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Dietary fat (virgin olive oil or sunflower oil) and physical training interactions on blood lipids in the rat
BASIC NUTRITIONAL INVESTIGATION
Dietary Fat (Virgin Olive Oil or Sunflower Oil) and Physical Training Interactions on Blood Lipids in the Rat Jose´ L. Quiles, PhD, Jesu´s R. Huertas, PhD, Julio J. Ochoa, PhD, Maurizio Battino, PhD, Jose´ Mataix, PhD, and Mariano Man˜as, PhD From the Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Granada, Spain; and the Institute of Biochemistry, Faculty of Medicine, University of Ancona, Ancona, Italy OBJECTIVE: We investigated whether the intake of virgin olive oil or sunflower oil and performance of physical exercise (at different states) affect plasma levels of triacylglycerols, total cholesterol, and fatty acid profile in rats. METHODS: The study was carried out with six groups of male rats subjected for 8 wk to a diet based on virgin olive oil (three groups) or sunflower oil (three groups) as dietary fat. One group for each diet acted as sedentary control; the other two groups ran in a treadmill for 8 wk at 65% of the maximum oxygen consumption. One group for each diet was killed 24 h after the last bout of exercise and the other was killed immediately after the exercise performance. Triacylglycerols, total cholesterol, and fatty acid profile were analyzed in plasma. Analysis of variance was used to test differences among groups. RESULTS: Animals fed on virgin olive oil had lower triacylglycerol and cholesterol values. Physical exercise reduced these parameters with both dietary treatments. Fatty acid profile showed higher monounsaturated fatty acid proportion in virgin olive fed oil animals and a higher -6 polyunsaturated fatty acid proportion in sunflower oil fed animals. Physical exercise reduced the levels of monounsaturated fatty acids with both diets and increased the proportions of -3 polyunsaturated fatty acids. CONCLUSIONS: Results from the present study supported the idea that physical exercise and the intake of virgin olive oil are very good ways of reducing plasma triacylglycerols and cholesterol, which is desirable in many pathologic situations. Concerning findings on fatty acid profile, we had results similar to those of other investigators regarding the effect of different sources of dietary fat on plasma. The most interesting results came from the effect of physical exercise, with significant increases in the levels of -3 polyunsaturated fatty acids, which may contribute to the antithrombotic state and lower production of proinflammatory prostanoids attributed to physical exercise. Nutrition 2003;19:363–368. ©Elsevier Science Inc. 2003 KEY WORDS: olive oil, sunflower oil, fatty acids, exercise, rats
INTRODUCTION The intake of high amounts of fat is a major risk factor in the etiology of cardiovascular disease and cancer, two of the main causes of death among populations in developed countries. However, it is evident that the fatty acid composition of a particular fat is more important that its absolute concentration regarding these diseases.1 Indeed, there is an important number of studies supporting the positive effects of the intake of fish oil (mainly rich in -3 polyunsaturated fatty acids, or PUFAs) and virgin olive oil (mainly monounsaturated fat) on these diseases.1 In the same way, many investigators have expressed their concern about the risk of an excessive intake of saturated fat or edible oils rich in -6 PUFAs (mainly seed oils such as sunflower or corn).2 Physical exercise is widely recommended as a way of reducing weight gain and ameliorating the risk of hyperlipemias and other
This work was supported by CICYT project ALI91-1113-C03-01. Correspondence to: Jose´ L. Quiles, PhD, Instituto de Nutricio´n y Tecnologı´a de Alimentos, Universidad de Granada, C/ Ramo´n y Cajal 4 (Edif. Fray Luis de Granada), 18071 Granada, Spain. E-mail: [email protected] Nutrition 19:363–368, 2003 ©Elsevier Science Inc., 2003. Printed in the United States. All rights reserved.
deleterious effects of a high fat intake.3 Thus, exercise training decreases the plasma levels of total cholesterol and low-density lipoprotein cholesterol, increases the levels of high-density lipoprotein cholesterol, reduces the concentration of triacylglycerols, reduces systolic and diastolic blood pressure, and is usually recommended to prevent or ameliorate the effects of cardiovascular diseases.4 Moreover, considerable epidemiologic evidence has been acquired linking increased physical activity with reduced occurrence of breast and colon cancers.5 In the same way, some experimental studies performed with animals have suggested that chronic exercise can retard, delay, or prevent the incidence, progression, or metastasis of experimental tumours.6 Some of the possible mechanisms supporting these results include reduced body fat, enhanced gut motility, stimulation of the immune system, and decreased time of exposure to estrogens and other hormones. We have already demonstrated some of the links between dietary fat and physical exercise in relation to health. In fact, previous studies have shown that a program of training for 8 wk leads to changes in the lipid profile of liver and skeletal muscle mitochondrial membranes in rats.7 These changes depended on the type of fat consumed in affecting the functionality of such membranes8 (contributing in some cases to the general benefits 0899-9007/03/$30.00 PII S0899-9007(02)00949-8
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Nutrition Volume 19, Number 4, 2003 TABLE I.
FATTY ACID COMPOSITION OF DIETARY OILS (g/100 g) Fatty acid
Virgin olive oil
Sunflower oil
16:0 16:1(-9) 16:1(-7) 18:0 18:1(-9) 18:2(-6) 18:3(-3)
11.32 0.11 0.84 4.34 74.12 7.64 0.61
7.19 0.02 0.19 4.51 32.08 54.26 0.10
attributed to physical exercise) and modifying their susceptibility to be oxidized by free radicals.9 This evidence and our previous experience encouraged us to investigate whether the combination of physical exercise and the intake of monounsaturated fat (virgin olive oil) has any additional positive effect on blood parameters related to lipid metabolism. For the present study we used virgin olive oil and sunflower oil as dietary fats (the most commonly used vegetable oils in Spain) and a program of physical exercise involving a chronic model and a chronic plus acute model. We estimated lipid profile in plasma and the levels of total cholesterol, serum triacylglycerols, and other parameters related to physical exercise.
MATERIALS AND METHODS
and Technology approved the protocols. Animals were handled according to the guidelines for care and use of laboratory animals by the Spanish Society for Laboratory Animal Sciences. Sample Analysis The rats were killed by decapitation, blood was collected in tubes coated with ethylene-diaminetetraacetic acid, and plasma was separated by centrifuge. Triacylglycerols, total cholesterol, glucose, and lactate concentrations were determined in plasma by enzymatic methods using Boehringer-Mannheim kits (Munich, Germany). Fatty acid profile of plasma was measured by gas liquid chromatography as described by Lepage and Roy.11 A gas–liquid chromatograph (Model HP-5890, Series II, Hewlett Packard, Palo Alto, CA, USA) equipped with a flame ionization detector was used to analyze fatty acids as methyl esters. Chromatography was performed with a 60-m capillary column with 32 mm inner diameter and 20 mm thickness impregnated with Sp 2330 FS (Supelco Inc., Bellefonte, Palo Alto, CA, USA). The injector and detector were maintained at 250°C and 275°C, respectively; nitrogen was used as the carrier gas, and the split ratio was 29:1. Temperature programming (for 40 min) was as follows: initial temperature, 160°C for 5 min, 6°C/min to 195°C, 4°C/min to 220°C, 2°C/min to 230°C, hold 12 min, 14°C/min to 160°C. All chemical products and solvents, of the highest quality available, were acquired from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany). The homologs of coenzyme Q were gifts from Eisai Co. (Tokyo, Japan). Virgin olive oil and sunflower oil were kindly provided by Coosur S.A. (Jaen, Spain).
Experimental Protocol
Statistical Analysis
Male Wistar rats, initially weighing 80 to 90 g, were allocated in groups of 10 per cage and maintained on a 12-h light/12-h darkness cycle, with free access to food and drinking water. The study lasted 9 wk (1 wk for animal selection followed by 8 wk for the experiment). During the selection week, all rats were fed a nonpurified diet and subjected to daily sessions on an exercise treadmill at a speed of 15 m/min for 15 min. The rats were fed semisynthetic and isoenergetic diets composed of (g/kg of diet): 267 casein, 135.3 starch, 453 sucrose, 80 edible oil, 37 mineral supplement, 10 vitamin supplement, 1.8 cellulose, 0.9 choline, and 3 methionine. Three groups of eight rats each received sunflower oil as dietary fat and the other groups (one of eight rats and two of six rats each) received virgin olive oil (the lipid profile of both oils are showed in Table I). For each diet one group was made up of sedentary rats (no exercise) and two groups followed a physical exercise program (as described below). Thus, the groups were as follow: rats fed virgin olive oil and sedentary (VS; n ⫽ 8); rats fed virgin olive oil and exercised (VT; n ⫽ 6); rats fed virgin olive oil and exercised to exhaustion (VE; n ⫽ 6); rats fed sunflower oil and sedentary (SS; n ⫽ 8); rats fed sunflower oil and exercised (ST; n ⫽ 8), and rats fed sunflower oil and exercised to exhaustion (SE; n ⫽ 8). The exercised animals (VT, VE, ST, and SE) underwent training sessions on a horizontal treadmill throughout the 8 wk of the experiment: for the first 2 wk, the rats were exercised 5 d/wk, once a day at a steadily increased rate, until they could run 40 min/d at 35 m/min. These conditions, equivalent to 65% to 70% of maximum oxygen consumption,10 were maintained during the remaining 6 wk. The VT and ST groups were killed 24 h after the last exercise bout. The VE and SE groups performed a final exercise test until exhaustion and immediately killed. Intakes for each group were monitored daily. The rats were killed at the same time of the day in all cases (between 12:00 and 1:00 PM) to avoid circadian fluctuations. The Ethical Committee of the Interministerial Commission of Science
The results represent the mean and standard error of six (VT and VE) or eight (VS, SS, ST and SE) animals. A two-way analysis of variance was performed for effects of dietary fat and physical activity on each variable. Significant (P ⬍ 0.05) interaction terms were evaluated by Scheffe´ ’s F test. Previous to any statistical analysis, all variables were checked for normal and homogeneous variance with Levene’s test. When a variable was found not to be normal, it was log-transformed and reanalyzed.
RESULTS Dietary intake did not vary significantly among groups (data not shown). The sedentary animals reached a higher weight gain than did exercised groups at the end of the study (Fig. 1A). According to the two-way analysis of variance, physical exercise was the only factor responsible for differences in rat weight gain. Sedentary animals fed sunflower oil reached the highest levels of triacylglycerols (Fig. 1B), and physical exercise (both models) led to a lower concentrations of serum triacylglycerols in the animals fed with both diets. Total cholesterol analysis (Fig. 1C) showed that physical exercise was responsible for lower levels of that molecule in both dietary groups. However, sedentary animals fed virgin olive oil had a lower cholesterol amount than did the sedentary animals fed sunflower oil. Physical exercise and the interaction between exercise and dietary fat were responsible for the levels of total cholesterol, according to the two-way analysis of variance. For both dietary treatments, levels of lactate (Fig. 2A) were similar and both types of exercise led to higher values, with no differences between fats. No differences were found between the sedentary groups for glucose (Fig. 2B), and physical exercise led to higher levels in exhausted rats, with no differences between diets. In relation to the plasma lipid profile (Table II), for the saturated fatty acids, there were differences among groups only for
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FIG. 2. Effects of physical exercise and dietary fat on plasma lactate and glucose of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects are considered significant at P ⬍ 0.05. The P values for this two-way analysis were as follows. For lactate: fat, 0.559; exercise, 0.001; fat ⫻ exercise, 0.359. For glucose: fat, 0.290; exercise, 0.000; fat ⫻ exercise, 1.000. SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
FIG. 1. Effects of physical exercise and dietary fat on weight gain, plasma triacylglycerols, and plasma total cholesterol of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects were considered significant at P ⬍ 0.05. The P values for this two-way analysis were as follows. For weight gain: fat, 0.454; exercise, 0.000; fat ⫻ exercise, 0.341. For triacylglycerols: fat, 0.690; exercise, 0.000; fat ⫻ exercise, 0.015. For total cholesterol: fat, 0.741; exercise, 0.000; fat ⫻ exercise, 0.016. SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
stearic acid (18:0), with all animals subjected to physical exercise reaching higher levels (there was no difference between diets). For all the monounsaturated fatty acids (MUFAs), the highest levels were found in animals fed virgin olive oil. In addition, physical exercise led to lower levels of MUFA in both dietary groups. Dietary fat and physical exercise were responsible for these changes according to analysis of variance. In relation to the -6
PUFAs, animals fed sunflower oil showed the highest levels. Physical exercise reached higher levels compared with their controls for 20:4(-6) in both diets and for total -6 PUFA in animals fed virgin olive oil. Concerning -3 PUFA, animals from both dietary groups had similar levels of 22:6(-3), and physical exercise led to higher proportions than in sedentary animals. Physical exercise affected total -3 PUFA in animals fed olive oil. The total PUFA index showed higher levels in all the animals fed sunflower oil and those subjected to physical exercise (for both diets). The highest 20:4(-6)/22:6(-3) ratio (Fig. 3A) belonged to animals fed sunflower oil. This ratio was lower in trained animals than in controls but not in the exhausted groups. Figure 3B shows the ratio -6 PUFA to -3 PUFA, with results similar to those described for the ratio of arachidonic to docosahexaenoic acid, but in this case physical exercise decreased this ratio in exhausted animals fed sunflower oil.
DISCUSSION There is contrasting evidence about the benefits of specific dietary fats and physical exercise on pathologic states such as cardiovascular disease and certain types of cancer. The goal of the present work was to investigate whether the combination of both factors
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Nutrition Volume 19, Number 4, 2003 TABLE II.
PLASMA FATTY ACID COMPOSITION (g/100 g) OF RATS SUBJECTED TO PHYSICAL EXERCISE AND DIFFERENT DIETARY FATS* Experimental groups
Two-way ANOVA†
Fatty acid or index
VS (n ⫽ 8)
VT (n ⫽ 6)
VE (n ⫽ 6)
SS (n ⫽ 8)
ST (n ⫽ 8)
SE (n ⫽ 8)
Fat
Exercise
Fat ⫻ exercise
16:0 18:0 ⌺Saturated 18:1(-7) 18:1(-9) ⌺MUFA 18:2(-6) 20:4(-6) ⌺PUFA(-6) 22:6(-3) ⌺PUFA(-3) ⌺PUFA
17.4 ⫾ 0.7a 8.7 ⫾ 1.1a 36.0 ⫾ 5.2b 4.5 ⫾ 0.2c 35.4 ⫾ 1.8c 43.8 ⫾ 2.6d 6.6 ⫾ 0.7a 10.5 ⫾ 1.2a 18.6 ⫾ 1.5a 0.9 ⫾ 0.1a 1.2 ⫾ 0.1a 19.1 ⫾ 2.8a
17.1 ⫾ 0.7a 10.2 ⫾ 0.8b 30.3 ⫾ 0.5b 3.6 ⫾ 0.3b 32.3 ⫾ 0.3b 38.1 ⫾ 1.4c 7.9 ⫾ 0.4a 14.1 ⫾ 0.7b 23.4 ⫾ 1.2b 1.6 ⫾ 0.3b 1.9 ⫾ 0.4b 27.2 ⫾ 2.2b
17.3 ⫾ 0.7a 11.5 ⫾ 0.9b 31.7 ⫾ 0.6b 3.1 ⫾ 0.1b 31.8 ⫾ 2.1b 35.2 ⫾ 2.2c 8.8 ⫾ 1.4a 18.5 ⫾ 1.5bc 28.2 ⫾ 1.7b 1.5 ⫾ 0.3b 1.9 ⫾ 0.3b 31.1 ⫾ 2.1b
17.1 ⫾ 0.4a 7.1 ⫾ 0.3a 26.1 ⫾ 0.2a 3.4 ⫾ 0.2b 18.6 ⫾ 0.4b 25.7 ⫾ 0.8b 28.7 ⫾ 1.6b 15.3 ⫾ 1.1b 46.9 ⫾ 0.7c 0.6 ⫾ 0.2a 0.9 ⫾ 0.1a 48.2 ⫾ 0.9c
17.1 ⫾ 0.4a 10.1 ⫾ 0.6b 29.4 ⫾ 0.5b 2.1 ⫾ 0.1a 15.3 ⫾ 1.1a 19.1 ⫾ 1.3a 29.2 ⫾ 1.1b 19.1 ⫾ 1.6c 49.4 ⫾ 1.1c 1.3 ⫾ 0.4b 1.6 ⫾ 0.2b 51.5 ⫾ 1.1d
17.5 ⫾ 0.3a 9.2 ⫾ 1.3b 28.9 ⫾ 1.4b 2.27 ⫾ 0.18a 14.6 ⫾ 1.2a 20.9 ⫾ 1.6a 25.1 ⫾ 2.1b 20.8 ⫾ 1.7c 48.4 ⫾ 1.1c 1.3 ⫾ 0.3b 1.6 ⫾ 0.2b 51.1 ⫾ 1.1d
0.132 0.132 0.133 0.000 0.000 0.000 0.000 0.011 0.000 0.102 0.109 0.000
0.312 0.045 0.898 0.000 0.015 0.012 0.185 0.001 0.103 0.012 0.010 0.002
0.103 0.353 0.029 0.169 0.421 0.423 0.300 0.762 0.036 0.617 0.123 0.071
* Results are presented as means ⫾ standard error of the mean. For each fatty acid or index, values in a row not sharing superscript letters are significantly different (P ⬍ 0.05). † P values for the two-way ANOVA for the effects of fat, exercise, and the interaction between fat and exercise (significant at P ⬍ 0.05). ANOVA, analysis of variance; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
(fat and exercise) affects blood lipid metabolism, thus modifying the predisposition to these diseases. Animals adapted to diet, as showed by plasma fatty acid profiles (animals fed virgin olive oil had a higher proportion of MUFA and the highest -6 PUFA was found in animals fed sunflower oil). Animals also adapted to the training program, as show by the significantly higher plasma triacylglycerols and total cholesterol levels in sedentary animals and by the levels of lactate and glucose. This was also confirmed by the lesser weight gain produced by exercise, which has been well documented in humans and animals.3,12 Regarding triacylglycerols, both models of exercise reduced serum levels of this molecule. Others previously reported the same effect in rats and humans.13,14 The hypotriglyceridemic effect may be secondary to reduced hepatic triacylglycerol secretion and/or enhanced removal of triacylglycerol-rich lipoproteins by the extrahepatic tissues, according to Tan et al.15 Altered activity of lipoprotein lipase, which is found in several tissues including skeletal muscle, heart, and adipose tissue, also may be involved because it is responsible for the clearance of postprandial chylomicrons and endogenous very low-density lipoprotein.16 With respect to the effect of dietary fat on serum triacylglycerols, we found that sedentary animals fed sunflower oil had higher levels than those fed virgin olive oil. After physical exercise the values reached with both diets were the same, but in terms of reduction, sunflower oil reduced triacylglycerols more than virgin olive oil after exercise. The reduction of serum triacylglycerols after intake of specific dietary fats such as fish oil have been described,17 but there are no reports describing differences between olive oil and sunflower oil. Our study supported the idea that physical exercise and the intake of unsaturated oils as the fat source reduce triacylglycerols, which is desirable in many pathologic situations. Because sunflower oil produces a higher proportion of triacylglycerols in sedentary animals than does virgin olive oil, the practice of physical exercise could be recommended more often if the consumed diet is rich in -6 PUFAs. Many studies,18 but not all,15 have shown that training lowers serum cholesterol in rats, and our data supported this hypothesis. In humans, the type of exercise performed appears to affect the
hypocholesterolemic effect of training.18 However, in the present work both types of exercise led to a reduction in the level of total cholesterol to the same extent. This means that exhaustive exercise in the VE and SE groups not compound the hypocholesterolemic effect in comparison with that obtained with the 8-wk training in the VT and ST groups. Regarding the effects of dietary fat, controversy exists about the effect of olive oil and sunflower oil on total cholesterol. Our results showed that, even if a diet enriched with olive oil leads to lower levels of serum cholesterol than with sunflower oil, the concentration of cholesterol in both diets may be considered normal and the intake of any one of the two diets led to similar levels of cholesterol after physical exercise. Plasma lipid profile of the rats showed that the proportion of saturated fatty acids (SFA) is barely influenced by the ingestion of any of the studied fat sources. SFA appears to be a relatively stable fraction within the general composition of plasma which is in accordance with the findings of others.19 In relation to physical exercise, Rocquelin and Juaneda20 investigated SFA and found that 16:0 but not 18:0 fatty acids decrease in the epididymal fat of trained rats. Our results showed that 18:0 increased in both dietary groups after physical exercise, as did the total SFA index for the sunflower oil group. Many studies have reported that plasma positively adapts itself to the consumption of a MUFA-rich diet, demonstrating in its composition the type of diet ingested.21 We confirmed those results in rats fed the diet rich in olive oil. Physical activity led to a decrease in all MUFAs in all animals. With regard to the -6 PUFA, the highest levels were obtained in animals fed the polyunsaturated fat source, as reported by other.22 Physical exercise increased arachidonic (20:4-6) acid in both dietary groups, as described by Vapaatalo et al.23 in the plasma of young subjects after short-term heavy exercise. In relation to -3 PUFA, both dietary treatments led to similar values, and physical exercise increased the amount of these fatty acids. In the present study, the effects of dietary fat on plasma fatty acid profiles were in agreement with those found by others,21 but the most interesting results concern the effect of physical exercise. It is very difficult to know whether the changes found among the different fractions of fatty acids after physical exercise are related to changes in a particular metabolic function, a higher or lower
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FIG. 3. Effects of physical exercise and dietary fat on 20:4(-6)/22:6(-3) and PUFA (-6)/PUFA(-3) indices in plasma of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects were considered significant for P ⬍ 0.05. The P values for this two-way analysis were as follows. For 20:4(-6)/22:6(-3): fat, 0.000; exercise, 0.005; fat ⫻ exercise, 0.349. For PUFA(-6)/PUFA(-3): fat, 0.000; exercise, 0.015; fat ⫻ exercise, 0.175. PUFA, polyunsaturated fatty acid; SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
disposability of the fatty acids, or if this is due to a higher regulation mechanism. It has been documented that 18:1-9 and other MUFAs have preferential mobilization and oxidation rates than other fatty acids.21 Leyton et al.24 demonstrated that rats oxidize fatty acids in the following order: oleic (18:1-9) ⬎ linoleic (18:2-6) ⬎ palmitic (16:0) and stearic (18:0). Similar differences exist in humans.25 This could justify the decrease of MUFA in our model after physical exercise because of the higher energetic requirements it caused. It could be mediated, according to Helge et al.,26 by an increase in the solubility of lipids in circulating lipoproteins and, hence, an enhanced enzyme-substrate contact, due to the unsaturation of the lipids. Another potential mechanism could be related to changes in lipoprotein lipase activity in heart and skeletal muscle mediated by adaptations to different diets, as suggested by Shimomura et al.27 If this hypothesis is true, the increase in some SFAs and PUFAs together with a decrease in MUFAs as a consequence of physical exercise could represent a change in the relative proportions of fatty acids in plasma. That is easy to see if we keep in mind that lipid profile results are expressed as relative proportions of the different fatty acids, and a decrease in any of the fractions could lead to a relative increase in all the others.
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Considerable controversy exists concerning the relative importance of -3 and -6 PUFAs in the prevention of cardiovascular diseases and cancer. Increase in -3 fatty acids has been related to a decrease in serum triacylglycerols and an increase in highdensity lipoprotein cholesterol, whereas increases in linoleic acid and ␥-linoleic acid mainly decrease total cholesterol and lowdensity lipoprotein cholesterol.28 In a similar way, high levels of -3 PUFA may decrease cancer progression and metastasis, and the opposite is true for -6 PUFA.29 Eicosapentaenoic acid (20: 5-3) inhibits platelet aggregation induced by arachidonic acid and docosahexaenoic acid (22:6-3) reduces platelet aggregating prostanoids (prostaglandins G2 and H2, thromboxane A2).30 Thus, low levels of 20:4-6/22:6-3 or, by extension, -6 PUFA/-3 PUFA in plasma reflect a state of antithrombogenesis. In fact, a high dietary intake of eicosapentaenoic and docosahexaenoic acid in volunteers reported by decreased plasma triacylglycerols and/or plasma total cholesterol, prolonging bleeding time, and also led to an antithrombotic state.30 In our study the lower levels of 20:46/22:6-3 and -6 PUFA/-3 PUFA were present in animals fed virgin olive oil. Moreover, regular training reduced these levels with both dietary fats. Thus, we could say that virgin olive oil is less prothrombotic than sunflower oil and that physical exercise promotes an antithrombotic state. In addition, an increase in the ratio of -3 PUFA to -6 PUFA may contribute to a lower production of proinflammatory prostanoids and a higher production of the anti-inflammatory ones. In conclusion, under our experimental conditions, physical exercise and intake of virgin olive oil were very effective in reducing plasma triacylglycerols and cholesterol. Concerning findings on the fatty acid profile, the most interesting results come from the effect of physical exercise, with significant increases in the levels of -3 PUFAs. These effects are very desirable in many pathologic situations.
ACKNOWLEDGMENTS Special thanks to Ms. Monica Glebocki for elaboration of the manuscript.
REFERENCES 1. Willett WC, Sacks F, Trichopoulou A, et al. Mediterranean diet pyramid: a cultural model for healthy eating. Am J Nutr 1995;61:1321S 2. Ramirez-Tortosa MC, Urbano G, Lopez-Jurado M, et al. Extra-virgin more than refined olive oil increases the resistance of LDL to oxidation in free-living men with peripheral vascular disease. J Nutr 1999;129:2177 3. Bell RR, Spencer MJ, Sherriff JL. Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat. J Nutr 1997;127:2006 4. Brooks GA, Fahey TD, White TP, Baldwin KM. Exercise physiology. Human bioenergetics and its applications, 3th ed. Mountain View, California: Mayfield Publishing Company, 1999 5. Thune I, Furberg AS. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific. Med Sci Sports Exerc 2001;33:S530 6. Woods JA. Exercise and resistance to neoplasia. Can J Physiol Pharmacol 1998;76:581 7. Quiles JL, Huertas JR, Man˜ as M, Battino M, Mataix J. Physical exercise affects the lipid profile of mitochondrial membranes in rats fed with virgin olive oil or sunflower oil. Br J Nutr 1999;81:21 8. Quiles JL, Huertas JR, Man˜ as M, et al. Dietary fat type and regular exercise affect mitochondrial composition and function depending on specific tissue in rat. J Bioenerg Biomembr 2001;33:127 9. Mataix J, Quiles JL, Huertas JR, Battino M, Man˜ as M. Tissue specific interactions of exercise, dietary fatty acids, and vitamin E in lipid peroxidation. Free Radic Biol Med 1998;24:511 10. Amstrong RB, Laughlin MH, Rome L, Taylor CL. Metabolism of rats running up and down an incline. J Appl Physiol 1983;55:518 11. Lepage G, Roy CC. Direct transesterification of all classes of lipids in one-step reaction. J Lipid Res 1986;27:114 12. Blair SN. Evidence for success of exercise in weight loss and control. Ann Intern Med 1993;119:702
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13. Narayan AK, McMullen JJ, Butler DP, Wakefield T, Calhoun WK. Effect of exercise on tissue lipids and serum lipoproteins of rats fed two levels of fat. J Nutr 1975;195:581 14. Costill DL, Cleary P, Fink WJ, et al. Training adaptations in skeletal muscle of juvenile diabetics. Diabetes 1979;28:818 15. Tan MH, Bonen A, Garner JB, Belcastro AN. Physical training in diabetic rats: effect on glucose tolerance and serum lipids. J Appl Physiol 1982;52:1514 16. Deshaies Y, Vallerand AL, Bukowiecki LJ. Serum lipids and cholesterol distribution in lipoproteins of exercise-trained female rats fed sucrose. Life Sci 1983;33:75 17. Durrington PN, Bhatnagar D, Mackness MI, et al. An omega-3 polyunsaturated fatty acid concentrate administered for one year decreased triacylglycerols in simvastatin treated patients with coronary heart disease and persisting hypertriglyceridaemia. Heart 2001;85:544 18. Bjo¨ rtorp P, Berchtold P, Grimby G. Effects of physical training on glucose tolerance plasma insulin and lipids on body composition in men after myocardial infarction. Acta Med Scand 1972;192:439 19. Rueda P, Lopez-Frias M, Llopis J, Mataix J, Urbano G. Influence of dietary fat on plasma fatty acid composition in rats. Nutr Res 1992;12:757 20. Rocquelin G, Juaneda P. Influence of prolonged physical training on the composition of fatty acids of epididymal adipose tissue and of the carcass in the young rat on a dietary regime of sunflower, rapeseed or primor oil. Rep Nutr Dev 1981;21:1015
Nutrition Volume 19, Number 4, 2003 21. Seiquer I, Man˜ as M, Martinez-Victoria E, Ballesta MC, Mataix FJ. Long-term influence of dietary fat (sunflower oil, olive oil, lard and fish oil) in the serum fatty acid composition and in the different lipidic fractions, in miniature swine. Int J Vitam Nutr Res 1996;66:171 22. Jacotot B, Lasserre M, Mendy F. Effects of different diets rich in polyunsaturated fatty acids on plasma phospholipids in the human. Progr Lipid Res 1986;25:185 23. Vapaatalo H, Laustiola K, Seppala E, et al. Exercise, ethanol and arachidonic acid metabolism in healthy men. Biomed Biochem Acta 1984;43:S413 24. Leyton J, Drury PJ, Crawford MA. Differential oxidation of saturated and unsaturated fatty acids in vivo in the rat. Br J Nutr 1987;57:383 25. Jones PJH, Pencharz PB, Clandinin MT. Whole body oxidation of dietary fatty acids: implications for energy utilization. Am J Clin Nutr 1985;42:769 26. Helge JW, Ayre K, Chaunchaiyakul S, et al. Endurance in high-fat-fed rats: effects of carbohydrate content and fatty acid profile. J Appl Physiol 1998;85:1342 27. Shimomura Y, Tamura T, Suzuki M. Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet. J Nutr 1990;120:1291 28. Haglund O, Wallin R, Wretling S, Saldeen T. Effects of fish oil alone and combined with long chain (n-6) fatty acids on some coronary risk factors in male subjects. J Nutr Biochem 1998;9:629 29. Lipworth L, Martinez ME, Angell J, Hsieh C-C, Trichopoulos D. Olive oil and human cancer: an assessment of the evidence. Prev Med 1997;26:181 30. Masumura S, Furui H, Hashimoto M, Watanabe Y. The effects of season and exercise on the levels of plasma polyunsaturated fatty acids and lipoprotein cholesterol in young rats. Biochim Biophys Acta 1992;1125:292
Dietary Fat (Virgin Olive Oil or Sunflower Oil) and Physical Training Interactions on Blood Lipids in the Rat Jose´ L. Quiles, PhD, Jesu´s R. Huertas, PhD, Julio J. Ochoa, PhD, Maurizio Battino, PhD, Jose´ Mataix, PhD, and Mariano Man˜as, PhD From the Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Granada, Spain; and the Institute of Biochemistry, Faculty of Medicine, University of Ancona, Ancona, Italy OBJECTIVE: We investigated whether the intake of virgin olive oil or sunflower oil and performance of physical exercise (at different states) affect plasma levels of triacylglycerols, total cholesterol, and fatty acid profile in rats. METHODS: The study was carried out with six groups of male rats subjected for 8 wk to a diet based on virgin olive oil (three groups) or sunflower oil (three groups) as dietary fat. One group for each diet acted as sedentary control; the other two groups ran in a treadmill for 8 wk at 65% of the maximum oxygen consumption. One group for each diet was killed 24 h after the last bout of exercise and the other was killed immediately after the exercise performance. Triacylglycerols, total cholesterol, and fatty acid profile were analyzed in plasma. Analysis of variance was used to test differences among groups. RESULTS: Animals fed on virgin olive oil had lower triacylglycerol and cholesterol values. Physical exercise reduced these parameters with both dietary treatments. Fatty acid profile showed higher monounsaturated fatty acid proportion in virgin olive fed oil animals and a higher -6 polyunsaturated fatty acid proportion in sunflower oil fed animals. Physical exercise reduced the levels of monounsaturated fatty acids with both diets and increased the proportions of -3 polyunsaturated fatty acids. CONCLUSIONS: Results from the present study supported the idea that physical exercise and the intake of virgin olive oil are very good ways of reducing plasma triacylglycerols and cholesterol, which is desirable in many pathologic situations. Concerning findings on fatty acid profile, we had results similar to those of other investigators regarding the effect of different sources of dietary fat on plasma. The most interesting results came from the effect of physical exercise, with significant increases in the levels of -3 polyunsaturated fatty acids, which may contribute to the antithrombotic state and lower production of proinflammatory prostanoids attributed to physical exercise. Nutrition 2003;19:363–368. ©Elsevier Science Inc. 2003 KEY WORDS: olive oil, sunflower oil, fatty acids, exercise, rats
INTRODUCTION The intake of high amounts of fat is a major risk factor in the etiology of cardiovascular disease and cancer, two of the main causes of death among populations in developed countries. However, it is evident that the fatty acid composition of a particular fat is more important that its absolute concentration regarding these diseases.1 Indeed, there is an important number of studies supporting the positive effects of the intake of fish oil (mainly rich in -3 polyunsaturated fatty acids, or PUFAs) and virgin olive oil (mainly monounsaturated fat) on these diseases.1 In the same way, many investigators have expressed their concern about the risk of an excessive intake of saturated fat or edible oils rich in -6 PUFAs (mainly seed oils such as sunflower or corn).2 Physical exercise is widely recommended as a way of reducing weight gain and ameliorating the risk of hyperlipemias and other
This work was supported by CICYT project ALI91-1113-C03-01. Correspondence to: Jose´ L. Quiles, PhD, Instituto de Nutricio´n y Tecnologı´a de Alimentos, Universidad de Granada, C/ Ramo´n y Cajal 4 (Edif. Fray Luis de Granada), 18071 Granada, Spain. E-mail: [email protected] Nutrition 19:363–368, 2003 ©Elsevier Science Inc., 2003. Printed in the United States. All rights reserved.
deleterious effects of a high fat intake.3 Thus, exercise training decreases the plasma levels of total cholesterol and low-density lipoprotein cholesterol, increases the levels of high-density lipoprotein cholesterol, reduces the concentration of triacylglycerols, reduces systolic and diastolic blood pressure, and is usually recommended to prevent or ameliorate the effects of cardiovascular diseases.4 Moreover, considerable epidemiologic evidence has been acquired linking increased physical activity with reduced occurrence of breast and colon cancers.5 In the same way, some experimental studies performed with animals have suggested that chronic exercise can retard, delay, or prevent the incidence, progression, or metastasis of experimental tumours.6 Some of the possible mechanisms supporting these results include reduced body fat, enhanced gut motility, stimulation of the immune system, and decreased time of exposure to estrogens and other hormones. We have already demonstrated some of the links between dietary fat and physical exercise in relation to health. In fact, previous studies have shown that a program of training for 8 wk leads to changes in the lipid profile of liver and skeletal muscle mitochondrial membranes in rats.7 These changes depended on the type of fat consumed in affecting the functionality of such membranes8 (contributing in some cases to the general benefits 0899-9007/03/$30.00 PII S0899-9007(02)00949-8
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Nutrition Volume 19, Number 4, 2003 TABLE I.
FATTY ACID COMPOSITION OF DIETARY OILS (g/100 g) Fatty acid
Virgin olive oil
Sunflower oil
16:0 16:1(-9) 16:1(-7) 18:0 18:1(-9) 18:2(-6) 18:3(-3)
11.32 0.11 0.84 4.34 74.12 7.64 0.61
7.19 0.02 0.19 4.51 32.08 54.26 0.10
attributed to physical exercise) and modifying their susceptibility to be oxidized by free radicals.9 This evidence and our previous experience encouraged us to investigate whether the combination of physical exercise and the intake of monounsaturated fat (virgin olive oil) has any additional positive effect on blood parameters related to lipid metabolism. For the present study we used virgin olive oil and sunflower oil as dietary fats (the most commonly used vegetable oils in Spain) and a program of physical exercise involving a chronic model and a chronic plus acute model. We estimated lipid profile in plasma and the levels of total cholesterol, serum triacylglycerols, and other parameters related to physical exercise.
MATERIALS AND METHODS
and Technology approved the protocols. Animals were handled according to the guidelines for care and use of laboratory animals by the Spanish Society for Laboratory Animal Sciences. Sample Analysis The rats were killed by decapitation, blood was collected in tubes coated with ethylene-diaminetetraacetic acid, and plasma was separated by centrifuge. Triacylglycerols, total cholesterol, glucose, and lactate concentrations were determined in plasma by enzymatic methods using Boehringer-Mannheim kits (Munich, Germany). Fatty acid profile of plasma was measured by gas liquid chromatography as described by Lepage and Roy.11 A gas–liquid chromatograph (Model HP-5890, Series II, Hewlett Packard, Palo Alto, CA, USA) equipped with a flame ionization detector was used to analyze fatty acids as methyl esters. Chromatography was performed with a 60-m capillary column with 32 mm inner diameter and 20 mm thickness impregnated with Sp 2330 FS (Supelco Inc., Bellefonte, Palo Alto, CA, USA). The injector and detector were maintained at 250°C and 275°C, respectively; nitrogen was used as the carrier gas, and the split ratio was 29:1. Temperature programming (for 40 min) was as follows: initial temperature, 160°C for 5 min, 6°C/min to 195°C, 4°C/min to 220°C, 2°C/min to 230°C, hold 12 min, 14°C/min to 160°C. All chemical products and solvents, of the highest quality available, were acquired from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany). The homologs of coenzyme Q were gifts from Eisai Co. (Tokyo, Japan). Virgin olive oil and sunflower oil were kindly provided by Coosur S.A. (Jaen, Spain).
Experimental Protocol
Statistical Analysis
Male Wistar rats, initially weighing 80 to 90 g, were allocated in groups of 10 per cage and maintained on a 12-h light/12-h darkness cycle, with free access to food and drinking water. The study lasted 9 wk (1 wk for animal selection followed by 8 wk for the experiment). During the selection week, all rats were fed a nonpurified diet and subjected to daily sessions on an exercise treadmill at a speed of 15 m/min for 15 min. The rats were fed semisynthetic and isoenergetic diets composed of (g/kg of diet): 267 casein, 135.3 starch, 453 sucrose, 80 edible oil, 37 mineral supplement, 10 vitamin supplement, 1.8 cellulose, 0.9 choline, and 3 methionine. Three groups of eight rats each received sunflower oil as dietary fat and the other groups (one of eight rats and two of six rats each) received virgin olive oil (the lipid profile of both oils are showed in Table I). For each diet one group was made up of sedentary rats (no exercise) and two groups followed a physical exercise program (as described below). Thus, the groups were as follow: rats fed virgin olive oil and sedentary (VS; n ⫽ 8); rats fed virgin olive oil and exercised (VT; n ⫽ 6); rats fed virgin olive oil and exercised to exhaustion (VE; n ⫽ 6); rats fed sunflower oil and sedentary (SS; n ⫽ 8); rats fed sunflower oil and exercised (ST; n ⫽ 8), and rats fed sunflower oil and exercised to exhaustion (SE; n ⫽ 8). The exercised animals (VT, VE, ST, and SE) underwent training sessions on a horizontal treadmill throughout the 8 wk of the experiment: for the first 2 wk, the rats were exercised 5 d/wk, once a day at a steadily increased rate, until they could run 40 min/d at 35 m/min. These conditions, equivalent to 65% to 70% of maximum oxygen consumption,10 were maintained during the remaining 6 wk. The VT and ST groups were killed 24 h after the last exercise bout. The VE and SE groups performed a final exercise test until exhaustion and immediately killed. Intakes for each group were monitored daily. The rats were killed at the same time of the day in all cases (between 12:00 and 1:00 PM) to avoid circadian fluctuations. The Ethical Committee of the Interministerial Commission of Science
The results represent the mean and standard error of six (VT and VE) or eight (VS, SS, ST and SE) animals. A two-way analysis of variance was performed for effects of dietary fat and physical activity on each variable. Significant (P ⬍ 0.05) interaction terms were evaluated by Scheffe´ ’s F test. Previous to any statistical analysis, all variables were checked for normal and homogeneous variance with Levene’s test. When a variable was found not to be normal, it was log-transformed and reanalyzed.
RESULTS Dietary intake did not vary significantly among groups (data not shown). The sedentary animals reached a higher weight gain than did exercised groups at the end of the study (Fig. 1A). According to the two-way analysis of variance, physical exercise was the only factor responsible for differences in rat weight gain. Sedentary animals fed sunflower oil reached the highest levels of triacylglycerols (Fig. 1B), and physical exercise (both models) led to a lower concentrations of serum triacylglycerols in the animals fed with both diets. Total cholesterol analysis (Fig. 1C) showed that physical exercise was responsible for lower levels of that molecule in both dietary groups. However, sedentary animals fed virgin olive oil had a lower cholesterol amount than did the sedentary animals fed sunflower oil. Physical exercise and the interaction between exercise and dietary fat were responsible for the levels of total cholesterol, according to the two-way analysis of variance. For both dietary treatments, levels of lactate (Fig. 2A) were similar and both types of exercise led to higher values, with no differences between fats. No differences were found between the sedentary groups for glucose (Fig. 2B), and physical exercise led to higher levels in exhausted rats, with no differences between diets. In relation to the plasma lipid profile (Table II), for the saturated fatty acids, there were differences among groups only for
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FIG. 2. Effects of physical exercise and dietary fat on plasma lactate and glucose of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects are considered significant at P ⬍ 0.05. The P values for this two-way analysis were as follows. For lactate: fat, 0.559; exercise, 0.001; fat ⫻ exercise, 0.359. For glucose: fat, 0.290; exercise, 0.000; fat ⫻ exercise, 1.000. SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
FIG. 1. Effects of physical exercise and dietary fat on weight gain, plasma triacylglycerols, and plasma total cholesterol of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects were considered significant at P ⬍ 0.05. The P values for this two-way analysis were as follows. For weight gain: fat, 0.454; exercise, 0.000; fat ⫻ exercise, 0.341. For triacylglycerols: fat, 0.690; exercise, 0.000; fat ⫻ exercise, 0.015. For total cholesterol: fat, 0.741; exercise, 0.000; fat ⫻ exercise, 0.016. SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
stearic acid (18:0), with all animals subjected to physical exercise reaching higher levels (there was no difference between diets). For all the monounsaturated fatty acids (MUFAs), the highest levels were found in animals fed virgin olive oil. In addition, physical exercise led to lower levels of MUFA in both dietary groups. Dietary fat and physical exercise were responsible for these changes according to analysis of variance. In relation to the -6
PUFAs, animals fed sunflower oil showed the highest levels. Physical exercise reached higher levels compared with their controls for 20:4(-6) in both diets and for total -6 PUFA in animals fed virgin olive oil. Concerning -3 PUFA, animals from both dietary groups had similar levels of 22:6(-3), and physical exercise led to higher proportions than in sedentary animals. Physical exercise affected total -3 PUFA in animals fed olive oil. The total PUFA index showed higher levels in all the animals fed sunflower oil and those subjected to physical exercise (for both diets). The highest 20:4(-6)/22:6(-3) ratio (Fig. 3A) belonged to animals fed sunflower oil. This ratio was lower in trained animals than in controls but not in the exhausted groups. Figure 3B shows the ratio -6 PUFA to -3 PUFA, with results similar to those described for the ratio of arachidonic to docosahexaenoic acid, but in this case physical exercise decreased this ratio in exhausted animals fed sunflower oil.
DISCUSSION There is contrasting evidence about the benefits of specific dietary fats and physical exercise on pathologic states such as cardiovascular disease and certain types of cancer. The goal of the present work was to investigate whether the combination of both factors
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Nutrition Volume 19, Number 4, 2003 TABLE II.
PLASMA FATTY ACID COMPOSITION (g/100 g) OF RATS SUBJECTED TO PHYSICAL EXERCISE AND DIFFERENT DIETARY FATS* Experimental groups
Two-way ANOVA†
Fatty acid or index
VS (n ⫽ 8)
VT (n ⫽ 6)
VE (n ⫽ 6)
SS (n ⫽ 8)
ST (n ⫽ 8)
SE (n ⫽ 8)
Fat
Exercise
Fat ⫻ exercise
16:0 18:0 ⌺Saturated 18:1(-7) 18:1(-9) ⌺MUFA 18:2(-6) 20:4(-6) ⌺PUFA(-6) 22:6(-3) ⌺PUFA(-3) ⌺PUFA
17.4 ⫾ 0.7a 8.7 ⫾ 1.1a 36.0 ⫾ 5.2b 4.5 ⫾ 0.2c 35.4 ⫾ 1.8c 43.8 ⫾ 2.6d 6.6 ⫾ 0.7a 10.5 ⫾ 1.2a 18.6 ⫾ 1.5a 0.9 ⫾ 0.1a 1.2 ⫾ 0.1a 19.1 ⫾ 2.8a
17.1 ⫾ 0.7a 10.2 ⫾ 0.8b 30.3 ⫾ 0.5b 3.6 ⫾ 0.3b 32.3 ⫾ 0.3b 38.1 ⫾ 1.4c 7.9 ⫾ 0.4a 14.1 ⫾ 0.7b 23.4 ⫾ 1.2b 1.6 ⫾ 0.3b 1.9 ⫾ 0.4b 27.2 ⫾ 2.2b
17.3 ⫾ 0.7a 11.5 ⫾ 0.9b 31.7 ⫾ 0.6b 3.1 ⫾ 0.1b 31.8 ⫾ 2.1b 35.2 ⫾ 2.2c 8.8 ⫾ 1.4a 18.5 ⫾ 1.5bc 28.2 ⫾ 1.7b 1.5 ⫾ 0.3b 1.9 ⫾ 0.3b 31.1 ⫾ 2.1b
17.1 ⫾ 0.4a 7.1 ⫾ 0.3a 26.1 ⫾ 0.2a 3.4 ⫾ 0.2b 18.6 ⫾ 0.4b 25.7 ⫾ 0.8b 28.7 ⫾ 1.6b 15.3 ⫾ 1.1b 46.9 ⫾ 0.7c 0.6 ⫾ 0.2a 0.9 ⫾ 0.1a 48.2 ⫾ 0.9c
17.1 ⫾ 0.4a 10.1 ⫾ 0.6b 29.4 ⫾ 0.5b 2.1 ⫾ 0.1a 15.3 ⫾ 1.1a 19.1 ⫾ 1.3a 29.2 ⫾ 1.1b 19.1 ⫾ 1.6c 49.4 ⫾ 1.1c 1.3 ⫾ 0.4b 1.6 ⫾ 0.2b 51.5 ⫾ 1.1d
17.5 ⫾ 0.3a 9.2 ⫾ 1.3b 28.9 ⫾ 1.4b 2.27 ⫾ 0.18a 14.6 ⫾ 1.2a 20.9 ⫾ 1.6a 25.1 ⫾ 2.1b 20.8 ⫾ 1.7c 48.4 ⫾ 1.1c 1.3 ⫾ 0.3b 1.6 ⫾ 0.2b 51.1 ⫾ 1.1d
0.132 0.132 0.133 0.000 0.000 0.000 0.000 0.011 0.000 0.102 0.109 0.000
0.312 0.045 0.898 0.000 0.015 0.012 0.185 0.001 0.103 0.012 0.010 0.002
0.103 0.353 0.029 0.169 0.421 0.423 0.300 0.762 0.036 0.617 0.123 0.071
* Results are presented as means ⫾ standard error of the mean. For each fatty acid or index, values in a row not sharing superscript letters are significantly different (P ⬍ 0.05). † P values for the two-way ANOVA for the effects of fat, exercise, and the interaction between fat and exercise (significant at P ⬍ 0.05). ANOVA, analysis of variance; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
(fat and exercise) affects blood lipid metabolism, thus modifying the predisposition to these diseases. Animals adapted to diet, as showed by plasma fatty acid profiles (animals fed virgin olive oil had a higher proportion of MUFA and the highest -6 PUFA was found in animals fed sunflower oil). Animals also adapted to the training program, as show by the significantly higher plasma triacylglycerols and total cholesterol levels in sedentary animals and by the levels of lactate and glucose. This was also confirmed by the lesser weight gain produced by exercise, which has been well documented in humans and animals.3,12 Regarding triacylglycerols, both models of exercise reduced serum levels of this molecule. Others previously reported the same effect in rats and humans.13,14 The hypotriglyceridemic effect may be secondary to reduced hepatic triacylglycerol secretion and/or enhanced removal of triacylglycerol-rich lipoproteins by the extrahepatic tissues, according to Tan et al.15 Altered activity of lipoprotein lipase, which is found in several tissues including skeletal muscle, heart, and adipose tissue, also may be involved because it is responsible for the clearance of postprandial chylomicrons and endogenous very low-density lipoprotein.16 With respect to the effect of dietary fat on serum triacylglycerols, we found that sedentary animals fed sunflower oil had higher levels than those fed virgin olive oil. After physical exercise the values reached with both diets were the same, but in terms of reduction, sunflower oil reduced triacylglycerols more than virgin olive oil after exercise. The reduction of serum triacylglycerols after intake of specific dietary fats such as fish oil have been described,17 but there are no reports describing differences between olive oil and sunflower oil. Our study supported the idea that physical exercise and the intake of unsaturated oils as the fat source reduce triacylglycerols, which is desirable in many pathologic situations. Because sunflower oil produces a higher proportion of triacylglycerols in sedentary animals than does virgin olive oil, the practice of physical exercise could be recommended more often if the consumed diet is rich in -6 PUFAs. Many studies,18 but not all,15 have shown that training lowers serum cholesterol in rats, and our data supported this hypothesis. In humans, the type of exercise performed appears to affect the
hypocholesterolemic effect of training.18 However, in the present work both types of exercise led to a reduction in the level of total cholesterol to the same extent. This means that exhaustive exercise in the VE and SE groups not compound the hypocholesterolemic effect in comparison with that obtained with the 8-wk training in the VT and ST groups. Regarding the effects of dietary fat, controversy exists about the effect of olive oil and sunflower oil on total cholesterol. Our results showed that, even if a diet enriched with olive oil leads to lower levels of serum cholesterol than with sunflower oil, the concentration of cholesterol in both diets may be considered normal and the intake of any one of the two diets led to similar levels of cholesterol after physical exercise. Plasma lipid profile of the rats showed that the proportion of saturated fatty acids (SFA) is barely influenced by the ingestion of any of the studied fat sources. SFA appears to be a relatively stable fraction within the general composition of plasma which is in accordance with the findings of others.19 In relation to physical exercise, Rocquelin and Juaneda20 investigated SFA and found that 16:0 but not 18:0 fatty acids decrease in the epididymal fat of trained rats. Our results showed that 18:0 increased in both dietary groups after physical exercise, as did the total SFA index for the sunflower oil group. Many studies have reported that plasma positively adapts itself to the consumption of a MUFA-rich diet, demonstrating in its composition the type of diet ingested.21 We confirmed those results in rats fed the diet rich in olive oil. Physical activity led to a decrease in all MUFAs in all animals. With regard to the -6 PUFA, the highest levels were obtained in animals fed the polyunsaturated fat source, as reported by other.22 Physical exercise increased arachidonic (20:4-6) acid in both dietary groups, as described by Vapaatalo et al.23 in the plasma of young subjects after short-term heavy exercise. In relation to -3 PUFA, both dietary treatments led to similar values, and physical exercise increased the amount of these fatty acids. In the present study, the effects of dietary fat on plasma fatty acid profiles were in agreement with those found by others,21 but the most interesting results concern the effect of physical exercise. It is very difficult to know whether the changes found among the different fractions of fatty acids after physical exercise are related to changes in a particular metabolic function, a higher or lower
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FIG. 3. Effects of physical exercise and dietary fat on 20:4(-6)/22:6(-3) and PUFA (-6)/PUFA(-3) indices in plasma of rats. Values are means ⫾ standard error of the mean. For each chart, columns not sharing superscript letters are significantly different (P ⬍ 0.05). A two-way analysis of variance was performed to test the effects of fat, exercise, and the interaction between fat and exercise. Effects were considered significant for P ⬍ 0.05. The P values for this two-way analysis were as follows. For 20:4(-6)/22:6(-3): fat, 0.000; exercise, 0.005; fat ⫻ exercise, 0.349. For PUFA(-6)/PUFA(-3): fat, 0.000; exercise, 0.015; fat ⫻ exercise, 0.175. PUFA, polyunsaturated fatty acid; SE, sunflower oil fed animals and exercised; SS, sunflower oil fed animals and sedentary; ST, virgin olive oil fed animals and training exercised; VE, virgin olive oil fed animals and exercised; VS, virgin olive oil fed animals and sedentary; VT, virgin olive oil fed animals and training exercised.
disposability of the fatty acids, or if this is due to a higher regulation mechanism. It has been documented that 18:1-9 and other MUFAs have preferential mobilization and oxidation rates than other fatty acids.21 Leyton et al.24 demonstrated that rats oxidize fatty acids in the following order: oleic (18:1-9) ⬎ linoleic (18:2-6) ⬎ palmitic (16:0) and stearic (18:0). Similar differences exist in humans.25 This could justify the decrease of MUFA in our model after physical exercise because of the higher energetic requirements it caused. It could be mediated, according to Helge et al.,26 by an increase in the solubility of lipids in circulating lipoproteins and, hence, an enhanced enzyme-substrate contact, due to the unsaturation of the lipids. Another potential mechanism could be related to changes in lipoprotein lipase activity in heart and skeletal muscle mediated by adaptations to different diets, as suggested by Shimomura et al.27 If this hypothesis is true, the increase in some SFAs and PUFAs together with a decrease in MUFAs as a consequence of physical exercise could represent a change in the relative proportions of fatty acids in plasma. That is easy to see if we keep in mind that lipid profile results are expressed as relative proportions of the different fatty acids, and a decrease in any of the fractions could lead to a relative increase in all the others.
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Considerable controversy exists concerning the relative importance of -3 and -6 PUFAs in the prevention of cardiovascular diseases and cancer. Increase in -3 fatty acids has been related to a decrease in serum triacylglycerols and an increase in highdensity lipoprotein cholesterol, whereas increases in linoleic acid and ␥-linoleic acid mainly decrease total cholesterol and lowdensity lipoprotein cholesterol.28 In a similar way, high levels of -3 PUFA may decrease cancer progression and metastasis, and the opposite is true for -6 PUFA.29 Eicosapentaenoic acid (20: 5-3) inhibits platelet aggregation induced by arachidonic acid and docosahexaenoic acid (22:6-3) reduces platelet aggregating prostanoids (prostaglandins G2 and H2, thromboxane A2).30 Thus, low levels of 20:4-6/22:6-3 or, by extension, -6 PUFA/-3 PUFA in plasma reflect a state of antithrombogenesis. In fact, a high dietary intake of eicosapentaenoic and docosahexaenoic acid in volunteers reported by decreased plasma triacylglycerols and/or plasma total cholesterol, prolonging bleeding time, and also led to an antithrombotic state.30 In our study the lower levels of 20:46/22:6-3 and -6 PUFA/-3 PUFA were present in animals fed virgin olive oil. Moreover, regular training reduced these levels with both dietary fats. Thus, we could say that virgin olive oil is less prothrombotic than sunflower oil and that physical exercise promotes an antithrombotic state. In addition, an increase in the ratio of -3 PUFA to -6 PUFA may contribute to a lower production of proinflammatory prostanoids and a higher production of the anti-inflammatory ones. In conclusion, under our experimental conditions, physical exercise and intake of virgin olive oil were very effective in reducing plasma triacylglycerols and cholesterol. Concerning findings on the fatty acid profile, the most interesting results come from the effect of physical exercise, with significant increases in the levels of -3 PUFAs. These effects are very desirable in many pathologic situations.
ACKNOWLEDGMENTS Special thanks to Ms. Monica Glebocki for elaboration of the manuscript.
REFERENCES 1. Willett WC, Sacks F, Trichopoulou A, et al. Mediterranean diet pyramid: a cultural model for healthy eating. Am J Nutr 1995;61:1321S 2. Ramirez-Tortosa MC, Urbano G, Lopez-Jurado M, et al. Extra-virgin more than refined olive oil increases the resistance of LDL to oxidation in free-living men with peripheral vascular disease. J Nutr 1999;129:2177 3. Bell RR, Spencer MJ, Sherriff JL. Voluntary exercise and monounsaturated canola oil reduce fat gain in mice fed diets high in fat. J Nutr 1997;127:2006 4. Brooks GA, Fahey TD, White TP, Baldwin KM. Exercise physiology. Human bioenergetics and its applications, 3th ed. Mountain View, California: Mayfield Publishing Company, 1999 5. Thune I, Furberg AS. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific. Med Sci Sports Exerc 2001;33:S530 6. Woods JA. Exercise and resistance to neoplasia. Can J Physiol Pharmacol 1998;76:581 7. Quiles JL, Huertas JR, Man˜ as M, Battino M, Mataix J. Physical exercise affects the lipid profile of mitochondrial membranes in rats fed with virgin olive oil or sunflower oil. Br J Nutr 1999;81:21 8. Quiles JL, Huertas JR, Man˜ as M, et al. Dietary fat type and regular exercise affect mitochondrial composition and function depending on specific tissue in rat. J Bioenerg Biomembr 2001;33:127 9. Mataix J, Quiles JL, Huertas JR, Battino M, Man˜ as M. Tissue specific interactions of exercise, dietary fatty acids, and vitamin E in lipid peroxidation. Free Radic Biol Med 1998;24:511 10. Amstrong RB, Laughlin MH, Rome L, Taylor CL. Metabolism of rats running up and down an incline. J Appl Physiol 1983;55:518 11. Lepage G, Roy CC. Direct transesterification of all classes of lipids in one-step reaction. J Lipid Res 1986;27:114 12. Blair SN. Evidence for success of exercise in weight loss and control. Ann Intern Med 1993;119:702
368
Quiles et al.
13. Narayan AK, McMullen JJ, Butler DP, Wakefield T, Calhoun WK. Effect of exercise on tissue lipids and serum lipoproteins of rats fed two levels of fat. J Nutr 1975;195:581 14. Costill DL, Cleary P, Fink WJ, et al. Training adaptations in skeletal muscle of juvenile diabetics. Diabetes 1979;28:818 15. Tan MH, Bonen A, Garner JB, Belcastro AN. Physical training in diabetic rats: effect on glucose tolerance and serum lipids. J Appl Physiol 1982;52:1514 16. Deshaies Y, Vallerand AL, Bukowiecki LJ. Serum lipids and cholesterol distribution in lipoproteins of exercise-trained female rats fed sucrose. Life Sci 1983;33:75 17. Durrington PN, Bhatnagar D, Mackness MI, et al. An omega-3 polyunsaturated fatty acid concentrate administered for one year decreased triacylglycerols in simvastatin treated patients with coronary heart disease and persisting hypertriglyceridaemia. Heart 2001;85:544 18. Bjo¨ rtorp P, Berchtold P, Grimby G. Effects of physical training on glucose tolerance plasma insulin and lipids on body composition in men after myocardial infarction. Acta Med Scand 1972;192:439 19. Rueda P, Lopez-Frias M, Llopis J, Mataix J, Urbano G. Influence of dietary fat on plasma fatty acid composition in rats. Nutr Res 1992;12:757 20. Rocquelin G, Juaneda P. Influence of prolonged physical training on the composition of fatty acids of epididymal adipose tissue and of the carcass in the young rat on a dietary regime of sunflower, rapeseed or primor oil. Rep Nutr Dev 1981;21:1015
Nutrition Volume 19, Number 4, 2003 21. Seiquer I, Man˜ as M, Martinez-Victoria E, Ballesta MC, Mataix FJ. Long-term influence of dietary fat (sunflower oil, olive oil, lard and fish oil) in the serum fatty acid composition and in the different lipidic fractions, in miniature swine. Int J Vitam Nutr Res 1996;66:171 22. Jacotot B, Lasserre M, Mendy F. Effects of different diets rich in polyunsaturated fatty acids on plasma phospholipids in the human. Progr Lipid Res 1986;25:185 23. Vapaatalo H, Laustiola K, Seppala E, et al. Exercise, ethanol and arachidonic acid metabolism in healthy men. Biomed Biochem Acta 1984;43:S413 24. Leyton J, Drury PJ, Crawford MA. Differential oxidation of saturated and unsaturated fatty acids in vivo in the rat. Br J Nutr 1987;57:383 25. Jones PJH, Pencharz PB, Clandinin MT. Whole body oxidation of dietary fatty acids: implications for energy utilization. Am J Clin Nutr 1985;42:769 26. Helge JW, Ayre K, Chaunchaiyakul S, et al. Endurance in high-fat-fed rats: effects of carbohydrate content and fatty acid profile. J Appl Physiol 1998;85:1342 27. Shimomura Y, Tamura T, Suzuki M. Less body fat accumulation in rats fed a safflower oil diet than in rats fed a beef tallow diet. J Nutr 1990;120:1291 28. Haglund O, Wallin R, Wretling S, Saldeen T. Effects of fish oil alone and combined with long chain (n-6) fatty acids on some coronary risk factors in male subjects. J Nutr Biochem 1998;9:629 29. Lipworth L, Martinez ME, Angell J, Hsieh C-C, Trichopoulos D. Olive oil and human cancer: an assessment of the evidence. Prev Med 1997;26:181 30. Masumura S, Furui H, Hashimoto M, Watanabe Y. The effects of season and exercise on the levels of plasma polyunsaturated fatty acids and lipoprotein cholesterol in young rats. Biochim Biophys Acta 1992;1125:292