Energy substrate oxidation during exercise is affected during both short- and long-term exposures to high altitude (9,35,41,42). Specifically, carbohydrate oxidation in men has been reported to be increased during exercise, conducted at the same absolute work rate, during acute and chronic exposures to altitude compared with sea level (9,35). These findings have been attributed to optimizing the energy yield per unit of O2 consumed (9). However, the interpretation of findings from these studies is complicated by the use of a higher relative percentage of V̇O2peak in the altitude condition, which would tend to increase reliance on glucose as a fuel (10).
Other studies on men have reported that fat oxidation is increased during exercise, conducted at the same relative exercise intensity, after chronic but not acute altitude exposure compared with sea level (41,42). Increased activation of the sympathoadrenal system with chronic altitude exposure may contribute to the observed increase in fat oxidation (34). The interpretation of findings after chronic altitude exposure, however, is complicated by the occurrence of body weight loss, which would tend to increase reliance on fat as a fuel (15). Thus, although an acute altitude exposure appears to be associated with increased carbohydrate oxidation during exercise in men, substrate oxidation during exercise after chronic altitude exposure remains unresolved and likely depends on exercise intensity, duration of altitude exposure, prior caloric intake, and time elapsed since last meal.
Women may not adjust their substrate oxidation upon acute or chronic exposure to altitude in the same manner as men. First, women exhibit a smaller increase in carbohydrate oxidation compared with men in other stressful situations that stimulate catecholamine release (i.e., hypoglycemia and exercise) (2,40). Second, high estradiol levels, which occur in the midluteal phase of the menstrual cycle, may redirect substrate oxidation toward fats (3,38,39). Braun et al. (8) recently reported greater resting fat oxidation in women during acute (∼24 h) and short-term (∼2 to 7-d) exposures to 4300 m compared with sea level. They also reported greater fat oxidation during exercise, conducted at the same relative exercise intensity, after 10 d at 4300-m altitude compared with sea level (8). McClelland et al. (31,32) reported similar fat and carbohydrate oxidation in female rats during exercise, conducted at the same relative exercise intensity, after chronic altitude exposure compared with sea level . Reasons for this gender-specific response to acute and chronic altitude exposures are not readily apparent but most likely are due to the effects of high estradiol levels promoting increased fat and decreased carbohydrate oxidation. Although the study by Braun et al. (8) controlled for menstrual cycle phase, cycle phase differences in energy substrate oxidation during exercise at altitude were not presented.
The purpose of this study was to determine whether substrate oxidation during submaximal exercise in women is affected by an acute exposure to 4300-m altitude and menstrual cycle phase. We hypothesized that, similar to data on altitude-acclimatized women, women acutely exposed to 4300-m altitude would exhibit a greater fat oxidation during exercise compared with sea level. We further hypothesized that substrate oxidation during exercise would be shifted toward fat oxidation during the midluteal phase of the menstrual cycle both at sea level and during an acute exposure to 4300-m altitude.
Volunteer test subjects.
The same eight female volunteers previously described (4) were enrolled in this study after giving written acknowledgment of their voluntary consent. All had normal hemoglobin ([Hb]) and serum ferritin levels. Investigators adhered to Army Regulation 70–25 and USAMRDC Regulation 70–25 on the use of volunteers in research . Menstrual cycle phase documentation in these volunteers has also been previously reported (4).
The study used an unblinded balanced experimental design in which several indicators of substrate utilization were measured during submaximal exercise in the early follicular (3–6 d after the beginning of menstruation) and midluteal (6–9 d after the luteinizing hormone surge) menstrual cycle phase at sea level and during an acute exposure to the altitude equivalent of 4300 m. The test conditions were defined as sea-level, follicular (SL/F); sea-level, luteal (SL/L); acute-altitude, follicular (AA/F); and acute-altitude, luteal (AA/L). During each test condition, 2 d were used to complete all exercise tests. On the first day, volunteers completed a treadmill peak oxygen uptake (V̇O2peak) test. After a 48-h rest period with no exercise, the second day of testing involved a treadmill submaximal exercise to exhaustion (EXH) test conducted at 70% of their altitude-specific V̇O2peak. Each exercise test was performed at the same time of day for each volunteer in all four testing conditions. The order of menstrual cycle phase and altitude combination in which testing was performed was randomized.
All exercise testing was performed in a hypobaric chamber, maintained at a temperature and relative humidity of 22 ± 3°C and 45 ± 5%, respectively. After the volunteers entered the hypobaric chamber for the V̇O2peak and EXH tests during AA exposures, the chamber was decompressed to the barometric equivalent of 4300 m (446 mm Hg) over a period of approximately 12 min. All exercise tests in the hypobaric chamber were initiated within 30 min of arriving at 4300 m and completed within 1–3 h.
Ovarian hormone analysis.
Resting ovarian hormone levels were measured to document menstrual cycle phase. A resting blood sample was obtained before the EXH tests for analyses of resting estradiol and progesterone levels. A serum progesterone value greater than 5 ng·mL−1 was accepted as confirmation of the midluteal phase (27).
Before all exercise tests, the volunteers were required to abstain from alcohol and caffeine for at least 24 h and not exercise on the testing day. The volunteers also maintained the same diet for 24 h before each of the four EXH tests. All testing was done in the morning after an overnight fast. This minimized variations in substrate availability before each test. Twenty-four-hour dietary logs were analyzed for energy content and percent contribution of macronutrients by computer using the Food Intake Analysis System (University of Texas Health Science Center, Houston, TX).
Before each exercise test, the volunteer was weighed (wearing t-shirt, shorts, and socks) to the nearest 0.1 kg. Respiratory gas measurements were made intermittently during the EXH tests by using open-circuit calorimetry (Model 2900 metabolic cart; SensorMedics Corporation, Yorba Linda, CA) calibrated with certified gases and volume standard. The metabolic cart provided values for V̇E, oxygen uptake (V̇O2), and carbon dioxide output (V̇CO2). Gas samples were collected simultaneously with the blood samples during the EXH tests. The respiratory exchange ratio (RER) was calculated from the V̇O2 and V̇CO2 measurements, and used to estimate the percent contribution of fat and carbohydrate (%) to energy metabolism during submaximal exercise (29). Carbohydrate oxidation (mg carbohydrate·L O2 −1) and fat oxidation (mg fat·L O2 −1) was also calculated from RER values by using standard values (29) and multiplied by V̇O2 (L·min−1) to obtain carbohydrate and fat oxidation rates in mg of carbohydrate or fat per minute. To account for differences in energy expenditure at SL and AA, carbohydrate and fat oxidation rates per unit energy (mg·kcal−1·min−1) were calculated. Although the RER was obtained without urinary nitrogen analysis, the error introduced should be minimal given the small contribution of protein to substrate oxidation during exercise.
Peak exercise testing.
The V̇O2peak was determined by a progressive-intensity, continuous treadmill running test to exhaustion. After a 3-min warm-up, the speed and grade were increased to 2.2 to 2.7 m·s−1 and 2.0%, respectively, for the first 2 min. Thereafter, the speed was kept constant and grade was increased by 2% every 2 min. The highest oxygen uptake achieved for one min before exhaustion was recorded as V̇O2peak.
Submaximal exercise testing.
Forty minutes before the EXH test, an indwelling catheter was inserted into a vein of the mid-forearm. Blood samples were collected in the standing position at rest, every 15 min during the test, and at exhaustion. After a 3-min warm-up, each volunteer exercised at 70% of her altitude-specific V̇O2peak until exhaustion. Treadmill speed and/or grades were adjusted to reach the desired percentage of V̇O2peak for each volunteer. In all cases, the target V̇O2 was obtained within 10 min of the beginning of exercise.
Exercise energy substrate measurements were measured to determine whether they were consistent with patterns of substrate oxidation suggested by the respiratory gas measurements. Aliquots of heparinized blood were used to measure blood lactate concentrations [La] and glucose concentrations [GLU] in duplicate (Model 2300 YSI analyzer; Yellow Springs Instruments, Yellow Springs, OH). Serum free fatty acid concentrations [FFA] (Wako Chemicals, Richmond, VA) and glycerol concentrations [GLY] (Sigma Diagnostics, St. Louis, MO) were determined by an enzymatic spectrophotometric method. Whole blood [Hb] and hematocrit (Hct) were measured in triplicate by using absorbance wavelength (Model OSM3 Hemoximeter, Radiometer, Inc. Copenhagen) and microcapillary methods, respectively. The [Hb] and hematocrit determinations were used to calculate changes in plasma volume (12). All blood substrate concentrations were corrected for changes in plasma volume during exercise.
Serum insulin, cortisol, growth hormone, estradiol and progesterone, and plasma glucagon were thawed once after storage at −70°C and measured using commercial radioimmunoassay kits (Diagnostic Products Corporation, Los Angeles, CA). The intra-assay coefficients of variation (CV) for insulin, cortisol, growth hormone, estradiol, progesterone, and glucagon were 9.0%, 5.2%, 5.0%, 4.7%, 7.4%, and 8.9%, respectively. All hormonal samples for each of the volunteers were analyzed in duplicate in the same assay to avoid interassay variations. Catecholamines were extracted from plasma (Alko Diagnostics Co., Holliston, MA) and measured by high-performance liquid chromatography (Model 2345, Waters Co., Norwell, MA). The percent recovery for the extraction of the catecholamines was 82.0%. The inter- and intra-assay CVs for both epinephrine and norepinephrine were less than 3.0% and 2.0%, respectively. All metabolic hormone values were corrected for changes in plasma volume during exercise. Because each volunteer’s time to exhaustion and number of blood samples differed during the EXH tests, energy substrate, and metabolic hormone data are presented at rest, at 20%, 40%, 60%, and 80% of the time to exhaustion, and at exhaustion.
Three-way ANOVAs with repeated measures on each factor were used to analyze the differences between altitude (SL and AA), menstrual cycle phase (early follicular and midluteal), and duration of exercise (before, during, and after the EXH tests) for all respiratory gas, energy substrate, and metabolic hormone measurements. Significant main effects and interactions were analyzed using Tukey’s least significant difference test. Pearson product-moment correlation coefficients were calculated for relationships between respiratory gas, energy substrate, and metabolic hormone measurements. Statistical significance was set at P < 0.05. All data are presented as means ± SD.
Volunteer test subjects.
Mean body weight (58 ± 6 kg) and energy intake (2023 ± 535) in all four test conditions were not different. Mean percent contribution of carbohydrate (53 ± 9), fat (29 ± 7), and protein (18 ± 5) to the diet were not different between testing conditions. Mean cycle length was 28 ± 2 d during the 4 months of testing, and ovulation occurred 14 ± 6 d after the onset of menstruation. In the early follicular phase, resting estradiol levels ranged from 22 to 73 pg·mL−1, whereas progesterone levels ranged from 0.2 to 1.1 ng·mL−1. In the midluteal phase, resting estradiol levels ranged from 62 to 200 pg·mL−1, whereas progesterone levels ranged from 5 to 27 ng·mL−1. Resting estradiol and progesterone levels in all four test conditions are presented in Table 1 and are within previously reported normal ranges for eumenorrheic women (27).
To maintain the same relative exercise intensity in both the SL and AA testing conditions, the mean V̇O2 for both phases combined at SL (1.89 ± 0.28 L·min−1) was higher than the mean V̇O2 for both phases combined at AA (1.35 ± 0.18 L·min−1). The relative intensities of exercise (range: 68.1–69.1%) during the submaximal exercise test in all four testing conditions were the same and have been previously reported (4).
The mean resting [Hb]s (range: 12.8–13.1 mg·dL−1) and Hcts (range: 39.4–40.3%) were not different and did not increase from rest with duration of exercise in any of the four test conditions. Mean plasma volume changes from rest during exercise (range: −1.6% to −3.7%) were also similar in all four test conditions. Mean plasma volume changes from SL/F to AA/F (2.5 ± 10.0%) and SL/L to AA/L (−3.2 ± 8.4%) were not different.
Primary indicator of substrate utilization.
The RER was used as a primary indicator of energy substrate utilization patterns during submaximal exercise. The RER values and percent contributions of carbohydrate and fat to energy metabolism did not change with duration of exercise in any of the four test conditions. Therefore, the mean of the five exercise time points (i.e., 20%, 40%, 60%, and 80% of the time to exhaustion, and at exhaustion) for RER and percent utilizations of both fat and carbohydrate are presented in Figure 1. The RER values were lower during exercise at AA/F and AA/L compared with both SL/F and SL/L. The percent utilization of carbohydrate was decreased and fat increased during submaximal exercise at AA compared with SL in both phases. The RER values were not affected by cycle phase at SL or AA. Carbohydrate and fat oxidation rates per unit energy (mg·kcal−1·min−1) did not change with time of exercise in any of the four test conditions. Therefore, the mean of the five exercise time points (i.e., 20%, 40%, 60%, and 80% of the time to exhaustion, and at exhaustion) for carbohydrate and fat oxidation rates per unit energy are presented in Figure 1. Carbohydrate and fat oxidation rates per unit energy were decreased and increased, respectively, during submaximal exercise at AA compared with SL in both phases. There were no menstrual phase differences in fat and carbohydrate oxidation rates per unit energy at SL or AA.
Resting [GLU], [La], [GLY], and [FFA] were not affected by AA or cycle phase (Table 1). During submaximal exercise, [GLU] and [GLY] were also not affected by AA or cycle phase, but [La] was increased in both phases at AA compared with SL at every time point. The [FFA] was also increased during submaximal exercise in both phases at AA compared with SL/F at 80% of the time to exhaustion and at exhaustion.
Resting insulin, glucagon, growth hormone, cortisol, epinephrine, and norepinephrine concentrations were not affected by AA or cycle phase (Table 1;Fig. 2). During submaximal exercise, insulin, glucagon, growth hormone, epinephrine, and norepinephrine concentrations were also not affected by AA or cycle phase, but cortisol concentrations from 60% of the time to exhaustion to exhaustion were increased in both phases at AA compared with both phases at SL (Table 1;Fig. 2). Plasma norepinephrine and serum [FFA] were positively correlated during submaximal exercise at SL/F (P = 0.05), SL/L (P = 0.06), AA/F (P = 0.05), and AA/L (P = 0.07). None of the other metabolic hormones were correlated with energy substrates during submaximal exercise.
Resting and exercise estradiol and progesterone concentrations were not affected by AA in either phase of the menstrual cycle but were increased in the midluteal phase in both the SL and AA conditions (Table 1). Neither progesterone, estradiol, nor the ratio of progesterone/estradiol was correlated with any of the energy substrates or metabolic hormones in any of the four test conditions.
Our primary hypothesis was that fat oxidation would be increased during exercise in women acutely exposed to 4300-m altitude. In this study, based on the RER values, fat oxidation was increased during exercise, conducted at the same percentage of altitude-specific V̇O2peak, during an AA exposure compared with SL in both menstrual cycle phases. Our second hypothesis was that substrate oxidation during exercise would be shifted toward fat oxidation during the midluteal phase of the menstrual cycle both at SL and during an AA exposure to 4300 m. In this study, substrate oxidation during exercise was not altered by menstrual cycle phase at SL or during an AA exposure.
Sea level versus altitude comparisons.
The lower RER values observed in these women during the same relative exercise intensity upon AA exposure compared with SL disagrees with previous reports in men exercising at the same relative exercise intensity during an acute altitude exposure (7,23,41). Thus, we questioned our RER values. However, our analyzers were carefully calibrated with certified gases and the lower RER at AA compared with SL in our study was reproducible on two random-order tests conducted at altitude over the course of a few months. Thus, women may rely more on fat oxidation than men during exercise upon AA exposure, which supports previous research on exercising men and women at SL (40).
Our substrate oxidation conclusions are limited due to the fact that calculations were based solely on RER measurements. Indirect calorimetry has often been criticized for accuracy during intense exercise due to hyperventilation, which may cause an overestimation of tissue CO2 production (14). However, Romijn et al. (36) validated indirect calorimetry measurements, even at exercise intensities of 80–85% of V̇O2max, with an isotopic tracer method that was completely independent of V̇CO2 measurements. In addition, a recent study reported that the sum of isotopic measures of working muscle intramuscular triglyceride and plasma free fatty acid oxidation rates is not different from fat oxidation rates calculated from indirect calorimetry (18). Therefore, we believe that RER measurements provide a valid estimate of whole body carbohydrate and fat oxidation rates.
Because RER values during exercise can be influenced by the preceding 24-h diet and overnight fast (24), care was taken to ensure that volunteers ate the same diet 24-h and exercised at the same time of day before each submaximal exercise session. The RER value can also be affected by energy balance conditions (i.e., losing or gaining body weight) with a tendency to burn more fat during weight loss conditions (15). Our volunteers were likely in energy balance as reflected by their nearly identical body weights on each of the four submaximal exercise test sessions. The RER can also be affected by increases or decreases in ventilation (14). Even though hyperventilation, which did occur in these subjects upon AA exposure (4), may cause an overestimation tissue CO2 production, this would tend to overestimate the rate of carbohydrate oxidation and underestimate the rate of fat oxidation (14). Thus, the observed increase in fat oxidation in these women during submaximal exercise upon AA exposure compared with SL may be even greater than what is reported.
Several reasons may explain the reason for the lower RER value during exercise upon AA exposure in our women compared with higher or similar RER responses reported from studies on men (7,23,41). Regardless of menstrual cycle phase, the clearest difference between men and women is the hormonal milieu surrounding the reproductive process. In females, the ovarian hormones, progesterone and estradiol, play a dominant role in reproductive functions, whereas in males, the testicular hormone, testosterone, plays a dominant role. A review of evidence from human and animal studies suggests that estradiol stimulates fat oxidation and spares glycogen utilization in the exercising muscle (3,38,39). Thus, underlying hormonal differences may be one of the causes for the divergent gender-specific RER responses during exercise upon AA exposure.
Gender-specific RER responses may also be related to higher resting total fat content found in female skeletal muscle compared with males (16). Unfortunately, RER values do not allow us to identify the specific energy source responsible for the observed increase in whole body fat oxidation in these women upon AA exposure. Recently, McClelland et al. (32) found an increased use of intramuscular triglycerides during exercise in female rats acclimatized to altitude. Whether this same increased utilization of intramuscular triglycerides during exercise holds true for females acutely exposed to altitude awaits further investigation.
Another potential reason for the RER differences during exercise between men and women may be the sympathoadrenal response elicited upon AA exposure. Given the recently documented findings of sex differences in adrenergic regulation of lipid mobilization during exercise (i.e., men activate beta- and alpha2-adrenergic receptors in adipose tissue, whereas women only activate stimulatory beta-adrenergic receptors) (22), perhaps women, compared with men, also tend to increase fat lipolysis to a greater degree in response to a relatively similar sympathetic stimulation during an AA exposure. If increased fat lipolysis increases fat availability, as our serum FFA levels indicate, an increase in fat oxidation may result. A positive correlation between [FFA] and free fatty acid oxidation has been established in some studies (1,21) but not others (30,35). The significant correlation between [FFA] and norepinephrine concentrations during exercise in this study also suggests that sympathetic nerve stimulation may play a role in mobilizing fats in these women.
Although some of our metabolic substrate and hormonal findings support an increase in fat oxidation during exercise during an AA exposure, others were not consistent with these findings. In support of our findings, cortisol, a weak stimulator of lipolysis and major stimulator of proteolysis, was elevated upon AA exposure in these women. Larsen et al. (28) recently reported a decrease in insulin action and glucose utilization in men after 2 d of exposure to 4559 m, which they partly attributed to elevated cortisol levels. Cortisol is known to induce peripheral insulin resistance (37), and the higher cortisol levels upon AA exposure in both this study and the study by Braun et al. (8) may have contributed to the observed increase in fat oxidation. Alternatively, cortisol may have affected proteolysis during the AA exposure. We assumed that protein oxidation did not contribute significantly to substrate oxidation during exercise at SL or AA, but this may not be the case. Because we did not measure urinary nitrogen excretion, our RER values are not only reflective of fat and carbohydrate but also protein oxidation. Thus, we cannot rule out the possibility that increased protein and not fat oxidation accounts for the lower RER upon AA exposure. Nonetheless, whether the source is increased protein or fat, substrate oxidation in women is altered differently from men during exercise upon AA exposure, which supports previous work on women at altitude (8). Given that our epinephrine, insulin, lactate, and glycerol responses did not provide supportive mechanistic evidence for the observed substrate oxidation changes, further studies are warranted to determine mechanisms responsible for these changes.
Menstrual cycle phase comparisons.
Our second hypothesis was that fat oxidation would be enhanced in the midluteal phase of the menstrual cycle at both SL and AA. Despite significantly increased estradiol and progesterone levels in the midluteal phase, RER values, energy substrates, and metabolic hormones were not affected by menstrual cycle phase during submaximal exercise at SL or AA. This finding agrees with some (11,17,19,25,33) but not all (5,6,13,20) research on substrate oxidation at sea level. In some studies reporting cycle-phase differences at SL (5,6,20), the dietary conditions were manipulated such that the subjects were 24-h fasted or consuming an oral glucose load (6), under a low-carbohydrate diet (5), or exercising at a lower exercise intensity (20). However, Dombovy et al. (13) found lower RER values at 67% and 75%VO2max but not at lower exercise intensities. Despite conflicting evidence at SL, this study found no cycle phase differences in substrate oxidation during exercise, conducted at ∼70% of altitude-specific VO2peak, during an AA exposure.
Although there were no menstrual cycle phase differences in metabolic substrate and hormonal responses to exercise when the early-follicular (i.e., low estradiol and progesterone levels) and midluteal phases (i.e., high estradiol and progesterone levels) of the menstrual cycle were compared, this finding does not rule out the possibility that cycle phase differences may exist when the ovulatory (i.e., high estradiol levels only) phase of the menstrual cycle is studied. There is evidence in humans and animals to suggest that estradiol-mediated metabolic effects are antagonized by progesterone (19,26). Thus, when levels of both hormones are high, the opposing metabolic effects may cancel each other. Hackney et al. (19) found that RER values were decreased and fat oxidation increased in the ovulatory but not midluteal phase of the menstrual cycle when compared with the follicular phase. They attributed the increased fat oxidation in the ovulatory phase to the unopposed estradiol-mediated effects on adipose tissue lipolysis. Use of exogenous estradiol or progesterone administration to either postmenopausal or amenorrheic women or men in future studies might be a useful approach for studying the isolated metabolic effects of these ovarian hormones.
In summary, the present findings suggest that substrate oxidation is altered in women during submaximal exercise, conducted at the same relative percentage of altitude-specific V̇O2peak, during an acute exposure to 4300-m altitude compared with SL. Whether the lower RER values during exercise upon AA exposure represent increased fat or increased protein oxidation cannot be determined from this study and warrants further investigation. In addition, substrate oxidation, energy substrates, and metabolic hormones were not affected by menstrual cycle phase during exercise at SL or AA. This study, conducted during an AA exposure, corroborates some but not all previous SL findings that reported a lack of cycle-phase differences in substrate oxidation during moderate-intensity exercise under normal dietary conditions in women.
We would like to thank the test volunteers for their participation and cooperation in this study. The authors gratefully acknowledge the technical and logistical support provided by Mr. Jim Devine, Mr. James Bogart, CPT Timothy Lyons, SGT Sinclair Smith, and PVT Keesha Miller.
The views, opinions, and/or findings in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision, unless so designated by other official documentation.
Citations of commercial organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of these organizations.
Address for correspondence: Beth A. Beidleman, Thermal and Mountain Medicine Division, United States Army Research Institute of Environmental Medicine, Natick, MA 01760; E-mail: beth. firstname.lastname@example.org.
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