J. Biochem. 112, 562-567 (1992)

Fatty Acid Composition and Properties of the Liver Microsomal Membrane of Rats Fed Diets Enriched with Cholesterol Francisco J.G. Muriana,* Carmen M. Vazquez,** and Valentine Ruiz-Gutierrez*'1 'Institute de la Grasa y sus Derivados (CSIC), Apartado 1078, and * *Departamento de Fisiologta y Biologia Animal, Facultad de Farmacia, 41012 Sevilla, Spain

Male rats were fed diets containing olive (OO) or evening primrose (EPO) oil (10% w/w), with or without added cholesterol (1% w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids.respectively, of rat liver microsomes. The results were consistent with the increases in J* and A6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased zJ* desaturase activity as well as membrane microviscosity, whereas it decreased A" and As desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA:cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups. This seems to indicate that the responses of these enzyme activities to the different diets take place in order to maintain the homeostasis of membranes.

It is established that the composition of dietary fat influences the membrane structural lipid composition and metabolic functions (1), including the plasma cholesterol level, in mammals. Thus, saturated dietary fatty acids (SFA) are hypercholesterolemic (2). Conversely, monounsaturated fatty acids (MUFA) (3, 4) and polyunsaturated fatty acids (PUFA) (5, 6) lower the plasma cholesterol level, playing an important role in the prevention of coronary heart disease. Recent studies have suggested that MUFA, especially in the form of olive oil,reducethe plasma cholesterol concentration but also have a specific liver cholesteryl ester elevating effect in rats (2, 7). In addition, we have found that heart free cholesterol levels decreased in animals fed olive oil (6), this effect being greater than those of corn and soybean oils and the same as that of marine fish oil. Regarding PUFA, linoleic acid [LA, 18:2(n-6)] appears to 1 To whom correspondence should be addressed. Abbreviations: AA, arachidonic acid; ACAT, acyl-coenzyme A: cholesterol acyltransferase; DGLA, dihomo-y-linolenic acid; DPH, diphenylhexatriene; EPO, evening primrose oil; GLA, y-linolenic acid; HMG-CoA, hydroxymethylglutaryl-coenzyme A; LA, linoleic acid; MUFA, monounsaturated fatty acids; OA, oleic acid; 0 0 , olive oil; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipids; PS, phoaphatidylserine; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; SM, sphingomyelin.

be the major cholesterol lowering fatty acid (8, 9) and is metabolized through a variety of pathways, one of which involves its conversion to y-linolenic acid [GLA, 18:3(n6)] by A6 desaturase (10). GLA is rapidly elongated to dihomo-y-linolenic acid [DGLA, 20:3(n-6)] and subsequently desaturated by As desaturase to arachidonic acid [AA, 20:4(n-6)] (20). These two acids are metabolized to different groups of compounds with diverse and often contrasting effects. Therefore, the usefulness of the composition of dietary fat for predicting the risk of coronary heart disease suggests that control of fatty acid desaturating and cholesterol/esterification related enzymes might be relevant as to human medicine. The purpose of the present study is to examine the specific effects of dietary supplementation of olive oil (source of MUFA, mainly oleic acid [OA, 18:l(n-9)]) or evening primrose oil (source of LA and GLA), with or without added cholesterol, on several liver microsomal enzymes, and the lipid and fatty acid compositions of liver membrane. The enzymes studied were A9, A6, and A5 desaturases (which are key enzymes that regulate unsaturated fatty acid biosynthesis) (11), hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase (which catalyzes the rate-limiting step in the biosynthesis of cholesterol), and acyl-coenzyme A:cholesterol acyltransferase (ACAT) (which is the major rate-controlling microsomal enzyme in cholesterol esterification) (12). Since cholesterol is known

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Received for publication, March 30, 1992

563

Lipids and Properties of Liver Membranes from Rats Fed with Cholesterol to influence the fluidity of biological membranes (13), we also measure the fluorescence anisotropy of a probe incorporated into the microsomal membrane. MATERIALS AND METHODS

TABLE I. Fatty acid compositions of dietary lipids. OO, olive oil; EPO, evening primrose oil; "y-linolenic acid (GLA). Fatty acid 00 EPO (weight %) 16:0 10.4 8.5 16:l(n-7) 0.9 0.1 18:0 3.0 2.3 18:l(n-9) 77.8 9.8 18:2(n-6) 6.9 70.8 — 18:3(71-6)' 7.8 — 18:3(n-3) 0.6 — 18:4(71-3) 0.2 0.4 20:0 0.2 — 20:l(n-9) 0.3 — 22:0 0.1 — 24:0 0.4 Others 0.3 0.2 Vol. 112, No. 4, 1992

r — xvv —

where Jvv and JVH are the polarized fluorescence intensities measured horizontally and perpendicularly to the polarized exciting light, G is a "grating factor" which corrects for instrument artifacts and is equal to IHV/IHH • Statistical Analysis—The results shown are means ± standard deviation. The effects of the dietary treatments were examined by means of analysis-of-variance procedures. Differences between individual diets were established by means of the unpaired Student's t test. RESULTS Average Body Weight, Food Intake, and Liver Weight of Rats—All groups of nnimnlR consumed similar amounts of food, irrespective of the dietary regimen (Table II). The average body weight and liver weight were slightly increased, but not significantly, in the cholesterol-fed animals. The liver-weight to average-body-weight ratios were also not affected in the groups fed the different fats, with or without cholesterol supplementation. Effects of Dietary Fats on the Lipid and Fatty Acid Compositions of Liver Microsomes—The lipid composition was similar in the liver microsomes from nnimnla fed the OO and EPO diets, except for the content of phosphatidyl-

TABLE II. Effects of dietary fats on the average body weight, food intake, and liver weights of rats. The results are expressed as means± standard deviation for 6 animals per group. Average body weight is defined as the difference between the body weight at entry and that at study; OO, olive oil (10%, w/w); EPO, evening primrose oil (10%, w/w); Choi, cholesterol (1%, w/w); LW, Uver weight; ABW, average body weight. Food Liver Average 100X Diet intake body wt. wt. (LW/ABW) (g/day) (g) (g) OO 216±9.3 17.3±1.0 12.0±1.4 5.5±0.5 OO + Chol 220±7.1 17.9±1.4 12.9±1.8 5.8±0.6 EPO 218±8.8 17.4±1.2 11.7±1.2 5.4±0.6 EPO + Chol 224±8.7 16.9±1.0 12.8±1.5 5.7±0.4

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Chemical Reagents—All cofactors, NADH, CoA (sodium salt), and bovine serum albumin (essentially free of fatty acid) were obtained from Sigma Chem. 1,6-Diphenylhexatriene was from Aldrich Chem. All other chemicals were of analytical grade. [l- u C]Palmitic acid (sp. act., 56.0 mCi/mmol), [l- u C]linoleic acid (sp. act., 50.5 mCi/ mrnol), and [l- 14 C]eicosa-8,ll,14-trienoic acid (sp. act., 54.9 mCi/mmol) were purchased from New England Nuclear. DL-3-Hydroxy-[3-'4C]methylglutaryl-CoA (sp. act., 50.1 mCi/mmol) and [l-MC]oleoyl-CoA (sp. act., 50.3 mCi/mmol) were from Amersham International. Animals and Diets—Male Wistar rats (Iffa-Credo, Lyon, France), obtained at 3 weeks of age, were randomly divided into four groups of six animals. The animals were housed in a well-ventilated room maintained at 22±2'C with a 12 h light/dark cycle. The composition of the basal diet was, in weight percent: milk casein, 20.8; corn starch, 19.6; glucose, 37.0; non nutritive cellulose, 5.3; mineral mix, 6.3; and vitamin mix, 1.0. Each group was fed the same basal diet but with different 10% (w/w) fat supplements: olive oil (OO), olive oil with added cholesterol (1% w/w), evening primrose oil (EPO), and evening primrose oil with added cholesterol. The fatty acid compositions of the different diets are shown in Table I. The great differences between OO and EPO are the concentration of oleic acid (77.8%) in the OO, and the presence of considerable amounts of linoleic acid (70.8%) and y-linolenic acid (7.8%) in EPO. The experimental diets and tap water were provided ad libitum. To minimize oxidation, all diets were prepared weekly and stored at 4°C under an atmosphere of nitrogen until needed. After consuming the experimental diets for 6 weeks, the animals were sacrificed by decapitation. Their livers were immediately excised, trimmed of connective tissue, weighed, and then homogenized in an ice-cold medium comprising 10 mM Tris-HCl (pH7.4), 0.25 M sucrose, 20 mM EGTA (ethylene-bis[oxyethylenenitrilo]tetraacetic acid), and 5 mM DTT (dithiothreitol), using a Potter-Elvehjem tissue homogenizer. Preparation of microsomal fractions was carried out as described previously (14). Protein was

assayed by the method of Lowry et al. (15), with bovine serum albumin as a standard. Enzyme Assays—The activities of A9, A*, and Ai desaturases, HMG-CoA reductase and ACAT were assayed as previously described (14, 16). Lipid Analysis—Lipids from liver microsomal preparations were extracted by the method of Folch et al.(17). The lipid composition was determined by means of the Iatroscan TLC/FID technique (18). The fatty acid composition was determined by gas-liquid chromatography (HewlettPackard, model 5710 A) of methyl esters on a 60 m X 0.25 m m Ld. fused silica capillary column (0.25 ftm Supelcowax 10 film), as reported (29, 20). Polarization Studies—Steady-state fluorescence polarization measurements were made using a 1 cm light and excitation-emission wavelengths of 360-440 n m with a Perkin-Elmer 650-40 spectrofluorometer adapted for fluorescence polarization, as previously described (19). The hydrophobic, fluorescence probe, diphenylhexatriene (DPH), was used to label microsomes. The degree of fluorescence anisotropy, r, was calculated (21):

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TABLE IV. Effects of dietary fats on the fatty acid composition of rat liver microsomes. The results are expressed as means ± standard deviation for 6 animals per group. Values with a superscript are significantly different from in the respective no cholesterol diet-fed animals: *p