Interaction of diet and training on endurance performance in rats

Transcription

1 Interaction of diet and training on endurance performance in rats Jong Sam Lee, Clinton R. Bruce, Lawrence L. Spriet* and John A. Hawley Exercise Metabolism Group, School of Medical Sciences, RMIT University, Bundoora 3083, Victoria, Australia and *Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Manuscript received 1 November 2000; accepted 4 May 2001) We determined the interaction of diet and training on metabolic adaptations in skeletal muscle and liver, and the consequences of these adaptations for endurance. Eighty rats performed a baseline treadmill run to exhaustion at 16 m min _1 (RUN1) and were then divided into two groups and given one of two diets: high carbohydrate (CHO) or high fat (FAT). Each dietary group was then divided into one of four subgroups: sedentary control that performed no training (NT); low-intensity running (8 m min _1 ; LOW) and two groups who trained at their maximal voluntary running speed without electrical stimulation (28 m min _1 ; VMAX). Training volume was identical for LOW and VMAX (1000 m session _1 ) and animals ran 4 days week _1 for 8 weeks. To assess the interaction of the higher intensity exercise with diet, a second endurance test (RUN2) was undertaken after 6 weeks at either 16 m min _1 or 28 m min _1. The NT group ran for a longer duration (increase of 77%) after FAT than CHO (239 ± 28 vs. 135 ± 30 min, P < 0.05) at 16 m min _1. There were no differences in RUN2 for the LOW group when rats ran at 16 m min _1 (454 ± 86 vs. 427 ± 75 min for CHO and FAT groups, respectively), but rats in the VMAX group fed FAT ran longer than rats fed CHO at 28 m min _1 (100 ± 28 vs. 58 ± 11 min, respectively, P < 0.05). FAT increased the activities of the enzymes citrate synthase, b-hydroxyacyl-coa dehydrogenase and carnitine palmitoyl-transferase compared to CHO (P<0.01), but there was no systematic effect of training. We conclude: (1) there was no additive effect of a high-fat diet on endurance performance when rats performed low-intensity training; (2) running performance at 28 m min _1 was only enhanced by a high-fat diet after more intense training; (3) diet-induced and training-induced adaptations that increase exercise capacity may be under independent control. Experimental Physiology (2001) 86.4, Regular endurance training evokes a multitude of biochemical adaptations in skeletal muscle that result in a decreased utilisation of carbohydrate and a concomitant increase in fat oxidation during exercise undertaken at the same absolute pre-training workload (Holloszy & Coyle, 1984). In addition to this training-induced glycogen sparing effect, the proportions of carbohydrate and fat in the diet can also markedly alter substrate utilisation during exercise: high-carbohydrate diets will elevate muscle and liver glycogen content and increase the proportion of energy derived from carbohydrate during exercise, while high-fat diets will increase the relative contribution of lipid to oxidative metabolism (Helge et al. 1998). There is strong evidence that in rats, the consumption of a high-fat diet elicits adaptive responses that are favourable for the enhancement of endurance exercise. Miller et al. (1984) were the first to report that untrained rats exposed to a highfat diet ran significantly longer distances than animals fed a high-carbohydrate diet, despite significantly lower preexercise muscle and liver glycogen stores. Subsequently Simi et al. (1991) showed an additive effect of high-intensity training on endurance exercise performance when combined with a high-fat diet. On the other hand, Helge et al. (1998) reported a training-induced increase in endurance running capacity in rats, but no effect of diet. It is interesting to note that Simi et al. (1991) and others (Conlee et al. 1990; Lapachet et al. 1996; Helge et al. 1998; Nakamura et al. 1998) all employed high-intensity (> 80 % of maximal oxygen uptake (V O2,max)) training protocols in conjunction with assorted dietary interventions: such intense training increases contraction-induced muscle glycogenolysis and glycolysis, increases recruitment of fast-twitch muscle fibres, and downregulates mitochondrial fatty acid (FA) uptake and oxidation (Brooks, 1997). Indeed, during high-intensity treadmill running in rats, the respiratory exchange ratio (RER) is typically > 0.95 (Brooks & White, 1978; Bedford et al. 1979; Divine-Patch & Brooks, 1980) indicating a dependence on carbohydrate-based fuels for energy metabolism. It seems reasonable to suggest that a combination of a less intense training stimulus (that would favour lipid oxidation) with adaptations induced by a highfat diet might result in an additive effect in maximising fat Publication of The Physiological Society Corrresponding author: john.hawley@rmit.edu.au 2158

2 500 J. S. Lee, C. R. Bruce, L. L. Spriet and J. A. Hawley Exp. Physiol metabolism and subsequently lead to the greatest improvements in endurance exercise capacity. To our knowledge, no previous study has systematically examined the effect of a high-carbohydrate or a high-fat diet, with low or more intense training, on skeletal muscle and liver adaptations and their metabolic consequences for exercise performance. In the present investigation we determined the interaction of different exercise (low and high intensity) and diet (high-fat and high-carbohydrate) regimens on endurance performance in rats. We hypothesised that a high-fat diet in combination with low-intensity training would evoke the greatest metabolic adaptations for fat metabolism in skeletal muscle and improve endurance running capacity to a greater extent than when either low or more intense training was undertaken on a high-carbohydrate diet. METHODS Overview of experimental design All experimental procedures were approved by the Animal Experimentation Ethics Committee of RMIT University, Australia. After 1 week of familiarisation with the laboratory conditions and treadmill running, rats performed a baseline endurance running test to exhaustion. Rats were then allocated to one of two diets: high-carbohydrate diet (standard laboratory chow; CHO) or a high-fat diet (FAT) (Fig. 1). Rats from each dietary group were further assigned to one of three subgroups: a sedentary control group that performed no exercise; a group that performed low-intensity running; and a group that trained at their maximal voluntary running velocity without electrical stimulation (Fig. 1). For the next 6 weeks animals followed a prescribed exercise rest programme (see below). After 6 weeks of prescribed training, all rats performed a further endurance run to exhaustion. Animals then rested for 72 h before recommencing their training schedule for a further 2 weeks. After 8 weeks, all animals were humanely killed. Animal care, diet, training programme and endurance running performance measures Eighty female Sprague-Dawley rats (initial body mass (BM) g) were obtained from the Animal Resources Centre, Monash University, Victoria, Australia, and housed two per cage in an environmentally controlled laboratory (temperature 22 ± 1 C, relative humidity 50 ± 2 %) with a 12 h light dark cycle (light h). For the first 7 days, all rats were fed CHO and were familiarised with exercise by running for 10 min day _1 for 4 days at a speed of 16 m min _1 (at an incline of 0 deg) on a custom-built eight-lane motorised treadmill in the hours before dark. Because of ethical considerations, the treadmill was not equipped with any form of electric shock device. All rats undertook a baseline endurance running performance test until exhaustion (RUN1), 24 h after their last familiarisation run, at a speed of 16 m min _1 with 0 deg incline. Rats were run until they could no longer maintain the pace of the treadmill, with exhaustion being defined as the inability of a rat to run despite being placed at the front of the treadmill by an investigator on three separate occasions. All performance tests were undertaken by the principal investigator to avoid bias in assessing exhaustion criteria. RUN1 for each animal was taken as the final exercise time completed (in minutes). In order to avoid any possible effects induced by diurnal changes in liver and muscle glycogen concentration, all performance tests commenced between and h (Clark & Conlee, 1979; Lapachet et al. 1996). After RUN1, animals were divided into one of two dietary groups. One group (n =40) was fed CHO (64.0 % of energy as carbohydrate; Ridley Agriproducts Pty Ltd, Victoria, Australia) while the other group (n =40) received FAT (78.1 % of energy as fat). The nutritional composition and energy content of both diets are shown in Table 1. The high-fat diet was freshly prepared every 2 weeks and stored at 4 C. The vitamin and mineral content of both diets was in accordance with the guidelines of the American Institute of Nutrition (Bieri et al. 1977). Rats were provided with food and water ad libitum. All food and fluid intake, as well as BM, was recorded at regular intervals (twice a week) throughout the experiment. Food consumption was estimated by measuring the difference between the weighed portions of food provided for the animals and the uneaten food, and dividing by two, as there were two animals per cage. Animals were also weighed twice a week before and after randomly selected training sessions (see below) to assess the impact of exercise intensity and duration on changes in BM. Animals in each diet group were divided into one of three subgroups: a sedentary (control) group that performed no training (NT, n=8); a group that performed low-intensity running (LOW, n =16); a group that trained at their maximal voluntary running speed (VMAX, n=16). In order to determine the effect of the two different training protocols on muscle and liver glycogen utilisation during exercise, 8 animals from the LOW and VMAX groups were killed after a single training bout. This occurred at the completion of the training programme. All rats were ranked in descending order according to their times from RUN1 (with the animal that ran for the longest duration ranking no. 1). Thirty per cent of the animals ran for 100 min, 26 % ran for > 100 but 200 min, 19 % ran for > 200 but 300 min, 13 % ran for > 300 but 400 min, 6 % ran for > 400 but 500 min, while 6 % ran for > 500 but 600 min. Animals were then allocated to the various treatment groups so that baseline endurance performance times were similar between groups ( min). Animals assigned to the NT group performed 10 min of treadmill running at a speed of 16 m min _1 on a 0 deg incline 2 days week _1 for the duration of the study. The purpose of this exercise regimen was to ensure that all animals were familiarised with treadmill running in order to obtain a valid and reliable performance on the subsequent endurance test. Over the following 4 weeks, rats from both LOW and VMAX groups had their training duration and intensity progressively increased so that they could complete 1000 m of treadmill running 4 days week _1. Once this training distance had been reached, animals maintained their training volume for the next 4 weeks. Rats in the LOW group ran for 125 min at a speed of 8 m min _1. The LOW running programme was chosen because previous investigations have shown this speed to elicit ~ % of V O2,max in rats (Shepherd & Gollnick, 1976; Bedford et al. 1979; Divine- Patch & Brooks, 1980). On the other hand we specifically wanted to examine the metabolic adaptations and performance changes induced by a running programme that all animals could complete without the use of an electric shock grid or other external motivation. Furthermore, we sought to maximise this stimulus so that the effects of the two distinct training programmes and their interaction with the two diets could be examined. Pilot testing revealed that all rats could run at a speed of 28 m min _1 (a velocity 3.5-fold faster than the speed of running in LOW) which corresponds to >75 % of V O2,max (Shepherd & Gollnick, 1976; Brooks & White, 1978; Bedford et al. 1979; Divine-Patch & Brooks, 1980). In order for animals

3 Exp. Physiol Exercise diet interaction and endurance 501 Table 1. Composition and energy content of the experimental diets CHO, high-carbohydrate diet; FAT, high-fat diet. from the VMAX group to complete the same training distance (1000 m session _1 ) as rats from the LOW group, they only had to run for 36 min session _1. All running sessions were performed with 0 deg incline, and rats trained 4 days week _1. After 6 weeks of training, all animals performed a further endurance run to exhaustion (RUN2). During RUN2, some rats were able to complete 630 min of continuous running. Therefore, the incline of the treadmill was increased by 5 deg every 30 min until all animals became exhausted. However, in order to assess the interaction of the higher intensity training undertaken by all animals in the VMAX groups (n =32), endurance performance in RUN2 was assessed at two different running speeds: 16 and 28 m min _1 (n=8 for both speeds) for each diet. After RUN2 animals rested for 72 h before recommencing the normal training programme for a further 2 weeks. At the end of the 8 week programme, all animals were killed 48 h after the last training bout. Tissue preparation and biochemical analyses All animals were killed at the same time of day in order to avoid any diurnal variation in muscle and liver glycogen content and plasma insulin concentration (Conlee et al. 1976). Animals were Figure 1 Allocation of animals to the treatment conditions after they had all undertaken a baseline test of running performance (RUN1). CHO, high-carbohydrate diet (standard laboratory chow); FAT, a high-fat diet. Rats from each dietary group were divided into one of three subgroups: a sedentary (control) group that performed no training (NT); a group that performed low-intensity running (LOW); a group that trained at their maximal voluntary velocity without electrical stimulation (VMAX). In order to assess the interaction of the higher intensity training undertaken by all animals in the VMAX groups, endurance performance was assessed at two different running speeds, 16 and 28 m min _1, for each diet.

4 502 J. S. Lee, C. R. Bruce, L. L. Spriet and J. A. Hawley Exp. Physiol anaesthetised by intraperitoneal injection of sodium pentobarbital (60 mg (kg body wt) _1 ). Once the anaesthesia took effect, hindlimb muscles from the right leg were exposed and the soleus (84 % type I fibres), the red vastus lateralis (RVL; 33 % type IIa fibres, 32 % type IId/x) and the white vastus lateralis (WVL; 100 % type IIb fibres; Delp & Duan, 1996) were dissected out, rapidly frozen and stored in liquid nitrogen. The abdomen was then opened and a portion of liver was excised and frozen. Muscle and liver samples were stored at _80 C until required for analysis. Blood (~1 ml) was drawn from the iliac artery and immediately analysed in duplicate for blood lactate and blood glucose concentrations using an automated analyser (Yellow Springs Instruments 2300 Stat Plus Glucose and L-Lactate Analyzer, Yellow Springs, OH, USA). Whole blood (~1 ml) was then placed into tubes containing EDTA and spun in a centrifuge at 900 g for 15 min at 4 C. The plasma was stored at _80 C and subsequently analysed for plasma free fatty acid (FFA) concentration in a spectrophotometer using an enzymatic colorimetric method (NEFA C test kit, Wako, Richmond, VA, USA). Skeletal muscle and liver glycogen concentrations were determined as glucose residues after hydrolysis in 2 M HCl at 100 C for 2 h (Lowry & Passonneau, 1972). Citrate synthase (CS) activity was determined in a portion of the muscle sample (5 10 mg) and was homogenised in 1:50 dilution (w/v) of a 175 mm potassium buffer solution and assayed spectrophotometrically at 25 C as previously described (Srere, 1969). b- Hydroxy-acyl-CoA dehydrogenase (b-had) activity was assayed spectrophotometrically at 25 C measuring the disappearance of NADH using the same homogenate as for CS (Lowry & Passonneau, 1972). Total carnitine palmitoyl transferase (CPT) activity was determined in muscle samples (5 10 mg) that were powdered in liquid nitrogen and homogenised in 1:40 dilution (w/v) of sucrose (0.25 M) buffer containing 0.2 mm EDTA adjusted to ph 7.4 using a polytron. The assay was based on measuring the initial rates of total CoASH formation in the spectrophotometer at 412 nm by the Figure 2 Endurance running performance determined at a treadmill speed of 16 m min _1 for rats who performed no training (NT) or were trained at a running speed of either 8 m min _1 (LOW) or 28 m min _1 (VMAX) while consuming either a highcarbohydrate diet (CHO) or a high-fat diet (FAT). * Significant effect of diet (P < 0.05). Significant effect of training (P < 0.05). VMAX significantly greater than LOW (P < 0.05). Values are means ± S.E.M. of n =8 rats per group. reduction of DTNB from palmitoyl CoA by mitochondrial carnitine (Bieber et al. 1972). Statistical analyses Data were analysed using a 2 w 3 factorial analysis of variance (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 ± S.E.M. RESULTS Body mass, energy and water intake At the end of the 8 week experimental period, there was no significant difference in the BM of any group of animals (see Table 2). This was despite the estimated energy intake being significantly greater (P < 0.01) in the animals fed CHO than in those fed FAT (Table 2). Sedentary animals fed FAT had significantly greater estimated energy intakes than animals that undertook VMAX running training (P < 0.05, Table 2). In accordance with the greater estimated energy intake of animals fed CHO compared to FAT, estimated water intake was also higher with the CHO (P < 0.01). Furthermore, animals in the NT group fed FAT had significantly greater estimated water intakes than animals that undertook VMAX training (P < 0.05). Endurance running performance The results of the endurance running performance test (RUN2) undertaken at 16 m min _1 for rats in the NT, LOW and VMAX groups are displayed in Fig. 2. There were significant effects of both diet and training on running performance. The running duration in the NT group was 77% longer after FAT compared to CHO (239 ± 28 vs. 135 ± 30 min, respectively; P < 0.05) at 16 m min _1. There was also a significant training effect such that both LOW and VMAX increased running performance for both dietary conditions compared to NT (P < 0.05; Fig. 2). Animals in the VMAX group also ran for a longer duration than animals in the LOW group on FAT, but not on CHO (P < 0.05, Fig. 2). Figure 3 displays the running performances of VMAX rats tested at 16 m min _1. Although there were no effects of diet on endurance exercise time (641 ± 26 vs. 583 ± 33 min for FAT and CHO, respectively), a greater proportion of fat-fed rats were able to maintain exercise for > 400 min compared to carbohydrate-fed rats. When fed CHO, only five animals were able to complete 500 min of running, compared to all eight animals fed FAT. Similarly, only three rats in the CHO group were able to complete 630 min of running, compared to 5 rats in the FAT group. After 630 min the incline of the treadmill was increased by 5 deg. Only one rat fed CHO completed this stage compared to three fat-fed rats. Because we originally hypothesised that there would be an interactive effect of training intensity with diet, one group of animals from each dietary group that undertook VMAX training were run to exhaustion at 28 m min _1 (Fig. 4). As expected, there was a significant effect of diet when endurance was determined at the higher treadmill speed. Thus, VMAX animals fed FAT ran for a significantly longer duration than

5 Exp. Physiol Exercise diet interaction and endurance 503 Table 2. Final body mass and estimated daily energy and water intake during the experimental period CHO, high-carbohydrate diet; FAT, high-fat diet; NT, no training; LOW, low intensity training (8 m min _1 ); VMAX, maximal voluntary running intensity (28 m min _1 ). a Significantly greater than FAT (P < 0.01). b Significantly greater than VMAX (P < 0.05). Values are means ± S.E.M., n =8 animals per group. animals fed CHO (58 ± 11 vs. 100 ± 28 min, respectively; P < 0.05). Blood substrate concentrations There were significant main effects of diet (P < 0.05) and training (P < 0.01) on blood glucose concentration (Table 3). Thus, in animals fed CHO, resting blood glucose concentration was significantly greater in the NT than the VMAX group (5.0 ± 0.23 vs ± 0.17 mm, respectively, P < 0.05), but not significantly different in the LOW group (4.54 ± 0.15 mm). On the other hand, blood glucose concentration was significantly greater in both the NT and LOW groups fed FAT than the VMAX group (5.12 ± 0.28 and 5.16 ± 0.36 vs ± 0.23 mm, respectively, P<0.05). There was a main effect of diet on resting blood glucose concentration such that animals receiving LOW training had significantly greater glucose concentrations in the FAT than in the CHO subgroups (5.16 ± 0.36 vs ± 0.15 mm, respectively, P<0.05). There was a significant main effect of training (P < 0.01) on resting blood lactate concentration (Table 3). Lactate concentration was Figure 3 The percentage of animals from each diet and the corresponding exercise time that they completed. Endurance running performance determined at a treadmill speed of 16 m min _1 for rats trained at a running speed of 28 m min _1 (VMAX) while consuming either a high-carbohydrate diet (CHO) or a high-fat diet (FAT). After 630 min the treadmill incline was increased by 5 deg every 30 min until exhaustion. Values are means of n =8 rats per group. Figure 4 Endurance running performance determined at a treadmill speed of 28 m min _1 for rats trained to run at 28 m min _1 (VMAX) while consuming either a high-carbohydrate diet (CHO) or a high-fat diet (FAT). * Significant effect of diet (P < 0.05). Values are means ± S.E.M. of n =8 rats per group.

6 504 J. S. Lee, C. R. Bruce, L. L. Spriet and J. A. Hawley Exp. Physiol Table 3. Resting concentrations of blood-borne substrates after the 8 week training programme NT, no training; LOW, low intensity training (8 m min _1 ); VMAX, maximal voluntary running intensity (28 m min _1 ); CHO, high-carbohydrate diet; FAT, high-fat diet; FFA, free fatty acid. a Significantly greater than VMAX (P < 0.05) when diets are the same. b Significant effect of diet (P < 0.05). c Significant effect of training (P < 0.05). d Significant effect of training intensity (P < 0.05). NS, not significant. Values are means ± S.E.M., n = 7 8 animals per group, expressed in mmol l _1. Table 4. Muscle and liver glycogen concentrations at rest and after a single training session (8 weeks) NT, no training; LOW, low intensity training (8 m min _1 ); VMAX, maximal voluntary running intensity (28 m min _1 ); CHO, high-carbohydrate diet; FAT, high-fat diet; RVL, red vastus lateralis; WVL, white vastus lateralis. a Significant effect of diet (P < 0.05). b Significant effect of training (P < 0.05). c Significantly lower than rest (P < 0.05). Values are means ± S.E.M., n = 7 8 animals per group, expressed in µmol (g wet wt) _1. also greater in rats fed FAT in the LOW group compared to animals fed FAT in the VMAX group (2.01 ± 0.15 vs ± 0.12 mm, respectively, P < 0.05). There were no differences in FFA concentration in the animals on the two diets, or following the various training interventions (Table 3). Skeletal muscle and liver glycogen concentration Soleus muscle. There was a significant interaction of diet with training (P<0.05; Table 4) on resting glycogen content in the soleus muscle. Thus, animals fed CHO that performed either LOW or VMAX training had a significantly greater glycogen concentration than trained animals fed FAT. Animals fed CHO that performed LOW training had a significantly greater muscle glycogen content than animals in the NT group (29.7 ± 2.3 vs ± 1.4 µmol (g wet wt) _1 ; P < 0.05). There were no training effects on resting muscle glycogen content in animals fed FAT. In animals fed CHO, a single bout of training resulted in a significant reduction in muscle glycogen content in both LOW (from 29.7 ± 1.8 to 20.3 ± 1.4 µmol (g wet wt) _1 ; P < 0.05) and VMAX (from 27.4 ± 1.8 to 19.1 ± 1.4 µmol (g wet wt) _1 ; P < 0.05, Table 4) groups. The decreases in muscle glycogen content in animals fed FAT were not different during either LOW or VMAX. Red vastus lateralis. There were significant main effects (P < 0.01) on resting muscle glycogen concentration in the RVL of both diet and training. Accordingly, animals that performed training on CHO had a significantly greater muscle glycogen content than trained animals fed FAT (Table 4). As with the soleus muscle, animals fed CHO that performed LOW had significantly greater glycogen concentration than the CHO-fed NT group (47.5 ± 2.7 vs ± 2.5 µmol (g wet wt) _1, P<0.05). There was no

7 Exp. Physiol Exercise diet interaction and endurance 505 Table 5. Enzyme activity in selected skeletal muscles after the 8 week intervention period NT, no training; LOW, low intensity training (8 m min _1 ); VMAX, maximal voluntary running intensity (28 m min _1 ); CHO, high-carbohydrate diet; FAT, high-fat diet; RVL, red vastus lateralis; WVL, white vastus lateralis. a Significant effect of diet (P < 0.05). b Significant effect of training (P < 0.05). Values are means ± S.E.M., n = 7 8 animals per group, expressed in mmol (kg wet wt) _1. increase in resting glycogen content with training for animals fed FAT (Table 4). Muscle glycogen content was significantly reduced after a single bout of training in all groups (P < 0.05, Table 4) except VMAX animals fed CHO. White vastus lateralis. There was a significant interaction of diet with training on resting muscle glycogen content in the WVL (P <0.05, Table 4). Thus, animals fed FAT that performed training had a significantly greater muscle glycogen content than NT. NT animals fed CHO had a significantly greater muscle glycogen content than those fed FAT. There were no consistent patterns with regard to muscle glycogen utilisation after a single bout of either LOW or VMAX training (Table 4). Liver. There were significant main effects (P <0.01) on resting liver glycogen concentration of both diet and training (Table 4). Accordingly, liver glycogen levels were significantly greater in animals fed CHO than FAT (P <0.05). There were significant effects of training (P < 0.01; Table 4) such that animals fed CHO that performed LOW (but not VMAX) had significantly greater levels of glycogen in the liver than animals in the NT group. On the other hand, both training interventions in animals fed FAT resulted in greater resting liver glycogen content than NT. Liver glycogen content was significantly reduced after an acute bout of LOW in animals on both FAT and CHO (P <0.05, Table 4). However, only FAT-fed animals showed a significant decline in liver glycogen after VMAX. Enzyme activities Citrate synthase. There were significant main effects of both diet (P < 0.01) and training (P < 0.05) on CS activity in the soleus and RVL muscles (Table 5). However, only diet had a significant effect on CS activity in the WVL with training causing only small increases in enzyme activity in this muscle. b-had. There were significant main effects of diet (P < 0.01) on b-had activity in all three skeletal muscles examined (Table 5). There was also a significant effect of training (P < 0.05) in the RVL but not the soleus or WVL (Table 5). CPT. There were significant main effects of diet on total CPT activity in the RVL (P < 0.01, Table 5). b-had/cs ratio. The ratio of b-had/cs activity has previously been used as an index of the relative potentials for FA oxidation versus overall aerobic metabolism (Pette & Dölken, 1975; Simi et al. 1991). Such ratios for the skeletal muscles under investigation are reported in Table 5. For the soleus, there was a significant interaction between diet and training, but there was no diet training interaction on the b-had/cs ratio for either the RVL or WVL. Effects of diet (RVL, P<0.05; WVL, P<0.01) and training (WVL, P<0.05) were also evident (Table 5). DISCUSSION Over 15 years ago Miller et al. (1984) reported that sedentary rats consuming a high-fat diet for 1 week ran for a longer duration than animals fed a high-carbohydrate diet for the same period. These workers also showed that this improvement in endurance was more marked the longer the dietary period, such that after 5 weeks, performance times for animals fed a high-fat diet were 33 % greater than for those fed a high-carbohydrate diet, compared to just 8 %

8 506 J. S. Lee, C. R. Bruce, L. L. Spriet and J. A. Hawley Exp. Physiol after 1 week (Miller et al. 1984). Accompanying the enhanced endurance capacity was a reduction in resting muscle and liver glycogen stores and an increase in skeletal muscle b-had activity (an indirect index of the capacity to oxidise fat). These results suggest that a high-fat diet alone was sufficient stimulus to induce adaptations which apparently increase the ability of muscle to oxidise FA, spare glycogen and thereby enhance submaximal endurance performance. Subsequently, Simi et al. (1991) reported that when highintensity exercise training was superimposed on a high-fat diet, the effects on endurance performance were additive. Of interest was the finding that the training-induced and dietinduced muscle enzyme adaptations in that study were also cumulative (Simi et al. 1991). Taken collectively, the results from those investigations (Miller et al. 1984; Simi et al. 1991) suggest that diet-induced and training-induced adaptations that increase FA oxidation during exercise may be under independent control. However, it should be noted that all previous investigations that have examined diet training responses in rats used intense (> 80% of V O2,max) running programmes (Miller et al. 1984; Simi et al. 1991; Lapachet et al. 1996; Helge et al. 1998) that rely primarily on carbohydrate-based fuels (muscle and liver glycogen, blood glucose, muscle, blood and liver lactate) rather than lipids (adipose and intramuscular triacyglycerol (TG), blood-borne FFA and TGs) for oxidation for muscular energy (Brooks & White, 1978; Bedford et al. 1979; Divine-Patch & Brooks, 1980; Brooks & Mercier, 1994). The possibility exists that the high glycolytic flux sustained during such training might counteract any diet-induced shifts towards lipid metabolism. Therefore, the protocol in the present study was designed to maximise both diet-induced and training-induced responses that would favour FA oxidation and improve exercise capacity. We hypothesised that a high-fat diet in combination with low-intensity training would evoke the greatest metabolic adaptations for FA metabolism in skeletal muscle and result in the greatest enhancement of submaximal, lowintensity endurance running performance. In agreement with previous studies (Miller et al. 1984; Simi et al. 1991), we found that sedentary animals fed a high-fat diet ran for a significantly longer duration than animals fed carbohydrate. On the other hand, sedentary rats fed a carbohydrate-rich diet had a reduced exercise capacity compared to baseline. This latter finding is in agreement with the results of Miller et al. (1984) who reported that rats fed a high-carbohydrate diet for 5 weeks had a 15% reduction in run time to exhaustion. These findings suggest that a carbohydrate-rich diet is of benefit to performance only when combined with training. However, in contrast to our original hypothesis, rats that performed low-intensity training and were fed the high-fat diet did not run for longer than animals fed carbohydrate when tested at the lower (16 m min _1 ) treadmill speed (Fig. 2). If adaptations accompanying the high-fat diet were not additive with any training-induced responses, at least in animals trained and tested at the lower running velocities, then it would be reasonable to suggest that there would also be no difference in endurance performance for fat-fed animals when tested at the faster speed. However, the second finding of this study was a significant enhancement of endurance in animals fed the high-fat diet tested at their specific training speed of 28 m min _1 (Fig. 3). The increase in endurance time observed in the present study (~70% in fat-fed animals) was almost 2-fold greater than reported in previous studies (Miller et al. 1984; Lapachet et al. 1996). Furthermore, unlike in previous studies, these rats trained at their maximal voluntary running speed without electrical stimulation. As both LOW and VMAX training programmes were of the same intensity (1000 m day _1 ), and as the high-fat diet had no additive effect on performance when combined with lowintensity running, this would suggest that exercise intensity exerts the predominant influence in determining the traininginduced responses and the balance of substrate utilisation during exercise. The precise mechanisms underlying improvements in endurance seen with the high-fat diet together with the more intense training programme in the present investigation are difficult to elucidate. Although we did not measure whole body rates of substrate oxidation during exercise, Helge et al. (1998) have previously reported lower RER values after a high-fat compared with a high-carbohydrate diet (i.e. carbohydrate sparing). In that study, however, any shift to a greater level of oxidation of fat during submaximal exercise was predominantly a diet-induced rather than a training-induced adaptation as Helge et al. (1998) failed to find a significant training effect on RER values determined during their standardised exercise test. In the present study, the major increases in CS and b-had activity were diet rather than training induced, a somewhat paradoxical finding given that endurance performance was only enhanced after the more intense training in rats fed a highfat diet. In the present study, the significantly lower resting muscle and liver glycogen levels (Table 4) provide indirect evidence of a greater reliance on FA than on carbohydrate oxidation during exercise in fat-fed animals. However, our data on muscle and liver glycogen utilisation before and after an acute bout of either low or high-intensity training are inconsistent with regard to any training-induced glycogen sparing effect. Although reduced rates of muscle glycogen breakdown during exercise have been reported in rats fed high-fat diets (Miller et al. 1984; Simi et al. 1991), such observations might just as readily be explained by low initial concentrations of muscle glycogen after these diets and the concomitantly lower absolute rates of glycogen oxidation, rather than glycogen sparing per se. Indeed, it is difficult to explain the magnitude of enhancement in exercise time to exhaustion (often 2-fold longer) in rats fed high-fat diets on the basis of glycogen sparing alone. Support for this contention comes from the recent study of Nakamura et al. (1998) who reported that rates of glycogen utilisation in trained rats fed a high-fat diet were only significantly lower than animals fed a highcarbohydrate diet in the early stages (i.e. the first 20 min) of submaximal exercise. These workers suggested that this dietinduced glycogen sparing could not be the single adaptive mechanism that enables fat-fed rats to be able to continue

9 Exp. Physiol Exercise diet interaction and endurance 507 exercise well beyond the fatigue point of carbohydrate-fed animals (Nakamura et al. 1998). It is tempting to speculate that increased adipose tissue or intramuscular TG (IMTG) lipolysis and FA provision stimulated FA oxidation during submaximal exercise, and postponed the development of fatigue in fat-fed rats. However, several studies in both animals and humans have shown no effect of training and/or diet (i.e. high-fat vs. highcarbohydrate) on either resting IMTG content (Lapachet et al. 1996; Helge et al. 1998; Simi et al. 1991) or subsequent FFA uptake and IMTG utilisation during submaximal exercise (Bergman et al. 1999). We did not determine IMTG concentration, but measured no effect of training or diet on resting plasma FFA concentration. Static measurements of plasma FFA do not provide information regarding FFA turnover and more studies measuring IMTG use and FFA appearance and disappearance following training and dietary manipulations are needed to investigate this further. To determine the interactive effects of diet and training on the metabolic potential of skeletal muscle, we determined selected enzyme activities. While the high-fat diet resulted in elevations in CS, b-had and total CPT activity, training had only a minor (non-additive) impact on the activities of these oxidative enzymes. This observation is in contrast with the data of Simi et al. (1991) who reported that training induced a 12 % elevation of CS activity above the effects of a high-fat diet alone in the red quadriceps. With respect to b-had and total CPT activity (an indirect marker of FA metabolism), we, like others (Miller et al. 1984; Simi et al. 1991; Helge et al. 1998) found that both red and white muscles of rats fed a high-fat diet had significantly higher levels of these enzymes than rats fed a diet rich in carbohydrate. Although individual enzyme capacities provide an indirect measurement of the metabolic profile of muscle, Staudte & Pette (1972) have proposed a concept of constant proportion of groups of enzymes to facilitate comparisons of tissue displaying disparate absolute enzyme activities. Thus, the ratio of b-had/cs activity provides an index of the relative potentials for FA oxidation versus overall aerobic metabolism (Pette & Dölken, 1975). Our data show that although the b-had/cs ratios were similar across the different training regimens, there were significant main effects of diet. It must be acknowledged that the training programme utilised in the current study did not uniformly enhance enzyme activity, possibly because the stimulus was too low. Given the constraints imposed by the ethical considerations of not been able to shock or prod animals while training, we feel that the current data are an important addition to the scientific literature because they represent the upper limit of voluntary exercise that animals are likely to undertake. Despite an estimated energy intake that was ~10% greater in animals fed CHO compared to FAT, there were no significant changes in body mass over the study period between the two groups (Table 2). Such an observation is difficult to explain, although others have reported the same phenomenon. For example, Donato & Hegsted (1985) found that rats fed a highfat diet increased body fat deposition despite total energy intake being severely limited. Oscai et al. (1987) also reported that rats fed a high-fat diet reduced their voluntary energy intake by 14 % compared with animals with access to carbohydrate ad libitum. Lapachet et al. (1996) also reported that rats fed a fat-rich diet voluntarily reduced their energy intake by 12% compared with animals fed a carbohydraterich diet, despite an intense exercise-training programme. A possible explanation for this paradox could be the efficiency of storing energy from dietary fat. Based on direct measurements of energy intake in young rats in which the relative energy efficiency of dietary carbohydrate and fats was determined, Donato & Hegsted (1985) have proposed the dietary value of fat as an energy substrate to be 46.5 kj g _1 rather than the more frequently employed value of 37.7 kj g _1 (Atwater & Bryand, 1900). When we applied the value of Donato & Hegsted (1985) to our data, there were no differences in the estimated daily energy intakes between rats fed either high-carbohydrate or high-fat diets (301.2 vs kj d _1 for CHO and FAT, respectively). Alternatively, it could simply be that rats fed high-carbohydrate diets undergo more voluntary exercise while in their cages compared to high fat-fed animals. In conclusion, this is the first investigation to examine the interaction of different exercise (low vs. intense) and diet (high-fat vs. high-carbohydrate) regimens on adaptations in skeletal muscle and liver, and their metabolic consequences for endurance performance in rats. In agreement with a previous study (Miller et al. 1984), we found that feeding untrained animals a high-fat diet enhanced their endurance running capacity to a greater extent than feeding a highcarbohydrate diet. Furthermore, low-intensity training enhanced endurance time to exhaustion when compared to no training. However, in contrast to our original hypothesis, there was no additive effect of the high-fat diet on exercise performance when rats performed low-intensity training. Indeed, running performance was only enhanced by a highfat diet after more intense training. This, presumably, was in spite of a greater contribution from FA than carbohydrate to energy metabolism during the less intense training sessions, a condition that might have been expected to amplify the potential for lipid oxidation and cause metabolic adaptations that would enhance subsequent endurance capacity. Taken collectively, our findings suggest that diet-induced and training-induced adaptations that increase exercise capacity may be under independent control. ATWATER, W. O. & BRYAND, A. P. (1900). Agricultural Experimental Station, 12th Annual Report (Storrs, CT, USA), pp BEDFORD, T. G., TIPTON, C. M., WILSON, N. C., OPPLIGER, R. A. & GISOLFI, C. V. (1979). 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