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1Metabolic Research Centre and Departments of 2Biomedical and 4Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522; and 3Exercise Metabolism Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia
Submitted 15 September 2003 ; accepted in final form 17 November 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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phospholipids; training; dietary fat; insulin sensitivity
With regard to the effects of alterations in dietary macronutrient content in modifying membrane FA composition, the turnover of FA in rodent skeletal and heart muscle is rapid, with major changes observed after 2–3 days (6) and approaching a plateau within 3–4 wk (32). Exercise has a more subtle effect on skeletal muscle FA profile. Some studies (4, 5, 15, 17) demonstrate alterations in phospholipid FA composition after short-term (4–6 wk) exercise-training programs, whereas others (25) report no measurable changes. As FA oxidation is increased during exercise at the same absolute workload following endurance training (30), it might be expected that contraction-induced changes may have resulted from a mobilization and subsequent oxidation of phospholipids during exercise. However, in rodents, exercising muscles rely mainly on FA derived from outside the muscle as a fuel source and do not oxidize intracellular lipids, such as membrane phospholipids or triglycerides, to any great extent (27, 28). Hence differences in results between various investigations may be related to methodological factors, the species and sex of the population studied, the fiber type of the muscles under investigation, and/or the training stimulus (intensity x duration x frequency).
To date, the effect of different training stimuli on membrane FA composition has received little attention. Furthermore, only limited information is available to show how changes in the supply of dietary FA interact with regular exercise, and whether this affects muscle phospholipid FA composition (15). As high-fat diets and physical inactivity are both independent predictors of insulin sensitivity (34), determining how diet and exercise programs may modify this disorder is of high priority. Accordingly, in the present study, we determined the interaction of diet (high carbohydrate vs. high fat) and exercise-training intensity (low vs. high) on FA profile of muscle phospholipid. This analysis was extended with the investigation of two muscles [red (RVL) and white vastus lateralis (WVL)] comprising different fiber types. Differences in responses to diet and exercise manipulations might be anticipated between these muscles, as oxidative fibers have a greater GLUT-4 protein content (19, 27), are more insulin sensitive (19), have greater rates of maximal glucose transport (33), and possess greater amounts of plasma membrane FA binding protein (13) than muscles with predominantly glycolytic fibers. Furthermore, as glycolytic fibers are primarily activated during more intense exercise (14), it might be anticipated that any potential exercise-related changes in membrane FA composition would only occur in the WVL in response to high-intensity exercise.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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After 1 wk, animals were randomly divided into one of two dietary conditions. One group (n = 18) was fed a high-carbohydrate diet (CHO; 64.0% carbohydrate by energy; Ridley Agriproducts Pty, Victoria, Australia), whereas the other group (n = 18) received a high-fat diet (Fat; 78.1% fat by energy). The nutritional composition and energy content of these diets are shown in Table 1, whereas the FA composition is presented in Table 2. The Fat diet was prepared from fresh ingredients every 2 wk and stored at 4°C. The vitamin and mineral content of both diets was in accordance with the guidelines reported by the American Institute of Nutrition (9). Rats were provided with ad libitum access to food and water. Both food and fluid intake, along with the animal's BM, was recorded every 2 wk during the experiment. Food consumption was estimated by measuring the difference between the weighed portions of food provided to animals and the uneaten food, and then dividing by two (as there were two animals per cage).
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Animals in each diet condition were divided into one of three subgroups: a sedentary (control) group (n = 6) that performed no training (NT); a group (n = 6) that performed low-intensity running (Low); and a group (n = 6) that trained at their maximal voluntary running speed (Vmax). The Low program was chosen because previous investigations have shown this speed to elicit 
45% of maximal O2 uptake in rats, an intensity at which lipid oxidation predominates over carbohydrate oxidation (11). We chose to examine the metabolic adaptations induced by a running program that all animals could complete without the use of external motivation (e.g., electric shock). As noted previously (25), this type of exercise provides a more physiological demand on the muscles. Furthermore, we sought to maximize this stimulus so that the effects of the two distinct training programs and their interaction with the two diet conditions could be examined. In determining the appropriate speed for the Vmax group, pilot testing revealed that all rats could run at a speed of 28 m/min (a velocity 3.5 times faster than the speed of running in Low), which corresponds to 
75% of maximal O2 uptake (11). Between weeks 2 and 5, rats from both Low and Vmax groups had their training duration and intensity progressively increased so that they could complete 1,000 m of treadmill running 4 days/wk. Once this training distance had been reached, animals maintained their training volume for the next 4 wk. Rats undertaking Low ran for 125 min at a speed of 8 m/min. In order for animals from the Vmax group to complete the same training distance (1,000 m/session) as rats from Low, they only had to run 36 min/session. All running sessions were performed with 0° incline, and rats trained 4 days/wk.
Animal death and tissue preparation. At the end of 8 wk, all animals were killed 48 h after the last training bout by heart removal under anesthesia (intraperitoneal injection of pentobarbital sodium, 60 mg/kg BM). Hindlimb muscles from the right leg were exposed, and the RVL (16% type I, 33% type IIa, 50% type IIb) and the WVL (100% type IIb fibers) were dissected out, rapidly frozen, and stored in liquid nitrogen. Muscle samples were stored at -80°C until analysis.
Analysis of FA composition. All solvents used in the lipid analysis were of ultra-pure grade and were from Merck Pty (Kilsyth, Victoria, Australia). Analytical grade butylated hydroxytoluene was from Sigma Aldrich (Castle Hill, NSW, Australia). Skeletal muscle lipids were extracted by standard methods (12) using chloroform-methanol (2:1 vol/vol) containing butylated hydroxytoluene (0.01% wt/vol) as an antioxidant. Phospholipids were separated by solid-phase extraction on Strata SI-2 silica cartridges (Phenomenex, Pennant Hills, NSW, Australia). FA analysis of the phospholipid fraction was determined, as described in detail previously (32). Briefly, phospholipid fractions were transmethylated with 14% (wt/vol) boron trifluoride in methanol, and FA methyl esters were separated by gas-liquid chromatography on a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) with a fused silica capillary column.
Individual FAs were identified by comparing each peak's retention time to those of external standards. The relative amount of each FA (percentage of total FA) was determined by integrating the area under the peak and dividing the result by the total area for all FAs. The sum of saturates, monounsaturates, and polyunsaturates (PUFA) was calculated, and total proportions of n-6 and n-3 FAs were also determined. Unsaturation index, which represents the average number of double bonds per 100 FA molecules, was calculated by summing the products of the proportion (mol%) of each unsaturated FA multiplied by its number of double bonds. The product-precursor ratio of several FAs was used to gain an indication of enzyme activity. The estimated enzyme activities included those of elongase, calculated as the stearic acid (18:0)-to-palmitic acid (16:0) ratio; 
5 desaturase, calculated as the arachidonic acid [20:4(n-6)]-to-di-homo-
-linolenic acid [20:3(n-6)] ratio; 6 desaturase, calculated as the di-homo--linolenic acid [20:3(n-6)]-to-linoleic acid [18:2(n-6)] ratio (assuming that 6 desaturase and not elongase is rate limiting); and 9 desaturase, calculated as the oleic acid [18:1(n-9)]-to-stearic acid (18:0) ratio.
Statistical analyses. Data were analyzed using a 2 x 3 factorial ANOVA, with diet and training as fixed factors. Where ANOVA revealed a significant effect, Tukey's post hoc test was administered to identify differences between treatments. Significance was accepted at the level of P < 0.05, and all results are reported as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Energy intake and BM. Energy intake in the CHO-fed animals was significantly higher than the intake in the Fat-fed rats (288–292 vs. 243–255 kJ/day; P < 0.05) (26). There were no differences in the BM of animals from any of the experimental groups at the end of the 8-wk intervention period (range 248–272 g).
Phospholipid FA composition. The FA composition of RVL and WVL phospholipids from the different treatments are presented in Tables 3 and 4, respectively. For both muscles, the estimated activities of the desaturase (5, 6, 9) and elongase enzymes are presented in Table 5.
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Diet effects. There was a significant main effect of diet on FA composition in RVL phospholipids for all individual FAs, except 16:0 and 18:1(n-7) (Table 3). In WVL phospholipids, there were also significant effects of diet for all but two FAs [18:1(n-9) and 20:3(n-6); Table 4]. Despite the large effect of diet on individual FAs, the general characteristics of the phospholipids (percent saturates, monounsaturates, PUFA) did not differ between diets in either RVL or WVL (results not shown). The most notable difference between the CHO and Fat diets in both muscles was the content of n-3 and n-6 PUFA. Phospholipids from the CHO-fed animals had greater amounts of n-3 PUFA, particularly docosahexaenoic acid [22:6(n-3)], which was present in very high concentrations, and also 20: 5(n-3), which was only found in this group. In Fat-fed animals, there was an increase in the proportions of n-6 PUFA, including arachidonic acid [20:4(n-6)], which was the major constituent in their membranes, and 22:4(n-6) and 22:5(n-6), which were not detected in the CHO-fed animals. As a result of this, the n-6-to-n-3 ratio (n-6/n-3) was significantly elevated in the Fat-fed animals for both RVL and WVL phospholipids (P < 0.01; Fig. 1). The Fat diet was also associated with significantly higher estimated activity in the elongase and desaturase (5, 6, 9) enzymes (P < 0.01, Table 5). The unsaturation index was significantly higher in RVL phospholipids from CHO-fed animals (P < 0.01, Table 3), whereas WVL phospholipids from the Fat-fed animals contained higher proportions of FA with 20–22 carbons (P < 0.01; Table 4).
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Training effects. Changes in phospholipid FA composition as a result of either training protocol were small compared with the substantial alterations resulting from the Fat intervention. In RVL phospholipids, 18:2(n-6) was the only individual FA that changed after training, with an increased content observed after both training protocols (P < 0.01; Table 3). A main effect of training (P < 0.01), along with a significant interaction of diet with training (P < 0.05), was observed for n-6/n-3 (Fig. 1). Thus Fat-fed animals that performed either Low or Vmax had a greater ratio of n-6/n-3 FA than the NT animals. There was a significant interaction of diet with training on the 18:0-to- 16:0 ratio (P < 0.05, Table 5), which is an estimate of elongase activity. Vmax animals that were Fat fed displayed greater elongase activity than NT or Low animals from the same group (P < 0.05). In WVL, there was a significant (P < 0.05) main effect of training for 20:3(n-6) and 22:4(n-6) (Table 4). For 20:3(n-6), there was also a significant interaction of diet with training (P < 0.05), such that Fat-fed animals from the NT, Low, and Vmax groups all had significantly different amounts. There was a significant main effect of training on the estimated activity of the 5 desaturase enzyme [20:4(n-6)/20:3(n-6)] in WVL (P < 0.01, Table 5), with training reducing this activity. Also evident was a significant diet-training interaction (P < 0.01), with Low and Vmax animals from the Fat-fed group showing reduced activity compared with the NT animals. A significant effect of training (P < 0.01) along with a significant interaction of diet with training (P < 0.05) was also observed for the estimated 6 desaturase activity [20:3(n-6)/18:2(n-6)]. Vmax animals that consumed the Fat diet had elevated 6 desaturase activity compared with the Low and NT animals.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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4 wk) exercise training (4, 5, 15, 17). Both of these factors can also influence endurance capacity. Consumption of a high-fat diet for short periods has been shown to result in adaptive responses that enhance exercise endurance (26). These responses depend on not only the total content of fat, but also the FA profile of the diet, with those diets containing a high amount of n-6 PUFA showing a greater improvement in exercise capacity in rats (7). In the present investigation, the effect of diet (CHO vs. Fat) and exercise intensity (low vs. high) on phospholipid FA composition was examined. The first important finding was that there was a significant and widespread effect of diet on phospholipid FA composition in skeletal muscles with diverse metabolic profiles. In contrast, a chronic program of exercise training that resulted in substantial increases in endurance capacity in these rats (26) was associated with only minor changes in muscle FA profile. Alterations in muscle membrane FA profile resulting from diet and exercise have important implications for insulin sensitivity, as skeletal muscle is one of the major sites for insulin-stimulated glucose disposal (35). A number of factors are associated with insulin sensitivity in skeletal muscle, including the FA composition of membrane lipids (10, 36, 37) and the concentration of intramuscular triglyceride. Increased membrane levels of PUFA, particularly n-3, along with decreased levels of saturated FA, have been associated with improved insulin action (36), whereas studies in both rodents (22) and humans (24, 31) have demonstrated strong negative relationships between muscle triglyceride content and whole body insulin action. A chronic high-fat diet, composed of predominantly saturated fat, is associated with abnormal lipid metabolism in skeletal muscle (38) and induces widespread insulin resistance (23, 35). In contrast, chronic exercise training is associated with improved lipid profiles and enhanced insulin sensitivity (18, 21). Of interest is the finding that exercise training compensates for, but does not completely reverse, the deleterious effects of a high-fat diet on insulin action (19, 20, 23).
In the present study, the most striking effect of diet, in both oxidative and glycolytic skeletal muscles, was on the n-6/n-3, which was increased in animals fed the Fat diet (Fig. 1). As noted previously, Ayre and Hulbert (7) found that a high n-6/n-3 is associated with improved performance during endurance exercise in rats. A high n-6/n-3 also appears to contribute to the development of insulin resistance (36); however, of interest is the fact that administration of dehydroepiandrosterone improves insulin action and also increases n-6/n-3 in Zucker rat skeletal muscle (1, 2), indicating that other mechanisms may be involved. The observed difference in the n-6/n-3 in the present study was primarily a reflection of the content of docosahexaenoic acid [22:6(n-3)] and arachidonic acid [20:4(n-6)] in the phospholipids from the rats fed the different diets. The CHO diet was associated with very high concentrations of 22:6(n-3) and also the presence of eicosapentanoic acid [20:5(n-3)], with their elevated contents a reflection of the FA profile of the diet (Table 2). The phospholipids from rats that consumed the Fat diet contained very high levels of 20:4(n-6) and also contained 22:4(n-6) and 22:5(n-6), which were two FA not detected in the muscles of animals that were fed the CHO diet. This high proportion of n-6 FA is indicative of the fact that no 18:3(n-3) was detected in the Fat diet (Table 2), and thus much of the long-chain PUFA would have been derived from 18:2(n-6). The increased elongase and desaturase enzyme activity observed in the Fat-fed rats (Table 5) would tend to support such a contention. However, it should be noted that enzyme activities were not directly measured, but were estimated from product precursor ratios and thus may have been influenced by preferential incorporation of particular dietary FA into membrane.
Exercise training had little effect on phospholipid FA composition in rats fed the CHO diet, which is consistent with previous findings (25). In rats fed the Fat diet, however, a number of small changes in phospholipid composition were observed that were likely to be related to the exercise-training intervention. In the RVL phospholipids, the content of 18: 2(n-6) was increased after both exercise-training regimens (Table 3). Helge et al. (15) reported a similar increase in 18:2(n-6) in three different muscle fiber types from rats after exercise training. WVL phospholipids from rats fed a high-fat diet were also modified by exercise, with increased levels of 20:3(n-6) and 22:4(n-6) seen following training (Table 4). Borkman et al. (10) reported negative correlations between the 18:2(n-6) and 20:3(n-6) content of skeletal muscle phospholipids and insulin sensitivity, whereas the content of 22:4(n-6) was positively correlated.
The exercise-induced changes observed in individual FA resulted in a significantly elevated n-6/n-3 in RVL phospholipids from the Fat-fed animals, with a similar trend (P = 0.075) also observed in WVL (Fig. 1). Kriketos et al. (25) reported an increased n-6/n-3 in an oxidative muscle (soleus) as a result of exercise training. Ayre and Hulbert (7) found that dietary manipulation of muscle phospholipid composition, resulting in an increased n-6/n-3, significantly improved endurance capacity in sedentary rats. Of interest in the present study was that consumption of a Fat diet and exercise training, both of which independently increased endurance capacity in these rats (26), also independently increased n-6/n-3 in the muscle phospholipids. Thus, although the mechanism is unclear, it appears that, in rodents, a high proportion of n-6 PUFA relative to n-3 PUFA is beneficial for endurance exercise.
The estimated activity of the desaturase and elongase enzymes also changed slightly in response to training in the rats fed a Fat diet. In RVL phospholipids, the estimated activity of the elongase enzyme was increased following the higher intensity training, whereas increased 6 and decreased 5 desaturase activity was also seen in WVL following training. These exercise-induced changes have previously been reported in rats (8, 15) and are generally associated with reduced insulin sensitivity (4, 10). Thus it appears that, whereas both RVL and WVL responded to diet in a similar fashion, there may be some small differences in their response to exercise training. As noted previously, the changes in phospholipids resulting from exercise training were only minor and were not affected by the intensity of the exercise intervention (high vs. low). Thus it remains to be determined whether they are of functional significance.
We did not assess the impact of exercise and diet manipulations on intramuscular triglyceride concentration in the present study. However, our laboratory (27) has previously reported that a high-fat, low-carbohydrate diet significantly increased muscle lipid content in both type I and type II muscle fibers in sedentary rodents in the face of normal glycogen stores. Furthermore, in that investigation, we also showed that an 8-wk program of treadmill-run training did not attenuate the increase in muscle triglyceride levels seen after the high-fat diet (27). Others have also reported that muscle triglyceride content is not altered by short-term (4–8 wk) training interventions (5, 16, 17), although these same studies do find alterations in muscle phospholipid FA composition after exercise training. For example, Helge and Dela (16) have recently reported that 8 wk of endurance training in healthy humans did not affect muscle triglyceride content or total triacylglycerol FA composition, but did induce changes in the content of phospholipid FA membrane PUFA. These workers found that the sum of phospholipid long-chain PUFA was significantly correlated with leg glucose uptake during a hyperinsulinemic clamp (r = 0.57, P < 0.05), indicating that membrane lipids may have a role in the training-induced increase in insulin sensitivity (16). Taken collectively, these findings suggest that changes in muscle phospholipid status are modified independently by diet and exercise. Indeed, the results from the present study indicate that, although diet and exercise act independently to modify FA composition of skeletal muscle, there may be subtle but important interactions between the two variables, as changes with training were only observed in the Fat diet and not the CHO diet group. The majority of these changes, however, appear to be negatively associated with insulin sensitivity, indicating that the compensatory effect of exercise training on insulin action previously observed in rats (19, 20, 23) may not be via any direct effects on the FA composition of skeletal muscle phospholipids.
In conclusion, we have demonstrated that, compared with a program of either low- or high-intensity exercise training, diet exerted a greater effect on the phospholipid FA composition of both oxidative and glycolytic rodent skeletal muscle. Although the Fat diet employed in the present investigation was extreme, our results demonstrate that a program of exercise training did little to attenuate the negative effects of high-fat feeding on membrane phospholipids.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This study was funded by an RMIT Faculty Research Grant (to J. A. Hawley) and an Australian Research Council grant (to J. A. Hawley and P. L. Else).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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O2 max and O2 during two running intensities in rats. Pflügers Arch 386: 215-219, 1980.
-3 fatty acids in muscle phospholipid. Diabetes 40: 280-289, 1991.
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training, P < 0.01.