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Effect of cholesterol feeding and estrogen treatment on synthesis of fatty acids in liver
407
Atherosclerosis, 27 (1977) 407-417 @ Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECT OF CHOLESTEROL FEEDING AND ESTROGEN TREATMENT ON SYNTHESIS OF FATTY ACIDS IN LIVER
K. SRINIVASAN and THOMAS I. PYNADATH Department
of Chemistry,
Kent State University,
Kent, Ohio 44242
(U.S.A.)
(Received 26 October, 1976) (Revised, received 7 March, 1977) (Accepted 7 March, 1977)
Summary
The effect of cholesterol feeding and estrogen administration on synthesis of fatty acids in liver mitochondria, microsomes and cytoplasm of male rabbits has been investigated. The synthesis was measured by the incorporation of [1-14C] acetyl CoA or [2-14C]malonyl CoA into long chain fatty acids under optimal conditions. It was found that atherogenesis markedly decreased the fatty acid synthesis in cytoplasm. The mitochondrial fatty acid synthesis was not affected by the disease. There was a small but measurable decrease in the synthesis of fatty acids in microsomes. Estrogen had no effect on the synthesis of fatty acids in mitochondria or microsomes. But if effectively counteracted, after a short lag period, the decreased synthesis of cytoplasmic fatty acids observed in atherosclerosis. It is possible that liver fatty acid synthetase is one of the enzyme systems through which estrogens exert their atherosclerosis-retarding effect. The decreased cytoplasmic fatty acid synthesis observed in atherosclerosis might account for the low levels of saturated fatty acids reported in liver and plasma lipids of atherosclerotic animals. Key words:
Effect of cholesterol -Rabbit
feeding
- Effect
of estrogen
- Liver fatty acid synthesis
Introduction
Atherosclerosis and composition
has been shown to have a significant effect on the content of tissue lipids [l-5]. It has been found that atherosclerosis
This investigation was supported by a Research Grant from the Akron District Chapter of American Heart Association, Akron. Ohio.
markedly alters the fatty acid composition of many tissue lipids, especially the plasma and liver lipids. Studies have shown that changes in fatty acid esterifying activities of the tissues could partly account for this alteration [ 5-71. It is possible that a change in the fatty acid synthesizing activities of the tissues might be a more important contributing factor for this alteration. Whereat [8] has studied the changes in fatty acid synthesis in atherosclerotic rabbit aorta. The results showed that in atherosclerotic aorta there was a 4-fold increase in the incorporation of acetate into long chain fatty acids. However, when acyl CoA compounds were used as substrates, under optimal conditions, no increase in synthesis was observed [9]. The most drastic changes in fatty acid composition, in atherosclerosis, occur in liver and plasma lipids. Hence it is conceivable that the fatty acid synthesizing systems most affected by atherosclerosis are the systems in liver. The present investigation is a study to determine the changes in fatty acid synthesis in mitochondria, microsomes and cytoplasm of rabbit liver during development of atherosclerosis. Optimal conditions for fatty acid synthesis were first established for each enzyme system and these conditions were used for all subsequent studies. Since estrogens have long been known to retard development of atherosclerosis in human [lo] and in animals [11,12], the effect of estrogen treatment on liver fatty acid synthesis of cholesterol-fed rabbits has also been investigated. Materials and methods Chemicals
Acetyl CoA and malonyl CoA were purchased from P-L Biochemicals. NADH, NADPH and ATP were from Sigma Chemical Co. [2-14C]malonyl CoA was purchased from New England Nuclear Corp. and [l-14C]acetyl CoA was obtained from International Chemical and Nuclear Corporation. Experimental
animals
Eighty New Zealand White male rabbits were divided into three groups. Group I containing 32 rabbits, fed Purina Lab Rabbit Chow, served as the control group. Group II, with 24 rabbits, was fed Purina Chow to which 1% (w/w) cholesterol was added. Group III rabbits, 24 in number, also were fed the 1% cholesterol diet, and in addition were given subcutaneous injections of 1.4 mg estradiol benzoate in 0.4 ml sesame oil on alternate days. Group I and II rabbits received placebo injections of 0.4 ml sesame oil on the same days on which Group III received estrogen injections. The rabbits were fed and watered ad libitum
.
Eight animals from Group I were sacrificed at the commencement of the project and this served as the zero week control. Thereafter, from each group eight animals were sacrificed at the end of three, 6- and g-week periods. All the animals were killed by air embolism. The physiological effect of the estrogen administered was determined by the atrophy of the testes.
409
Isolation
of su bcellular
fractions
The liver, removed immediately after the sacrifice, was washed with an icecold solution of 0.28 M sucrose containing 5 X lo-‘M EDTA (SE medium). The tissue was then sliced into thin pieces and homogenized in four volumes (w/v) of SE medium in a motor-driven Potter Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 800 X g for 15 min in a refrigerated centrifuge to sediment the nuclei and the cell debris. The supernatant, freed of any floating layer of lipids by filtration through a pad of glass wool, was centrifuged at 10,000 X g for 10 min to sediment the mitochondria. The mitochondria were washed once by resuspending the pellet in SE medium and left after the resedimenting it at 10,000 X g for 10 min. The supernatant removal of mitochondria, was centrifuged at 31,000 X g for 30 min. The pellet which contained mostly lysosomes was discarded and the supematant was centrifuged at 125,000 X g for 1 h to sediment the microsomes. The supernatant from this was used as the cytoplasmic fraction. All fractions were stored at -20°C and thawed just before they were assayed for their enzyme activity. Protein
estimation
The protein content of the subcellular fractions was determined method of Lowry et al. [13] using bovine serum albumin as standard. Assay of fatty acid synthesizing
by the
activities
The fatty acid synthesizing activities of the enzyme fractions were determined by the incorporation of 14C-labeled substrates into long chain acids when the enzyme fraction was incubated with these substrates and necessary cofactors. The incubations were carried out under optimal conditions of enzyme activity determined previously for each enzyme fraction. Fatty acid synthesis in mitochrondria was determined according to the method of Donaldson et al. [14] with minor modifications. The standard incubation mixture contained 330 nmoles [l-14C]acetyl CoA (310,000 counts/ min), 5.0 nmoles myristyl CoA, 1.0 E.tmole NADH, 2.0 pmoles ATP, 2.0 pmoles MgC12, 1.0 pmole KCN and 0.75-1.0 mg mitochondrial protein in 1.0 ml 0.05 M potassium phosphate buffer, pH 7.4. The incubation was at 37°C. Microsomal fatty acid synthesizing activity was measured essentially by the method of Nugteren [15]. Magnesium chloride was not included in the incubation mixture since a slightly enhanced synthesis was observed in the absence of MgCl*. The incubation mixture contained 95 nmoles [2-14C]malonyl CoA (351,000 counts/min), 5.0 pmoles myristyl CoA, 5.0 pmoles ATP, 1.0 pmole NADPH, 5.0 pmoles /3-mercaptoethanol, and 0.6-0.8 mg microsomal protein in a total volume of 1.0 ml 0.1 M potassium phosphate buffer, pH 7.0. The incubation was carried out at 37°C. Cytoplasmic fatty acid synthesis was measured by the method of Donaldson et al. [14]. Addition of ATP was found to increase the incorporation of 14Clabeled malonate into fatty acids by about 20%. A pH of 6.5 was found to be more favorable than the 7.4 used by Donaldson for the rat liver system. The incubation mixture contained 53 nmoles [2-14C]malonyl CoA (178,000 counts/ min), 40 nmoles acetyl CoA, 5.0 pmoles ATP, 1.0 I.tmol NADPH, 5.0 pmoles /3-mercaptoethanol, and 0.1 mg cytoplasmic protein in 1.0 ml 0.1 M potassium
410
phosphate buffer, pH 6.5. The incubation was done at 37°C. In all the incubations described above, the reaction was initiated by the addition of the enzyme fraction. At the end of the incubation period, the reaction was terminated by the addition of 0.5 ml 3 M KOH followed by 2 mg palmitic acid in 0.1 ml pentane, as carrier. The contents were then saponified at 8590°C in a water bath for 1 h. The pH of the resulting mixture was adjusted to 1.5-2 by addition of 4 N sulfuric acid and the long chain fatty acids were extracted three times with 3 ml portions of pentane. The extracts were pooled, and washed twice with 2 ml portions of 1% acetic acid to remove any contaminating 14C-acetate in the extract. The washed extracts were then quantitatively transferred to counting vials, the pentane carefully evaporated off by a stream of nitrogen, and the radioactivity in the residue determined in a liquid scintillation spectrometer after addition of 5 ml Bray’s solution [ 161. Results (A) The effect of cholesterol feeding and estrogen administration on aortic lesions The severity of atherosclerosis induced in the experimental rabbits by cholesterol feeding was graded on a O-4 scale, with 0 representing no lesions and 4 representing lesions over the entire aorta. Table 1 shows the extent of aortic lesions observed in each group of rabbits. It can be seen that the degree of aortic involvement in the cholesterol-fed rabbits steadily increased with time during the g-week period of the experiment, while in the control and the estrogen-treated rabbits the aortas were essentially free of lesions. The marked increase in the ratio of the body weight to testes weight in the estrogen-treated animals indicated that the administered hormone was physiologically active. TABLE 1 PHYSICAL
PARAMETERS
Exptl. Group
Group I (control)
Group II (cholesterol-fed)
Group III (cholesterol-fed and estrogentreated)
OF THE EXPERIMENTAL
Period of treatment (weeks)
No. of rabbits in group
0 3 6 9
ANIMALS
(kg)
Ave. aortic lesions
a a 8 7e
0.4 0.6 0.8
0 0 0 0
9.4 7.1 7.8 7.3
3 6 9
8 8 7a
0.3 0.7 0.5
0.3 1.4 1.7
6.6 7.4 8.4
3 6 9
8 8 gb
0.5 0.6 0.6
0 0 0.5
a One animal in the group died b Two animals in the group died
Average wt. increase
Ave. body wt. testes wt. (x10-2)
12 22 29
411
(B) Requirements and optimum conditions for fatty acid synthesis in liver mitochondria Mitochondrial fatty acid synthesis required exogenous myristyl CoA for maximal synthesis (Table 2). Elimination of exogenous primer reduced the synthesis by about 20%. NADH was the reducing cofactor most favorable for the synthesis. Addition of NADPH to NADH did not show any additive effect in the synthesis. ATP stimulated the synthesis even when the thiol esters of the substrates were used. The stimulation of ATP was probably due to an ATPdependent resynthesis of the thiol ester substrates hydrolyzed by deacylase in mitochondrial prepartion. Citrate did not show any significant stimulation. Freezing and thawing of the mitochondria showed an enhanced activity for the enzyme. Hence in all subsequent studies the mitochondria were frozen and thawed before used in incubations. Microsomes Table 3 summarizes the cofactor requirements for the synthesis of fatty acids by the microsomal system. As in the mitochondrial system, omission of myristyl CoA resulted in a small decrease in the synthesis. Both ATP and NADPH were found to be essential for maximum synthesis. Mg” was not required for this synthesis. Acetyl CoA could not replace malonyl CoA as substrate in this sytem. Frozen and thawed microsomes incorporated more malonyl CoA than fresh microsomal preparations, and hence were used in all subsequent studies. Cytoplasm The rate of cytoplasmic fatty acid synthesis as a function of malonyl CoA concentration’is shown in Fig. 1. The concentration of acetyl CoA was constant at 40 w in these studies. The synthesis increased up to a concentration of 75 /.LMmalonyl CoA and then leveled off. Addition of ATP did show a small but definite increase in synthesis. Under the conditions used 4 w concentration of ATP showed maximum synthesis (Fig. 2). ATP was probably involved in the resynthesis of the thiol ester substrates hydrolyzed by deacylase in the enzyme preparation.
TABLE
2
COFACTOR
REQUIREMENTS
Incubation
conditions
FOR
are described
FATTY in the
ACID
SYNTHESIS
IN MITOCHONDRIA
text. nmoles
System
[ 1-‘4C3
incorporated/mg Complete
system
(with
fresh
Complete
system
(with
frozen
mitochondrlal) and
thawed
5.220 mitochondrla)
5.970
Minus
NADH
2.496
Minus
ATP
2.224
Minus
myristyl
CoA
Minus
myristyl
CoA
4.222 and
Plus NADPH Plus NADPH
ATP
1.828 5.970
and
citrate
6.150
acetyl
CoA
protein/30
min
412 TABLE 3 COFACTOR
REQUIREMENTS
FOR FATTY
ACID SYNTHESIS
IN MICROSOMES
Incubation conditions are described in the text.
System
mnoles malonyl CoA incorporated/ mg protein/20 min
Complete system (with fresh microsomes) Complete system (with frozen and thawed microsomes) Minus Mg* Minus myristyl CoA Minus ATP Minus NADPH Minus Malonyl CoA, Plus acetyl CoA
3.769 4.870 4.205 3.448 1.880 2.059 0.296
Effect of exogenous primer and pH Both mitochondria and microsomes required exogenous acyl CoA primer for maximum synthesis of fatty acids. Myristyl CoA was the primer used in both the systems. In mitochondria the incorporation of acetyl CoA into fatty acids increased proportionately with the concentration of the primer until a maximum incorporation was obtained at 5.0 @l4 concentration (Fig. 3). In microsomes 5.0 PM concentration of myristyl CoA showed maximum synthesis, as shown in Fig. 3. The pH optima for fatty acid synthesis were different for the three enzyme
0
20 Cone
40
60
of Malonyl
SO
100
CoA (PM)
Fig. 1. Effect of malonyl CoA concentration on fatty acid synthesis in cytoplasm. The incubation mixture contained [2- 14Clmalonyl CoA, 40 nmoles acetyl CoA, 1.0 pmole NADPH, 5.0 flumoles ATP. 5.0 pmoles @-mercaptoethanol, and 0.1 mg cytoplasmic protein in 1.0 ml 0.1 M potassium phosphate buffer at pH 6.5. The incubation was for 30 min at 37’C.
413
d _
SE =
I h(
7.0,
6
2.5
0.0
I
5.0
7.5
Cont. of ATP (mM) Fig. 2. Effect of ATP concentration on fatty acid synthesis in cytoplasm. The incubation conditions were the same as in Fig. 1 except that the concentration of [2- 14Clmalon~l CoA was 53 pVf and the concentration of ATP was varied.
systems. The optimal pH for mitochondria microsomes and cytoplasm, respectively.
was 7.5 while it was 7.0 and 6.5 for
(C) Effect of cholesterol feeding and estrogen treatment on synthesis’of fatty acids in liver subcellular fractions Effect of cholesterol feeding and estrogen treatment of synthesis of fatty acids in mitochondria, microsomes and cytoplasm of liver was studied under optimal conditions. The results are given in Table 4 and graphically presented in Fig. 4. In mitochondria neither cholesterol feeding nor estrogen treatment had any effect on the synthesis of fatty acids. On the other hand, in microsomes and especially in cytoplasm, cholesterol feeding showed an inhibitory
7
Cont.
of Myrlstyl CoA (PM)
Fig. 3. Effect of myristyl CoA concentration on synthesis of fatty acids in mitochondria and microsomes.
414 TABLE
4
EFFECT
OF
ACIDS The
CHOLESTEROL
IN LIVER
incubation
Exptl.
FEEDING
SUBCELLULAR
conditions
are described Period
Group
AND
ESTROGEN
of
I
Group
II
(cholesterol-fed)
Group III
SYNTHESIS
OF
of rabbits
Fatty
acid
synthesis
in
Mitochondria
a
Microsomes
b
Cytoplasm
0
8
2.81
i 0.15
10.4
f 0.9
152
f 18
3
a
2.82
+ 0.11
11.4
* 0.6
160
+
7
6
8
2.71
i- 0.23
10.3
f 0.6
161
f
9
9
7
2.64
? 0.14
10.3
* 0.5
166
* 19
3
8
2.92
+ 0.15
10.04
f 0.90
126
+ 4
6
8
2.92
+ 0.44
7.95
i: 0.60
112
* 4
9
7
2.51
f 0.38
5.94
f 0.76
63
+ 3
3
8
2.33
f 0.11
a.02
f 0.71
113
? 4
(cholesterol-fed
6
8
3.05
f 0.47
5.95
* 0.26
121
f 4
and
9
6
2.51
? 0.28
5.92
r 0.46
174
+ 6
estrogen-
FATTY
in group
(weeks)
(control)
ON
in the text.
No.
treatment
Group
TREATMENT
FRACTIONS
c
treated) a nmoles
[1-14C1
b nmoles
[2-14Clmalonyl
acetyl
CoA
CoA
incorporated/mg
protein/30
min
+ SE
c mnoles
[2-14Cl
CoA
incorporated/mg
protein/30
min
i SE
malonyl
incorporated/mg
protein/l2
min
f SE
3.5
A. MITOCHONDRIA
12
c
6
B. MICROSOMES 7
\
I C. CYTOPLASM
1801
I 3
6Ol 0
Period Fig.
4.
Fatty
estrogen-treated L
acid
in
rabbits.
A = mitochondria; ? -?
liver -
9
(weeks)
synthesis
- - - - -a = cholesterol-fed;
‘\
4 6
--O
subcellular
fractions
of
normal.
B = microsomes;
= cholesterol-fed
cholesterol-fed, C = cytoplasm.
and estrogen-treated.
and
cholesterol-fed
o-------o
= controls
415
effect on the synthesis of fatty acids. In the cholesterol-fed animals the microsomal fatty acid synthesis showed a significant decrease at the end of the g-week period. At the end of the g-week period this decrease was as much as 40%. Thus, atherosclerosis appears to have a considerable inhibitory effect on liver microsomal fatty acid synthesis. Estrogen treatment did not have any noticeable effect on this inhibition. In cytoplasm, fatty acid synthesis was drastically reduced in the cholesterol-fed animals. At the end of the g-week period the synthesis was only 38% of that in the control animals. In the cholesterol-fed and estrogen-treated animals, however, a different phenomenon was observed. In this group, as in the cholesterol-fed group, a decrease in synthesis was observed during the first three weeks of the experiment. But after the 3-week period the synthesis showed a remarkable recovery, presumably due to the estrogen treatment, such that the synthesis at the end of the g-week period was comparable to that in the control group. Estrogen thus appeared to counteract, after a short lag period, the inhibitory effect of atherogenesis on cytoplasmic fatty acid synthesis in liver. Discussion Fatty acid synthesis has been studied in great detail in tissues of various animals, and in many cases the optimum conditions for the synthesis have been established [ 17-261. However, very little is known about the synthesis of fatty acids in rabbit, an animal very commonly used in the study of atherosclerosis. The present study has established the requirements and optimum conditions for fatty acid synthesis in rabbit liver and has investigated the effect of atherosclerosis and estrogen treatment on this synthesis. The cofactor requirements for fatty acid synthesis in rabbit liver, to a great extent, resemble the requirements reported for rat liver. In mitochondria and microsomes, addition of an exogenous primer, myristyl CoA, increased the synthesis only to a small extent indicating the availability of significant amounts of endogenous primer in the system. Higher concentrations of acyl CoA had an inhibitory effect in both these systems, but the effect was more pronounced in the microsomes. Podack et al. [27] have reported that high levels of long chain acyl CoA inhibited fatty acid synthesis in beef adrenal cortex. Acetyl CoA was the source of the condensing &-unit in mitochondria while malonyl CoA provided the &-unit in both microsomes and cytoplasm. In agreement with several previous reports, acetyl CoA could not serve as substrate for fatty acid synthesis in microsomes. Mitochondria used NADH as the reducing cofactor for the synthesis while the other two systems required NADPH. Some investigators have attributed the NADH requirement of mitochondrial fatty acid synthesizing system to a possible role of this mitochondrial system in the regulation of the NAD’-requiring oxidative qeamination of glutamate [26]. ATP stimulated to varying extents, the synthesis of fatty acids in all the three fractions. Mitochondria required ATP for optimal synthesis probably because of the presence of deacylase in the preparation, as suggested by other investigators [18-201. The need for ATP in the microsomes, on the other hand, might possibly be for the incorporation of synthesized fatty acids into phospholipids [28], for the transport of substrates or cofactors to the active site of the enzyme [29], or
416
for the resynthesis of malonyl CoA hydrolyzed by deacylase. The pH optima for the synthesis were different for the three fractions. It has been known for years that atherosclerosis can be induced in rabbits by feeding a diet rich in cholesterol. In the present study, almost all the cholesterol-fed rabbits developed severe aortic lesions by the end of the six-week period. However, treatment of the cholesterol-fed rabbits with estrogen very effectively retarded or prevented the development of aortic lesions in these animals. This has once again substantiated the reported ability of estrogen for retarding atherosclerosis. Mitochondrial fatty acid synthesis was not found to be affected by either cholesterol feeding or estrogen administration. If one of the primary functions of mitochondrial fatty acid synthesis is to regenerate NAD’ for oxidative deamination of glutamate [26] one would not expect this process to be significantly affected by atherosclerosis. It has been shown that there is a decrease in the ratio of phospholipids to cholesterol ester during development of atherosclerosis. This could be interpreted as being due to an increase in the synthesis of cholesterol esters or a decrease in the synthesis of phospholipids, or both. Since microsomes are known to be the primary site for the synthesis of phospholipids, it is conceivable that any change in the synthesis of fatty acids in microsomes or cytoplasm (since fatty acids synthesized in cytoplasm are easily accessible to microsomes) would have an effect on the synthesis of phospholipids. In the present investigation, a decrease in the synthesis of fatty acids in microsomes was observed during atherogenesis. The effect was much more noticeable in cytoplasm where there was approximately a 60% decrease in fatty acid synthesis by the end of the g-week period. It is known that the principal product of cytoplasmic fatty acid synthesis, palmitic acid, is also a principal fatty acid found in phospholipids. The data would then tend to suggest that a decreased phospholipid synthesis could, at least in part, result from a decreased fatty acid synthesis in microsomes and cytoplasm. Estrogen, in the present study, was found to counteract the decreased synthesis of fatty acids in cytoplasm. Morrin [30] demonstrated that estrogens increased the incorporation of 14C-acetate into phosphatidyl choline fraction of human peripheral arteries, presumably by an increased incorporation of fatty acids into this lipid fraction. It is likely that estradiol benzoate enhanced fatty acid synthesis indirectly by increasing the incorporation of fatty acids into phospholipids, thereby depleting the endogenous pool of fatty acids. Further experiments are in progress to determine the mode by which cytoplasmic fatty acid synthesis is influenced by estrogen. References Swell, L., Law. M.D., Schools, P.E. and Treadwell. C.R., Tissue lipid fatty acid changes following the feeding of high cholesterol, essential fatty acid-supplemented diets in rabbits, J. Nutr., 75 (1961) 181. Swell, L.. Law. M.D. and Treadwell. C.R., Tissue cholesterol ester and triglyceride fatty acid composition of rabbits fed cholesterol diets high and low in linoleic acid, J. Nutr., 76 (1962) 429. Evrard, E., Van Den Bosch, J.. Joossens, J.V. and De Some& P., Fatty acid composition of plasma lipids of normal, triton-treated and cholesterol-fed rabbits, Amer. J. Clin. Nutr.. 10 (1962) 240. Smith, E.B., The influence of age and atherosclerosis on the chemistry of aortic intima-lipids. J. Atheroscler. Res.. 5 (1965) 224.
417 5 St. Clair, R.W.. Lofland. H.B. and Clarkson, T.B., Composition and synthesis of fatty sclerotic aortas of the pigeon, J. Lipid Res., 9 (1968) 739. 6 Patelski. J., Bowyer. D.E., Howard, A.N.. Jenning, I.W., Thorne, C.J.R. and Gresham, tion of enzyme activities in experimental atherosclerosis in the rabbit, Atherosclerosis, 7 Day, A.J., Wahlquist, M.L. and Tume. R.K., Incorporation of different fatty acids
acids in atheroG.A., Modifica12 (1970) 41. into combined
lipids in rabbit atherosclerotic lesions, Atherosclerosis. 12 (1970) 253. 8 Whereat, A.F.. Fatty acid synthesis in cell-free system from rabbit aorta, J. Lipid Res., 7 (1966) 671. of cholesterol-fed and 9 Cathcart, R. and Pynadath. T.I., Fatty acid synthesis in aortic mitochondria estrogen-treated rabbits. Atherosclerosis. 23 (1976) 191. 10 Stamler. J.. Pick, R.. Katz. L.N., Pick, A. and Kaplan. B.M., Effectiveness of estrogens for therapy of myocardial infarction in middle-age men, J. Amer. Med. Ass., 183 (1963) 632. 11 Pick, R., Stamler, J., Rodbard, C. and Katz, L.N.. Inhibition of coronary atherosclerosis by estrogens in cholesterol-fed chicks, Circulation, 6 (1952) 276. 12 Malinow, M.R., PeIIegrino. A.A. and Ramos, E.H., Prevention of aortic atherosclerosis in the rabbit by intravenous microcrystaIIized estradiol benzoate and dextran. Proc. Exp. Biol. Med., 97 (1958) 449. 13 Lowry, O.H.. Rosebrough, N.J.. Farr, A.L. and Randall. R.J., Protein measurement with Folin phenol reagent. J. Biol. Chem.. 193 (1951) 265. 14 Donaldson, W.E., Wit-Peeters. E.M. and Scholte, H.R., Fatty acid synthesis in rat liver - Relative contributions of the mitochondrial, microsomal and non-particulate systems, Biochim. Biophys. Acta, 202 (1970) 35. 15 16 17 18 19 20 21 22 23 24 25
26 27 28 29 30
Nugteren. D.H., The enzymic chain elongation of fatty acids by rat-liver microsomes. Biochim. Biophys. Acta. 106 (1965) 280. Bray, G.A., A simple efficient liquid scinthlator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem., 1 (1960) 279. Howard, Jr., C.F., Synthesis of fatty acids in outer and inner membranes of mitochondria, J. Biol. Chem., 245 (1970) 462. Barron, E.J. and Mooney, L.A. Identification of possible intermediates in the mitochondriai fatty acid chain elongation system, Biochemistry, 9 (1970) 2143. Mooney, L.A. and Barron. E.J.. Cofactor requirements and general characteristics of a soluble fatty acid elongating system from mitochondria, Biochemistry, 9 (1970) 2138. QuagIiarieIIo. E., Landriscina. C. and Coratelh. P.. Fatty acid synthesis by chain elongation in rat-liver mitochondria, Biochim. Biophys. Acta. 164 (1968) 12. Wit-Peeters, E.M.. Synthesis of long chain fatty acids in mitochondria, Biochim. Biophys. Acta, 176 (1969) 453. Nugteren, D.H., The enzymic conversion of y-Iinolenic acid into homey-Iinolenic acid, Biochem. J., 89 (1963) 28P. WakiI. S.J., Mechanism of fatty acid synthesis, J. Lipid Res.. 2 (1961) 1. Katiyar. S.S. and Porter. J.W., Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for overall synthetase activity, Arch. Biochem. Biophys., 163 (1974) 324. Phillips, G.T., Nixon, J.E., Abramovitz, A.S. and Porter, J.W. Identification of the sites of binding of acetyl and malonyl groups to the pigeon liver fatty acid synthetase complex, Arch. Biochem. Biophys., 138 (1970) 357. Seubert, W. and Podack. E.R., Mechanisms and physiological roles of fatty acid chain elongation in microsomes and mitochondria, Mol. CeII. Biochem., 1 (1973) 29. Podack, E.R., Lakomek, M., SaaIhoff. G. and Seubert, W., On the mechanism and control of the malonyl CoA-dependent chain elongation of fatty acids, Europ. J. Biochem., 45 (1974) 13. Guchhait, R.B., Putz, G.R. and Porter, J.W., Synthesis of long chain fatty acids by microsomes of pigeon liver, Arch. Biochem. Biophys., 117 (1966) 541. Plate, C.A., Joshi, V.C., Sedgwick, B. and WakiI, S.J.. Studies on the mechanism of fatty acid synthesis, J. Biol. Chem.. 243 (1968) 5439. Morin. R.J., Effects of estradiol on the in vitro incorporation of acetate-l-% and choline l-2-% into phospholipids of human peripheral arteries, Experientia (Basel). 26 (1970) 829.
Atherosclerosis, 27 (1977) 407-417 @ Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECT OF CHOLESTEROL FEEDING AND ESTROGEN TREATMENT ON SYNTHESIS OF FATTY ACIDS IN LIVER
K. SRINIVASAN and THOMAS I. PYNADATH Department
of Chemistry,
Kent State University,
Kent, Ohio 44242
(U.S.A.)
(Received 26 October, 1976) (Revised, received 7 March, 1977) (Accepted 7 March, 1977)
Summary
The effect of cholesterol feeding and estrogen administration on synthesis of fatty acids in liver mitochondria, microsomes and cytoplasm of male rabbits has been investigated. The synthesis was measured by the incorporation of [1-14C] acetyl CoA or [2-14C]malonyl CoA into long chain fatty acids under optimal conditions. It was found that atherogenesis markedly decreased the fatty acid synthesis in cytoplasm. The mitochondrial fatty acid synthesis was not affected by the disease. There was a small but measurable decrease in the synthesis of fatty acids in microsomes. Estrogen had no effect on the synthesis of fatty acids in mitochondria or microsomes. But if effectively counteracted, after a short lag period, the decreased synthesis of cytoplasmic fatty acids observed in atherosclerosis. It is possible that liver fatty acid synthetase is one of the enzyme systems through which estrogens exert their atherosclerosis-retarding effect. The decreased cytoplasmic fatty acid synthesis observed in atherosclerosis might account for the low levels of saturated fatty acids reported in liver and plasma lipids of atherosclerotic animals. Key words:
Effect of cholesterol -Rabbit
feeding
- Effect
of estrogen
- Liver fatty acid synthesis
Introduction
Atherosclerosis and composition
has been shown to have a significant effect on the content of tissue lipids [l-5]. It has been found that atherosclerosis
This investigation was supported by a Research Grant from the Akron District Chapter of American Heart Association, Akron. Ohio.
markedly alters the fatty acid composition of many tissue lipids, especially the plasma and liver lipids. Studies have shown that changes in fatty acid esterifying activities of the tissues could partly account for this alteration [ 5-71. It is possible that a change in the fatty acid synthesizing activities of the tissues might be a more important contributing factor for this alteration. Whereat [8] has studied the changes in fatty acid synthesis in atherosclerotic rabbit aorta. The results showed that in atherosclerotic aorta there was a 4-fold increase in the incorporation of acetate into long chain fatty acids. However, when acyl CoA compounds were used as substrates, under optimal conditions, no increase in synthesis was observed [9]. The most drastic changes in fatty acid composition, in atherosclerosis, occur in liver and plasma lipids. Hence it is conceivable that the fatty acid synthesizing systems most affected by atherosclerosis are the systems in liver. The present investigation is a study to determine the changes in fatty acid synthesis in mitochondria, microsomes and cytoplasm of rabbit liver during development of atherosclerosis. Optimal conditions for fatty acid synthesis were first established for each enzyme system and these conditions were used for all subsequent studies. Since estrogens have long been known to retard development of atherosclerosis in human [lo] and in animals [11,12], the effect of estrogen treatment on liver fatty acid synthesis of cholesterol-fed rabbits has also been investigated. Materials and methods Chemicals
Acetyl CoA and malonyl CoA were purchased from P-L Biochemicals. NADH, NADPH and ATP were from Sigma Chemical Co. [2-14C]malonyl CoA was purchased from New England Nuclear Corp. and [l-14C]acetyl CoA was obtained from International Chemical and Nuclear Corporation. Experimental
animals
Eighty New Zealand White male rabbits were divided into three groups. Group I containing 32 rabbits, fed Purina Lab Rabbit Chow, served as the control group. Group II, with 24 rabbits, was fed Purina Chow to which 1% (w/w) cholesterol was added. Group III rabbits, 24 in number, also were fed the 1% cholesterol diet, and in addition were given subcutaneous injections of 1.4 mg estradiol benzoate in 0.4 ml sesame oil on alternate days. Group I and II rabbits received placebo injections of 0.4 ml sesame oil on the same days on which Group III received estrogen injections. The rabbits were fed and watered ad libitum
.
Eight animals from Group I were sacrificed at the commencement of the project and this served as the zero week control. Thereafter, from each group eight animals were sacrificed at the end of three, 6- and g-week periods. All the animals were killed by air embolism. The physiological effect of the estrogen administered was determined by the atrophy of the testes.
409
Isolation
of su bcellular
fractions
The liver, removed immediately after the sacrifice, was washed with an icecold solution of 0.28 M sucrose containing 5 X lo-‘M EDTA (SE medium). The tissue was then sliced into thin pieces and homogenized in four volumes (w/v) of SE medium in a motor-driven Potter Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged at 800 X g for 15 min in a refrigerated centrifuge to sediment the nuclei and the cell debris. The supernatant, freed of any floating layer of lipids by filtration through a pad of glass wool, was centrifuged at 10,000 X g for 10 min to sediment the mitochondria. The mitochondria were washed once by resuspending the pellet in SE medium and left after the resedimenting it at 10,000 X g for 10 min. The supernatant removal of mitochondria, was centrifuged at 31,000 X g for 30 min. The pellet which contained mostly lysosomes was discarded and the supematant was centrifuged at 125,000 X g for 1 h to sediment the microsomes. The supernatant from this was used as the cytoplasmic fraction. All fractions were stored at -20°C and thawed just before they were assayed for their enzyme activity. Protein
estimation
The protein content of the subcellular fractions was determined method of Lowry et al. [13] using bovine serum albumin as standard. Assay of fatty acid synthesizing
by the
activities
The fatty acid synthesizing activities of the enzyme fractions were determined by the incorporation of 14C-labeled substrates into long chain acids when the enzyme fraction was incubated with these substrates and necessary cofactors. The incubations were carried out under optimal conditions of enzyme activity determined previously for each enzyme fraction. Fatty acid synthesis in mitochrondria was determined according to the method of Donaldson et al. [14] with minor modifications. The standard incubation mixture contained 330 nmoles [l-14C]acetyl CoA (310,000 counts/ min), 5.0 nmoles myristyl CoA, 1.0 E.tmole NADH, 2.0 pmoles ATP, 2.0 pmoles MgC12, 1.0 pmole KCN and 0.75-1.0 mg mitochondrial protein in 1.0 ml 0.05 M potassium phosphate buffer, pH 7.4. The incubation was at 37°C. Microsomal fatty acid synthesizing activity was measured essentially by the method of Nugteren [15]. Magnesium chloride was not included in the incubation mixture since a slightly enhanced synthesis was observed in the absence of MgCl*. The incubation mixture contained 95 nmoles [2-14C]malonyl CoA (351,000 counts/min), 5.0 pmoles myristyl CoA, 5.0 pmoles ATP, 1.0 pmole NADPH, 5.0 pmoles /3-mercaptoethanol, and 0.6-0.8 mg microsomal protein in a total volume of 1.0 ml 0.1 M potassium phosphate buffer, pH 7.0. The incubation was carried out at 37°C. Cytoplasmic fatty acid synthesis was measured by the method of Donaldson et al. [14]. Addition of ATP was found to increase the incorporation of 14Clabeled malonate into fatty acids by about 20%. A pH of 6.5 was found to be more favorable than the 7.4 used by Donaldson for the rat liver system. The incubation mixture contained 53 nmoles [2-14C]malonyl CoA (178,000 counts/ min), 40 nmoles acetyl CoA, 5.0 pmoles ATP, 1.0 I.tmol NADPH, 5.0 pmoles /3-mercaptoethanol, and 0.1 mg cytoplasmic protein in 1.0 ml 0.1 M potassium
410
phosphate buffer, pH 6.5. The incubation was done at 37°C. In all the incubations described above, the reaction was initiated by the addition of the enzyme fraction. At the end of the incubation period, the reaction was terminated by the addition of 0.5 ml 3 M KOH followed by 2 mg palmitic acid in 0.1 ml pentane, as carrier. The contents were then saponified at 8590°C in a water bath for 1 h. The pH of the resulting mixture was adjusted to 1.5-2 by addition of 4 N sulfuric acid and the long chain fatty acids were extracted three times with 3 ml portions of pentane. The extracts were pooled, and washed twice with 2 ml portions of 1% acetic acid to remove any contaminating 14C-acetate in the extract. The washed extracts were then quantitatively transferred to counting vials, the pentane carefully evaporated off by a stream of nitrogen, and the radioactivity in the residue determined in a liquid scintillation spectrometer after addition of 5 ml Bray’s solution [ 161. Results (A) The effect of cholesterol feeding and estrogen administration on aortic lesions The severity of atherosclerosis induced in the experimental rabbits by cholesterol feeding was graded on a O-4 scale, with 0 representing no lesions and 4 representing lesions over the entire aorta. Table 1 shows the extent of aortic lesions observed in each group of rabbits. It can be seen that the degree of aortic involvement in the cholesterol-fed rabbits steadily increased with time during the g-week period of the experiment, while in the control and the estrogen-treated rabbits the aortas were essentially free of lesions. The marked increase in the ratio of the body weight to testes weight in the estrogen-treated animals indicated that the administered hormone was physiologically active. TABLE 1 PHYSICAL
PARAMETERS
Exptl. Group
Group I (control)
Group II (cholesterol-fed)
Group III (cholesterol-fed and estrogentreated)
OF THE EXPERIMENTAL
Period of treatment (weeks)
No. of rabbits in group
0 3 6 9
ANIMALS
(kg)
Ave. aortic lesions
a a 8 7e
0.4 0.6 0.8
0 0 0 0
9.4 7.1 7.8 7.3
3 6 9
8 8 7a
0.3 0.7 0.5
0.3 1.4 1.7
6.6 7.4 8.4
3 6 9
8 8 gb
0.5 0.6 0.6
0 0 0.5
a One animal in the group died b Two animals in the group died
Average wt. increase
Ave. body wt. testes wt. (x10-2)
12 22 29
411
(B) Requirements and optimum conditions for fatty acid synthesis in liver mitochondria Mitochondrial fatty acid synthesis required exogenous myristyl CoA for maximal synthesis (Table 2). Elimination of exogenous primer reduced the synthesis by about 20%. NADH was the reducing cofactor most favorable for the synthesis. Addition of NADPH to NADH did not show any additive effect in the synthesis. ATP stimulated the synthesis even when the thiol esters of the substrates were used. The stimulation of ATP was probably due to an ATPdependent resynthesis of the thiol ester substrates hydrolyzed by deacylase in mitochondrial prepartion. Citrate did not show any significant stimulation. Freezing and thawing of the mitochondria showed an enhanced activity for the enzyme. Hence in all subsequent studies the mitochondria were frozen and thawed before used in incubations. Microsomes Table 3 summarizes the cofactor requirements for the synthesis of fatty acids by the microsomal system. As in the mitochondrial system, omission of myristyl CoA resulted in a small decrease in the synthesis. Both ATP and NADPH were found to be essential for maximum synthesis. Mg” was not required for this synthesis. Acetyl CoA could not replace malonyl CoA as substrate in this sytem. Frozen and thawed microsomes incorporated more malonyl CoA than fresh microsomal preparations, and hence were used in all subsequent studies. Cytoplasm The rate of cytoplasmic fatty acid synthesis as a function of malonyl CoA concentration’is shown in Fig. 1. The concentration of acetyl CoA was constant at 40 w in these studies. The synthesis increased up to a concentration of 75 /.LMmalonyl CoA and then leveled off. Addition of ATP did show a small but definite increase in synthesis. Under the conditions used 4 w concentration of ATP showed maximum synthesis (Fig. 2). ATP was probably involved in the resynthesis of the thiol ester substrates hydrolyzed by deacylase in the enzyme preparation.
TABLE
2
COFACTOR
REQUIREMENTS
Incubation
conditions
FOR
are described
FATTY in the
ACID
SYNTHESIS
IN MITOCHONDRIA
text. nmoles
System
[ 1-‘4C3
incorporated/mg Complete
system
(with
fresh
Complete
system
(with
frozen
mitochondrlal) and
thawed
5.220 mitochondrla)
5.970
Minus
NADH
2.496
Minus
ATP
2.224
Minus
myristyl
CoA
Minus
myristyl
CoA
4.222 and
Plus NADPH Plus NADPH
ATP
1.828 5.970
and
citrate
6.150
acetyl
CoA
protein/30
min
412 TABLE 3 COFACTOR
REQUIREMENTS
FOR FATTY
ACID SYNTHESIS
IN MICROSOMES
Incubation conditions are described in the text.
System
mnoles malonyl CoA incorporated/ mg protein/20 min
Complete system (with fresh microsomes) Complete system (with frozen and thawed microsomes) Minus Mg* Minus myristyl CoA Minus ATP Minus NADPH Minus Malonyl CoA, Plus acetyl CoA
3.769 4.870 4.205 3.448 1.880 2.059 0.296
Effect of exogenous primer and pH Both mitochondria and microsomes required exogenous acyl CoA primer for maximum synthesis of fatty acids. Myristyl CoA was the primer used in both the systems. In mitochondria the incorporation of acetyl CoA into fatty acids increased proportionately with the concentration of the primer until a maximum incorporation was obtained at 5.0 @l4 concentration (Fig. 3). In microsomes 5.0 PM concentration of myristyl CoA showed maximum synthesis, as shown in Fig. 3. The pH optima for fatty acid synthesis were different for the three enzyme
0
20 Cone
40
60
of Malonyl
SO
100
CoA (PM)
Fig. 1. Effect of malonyl CoA concentration on fatty acid synthesis in cytoplasm. The incubation mixture contained [2- 14Clmalonyl CoA, 40 nmoles acetyl CoA, 1.0 pmole NADPH, 5.0 flumoles ATP. 5.0 pmoles @-mercaptoethanol, and 0.1 mg cytoplasmic protein in 1.0 ml 0.1 M potassium phosphate buffer at pH 6.5. The incubation was for 30 min at 37’C.
413
d _
SE =
I h(
7.0,
6
2.5
0.0
I
5.0
7.5
Cont. of ATP (mM) Fig. 2. Effect of ATP concentration on fatty acid synthesis in cytoplasm. The incubation conditions were the same as in Fig. 1 except that the concentration of [2- 14Clmalon~l CoA was 53 pVf and the concentration of ATP was varied.
systems. The optimal pH for mitochondria microsomes and cytoplasm, respectively.
was 7.5 while it was 7.0 and 6.5 for
(C) Effect of cholesterol feeding and estrogen treatment on synthesis’of fatty acids in liver subcellular fractions Effect of cholesterol feeding and estrogen treatment of synthesis of fatty acids in mitochondria, microsomes and cytoplasm of liver was studied under optimal conditions. The results are given in Table 4 and graphically presented in Fig. 4. In mitochondria neither cholesterol feeding nor estrogen treatment had any effect on the synthesis of fatty acids. On the other hand, in microsomes and especially in cytoplasm, cholesterol feeding showed an inhibitory
7
Cont.
of Myrlstyl CoA (PM)
Fig. 3. Effect of myristyl CoA concentration on synthesis of fatty acids in mitochondria and microsomes.
414 TABLE
4
EFFECT
OF
ACIDS The
CHOLESTEROL
IN LIVER
incubation
Exptl.
FEEDING
SUBCELLULAR
conditions
are described Period
Group
AND
ESTROGEN
of
I
Group
II
(cholesterol-fed)
Group III
SYNTHESIS
OF
of rabbits
Fatty
acid
synthesis
in
Mitochondria
a
Microsomes
b
Cytoplasm
0
8
2.81
i 0.15
10.4
f 0.9
152
f 18
3
a
2.82
+ 0.11
11.4
* 0.6
160
+
7
6
8
2.71
i- 0.23
10.3
f 0.6
161
f
9
9
7
2.64
? 0.14
10.3
* 0.5
166
* 19
3
8
2.92
+ 0.15
10.04
f 0.90
126
+ 4
6
8
2.92
+ 0.44
7.95
i: 0.60
112
* 4
9
7
2.51
f 0.38
5.94
f 0.76
63
+ 3
3
8
2.33
f 0.11
a.02
f 0.71
113
? 4
(cholesterol-fed
6
8
3.05
f 0.47
5.95
* 0.26
121
f 4
and
9
6
2.51
? 0.28
5.92
r 0.46
174
+ 6
estrogen-
FATTY
in group
(weeks)
(control)
ON
in the text.
No.
treatment
Group
TREATMENT
FRACTIONS
c
treated) a nmoles
[1-14C1
b nmoles
[2-14Clmalonyl
acetyl
CoA
CoA
incorporated/mg
protein/30
min
+ SE
c mnoles
[2-14Cl
CoA
incorporated/mg
protein/30
min
i SE
malonyl
incorporated/mg
protein/l2
min
f SE
3.5
A. MITOCHONDRIA
12
c
6
B. MICROSOMES 7
\
I C. CYTOPLASM
1801
I 3
6Ol 0
Period Fig.
4.
Fatty
estrogen-treated L
acid
in
rabbits.
A = mitochondria; ? -?
liver -
9
(weeks)
synthesis
- - - - -a = cholesterol-fed;
‘\
4 6
--O
subcellular
fractions
of
normal.
B = microsomes;
= cholesterol-fed
cholesterol-fed, C = cytoplasm.
and estrogen-treated.
and
cholesterol-fed
o-------o
= controls
415
effect on the synthesis of fatty acids. In the cholesterol-fed animals the microsomal fatty acid synthesis showed a significant decrease at the end of the g-week period. At the end of the g-week period this decrease was as much as 40%. Thus, atherosclerosis appears to have a considerable inhibitory effect on liver microsomal fatty acid synthesis. Estrogen treatment did not have any noticeable effect on this inhibition. In cytoplasm, fatty acid synthesis was drastically reduced in the cholesterol-fed animals. At the end of the g-week period the synthesis was only 38% of that in the control animals. In the cholesterol-fed and estrogen-treated animals, however, a different phenomenon was observed. In this group, as in the cholesterol-fed group, a decrease in synthesis was observed during the first three weeks of the experiment. But after the 3-week period the synthesis showed a remarkable recovery, presumably due to the estrogen treatment, such that the synthesis at the end of the g-week period was comparable to that in the control group. Estrogen thus appeared to counteract, after a short lag period, the inhibitory effect of atherogenesis on cytoplasmic fatty acid synthesis in liver. Discussion Fatty acid synthesis has been studied in great detail in tissues of various animals, and in many cases the optimum conditions for the synthesis have been established [ 17-261. However, very little is known about the synthesis of fatty acids in rabbit, an animal very commonly used in the study of atherosclerosis. The present study has established the requirements and optimum conditions for fatty acid synthesis in rabbit liver and has investigated the effect of atherosclerosis and estrogen treatment on this synthesis. The cofactor requirements for fatty acid synthesis in rabbit liver, to a great extent, resemble the requirements reported for rat liver. In mitochondria and microsomes, addition of an exogenous primer, myristyl CoA, increased the synthesis only to a small extent indicating the availability of significant amounts of endogenous primer in the system. Higher concentrations of acyl CoA had an inhibitory effect in both these systems, but the effect was more pronounced in the microsomes. Podack et al. [27] have reported that high levels of long chain acyl CoA inhibited fatty acid synthesis in beef adrenal cortex. Acetyl CoA was the source of the condensing &-unit in mitochondria while malonyl CoA provided the &-unit in both microsomes and cytoplasm. In agreement with several previous reports, acetyl CoA could not serve as substrate for fatty acid synthesis in microsomes. Mitochondria used NADH as the reducing cofactor for the synthesis while the other two systems required NADPH. Some investigators have attributed the NADH requirement of mitochondrial fatty acid synthesizing system to a possible role of this mitochondrial system in the regulation of the NAD’-requiring oxidative qeamination of glutamate [26]. ATP stimulated to varying extents, the synthesis of fatty acids in all the three fractions. Mitochondria required ATP for optimal synthesis probably because of the presence of deacylase in the preparation, as suggested by other investigators [18-201. The need for ATP in the microsomes, on the other hand, might possibly be for the incorporation of synthesized fatty acids into phospholipids [28], for the transport of substrates or cofactors to the active site of the enzyme [29], or
416
for the resynthesis of malonyl CoA hydrolyzed by deacylase. The pH optima for the synthesis were different for the three fractions. It has been known for years that atherosclerosis can be induced in rabbits by feeding a diet rich in cholesterol. In the present study, almost all the cholesterol-fed rabbits developed severe aortic lesions by the end of the six-week period. However, treatment of the cholesterol-fed rabbits with estrogen very effectively retarded or prevented the development of aortic lesions in these animals. This has once again substantiated the reported ability of estrogen for retarding atherosclerosis. Mitochondrial fatty acid synthesis was not found to be affected by either cholesterol feeding or estrogen administration. If one of the primary functions of mitochondrial fatty acid synthesis is to regenerate NAD’ for oxidative deamination of glutamate [26] one would not expect this process to be significantly affected by atherosclerosis. It has been shown that there is a decrease in the ratio of phospholipids to cholesterol ester during development of atherosclerosis. This could be interpreted as being due to an increase in the synthesis of cholesterol esters or a decrease in the synthesis of phospholipids, or both. Since microsomes are known to be the primary site for the synthesis of phospholipids, it is conceivable that any change in the synthesis of fatty acids in microsomes or cytoplasm (since fatty acids synthesized in cytoplasm are easily accessible to microsomes) would have an effect on the synthesis of phospholipids. In the present investigation, a decrease in the synthesis of fatty acids in microsomes was observed during atherogenesis. The effect was much more noticeable in cytoplasm where there was approximately a 60% decrease in fatty acid synthesis by the end of the g-week period. It is known that the principal product of cytoplasmic fatty acid synthesis, palmitic acid, is also a principal fatty acid found in phospholipids. The data would then tend to suggest that a decreased phospholipid synthesis could, at least in part, result from a decreased fatty acid synthesis in microsomes and cytoplasm. Estrogen, in the present study, was found to counteract the decreased synthesis of fatty acids in cytoplasm. Morrin [30] demonstrated that estrogens increased the incorporation of 14C-acetate into phosphatidyl choline fraction of human peripheral arteries, presumably by an increased incorporation of fatty acids into this lipid fraction. It is likely that estradiol benzoate enhanced fatty acid synthesis indirectly by increasing the incorporation of fatty acids into phospholipids, thereby depleting the endogenous pool of fatty acids. Further experiments are in progress to determine the mode by which cytoplasmic fatty acid synthesis is influenced by estrogen. References Swell, L., Law. M.D., Schools, P.E. and Treadwell. C.R., Tissue lipid fatty acid changes following the feeding of high cholesterol, essential fatty acid-supplemented diets in rabbits, J. Nutr., 75 (1961) 181. Swell, L.. Law. M.D. and Treadwell. C.R., Tissue cholesterol ester and triglyceride fatty acid composition of rabbits fed cholesterol diets high and low in linoleic acid, J. Nutr., 76 (1962) 429. Evrard, E., Van Den Bosch, J.. Joossens, J.V. and De Some& P., Fatty acid composition of plasma lipids of normal, triton-treated and cholesterol-fed rabbits, Amer. J. Clin. Nutr.. 10 (1962) 240. Smith, E.B., The influence of age and atherosclerosis on the chemistry of aortic intima-lipids. J. Atheroscler. Res.. 5 (1965) 224.
417 5 St. Clair, R.W.. Lofland. H.B. and Clarkson, T.B., Composition and synthesis of fatty sclerotic aortas of the pigeon, J. Lipid Res., 9 (1968) 739. 6 Patelski. J., Bowyer. D.E., Howard, A.N.. Jenning, I.W., Thorne, C.J.R. and Gresham, tion of enzyme activities in experimental atherosclerosis in the rabbit, Atherosclerosis, 7 Day, A.J., Wahlquist, M.L. and Tume. R.K., Incorporation of different fatty acids
acids in atheroG.A., Modifica12 (1970) 41. into combined
lipids in rabbit atherosclerotic lesions, Atherosclerosis. 12 (1970) 253. 8 Whereat, A.F.. Fatty acid synthesis in cell-free system from rabbit aorta, J. Lipid Res., 7 (1966) 671. of cholesterol-fed and 9 Cathcart, R. and Pynadath. T.I., Fatty acid synthesis in aortic mitochondria estrogen-treated rabbits. Atherosclerosis. 23 (1976) 191. 10 Stamler. J.. Pick, R.. Katz. L.N., Pick, A. and Kaplan. B.M., Effectiveness of estrogens for therapy of myocardial infarction in middle-age men, J. Amer. Med. Ass., 183 (1963) 632. 11 Pick, R., Stamler, J., Rodbard, C. and Katz, L.N.. Inhibition of coronary atherosclerosis by estrogens in cholesterol-fed chicks, Circulation, 6 (1952) 276. 12 Malinow, M.R., PeIIegrino. A.A. and Ramos, E.H., Prevention of aortic atherosclerosis in the rabbit by intravenous microcrystaIIized estradiol benzoate and dextran. Proc. Exp. Biol. Med., 97 (1958) 449. 13 Lowry, O.H.. Rosebrough, N.J.. Farr, A.L. and Randall. R.J., Protein measurement with Folin phenol reagent. J. Biol. Chem.. 193 (1951) 265. 14 Donaldson, W.E., Wit-Peeters. E.M. and Scholte, H.R., Fatty acid synthesis in rat liver - Relative contributions of the mitochondrial, microsomal and non-particulate systems, Biochim. Biophys. Acta, 202 (1970) 35. 15 16 17 18 19 20 21 22 23 24 25
26 27 28 29 30
Nugteren. D.H., The enzymic chain elongation of fatty acids by rat-liver microsomes. Biochim. Biophys. Acta. 106 (1965) 280. Bray, G.A., A simple efficient liquid scinthlator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem., 1 (1960) 279. Howard, Jr., C.F., Synthesis of fatty acids in outer and inner membranes of mitochondria, J. Biol. Chem., 245 (1970) 462. Barron, E.J. and Mooney, L.A. Identification of possible intermediates in the mitochondriai fatty acid chain elongation system, Biochemistry, 9 (1970) 2143. Mooney, L.A. and Barron. E.J.. Cofactor requirements and general characteristics of a soluble fatty acid elongating system from mitochondria, Biochemistry, 9 (1970) 2138. QuagIiarieIIo. E., Landriscina. C. and Coratelh. P.. Fatty acid synthesis by chain elongation in rat-liver mitochondria, Biochim. Biophys. Acta. 164 (1968) 12. Wit-Peeters, E.M.. Synthesis of long chain fatty acids in mitochondria, Biochim. Biophys. Acta, 176 (1969) 453. Nugteren, D.H., The enzymic conversion of y-Iinolenic acid into homey-Iinolenic acid, Biochem. J., 89 (1963) 28P. WakiI. S.J., Mechanism of fatty acid synthesis, J. Lipid Res.. 2 (1961) 1. Katiyar. S.S. and Porter. J.W., Substrate inhibition of pigeon liver fatty acid synthetase and optimum assay conditions for overall synthetase activity, Arch. Biochem. Biophys., 163 (1974) 324. Phillips, G.T., Nixon, J.E., Abramovitz, A.S. and Porter, J.W. Identification of the sites of binding of acetyl and malonyl groups to the pigeon liver fatty acid synthetase complex, Arch. Biochem. Biophys., 138 (1970) 357. Seubert, W. and Podack. E.R., Mechanisms and physiological roles of fatty acid chain elongation in microsomes and mitochondria, Mol. CeII. Biochem., 1 (1973) 29. Podack, E.R., Lakomek, M., SaaIhoff. G. and Seubert, W., On the mechanism and control of the malonyl CoA-dependent chain elongation of fatty acids, Europ. J. Biochem., 45 (1974) 13. Guchhait, R.B., Putz, G.R. and Porter, J.W., Synthesis of long chain fatty acids by microsomes of pigeon liver, Arch. Biochem. Biophys., 117 (1966) 541. Plate, C.A., Joshi, V.C., Sedgwick, B. and WakiI, S.J.. Studies on the mechanism of fatty acid synthesis, J. Biol. Chem.. 243 (1968) 5439. Morin. R.J., Effects of estradiol on the in vitro incorporation of acetate-l-% and choline l-2-% into phospholipids of human peripheral arteries, Experientia (Basel). 26 (1970) 829.