25

Biochimico et Biophysics Acta, 441 (1976) 25-31 0 Elsevier Scientific Publishing Company, Amsterdam

- Printed

in The Netherlands

BBA 56818

LINOLEIC ACID DESATURATION ACTIVITY OF LIVER MICROSOMES OF ESSENTIAL FATTY ACID DEFICIENT AND SUFFICIENT RATS

RACL

0. PELUFFO

*, ANiBAL

M. NERVI

* and RODOLFO

R. BRENNER

*

Cdtedra de Bioquimica, Instituto de Fisiologia, Facultad de Ciencias Mbdicas, Universidad National de La Plats, La Plats (Argentina) (Received

December

24th,

1975)

Summary Studies were carried out to relate the changes of the fatty acid and lipid composition of rat microsomes with the modification of the activity of the linoleic acid desaturation evoked by an essential fatty acid deficient diet. Two steps were shown in the progression of the essential fatty acid deficiency. In a first step shown at three days of essential fatty acid deficiency the fatty acid composition was changed by decreasing linoleic and arachidonic acids and increasing oleic and eicosatrienoic (n-9) acids. No change was found in the lipid distribution and approximate V and K, of the linoleic acid desaturation. In this first step the unsaturated/saturated fatty acids ratio fell in spite of the synthesis of eicosatrienoic (n-9) acid that was produced without any change of enzyme activity. In a second step shown at 15 days of essential fatty acid deficiency the change of the fatty acid composition was greater but the unsaturated/saturated acid ratio was restored. An increase of triacylglycerols and a decrease of phospholipids was also detected together with an enhanced activity of linoleic acid desaturation (higher approximate V) and a higher approximate

K III*

The increase of the V of linoleic acid desaturation is considered to be evoked by an increased level of active A” desaturase. The increased activity of the A6 desaturase in this second period is a secondary and important response of the cell to maintain the unsaturated : saturated acid ratio and fluidity of the membrane .

Introduction Both stearoyl-CoA desaturase bound to the cellular endoplasmic * Member of the “Carrera y T6cnicas”. Argentina.

de1 InvestiRador”

and linoleoyl-CoA desaturase are enzymes reticulum [ 1,2]. However, they show differof the “Consejo

National

de lnvestigaciones

Cientfficas

26

ent responses to some dietary and hormonal stimuli. The A9 desaturase is stimulated by a carbohydrate-rich diet and is not modified by a diet containing excess protein [3,4], On the other hand, the activity of the A6 desaturase is enhanced by the excess protein diet and inhibited by the carbohydrate-rich diet [3,4]. High insulin doses reactivate better the stearoyl-CoA desaturase [5,6] than the linoleoyl-CoA desaturase of diabetic rats. Glucagon, by means of its second messenger, cyclic AMP, inhibits the reactivation of the A6 desaturase evoked by refeeding fasted animals, whereas the A9 desaturase is not affected 171. However, both enzymes respond similarly to other variables. They are stimulated by an essential fatty acid deficient diet and depressed by fasting [ 8101. Stearic acid desaturation is associated with a NADH-linked microsomal electron transport system constituted by the NADH : cytochrome b5 reductase, cytochrome b5 and a cyanide sensitive factor that is the desaturase [ll131. The electron transport system requires lipids [ 14-161. However, Holloway and Holloway [9] have shown that the activation of stearoyl-Coil, desaturation evoked by an essential fatty acid deficient diet compared to animals fed a sunflower oil supplemented diet is not a direct consequence of the different fatty acid composition of the microsomes. When this last work appeared we were engaged in the study of the effect of essential fatty acids on the A* desaturase. The A6 desaturase is also apparently linked to the NADH microsomal electron transport system and is inhibited by CN- [17]. Since liver microsomes from rats fed an essential fatty acid deficient diet contain more A* desaturase activity than microsomes derived from animals fed a sunflower oil supplemented diet [lo] a comparative study is made here to relate the kinetic properties of the enzyme with changes of microsomd lipid composition induced by the aforementioned diets. Materials and Methods An~~u~~. Immediately after weaning male Wistar rats were divided into groups of five animals each. A control diet containing sucrose (75 g), lipid free casein (16 g), sunflower seed oil (3 g), minerals (4 g) and a mixture of vitamins and casein (2 g) [lo] was administered ad libitum to two groups of rats. The other four groups were fed on essential fatty acids free diet in which 3 g hydrogenated coconut oil was used instead of the sunflower seed oil. After three days the two control groups and two of the essential fatty acid deficient groups were killed, while the other two deficient groups were killed 15 days later. No significant difference was found in the weight of the rats fed on the different diets. Preparation of liver microsomes. Rat livers were immediately washed in icecold 0.25 M sucrose after decapitation, dried on filter paper and weighed. The liver of each animal was homogenized and the microsomes separated by differential centrifugation at 100 000 X g [19]. Proteins were assayed by the biuret method [IS]. A6 desuturuse assay. [l-‘4C]Linoleic acid (56 Ci/mol, 99% pure, Amersham Searle, Amersham, England) was diluted with unlabeled acid (3 : 7). 25, 50, 60 and 70 nmol were incubated with 5 mg microsomal protein for 10 min at 35°C in a total volume of 1.5 ml. The incubation solution contained in BmoI: ATP,

27

4; CoA, 0.2; NADH, 0.8; MgClz, 7.5; KCl, 75; N-acetylcysteine, 2.8; NaF, 62.5; nicotinamide, 0.5; phosphate buffer (pH 7.0), 62.5. The reaction was started by the addition of the microsomes and stopped with KOH solution. Livers of five animals for each diet were incubated individually in duplicate. After incubation the fatty acids were converted to the methyl esters and the radioactivity distribution measured by gas-liquid radiochromatography in a Packard apparatus provided with a proportional counter [lo]. The R, and V were calculated by the Lineweaver-Burk plot using a computer. Lipid co~~osi~io~ of the microsomes. The microsomes of each group of rats were alotted in pools of five specimens each. The total lipids were extracted with chloroform/methanol (2 : 1, v/v) by the method of Folch et al. [19] and weighed. No difference was found in the lipid : protein ratio among the groups. 10 mg of lipids dissolved in 95% ethanol were hydrogenated for 2 h with platinum oxide catalyst [20]. The hydrogenated lipids were extracted with chlorofo~/methano~ (2 : 1, v/v), washed a&taken to a final volume of 1.2 ml. 20 crl of the hydrogenated lipids were separated in a chromatoplate (20 X 40 cm) with a 0.25 mm layer of Silica Gel G. Standards of lysophospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, diacylglycerol, triacylglycerol, cholesterol, and cholesterol esters were also added. The plate was developed first with chlorofo~~meth~ol~water (65 : 25 : 4, v/v). When the solvent reached a distance of 19 cm the plate was dried and developed again with light petroleum (b.p. 30-60”C)/ethyl ether/acetic acid (80 : 20 : 2, v/v) until the top mark was reached. The spots were charred by spraying with sulphuric acid/potassium dichromate reagent and heating 1201. Once the spots were identified, a new plate previously washed with chloroform/meth~ol (80 : 20, v/v) was used for the qu~titative.~~ysis. lo,20 and 30 ~1 of the hydrogenated samples were separated by the same thin layer chromatography system, using standards of stearic acid of 5, 10 and 15 c(g of carbon. After charring, densitometry was carried out with a Zeiss spectrometer specially designed for this purpose. Quantification was done on the basis of the areas of peaks 1201 and good linearity was obtained for the amounts tested. The I.rg carbon were converted to mg lipids in considering that the mean molecular weight of the attached fatty acids corresponded to stearic acid. When cholesterol and free fatty acids showed a bad separation, they were determined by thin layer chromatography using 20 X 20 cm plates and developed with light petroleum/ethyl ether/acetic acid (80 : 20 : 2, v/v). Fatty acid composition of lipid fractions. Non-hydrogena~d microsomal lipids were separated by thin-layer chromatography. Neutral lipids were separated with light petroleum/ethyl ether/acetic acid (80 : 20 : 2, v/v). Polar lipids were fractionated with chloroform/methanol/water (65 : 25 : 4, V/V). The spots were sucked off and the lipids saponified under nitrogen and converted to the methyl esters. The fatty acid composition was determined by gas-liquid chromato~phy in a Packard apparatus. Results The early effects of an essential fatty acid deficient diet compared to an essential fatty acid sufficient diet on the approximate fi;, and V of the linoleic

28 TABLE

I

APPROXIMATE RATS

FED

c’

K,

AND

A SUFFICIENT

OF

+INOLEIC

DIET

UR

ACID

AN

DESATURATION

ESSENTIAL

FATTY

IN

ACID

LIVER

MICROSOMES

DEFICIENT

DIET

OF

FOR

3 AND

15 DAYS Results mals

are calculated

analysed

--

by

individually

computation

from

in duplicate

C S.D.

four

different

concentrations.

They

are the mean

five

.

-. Sufficient

Deficient

diet

15

__._...

mol/min/mg

protein)

Km (10-5M)

ani-

__ diet

3 days V (lo-’

of

days

0.70

’ 0.05

0.8

t 0.04

3.5

* 0.3

2.80

7 0.05

2.6

+ 0.04

10.7

- 0.3

acid desaturation of rat microsomes are reported in Table I. Although the Km and V values measured are only approximate due to the complexity of reactions taking place in the microsomes, it has been repeatedly shown that they are adequate to evaluate enzyme behaviour [21,22]. Administration of an essential fatty acid deficient diet for three days, compared to animals fed on an essential fatty acid sufficient diet, did not bring about changes in Km or V of linoleic acid desaturation. A longer period was necessary: at 15 days an increase of both Km and V was detected in the animals fed an essential fatty acid deficient diet. These changes may suggest a decrease in affinity of the enzymes for the substrate and at the same time an increase in the activity of the reaction. The lipid composition of the microsomes is shown in Table II. Changes in the lipid distribution in the microsomes of the animals that received either of the two diet8 during three days were not apparent. However, significant changes were detected when the animals were administered the essential fatty acid deficient diet for 15 days. The essential fatty acid deficient diet evoked an increase of the triacylglycerol and a decrease of the phosphatidylcholine.

TABLE

II

LIPID

DISTRIBUTION

IN

RAT

LIVER

of two

pools

analyzed

MICROSOMES

IN

EARLY

ESSENTIAL

FATTY

ACID

DE-

FICIENCY Results --.

-

are the mean -.

.

_

in triplicate

t S.D.

. .Sufficient

diet

Deficient

diet

(%)

(%b) 15 days

3 days

--

-_ 4.0

Lysophospholipids

50.0

Phosphatidylcholine Phosphatidylethanolamine

7.0 0.8

Phosphatidylserine

19.5

Triac~lgl~cerol

.! 0.3 i

3.9

1.2

49.8

f 0.7

6.3 0.8

’ 0.05

20.3

? 0.8

.

0.1

3.4

? 0.9

42.0

I 1.7

6.4

+ 0.5

’

0.3

5 0.05 + 0.9

0.8 26.1

+ 0.3

5 0.03 ? 0.4

Diacyklycerol

1.5

+ 0.3

1.4

f

0.1

2.1

* 0.3

Cholesterol

7.5

+ 1.0

7.7

+ 0.8

10.7

* 0.7

4.5

k 0.6

4.6

t 0.7

3.5

f 0.5

’

5.2

t 0.9

4.0

- 0.7

Cholesterol Free _____

fatty

esters acids ._._

.-....__--.

5.2 _._. _..._

0.6

._

TABLE III FATTY ACID COMPOSITION OF THE LIPIDS OF RAT LIVER MICROSOMES OF ESSENTIAL FATTY ACID DEFICIENCY

IN EARLY

PERiON

Minor components make up for 100% Fatty acids

Pha@atidylcholine

Phosphatidylethanolamine

Free acids

Macyl&cerol

Suffi-

Sufficient

Sufficient

Sufficient

Deficient

eient

--

-~

~_____“^___

14 : 0 16 : 0 16 : 1 18 : 0 18: 1 16: 2 20 : 3 20 : 4

_-_..

1.0 30.0 2.5 24.2 1’1.9 6.6 2.3 18.9

-,

3

16

days

days -.--.

1.0 32.2 4.6 24.7 17.9 3.6 5.9 10.3

0.7 26.6 5.0 23.2 19.0 2.7 13.5 9.2

.-._-.

Deficient

..__.

0.5 29.4 1.2 33.6 7.8 6.1 0.3 21.0

0.9 30.5 4.3 28.3 13.2 4.5 0.6 17.7

_ ..--.

0.4 26-O 2.7 22.6 13.9 1.7 14.1 16.3

.--.

3 days

15 days

1.6 31.3 3.5 31.1 17.6 3.7 3.8 2.9

1.8 29.3 3.5 35.7 10.7 2.9 3.7 2.9

--_,

-. 1.4 32.9 4.7 26.8 13.9 6.1 3.2 6.0

.~...

Deficient -

--

3 15 days days ..-... ----..

,.-_

Deficient

--_

1.3 41.0 6.4 13.5 27.8 4.1 2.0 1.6

3 days

15 days

3.6 37.7 8.5 10.7 29.9 3.0 1.6 0.9

3.1 40.0 8.2 6.# 37.0 3.0 3.1 0.8

* Period of essentiat fatty acid deficiency.

Therefore, it is significant to remark that the changes of the kinetic properties of the linoteic acid desatu~tiun were found when the lipid composition of the microsomes was altered and an increase of neutral lipids was evoked by the essential fatty acid deficient diet. The earliest changes evoked on the microsomes by the essential fatty acid deficient diet were the alterations of the fatty acid composition of the lipids (Table III). These modifications are already very significant on the third day of essential fatty acid deficiency, or even earlier, and increase with time of deficiency. The changes were typical of essential fatty acid deficiency and were expressed by an increase of oleic and eicosatrienoic acids and a decrease of linoleic and arachidonic acids. They were rather similar in ah the microsomal lipids. Three days of essential fatty acid deficiency evoked a drastic fall of the double bond index : saturated acid ratio of total fatty acids in the microsomes from 2.2 to 1.2. But at 15 days the 2.2 value was re-established. The ratios were calculated from the total fatty acid composition of the microsomes (not shown in the present work). The changes in the fatty acid composition caused the modification of the lipid distribution shown in Table II. However, the redistribution of the lipids with an increase of triacylglycerol : phospholipid ratio is only found at 15 days of essential fatty acid deficiency. A significant increase of the ~ia~y~lycerol of the liver evoked by a fat free diet has been shown to take place only after approximately IO days of deficiency [ 23 f . Therefore, comparing results from Tables I, II and III it is obvious that the change of the fatty acid composition of the microsomes is not the direct cause of the increase of the A6 desaturation activity of the membranes evoked by the essential fatty acid deficient diet.

30

Discussion Diets with different fatty acid compositions significantly alter hepatic lipogenesis [24] and A6 and A9 desaturation of fatty acids (Table I) [8-lo]. The increase of the A9 desaturation elicited by fat free diets, essential fatty acid deficient diets or diets poor in polyunsaturated acids [3,8] may lead to maintain the appropriate “fluidity” of membranes by an increased synthesis of monounsaturated fatty acids and therefore to maintain the proper unsaturated : saturated acid ratio. The increase of the A6 desaturase activity evoked by a fat-free or essential fatty acid free diet [lo] may play the same role. This mechanism may be relevant since, as shown by Brenner [ 251, the A6 desaturase is the main enzyme that regulates the biosynthesis of polyunsatvrated fatty acids. It has been already mentioned that both A6 and A’ desaturases are membrane-bound enzymes that receive the electron from NADH-linked microsomal electron transport system. Not only is the membrane a lipoproteic system but it has been also shown that two steps of the electron transport chain require lipids. One is the NADH : cytochrome b, reductase [ 14,151 and the other is located between the cytochrome b, and oxygen [ 161. The importance of lipids in the membrane structure and in the nonpolar interactions with the desaturating enzymes and electron transport system are proved by the necessity of detergents to solubilize or partially solubilize these components [ 11,26-281. Therefore, the kinetic properties of the linoleic acid desaturase or other components of the electron transport system may be modified by the composition of the lipid environment. Table III shows that the fatty acid composition of liver microsomes is significantly altered after three days of essential fatty acid deficiency but this change does not yet modify either the lipid distribution of the membrane (Table II) or the K, and V of linoleic acid desaturation (Table I). Therefore, it is obvious that the change of the fatty acid composition of microsomes to a typical essential fatty acid deficient composition ratio, in which there is also an important fall of the double bond index : saturation ratio, is not enough to evoke immediate and detectable changes in the desaturase. These results agree with those of IIolloway and Holloway [9]. They showed that the fractionation and reassemblage of the stearoyl-CoA desaturating system by addition of lipids with different fatty acid composition do not alter the activity of the reaction. After three days of essential fatty acid deficiency the rat shows an active synthesis of 20 : 3 (n-9) fatty acid without any activation of the Ah desaturase. The synthesis of arachidonic acid is decreased in the mean time due to a decrease of the level of substrate. Hence the increase of 20 : 3 (n-9) fatty acid and also of oleic acid is easily explained as a consequence of a decrease of the highly competitive acids linoleic and arachidonic 1251. This mechanism intends to maintain in an early step the double bond index : saturated acid ratio and the fluidity of the lipids without resorting to an enzymatic activation. However, it fails in some way since the ratio is decreased. A second mechanism that tends to maintain the fluidity of the membrane comes into action now and is apparent in the animals after 15 days of essential fatty acid deficiency. In this case the activity of the linoleic acid desaturase is enhanced by increasing V (Table I). At the same time K, is also increased. These changes, as have been

31

shown in Tables I1 and III, take place together with a more pronounced alteration of the fatty acid composition that now evokes an increase of the triacylglycerol : phosphatidylcholine ratio. The increase of the V corresponds to an increase in the level of the active enzyme provoking an enhanced synthesis of polyunsaturated fatty acids. The enhancement is shown by a re-establishment of the double bond index : saturated fatty acid ratio. The change of the activity of V and of K, may be evoked either on the A6 desaturase or on the components of the electron transport system in the case when they were the limiting factors of the reaction chain. Acknowledgements This work was supported in part by grants from the Consejo National de Investigaciones Cientificas y Tecnicas (Argentina) and Ministerio de Salud Publica de la Nation. Technical help was provided by Osvaldo Almeira. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 16 I7 18 19 20 21 22 23 24 25 26 27 28

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