Sciences,
and J.A. RAMOS
University of Madrid, Madrid (Spain)
1974)
Summary 1. In vitro elongation, saturation and desaturation reactions of frtty acids using labelled 10 : 0, 12 ?*O, 14 : 0, 18 : 0, 18 : 1 and 18 : 2 have been investigated with homogenates of larvae and pharste adults of the Dipterous Ceratitis capitata.
2. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. Direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. Unsaturated fatty acids are scarcely hydrogenated to stearic acid by larval homogenates; pharate adult homogenates does not exhibit any saturating activity. 3. Larval and pharate adult homogenates show a different pattern of incorporation of labelled fatty acids into the main classes of lipids. As a general rule, labelled fatty acids are preferentially incorporated into triacylglycerols by larval homogenates; C, 8 fatty acids exhibit a different incorporating pattern according to the degree of unsaturation. Labelled fatty acids remain mainly as free fatty acids in the presence of pharate adult homogenates. This different behaviour of larval and pharate adult homogenates can not be explained through differences in the fatty acid activating capacity of both stages of development. 4. In vivo experiments of feeding larvae with 16 : 0, 18 : 0, 18 : 1 and 18 : 2 have been also carried out. Transformations of these fatty acids are followed through the different stages of development of the insect. The observed elongation activity is in general agreement with the in vitro results. Palmitic and stearic acids are increasingly desaturated during the larval stage of development, that is also in agreement with the in vitro results. A de novo synthesis from labelled acetate, obtained from oxidative degradation of labelled fatty acids, may explain the presence in the lipid fraction of shorter-chain labelled fatty acids than the substrates. 5. Incorporation of 18 : 0 and 18 : 2 into the main classes of lipids was also determined in the ih vivo experiments. An incorporation of free fatty acids into triacylglycerols during larval and pharate adult stages of development is
290
noticed. Incorporation of 18 : 2 into phospholipids was 18 : 0. In the main, the in vivo results of incorporation do vitro experiments, as far as the triacylglycerols-free fatty concerned. ___ ~-- ._..._~ ~_.__
higher than that of not reproduce the in acids relationship is ___-_
.___
Introduction The fatty acid synthetic capabilities of the insects as a group and their changes during different stages of development constitute important aspects of the metabolic activity of lipids in insects. Experiments involving the fate of saturated fatty acids in the presence of homogenates of different stages of development of the insect Ceratitis capitatu have been previously undertaken [ 1,2] . The results obtained show clear differences in the behaviour of larval and pharate adult stages of development. Larval homogenates desaturate and elongate the labelled substrates according to their chain length, whereas direct unsaturation and elongation of fatty acids by pharate adult homogenates are insignificant. Microsomes isolated from the fat body of the migratory locust readily desaturated stearic to oleic acid when incubated aerobically in the presence of reduced pyridine nucleotides [ 31. The presence of a reversible desaturation reaction catalyzed by the microsomes of both hepatopancreas and muscle of some Crustacea has been suggested [ 41. The fatty acid metabolism in insects requires more investigation and to gather further information on this problem, the in vitro and in vivo behaviour of saturated, monounsaturated and polyunsaturated fatty acids was studied in the Dipterous C. capitata. Materials and Methods Materials ATP, CoA and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. [l-l 4 C] -1abelled fatty acids were obtained from The Radiochemical Centre, Amersham, Bucks. Rearing of inset ts C. capitata (Wiedemann) was used during the larval, pharate adult and adult stages of development. Diet, temperature and humidity conditions of culturing were carefully controlled, as reported previously [l] . For the in vitro experiments, larvae, pharate adults and adults were reared at different development times according to the particular experiments. Larvae were starved for 4-5 h before homogenization. Adults were collected during the first 24 h after emergence. For the in vivo experiments, larvae were reared at the corresponding times of development and fasted for 4-5 h before feeding [l-’ 4 C] -1abelled fatty acids.
291
Incubation mhture Insects were directly homogenized with 3 vols of cold homogenizing buffer (0.35 M sucrose-O.05 M Tris, pH 7.4) according to the method previously described [ 21. The assay mixture for incorporating [l-’ 4 C] -labelled fatty acids contained ATP (6 pmoles/ml), CoA (2 pmoles/ml), NADPH (1 E.tmole/ml), MgCl* (2 pmoles/ml) in 0.05 M potassium phosphate buffer, pH 7.4 (22 pmoles/ml), streptomycin (1 mg/ml) and 2 /.Si of the labelled fatty acid and 0.5 ml of the total homogenate. Labelled fatty acids (10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2) having a range of specific activity 20-40 Ci/mole, were suspended in potassium phosphate buffer and the mixture was sonicated for 2 min prior to the addition to the incubation mixture at 0.20 ymole/ml. Incubations were made in a shaker at 37°C for 180 min. Final volume 1 ml containing 10 mg/ml of proteins. ‘In vivo experiments Larvae reared at the corresponding times of development were fasted during 4-5 h and fed a single dose of the [l- ’ 4 C] -1abelled fatty acid (10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2) (1 mCi/2 g of larvae) for 5 h. After this time, larvae were allowed to follow the development feeding the normal diet; aliquots of the insect pool were taken for lipid analysis according to the experiment. Extraction and separation of lipids At the end of the incubation time, the reaction was stopped by the addition of 1.0 ml of chloroform and 2.1 ml of methanol. The crude lipid extract was obtained and purified by the Bligh and Dyer procedure [5]. Insects from the in vivo experiments were homogenized and extracted by the same method. Purified lipids were fractionated into lipid classes by thin-layer chromatography on silica gel G by the method described [l] . Thin-layer bands were scraped off the plate and either directly transferred to scintillation vials for counting or submitted to the methanolysis procedure. Fatty acid methyl esters Fatty acid methyl esters were obtained either from total lipids or silica gel bands after separating into lipid classes. Methanolysis was carried out with boron trifluoride-methanol reagent as described [ 11. Incorporation of radioactivity into lipid classes and Details of the experimental procedures for the activity in both cases were previously described quoted represent an average of three determinations
individual fatty acids determination of the radio[ 11. The analytical figures (P < 0.05).
Results and Discussion In vitro experiments In a series of in vitro experiments the fatty acids 10 : 0, 12 : 0, 14 : 0, 16 : 0, 18 : 0, 18 : 1 and 18 : 2 have been used. The fatty acid-incorporating
Fig. 1. Incorporation of [l- * 4C]-labelled fatty acids by &lay-old larval homogenates and B-day-old pharate adult homogenates into triacylglycerols (first bars from the left). free fatty acids (second bars), diacylglycerols (third bars) and phopholipids (fourth bars).
capacity of the larval homogenates (Fig. 1) appears to be unequally shown in the different classes of lipids, triacylglycerols, free fatty acids, diacylglycerols and phopholipids. Triacylglycerols exhibited the highest levels of incorporation of 10 : 0, 12 : 0, 14 : 0 and 16 :0 by larval homogenates whereas very low levels of these fatty acids remained as free acids. On the other hand, very sharp differences were observed in the fate of the C , B fatty acids. Stearic acid showed a sharp drop in the incorporation into triacylglycerols remaining mainly as free fatty acids. The incorporation ability of the Cl 8 fatty acids into triacylglycerols by larval homogenates depended on the unsaturation degree; the more unsaturated the fatty acid, the more the incorporating capacity into triacylglycerols and the lower the levels of free fatty acids. The incorporating capacity of labelled fatty acids into phospholipids by larval homogenates exhibited rather low values, oleic and linoleic acids showing the highest incorporation. It has been clearly established by earlier studies [2] that the incorporation of labelled acetate takes place mainly into phopholipids and at lower levels into triacylglycerols and as free fatty acids. From these facts, therefore, the existence of an inverse correspondence between the fatty acid incorporation into triacylglycerols and its presence as free fatty acids is clear.
293
Pharate
adult
homogenates show a completely different pattern of fatty activity. Fatty acids remained mainly present as free fatty acids whereas their incorporation into triacylglycerols was rather low; steak acid showed the lowest incorporating capacity into triacylglycerols by pharate adult homogenates, remaining present mainly as free fatty acid. Thus, the very high percentage of labelled fatty acids present in the lipids as free fatty acids is one of the most characteristic facts in the biochemical behaviour of the pharate adult homogenates. The incorporating capacity of fatty acids into phospholipids by pharate adult homogenates was very low, oleic and linoleic also being the acids exhibiting the highest values of incorporation. From these data, the existence of a different in vitro behaviour of larval and pharate adult homogenates, where the fatty acid incorporation into triacylglycerols is concerned, is again clear. This different behaviour can not be explained through the differences in the fatty acid activating capacity exhibited by either larval or pharate adult stages of development [6]. The changes that each labelled fatty acid undergoes during the in vitro incubation in the presence of homogenates of either larvae or pharate adult are given in Table I. These results are in general agreement with previous results on the fate of saturated fatty acids [2]. Larval homogenates desaturate and elongate the labelled substrates according to their chain length. The longer the length of the fatty acids used as substrates, the more pronounced the abundance of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Direct unsaturation and elongation of fatty acids by pharate adult homogenates were insignificant. The unsaturated fatty acids, 18 : 1 and 18 : 2, were scarcely hydrogenated to stearic acid by larval homogenates whereas pharate adult homogenates did not show any saturating activity. Oleic acid was elongated to 20 : 1 in the presence of larval homogenates.
acid-incorporating
TABLE I SATURATION, DESATURATION AND ELONGATION NATES OF THE INSECT C. CAPITATA Values
ACIDS
BY LARVAL
HOMOGE-
are given as percentages of the fatty acids used as substrates.
Recovered
Substrate lo:o
lo:o 1O:l 12:o 12:l 14:o 14:l 16:0 16:l 18:0 18:l 18:2 2O:l
OF FATTY
12:o
14:o
16:0
18:O
18:l
18:2
0.8
0.2
2.0 12.0 0.8
3.0 5.0 1.0
1.5 1.5 40.0 50.0 3.7
294
In vivo experiments In vivo experiments of feeding palmitic, stearic, oleic and linoleic acids to larve of C. capitata have been also carried out. Individual changes have been examined by radio gas-liquid chromatography in the fatty acids from total lipids of larvae (5- and 6-day old), pharate adults (l- and g-day old) and l-day-old adults. Results are given in Fig. 2. Palmitic and stearic acids were increasingly desaturated during the larval stage of development of the insect until the first day of the pharate adult stage; the levels of palmitoleic and oleic acids thus formed represent a rather high desaturation activity that, on the other side, is in agreement with the in vitro results. Palmitic acid was elongated to 18 : 0 and 20 : 0 and the elongation product, 18 : 0, was simultaneously desaturated to oleic acid. Stearic acid was elongated to 20 : 0. This elongation activity is also in general agreement with the in vitro results since the percentages of elongation products from 18 : 0 were smaller than those obtained from 16 : 0. It is interesting to observe that in vivo admimstration of labelled stearic acid yielded estimable amounts of both 16 : 0 and 16 : 1. Since fatty acids used in these experiments were labelled at the C, atom, the presence of labelled shorter fatty acids involves several possibilities that exclude, therefore, chain shortening by the direct reversal of chain elongation. Palmitate and stearate could be oxidized to acetate in the mitochondria and the radioactive acetate can be used for many synthetic reactions. Among these reactions acetate can be utilized for chain elongation of saturated and monounsaturated fatty acids in the mitochondria themselves [7] ; it can be utilized by the microsomes, previously transported out of the mitochondria under the form of citrate, to form polyunsaturated fatty acids by chain elongation of 18 : 2 and 18 : 3 [8] ; it can be used for de novo synthesis of fatty acids in the cytosol, palmitate being the major end product of this reaction [9,10]. Thus, direct desaturation, mitochondrial elongation and de novo synthesis from labelled acetate obtained from oxidative degradation might explain the origin of the fatty acids formed after feeding larve with either palmitic or
V c, 18 1
Fig. 2. Percentages of relative abundance of labelled fatty acids (calculated from used as substrates) at different stages of the insect development after feeding 16 : 0, 18 : 0, 18 : 1 or 18 : 2 (1.. larvae: ph.ad., pharate adults: ad., adults).
182
7
the labelled fatty acids B-day-old larvae either
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stearic acids. All these fatty acids remained at practically constant levels during the pharate adult stage of development. Administration of labelled oleic acid yielded the elongation product 20 : 1 and the shorter fatty acids palmitic and palmitoleic acids. Both these labelled fatty acids had to be formed by de novo synthesis. However, stearate could be synthesised by either a direct hydrogenation or elongation of a previously de novo synthesised palmitate. Administration of labelled polyunsaturated fatty acid, 18 : 2, yielded several labelled fatty acids, the polyunsaturated 20 : 4 being the fatty acid obtained in the highest proportion; this fatty acid could be formed through microsomal chain elongation. Palmitate and palmitoleate were also formed from linoleate although in lower proportions than they were from oleate. The presence of oleate would be explained by a partial hydrogenation of linoleate. The absence of labelled stearic acid after feeding linoleic acid is consistent with the hydrogenation reaction 18 : 2 -+ 18 : 1 since the formation of oleate from linoleate through an elongation mechanism of palmitate seemed to require the simultaneous presence of stearate. Direct hydrogenation would be, therefore, the simplest explanation although there are not many experimental results on enoyl-CoA reductases. A pans-2,3-enoyECoA reductase activity in microsomal fractions of lactating rabbit mammary gland and of rat liver [11,12] has been described. Nugteren [13] observed the conversion of 3,6,9,12-Cl s : 4 to 8,11,14-C2,, : 3 in rat liver microsomes and proposed a mechanism consisting of reduction of the first double bound and subsequent chain elongation. The saturation of oleic acid by microsomal preparations from muscle of Eriocheir sinensis [4] have been also described. In the mitochondrial fraction of rat liver, the presence of enzyme activities that carry out chain shortening and partial hydrogenation of double bonds has been shown [14]. It may be pointed here that the diet administration of [l-l 4 C] acetate gave rise to the saturated fatty acids 12 : 0, 14 : 0, 16 : 0 and 18 : 0, palmitic acid showing the highest levels. Nevertheless, the presence of C 16 fatty acids formed after feeding either 18 : 0 or 18 : 1 or 18 : 2 was not followed by both shorter fatty acids 12 : 0 and 14 : 0. This apparent lack of coincidence might be explained because’of a large dilution of the radioactive acetate pool by the acetate from carbohydrates and other metabolic sources. Under these circumstances the de novo synthesis of fatty acids could scarcely manifest itself. Nevertheless, the results obtained in the [l- ’ 4 C] acetate feeding experiments did not agree with this assumption. Another hypothesis would be the existence of a compartmentalized pool of acetyl-CoA, as suggested by Yates and Garland [15] , that could make the radioactivity acetate obtained from oxidative degradation of C, H fatty acids more utilizable for chain elongation than for de novo synthesis of palmitate. Fig. 3 shows the percentages of incorporation of 18 : 0 and 18 : 2 into the most abundant classes of lipids after feeding larvae with the labelled fatty acids. From these results is is possible to outline several features as to the in vitro and in vivo differences of fatty acid incorporation during the development of the insect.
296
In the in vitro experiments of fatty acid incorporation by larval homogenates, 18 : 2 showed a very high incorporation into triacylglycerols whereas 18 : 0 remained mainly as free fatty acid. This different behaviour was also observed in the in vivo experiments from a qualitative point of view; thus, 18 : 0 from the lipids of 5-day-old larvae was present as free fatty acid at a small higher level than it was incorporated into triacylglycerols whereas 18 : 2 was mainly incorporated into triacylglycerols. Nevertheless, the high percentages that 18 : 0 and 18 : 2 exhibited as free fatty acids after incubating with pharate adult homogenates were not present in this stage of development after feeding larvae with the labelled fatty acids. It is interesting to remark that, on the contrary, the in vivo fate of either 18 : 0 or 18 : 2 followed a similar pattern, the most outstanding fact being the incorporation of free fatty acids into triacylglycerols during larval and pharate adult stages of development. Incorporation of 18 : 2 into phospholipids was rather higher than of 18 : 0, in agreement with the in vitro results using either larval or pharate adult homogenates. The emergence of the adults was followed by a decrease in the levels of triacylglycerols that were counterbalanced by an increase of free fatty acids and phospholipids. These facts confirmed directly the use of reserves of triacylglycerols for the synthesis of phospholipids that require the rearrangement of tissues during metamorphosis; these biochemical changes take place, therefore, in the last period before adult emergence. It is clear, then, that in the main the 180
,
182
7 6 5 4 3 2 1 70 60
FFA
50 40 30 20 10
70 60 53 I
PL
Incorporation of 18 : 0 and 18 : 2 in the viva experiments into the main classes of lipids (triacylfree fatty acids and phospholipids) at different stages of insect development Cl., larvae: ph.ad., pharate adults: ad., adults). Fig. 3.
glycerols,
297
in vitro experiments using pharate adult homogenates did not reproduce the in vivo results, as far as triacylglycerols-free fatty acids relationship is concerned. References 1 Municm, A.M., Odriozola. J.M., Pitieiro, A. and Ribera, A. (1971) Biochim. Biophys. 212-225 2 Municio, A.M., Odriozola. J.M., Pifieiro. A. and Ribera. A. (1972) Biochnn. Biophys. 248-257 3 Tietz, A. and Stern, N. (1969) FEBS L&t. 2. 286-288 4 Heinen, E. and Dandrifosse, G. (1973) Arch. Int. Physiol. Biochem. 81,9-15 5 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 39. 911-917 6 P&z-Albarsanz, M.A. (1974) Doctoral Thesis, Unwersity of Madrid 7 Boone. S.C. and Wakil. S.J. (1970) Biochemistry 9. 1470-1479 8 Watson, J.A. and Lowenstein. J.M. (1970) J. Biol. Chem. 245, 5993-6002 9 Lynen, F. (1961) Fed. Proc. 20.941-951 10 Bressler, R. and Wakil, S.J. (1962) J. Biol. Chem. 237. 1441-1448 11 Lachance, J.P., Popjak, G. and de Waard, A. (1958) Biochem. J. 68. 7p 12 Matthes, K.J., Abraham, S. and Chaikoff, I.L. (1960) Biochnn. Biophys. Acta 37.180-181 13 Nugteren, D.H. (1965) Biochim. Biophys. Acta 106. 280-290 14 Kunau, W.H. and Couzens, B. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 352.1297-1305 15 Yates, D.W. and Garland, P.P. (1966) Biochem. Biophys. Res. Commun. 23. 460465
Acta 248, Acta 280,