Vol. 40, No. 4, November 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages

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1 Vol. 40, No. 4, November 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages SHORT-TERM EFFECTS OF DIETARY FATS ON THE LIPID COMPOSITION AND DESATURASE ACTIVITIES OF RAT LIVER MICROSOMES M.D. Gir6n*, A. Lara and M.D. Su(trez Department of Biochemistry and Molecular Biology. School of Pharmacy. University of Granada. Campus de Cartuja s/n GRANADA. Spain Received September 23, 1996 SUMMARY. Diets supplemented with 10% coconut, olive or sunflower oil were given to rats at weaning. After two, four and six days, the lipid composition and desaturase activities of liver microsomes were measured. The percentage of oleic acid and A9-desaturase activity were increased in animals fed an olive oil diet while animals given sunflower oil showed the highest content of linoleic and polyunsaturated n-6 fatty acids. On day 6, olive oil-fed rats had the highest levels of 22:6 n-3 in the phosphatidylethanolamine fraction. Saturated fatty acids were similar among dietary groups, despite the marked differences in the saturated fatty acid content of the three oils. Polyunsaturated n-6 and n-3 fatty acids decreased during the six days of feeding with coconut oil. Our results show that liver microsome membranes respond to different dietary fatty acids sources by changes in enzyme activities and relative content of some fatty acids after a period of dietary manipulation as brief as 6 days. KEY WORDS: Fatty acids, Liver, Desaturase, Microsomes, Coconut oil, Olive oil, Sunflower oil INTRODUCTION The stability and pelxneability of membranes in the endoplasmic reticulum depend on fatty acid composition and cholesterol content, the former being markedly influenced by the nature, degree of unsaturation, and arrangement of the constituent fatty acids. Liver endoplasmic reticulum an important role in fatty acid biosynthesis and in the processing of dietary fatty acids that reach the liver as remnant chylomicrons and other lipoproteins (1). The enzymes responsible for processing fatty acids are malonyl-coa-dependent elongases and desaturases, both located in the endoplasmic reticulum (2). Desaturases are key enzymes in the regulation of unsaturated fatty acids biosynthesis, their activity being regulated mostly by hormonal and dietary changes (3). Most dietary supplementation studies have involved long periods of time (more than 2-3 weeks) (2,5-9). Few studies have investigated the incorporation with time of fatty acids, into tissue phospholipids after adaptation to a fat-free diet (10,11). We studied the extent of modifications in liver fatty acid composition and desaturase activities during dietary manipulation in rats. The present study / /0 Copyright 1996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 evaluates the effect of a short period of feeding with qualitatively different lipid dietary intakes [coconut oil (rich in saturated fatty acids and devoid of n-6 and n-3 unsaturated fatty acids), olive oil (rich in monounsaturated fatty acids and main component of the Mediterranean diet) and sunflower oil (rich in n-6 polyunsaturated fatty acids)] on the fatty acid composition of liver microsomes. We also studied A 9- and A6-desaturase activities, since these two enzymes play key roles in the fatty acid metabolism. Moreover, the desaturation system is embebed in the lipid bilayer of the mierosomal membrane and the fatty acid composition of phospholipids may play a role in desaturase activity. Minor changes in fluidity of the lipid bilayer may be evoked by diet changes and other factors (2,3). MATERIAL AND METHODS Chemicals: [ 1-14C]Palmitic acid (58 mci/nmol; 97.1 radiochemical purity) and [ 1-14C]linoleic acid (59 mci/nmol; 98.8 radiochemieal purity) were purchased from Amersham International (Amersham, England). Unlabeled fatty acids, coenzyme A, bovine fatty acid free serum albumin, ATP and NADH were purchased from Sigma Chemicals Co. (St Louis, MO). All others chemicals were of analytical grade. Animals and Diets: Male weanling Wistar rats weighing g were housed with a 12-h lightdark cycle and randomly divided into three groups of 21 animals. Each group was fed the same basal diet with a different 10% (by weigh0 fat supplement: coconut oil (CO), olive oil (OO) or sunflower oil (SO). The composition of the basal diet was 65.6% starch, 10% sucrose, 10% vitamin-free casein, 8% cellulose, 4.5% mineral mix, 1% vitamin mix, 0.5% DL-methionine and 0.33% choline bitartrate (11). The fatty acid composition of the three diets is shown in Table 1. Rats were fed fresh food daily. All animals had free access to food and water. Animals were studied in compliance with our institution's guidelines for animal research. After feeding the experimental diets for two, four or six days, 12-h fasted rats were killed by decapitation and the livers were rapidly excised. A group of seven weanling rats were also killed under the same conditions. Immediately after the liver was removed, microsomes were prepared as described (6). Microsomal protein was assayed by the method of Lowry et al. (12) with bovine serum albumin as a standard. Microsomal lipid composition: Lipids were extracted with chloroform:methanol (2:1, v/v) containing butylated hydroxy-toluenei(50 mg/1) as an antioxidant (13). Main liver membrane phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) were separated by onedimensional thin layer chromatography on Silica Gel G 60 as described (5). The areas of silica gel containing PC and PE were visualized by exposure to iodine vapors, scraped off and analyzed for fatty acid composition. Fatty acids were transmethylated according to Morrison and Smith (15). Fatty acid methyl esters were analyzed by gas-liquid chromatography (Hewlett Packard, model 5880 A) as previously described (4) and expressed as the percent distribution of fatty acid methyl esters. Enzyme assays: The A9- and A6-desaturation of fatty acids of liver microsomes was measured by estimating the percentage conversion of [1-14C]palmitic acid to palmitoleic acid and [1-14C]linoleic to "t-linolenic acid, respectively, as described previously (5,7). Statistical analysis: The results shown are means + SEM. The effects of the dietary treatments and time were examined by one-way analysis of variance followed by a Tukey test. Analyses were done with the BMDP Computer Software program (BMDP Statistical Software, Cork, hx~land). 844

3 RESULTS The relative fatty acid composition of liver microsomal PC and PE in weanling rats and in rots fed the CO oil, OO and SO diets for two, four or six days are shown in Tables 2 and 3, respectively. Palmitic and stearic acids were the main saturated fatty acids in liver microsomes. Only changes in the total saturated fatty acids (SAT) were reported (Tables 2-3). Saturated fatty acids increased with CO feeding, and did not change with the SO diet. In PE (Table 3), these fatty acids decreased in OO-fed rats. Oleie acid (18:1 n-9) was approximately half as high in rats fed OO than in SO-fed animals (Tables 2-3). The major n-6 fatty acids in PC and PE were arachidonic acid (20:4 n-6) (between 10 and 30%) and linoleic acid (18:2 n-6) (between 2 and 14%). The levels of the other n-6 fatty acids were low. Therefot~e, only the changes of 18:2 n-6, 20:4 n-6 and docosapentaenoic acid (22:5 n-6), the end product of n-6 fatty acids, are reported. The relative content of linoleie acid was twice as high in microsomal PC and PE from animals fed the SO diet as in animals fed CO or OO (Tables 2-3). The levels of this fatty acid decreased with CO feeding in PE (Table 3) and did not change with OO or SO diet in PC (Table 2) and PE. Arachidonic acid decreased with the CO diet (between 28-35%); the changes were most pronounced after four days of treatment. 20:4 n-6 did not change in rats fed OO or SO. Both groups of animals had similar values of this fatty acid, except for the PC fraction (Table 2), where it was Table I. Fatty acid composition of diets Fatty acid Cocouut oil a Olive oil b Sunflower oil c 10: : :0 22:3 16: :1 n : :1 n :2 n :3 n : A 20: Results are expressed as file percentages of total fatty acid methyl esters, a Aeofar, Acofar SA, Barcelona, Spain. b Carbonell, Carbonell y Cia de Cordoba S.A., Cordoba, Spain. c Koipesol, Koipe SA, San Sebastian, Spain. 845

4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Table II. Fatty acid composition of liver microsomal phosphatidylcholine in weanling rats and rats fed coconut, olive or sunflower oil for two, four or six days. Fatty acid Weaning rats Two days Four days Six days SAT C ± 1.4 O 55.6 ± ± S :1 n C O a2d S 6.5 ± 0.7 c2 3.7 ± 0.2 b2c2 3.4 ± 0.6 b2c2 18:2 n ± 0.5 C O ± S 11.5 ± 1.3 c ± 0.8 b2c b2c2 20:4 n C ± ± 1.0 O 13.8 ± 1A 16.0 ± ± 0.8 S 21.3 ± ± 1.0 b2c ± 2.7 b2c2 22:5 n ± 0.2 C 1.6 ± ± ± 0.1 O S 1.9 ± 0.2(;2 1.9 :l: 0.2 c1 2.0 ± 0.2 b2c2 22:6 n ± 0.3 C 2A ± ± 0.3 dt dl O 1.5 ± ± a2 S 0.8 ± 0.1 b2cl blcl 0.5 ± 0.1 c2 PUFA n ± 1.2 C 18.0 ± ± ± 1.1 dl O ± A ± 1.2 S 24.6 ± 2.3 Cl b2c ± 2.7 b2c2 PUFA n ± 0.3 C 2.8 ± ± ± 0.1 dl O 1.6 ± 0.2 al 1.5 ± a2 S 1.3 ± 0.2 b2d2 0.8 ± 0.1 blcl 0.6 ± 0.1 c2 UNID ± 3.9 C ± ± ~ 4.1 O ± 6.5 d :l: S 138.7± b2c ± 11 b2c2dl The results are expressed as mean percent distribution + standard deviation for seven animals per group. a: Significance between the coconut and olive oil groups at the same age. b: Significance between coconut and sunflower oil groups at the same age. c: Significance between olive and sunflower oil groups at the same age. d: Significance between two consecutive ages in the same group and with respect to weanling rats. 1:p<0.05 and 2: p<0.01. UNID: unsatnra~n index. It is Y~ [ab] where a is the relative peru:enrage of each monou~*,aturated favy acid and b is the namber of double bonds for that particular fatty acid. 846

5 VoI., 40, No. 4, 1996 Table lil Fatty acid composition of liver microsomai phosphatidylethanolamine in weanling rats and rats fed coconut, olive or sunflower oil for two, four or six days. Fatty acid Weaning rats Two days Four days Six days SAT C O dl I S :1 n C 5.7± O S :2 n C O d S 5.9 ± 0.4 c2 4.7 ± 0.4 blc2 4.7 ± 0.4 b2 20:4 n _+ 0.9 C 23.5 ± ± ± 1.4 O 20.5 ± ± 1.2 S 23.9 ± ± :5 n ± 0A C ± ± 0.2 O 1.9 ± S 6.1 ± 0.5 blc2dl b2c bl 22:6 n C 6.6 ± ± 0.4 O ± a2 S 4.8 ± ± ± 0.3 c2 PUFA n ± 1.0 C 28.8 ± ± O 26.0 ± ± _+ 1.4 al S 38A ± ± 1.3 Cl PUFA n ± 0.6 C ± 0.4 O dl 8.9± ± 0.5 a2 S ± 0.3 c2 UNID ± 6.2 C ± O d ± a2 S ± 20.2 cl ± 5.6 b2 The results m-e expressed as mean percent distribution :t: standard deviation for seven animals per group. a: Significance between the coconut and olive oil groups at the same age. b: Significance between coconut and sunflower oil groups at the same age. c: Significance between olive and sunflower oil groups at the same age. d: Significance between two consecutive ages in the same group and with respect to weanling rats. 1:p<0.05 and 2: p<0.01. See Table 2 for abbreviations. 847

6 BIOCHEMISTRY ond MOLECULAR BIOLOGY INTERNATIONAL significantly increased in the SO group. The most unsaturated n-6 fatty acid, 22:5, was higher in the SO group than in the other two in PC (Table 2) and PE (Table 3). The higher amount of n-6 polyunsaturated fatty acid with more than 18 carbon atoms (PUFA) led to the greater degree of unsaturation in membranes from rats given SO. 22:6n-3 was the main n-3 fatty acid in microsomal phospholipids (Tables 2-3). PE (Table 3) showed the highest relative content. After six days of OO feeding this fatty acid increased a 50% in PE and did not change in PC (Table 2). Under the CO diet, the levels of 22:6n-3 decreased (between 38 and 72%) in all fractions assayed. This fatty acid usually decreased between days 4 and 6 in the SO group, and by day 6 was lower than the level in the OO group in PE. The unsaturation index (UNID) (Tables 2-3) reflected a fast response by the liver to dietary supplementation. After six days, CO fed rats showed a significant decrease in membrane unsatu~tion in PE (Table 3). Membrane unsaturation did not change in PC (Table 2) and increased in PE with OO feeding. In general, SO-fed rats had the highest membrane unsaturation in PC and PE except for the PE fraction on day 6 of the study. Table 4 shows the results for desaturation activity. A9. desaturase activity was lower after the onset of dietary treatment. This activity was higher after the OO diet than after the two other diets. In the SO group, A9. desaturase activity decreased significantly by day 4, and remained low on days 4 and 6. A9-Desaturase did not change with CO and OO diets. A6-desaturase activity was generally lower than A9-desaturase activity. The highest values were seen in the OO group. The CO group showed very low activity. With the SO diet, A6-desaturase activity increased, and by day 6 was similar to that found in the OO group. DISCUSSION We have previously found that the long-term manipulation of dietary fat resulted in alterations in fatty acid profiles of the liver and duodenal membrane lipids (5-7). Modifications in the composition of membranes lead to changes in the activities of bound enzymes (2,3,8). Therefore, the present study was designed to determine whether feeding with a particular diet for a shol~ period of time (up to 6 days) was associated with apparent changes in individual fatty acids or desaturasc activities in liver microsomes. 848

7 Table IV A9. and A6-desaturase activities of 'liver microsnmes in weanling rats and rats fed coconut, olive or sunflower oil for two, four or six days. Weanling Two days Four days Six days rats A _ C d dl O _ aldl _ :t: al S _+ 0.1 d _ ble2d ble2 A C d _ ( :1:0.018 O aldl al ~ a2 S :t: bleldl el b2d2 The results are expressed as means of the enzymatic activity (nmol of product produced per min per mg protein) + standard deviation, a: Significance between the coconut and olive oil groups at the same age. b: Significance between coconut and sunflower oil groups at the same age. c: Significance between ofive and sunflower oil groups at the same age. d: Significance between two consecutive ages in the same group and with respect to weanling rats. 1:p<0.05 and 2: p<0.01. Saturated fatty acids were similar in all groups despite the marked differences in the relative saturated fatty acid content of the three oils. This may he the result of an increased deposition of saturated fatty acids in response to the level of unsaturation of the OO and SO diets (16). When weanling rats were fed the OO oil diet, oleic acid was detected within 48 h in membrane phospholipids. This may be attributed to the high relative content of monounsaturated fatty acids in the OO diet, and to the increased A9-desaturase activity. An earlier study (17) reported that when endothelial cell cultures were supplemented with oleic acid, the 18:ln-9 content of phospholipids increased, while saturated fatty acid content decreased. In our study, PL and PE fractions in rats fed OO showed only a small decrease in total saturated fatty acid content. Similar results have been found by other authors in long-ten'n studies of the effects of OO supplementation on rat liver microsomes (4,8,18). The high oleic acid levels as a result of supplementation with CO may have been caused by the high content of stearic acid, a substrate for A9-desaturase, in CO. Linoleic acid appeared as early as two days in microsomes of rats given SO, as would be expected from the high dietary content of this fatty acid. Arachidonic acid is the most common fatty acid present in cellular phospholipids and can be obtained by desaturation and chain elongation of dietary linoleic acid. Its percentage in phospholipids is 849

8 normally dependent upon the desaturating capacity of the tissue under study, and the mechanisms regulating its incorporation into cell phospholipids (18). Also, this fatty acid serves as inmediate precursor of many eicosanoids with desirable as well undesirable effects. In humans, it is greatly dependent on dietary linoleic acid content, as A6-desaturation is slow and late limiting. The percentage of arachidonic acid was similar in all three groups, despite a significant increase in the proportion of linoleic acid in membrane lipids as a result of feeding the SO diet. These results areconsistent with earlier studies, and are concordant with the fact that linoleic acid inhibits A5-desaturase (9,10). In spite of the similar level ofarachidonic acid in all groups, 22:5 n-6 was higher after the SO diet in the two major classes of phospholipids, PC and PE. Polyunsaturated fatty acids of the n-3 series with more than 18 carbon atoms were decreased in all three groups in comparison with weanling rats, thus suggesting that this decrease could be related to differences in fatty acid composition between rat milk and experimental diets. In fact, rat milk presents 0.1% 20:5n-3, 0.1% 22"5n-3 and 0.1% 22:6n-3, which are absent from CO, OO and SO (19). Linoleic and tx-linolenic acids compete for desaturases, which show greater affinity for ct-linolenic acid (3). The level of n-3 and n-6 PUFA depends more on the linoleic/tx-linolenic ratio in the diet than on the absolute content of individual fatty acids. Because of the high content of linoleate, the 18:2n- 6/18:3n-3 ratio in SO is 219.7, a value greater than in OO (3.2). This could explain the lower levels of long-chain polyunsaturated fatty acids of the n-3 series found in the microsomes of rats fed SO diet as compared to animals fed OO. The low amounts of polyunsaturated n-6 and n-3 fatty acids in the liver microsomes from CO group may be due to the lack of essential precursors of 18-carbon n-6 and n-3 fatty acids in CO (3,4). Arachidonic acid in the different classes of phospholipids decreased more slowly than n-3 fatty acids. These results are suggestive of the physiological significance of arachidonic acid in membrane phospholipids for membrane functions (9). In accordance with the findings of Ifitani and Narita (9), our results show that in liver membranes, the percentages of polyunsaturated fatty acids with more than three double bonds is higher in phosphatidylethanolamine than in phosphatidylcholine or sphingomyelin. This may be related to the role of phosphatidylethanolamine in the transacylation of fatty acids between phospholipids. 850

9 Rats fed SO showed no changes in total saturated fatty acid content or in the unsaturation index. Animals fed the OO diet showed a decrease in saturated fatty acids together with an increase in the unsaturation index, although the value on day 6 was, in general, lower than in the SO group. These changes are reflected in desaturase activities. The increase in membrane unsaturation in the OO group may be due to high A9. and A6-desaturase activities. This seems to indicate that enzyme activities respond to the different diets take order to maintain membrane homeostasis. We conclude that the effects of dietary supplementation on the lipid composition and desaturase activities in rat liver microsomes reported in previous studies using longer treatments can be measured after a period of dietary manipulation as brief as 6 days. Acknowledgments : This study was supported by the CICYT through Project no. PB We thank Ms Karen Shashok for improving the English style of the manuscript. REFERENCES 1. MacDonald J.S.I. and Sprecher H. (1991). Biochim. Biophys. Acta 1084, Ulman, L., Blond, J.P., Maniongui, C., Poisson, J.P., Durand, G., Bezard, J. and Pascal, G. (1991). Lipids 26, Brenner, R.R. (1989) in The Role of Fats in Human Nutrition (Vergroesen A.J. and Crawford, M., eds.) pp Academic Press, London. 4. Periago, J.L., DeLucchi, C., Gil, A., Suhrez, M.D. and Pita, M.L. (1988). Biochim. Biophys. Acta 962, Gir6n, M.D., Mataix, F.J., Faus, M.L and Su~irez, M.D. (1989). Biochem. Int. 19, Periago, J.L., Pita, M.L., S~inchez del Castillo, M.A., Caamafio, G. and Su~irez, M.D. (1989). Lipids 24, Gir6n, M.D., Mataix, F.J., and Sutit~z, M.D. (1990). Biochim. Biophys. Acta 1045, Muriana, F.J.G., V~izquez, C.M. and Ruiz-Guti6rrez, V. (1992). J. Bioehem. 112, Iritani, N. and Narita, R. (1984). Biochim. Biophys. Acta 793, Huang, Y.S., Nassar, B.A. and Hon'obin, D.F. (1986). Biochim. Biophys. Acta 879, ILAR Committee on Laboratory Animal Diets (1979). Nutr. Abs. Rev. 49, Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). J. Biol. Chem. 193, Folch, L, Lees, M. and Stanley, G.H.S. (1957). J. Biol. Chem. 226, Vitiello, F. and Zanetta, J.P. (1978). J. Chromatogr. 166, Morrison, W.R. and Smith, L.M. (1964). J. Lipid Res. 5, Tripodi, A., Loria, P., Dilengite, M.A. and Carulli, N. (1991). Biochim. Biophys. Acta 1083, Vossen R.C.M.R., van Dam-Mieras M.C.E., Lemmens P.J.M.R., Hornstra G. and Zwaal R.F.A. (1991). Biochim. Biophys. Acta 1083, Navarro, M.D., Periago, J.L., Pita, M.L. and Hortelano, P. (1994). Lipids 29, Yokenubo, A., Honda, S., Okano, M., Takahashi, K. and Yamamoto, Y. (1993). J. Nutr. 123,