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In vitro elongation and desaturation of fatty acids during development of insects
BIOCHIMICA
248 BBA
ET BIOPHYSICA
ACTA
j6rog
IN VITRO
ELONGATION
DEVELOPMENT
AND DESATURATION
OF FATTY
ACIDS
DURING
OF INSECTS
I. Ix vitro elongation and desaturation reactions of fatty acids using labelled ro:o, 12: o, 14:o and 16: o have been investigated with homogenates of larvae and pharate adults of Cerutitis ca$itata. The obtained results show clear differences in the behaviour of larval and pharate adult stages of development. 2. Larval homogenates desaturate and elongate the labelled substrates arcording 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 are insignificant. 3, Labelled acetate incorporation into fatty acids by the larval homogenates is notably stimulated in the presence of malonate, whereas the fatty acid synthesis b! pharate adult homogenates is practically unaffected. The fatt). acids IO : o and 12 : o are elongated by larval homogenates more effectively in the presence of acetate than that
of malonate. 4. The larval homogenate incorporates considerably more labelled fatty acids into triglycerides than into phospholipids, whereas the radioactivity remains mainly as free fatty acids in the presence of pharate adult homogenates. 5. Pharate adult mitochondria preparations incorporate more acetate than larval mitochondria preparations. Incorporation by larval mitochondria was not in fluenced by the presence of malonate and the resulting fatt), acids are present mainl! as phospholipids, in a clear contrast with the incorporation achieved by the whole larval homogenates.
INTRODLCTIOK
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. Experimentsinvolving the fate of rr*C]acetate and incorporation of other labelled fatty acids by homogenates of different stages of devel-
FATTY
ACIDS
AND
INSECT
249
DEVELOPMENT
opment of the insect Ceratitis capitata have been previously
undertaken.
It was found
that the distribution patterns of [14C]acetate incorporation among free fatty acids, triglycerides and phospholipids were different in the larval and pharate adult stages of development. The time course of [lK]acetate incorporation showed some differences in the behaviour of larval and pharate adult homogenates; triglycerides and phospholipids become labelled in a different way by either larval or pharate adult homogenates’. The time course of [l*C]acetate incorporation also showed clear differences in the behaviour of larval and pharate adult homogenates regarding the specific activities of the fatty acids synthesised by them. Specific radioactivities of fatty acids synthesised by larval homogenates exhibited a net distinction between medium-chain and long-chain fatty acids; this distinction was not exhibited in the [Klacetate incorporation by pharate adult homogenates’. This different behaviour of these two developmental stages of C. capitata also applied to the specific radioactivity of the individual fatty acids present in the lipid classes2. Decanoic acid had the highest specific radioactivity of the fatty acids present in triglycerides from larval homogenates; the fatty acids of triglycerides from pharate adults, on the other hand, did not exhibit any incorporation of radioactivityz. In addition, Lr-Kjpalmitic and Lr-%]stearic acids were desaturated by larval homogenates, but not by pharate adult homogenates”. It was the intention of the present study to attempt to compare the desaturation and elongation of different fatty acids by either larval or pharate adult homogenates of C. capitata as well as the incorporation of these fatty acids into the most abundant classes of lipids. MATERIALS
AND
METHODS
!Vaterials ATP, CoA and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. [I-W]Acetate and I-l*C-labelled fatty acids (IO:O to 16:o) were obtained from The Radiochemical Centre, Amersham, Bucks. Rearing of insects C. capitata (Wiedemann) was used during the larval and pharate adult stages of development and the diet, temperature and humidity conditions of culturing were carefully controlled, as reported previously2. Larvae were reared 2-3 days before the larval-pupal apolysis and were starved 2-3 h before homogenization. Pharate adults were 5 days old from the larval-pupal apolysis. Both larvae and pharate adults were carefully washed with distilled water before use. Preparation of homogenates Larvae and pharate adults were directly homogenized with 3 vol. of cold homogenizing buffer (0.35 M sucrose-0.05 M Tris, pH 7.4) in a glass homogenizer fitted with a Teflon pestle. The total homogenate was filtered through two layers of cheese-cloth and the filtrate was centrifuged at 900 x g for IO min; to free the 900 x g supernatant from loosely bound chitinous particles centrifugation was carried out twice at 1200 x g Biochim.
Biophys.
Acta, Z?O (1972)
248-257
for IO min. The 1200 xg supernatant was used as total homogenate and as the source of the mitochondrial fraction. Mitochondria were removed from the 12ooxg supernatant by centrifugation at 12000 x g for IO min; the pellet was resuspended in the sucrose-Tris medium and recentrifuged for IO min at 12 ooo XS. Purity of the mitochondria fraction was assessed by electron microscopy. All operations were carried out at o--4 “C. Incubation mixture The assay mixture for incorporating [I-Xjacetate or 1-14C-labelled fatty acids contained ATP (6 ymoles/ml), CoA (2 pmoles/ml), NADPH (I pmole/ml) and l&Cl, (z pmoles/ml) in 0.05 M potassium phosphate buffer, pH 7.4 (22 ~moleslml), z pmoles, ml of either sodium acetate or sodium malonate, and 0.5 ml of total homogenate or mitochondrial preparation. ;r-14Cj_4cetate (spec. act. 57 mCi/mmole) was present in the incubation mixture at 60 &i/ml. Labelled fatty acids (spec. act. 16.4 mCi/mmole for 10: 0, 21.0 mCi/mmole for 12 :o, 15,4 mCi/mmole for 14:0, 48.0 mCi/mmole for 16 : o) were suspended in potassium phosphate buffer and the mixture was sonicated for z min prior to the addition to the incubation mixture at 4 &i/ml (0.20-0.24 pmole/ml). Incubations were made in a shaker at 37 “C for 180 min according to the Final volume 2 ml containing 10 mg/ml of time-course results previously obtainedl,l. proteins (total homogenate) or 2.5 mg/ml (mitochondrial preparations). into ilcetyl-CoA carboxylase was assayed by WO, fixation from NaHXO, acid-stable material. Preincubation of the reaction components was not carried out since previous experiments showed no influence on the radioactivity incorporation. Incubation mixture contained: 22.5 pmoles/ml potassium phosphate buffer, pH 7.0; 3 pmoles/ml sodium citrate; z pmoles/ml M&l,; 2.5 pmoles/ml mercaptoethanol; IO pmoles/ml potassium bicarbonate; 0.5 pmole/ml acetyl-CoA, (or 0.5 pmole/ml Co4 filus 3 pmoles/ml acetate) and 6 pmoles/ml ATE’; enzyme preparation, IO mg protein/ml. Final volume 0.5 ml. The reaction was started by the addition of I &i/ml NaHKO,. Incubations were in duplicate and incubations without enzyme preparations were always included as controls. The reaction was stopped with 0.1 ml of 6 nl HCl at 30 min and the tubes agitated and dried under vacuum; the residue was assayed for radioactivity by liquid scintillation counting. Extraction and separation of lieids At the end of the incubation time the reaction was stopped by the addition of 5 ml of chloroform and the mixture was made into a single phase by the addition of methanol. The crude lipid extract was purified as described2. Purified lipids were fractionated into lipid classes by thin-layer chromatography on silica gel G by the method described2. Thin-layer bands were scraped off the plate and either directly transferred to scintillation vials for counting or submitted to the methanolysis procedure. Fatt_y 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 trifluoridemethanol reagent as described”.
BLochim. Bi@hys.
Ada,
280 (1972) 2$-2jj
FATTY
ACIDS AKD INSECT DEVELOPMENT
Determination
of incorporation
251
of radioactivity
into lipid
classes
and individual
fatty
acids Lipids were located by a slight spray with Rhodamine B solution and the areas corresponding to each lipid were scraped from the plate and placed into counting vials with 15 ml of the scintillation mixture (4 g PPO and 0.1 g POPOP diluted to I 1 with toluene). Chromatographic separation of methyl esters and determination of radioactivity was carried out using a Perkin-Elmer Model RGC 170 flow-through reactor/ proportional counter connected to a gas-liquid chromatography unit (Perkin-Elmer F20FE flame-ionisation instrument). Details of the experimental procedure were previously describeds. Specific activities are conveniently expressed as counts per unit area under each mass peak. RESITLTS AND DISCUSSION LIesaturation and elongation of labelled IO: o, 12: o, 14: o and I 6: ofatty acids Figs. I and 2 represent the behaviour of larval and pharate adult homogenates, respectively, in a series of desaturation and elongation experiments using labelled IO: o, 12 : o, 14: o and 16 : o fatty acids as substrates. Incubation time was ISO min.
Fig. I. Elongation and desaturation of fatty acids by larval homogenates of Ceratitis cupitata. r-14C-labelled fatty acids used as substrates were IO : o (a), 12: o (b), 14: o (c) and 16 : o (d). Synthcsised fatty acids are expressed as percentages of the fatty acids used as substrates. Black bars, substrates. Stripped bars, desaturation products. Dotted bars, elongation products.
a
.:.:.::,: ..:=:.,:i “:::::
b
..:. c
cl
11111111
llll .... Ill1 :&
1111111111111 14:1 II:0
li: 12:1
~,~*$$*fg
/ 1z:n
1o:, ItO 100
50
I
0.5
relative abundant 50'
I05
50
1
0.5
i 50 T
Fig. 2. Elongation and desaturation of fatty acids by pharate adult homogenates of Cevatitis r-r4C-labelled fatty acids used as substrates were ro:o (a), 12: o (b), r.+: o (c) and 16: o (d). Synthesised fatty acids are expressed as percentages of the fatty acids used as substrates. Black bars, substrates. Striped bars, desaturation products. Dotted bars, elongation products. capztata.
Biochim.
Biophys.
Acta,
280 (1972) 248-257
252
A.
M.
ivI’SICIC)
zt al.
taking into consideration the previous time-course experiment+*, and that at this time the total radioactivity incorporation achieved by larval and pharate adult homogenates exhibited the most similar values’. Larval homogenates (Fig. I) show considerable differences in the desaturation and elongation products according to the chain length of the substrates. It should be noted that the longer the length of the fatty acids used as substrates the more pronounced the synthesis of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Thus, in the conditions of our experiments, larval homogenates do not elongate 16:o fatty acid, whereas the relative abundance of palmitoleic acid reaches values close to 50%; on the other hand, the low levels exhibited by IO : I as a consequence of desaturation of IO: o can be seen. Pig. 2 shows the results of a typical experiment in which the relative abundance of desaturation and elongation products formed by pharate adult homogenates from the same labelled fatty acids are compared as before. Inspection of these results clearly shows that, when present, the radioactivity levels are generally very small; thus, 14: o and 16:o do not exhibit any elongation and IO: o and IZ:O do not form their direct desaturation products. However, since under identical experimental conditions larval homogenates were shown to be much more active than the pharate adult homogenates, it seems reasonable to conclude that direct desaturation and elongation of fatty acids are not a major metabolic route in the pharate adult stage of development. This fact could be related with the invariability of the molar unsaturation degree observed during pharate adult development 3,3 in choline and ethanolamine phosphoglycerides, while a tendency to increase the unsaturation degree was found during larval and adult development. These findings reflect the existence of a different metabolic behaviour between these two developmental stages of the insect which had been previously establisheds. Bffectively, NADPH enhanced the incorporation of labelled acetate by larval and pharate adult homogenates in a different way; also [lK]acetate incorporation by larval and pharate adult homogenates in the presence of ATP-Mg was enhanced or decreased by citrate, respectively”. Incorporation of acetate @US CoA into malonyl-Co,4 through a YO,-carboxylation reaction by larval and pharate adult homogenates has been correlated with that of acetyl-CoA in order to prove the acetate activation in both systems. Results given in Table I show that acetate can be used as substrate for the synthesis of fatty acids in either larval or pharate adult homogenates. Influence of ATP concentrations on the incorporation of r%]acetate into long-
14COl~NCORPORATIONBY~AR”AI.ANDPHARATE ADULTHOMOGE~ATESI~THEPRESBPI'CEOPEITNER ACETATE I
PLUS
CO/l
,uCi NaH%O,
OR
ACETYL-Cd
was added to the reaction
Results are given in cpm/mg ATP
/v&i-l
mixture
as described
Larvae
-
Pharatr
adults
Acetate + CoA
Acetyl-CoA
Acetate
0
2600
2300
2800
.+200
2
2850 3270
2660 2830
25.50 2150
3125 2450 _____
Biochim.
Bi+hys.
A&,
280
in hlaterials
protein.
(1972)
248-2j7
t CoA
Acrtyl-CoA
.~
and Methods.
FATTY
ACIDS
AND
INSECT
DEVELOPMENT
253
chain fatty acids by larval and pharate adult homogenates was previously established5; maximal levels of incorporation were obtained at 6-12 mM ATP and this concentration has been used in this paper. In the past years the non-mitochondrial, the mitochondrial and the microsomal fatty acid-synthesising systems have been intensively studied in many tissues and organisms6p9 and it was found that elongation with C, units is one of the major reactions regardless of the chain length of the fatty acid used as substrate. The supernatants synthesise long-chain fatty acids by the repeated additions of malonyl-CoA on an initial acetyl-Co,%. The malonyl-CoA is synthesised from acetyl-CoA with acetylCoA carboxylase, in what has been shown to be generally the rate-limiting step for de GOZ’Ofatty acid synthesis. The mitochondrial system is involved in an elongation pathway in which acetate is added onto a previously formed fatty acid. Microsomes have been shown to synthesise fatty acids by elongation involving malonyl-CoAln-la. Malonate has been described to stimulate the acetyl-CoA carboxylationlS and the influence of malonate on [I-Xjacetate incorporation into fatty acids by larval and phara-te adult homogenates is shown in Table II. These results clearly show that the labelled acetate incorporation into fatty acids is notably stimulated by malonate in the presence of larval homogenates, whereas radioactivity incorporation is unaffected in the presence of pharate adult homogenates. These findings reflect again the different metabolic behaviour between these developmental stages. The clear stimulation of the specific activity of the fatty acids produced by malonate in the presence of larval homogenates (Table II) might be due to its influence on the de nova fatty acid synthesis or on the elongation system. However, Table III shows that malonate does not enhance the elongation of fatty acids by larval homogenates; effectively, IO:O and IZ:O fatty acids are elongated more efficiently in the presence of acetate than is the case with malonate, whereas elongation of 14: o is similarly stimulated by both factors. Elongation of 16 : o does not cocur. Incorporation of labelled ~o:o, 12:0, 14:o and 16:o into different lifiid classes Table IV shows the specific activities of the most abundant classes of lipids after incorporation of labelled fatty acids by larval and pharate adult homogenates TABLE
II
ISFLUEXcE
OF
MALONATE
j+C]ACET_4TE
ON
THE
INCORPORATION
BY
SPECIFIC LARVAL
RADIOACTIVITY
OF
AKD
ADULT
PHARATE
FATTY
ACIDS
RESULTING
HOMOGEXATES
OF
FROM
C‘VatitiS
capitata Distribution of label in fatty acid methyl esters was accomplished by gas-liquid radiochromatography. Specific radioactivities are given as the ratio of cpm. to the area under the mass peak (mean values of three individual determinations).
Fatty acids
Specific radioactivzty LWUCZf _____
A cetatc 1010
8.j
12:0
IO.3
1410
4.0
14:1 16~0 16:1 18:o
7.1 0.7
18:r
0.3
Phavate aldults Malon&?
‘4cetate
Malonatc
22.8
29.1
2.4
2.j
14.2
3.1
3.3
22.6 3.5
0.8
4.8
0.1
0.7 I.5
9.0
IO.7
I.1 1.3
1.2 I.3
0.1
0.1
B&him.
Biophys.
A&,
280
(1972)
Z-+8-2.57
TABLE
III
INFr_UEiYCE OF MALoNATE ON ELONGATION
OF
LABELLED
THE
FATTY
SPECIFIC ACIDS
RADIOACTIVITY
BY
LARVAL
OF
HOMOGENATES
FATTY OF
ACIDS
RESULTIKG
FROM
THE
CUfdZtU
CWUtitis
Distribution of label in fatty acid methyl esters was accomplished by gas-liquid radiochromatography. Specific radioactivities of the labelled fatty acid used as substrate and elongated fatty acids are given (mean values of three individual determinations).
_.__~.
______~ Fatty acids
TAHLE
r-14C-labelled fatty acids I/J:0
I.?:0
10.0 .___. .4 cd& Malonate ~~_~ ~~___
Malonate
Acetate
~~
~____ 16:o
Acetate ___-Malonatc~
_
.4 cetate
~~llalomate
I \-
SPECIFIC
ACTIVITY
ACETATE
AND
SHOWN
BY
I-“C-LABELLED
DIFFERENT FATTY
CLASSES
ACIDS
BY
OF
LIPIDS
LARVAL
AND
AFTER
INCORPORATION
PHARATE
ADULT
OF
HOMOGEXATES
L+‘c]OF
Cevatztis capitata. Labelled total lipids were extracted and fractionated in the lipid classes by thin-layer chromatography as described in Materials and Methods. Control plates were used for determining triglycerides*, phospholipids* * and free fatty acids * * * in both larval and pharate adult preparations. The corresponding levels were employed to calculate the specific activities. ._ ___._~~ ~~ .~~_~~. ~- ~~ Lipid classes Labelled substrates Fvee fatty acids Tviglycerides Phospholipzds _ ____._ .~ _..__ ~~~ Pharate adults Lamar Phavate adults Pharatr adults Larvae Larvae 1o:o
I.-i
0.15
cl..+1
0.13
0.08
TX0
I.3
0.24
0.46
0.22
0.25
I_+:0
1.4
0.20
0.60
043 0.83 x.3
1610
I.2
0.I.j
I.00
Acetate
T.o
0.01
L.j
* According ** According *** According
0.20
5.6 5
-i
5.3
0.26
j.1
0.06
O-4
to W. D. Skidmore (J. Lipid Res., 3 (1962) 356). to G. K. Bartlett (J. Bzol. Chew., 234 (1959) 466). to T. Ko and D. Iioyer (Anal. Biochem., 20 (1967) 205).
of C. ca;hitata. For comparative purposes Table IV gives also the specific activities of the lipid classes after the incorporation of 1r-X]acetate by both homogenates. The fatty acid-incorporating capacity of the larval homogenates appears to be unequally shown in the different classes of lipids, triglycerides exhibiting the highest specific activities. On the other hand, incorporation of ]~-~~C]acetate takes place mainly into phospholipids and at lower levels to triglycerides and free fatty acids. Palmitic acid exhibits the highest similarity in the distribution between triglycerides and phospholipids. Pharate adult homogenates exhibit a completely different pattern of fatty acid specific activity; radioactivity is mainly present as free fatty acids and a significantly lower level of activity is present in triglycerides and phospholipids. By contrast, the major portion of the Y-labelled acetate was incorporated by pharate adult homogenates to phospholipids and insignificant amounts into triglycerides. Several possible interpretations can be given for explaining these differences in the ! 14C] acetate and 13C-labelled fatty acid incorporation exhibited by larval and pharate adult homogenates. Among them, differences in either the level or the activity of long-chain BLochzm. Biophys.
Acta, 280
(1972)
24X-257
FATTY
fatty ATP
ACIDS
AND
INSECT
DEVELOPMENT
25.5
acid-activating enzymes and of fatty acid synthetase. The concentration of in the experiments has been shown to be adequate to achieve the maximal
radioactivity incorporation in both larval and pharate adult homogenates; on the other hand, acetyl-Co4 carboxylase activity exhibits no differences between larval and pharate adult homogenates that could account for the results found by comparison of the metabolic behaviour of these two developmental stages of the insect16. However, differences shown in Table IV are in general agreement with the influence exerted by the presence of added glycerophosphate on the ]~X]acetate incorporation. Inspection of Table V clearly shows the different behaviour of larval and pharate adult homogenates; incorporation of radioactivity increases with glycerophosphate concentration in the presence of larval homogenates, whereas there is no significant change in the total radioactivity incorporation by pharate adult homogenates. For explaining these differences it can be also born in mind that duringmetamorphosis from the larval to the adult stage extensive histolysis and rearrangement of tissues occurs and that the energy required for this transformation is derived from lipids, mainly reserves of triglycerides 17-19. Furthermore, morphological differences between tissues of larvae and pharate adult+ allow one to suggest a different participation of cytosol and subcellular particles in the fatty acid and lipid synthetic path ways. It would seem that in the larval stage the microsomal-soluble fraction exerts a predominant effect, whereas mitochondria would appear to be preferentially involved in the activity observed for the pharate adult stage. At present, the system for the de nova synthesis of fatty acids from acetate has been prepared in soluble form from several animal and plant tissues and microorganisms. On the other hand, as has been already mentioned, the elongation system for the stepwise addition of two-carbon units to the long-chain acyl-CoA is present in particulate fractions; in mitochondria acetyl-CoA4 and palmityl-CoA are the main substrates 21--23,whereas a similar pathway for fatty acid synthesis has been described in microsomes making use of malonylCOA’~~*~~~.In some cases26 mitochondria were by far the most active fraction in the promotion of [Xlacetate incorporation into long-chain fatty acids. The addition of microsomes to the soluble enzyme system, and according to the circumstances, can exert a stimulatory or inhibitory influence on the fatty acid synthesis27-30. X significant amount of the fatty acids synthesised by both mitochondria and microsomes are incorporated into triglycerides, phospholipids and cholesterol ester+. TAB12
V
INFI_UEPI‘CE OF GLYCEROPHOSPHATE ON THE [%]ACETATE LARVALA\‘D PHARATE ADULT HOMOGESATES
INCORPORATION
INTO FATTY
ACIDS BY
Disodium DL-cc-glycerophosphate (0.5-10.0 /cmoles/ml) was added to the incubation mixture as described in ;Clatcrials and Methods. Radioactivity incorporation is given as cpm/mg protein of the larval and pharate adult homogenates.
Glyccvophosphate (pmoles~rtzl)
Lavvae
Pharatc
0
15900
0.j
16 300 17800 21goo
11800 11700 11600 II500
23200 *jOOO
I1550 11500
1.0 3.0 i 0 10.0
adult
Biochim.
Biophys.
Acta,
280 (1972) 2-+8-2.j7
The fact that synthetic cyclopropane fatty acids, which retain many structural characteristics of the original unsaturated acids, are incorporated into adipose tissue lipids-triglycerides-and not into mitochondria and microsomes-mainly phospholipids-raises questions concerning the mechanisms for incorporating fatty acids into both classes of lipids3”. In this connection larval homogenates efficiently use acetate and fatty acids by the microsomal-supernatant system. This agrees with the activation that malonate produces (Table II) on the utilization of acetate for the fatty acid synthesis. The low activity of mitochondria in this stage of development agrees with the finding that the ratio of specific activities phospholipid/triglyceride attained by the larval homogenates is much less than that exhibited by pharate adult homogenates. The results obtained with pharate adult homogenates allow one to suggest major participation of mitochondrial systems; thus, malonate did not significantly modify the incorporation of [ r*C]acetate into fatty acids. On the other hand, this incorporation takes place mainly into phospholipids and at a higher rate than in the larval stage, previously shown in a series of time-course experimentsr. A study of [%]acetate incorporation by mitochondrial preparations from larvae and pharate adults (Table VI) adds some new data in the correlation of metabolic behaviour between larval and pharate adult homogenates. Pharate adult mitochondrial preparations incorporated more acetate than larval mitochondrial preparations; the incorporation by both mitochondrial preparations was not influenced b\r the presence of malonate. The newly synthesised labelled fatty acids from acetate were incorporated by larval mitochondrial preparations into different lipid classes (Table VII) according to a pattern different to that exhibited by the whole larval homogenates. Larval mitochondrial preparations incorporated fatty acids mainly into phospholipids, which, although not surprising, is in a clear contrast with the incorporation achieved by the total larval homogenates.
INCORPORATIOX ATE
AI)ULT
OF TOTAL
RADIOACTIVITY
Pvcpavation
Luruac
Total
1510-l 4.5450 I376 1229
homogenate
Plus malonate hlitochondrial pzus rnalollate T;\BLI’
TOTAL
LIPIDS
ASI>
MITOCHOKDRIAL
(cpm)
Pharute
FROM
[+'C]KETATE
BY
LARVALASD
PHAR-
PREPARATIOXS
adults (c-pm)
15558 1514.5 SSI8 5 700
\-II
DISTRIBUTION LIPIDSFROM
Lipid
INTO
HOMOGENATES
OF
RADIOACTIVITY
class
Triglycerides Phospholipids Free fatty acids
Biochim.
(PERCEKTAGES)
IPiCORPORATED
ISTO
[I-~~C]ACETATEBYLARVALMITO~HOIVDRIAAP;DWHOLEKOMOGE~ATEPREPARATIO~S
Biophys.
Acta,
Mitochondrial preparation
Whole homogenate
3.1 82.6
63.2
8.5
I.5 -______
280 (1972)
33.0
248-2j7
1)IFFEREXT
CLASSES
OF
FATTY
ACIDS ASD
INSECT DEPLOYMENT
257
ACKNOWLEDGMENT
This work was supported de la Investigation Cientifica.
by a grant from the Fonda National
para el Desarrollo
REFERENCES T *4-\. 31. Municio, J. M. Odriozola, A. Piiieiro and A. Ribera, Insect Biochem., 2 (1972). 2 A. M. Municio, J. M. Odriozola, A. Piiieiro and A. Ribera, Riochim Biophys. Acta, z+Y (1971)
212. 3 J. M. Fernandez Sousa, A. M. Municio and A. Ribera, Biochim. Biophys. Acta, 231 (1971) 527. 4 J. M. Fernandez Sousa, A. M. Municio a.nd A. Ribera, Bzochim. Bioph,ys. Acta, 248 (1971) 226. 5 A. M. ?ilunicio, J. M. Odriozola and A. Piiieiro, Cow@. Biochem.. Ph~~sioE., 37 (1970) 387. 6 1’. R. Vagelos, Annu. Rev. ~~ac~em., 3.3 (‘1964) 139. 7 D. H. Nugteren, Biochim. Biophys. Acta, 106 (1965) 280. 8 E. J. Christ, Biochim. Biophys. Acta, 152 (1~68) 50. g H. Schiller and Ii. Bensch, I. Lipid Bes., 12 (1971) 248. 10 IT J. Christ and W. C. Hiilsmann, Biochim. Riophys. Acta, 60 (1962) 72. II XV. C. Hiilsmann, Riochim. Biophys. Acta, 58 (1962) 417. ra W. C. Htilsmann and D. C. Dow, Biochim. Biophys. 4&z, 8-1 (1964) 486. 13 W. Stoffel, Hoppe-Seyler’s Z. Physiol. Chem., 333 (1963) 71. x4 W. Stoffel and K. I-. Ach, Hoppe-Seyler’s Z. Play&Z. Chem., 337 (1964) 123. 15 F. Lynen, M. Matsuhashi, S. Numa and E. Schweizer, in J. K. Grant, The cot&& of lipid mlptabolism, Academic Press, New York, 1963, p. 43. 16 A. RI. >lunicio, J. M. Odriozola and J. A. Ramos, Insect B&hem., z (1972). 17 J. L. Villeneuve and A. Lemonde, Arch. Int. Physinl. Biochem., 71 (1963) 141. IX G. H. Schmidt, J. Ins~d Physiol., 12 (1966) 237. rg P. G. Fast, in Progress in the Chemistry of Fats and Other Lipids, Vol. XI, Part 2, Pergamon Press, 1970, p. 181. 20 M. Ltischer, in J. W. L. Beamont and J. E. Treherne, Insects and Physiology, Oliver and Boyd, London, 1967, p. 39. 21 S. J. %Vakil, L. W. _&Lain and J. B. Warshaw, f. B&E. Chem., 235 (1960) PC 31. 22 S. 1. Wakil. I. LiPid Res.. z (146x1 I. 23 WYK. Harlak and S. J. wakii, >. hiol. Chem., 238 (1~63) 3216. rq S. Abraham, E. Larch and I. L. Chaikoff, R&hem. Biophys. Res. Commun., 7 (1962) IQO. 2.5 E. Larch, S. hbraham and I. L. Chaikoff, B&him. Riophys. 4&z, 70 (1963) 627. 26 E. G. Trombopoulos, Sciencr, 146 (1964) 1180. 27 S. Abraham, I. L. Chaikoff, W. M. Bortz, H. P. Klein and H. Den, Nature, 192 (1961) 1287. r8 E. J. Jfasoro, H. M. Korchak and E. Porter, B&him. Biophys. Acta, 58 (1~62) 407. 29 S. A%braham, K. J. Matthes and I. L. ChaikotI, B&him. Biophys. Acta, 70 (1963) 357. 30 D. \V. Foster and W. P. McWhorter, ,f, Biol. C&m., 244 (1969) 260. 31 E. Aeberhard and J. H. Jlenkes, J. Biol. Chem., 243 (1968) 3834. 32 RI. Guarnieri and R. M. Johnson, Adv. Lipid Reg., 8 (1970) 137. Biochim.
Bi~phys.
ilcta,
280 (1972) 248-257
248 BBA
ET BIOPHYSICA
ACTA
j6rog
IN VITRO
ELONGATION
DEVELOPMENT
AND DESATURATION
OF FATTY
ACIDS
DURING
OF INSECTS
I. Ix vitro elongation and desaturation reactions of fatty acids using labelled ro:o, 12: o, 14:o and 16: o have been investigated with homogenates of larvae and pharate adults of Cerutitis ca$itata. The obtained results show clear differences in the behaviour of larval and pharate adult stages of development. 2. Larval homogenates desaturate and elongate the labelled substrates arcording 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 are insignificant. 3, Labelled acetate incorporation into fatty acids by the larval homogenates is notably stimulated in the presence of malonate, whereas the fatty acid synthesis b! pharate adult homogenates is practically unaffected. The fatt). acids IO : o and 12 : o are elongated by larval homogenates more effectively in the presence of acetate than that
of malonate. 4. The larval homogenate incorporates considerably more labelled fatty acids into triglycerides than into phospholipids, whereas the radioactivity remains mainly as free fatty acids in the presence of pharate adult homogenates. 5. Pharate adult mitochondria preparations incorporate more acetate than larval mitochondria preparations. Incorporation by larval mitochondria was not in fluenced by the presence of malonate and the resulting fatt), acids are present mainl! as phospholipids, in a clear contrast with the incorporation achieved by the whole larval homogenates.
INTRODLCTIOK
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. Experimentsinvolving the fate of rr*C]acetate and incorporation of other labelled fatty acids by homogenates of different stages of devel-
FATTY
ACIDS
AND
INSECT
249
DEVELOPMENT
opment of the insect Ceratitis capitata have been previously
undertaken.
It was found
that the distribution patterns of [14C]acetate incorporation among free fatty acids, triglycerides and phospholipids were different in the larval and pharate adult stages of development. The time course of [lK]acetate incorporation showed some differences in the behaviour of larval and pharate adult homogenates; triglycerides and phospholipids become labelled in a different way by either larval or pharate adult homogenates’. The time course of [l*C]acetate incorporation also showed clear differences in the behaviour of larval and pharate adult homogenates regarding the specific activities of the fatty acids synthesised by them. Specific radioactivities of fatty acids synthesised by larval homogenates exhibited a net distinction between medium-chain and long-chain fatty acids; this distinction was not exhibited in the [Klacetate incorporation by pharate adult homogenates’. This different behaviour of these two developmental stages of C. capitata also applied to the specific radioactivity of the individual fatty acids present in the lipid classes2. Decanoic acid had the highest specific radioactivity of the fatty acids present in triglycerides from larval homogenates; the fatty acids of triglycerides from pharate adults, on the other hand, did not exhibit any incorporation of radioactivityz. In addition, Lr-Kjpalmitic and Lr-%]stearic acids were desaturated by larval homogenates, but not by pharate adult homogenates”. It was the intention of the present study to attempt to compare the desaturation and elongation of different fatty acids by either larval or pharate adult homogenates of C. capitata as well as the incorporation of these fatty acids into the most abundant classes of lipids. MATERIALS
AND
METHODS
!Vaterials ATP, CoA and NADPH were purchased from Sigma Chemical Co., St. Louis, MO. [I-W]Acetate and I-l*C-labelled fatty acids (IO:O to 16:o) were obtained from The Radiochemical Centre, Amersham, Bucks. Rearing of insects C. capitata (Wiedemann) was used during the larval and pharate adult stages of development and the diet, temperature and humidity conditions of culturing were carefully controlled, as reported previously2. Larvae were reared 2-3 days before the larval-pupal apolysis and were starved 2-3 h before homogenization. Pharate adults were 5 days old from the larval-pupal apolysis. Both larvae and pharate adults were carefully washed with distilled water before use. Preparation of homogenates Larvae and pharate adults were directly homogenized with 3 vol. of cold homogenizing buffer (0.35 M sucrose-0.05 M Tris, pH 7.4) in a glass homogenizer fitted with a Teflon pestle. The total homogenate was filtered through two layers of cheese-cloth and the filtrate was centrifuged at 900 x g for IO min; to free the 900 x g supernatant from loosely bound chitinous particles centrifugation was carried out twice at 1200 x g Biochim.
Biophys.
Acta, Z?O (1972)
248-257
for IO min. The 1200 xg supernatant was used as total homogenate and as the source of the mitochondrial fraction. Mitochondria were removed from the 12ooxg supernatant by centrifugation at 12000 x g for IO min; the pellet was resuspended in the sucrose-Tris medium and recentrifuged for IO min at 12 ooo XS. Purity of the mitochondria fraction was assessed by electron microscopy. All operations were carried out at o--4 “C. Incubation mixture The assay mixture for incorporating [I-Xjacetate or 1-14C-labelled fatty acids contained ATP (6 ymoles/ml), CoA (2 pmoles/ml), NADPH (I pmole/ml) and l&Cl, (z pmoles/ml) in 0.05 M potassium phosphate buffer, pH 7.4 (22 ~moleslml), z pmoles, ml of either sodium acetate or sodium malonate, and 0.5 ml of total homogenate or mitochondrial preparation. ;r-14Cj_4cetate (spec. act. 57 mCi/mmole) was present in the incubation mixture at 60 &i/ml. Labelled fatty acids (spec. act. 16.4 mCi/mmole for 10: 0, 21.0 mCi/mmole for 12 :o, 15,4 mCi/mmole for 14:0, 48.0 mCi/mmole for 16 : o) were suspended in potassium phosphate buffer and the mixture was sonicated for z min prior to the addition to the incubation mixture at 4 &i/ml (0.20-0.24 pmole/ml). Incubations were made in a shaker at 37 “C for 180 min according to the Final volume 2 ml containing 10 mg/ml of time-course results previously obtainedl,l. proteins (total homogenate) or 2.5 mg/ml (mitochondrial preparations). into ilcetyl-CoA carboxylase was assayed by WO, fixation from NaHXO, acid-stable material. Preincubation of the reaction components was not carried out since previous experiments showed no influence on the radioactivity incorporation. Incubation mixture contained: 22.5 pmoles/ml potassium phosphate buffer, pH 7.0; 3 pmoles/ml sodium citrate; z pmoles/ml M&l,; 2.5 pmoles/ml mercaptoethanol; IO pmoles/ml potassium bicarbonate; 0.5 pmole/ml acetyl-CoA, (or 0.5 pmole/ml Co4 filus 3 pmoles/ml acetate) and 6 pmoles/ml ATE’; enzyme preparation, IO mg protein/ml. Final volume 0.5 ml. The reaction was started by the addition of I &i/ml NaHKO,. Incubations were in duplicate and incubations without enzyme preparations were always included as controls. The reaction was stopped with 0.1 ml of 6 nl HCl at 30 min and the tubes agitated and dried under vacuum; the residue was assayed for radioactivity by liquid scintillation counting. Extraction and separation of lieids At the end of the incubation time the reaction was stopped by the addition of 5 ml of chloroform and the mixture was made into a single phase by the addition of methanol. The crude lipid extract was purified as described2. Purified lipids were fractionated into lipid classes by thin-layer chromatography on silica gel G by the method described2. Thin-layer bands were scraped off the plate and either directly transferred to scintillation vials for counting or submitted to the methanolysis procedure. Fatt_y 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 trifluoridemethanol reagent as described”.
BLochim. Bi@hys.
Ada,
280 (1972) 2$-2jj
FATTY
ACIDS AKD INSECT DEVELOPMENT
Determination
of incorporation
251
of radioactivity
into lipid
classes
and individual
fatty
acids Lipids were located by a slight spray with Rhodamine B solution and the areas corresponding to each lipid were scraped from the plate and placed into counting vials with 15 ml of the scintillation mixture (4 g PPO and 0.1 g POPOP diluted to I 1 with toluene). Chromatographic separation of methyl esters and determination of radioactivity was carried out using a Perkin-Elmer Model RGC 170 flow-through reactor/ proportional counter connected to a gas-liquid chromatography unit (Perkin-Elmer F20FE flame-ionisation instrument). Details of the experimental procedure were previously describeds. Specific activities are conveniently expressed as counts per unit area under each mass peak. RESITLTS AND DISCUSSION LIesaturation and elongation of labelled IO: o, 12: o, 14: o and I 6: ofatty acids Figs. I and 2 represent the behaviour of larval and pharate adult homogenates, respectively, in a series of desaturation and elongation experiments using labelled IO: o, 12 : o, 14: o and 16 : o fatty acids as substrates. Incubation time was ISO min.
Fig. I. Elongation and desaturation of fatty acids by larval homogenates of Ceratitis cupitata. r-14C-labelled fatty acids used as substrates were IO : o (a), 12: o (b), 14: o (c) and 16 : o (d). Synthcsised fatty acids are expressed as percentages of the fatty acids used as substrates. Black bars, substrates. Stripped bars, desaturation products. Dotted bars, elongation products.
a
.:.:.::,: ..:=:.,:i “:::::
b
..:. c
cl
11111111
llll .... Ill1 :&
1111111111111 14:1 II:0
li: 12:1
~,~*$$*fg
/ 1z:n
1o:, ItO 100
50
I
0.5
relative abundant 50'
I05
50
1
0.5
i 50 T
Fig. 2. Elongation and desaturation of fatty acids by pharate adult homogenates of Cevatitis r-r4C-labelled fatty acids used as substrates were ro:o (a), 12: o (b), r.+: o (c) and 16: o (d). Synthesised fatty acids are expressed as percentages of the fatty acids used as substrates. Black bars, substrates. Striped bars, desaturation products. Dotted bars, elongation products. capztata.
Biochim.
Biophys.
Acta,
280 (1972) 248-257
252
A.
M.
ivI’SICIC)
zt al.
taking into consideration the previous time-course experiment+*, and that at this time the total radioactivity incorporation achieved by larval and pharate adult homogenates exhibited the most similar values’. Larval homogenates (Fig. I) show considerable differences in the desaturation and elongation products according to the chain length of the substrates. It should be noted that the longer the length of the fatty acids used as substrates the more pronounced the synthesis of the corresponding monounsaturated fatty acids, as well as the smaller activity of the elongation pathway. Thus, in the conditions of our experiments, larval homogenates do not elongate 16:o fatty acid, whereas the relative abundance of palmitoleic acid reaches values close to 50%; on the other hand, the low levels exhibited by IO : I as a consequence of desaturation of IO: o can be seen. Pig. 2 shows the results of a typical experiment in which the relative abundance of desaturation and elongation products formed by pharate adult homogenates from the same labelled fatty acids are compared as before. Inspection of these results clearly shows that, when present, the radioactivity levels are generally very small; thus, 14: o and 16:o do not exhibit any elongation and IO: o and IZ:O do not form their direct desaturation products. However, since under identical experimental conditions larval homogenates were shown to be much more active than the pharate adult homogenates, it seems reasonable to conclude that direct desaturation and elongation of fatty acids are not a major metabolic route in the pharate adult stage of development. This fact could be related with the invariability of the molar unsaturation degree observed during pharate adult development 3,3 in choline and ethanolamine phosphoglycerides, while a tendency to increase the unsaturation degree was found during larval and adult development. These findings reflect the existence of a different metabolic behaviour between these two developmental stages of the insect which had been previously establisheds. Bffectively, NADPH enhanced the incorporation of labelled acetate by larval and pharate adult homogenates in a different way; also [lK]acetate incorporation by larval and pharate adult homogenates in the presence of ATP-Mg was enhanced or decreased by citrate, respectively”. Incorporation of acetate @US CoA into malonyl-Co,4 through a YO,-carboxylation reaction by larval and pharate adult homogenates has been correlated with that of acetyl-CoA in order to prove the acetate activation in both systems. Results given in Table I show that acetate can be used as substrate for the synthesis of fatty acids in either larval or pharate adult homogenates. Influence of ATP concentrations on the incorporation of r%]acetate into long-
14COl~NCORPORATIONBY~AR”AI.ANDPHARATE ADULTHOMOGE~ATESI~THEPRESBPI'CEOPEITNER ACETATE I
PLUS
CO/l
,uCi NaH%O,
OR
ACETYL-Cd
was added to the reaction
Results are given in cpm/mg ATP
/v&i-l
mixture
as described
Larvae
-
Pharatr
adults
Acetate + CoA
Acetyl-CoA
Acetate
0
2600
2300
2800
.+200
2
2850 3270
2660 2830
25.50 2150
3125 2450 _____
Biochim.
Bi+hys.
A&,
280
in hlaterials
protein.
(1972)
248-2j7
t CoA
Acrtyl-CoA
.~
and Methods.
FATTY
ACIDS
AND
INSECT
DEVELOPMENT
253
chain fatty acids by larval and pharate adult homogenates was previously established5; maximal levels of incorporation were obtained at 6-12 mM ATP and this concentration has been used in this paper. In the past years the non-mitochondrial, the mitochondrial and the microsomal fatty acid-synthesising systems have been intensively studied in many tissues and organisms6p9 and it was found that elongation with C, units is one of the major reactions regardless of the chain length of the fatty acid used as substrate. The supernatants synthesise long-chain fatty acids by the repeated additions of malonyl-CoA on an initial acetyl-Co,%. The malonyl-CoA is synthesised from acetyl-CoA with acetylCoA carboxylase, in what has been shown to be generally the rate-limiting step for de GOZ’Ofatty acid synthesis. The mitochondrial system is involved in an elongation pathway in which acetate is added onto a previously formed fatty acid. Microsomes have been shown to synthesise fatty acids by elongation involving malonyl-CoAln-la. Malonate has been described to stimulate the acetyl-CoA carboxylationlS and the influence of malonate on [I-Xjacetate incorporation into fatty acids by larval and phara-te adult homogenates is shown in Table II. These results clearly show that the labelled acetate incorporation into fatty acids is notably stimulated by malonate in the presence of larval homogenates, whereas radioactivity incorporation is unaffected in the presence of pharate adult homogenates. These findings reflect again the different metabolic behaviour between these developmental stages. The clear stimulation of the specific activity of the fatty acids produced by malonate in the presence of larval homogenates (Table II) might be due to its influence on the de nova fatty acid synthesis or on the elongation system. However, Table III shows that malonate does not enhance the elongation of fatty acids by larval homogenates; effectively, IO:O and IZ:O fatty acids are elongated more efficiently in the presence of acetate than is the case with malonate, whereas elongation of 14: o is similarly stimulated by both factors. Elongation of 16 : o does not cocur. Incorporation of labelled ~o:o, 12:0, 14:o and 16:o into different lifiid classes Table IV shows the specific activities of the most abundant classes of lipids after incorporation of labelled fatty acids by larval and pharate adult homogenates TABLE
II
ISFLUEXcE
OF
MALONATE
j+C]ACET_4TE
ON
THE
INCORPORATION
BY
SPECIFIC LARVAL
RADIOACTIVITY
OF
AKD
ADULT
PHARATE
FATTY
ACIDS
RESULTING
HOMOGEXATES
OF
FROM
C‘VatitiS
capitata Distribution of label in fatty acid methyl esters was accomplished by gas-liquid radiochromatography. Specific radioactivities are given as the ratio of cpm. to the area under the mass peak (mean values of three individual determinations).
Fatty acids
Specific radioactivzty LWUCZf _____
A cetatc 1010
8.j
12:0
IO.3
1410
4.0
14:1 16~0 16:1 18:o
7.1 0.7
18:r
0.3
Phavate aldults Malon&?
‘4cetate
Malonatc
22.8
29.1
2.4
2.j
14.2
3.1
3.3
22.6 3.5
0.8
4.8
0.1
0.7 I.5
9.0
IO.7
I.1 1.3
1.2 I.3
0.1
0.1
B&him.
Biophys.
A&,
280
(1972)
Z-+8-2.57
TABLE
III
INFr_UEiYCE OF MALoNATE ON ELONGATION
OF
LABELLED
THE
FATTY
SPECIFIC ACIDS
RADIOACTIVITY
BY
LARVAL
OF
HOMOGENATES
FATTY OF
ACIDS
RESULTIKG
FROM
THE
CUfdZtU
CWUtitis
Distribution of label in fatty acid methyl esters was accomplished by gas-liquid radiochromatography. Specific radioactivities of the labelled fatty acid used as substrate and elongated fatty acids are given (mean values of three individual determinations).
_.__~.
______~ Fatty acids
TAHLE
r-14C-labelled fatty acids I/J:0
I.?:0
10.0 .___. .4 cd& Malonate ~~_~ ~~___
Malonate
Acetate
~~
~____ 16:o
Acetate ___-Malonatc~
_
.4 cetate
~~llalomate
I \-
SPECIFIC
ACTIVITY
ACETATE
AND
SHOWN
BY
I-“C-LABELLED
DIFFERENT FATTY
CLASSES
ACIDS
BY
OF
LIPIDS
LARVAL
AND
AFTER
INCORPORATION
PHARATE
ADULT
OF
HOMOGEXATES
L+‘c]OF
Cevatztis capitata. Labelled total lipids were extracted and fractionated in the lipid classes by thin-layer chromatography as described in Materials and Methods. Control plates were used for determining triglycerides*, phospholipids* * and free fatty acids * * * in both larval and pharate adult preparations. The corresponding levels were employed to calculate the specific activities. ._ ___._~~ ~~ .~~_~~. ~- ~~ Lipid classes Labelled substrates Fvee fatty acids Tviglycerides Phospholipzds _ ____._ .~ _..__ ~~~ Pharate adults Lamar Phavate adults Pharatr adults Larvae Larvae 1o:o
I.-i
0.15
cl..+1
0.13
0.08
TX0
I.3
0.24
0.46
0.22
0.25
I_+:0
1.4
0.20
0.60
043 0.83 x.3
1610
I.2
0.I.j
I.00
Acetate
T.o
0.01
L.j
* According ** According *** According
0.20
5.6 5
-i
5.3
0.26
j.1
0.06
O-4
to W. D. Skidmore (J. Lipid Res., 3 (1962) 356). to G. K. Bartlett (J. Bzol. Chew., 234 (1959) 466). to T. Ko and D. Iioyer (Anal. Biochem., 20 (1967) 205).
of C. ca;hitata. For comparative purposes Table IV gives also the specific activities of the lipid classes after the incorporation of 1r-X]acetate by both homogenates. The fatty acid-incorporating capacity of the larval homogenates appears to be unequally shown in the different classes of lipids, triglycerides exhibiting the highest specific activities. On the other hand, incorporation of ]~-~~C]acetate takes place mainly into phospholipids and at lower levels to triglycerides and free fatty acids. Palmitic acid exhibits the highest similarity in the distribution between triglycerides and phospholipids. Pharate adult homogenates exhibit a completely different pattern of fatty acid specific activity; radioactivity is mainly present as free fatty acids and a significantly lower level of activity is present in triglycerides and phospholipids. By contrast, the major portion of the Y-labelled acetate was incorporated by pharate adult homogenates to phospholipids and insignificant amounts into triglycerides. Several possible interpretations can be given for explaining these differences in the ! 14C] acetate and 13C-labelled fatty acid incorporation exhibited by larval and pharate adult homogenates. Among them, differences in either the level or the activity of long-chain BLochzm. Biophys.
Acta, 280
(1972)
24X-257
FATTY
fatty ATP
ACIDS
AND
INSECT
DEVELOPMENT
25.5
acid-activating enzymes and of fatty acid synthetase. The concentration of in the experiments has been shown to be adequate to achieve the maximal
radioactivity incorporation in both larval and pharate adult homogenates; on the other hand, acetyl-Co4 carboxylase activity exhibits no differences between larval and pharate adult homogenates that could account for the results found by comparison of the metabolic behaviour of these two developmental stages of the insect16. However, differences shown in Table IV are in general agreement with the influence exerted by the presence of added glycerophosphate on the ]~X]acetate incorporation. Inspection of Table V clearly shows the different behaviour of larval and pharate adult homogenates; incorporation of radioactivity increases with glycerophosphate concentration in the presence of larval homogenates, whereas there is no significant change in the total radioactivity incorporation by pharate adult homogenates. For explaining these differences it can be also born in mind that duringmetamorphosis from the larval to the adult stage extensive histolysis and rearrangement of tissues occurs and that the energy required for this transformation is derived from lipids, mainly reserves of triglycerides 17-19. Furthermore, morphological differences between tissues of larvae and pharate adult+ allow one to suggest a different participation of cytosol and subcellular particles in the fatty acid and lipid synthetic path ways. It would seem that in the larval stage the microsomal-soluble fraction exerts a predominant effect, whereas mitochondria would appear to be preferentially involved in the activity observed for the pharate adult stage. At present, the system for the de nova synthesis of fatty acids from acetate has been prepared in soluble form from several animal and plant tissues and microorganisms. On the other hand, as has been already mentioned, the elongation system for the stepwise addition of two-carbon units to the long-chain acyl-CoA is present in particulate fractions; in mitochondria acetyl-CoA4 and palmityl-CoA are the main substrates 21--23,whereas a similar pathway for fatty acid synthesis has been described in microsomes making use of malonylCOA’~~*~~~.In some cases26 mitochondria were by far the most active fraction in the promotion of [Xlacetate incorporation into long-chain fatty acids. The addition of microsomes to the soluble enzyme system, and according to the circumstances, can exert a stimulatory or inhibitory influence on the fatty acid synthesis27-30. X significant amount of the fatty acids synthesised by both mitochondria and microsomes are incorporated into triglycerides, phospholipids and cholesterol ester+. TAB12
V
INFI_UEPI‘CE OF GLYCEROPHOSPHATE ON THE [%]ACETATE LARVALA\‘D PHARATE ADULT HOMOGESATES
INCORPORATION
INTO FATTY
ACIDS BY
Disodium DL-cc-glycerophosphate (0.5-10.0 /cmoles/ml) was added to the incubation mixture as described in ;Clatcrials and Methods. Radioactivity incorporation is given as cpm/mg protein of the larval and pharate adult homogenates.
Glyccvophosphate (pmoles~rtzl)
Lavvae
Pharatc
0
15900
0.j
16 300 17800 21goo
11800 11700 11600 II500
23200 *jOOO
I1550 11500
1.0 3.0 i 0 10.0
adult
Biochim.
Biophys.
Acta,
280 (1972) 2-+8-2.j7
The fact that synthetic cyclopropane fatty acids, which retain many structural characteristics of the original unsaturated acids, are incorporated into adipose tissue lipids-triglycerides-and not into mitochondria and microsomes-mainly phospholipids-raises questions concerning the mechanisms for incorporating fatty acids into both classes of lipids3”. In this connection larval homogenates efficiently use acetate and fatty acids by the microsomal-supernatant system. This agrees with the activation that malonate produces (Table II) on the utilization of acetate for the fatty acid synthesis. The low activity of mitochondria in this stage of development agrees with the finding that the ratio of specific activities phospholipid/triglyceride attained by the larval homogenates is much less than that exhibited by pharate adult homogenates. The results obtained with pharate adult homogenates allow one to suggest major participation of mitochondrial systems; thus, malonate did not significantly modify the incorporation of [ r*C]acetate into fatty acids. On the other hand, this incorporation takes place mainly into phospholipids and at a higher rate than in the larval stage, previously shown in a series of time-course experimentsr. A study of [%]acetate incorporation by mitochondrial preparations from larvae and pharate adults (Table VI) adds some new data in the correlation of metabolic behaviour between larval and pharate adult homogenates. Pharate adult mitochondrial preparations incorporated more acetate than larval mitochondrial preparations; the incorporation by both mitochondrial preparations was not influenced b\r the presence of malonate. The newly synthesised labelled fatty acids from acetate were incorporated by larval mitochondrial preparations into different lipid classes (Table VII) according to a pattern different to that exhibited by the whole larval homogenates. Larval mitochondrial preparations incorporated fatty acids mainly into phospholipids, which, although not surprising, is in a clear contrast with the incorporation achieved by the total larval homogenates.
INCORPORATIOX ATE
AI)ULT
OF TOTAL
RADIOACTIVITY
Pvcpavation
Luruac
Total
1510-l 4.5450 I376 1229
homogenate
Plus malonate hlitochondrial pzus rnalollate T;\BLI’
TOTAL
LIPIDS
ASI>
MITOCHOKDRIAL
(cpm)
Pharute
FROM
[+'C]KETATE
BY
LARVALASD
PHAR-
PREPARATIOXS
adults (c-pm)
15558 1514.5 SSI8 5 700
\-II
DISTRIBUTION LIPIDSFROM
Lipid
INTO
HOMOGENATES
OF
RADIOACTIVITY
class
Triglycerides Phospholipids Free fatty acids
Biochim.
(PERCEKTAGES)
IPiCORPORATED
ISTO
[I-~~C]ACETATEBYLARVALMITO~HOIVDRIAAP;DWHOLEKOMOGE~ATEPREPARATIO~S
Biophys.
Acta,
Mitochondrial preparation
Whole homogenate
3.1 82.6
63.2
8.5
I.5 -______
280 (1972)
33.0
248-2j7
1)IFFEREXT
CLASSES
OF
FATTY
ACIDS ASD
INSECT DEPLOYMENT
257
ACKNOWLEDGMENT
This work was supported de la Investigation Cientifica.
by a grant from the Fonda National
para el Desarrollo
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212. 3 J. M. Fernandez Sousa, A. M. Municio and A. Ribera, Biochim. Biophys. Acta, 231 (1971) 527. 4 J. M. Fernandez Sousa, A. M. Municio a.nd A. Ribera, Bzochim. Bioph,ys. Acta, 248 (1971) 226. 5 A. M. ?ilunicio, J. M. Odriozola and A. Piiieiro, Cow@. Biochem.. Ph~~sioE., 37 (1970) 387. 6 1’. R. Vagelos, Annu. Rev. ~~ac~em., 3.3 (‘1964) 139. 7 D. H. Nugteren, Biochim. Biophys. Acta, 106 (1965) 280. 8 E. J. Christ, Biochim. Biophys. Acta, 152 (1~68) 50. g H. Schiller and Ii. Bensch, I. Lipid Bes., 12 (1971) 248. 10 IT J. Christ and W. C. Hiilsmann, Biochim. Riophys. Acta, 60 (1962) 72. II XV. C. Hiilsmann, Riochim. Biophys. Acta, 58 (1962) 417. ra W. C. Htilsmann and D. C. Dow, Biochim. Biophys. 4&z, 8-1 (1964) 486. 13 W. Stoffel, Hoppe-Seyler’s Z. Physiol. Chem., 333 (1963) 71. x4 W. Stoffel and K. I-. Ach, Hoppe-Seyler’s Z. Play&Z. Chem., 337 (1964) 123. 15 F. Lynen, M. Matsuhashi, S. Numa and E. Schweizer, in J. K. Grant, The cot&& of lipid mlptabolism, Academic Press, New York, 1963, p. 43. 16 A. RI. >lunicio, J. M. Odriozola and J. A. Ramos, Insect B&hem., z (1972). 17 J. L. Villeneuve and A. Lemonde, Arch. Int. Physinl. Biochem., 71 (1963) 141. IX G. H. Schmidt, J. Ins~d Physiol., 12 (1966) 237. rg P. G. Fast, in Progress in the Chemistry of Fats and Other Lipids, Vol. XI, Part 2, Pergamon Press, 1970, p. 181. 20 M. Ltischer, in J. W. L. Beamont and J. E. Treherne, Insects and Physiology, Oliver and Boyd, London, 1967, p. 39. 21 S. J. %Vakil, L. W. _&Lain and J. B. Warshaw, f. B&E. Chem., 235 (1960) PC 31. 22 S. 1. Wakil. I. LiPid Res.. z (146x1 I. 23 WYK. Harlak and S. J. wakii, >. hiol. Chem., 238 (1~63) 3216. rq S. Abraham, E. Larch and I. L. Chaikoff, R&hem. Biophys. Res. Commun., 7 (1962) IQO. 2.5 E. Larch, S. hbraham and I. L. Chaikoff, B&him. Riophys. 4&z, 70 (1963) 627. 26 E. G. Trombopoulos, Sciencr, 146 (1964) 1180. 27 S. Abraham, I. L. Chaikoff, W. M. Bortz, H. P. Klein and H. Den, Nature, 192 (1961) 1287. r8 E. J. Jfasoro, H. M. Korchak and E. Porter, B&him. Biophys. Acta, 58 (1~62) 407. 29 S. A%braham, K. J. Matthes and I. L. ChaikotI, B&him. Biophys. Acta, 70 (1963) 357. 30 D. \V. Foster and W. P. McWhorter, ,f, Biol. C&m., 244 (1969) 260. 31 E. Aeberhard and J. H. Jlenkes, J. Biol. Chem., 243 (1968) 3834. 32 RI. Guarnieri and R. M. Johnson, Adv. Lipid Reg., 8 (1970) 137. Biochim.
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ilcta,
280 (1972) 248-257