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Aerobic fitness determines whole-body fat oxidation rate during exercise in the heat
Juan Del Coso, Nassim Hamouti, Juan Fernando Ortega, and Ricardo Mora-Rodriguez

Abstract: The purpose of this study was to determine whole-body fat oxidation in endurance-trained (TR) and untrained (UNTR) subjects exercising at different intensities in the heat. On 3 occasions, 10 TR cyclists and 10 UNTR healthy subjects (peak oxygen uptake = 60 6 vs. 44 3 mLkg1min1; p < 0.05) exercised at 40%, 60%, and 80% peak oxygen uptake in a hot, dry environment (36 8C; 25% relative humidity). To complete the same amount of work in all 3 trials, exercise duration varied (107 4, 63 1, and 45 0 min for 40%, 60%, and 80% peak oxygen uptake, respectively). Substrate oxidation was calculated using indirect calorimetry. Blood samples were collected at the end of exercise to determine plasma epinephrine ([EPI]plasma) and norepinephrine ([NEPI]plasma) concentrations. The maximal rate of fat oxidation was achieved at 60% peak oxygen uptake for the TR group (0.41 0.01 gmin1) and at 40% peak oxygen uptake for the UNTR group (0.28 0.01 gmin1). TR subjects oxidized absolutely (gmin1) and relatively (% of total energy expenditure) more fat than UNTR subjects at 60% and 80% peak oxygen uptake (p < 0.05). At these exercise intensities, TR subjects also had higher [NEPI]plasma concentrations than UNTR subjects (p < 0.05). In the heat, whole-body peak fat oxidation occurs at higher relative exercise intensities in TR than in UNTR subjects (60% vs. 40% peak oxygen uptake). Moreover, TR subjects oxidize more fat than UNTR subjects when exercising at moderate to high intensities (>60% peak oxygen uptake). Key words: catecholamines, carbohydrate oxidation, indirect calorimetry, cycling, body composition, endurance exercise. tude se propose danalyser le degre doxydation des gras dans tout lorganisme de sujets entra ne s en en sume : Cette e Re ne s en endurance (UNTR) au cours dexercices accomplis a ` diffe rentes intensite s dans durance (TR) et de sujets non entra (consommation doxyge ` ne un environnement chaud. En trois occasions, 10 cyclistes TR et 10 sujets UNTR en bonne sante ` 44 3 mLkg1min1; p < 0,05) font un effort dans un environnede pointe = 60 6 mLkg1min1 comparativement a s suivantes : 40 %, 60 % et 80 % du consommation doxyge ` ne de ment chaud (36 8C; 25 % HR), et ce, aux intensite de travail accompli, la dure e des efforts varie comme suit : 107 4 min, pointe. Afin de maintenir constante la quantite s respectives de 40 %, 60 % et de 80 % du consommation doxyge ` ne de 63 1 min et 45 0 min pour les intensite ` la fin des se doxydation des gras par calorime trie indirecte. A ances dexercice, on pre le ` ve des pointe. On mesure le degre chantillons sanguins pour en de terminer les concentrations plasmatiques de pine phrine ([EPI]plasma) et de nore pine phrine e ` 60 % du consommation doxyge ` ne de ([NEPI]plasma). On enregistre le plus haut taux doxydation des gras chez les TR a ` 40 % du consommation doxyge ` ne de pointe (0,28 0,01 gmin1). Les pointe (0,41 0,01 gmin1) et, chez les UNTR, a nergie sujets TR oxydent plus de gras que les sujets UNTR en valeur absolue (gmin1) et en valeur relative (% du total de ` ces intensite pense e) a ` 60 % et 80 % du consommation doxyge ` ne de pointe (p < 0,05). A s dexercice, la [NEPI]plasma de leve e que chez les sujets UNTR (p < 0,05). Dans un environnement chaud, loxydation des chez les sujets TR est plus e ` une plus haute intensite relative : 60 % comparativement a ` 40 % gras dans lorganisme se manifeste chez les sujets TR a ` ne de pointe chez les UNTR. De plus, les sujets TR oxydent plus de gras que les sujets UNTR du consommation doxyge mode re e a `e leve e (>60 % du consommation doxyge ` ne de pointe). au cours defforts dintensite cholamines, oxydation des sucres, calorime trie indirecte, cyclisme, composition corporelle, s : cate Mots-cle exercice dendurance. daction] [Traduit par la Re

Introduction
During exercise, factors such as exercise intensity and duration, environmental conditions, training status, adiposity, and the pre-exercise meal influence fat oxidation (Jeu-

kendrup 2003). From light to moderate exercise intensities, fat is the main energy substrate for muscle contraction, with carbohydrate making a lower contribution (Jones et al. 1980). The highest rate of fat oxidation during exercise (Fatmax) in a thermonetural environment has been found at

Received 8 April 2010. Accepted 12 August 2010. Published on the NRC Research Press Web site at apnm.nrc.ca on 5 November 2010. J. Del Coso, N. Hamouti, J.F. Ortega, and R. Mora-Rodriguez.1 Exercise Physiology Lab at Toledo, Universidad de Castilla La Mancha, Avda. Carlos III, s/n, Toledo, 45071, Spain.
1Corresponding

author (e-mail: Ricardo.Mora@uclm.es).

doi:10.1139/H10-068 Published by NRC Research Press

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exercise intensities ranging from 33% to 65% peak oxygen _ O2 peak) (Romijn et al. 1993, 2000; Bergman and uptake (V Brooks 1999; Achten et al. 2002; Venables et al. 2005; Nordby et al. 2006; Stisen et al. 2006; Riddell et al. 2008). The rate of fat oxidation declines at exercise intensities _ O2 peak, and it is almost negligible at higher than 70% V _ O2 max) (Achten et al. 90% maximal oxygen uptake (V 2002). As exercise progresses in duration, there is an increasing contribution of fat to energy production, while the use of carbohydrate decreases over time (Romijn et al. 1993). Because of this, individuals undergoing weight loss programs should target exercise intensity close to their Fatmax while exercising for the longest possible duration. However, this population is often unable to maintain exercise at Fatmax for a long time (Hulens et al. 2001). Exercise practitioners may then consider reducing exercise intensity to benefit from exercise duration. Data that address the best combination of exercise intensity and duration that maximizes total fat oxidation are scarce. One of the most prominent metabolic adaptations to endurance training is the higher reliance on fat oxidation during exercise. Several studies have shown that 1012 weeks of endurance training substantially increase the rate of fat _ O2 peak) oxidation at a given relative workload (65% V (Coggan et al. 1990, 1993; Friedlander et al. 1998). Endurance-trained subjects oxidize relatively (as a percentage of energy expenditure) and absolutely (gmin1) more fat than their untrained counterparts when exercising at the same relative intensity (Bergman and Brooks 1999; Stisen et al. 2006). In addition, endurance-trained individuals achieve Fatmax at higher relative exercise intensities than untrained _ O2 peak) (Nordby et al. 2006). subjects (50% vs. 43% V Thus, the exercise intensity prescribed to maximize fat oxidation may vary, depending on the aerobic capacity of the individual. Most studies focusing on fat oxidation during exercise have been conducted in thermoneutral environments. However, exercise is frequently performed outdoors, where ambient temperature can be high. It has been found that _ O2 max in a hot environment (40 8C) deexercise at 70% V creases the rate of fat oxidation in comparison to a thermoneutral environment (20 8C) (Febbraio et al. 1994). However, it is unclear if the reduction in fat oxidation in the heat is present at all exercise intensities, or if the heat affects the intensity at which Fatmax is achieved. The first purpose of this study was to determine the exercise intensity that elicits Fatmax in trained and untrained subjects in a hot environment in a fed state. The second purpose was to investigate the best combination of exercise intensity and duration to maximize total fat oxidation and energy expenditure during exercise in the heat. We hypothesized that endurance-trained individuals would maximize fat oxidation in the heat at a higher relative exercise intensity than their untrained counterparts.

tics is shown in Table 1. Each group was composed of 7 males and 3 females. TR subjects were licensed cyclists who routinely trained at least 2 h a day, 47 days a week, at the time of the study and during the previous 4 years. UNTR subjects were university students who regularly performed <2 h of physical activity per week during the previous year. All subjects were fully informed of any risks and discomforts associated with the experiment before giving their informed written consent to participate. Once the participants fulfilled the inclusion criteria and agreed to participate in the study, they underwent a physical examination (including rest and exercise electrocardiogram) to ensure that they were in good health. The study was approved by the local Hospital Research Ethics Committee of the Hospital Virgon de la Salud, Spain, and conformed to the Declaration of Helsinki. Preliminary testing TR and UNTR subjects were tested using a continuous incremental test to volitional exhaustion to determine their _ O2 peak. After a standardized 10-min warm-up, the initial V power output was set at 75 W for UNTR and 125 W for TR subjects, and it increased 25 Wmin1 until voluntary cessation (or when pedaling cadence dropped below _ O2) was measured using an 60 rmin1). Oxygen uptake (V automated breath-by-breath system (Quark b2, Cosmed, Rome, Italy), and the data were averaged each 15 s. The _ O2 value during the in_ O2 peak was defined as the highest V V cremental test (Balady et al. 2000). The test was carried out 1 week before the onset of the experimental trials; it was performed on a cycle ergometer (Ergoline 200, Cosmed). Subjects withdrew from all dietary sources of caffeine 48 h _ O2 peak test. The body composition of the subbefore the V jects was assessed using skinfold measurements (Jackson and Pollock 1978; Jackson et al. 1980). Experimental design On 3 occasions, each subject pedaled a cycle ergometer at _ O2 peak in a hot, dry ventilated 40%, 60%, or 80% of their V environment (36 0.5 8C; 25% 5% relative humidity; _ O2 peak test data, a V _ O22.5 ms1 wind speed). Using the V workload best-fit equation was generated for each subject to determine individual workloads corresponding to the desired _ O2 peak test was performed _ O2 peak. However, since the V %V under temperate conditions (25 8C; 40% relative humidity), exercise intensity for experimental protocols might be msson et al. slightly overestimated in both groups (Arngr 2003). To complete the same amount of work in each trial (658 17 kJ for TR and 508 11 kJ for UNTR subjects; p < 0.05), exercise duration was set at 107 4, 63 1, and _ O2 peak, respectively 45 0 min for 40%, 60%, and 80% V (see Table 2 for details). Total amount of work was set to _ O2 peak, since pilot data revealed that equal 45 min at 80% V UNTR subjects could barely finish that trial in our hot environment. The exercise durations for the remaining trials (i.e., _ O2 peak) were calculated by dividing the total 40% and 60% V amount of work by the individual workload assigned for these trials. Experimental trials were performed in a randomized order, and were separated by at least 4 days to ensure proper recovery and glycogen repletion. Data on carPublished by NRC Research Press

Materials and methods

Subjects Ten endurance-trained cyclists (TR) and 10 untrained (UNTR) healthy subjects volunteered to participate in this study. A detailed summary of subjects physical characteris-

Del Coso et al. Table 1. Morphological characteristics of endurance-trained cyclists (TR) and untrained healthy subjects (UNTR). Subjects TR UNTR Age (y) 22.96.6 24.14.4 Height (cm) 17410 17613 Weight (kg) 67.011.9* 76.815.5 Body mass index (kgm2) 21.81.9* 24.52.6 Body fat (%) 9.05.2* 18.46.4 O2 peak V (mLkg1min1) 59.65.7* 43.63.2

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O2 peak, peak oxygen uptake. Note: V *Different from UNTR (p < 0.05).

Table 2. Power output, exercise times, and total work during the 3 experimental trials at 40%, 60%, and 80% peak oxygen uptake for trained and untrained individuals. O2 peak Trial V (%) TR 40 60 80 UNTR 40 60 80 Intensity (W) 1099* 17614* 24420* 778 13210 18813 Time (min) 1014 621 450 50811 1125 641 450 Total work (kJ) 65817*

O2 peak, peak oxygen uptake. Note: V *Different from UNTR (p < 0.05).

diovascular and thermoregulatory variables of this experimental design have been published elsewhere (MoraRodriguez et al. 2010). Experimental protocol Experimental trials were carried out from February to June to avoid the possible effects of heat acclimation on substrate utilization (Kirwan et al. 1987). Two days before the experimental trials, subjects withdrew from all dietary sources of alcohol and caffeine. The day before, subjects were instructed to consume at least 7 gkg1 body weight of carbohydrate and to perform light exercise, if any. Food and training diaries were collected to aid in the replication of diet and exercise before each trial. Each subject performed all trials at the same time of day and in the postabsorptive state 4 h after a standardized breakfast (~3 g carbohydratekg1 body weight). Subjects also ingested 500 mL of tap water 2 h before the onset of the experimental trials to ensure euhydration, which was confirmed by urine specific gravity (pre-exercise urine sample <1.020). On arrival, a 22-gauge Teflon catheter was inserted into an antecubital vein of each subject for blood sampling. The catheter was frequently flushed with 3 to 4 mL of 0.9% sterile saline to ensure patency. Afterward, subjects entered the climatic chamber and sat quietly on the cycle ergometer for 5 min while pre-exercise variables were recorded. After that, a venous blood sample was withdrawn. Then, subjects pedaled in steady-state for the duration and at the exercise intensity selected for the trial; no fluid was provided during the trial. Indirect calorimetry measurement and calculations _ CO2) rates were _ O2 and carbon dioxide production (V V measured during the trials for 6-min periods, starting after

10 and 39 min, and at the end of trials. For these measurements, subjects wore a mouth piece and a noseclip while samples of the expired air were collected breath by breath to determine gas concentrations (Quark b2, Cosmed). The volume of inspired and expired air was also measured with a digital turbine flowmeter. Certified calibration gases (16.0% O2; 5.1% CO2) and a 3-L syringe were used to calibrate the gas analyser and turbine flowmeter before each trial. During the last 2 min of each collection period, the ventilatory parameters were averaged to achieve a represen_ O2 and V _ CO2, energy tative value for each period. From V expenditure and substrate oxidation (fat and carbohydrate) were calculated using the nonprotein respiratory quotient, with the assumption that the urinary nitrogen excretion rate was negligible (Brouwer 1957; Frayn 1983): Energy expenditure rate (kJmin1) was calculated as _ O2) + 91.195 (V _ CO2)] 4.186. [(3.869 V Fat oxidation rate (gmin1) was calculated as (1.67 _ CO2). _ O2) (1.67 V V Carbohydrate oxidation rate (gmin1) was calculated as _ CO2) (3.21 V _ O2), where V _ O2 and V _ CO2 are (4.55 V in Lmin1. Total energy expenditure and total fat oxidation were calculated using the rate of energy expenditure or fat oxidation multiplied by exercise duration. Blood analysis Venous blood samples (5 mL) were obtained before exercise and at the end of each trial. A portion of whole blood was introduced in a clinical blood gas analyzer (ABL 520, Radiometer, Copenhagen, Denmark) to determine H+ concentration in the plasma portion of the blood. The remaining blood (i.e., 3 mL) was added to a tube containing 0.3 mL of reduced glutathione (4.5 mg), sodium heparin (50 IU), and 20 mL of 0.24 molL1 EGTA. Tubes were then centrifuged at 2000g to separate the plasma. At a later date, plasma epinephrine ([EPI]plasma) and norepinephrine ([NEPI]plasma) concentrations were measured using high performance liquid chromatography with electrochemical detection (Hjemdahl 1984). Plasma catecholamine concentrations were not corrected for changes in plasma volume so that its real bioavailability in blood was presented. A limitation of this study is that [EPI]plasma and [NEPI]plasma concentrations were only measured at the end of each trial. Statistics Unpaired Students t tests were used to compare subjects physical and morphological characteristics. One-way repeated-measures analysis of variance (ANOVA) was used to compare the total energy expenditure and total carbohyPublished by NRC Research Press

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Appl. Physiol. Nutr. Metab. Vol. 35, 2010 Fig. 1. Average fat oxidation rate in endurance-trained (TR) and untrained (UNTR) individuals at 40%, 60%, and 80% peak oxygen _ O2 peak). Data are means standard error of measurement. uptake (V _ O2 peak, *, Different from UNTR, p < 0.05; {, different from 60% V _ p < 0.05; {, different from 40% V O2 peak, p < 0.05.

drate and fat oxidations among trials within the same group of subjects. Factorial ANOVA was used to compare energy expenditure and substrate oxidation rates between TR and UNTR subjects at the different exercise intensities. After a significant F test (GeisserGreenhouse correction for the assumption of sphericity), differences between means were identified using Tukeys HSD post hoc procedure. The significance level was set at p < 0.05. All data are presented as means standard error of the measurement.

Results
Energy expenditure As expected, the rate of energy expenditure increased along with the exercise intensity (i.e., 40% < 60% < 80% _ O2 peak) in both TR (39 3 < 55 4 < 76 5 kJmin1; V p < 0.05) and UNTR (33 3 < 46 3 < 60 4 kJmin1; p < 0.05) subjects. Total energy expenditure was higher _ O2 peak for a long time (107 when exercising at 40% V 4 min) than when the same amount of work was performed _ O2 peak in TR (3946 250 > 3445 at 60% and 80% V 226 & 3431 241 kJ; respectively; p < 0.05) and UNTR (3458 223 > 2955 170 > 2717 172 kJ; p < 0.05) subjects. Total energy expenditure (kJ) and rate (kJmin1) were higher for TR than UNTR subjects at all the relative exercise intensities tested (p < 0.05). Fat oxidation Figure 1 depicts the fat oxidation rate during each trial in TR and UNTR subjects. While TR subjects achieved Fatmax _ O2 peak (0.41 0.01 gmin1), UNTR subjects at 60% V _ O2 peak (0.28 0.01 gmin1). The achieved Fatmax at 40% V rate of fat oxidation was higher for TR than for UNTR subjects, although it only reached statistical difference at 60% _ O2 peak (p < 0.05; Fig. 1). The total fat oxidation and 80% V for the set amount of work was higher at 40% than at 60% _ O2 peak for TR (36 5 > 26 4 > 11 2 g; and at 80% V p < 0.05) and UNTR (29 3 > 14 3 > 2 1 g; p < 0.05) subjects; it was higher for TR than UNTR subjects only at _ O2 peak (p < 0.05). 60% and 80% V Carbohydrate oxidation The rate of carbohydrate oxidation increased along with _ O2 peak) in the exercise intensity (i.e., 40% < 60% < 80% V both TR (1.6 0.2 < 2.5 0.2 < 4.2 0.3 gmin1; p < 0.05) and UNTR (1.4 0.2 < 2.4 0.1 < 3.6 0.3 gmin1; p < 0.05) subjects. Despite the differences in the energy expenditure rates, the differences in the carbohydrate oxidation rates between TR and UNTR subjects did not reach statistical significance. Substrate contribution to total energy expenditure Figure 2 depicts the relative contribution of carbohydrate and fat to total energy expenditure in TR and UNTR subjects. Carbohydrate was the predominant substrate oxidized during exercise in the heat at all exercise intensities for both groups of subjects. Furthermore, the relative contribution of carbohydrate to total energy expenditure increased with exercise intensity (p < 0.05). However, TR individuals oxidized relatively more fat than UNTR subjects at 60%

Fig. 2. Relative fat (FAT) and carbohydrate (CHO) oxidation for endurance-trained and untrained individuals at 40%, 60%, and 80% _ O2 peak. Data are means standard error of measurement. *, DifV _ O2 peak, p < ferent from UNTR, p < 0.05; {, different from 60% V _ O2 peak, p < 0.05. 0.05; {, different from 40% V

_ O2 peak (28% 4% vs. 17% 3%, p < 0.05) and 80% V _ O2 peak (11% 2% vs. 4% 1%, p < 0.05), but not at V _ O2 peak (35% 5% & 33% 4%). 40% V Blood responses and respiratory exchange ratio From similar resting values (7.42 0.01 for TR and 7.41 0.01 for UNTR), blood pH was well maintained during exercise at all exercise intensities in both TR (7.44 0.01, 7.43 0.01, and 7.41 0.01 at 40%, 60%, and 80% _ O2 peak, respectively) and UNTR (7.44 0.01, 7.44 0.01, V _ O2 peak, and 7.41 0.01 for UNTR, at 40%, 60%, and 80% V respectively) subjects. At the end of exercise, [EPI]plasma concentrations increased along with the increases in exercise inPublished by NRC Research Press

Del Coso et al.

745 Fig. 3. (A) Plasma epinephrine and (B) plasma norepinephrine concentrations in trained and untrained individuals at the end of exer_ O2 peak. Data are means standard cise at 40%, 60%, and 80% V error of measurement. *, Different from UNTR, p < 0.05; {, differ_ O2 peak, p < 0.05; {, different from 40% V _ O2 peak, ent from 60% V p < 0.05.

_ O2 peak), but it was similar tensity (i.e., 40% < 60% < 80% V between groups (Fig. 3A). [NEPI]plasma concentrations also increased with exercise intensity, but it was higher in TR than in UNTR subjects at the end of the 60% and 80% _ O2 peak trials (Fig. 3B; p < 0.05). Finally, respiratory exV change ratios were 0.89 0.01 vs. 0.90 0.01, 0.91 0.01 vs. 0.94 0.01 (p < 0.05), and 0.96 0.01 vs. 0.99 0.01 (p < 0.05) for TR vs. UNTR subjects at 40%, 60%, and 80% _ O2 peak, respectively. V

Discussion
Several studies have been geared to elucidate the exercise intensity that elicits Fatmax in a thermoneutral environment (Romijn et al. 1993, 2000; Bergman and Brooks 1999; Achten et al. 2002; Venables et al. 2005; Nordby et al. 2006; Stisen et al. 2006; Riddell et al. 2008). The practical finding is that the exercise intensity that produces Fatmax _ O2 peak. This ample range of may range from 33% to 65% V exercise intensities for Fatmax can be explained by the different nature of the subjects participating in the cited studies (i.e., different aerobic capacities). When comparing data from Achten et al. (2002), who used TR subjects, and Venables and colleagues (2005), who used UNTR subjects, TR subjects obtained Fatmax at a higher relative exercise inten_ O2 max). Similarly, sity than UNTR subjects (64% vs. 48% V Nordby et al. (2006) found that Fatmax was attained at a higher relative exercise intensity in TR than in UNTR sub_ O2 max). Likewise, in a thermoneutral jects (50% vs. 43% V environment, we found in this study that Fatmax in the heat occurred at a higher relative intensity in TR than in UNTR _ O2 peak). In addition, during exersubjects (60% vs. 40% V cise in the heat, the curve depicting fat oxidation rate as a function of relative exercise workload was displaced upward and rightward in TR, compared with UNTR, individuals (Fig. 1). Exercise in the heat (40 8C) increases muscle glycogen oxidation and reduces whole-body fat oxidation (Febbraio et al. 1994), in comparison to the same exercise intensity performed at 20 8C. In agreement with data from Febbraio et al. (1994), Fatmax levels found in this study (0.41 and 0.28 gmin1 for TR and UNTR, respectively) were substantially lower than those found in temperate environments. For example, Achten and coworkers (2002) found that TR subjects oxidized fat at a rate as high as ~0.6 gmin1, whereas Venables and colleagues (2005) reported fat oxidation rates as high as ~0.4 gmin1 in UNTR subjects. In addition, fat _ O2 peak represented 35% 5% for TR oxidation at 40% V and 33% 4% for UNTR subjects of total energy expenditure (Fig. 2), whereas Bergman and Brooks (1999) reported a contribution of 40% of total energy derived from fat when _ O2 peak in a thermoneutral environment. exercising at 40% V It is then possible that our hot environmental conditions induced to the reported lower rates of fat oxidation, in comparison to those studies. To increase the applicability of our study, subjects ingested a standardized breakfast prior to each trial, containing 3 g of carbohydrate per kilogram of body weight. The ingestion of a similar breakfast has been shown to decrease exercise fat oxidation rates in both trained and untrained subjects (Bergman and Brooks 1999). However, in the Bergman and

Brooks (1999) study, the ingestion of pre-exercise carbohydrates did not affect the exercise intensity that produced Fatmax. Thus, the lower Fatmax found in our study, in comparison to other studies, might be due to a combination of the high heat load and the effect of the pre-exercise ingestion of carbohydrates. A limitation of our study is that we cannot distinguish between the effects of heat load and those of carbohydrate ingestion in reducing the fat oxidation rate. Some studies have used a single progressive exercise test to determine the exercise intensity that elicits Fatmax (Achten et al. 2002; Venables et al. 2005; Stisen et al. 2006). A different approach is to perform bouts of exercise at diverse intensities on different days, and then to compare the fat oxidation rate obtained at each exercise intensity (Romijn et al. 1993; Bergman and Brooks 1999; Meyer et al. 2007). For our study, we chose the latter approach (3 trials separated by 4 days) to avoid the influence of progressive fatigue and substrate depletion on fat oxidation during a progressive exercise test. Besides, this approach allowed us to calculate total energy expenditure and total fat oxidation required to complete the same amount of work despite exercising at difPublished by NRC Research Press

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ferent intensities. Interestingly, both groups of subjects expended more energy and oxidized significantly more fat _ O2 peak) for a when exercising at light intensity (i.e., 40% V long time (107 min), despite their differences in the relative exercise intensity for Fatmax (Fig. 1). In the case of TR sub_ O2 peak (0.41 gmin1), jects, they achieved Fatmax at 60% V but they needed only 62 min to complete the assigned work at this intensity. In contrast, the fat oxidation rate at 40% _ O2 peak was high (0.36 gmin1), but subjects were required V to exercise for more than 100 min to complete the trial. This information may be relevant for trained and healthy untrained subjects aiming to maximize energy cost and fat oxidation during training. Our data suggest that prolonged exercise at a low intensity is the best choice to increase total energy expenditure and fat oxidation during exercise in the heat. Figure 2 depicts the relative contribution of fat and carbohydrate oxidations to the total energy expended in each trial. During exercise in the heat, TR subjects relied more on fat oxidation than their UNTR counterparts when exercising at _ O2 peak; while there were no differences be60% and 80% V _ O2 peak. Our data are in agreement tween groups at 40% V with previous authors, who found a higher relative contribution of fat oxidation to the energy expenditure at moderate _ O2 peak (Stisen et al. 2006)) and high intensities (45%60% V _ O2 peak (Coggan et al. 2000)) in TR than in (75%80% V UNTR subjects when exercising in thermoneutral environments. In the case of light exercise intensities (20%40% _ O2 peak), Bergman and Brooks (1999) found a higher conV tribution of fat oxidation in TR subjects, whereas Stisen and coworkers (2006) failed to find differences between groups, as we did in this study. Our data confirm that TR subjects had a superior reliance on fat as fuel during exercise in the heat, compared with UNTR subjects, at least at moderate to high intensities. In addition, we speculate that TR subjects did not oxidize significantly more fat than UNTR subjects _ O2 peak, because UNTR subjects achieved their at 40% V Fatmax at this exercise intensity, whereas TR subjects did so _ O2 peak. when exercising at 60% V The mechanisms for a higher fat oxidation in TR than UNTR subjects may be related to different metabolic adaptations derived from aerobic training. During exercise, [EPI]plasma increases lipolysis (Mora-Rodriguez et al. 2001), although high levels of [EPI]plasma induce a higher rate of glycogen utilization, which in turn decreases fatty acid oxidation during exercise (Mora-Rodriguez and Coyle 2000). In our study, [EPI]plasma concentrations rose with the exercise intensity, but the increase was similar in TR and UNTR subjects (Fig. 3A). On the other hand, [NEPI]plasma concentrations were higher in TR than UNTR subjects at 60% and _ O2 peak (Fig. 3B), in agreement with data from a train80% V ing study (Greiwe et al. 1999) and during exercise in an uncompensable hot environment (Wright et al. 2010). Interestingly, these precise exercise intensities (60% and _ O2 peak) were the ones at which TR subjects had 80% V higher absolute and relative fat oxidation than UNTR subjects (Fig. 2). It has been found that a higher [NEPI]plasma concentration increases the rate of free fatty acid release from adipose tissue (Hodgetts et al. 1991), thus increasing the availability of free fatty acids and, potentially, fat oxidation during exercise.

Muscle pH reductions inhibit the transport of free fatty acids into the mitochondria by inhibiting CPT1 activity (Starritt et al. 2000). In our study, blood pH (a surrogate of muscle acidity (Juel et al. 2004)) was maintained to a similar degree between groups at the end of exercise. On the other hand, b-oxidative muscle enzyme activity (e.g., b-hydroxyacyl-CoA-dehydrogenase activity) is higher in TR than in UNTR subjects (Nordby et al. 2006; Stisen et al. 2006), which in turn facilitates higher fat oxidation at moderate and high exercise intensities in the TR population (Stisen et al. 2006). Thus, the higher [NEPI]plasma concentration and increased b-oxidative muscle enzyme activity induced by aerobic training may be responsible, in part, for the higher fat oxidation in TR than in UNTR subjects in our study. Since, for a given relative exercise intensity, TR subjects exercised at a higher absolute workload than UNTR subjects, their sweat rate during exercise was higher (table 3, in Mora-Rodriguez et al. 2010), which likely allowed them to remain at core temperatures similar to their UNTR counter_ O2 peak). The increased sweat rate parts (at least up to 80% V induced higher dehydration in TR than in UNTR subjects (p < 0.05). It could then be argued that the larger dehydration in the TR group may have influenced metabolism through an effect on muscle glycogen utilization (Hargreaves et al. 1996). Although the differences in dehydration between TR and UNTR subjects were statistically significant, the metabolic effect of the small absolute differences (~0.6% dehydration) is dubious. In addition, it has been found that a reduction in body-water content does not affect whole-body fat oxidation during exercise (Roy et al. 2000). One limitation of this study was the utilization of indirect calorimetry to determine fat and carbohydrate oxidation dur_ O2 reliably reflects tissue O2 uptake, ing exercise. While V _ V CO2 is a good estimation of tissue CO2 production up to moderate exercise intensities, where H+ production is minimal and the bicarbonate pool is maintained stable (Del Coso et al. 2009). At high exercise intensities, the increase in H+ production is buffered by bicarbonate, producing nonmetabolic CO2, which in turn will produce an overestimation of carbohydrate oxidation and an underestimation of fat oxidation. In our study, the acidbase balance was well maintained in both groups at all exercise intensities, and respiratory exchange ratio was always maintained at less than 1.0, indicating minimal production of nonmetabolic CO2. In addition, it has been reported that indirect calorimetry accurately determines substrate oxidation (compared with a 13C_ O2 max, to-12C ratio technique) during exercise up to 85% V at least in trained subjects (Romijn et al. 1992). For all these reasons, we consider that the substrate oxidation rates calculated in this study were minimally affected by nonmetabolic CO2 production.

Conclusion
When exercising in hot environments, whole-body peak fat oxidation occurs at higher relative exercise intensity in aerobically trained individuals than untrained counterparts _ O2 peak). In addition, trained individuals oxi(60% vs. 40% V dize more fat than their untrained counterparts when exercis_ O2 peak). This ing at moderate to high intensities (>60% V
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Del Coso et al.

747 tive intensity before and after endurance exercise training. J. Appl. Physiol. 86(2): 531535. PMID:9931187. Hargreaves, M., Dillo, P., Angus, D., and Febbraio, M.A. 1996. Effect of fluid ingestion on muscle metabolism during prolonged exercise. J. Appl. Physiol. 80(1): 363366. PMID:8847329. Hjemdahl, P. 1984. Catecholamine measurements by high-performance liquid chromatography. Am. J. Physiol. 247(1 Pt. 1): E13E20. PMID:6377918. Hodgetts, V., Coppack, S.W., Frayn, K.N., and Hockaday, T.D. 1991. Factors controlling fat mobilization from human subcutaneous adipose tissue during exercise. J. Appl. Physiol. 71(2): 445451. PMID:1938716. Hulens, M., Vansant, G., Lysens, R., Claessens, A.L., and Muls, E. 2001. Exercise capacity in lean versus obese women. Scand. J. Med. Sci. Sports, 11(5): 305309. doi:10.1034/j.1600-0838. 2001.110509.x. PMID:11696216. Jackson, A.S., and Pollock, M.L. 1978. Generalized equations for predicting body density of men. Br. J. Nutr. 40(3): 497504. doi:10.1079/BJN19780152. PMID:718832. Jackson, A.S., Pollock, M.L., and Ward, A. 1980. Generalized equations for predicting body density of women. Med. Sci. Sports Exerc. 12(3): 175181. PMID:7402053. Jeukendrup, A.E. 2003. Modulation of carbohydrate and fat utilization by diet, exercise and environment. Biochem. Soc. Trans. 31(Pt. 6): 12701273. doi:10.1042/BST0311270. PMID:14641041. Jones, N.L., Heigenhauser, G.J., Kuksis, A., Matsos, C.G., Sutton, J.R., and Toews, C.J. 1980. Fat metabolism in heavy exercise. Clin. Sci. (Lond.), 59(6): 469478. PMID:6777109. Juel, C., Klarskov, C., Nielsen, J.J., Krustrup, P., Mohr, M., and Bangsbo, J. 2004. Effect of high-intensity intermittent training on lactate and H+ release from human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 286(2): E245E251. doi:10.1152/ ajpendo.00303.2003. PMID:14559724. Kirwan, J.P., Costill, D.L., Kuipers, H., Burrell, M.J., Fink, W.J., Kovaleski, J.E., and Fielding, R.A. 1987. Substrate utilization in leg muscle of men after heat acclimation. J. Appl. Physiol. 63(1): 3135. PMID:3624132. ssler, N., and Kindermann, W. 2007. Determination Meyer, T., Ga of Fatmax with 1 h cycling protocols of constant load. Appl. Physiol. Nutr. Metab. 32(2): 249256. doi:10.1139/H06-108. PMID:17486166. Mora-Rodriguez, R., and Coyle, E.F. 2000. Effects of plasma epinephrine on fat metabolism during exercise: interactions with exercise intensity. Am. J. Physiol. 278(4): E669E676. Mora-Rodriguez, R., Hodgkinson, B.J., Byerley, L.O., and Coyle, E.F. 2001. Effects of b-adrenergic receptor stimulation and blockade on substrate metabolism during submaximal exercise. Am. J. Physiol. Endocrinol. Metab. 280(5): E752E760. PMID: 11287358. Mora-Rodriguez, R., Del Coso, J., Hamouti, N., Estevez, E., and Ortega, J.F. 2010. Aerobically trained individuals have greater increases in rectal temperature than untrained ones during exercise in the heat at similar relative intensities. Eur. J. Appl. Physiol. 109(5): 973981. doi:10.1007/s00421-010-1436-4. PMID: 20349316. Nordby, P., Saltin, B., and Helge, J.W. 2006. Whole-body fat oxidation determined by graded exercise and indirect calorimetry: a role for muscle oxidative capacity? Scand. J. Med. Sci. Sports, 16(3): 209 214. doi:10.1111/j.1600-0838.2005.00480.x. PMID:16643200. Riddell, M.C., Jamnik, V.K., Iscoe, K.E., Timmons, B.W., and Gledhill, N. 2008. Fat oxidation rate and the exercise intensity that elicits maximal fat oxidation decreases with pubertal status in young male subjects. J. Appl. Physiol. 105(2): 742748. doi:10.1152/japplphysiol.01256.2007. PMID:18535137.
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elevated reliance on fat as a fuel during exercise in the heat could be related to the higher plasma norepinephrine concentration observed in the aerobically trained individuals.

Acknowledgements
The authors thank the subjects for their invaluable contribution to the study. Juan Del Coso and Nassim Hamouti were supported by a predoctoral fellowship from the CastillaLa Mancha Government in Spain. Juan F. Ortega received funds from the Gatorade Sports Science Institute.

References
Achten, J., Gleeson, M., and Jeukendrup, A.E. 2002. Determination of the exercise intensity that elicits maximal fat oxidation. Med. Sci. Sports Exerc. 34(1): 9297. doi:10.1097/00005768200201000-00015. PMID:11782653. msson, S.A., Stewart, D.J., Borrani, F., Skinner, K.A., and Arngr Cureton, K.J. 2003. Relation of heart rate to percent VO2 peak during submaximal exercise in the heat. J. Appl. Physiol. 94(3): 11621168. PMID:12391114. Balady, G.J., Berra, K.A., Golding, L.A., Gordon, N.F., Mahler, D.A., Myers, J.N., and Sheldahl, L.M. 2000. ACSMs guidelines for exercise testing and prescription. Lippincott Williams and Wilkins, Baltimore, M.D. Bergman, B.C., and Brooks, G.A. 1999. Respiratory gas-exchange ratios during graded exercise in fed and fasted trained and untrained men. J. Appl. Physiol. 86(2): 479487. PMID:9931180. Brouwer, E. 1957. On simple formulae for calculating the heat expenditure and the quantities of carbohydrate and fat oxidized in metabolism of men and animals, from gaseous exchange (oxygen intake and carbonic acid output) and urine-N. Acta Physiol. Pharmacol. Neerl. 6(1): 795802. PMID:13487422. Coggan, A.R., Kohrt, W.M., Spina, R.J., Bier, D.M., and Holloszy, J.O. 1990. Endurance training decreases plasma glucose turnover and oxidation during moderate-intensity exercise in men. J. Appl. Physiol. 68(3): 990996. PMID:2111314. Coggan, A.R., Spina, R.J., Kohrt, W.M., and Holloszy, J.O. 1993. Effect of prolonged exercise on muscle citrate concentration before and after endurance training in men. Am. J. Physiol. 264(2 Pt. 1): E215E220. PMID:8447387. Coggan, A.R., Raguso, C.A., Gastaldelli, A., Sidossis, L.S., and Yeckel, C.W. 2000. Fat metabolism during high-intensity exercise in endurance-trained and untrained men. Metabolism, 49(1): 122128. doi:10.1016/S0026-0495(00)90963-6. PMID:10647075. Del Coso, J., Hamouti, N., Aguado-Jimenez, R., and MoraRodriguez, R. 2009. Respiratory compensation and blood pH regulation during variable intensity exercise in trained versus untrained subjects. Eur. J. Appl. Physiol. 107(1): 8393. doi:10. 1007/s00421-009-1101-y. PMID:19513741. Febbraio, M.A., Snow, R.J., Stathis, C.G., Hargreaves, M., and Carey, M.F. 1994. Effect of heat stress on muscle energy metabolism during exercise. J. Appl. Physiol. 77(6): 28272831. PMID:7896628. Frayn, K.N. 1983. Calculation of substrate oxidation rates in vivo from gaseous exchange. J. Appl. Physiol. 55(2): 628634. PMID:6618956. Friedlander, A.L., Casazza, G.A., Horning, M.A., Buddinger, T.F., and Brooks, G.A. 1998. Effects of exercise intensity and training on lipid metabolism in young women. Am. J. Physiol. 275(5 Pt. 1): E853E863. PMID:9815006. Greiwe, J.S., Hickner, R.C., Shah, S.D., Cryer, P.E., and Holloszy, J.O. 1999. Norepinephrine response to exercise at the same rela-

748 Romijn, J.A., Coyle, E.F., Hibbert, J., and Wolfe, R.R. 1992. Comparison of indirect calorimetry and a new breath 13C/12C ratio method during strenuous exercise. Am. J. Physiol. 263(1 Pt. 1): E64E71. PMID:1636700. Romijn, J.A., Coyle, E.F., Sidossis, L.S., Gastaldelli, A., Horowitz, J.F., Endert, E., and Wolfe, R.R. 1993. Regulation of endogenous fat and carbohydrate metabolism in relation to exercise intensity and duration. Am. J. Physiol. 265(3 Pt. 1): E380E391. PMID:8214047. Romijn, J.A., Coyle, E.F., Sidossis, L.S., Rosenblatt, J., and Wolfe, R.R. 2000. Substrate metabolism during different exercise intensities in endurance-trained women. J. Appl. Physiol. 88(5): 17071714. PMID:10797133. Roy, B.D., Green, H.J., and Burnett, M. 2000. Prolonged exercise after diuretic-induced hypohydration: effects on substrate turnover and oxidation. Am. J. Physiol. Endocrinol. Metab. 279(6): 13831390. PMID:11093927.

Appl. Physiol. Nutr. Metab. Vol. 35, 2010 Starritt, E.C., Howlett, R.A., Heigenhauser, G.J., and Spriet, L.L. 2000. Sensitivity of CPT I to malonyl-CoA in trained and untrained human skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 278(3): E462E468. PMID:10710500. Stisen, A.B., Stougaard, O., Langfort, J., Helge, J.W., Sahlin, K., and Madsen, K. 2006. Maximal fat oxidation rates in endurance trained and untrained women. Eur. J. Appl. Physiol. 98(5): 497 506. doi:10.1007/s00421-006-0290-x. PMID:17006714. Venables, M.C., Achten, J., and Jeukendrup, A.E. 2005. Determinants of fat oxidation during exercise in healthy men and women: a cross-sectional study. J. Appl. Physiol. 98(1): 160167. doi:10.1152/japplphysiol.00662.2003. PMID:15333616. Wright, H.E., Selkirk, G.A., and McLellan, T.M. 2010. HPA and SAS responses to increasing core temperature during uncompensable exertional heat stress in trained and untrained males. Eur. J. Appl. Physiol. 108(5): 987997. doi:10.1007/s00421-0091294-0. PMID:19967394.

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