Effects of dietary sucrose and environmental temperature on fatty acid synthesis in Drosophila melanogaster

Insect Biochem., 1977, Vol. 7, pp. 371 to 379. Pergamon Press. Printed in Great Britain

EFFECTS OF DIETARY SUCROSE AND ENVIRONMENTAL TEMPERATURE ON FATTY ACID SYNTHESIS IN DROSOPHILA MELANOGASTER B. W. GEER and T. J. PERILLE* Department of Biology, Knox College, Galesburg, Illinois 61401, U.S.A. (Received 19 January 1977)

Abstract--The proportion of dietary sucrose devoted to lipid synthesis in the larvae of Drosophila melanogaster increased when the concentration was raised from 9 mM to 145 mM. A high dietary sucrose concentration increased the microsomal 9-desaturase activity and altered the specificity for de novo fatty acid synthesis termination to shorter chain lengths (14 and 16-C). Both neutral and phospholipid fatty acid contents were modified by dietary sucrose. A low environmental temperature favored the synthesis of monounsaturated fatty acids but had little effect on the length of fatty acids formed by de novo synthesis. Dietary sucrose minimized the utilization of dietary long chain fatty acids for lipid synthesis and diminished dietary fatty acid effects on fatty acid desaturation and chain elongation.melanooaster. Larvae fed a synthetic diet supplemented with lipid exhibited lower rates of lipogenesis and tissue levels of the oxidative NADP-enzymes INFLUENCES of different nutrients on lipogenesis and than larvae fed a fat-free diet with a similar carbothe interconversion of fatty acids in insects have been the subjects of several investigations. The inclusion hydrate content (GEER et al., 1976). When larvae were fed fat-free diets, the tissue lipid content and levels of any of several fatty acids in the diet altered the of the oxidative pentose shunt enzymes and malic tissue fatty acid profile (KEITn, 1966, 1967), and two enzyme paralleled the dietary concentration of de novo routes for the desaturation of dietary fatty sucrose; the tissue level of NADP-isocitrate dehydroacids by the larvae of Drosophila melanooaster were proposed (KEITH, 1967; KIYOMOTO and KEITH, 1970). genase was negatively correlated with the dietary sucrose concentration (GEER et al., 1977b). GEER et Pathways for the chain shortening of endogenous al. (1976) proposed that a derivative of r-oxidation fatty acids have been postulated for D. melanogaster (KL~rn, 1967) and Anthonomus grandis (LAMBREMONT of fatty acids was the mediator of the negative modulation of the oxidative NADP-enzymes; whereas, the et al., 1976). De novo synthesis of fatty acids occurs by the elongation of short chain precursors, and the inducer of the oxidative pentose shunt enzymes was postulated to be a derivative of glycolysis. elongation of endogenous long chain fatty acids has Because of the correlations between the tissue levels been demonstrated in the homogenates of several insects (LAMBREMONTet al., 1965; KEITH, 1967; BRIDGES, of the oxidative NADP-enzymes, which provide 1971 ; THOMPSON and BARLOW, 1971, 1972; MUNICIO N A D P H for fatty acid synthesis, and dietary carbohydrate; the present study was conducted to deteret al., 1972a; PENNER and BARLOW, 1972). De novo fatty acid synthesis in Ceratitis capitata is blocked mine the influence of dietary carbohydrate on fatty during periods of fasting (MUNIClO et al., 1973). Intra- acid synthesis and utilization in D. melanogaster. cellular mechanisms for the regulation of de novo synthesis of fatty acids in insects were suggested by the MATERIALS AND METHODS observations that CoASH inhibited fatty acid synthesis by homogenates of Musca domestica (BRIDGES Animals and WATTS, 1976); whereas, N A D P H activated aceThe wild-type strain of D. melanogaster employed for tyl-CoA carboxylase, the rate-limiting enzyme for most of the experiments was derived from the Canton-S fatty acid synthesis, in homogenates of C. capitata and Zw- strains (GEER et al., 1976) and maintained under continuous axenic culture on modified Sang's medium C (MuNICIO et al., 1972b). Previous studies have established close correlations (GEER et al., 1977b) for 2½ years before the initiation of between the rate of lipogenesis, the tissue levels of the current experimentation. The other wild-type strains that were examined for comparative purposes were estabthe oxidative NADP-enzymes, and the lipid and lished from single wild-caught females (Galesburg) and carbohydrate content of the diet of the larvae of D. placed in axenic culture by the methods described previously (GEER and NEWBURGH, 1970). Test cultures were * Present address: Northwestern University School of set up by placing 10 to 15 female-male pairs of adults in 6-dram shell vial cultures containing 5 ml of modified Medicine, Evanston, Illinois 60204, U.S.A.

INTRODUCTION

371

372

B.W. GEER AND T. J. PERILLE

Sang's medium C for 24 hr. Four days after the removal of the egg-laying adults the larvae were transferred by the previously described methods (GEER et al., 1976) to vials containing the final test diets. The final diets contained different amounts of sucrose, fatty acids or acetate as indicated in the tables of the Results section. Third instar larvae were removed from the vials containing the final diet after two days of feeding for analysis. Cultures were maintained at 22.8°C for the first 4 days. During the final 2 day feeding period cultures were kept at 18 °, 22.8 ° or 28°C. A I5 hr light-9 hr dark lighting schedule was maintained throughout the 6 day feeding period. Cultures contaminated with microorganisms were discarded. Total lipid and protein determination The total lipid content of larvae was quantified gravimetrically using the extraction methods of GEER and DOWNING (1972). Protein was measured by the method of LOWRY et al. (1951) using crystalline bovine albumin as the standard. Chromatography methods For chromatographic analysis 100 larvae were homogenized in 0.5 ml of glass-distilled water and the lipid extracted with chloroform-methanol (FOLcH et al., 1957). In some experiments neutral lipids and phospholipids were separated by column chromatography by eluting the neutral lipids initially from a silicic acid (10g) column with 15 ml of chloroform followed by the elution of the phospholipids with 10 ml of chloroform-methanol (2:1, v/v) and 20 ml of methanol. After evaporation of the lipid preparation to dryness, the lipid was saponified and methyl esters of the fatty acids prepared by the method of METCALFE et al. (1966). The fatty acid methyl esters were dissolved in 0.1 ml carbon disulfide for chromatographic analysis and the antioxidant, butylated dehydroxyanisole, was added at a concentration of 5 to l0 mg ~oSeparation of the fatty acid methyl esters was performed with a Carle Model 211 Analytical Gas Chromatograph with a 1/8 in. × 6 ft column containing 20~o DEGS on 80-100 mesh Chromatosorb W. Nitrogen at a 40 ml/min flow rate was used as the carrier gas and the oven temperature was maintained at 190°C. Fatty acid methyl ester standards were used to identify peaks. For individual separations retention times were calculated relative to the retention time of methyl palmitate and the amount of each ester (~, of total) was quantified using an OmniScribe Model 5211-15 recorde~integrater. The microsomal Jatty acid ,/,'~aturase assay The microsomal fraction xx,;, prepared by homogenizing 300 larvae in 1.5 ml o~ t~.25M sucrose4).l M Tris-HC1 l mM Na2HzEDTA. iql 7.4 solution. The homogenate was centrifuged at 13,000 9 I),r 30 min at 4°C and the supernatant recentrifuged at 45.t,lo.q for 2 h r at 4'~C. The microsomal pellet was resuspendcd in 0.5 ml of homogenizing solution. The 9-desaturase activity of the microsomal preparation was assayed in a 1 ml mixture containing 84,umoles KH2PO4 K2HPO4 buffer, pH 7.4; 7#moles MgCI2; 7/~moles ATP; 1/1mole N-acetylcysteine; 0.4 #mole NADPH ; 0.2/~mole CoASH; 300 nmoles [-1-~4C]palmitic acid (1 ,uCi/#mole) in 0.1 ml propylene glycol; and 0.1 ml of the microsomal preparation containing 1 to 3 mg protein. The reaction mixture was incubated at 30°C for 1 hr in a shaking water bath and the reaction terminated with 1 ml of 1.8 N ethanolic KOH. Reaction mixtures contain-

ing heat-inactivated microsomal preparation were run in parallel with the test mixtures for control purposes. The fatty materials in the reaction mixture were saponifled and methyl esters of the fatty acids prepared according to METCALFEet al. (1966). The saturated and monounsaturated esters were separated on 10% silver nitrate-silica gel G thin layer chromatography plates using a petroleum ether (30-60°C)-diethyl ether (95:5, v/v) solvent. The fatty acid esters were visualized by spraying the plates with a 2',7'-dichlorofluorescein 95"L ethanol solution and viewing the plates under u.v.-light. Methyl palmitoleate and methyl palmitate were identified by comparison with standards. The lipid spots were scrapped into scintillation vials and 10ml of scintillation fluid (5 g PPO and 0.3 g dimethyl P O P O P per liter of toluene) added. The radioactivity of the samples was determined with a Beckman LS-100C Scintillation Counter and corrected for quenching by the external standard method. The desaturase activity is expressed as the percentage of palmitic acid in the reaction mixture desaturated per mg of microsomal protein. Lipid and protein synthesis To measure the incorporation of label from dietary sucrose and acetate into protein and lipid, larvae were transferred from the standard synthetic medium to test diets containing [U-14C]glucose (2 x 104dpm//~mole of sucrose) or [2-14C]acetate (4.3 x 104dis/min/pmole of acetate) for two days. For computation purposes the sucrose was assumed to be degraded to two glucose equivalents by the test larvae. Lipid and protein were extracted sequentially from the test larvae and the label incorporated into these tissue constituents determined by the liquid scintillation methods described by GEER and DOWNING (1972). The rates of lipid and protein synthesis are given as the nanomoles of glucose equivalents or acetate incorporated into lipid or protein per mg of larval protein during the test feeding period. Reagents Vitamin-free casein for the test diets was obtained from the Gentosan Division, Fisons Pharmaceutical, Ltd., Leicestershire, England. Other biochemicals were purchased from the Sigma Chemical Co., St. Louis, Missouri. Labelled compounds were obtained from Amersham/ Searle Corp., Arlington Heights, Illinois. The materials for the chromatographic procedures were acquired from Analabs, New Haven, Connecticut. RESULTS Dietary sucrose stimulates lipid synthesis The incorporation of labelled c a r b o h y d r a t e from the diet into lipid a n d protein by larvae was examined to determine the degrees of dependency of lipid and protein synthesis upon c a r b o h y d r a t e when fed at different concentrations. The a m o u n t of glucose equivalent incorporated into lipid paralleled the dietary concentration of sucrose to 1 4 5 m M (Table l). The a m o u n t s of dietary carbohydrate utilized for lipid and protein synthesis were similar when a 9 m M sucrose diet was fed but an increasingly greater proportion of the dietary carbohydrate was employed in lipid synthesis as the concentration of sucrose was elevated in the diet. Almost 15 times more glucose was incorporated into lipid when a 145 m M sucrose diet was

Modulation of fatty acid synthesis

373

Table 1. The incorporation of label from dietary [U-14C]glucose and [2-14C]acetate into the lipid and protein of modified Canton-S larvae Dietary supplement

No. samples

mM Sucrose 9 29 145 290 Acetate 29 58 116 145

Incorporation into lipid

Incorporation into protein

nmoles/mg protcin 6 6 6 6

331 ± 1048 ± 4848 ± 4903 ±

48* ll6t 575 t 799t

360 ± 699 ± 1877 ± 2123 ±

31 72t 305t 240t

3 3 3 3

11074 ± 9654 ± 7864± 8346 ±

1276 1132 845 911

1370 ± 353 1688 ± 485 1542 ± 4 0 2 1059 ± 289

* The mean + S.D. "~Different at the 0.01 level from the corresponding mean for larvae fed a 9 mM sucrose diet (t-test). fed as when a 9 m M sucrose diet was administered but there was only a 5-fold difference in glucose incorporation into protein for larvae fed the two diets. Labelled acetate was fed at different concentrations to larvae to see if the acetate utilization pattern is similar to that of sucrose. Despite being efficiently utilized for both protein and lipid synthesis, the amount of label from acetate incorporated into lipid and protein by test larvae was only slightly influenced by the concentration of acetate in the diet (Table 1). Thus, the mechanism by which sucrose serves as a variable source of substrate for lipid and protein synthesis is not triggered by dietary acetate in D. melano-

gaster. Dietary sucrose alters the fatty acid composition of larvae The fatty acid profiles of the lipid extracts of larvae fed diets containing different sucrose concentrations indicated that dietary sucrose specifically modifies fatty acid metabolism (Table 2). Feeding a 290 m M sucrose diet to larvae produced a 27~o increase in

palmitoleic acid (16:t), a 40~o increase in myristic acid (14:0) and a 25~o decrease in oleic acid (18:1) relative to the amounts in larvae fed a 9 m M sucrose diet. Comparisons of the 18:1/18:0 and 16:1/16:0 ratios for larvae fed different sucrose concentrations indicated that high dietary sucrose concentrations stimulated increases of the monounsaturated fatty acids relative to their saturated counterparts. The increases in myristic acid and palmitoleic acid were more substantial when the lipid contents of the test larvae were considered (Table 2). Larvae fed a diet containing 290 m M sucrose had about one-third more lipid per mg of soluble protein as larvae fed a 9 m M sucrose diet. Consequently, the increases in myristic acid and palmitoleic acid relative to the other fatty acids in larvae fed a high sucrose diet were superimposed on top of a marked increase in total lipid content. That the changes in the fatty acid profile of the test animals were not limited to one lipid class was demonstrated by the analysis of the phospholipid and neutral lipid fractions (Table 3). Although the phos-

Table 2. The fatty acid contents of modified Canton-S larvae fed different concentration of sucrose Fatty acid

9

12:0 14:0 14:1 16:0 16:1 18:0 18:1 lipid:~

2.5 + 0.3* 14.2 ± 0.6 7.5 ± 1.2 19.0 ± 0.7 22.2 _ 2.1 2.7 _ 1.3 27.9 ± 2.9 1.10

Sucrose concentration (mM) 29 145 2.6 16.3 5.0 20.1 24.9 2.6 26.0

~o of total fatty acid _ 0.4 3.0 _ 0.1 ± 0.7 18.1 ± 1.6 + 1.0 6.7 _ 0.7 ± 0.9 20.0 ± 0.6 _ 0.5 26.5 + 1.2 _ 0.5 1.9 _ 0.6 ± 1.6 22.2 ± 1.7 1.29 1.62

290 2.9 _ 0.2 19.9 ± 0.6t 7.2 ± 1.2 17.9 ± 1.2 28.3 ±_ 0.3~" 1.7 _ 0.1 21.0 ± 0.51" 1.64

* The mean _ S.D. of four determinations. # Different at the 0.05 level from the corresponding mean for larvae fed 9 mM sucrose (t-test). :~mg lipid/mg soluble protein.

374

B.W. GEER AND T. J. PERILLE Table 3. The fatty acid contents of the neutral lipid and phospholipid fractions of modified Canton-S larvae fed different concentrations of sucrose Fraction and fatty acid

9

Sucrose concentration (mM) 29 145

290

Neutral lipid 12:0 14:0 14:1 16:0 16:1 18:0 18:1

2.7* 16.9 5.9 21.8 22.6 2.7 24.9

~o of total fatty acid 3.2 3.1 18.5 20.7 5.9 6.5 20.5 19.1 22.0 28.1 2.3 1.5 22.7 19.6

3.3 21.4 7.1 17.6 29.0 1.4 18.7

2.3 8.2 4.2 19.0 22.0 3.7 34.9

2.0 8.4 5.7 18.0 27.0 3.5 29.3

Phospholipid 12:0

1.0

14:0 14:1 16:0 16: 1

5.8 2.3 19.7 22.1 3.9 40.9

t 8:0

18 : 1

2.4 8.4 5.l t8.5 24.2 3.3 30.6

* The mean of two samples. pholipid fraction had higher total concentrations of the unsaturated fatty acids than the neutral lipid fraction at all dietary sucrose concentrations, both fractions had greater quantities of myristic acid and palmitoleic acid and less oleic acid when a high sucrose diet was fed to larvae compared to the fatty acid contents of larvae fed a low sucrose diet. Dietary sucrose influenced fatty acid synthesis and not the partitioning of fatty acids into lipid classes during lipogenesis.

Dietary sucrose and environmental temperature alter the fatty acid composition of larvae in different ways To examine the influence of an exogenous factor other than the diet on the fatty acid metabolism of larvae, larvae were exposed to different environmental temperatures during the final two day feeding period. The effects of dietary sucrose alteration and environmental temperature change on the fatty acid profiles of the test larvae were different (Table 4). Both a sucrose increase and temperature reduction increased the relative amounts of myristic acid and palmitoleic acid. Also, modification of the dietary sucrose concentration did not influence the relative amount of palmitic acid in the larval lipids but temperature alteration affected it markedly. Change in the dietary sucrose concentration altered the oleic acid content but temperature change exerted little, if any, effect on the oleic acid level in the larvae. Consideration of the total amount of fatty acid of each chain length (saturated plus unsaturated) showed that temperature alteration exerted little influence upon fatty acid elongation, whereas a change in the dietary sucrose concentration affected it markedly. These observations suggest that dietary sucrose and environmental temperature modify larval fatty acid metabolism by different routes.

Dietary sucrose modifies the microsomal palmitic acid desaturase system Because the alteration of the dietary sucrose concentration influenced the palmitoleic acid content of larval lipids, the microsomal desaturase system (9-desaturase) that catalyzes the conversion of palmitic acid to palmitoleic acid was examined (Table 5). The desaturase activity was 13-fold higher in the microsomal fraction from larvae fed a 290mM sucrose diet than in larvae fed a 9 m M sucrose diet. The microsomal fraction from larvae fed a diet containing 145 m M sucrose had an intermediate desaturase activity. Thus, the palmitic acid desaturase system is induced and/or activated in the microsomal fraction proportionate to the concentration of dietary sucrose.

Dietary sucrose inhibits the utilization of dietary long chain fatty acids To examine the metabolic interconversions of fatty acids in larvae cultured on a defined medium, larvae were grown on a sucrose-free medium supplemented with a fatty acid and a 145 mM sucrose diet containing a fatty acid. The rationale was that intracellular fatty acid synthesis would be limited by the carbohydrate supply in animals fed the former diet but that dietary substrate for de novo fatty acid synthesis would be plentiful for the second group of larvae. Dietary saturated fatty acids of ten carbons or less exerted little influence on the fatty acid profile (Table 6). Dietary 12:0 promoted a 7.5-fold increase in the 12:0 content of larvae fed a sucrose-free diet and the amount of myristic acid was about 1.6-times greater in larvae fed a 14:0 supplemented sucrose-free diet compared to larvae fed an unsupplemented sucrosefree diet. Inclusion of 145 mM sucrose with dietary

375

Modulation of fatty acid synthesis Table 4. The fatty acid contents of modified Canton-S larvae cultured at different temperatures and fed different concentrations of sucrose Temperature and fatty acid

9

Sucrose concentration (mM) 29 145

290

18°C 12:0 14:0 14:1 16:0 16:1 18:0 18:1

3.1 13.8 6.4 17.9 24.6 2.1 27.9

+__ 1.0" ___ 1.3 _+ 0.5 + 0.8 _ 0.9 _ 0.4 + 2.4

of total fatty acid 3.0 _ 0.4 3.1 + 0.5 15.5 ___ 1.8 17.6 + 0.6 5.8 + 0.5 7.2 + 0.9 19.3 + 1.4 15.4 ___ 1.4 25.3 ___ 1.2 28.5 _ 0.4 1.9 + 0.5 2.0 + 0.3 25.6 + 1.4 23.4 + 0.9

3.4 _+ 0.6 20.1 _+ 1.2 7.1 _+ 0.5 16.7 + 0.8 29.6 + 0.7 1.4 + 0.3 20.7 -t- 3.0

28 °C 12:0 14:0 14:1 16:0 16:1 18:0 18:1

2.3 16.2 4.7 24.0 21.8 2.0 26.8

+ 0.1 + 0.3 _+ 0.5 + 1.0~ _ 0.8 + 0.1 _ 0.9

2.6 18.6 4.6 21.9 23.2 1.7 25.8

2.9 22.0 6.4 21.0 25.5 1.3 19.7

* The mean + S.D. for four t Different at the 0.05 level same concentration of sucrose :~ Different at the 0.01 level same concentration of sucrose

_+ 0.3 _+ 0.8 _+ 0.4 _ 0.9 -t- 2.3 _ 0.2 _ 2.6

2.7 20.6 6.7 21.1 24.5 1.6 22.2

_+ 1.2 + 0.7 + 0.5 _+ 0.7? _+ 0.8I" _+ 0.3 + 1.0

determinations. from the corresponding mean for larvae exposed to 18°C and fed the (t-test). from the corresponding mean for larvae exposed to 18°C and fed the (t-test).

12:0 a n d 14:0 reduced the relative a m o u n t s of 12:0 a n d 14:0 in the larval lipids c o m p a r e d to the contents of larvae fed the fatty acids in sucrose-free diets. Dietary palmitic acid increased the tissue contents of palmitic acid a n d palmitoleic acid to a greater extent in the absence of dietary sucrose than when sucrose was present. The oleic acid contents of larvae fed the corresponding fatty acid-free diets. Supplementation of the diet with stearic acid (18:0) decreased 16:0 a n d 16:1 a n d elevated the relative p r o p o r t i o n s of 14:0 a n d 14:1 c o m p a r e d to the fatty acid contents of larvae fed fatty acid-free diets. Thus, dietary stearic acid inhibited chain elongation of fatty acids during synthesis. U n s a t u r a t e d fatty acids were also tested as dietary supplements (Table 7). C o m p a r e d to the fatty acid profile of larvae fed a n unsupplemented sucrosefree diet (Table 6), dietary palmitoleic acid fed in a sucrose-free diet caused a n increase in b o t h the 16:1 and 14:1 contents of the larval lipids but the level

of 18:1 was lowered. W h e n added to a sucrose-free diet, oleic acid effected an increase in the oleic acid content of the larval lipids but the 14:0, 16:0 a n d 16:1 contents were decreased. Linoleic acid (18:2), when administered in a sucrose-free diet, decreased the relative a m o u n t s of 14:1, 16:1 a n d 18:1 fatty acids while increasing the 16:0 and 18:0 contents. Thus, dietary 18:2 effectively inhibited the desaturation of long chain fatty acids in larvae. ~-Linolenic acid and ~-linolenic acid (18:3) also decreased the amounts of 18:1 and 16:1 and increased the levels of 16:0 and 18:0 in the larval lipids when fed in a sucrose-free diet. The influence of dietary sucrose in all cases was to minimize the effect of the dietary fatty acid on fatty acid metabolism. The 15:0 a n d 17:0 fatty acids were extensively incorporated into lipid when larvae were fed a sucrosefree m e d i u m (Table 8). Both of the fatty acids were converted to 13, 15, a n d 17-carbon saturated a n d m o n o u n s a t u r a t e d fatty acids. W h e n 17:0 was fed, the

Table 5. The desaturase activity in the microsomal fraction of modified Canton-S larvae fed different sucrose concentrations (mean + S.D.)

I.B. 7 / ~ E

_+ 0.6 _+ 1.3 _+ 0.6 _+ 0.8? _+ 1.1I" _+ 0.2 _+ 1.7

Sucrose concentration (mM)

No. samples

~o desaturation of [1-14C]palmitate per mg microsomal protein

9 145 290

3 3 3

0.3 -t- 0.1 2.5 + 0.5 4.0 _+ 0.7

376

B.W. GEER AND T. J. PERILLE Table 6. The fatty acid contents of modified Canton-S larvae fed sucrose-free and 145 mM sucrose diets supplemented with different saturated fatty acids Dietary fatty acid

Dietary sucrose level

12:0

14:0

Tissue fatty acids 14:1 16:0 16: l

18:0

18:1

mM 0 145 0 145 0 145 0 145 0 145 0 145 0 145

2.7* 3.1 2.8 3.2 19.6 9.1 3.9 3.4 2.7 3.3 3.1 3.3 2.6 3.0

13.5 16.6 12.5 19.7 12.7 18.4 31.7 27.0 13.8 18.9 21.2 21.5 12.0 18.1

~o of total fatty acid 5.9 20.9 22.2 6.9 18.7 24.0 5.5 22.1 21.2 5.9 20.2 26.0 4.4 16.8 18.8 5.9 17.3 24.8 6.4 13.4 16.5 6.5 16.1 23.6 5.7 26.0 29.9 6.7 22.1 28.0 12.1 12.7 11.7 10.8 16.7 21.6 3.4 18.3 22.4 6.7 20.0 26.5

2.5 2.6 3.5 1.6 3.0 1.6 2.2 1.6 2.5 1.6 6.2 3.5 3.8 1.9

29.1 24.2 27.3 21.9 22.5 20.8 21.7 18.2 16.9 18.2 28.8 22.1 30.7 22.2

8:0 10:0 12:0 14:0 16:0 18:0 none

* The mean of three determinations. The relative S.D. (100 x S.D./mean) did not exceed 12~, for any fatty acid component. most a b u n d a n t fatty acid to appear in the profile was 17:1. Because the 16:1/16:0 ratio was also low, the dietary 17:0 apparently competed well with endogenous 16:0 for the desaturase complex. The relative amounts of odd-chain fatty acids incorporated into the larval lipids were greatly reduced when sucrose was fed with the fatty acids.

Lipid modification by dietary sucrose has a genetic component To examine the possibility that the modified Canton-S wild-type strain used for the current experimentation was unique in regard to carbohydrate-induced changes in fatty acid metabolism, four other wild-type strains were examined. The fatty acid profiles of the lipids of the larvae of each of the four strains were influenced in the ways described for the modified

Canton-S strain (Table 9). That is, an increase in dietary sucrose concentration increased the relative amounts of 14:0, 14:1, and 16:1 and decreased the a m o u n t s of 16:0 and 18:1. Nevertheless, strain differences became obvious when the ratios of the total 14:0, 16:0, and 18:0 content to the total 14:1, 16:1, and 18:1 content for the strains were compared. The modified Canton-S strain (Table 2) and strain 1 possessed a greater proportion of unsaturated fatty acids in their lipids than did strains 2, 3, and 4 when fed a 9 m M sucrose diet. Different responses were also noted for the wild-type strains to a high dietary sucrose concentration. The larvae of strains 2, 3, and 4 exhibited less total saturated fatty acid and more total unsaturated fatty acid when fed a 2 9 0 m M sucrose diet than when a 9 m M sucrose diet was fed. A high dietary sucrose concentration promoted in-

Table 7. The fatty acid composition of modified Canton-S larvae fed sucrose-free and 145 mM sucrose diets supplemented with different unsaturated fatty acids

Dietary fatty acid 16:1 18:1 18:2 ct-18:3 7-18:3

Dietary sucrose concentration

12:0

14:0

mM 0 145 0 145 0 145 0 145 0 145

2.1" 2.8 1.8 3.1 2.1 3.2 1.8 3.1 1.8 3.1

9.0 7.3 19.2 6.3 9.3 14.2 19.0 9.0 11.1 2.0 20.6 4.2 10.5 3.1 18.9 5.1 10.5 3.5 19.4 4.9

14:l

14:2 ---3.0 0.8 -1.7 0.5

Tissue fatty acids 16:1 16:2

18:0

18:1

~ of total fatty acid 18.8 35.7 18.8 29.9 -17.9 14.0 -18.4 24.7 24.3 8.1 4.3 21.8 22.0 0.8 24.8 10.8 21.8 21.1 22.6 13.7 20.7 23.3 --

4.0 1.5 2.2 1.5 5.2 2.1 4.1 2.2 5.8 2.7

18.8 21.1 38.6 23.9 16.5 21.1 23.6 21.4 21.1 21.2

16:0

18:2

18:3

--21.8 4.2 ---

19.8 6.0 13.7 4.0

* The mean of three determinations. The relative S.D. (100 x S.D./mean) did not exceed 10~o for any fatty acid component.

Modulation of fatty acid synthesis

377

Table 8. The fatty acid composition of modified Canton-S larvae fed sucrosefree and 145 mM sucrose diets supplemented with different odd-chain fatty acids Dietary fatty acid 15:0

17:0

Tissue fatty acids

No sucrose

12:0 13:0 14:0 14:1 15:0 15:1 16:0 16:1

1.7" 10.8 0.6 9.7 3.8 24.0 5.5 8.0 12.5

17: 0 17: 1

0.9 3.4

1.4 1.0

18:0 18:1

1.4 16.4

2.1 18.8

13 : 1

145 mM sucrose

No sucrose

of total fatty acid 2.5 1.4 3.7 7.2 0.4 1.7 17.6 8.7 5.4 1.8 7.1 3.5 1.7 1.5 15.2 14.4 22.9 10.8 10.9

19.4 2.2 14.5

145 mM sucrose 2.6 5.1 1.6 17.1 5.1 1.1 1.0 16.3 18.6 4.4 7.8 2.0 17.1

* The mean of three determinations. The relative S.D. (100 x S.D./mean) did not exceed 12°/, for any fatty acid component. creases of both total ~lturated fatty acid and total unsaturated fatty acid in strain 1 larvae, whereas the modified Canton-S larvae responded to an increase in dietary sucrose concentration with an increase in total saturated fatty acid and a decrease in total unsaturated fatty acid. The net effect of a high dietary sucrose concentration was to minimize strain differences in fatty acid content. DISCUSSION The current studies in which the larvae of D.

melanogaster were fed fatty acids in a fat-free synthetic medium under axenic conditions supported the interconversions postulated by KEITH (1966, 1967). Short chain saturated intermediates are elongated by twocarbon additions with 18 carbons being the maximum length. Myristic acid (14:0), palmitic acid (16:0) and

stearic acid (18:0) may be desaturated to their monounsaturated counterparts and these in turn elongated to 18:1. Shortening of the long chain saturated and monounsaturated fatty acids may occur by the removal of two-carbon units. The polyunsaturated fatty acids do not appear in the lipids of the larvae of D. melanogaster unless they are included in the diet. Linoleic acid (18:2) is shortened to 16:2 and 14:2, but neither ~-linolenic acid or 7-1inolenic acid (18:3) is efficiently modified by D. melanooaster. Dietary sucrose influences lipid metabolism in several ways in Drosophila. Dietary sucrose stimulates lipid synthesis and the radioisotope data of the present study showed that the amount of dietary carbohydrate convertible to lipid increased greatly when the dietary sucrose concentration was raised from 9 to 145 mM. In contrast, although dietary acetate is efficiently utilized as a substrate for lipid syn-

Table 9. The fatty acid contents of larvae of different Galesburg wild-type strains fed different concentrations of dietary sucrose

Strain 1 2 3 4

Dietary sucrose level

12:0

14:0

Tissue fatty acids 14:1 16:0 16:1

18:0

18:1

mM 9 290 9 290 9 290 9 290

3.7* 3.3 2.9 3.5 2.9 3.5 2.8 3.6

15.7 19.6 18.7 22.2 19.3 22.1 18.2 -21.0

~o of total fatty acid 8.8 18.6 16.8 9.2 16.9 24.8 5.5 22.1 20.3 7.0 17.2 26.9 7.6 20.9 19.8 8.6 15.9 28.3 7.3 20.7 20.1 9.6 15.8 28.3

2.7 2.0 1.8 1.5 1.6 1.5 1.7 1.4

28.2 21.7 26.8 20.8 25.2 18.7 26.8 19.1

* The mean of two determinations.

378

B.W. GEER AND T. J. PERILLE

thesis by the larvae of D. melanoyaster, increasing the concentration of dietary acetate did not increase the incorporation of label from acetate into lipid. Apparently, a product of sucrose degradation other than acetyl-CoA stimulates lipid synthesis in D. melanooaster. Dietary sucrose modifies fatty acid desaturation and elongation in D. melano#aster. When measured directly in the present study, the microsomal palmitic acid desaturase activity was found to increase as the concentration of sucrose in the larval diet was raised. Using the indirect approach of computing 14: 1/14:0, 16:1/16:0, and 18:1/18:0 ratios as indicators of myristic acid, palmitic acid and stearic acid desaturase activities and employing 16:0/14:0 and 18:0/16:0 ratios to quantify myristic acid and palmitic acid elongation, more subtle aspects of dietary sucrose and environmental temperature modulation in D. melano#aster are evident. The palmitic acid and stearic acid desaturase activities were increased in the test larvae in proportion to the increase of dietary sucrose, whereas myristic acid desaturation was uninfluenced. Also, the elongation of myristic acid and palmitic acid declined as dietary sucrose was elevated. Environmental temperature changes influenced fatty acid metabolism in the larvae of D. melano#aster in the present study. A low environmental temperature favored the desaturation of myristic acid and palmitic acid but stearic acid desaturation was only slightly affected by temperature. When the saturated and unsaturated fatty acids of each carbon length were considered together, the elongation process in D. melano#aster was found to be altered only slightly by a change in environmental temperature. Thus a low environmental temperature, like a high dietary sucrose concentration, sponsored fatty acid desaturation but the specificities of the desaturase systems were different. The influence of dietary sucrose on fatty acid desaturation in D. melanooaster appears to reside in activation and/or induction of some component of the microsomal desaturase systems involved in the direct desaturation of the 14:0, 16:0, and 18:0 fatty acids and in the alteration of the specificities of these systems. The number of systems involved in the direct desaturation of long chain saturated fatty acids in D. melano#aster were not indicated by the present study. Two observations suggest that the influence of dietary sucrose on the desaturase mechanisms is dependent upon a product of the pentose shunt pathway. Firstly, dietary acetate did not stimulate palmitic acid desaturation nor did it inhibit chain elongation in the present study. The amount of pentose shunt substrate provided by dietary acetate is minimal and consequently shunt products were at a low level in the larvae fed an acetate diet. The second evidence is that the desaturase systems in a strain of D. melano#aster in which the pentose shunt pathway is genetically blocked were not influenced by dietary sucrose (GEER et al., 1977a). The influences of dietary fatty acids on desaturation

and elongation indicate other aspects of control mechanisms in D. melanogaster. The intermediate length fatty acids stimulated elongation when fed in a sucrose-free diet. In contrast, dietary stearic acid inhibited 16:0 elongation and desaturation. Dietary palmitoleic acid (16:1) inhibited 18:0 desaturation and oleic acid (18:1) hindered chain elongation. The polyunsaturated fatty acids were potent inhibitors of fatty acid desaturation in D. melano#aster but had little influence on chain elongation. Dietary linoleic acid effectively inhibited the desaturation of 14:0, 16:0 and 18:0. ~-Linolenic acid and 7-1inolenic acid were less efficient inhibitors of 16:0 and 18:0 desaturation than linoleic acid and exerted little influence on 14:0 desaturation. In all instances sucrose, when included in the diet with a fatty acid, minimized the influence of the dietary fatty acid on the larval fatty acid profile. Apparently, fatty acid intermediates generated from dietary carbohydrate largely preempt the utilization of dietary fatty acids for lipid synthesis. This is not surprising since D. melanogaster has no dietary fatty acid requirements, and this type of control mechanism would insure a relatively constant fatty acid profile for the tissues of the animal when dietary carbohydrate is available. The present study showed that chain length specificity for synthesis termination of fatty acids is dietinfluenced in the larvae of D. melano#aster. MUNICIO et al. (1971) found that the specificity of the fatty acid synthetase complex for synthesis termination in larval homogenates of C. capitata was for shorter chain fatty acids than the specificity of the fatty acid synthetase complex of adult homogenates, suggesting that there is flexibility in the specificity of the insect fatty acid synthetase complex. It has been suggested that in mammary glands rapid lipid synthesis increases the ratio of malonyl-CoA to acetyl-CoA and that this promotes premature deacylation of the long chain acyl group bound to the fatty acid synthetase complex (CAREYand DILS, 1973). BRIDGES and WATTS (1976) reported that larval homogenates of M. domestica incorporated a greater proportion of label from [U-14C]acetate into 16 and 18 carbon fatty acids when CoASH was added and a greater proportion into intermediate length fatty acids when CoASH was omitted from homogenates. Dietary sucrose may alter fatty acid chain elongation in D. melanogaster by altering the tissue concentrations of short chain-CoA compounds and CoASH. The influence of dietary carbohydrate on microsomal stearyl-CoA (9-C) desaturase in mammalian liver is well documented (INKPEN et al., 1969; OSHINO, 1972; OSHINO and SAaO, 1972). The increase in desaturase activity due to dietary carbohydrate is linked to protein synthesis and it has been postulated that the terminal cyanide sensitive factor is the inducible protein of the microsomal desaturase system (OsmYO, 1972; OSmNO and SAT#, 1972). KIYOMOTOand KE1Tn (1970) have presented evidence that D. melanoaster produces monounsaturated fatty acids by direct

Modulation of fatty acid synthesis

379

feeding by microsomal systems of rat liver 6- and 9-desaturation of fatty acids. J. Lipid Res. 10. 227-282. KEnYa A. D. (1966) Fatty acid metabolism in D. melanogaster: formation of palmitoleate. Life Sci. 6. 213-218. KElaIJ A. D. (1967) Fatty acid metabolism in Drosophila melanogaster: interaction between dietary fatty acids and de novo synthesis. Comp. Biochem. Physiol. 21, 587-600. KIYOMOTOR. K. and KEIa~J A. D. (1970) Fatty acid metabolism in Drosophila melanogaster: II. Metabolic origin of monoenes. Lipids 5. 617-620. LAMBREMONTE. N., ERNST N. R., FERGUSON J. R., and DIAL P. F. (1976) Lipid metabolism of insects: chain Acknowledgements--We would like to thank LEE FARshortening of a long-chain dietary fatty acid. Comp. BioRAR, KARENMcGINNlS and DAWN LINDELfor their technichem. Physiol. 54B, 167-169. cal assistance. This research was supported by National LAMBREMONTE. N., STEIN C. I., and BENNETTA. F. (1965) Institute of Health Grant GM20393. Synthesis and metabolic conversion of fatty acids by the larval boll weevil. Comp. Biochem. Physiol. 16. 289-302. LOWRY O. H., ROSEBROUGHN. J., FARR A. L,, and RANREFERENCES DALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193. 265-275. BRIDOESR. G. (1971) Incorporation of fatty acids into the METCALEE L. D., SCHM1TZA. A., and PELKA J. R. (1966) lipids of the housefly, Musca domestica, d. Insect Physiol. Rapid preparation of fatty acid esters from lipids for 17, 881-895. gas chromatographic analysis. Anal. Chem. 38, 514-515. BmDGES R. (3. and WATTS S. (3. (1976) Synthesis of fatty acids in vitro by choline-deficient and normal larvae of MUNICIOA. M., ODRIOZOLAJ. M., PINE1ROA., and RIBERA A. (1971)In vitro fatty acid and lipid biosynthesis during the housefly, Musca domestica. J. Insect Physiol. 22. development of insects. Biochim. biophys. Acta 248. 101-106. 212-225. CARET E. M. and DILS R. (1973) Regulation of the chain MUNICIOA. M., ODRIOZOLAJ. M., PINEIROA., and RIBERA length of fatty acids synthesized by cell-free preparations A. (1972a) In vitro elongation and desaturation of fatty of lactating rabbit, rat and guinea pig mammary gland. acids during development of insects. Biochim. biophys. Comp. Biochem. Physiol. 44B. 989-1000. Acta 280, 248-257. FOLCH J., LEES M., and SLOANESTANLEYG. H. (1957) A simple method for the isolation and purification of total MUNICIOA. M., ODRIOZOLAJ. M., and RAMOSJ. A. (1972b) In vitro regulation by NADPH of fatty acid biosynthesis lipides from animal tissues. J. biol. Chem. 226, 497-509. with larval and pharate adult homogenates of the fly, GEER B. W. and DOWNING B. C. (1972) Changes in lipid Ceratitis capitata. Insect Biochem. 2. 353-360. and protein syntheses during spermatozoan development and thoracic tissue maturation in Drosophila hydei. Wil- MUNICIO A. M., ODRIOZOLAJ. M., and RAMOSJ. A. (1973) Effect of diet on lipogenesis of larvae of Ceratitis capihelm RouT Arch. EntwMech. Org. 170. 83-89. tara. Insect Biochem. 3. 359 366. GEER B. W., KAYAK S. N., KIDD K. R., NISHIMURAR. A., and YEMM S. J. (1976) Regulation of the oxidative OSH1NO N. (1972) The dynamic behavior during dietary induction of the terminal enzyme (cyanide-sensitive facNADP-enzyme tissue levels in Drosophila melanogaster. tor) of the stearyl-CoA desaturation system of rat liver I. Modulation by dietary carbohydrate and lipid. J. exp. microsomes. Arch. Biochem. Biophys. 149. 378-387. Zool. 195. 15-32. OSmNO N. and SATO R. (1972) the dietary control of the GEER B. W., LINDELD. L., and LINDEL D. M. (1977a) The microsomal stearyl-CoA desaturation enzyme system in relationship of the pentose shunt pathway to fatty acid rat liver. Arch. Biochem. Biophys. 149. 369-377. synthesis and the regulation of the oxidative NADPPENNER K. R. and BARLOW J. S. (1972) The composition enzyme tissue levels in Drosophila melanogaster. Biochem. and metabolism of fatty acids in Ips paraconfusus (ColGenetics. In press. eoptera: Scoytidae). Can. J. Zool. 50, 1263-1267. GEER B. W. and NEWBURGHR. W. (1970) Carnitine acetyltransferase and spermatozoan development in Droso- THOMPSONS. N. and BARLOWJ. S. (1971) Aspects of fatty acid metabolism in Galleria mellonella (L.) (Lepidoptra: phila melanogaster. J. biol. Chem. 245, 71-79. Pryalidae): isolation of the elongation system. Comp. GEER B. W., WOODWARD C. G., and MARSHALL S. D. Biochem. Physiol. 38B, 333 346. (1977b) Regulation of the oxidative NADP-enzyme tissue levels in Drosophila melanogaster. II. The biochemi- THOMPSON S. N. and BARLOW J. S. (1972) Presence and synthesis of a 20 carbon monounsaturated fatty acid, cal basis of dietary carbohydrate and D-glycerate modu9-eicosenoic acid, and other fatty acids, in Galleria mellation. J. exp. Zool. In press. lonella (Lepidoptra: Pryalidae). Ann. ent. Soc. Am. 65. INKPEN C. A., HARRIS A. A., and QUACKENBUSHF. W. 1020-1023. (1969) differential responses to fasting and subsequent

desaturation of long chain precursors and by the desaturation of an intermediate length precursor followed by elongation. The latter system was not repressed by dietary linoleic acid. Because of the experimental design of the current study, it can only be concluded that the direct desaturase systems are modified in D. melanogaster by dietary sucrose. The effect of the diet on the indirect monounsaturated fatty acid biosynthetic pathway is unknown.