Interrelationship between the Dietary Regulation of Fatty Acid Synthesis and the Fatty AcyI-CoA Desaturases R. JEFFCOAT and A.T. JAMES, Basic Studies Unit, Biosciences Division, Unilever Research, Colworth/Welwyn Laboratory, Sharnbrook, Bedford MK44 1LQ, U.K.


the level of enzyme synthesis which could be considered as a long term control mechanism. Similarities in the behavior of acetyl-CoA carboxylase, fatty acid synthetase, and stearoylCoA desaturase to changes in the diet suggest that these may be linked as a unit by a common control mechanism. However, detailed studies have not yet been carried out, and this paper describes work on the direct comparison of t w o of these enzymes as influenced by dietary carbohydrate and lipid.

In this paper we present further evidence for the close control of fatty acid synthetase and stearoyl-CoA desaturase. Furthermore, we have established that whereas .dietary palmitic acid may influence the activity of this desaturase but not of fatty acid synthetase, dietary linoleic acid appears to control both these enzymes. Finally, we have studied the influence of dietary fat and carbohydrate on the activities of the A6 and AS desatu r a s e s . The former is only slightly affected by these dietary components. The AS desaturase activity is stimulated as the dietary fat content rises but is unaffected by dietary carbohydrate. The control of these enzymes is therefore independent of the control of fatty acid synthetase and stearoyl-CoA desaturase. From the data presented, the magnitude of the controlling effect of polyunsaturated fatty acids on fatty acid synt h e t a s e a n d stearoyl-CoA desaturase activity is determined and its relevance to lipogenesis in man based on daily intake o f carbohydrate and linoleic acid is discussed.



A recent review by Volpe and Vagelos (1) discusses at length the regulation of the biosynthesis of saturated fatty acids via acetyl-CoA carboxylase and fatty acid synthetase. It is these enzymes which provide, in part, the endogenous substrates for the microsomal A9 desaturase. Although it has been known for many years that dietary carbohydrate can result in the induction of stearoyl-CoA desaturase (2) as well as several other lipogenic enzymes (3), little is known about the detailed control of desaturases. In general terms, the control of fatty acid biosynthesis appears to be at two levels. First is the metabolic or allosteric control of acetyl-CoA carboxylase. This is manifested by the competition between citrate and palmitate for binding sites on the enzyme protomers, which results in activation (association) and deactivation (dissociation) of the enzyme, respectively. Second is the control at

All feeding experiments were carried out with groups of six male litter weanling rats of the Colworth-Wistar strain. Animals were fed ad libitum for 14 days on either a control diet consisting of 64% starch, 25% casein, 4% minerals, 6% cellulose powder, 0.15% cystine, and vitamins 0.53% or a test diet in which some of the starch had been replaced by other carbohydrates or lipid. In the preliminary studies to examine the effect of different lipid types on enzyme activities, dietary starch was replaced by an equal weight of the lipid under investigation. C o n s e q u e n t l y , the calorie content of the control starch based diet was lower than the l i p i d supplemented diets, which were all comparable. In subsequent studies designed to investigate the specific effect of linoleic and palmitic acids on the control of enzyme activities, all diets were essentially isocatoric. The total amount of fat ingested was determined from the food intakes and the percentage fat added to the diet. Enzyme Assays

Fatty acid synthetase was assayed according to the method described by Bruckdorfer et al. (4) and the A9 desaturase as described by Jeffcoat et al. (5). The A6 and A 5 desaturases were assayed for 10 min at 37 C using 2.5 mg and 1 mg of microsomal protein, respectively. The incubations in a total volume of 2.5 ml of 0.1 M potassium phosphate buffer pH 7.4, contained 20 nmoles of either [1A4C]linoleic acid (The Radiochemical Centre, Amersham, England) or [1-14C]eicosatrienoic acid (New



470 A



9 !

turn might result in enhanced stearoyl-CoA desaturase activity via adaptive enzyme formation and indeed there is some evidence to support this hypothesis. Mercuri et al. (10) have shown that in the case of streptozotocin induced diabetic rats the decreased level of the A9 desaturase can be elevated by feeding fructose, glycerol, or saturated fatty acids. This is in contrast to our own observations reported here where we have attempted to understand the dietary factors which influence the activities of the fatty acid synthetase, and the A 9, A 6, and As desaturases. The Influence of Carbohydrate Diets on ~ 9 Desaturase

Bruckdorfer et al. (4) demonstrated that feeding rats a high carbohydrate diet resulted in enhanced levels of fatty acid synthetase. In particular, they showed that of the sugars tested (maltose, glucose, sucrose, starch, and fructose) fructose gave the highest levels of fatty acid synthetase activity in the liver. Although Oshino and Sato (2) had demonstrated that diets rich in sucrose were also England Nuclear, Winchester, England). The effective in raising the levels of A9 desaturase, following concentrations of cofactors were also no work has been reported on the effect of u s e d : 9 / J m o l e s A T P , 10ktmoles MgC12, various dietary sugars on the A9 desaturase. We 700 nmoles NADH, 360 nmoles NADPH, and therefore investigated the effect of feeding rats 130 n m o l e s coenzyme A. The percentage diets containing 15% casein, 4% minerals, 5% desaturation was determined by gas liquid vitamin mix, 2% cellulose powder, and either chromatography of the methyl esters of the 74% sucrose, glucose, or fructose. The results from a 14 day continuous feeding study are fatty acids (5). All enzyme assays were carried out using shown in Figure 1A and from a 24 hr starvation either the 1 0 0 , 0 0 0 x g supernatant or the followed by a 17 hr refeeding period after 14 microsomal fraction from six livers from rats on days continuous feeding are shown in Figure each of the various diets. Each incubation was lB. The close correlation between fatty acid carried out with two or three concentrations of synthetase and A9 desaturase activity would protein to ensure the linear enzyme dependence support the hypothesis that the latter was of the reaction. induced by adaptive enzyme formation. To investigate this in more detail, the effect of saturated as well as unsaturated dietary fatty RESULTS A N D DISCUSSION acids on the level of activity of fatty acid It is now generally accepted that in mam- synthetase A9, A6, and AS desaturase was malian systems a high carbohydrate/low fat diet studied. results in enhanced activity of acetyl-CoA carboxylase (1), fatty acid synthetase (1), The Influence of Dietary Fats on Desaturase Activity elongase (6), stearoyl-CoA desaturase (2), and Rats were fed ad libitum for fourteen days the biosynthesis of triacylglycerol (7,8). How- either a control diet or diets in which 20% of ever, little is known about the detailed control the diet as starch had been replaced by an equal mechanisms which enhance enzyme activity or weight of various lipids or sucrose. The specific result in the biosynthesis of new enzyme pro- activities of the various enzymes relative to tein. It has been suggested that high carbo- their value on the purified diet are given in hydrate diets could result in elevated levels of Table I. The most pronounced effects are seen citrate which is known to activate acetyl-CoA with the high carbohydrate]low fat diets which carboxylase (9). This enzyme is thought by elevate the levels of fatty acid synthetase and some to be the rate-limiting step in the bio- the A9 desaturase but have comparatively little synthesis of saturated fatty acids (9) and thus effect on the activities of the A6 and the A5 an increase in this activity would result in an desaturases. Conversely, a high fat diet seems to overall increase in fatty acid synthesis. This in stimulate the activity of the A5 desaturase and FIG. 1. The effect of dietary carbohydrates on the activity of fatty acid synthetase and stearoyl-CoA desaturase. In the continuous feeding (A) and starving/ refeeding (B) situation fatty acid synthetase activity is expressed as #moles NADPH oxidized per min per g liver (tJ) and stearoyl-CoA desaturase as nmoles oleate produced per min per mg microsomal protein (-). 1. Purified diet, 2. 74% (w/w) sucrose, 3. 74% (w/w) glucose, 4. 74% (w/w) fructose.

LIPIDS, VOL. 12, NO. 6



TABLE I The Influence of Dietary Fat on the Levels of Hepatic Fatty Acid Synthetase, A9, A6, and A5 Desaturase Diet


A9 b

A6 c

A5 d

Control Sucrose Tallow Hydrogenated tallow Corn oil Triolein Spital

1.00 2.29 0.34 0.46 0.19 0.40 0.34

1.00 1.54 0.41 0.81 0.25 0.56 0.30

1.00 1.25 0.99 1.07 0.75 0.88 0.59

1.00 1.08 1.18 1.58 1.72 2.45

aFatty acid synthetase 1.35 #moles NADPH oxidized per min per g liver. bStearoyl-CoA desaturase 1.33 nmoles oleate produced per min per mg protein. CLinoleoyl CoA desaturase 284 ttmoles 3'-linolenate produced per min per mg protein. dEicosatrienoic acid desaturase. 893 pmoles arachidonate produced per m i n per mg protein. TABLE II Dietary Intake Expressed as g Fatty Acid Ingested per Rat Over 14 Days




Control Sucrose Tallow Hydrogenated tallow Corn oil Triolein Spital

0.17 0.12 8.70 11.86 3.56 0.48 2.09

0.06 0.04 6.91 24.98 0.51 0.48 0.24

i n h i b i t t h e activity of t h e f a t t y acid s y n t h e t a s e a n d t h e A 9 desaturase. It is p e r h a p s n o t surprising t h a t d i e t a r y fats limit t h e activity of t h e f a t t y acid s y n t h e t a s e , b u t a n o b v i o u s e x p l a n a t i o n f o r t h e a p p a r e n t s t i m u l a t i o n of t h e A 5 d e s a t u r a s e activity is less clear. However, f r o m the k n o w n i n h i b i t o r y effect of p a l m i t i c acid a n d p a l m i t o y l - C o A o n t h e f a t t y acid s y n t h e t a s e a n d a c e t y l - C o A c a r b o x y l a s e , it m i g h t b e predicted t h a t diets w i t h high levels of this f a t t y acid w o u l d be t h e m o s t effective in c o n t r o l l i n g t h e activities of these t w o e n z y m e s . F r o m T a b l e II, it is a p p a r e n t t h a t this is n o t t h e case a n d in fact t h o s e rats fed a c o r n oil s u p p l e m e n t e d diet s h o w e d t h e l o w e s t activity. F u r t h e r m o r e , t h e rats fed a Spital diet (a c o m m e r c i a l diet comp o s e d o f high c a r b o h y d r a t e a n d low fat) s h o w e d c o n s i d e r a b l y depressed levels of f a t t y acid s y n t h e t a s e unlike t h o s e a n i m a l s fed a sucrose s u p p l e m e n t e d diet. Since t h e h i g h c a r b o h y d r a t e effect was b e i n g c o m p e n s a t e d b y s o m e o t h e r c o m p o n e n t , a t t e n t i o n was f o c u s e d u p o n t h e f a t t y acid intake. C o n s i d e r i n g t h a t rats o n a c o r n oil s u p p l e m e n t e d diet s h o w e d t h e lowest f a t t y acid s y n t h e t a s e activity, it s e e m e d likely t h a t t h e relatively h i g h linoleic acid i n t a k e m i g h t be playing a c o n t r i b u t o r y role.

Fatty acid 18:1



0.15 0.12 13.75

0.10 0.05 1.15

8.45 19.46 2.0


0.60 0.40 33.9 40.6 31.8 21.0 8.0


The Influence of Dietary Ethyl Linoleate on Fatty Acid Synthetase and A 9 Desaturase

Six male l i t t e r weanlings were a l l o c a t e d to f o u r d i e t a r y groups. G r o u p 1 was fed ad l i b i t u m a 20% ( w / w ) s u p p l e m e n t e d diet a n d G r o u p s 2-4 20% ( w / w ) sucrose w i t h 0.5, 1.0 a n d 1.5% ( w / w ) e t h y l linoleate, respectively. A t t h e e n d o f 14 days t h e rats were killed, t h e i r livers removed, homogenized and fractionated to allow m e a s u r e m e n t s of t h e f a t t y acid s y n t h e tase a n d s desaturase to be m a d e . T h e results are s h o w n in Figure 2. A c o n t r o l e x p e r i m e n t was also set u p in w h i c h t h e e t h y l linoleate was replaced by ethyl palmitate. In b o t h series of e x p e r i m e n t s , a f i f t h g r o u p were fed o n Spital. F r o m these results, it was apparent that dietary ethyl linoleate could i n f l u e n c e t h e a c t i v i t y of b o t h f a t t y acid synt h e t a s e a n d t h e A9 desaturase. E t h y l p a l m i t a t e over a similar c o n c e n t r a t i o n range h a d n o e f f e c t at all o n t h e f a t t y acid s y n t h e t a s e b u t p a r t i a l l y i n h i b i t e d t h e A 9 d e s a t u r a s e activity. T h e s e results are in a g r e e m e n t w i t h t h e view t h a t s h o r t t e r m c o n t r o l o f f a t t y acid s y n t h e t a s e is at t h e level of a c e t y l c a r b o x y l a s e m a n i f e s t e d via t h e d i s s o c i a t i o n / a s s o c i a t i o n o f t h e e n z y m e p r o t o m e r s whereas the long t e r m c o n t r o l is at LIPIDS, VOL. 12, NO. 6




A 9 desat a









20 s





Fatty add ingested (gl

0 1 2 3 Fatty acid ingested (g)

FIG. 2. The effect of dietary fatty acid ethyl esters on the activity of fatty acid synthetase and stearoylCoA desaturase. Data are expressed as percentage activity, relative to the enzyme activity determined from rats on a fat-free diet, as a function o f the total amount of the specific fatty acid ingested per rat during the fourteen day feeding trial. The open symbols (o) refer to the enzyme activities when the diets were supplemented with ethyl palmitate and the closed symbols (e) ethyl linoleate. The 100% value is obtained from animals fed a diet consisting of 20% (w/w) sucrose at the expense o f the starch. The closed symbols corresponding to 2.7 g of ingested ethyl linoleate (s) represent the value of ingested palmitic and linoleic acids from the Spital diet.

the level of the fatty acid synthetase. The former was brought about by competition between palmitic acid and citric acid and the latter by induced enzyme synthesis possibly under hormonal control. The A9 desaturase is thought to be induced via adaptive enzyme formation independently of hormonal control by the increased flux of saturated fatty acids

produced by the enhanced activity of fatty acid synthetase. These observations have come from the work of Mercuri et al. (10) who showed that in the normal or diabetic rat the level of A9 desaturase activity could be raised by f e e d i n g saturated fatty acids. Our results reported here suggest that palmitic acid, while not affecting fatty acid synthetase activity, actually diminished the activity of the A9 desaturase. However, it was only about onefifth as effective as linoleic acid, and it is clear from the data shown in Figure 2 that it is not the palmitic acid content of the Spiral diet which is contributing to the depressed level of the enzyme activities. Extrapolation of the experimental data would indicate that linoleic acid is the major contributing fatty acid. A recent report by Huntley (11) refers to the close correlation between the activities of acetyl-CoA carboxylase, fatty acid synthetase, and the A9 desaturase in maternal, foetal, and neonatal rats. Furthermore, Huntley also refers to the linoleic acid sensitivity of the desaturase, an observation which has been referred to by Muto and Gibson (12) and tentatively by Inkpen et al. (13). In these last experiments, the level of A9 desaturase was compared in groups of rats fed either stimulatory amounts of hydrogenated coconut oil or diets in which this fat had been replaced by 5% increments of safflower oil. The authors conclude that the decrease in desaturase activity is a function of the increasing linoleic acid content of the diet due to the safflower oil although the results could equally well be explained in terms of decreasing amounts of coconut oil. Our data based on feeding experiments utilizing carboh y d r a t e fat free diets supplemented with increasing amounts of ethyl linoleate enable us

TABLE III Food Intakes and Body Weights of Rats Fed a High Carbohydrate Diet Supplemented with Either (A) Ethyl Palmitate or (B) Ethyl Linoleate a Control


Fatty acid supplemented diet

(A) Ethyl Paimitate Food intake (g) Initial body wt. (g) Final body wt. (g) Increase in body wt.

238 59 135 2.3

282 58 141 2.4

232 58 138 2.4

226 60 140 2.3

217 59 134 2.3

(B) Ethyl Linoleate Food intake (g) Initial body wt. (g) Final body wt. (g) Increase in body wt.

176 42 106 2.5

206 42 117 2.8

170 41 102 2.5

173 41 104 2.5

153 40 100 2.5

aAll values represent an average of six determinations--one from each of the six animals in each dietary group. Food intakes are expressed as the average value of food ingested per rat over the fourteen day feeding trial. The three sets of data for fatty acid supplemented diets represent diets supplemented with ca. 0.5, 1.0, and 1.5% fatty acid ethyl ester. LIPIDS, VOL. 12, NO. 6



TABLE IV to quantitate the magnitude of the effect of this fatty acid on the fatty acid synthetase and The Inhibitory Effect o f Dietary Corn Oil on A9 desaturase. It can be calculated from the the Induction o f Fatty Acid Synthetase and known food intakes, Table III, and the perA9 Desaturase by Glucose and Fructose centage of fatty acid composition of the diet, Diet FASa A9 Desaturase b that for every 3 g of linoleic acid ingested, each rat also ingest 54 g of sucrose during the 14 day 20% glucose 2.1 2.9 feeding period. From these observations, it can glucose 0.8 1.5 be shown that linoleic acid on a weight for 20% 3% corn oil weight, calorie for calorie, or mole for mole 20% fructose 2.2 4.6 basis is 18, 8, and 15 times more effective, 20% fructose respectively, in repressing these enzymes than 3% corn oil 1,2 3.2 sucrose is in inducing them. aFatty acid synthetasr is expressed as ~moles The nature of the linoleic acid control is not NADPH oxidized per min per g liver. yet understood and so a possible link between bA9 Desaturase is expressed as n m o l e s oleate prothe inhibitory effect of this essential fatty acid duced per rain per mg microsomal protein. and the stimulatory effect of carbohydrate was sought. Mercuri et al. (10) have shown that in average U.K. food intakes calculated from the t h e diabetic rate 75% of the normal stearoylHousehold Food Composition and ExpendiCoA desaturase activity can be restored by ture, HMSO, 197 I, show that the actual value is feeding fructose which is known to be metabonly 10-15 g of linoleic acid per day. The daily olized by insulin independent reactions in the carbohydrate intake calculated from the same liver (14,15). It has also been demonstrated source indicated ca. 30% of the 300 g of that during fasting the blood insulin levels ingested carbohydrate is m o n o - a n d disacchadecrease as does the liver fatty acid synthetase ride. From Figure 2 it has been calculated that and the A9 desaturase activities. After feeding, 1 g of linoleic acid can completely suppress the t h e levels of the enzymes and the hormone rise inductive effect of 18 g of sucrose. The 10-15 g in parallel. If the inhibition of the lipogenic of linoleic acid ingested daily by man should, enzymes by dietary linoleic acid is a reflection on this basis, assuming a similar metabolism and of the insulin production, then elevated levels control as in the rat, be capable of suppressing of fatty acid synthetase and the A9 desaturase the inductive effect of 180-270g of dietary brought about by dietary fructose should not sucrose. From the data quoted for the dietary be repressed by dietary linoleic acid. In order to i n t a k e o f m o n o - a n d disaccharides (ca. test this possibility, four groups of rats were fed 100-200 g per day), it becomes apparent that either 20% glucose, 20% glucose and 3% corn the daily intake of essential fatty acid should be oil, 20% fructose, or 20% fructose and 3% corn adequate to prevent over-synthesis of fatty acid oil. The results are shown in Table IV. They and synthetase and A9 desaturase. show that dietary corn oil (60% linoleic acid) is less inhibitory when fructose rather than glucose is used to induce these two enzymes. REFERENCES However, the data do suggest that the major inhibitory effect of the linoleic acid cannot be I. Volpe, J.J., and P.R. Vagelos, Physiol. Rev. 56:339 (1976). explained by a mechanism which involves the 2. Oshino, N., and R. Sato, Arch. Biochem. Biophys. control of insulin. 149:369 (1972). Finally, it is worth considering the quantita3. Gibson, D.M., R.T. Lyons, D.F. Scott, and Y. tive controlling effect of the dietary linoleic Muto, Adv. Enz. Regul. 8:187 (1972). 4. Bruckdorfer, K.R., 1.H. Khan, and J. Yudkin, acid and its possible relevance to the human Biochem. J. 129:439 (1972). situation. From Figure 2 it has already been 5. Jeff coat, R., P.R. Brawn, and A.T. James, calculated that dietary linoleic acid has a sensiBiochim. Biophys. Acta 431:33 (19"/6). tive control over certain lipogenic enzymes. On 6. Sprecher, H., Ibid. 360:113 (1974). 7. Waddell, M., and H.J. Fallon, J. Clin. Invest. a weight for weight basis, it can be shown that 52:2725 (I 973). 2-2.5 g linoleic acid ingested per day per kilo 8. Aas, M., and L.N.W. Daae, Biochim. Biophys. b o d y weight can completely suppress the Acta 239:208 (1971). inductive effect of 20% dietary sucrose on the 9. Vagelos, R.R., MTP International Review of Science, Biochemistry Series !, Biochemistry o f hepatic fatty acid synthetase and A9 desaturase Lipids 4:99 (I 974). activity in the rat. Extrapolation of these values 10. Mercuri, O., R.O. Peluffo, and M.E. de Tomas, would mean that the daily intake of a 75 kiloBiochim. Biophys. Acta 369:264 (1974). gram man would have to be 150-190 g linoteic 11. Huntley, T.E., Fed. Proc. Fed. Am. Soc. Exp. Biol. 35:1526 (1976). acid per day. This is clearly meaningless since LIPIDS, VOL. 12, NO. 6



12. Muto, Y., and D.M. Gibson, Biochem. Biophys. Res. C o m m u n . 38:9 (1970). 13. Inkpen, C.A., R.A. Harris, and F.W. Quackenbush, J. Lipid Res. 10:277 (1969). 14. Renold, A.E., A.B. Hastings, and F.B. Nesbett, J.

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Biol. Chem. 209:687 (I 954). 15. W i c k , A.N., J.W. Sherrill, Diabetes 2:465 (1963).



[ Received November 8, 1976]


Interrelationship between the dietary regulation of fatty acid synthesis and the fatty acyl-CoA desaturases.

Interrelationship between the Dietary Regulation of Fatty Acid Synthesis and the Fatty AcyI-CoA Desaturases R. JEFFCOAT and A.T. JAMES, Basic Studies...
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