Departments of 1 Human Physiology and 2 Medical Physiology, Copenhagen Muscle Research Centre, University of Copenhagen; and 3 Department of Clinical Physiology and Nuclear Medicine, Herlev Hospital, DK-2100 Copenhagen, Denmark
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ABSTRACT |
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Substrate utilization across the leg
during 90 min of bicycle exercise at 58% of peak oxygen uptake
(O2 peak) was studied in seven
endurance-trained males and seven endurance-trained, eumenorrheic
females by applying arteriovenous catheterization, stable isotopes, and
muscle biopsies. The female and male groups were matched according to
O2 peak per kilogram of lean body mass,
physical activity level, and training history of the subjects. All
subjects consumed the same diet, well controlled in terms of nutrient
composition as well as energy content, for 8 days preceding the
experiment, and all females were tested in the midfollicular phase of
the menstrual cycle. During exercise, respiratory exchange ratio (RER)
and leg respiratory quotient (RQ) were similar in females and males.
Myocellular triacylglycerol (TG) degradation was negligible in males
but amounted to 12.4 ± 3.2 mmol/kg dry wt in females and
corresponded to 25.0 ± 6.0 and 5.0 ± 7.3% of total oxygen
uptake in females and males, respectively (P < 0.05).
Utilization of plasma fatty acids (12.0 ± 2.5 and 9.6 ± 1.5%), blood glucose (13.6 ± 1.5 and 14.3 ± 1.5%), and
glycogen (48.5 ± 4.9 and 42.8 ± 2.1%) were similar in
females and males. Thus, in females, measured substrate oxidation
accounted for 99% of the leg oxygen uptake, whereas in males 28% of
leg oxygen uptake was unaccounted for in terms of measured oxidized
lipid substrates. These findings may indicate that males utilized
additional lipid sources, presumably very low density lipoprotein-TG or
TG located between muscle fibers. On the basis of RER and leg RQ, it is
concluded that no gender difference existed in the relative
contribution from carbohydrate and lipids to the oxidative metabolism
across the leg during submaximal exercise at the same relative
workload. However, an effect of gender appears to occur in the
utilization of the different lipid sources.
[13C]palmitate; plasma fatty acids; myocellular triacylglycerol; glucose; glycogen
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INTRODUCTION |
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RECENTLY, IT HAS BEEN INVESTIGATED in several studies whether a gender difference exists in the relative utilization of carbohydrates and lipids as fuel sources during submaximal exercise. Some studies have shown that females derive a relatively larger contribution from lipids to oxidative metabolism during exercise than males (6, 11, 19, 36). However, others have observed a similar relative utilization of carbohydrates and lipids in females and males exercising at the same relative workload (3, 17, 26).
The relative contribution from carbohydrates and lipids as fuel during submaximal exercise is a result of the sum of utilization of the different carbohydrates (blood glucose and muscle glycogen) and lipids [albumin-bound long-chain fatty acids (FA) from the blood plasma, FA from circulating very low density lipoprotein-triacylglycerols (VLDL-TG), and FA from myocellular triacylglycerols (MCTG)]. It has previously been observed in a few studies that females and males have a similar systemic turnover of plasma FA during exercise expressed relative to body mass (BM) (3, 7) or lean body mass (LBM) (26). Furthermore, Friedlander et al. (7) found that systemic plasma FA oxidation during exercise did not differ between untrained females and males. It has also previously been observed that systemic turnover of glucose was similar in females and males during submaximal exercise at the same relative workload (6, 17, 26).
The study of substrate utilization locally in the active muscles
provides a much more detailed picture than whole body measurements, which do not reflect metabolism only in skeletal muscle. Recently, Burguera et al. (3) observed that plasma FA total uptake
and release across the leg were similar in untrained females and males during bicycle exercise at 45% of peak oxygen uptake
(O2 peak). However, whether this gender
similarity exists at a higher relative workload and/or in trained
individuals is not known. Furthermore, the oxidation of plasma FA in
the exercising leg has not yet been compared in females and males.
So far, the three methods most often applied to quantify the respective utilization of blood glucose, glycogen, plasma FA, VLDL-TG, and MCTG during exercise are the muscle biopsy technique, the stable isotope tracer technique, and net balances across the active muscle tissue bed, respectively. However, up to now, the relative utilization of these energy sources during exercise has not been determined by applying the three mentioned methods simultaneously in a single gender comparative study. Particularly, gender comparisons of net balances across the active muscles are lacking. Such studies would be expected to yield valuable information on possible gender differences in substrate utilization during exercise.
Therefore, the purpose of the present study was specifically to determine the utilization of blood glucose, glycogen, plasma FA, and MCTG, when applying simultaneously the muscle biopsy technique, the stable isotope tracer technique, and net balances across the active muscles.
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MATERIALS AND METHODS |
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From screening of 48 young endurance-trained females and males,
seven females and seven males were recruited to participate in the
study (Table 1) on the basis of the
training and oxidative capacity criteria given in
Prescreening that all females were eumenorrheic with a cycle
length between 28 and 35 days and that none of them were taking oral
contraceptives. Preliminary tests and the main exercise experiment in
females were carried out in the midfollicular phase of their menstrual
cycle [determined as days 7-11 from onset of
menstruation, mean day 9 ± 1 (mean ± SE) for the exercise
experiment].
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All subjects were fully informed of the risks associated with the study, and all of them gave written, informed consent. The study was approved by the Copenhagen Ethics Committee and was carried out in accordance with the Declaration of Helsinki II.
Experimental Design
Prescreening.
O2 peak was initially determined in an
incremental bicycle exercise test, where respiratory measurements were
carried out with the Douglas bag technique. Training history and
present weekly training were determined from self-reports
(questionnaire and activity record). Furthermore, body composition was
calculated from body density (31) determined by
hydrostatic weighing with a correction for residual lung volume
measured by the oxygen dilution method (16). Single-leg
composition was determined by means of dual-energy X-ray absorptiometry
(Lunar, Madison DPX-IQ version 4.6.6) from a whole body scan by a
pelvis cut angled through the femoral necks according to the
manufacturer's directions (Table 1).
Dietary control. When enrolled in the study, all subjects recorded their food intake by weighing on 5 nonconsecutive days (consisting of 1 day without training, 1 day with heavy training, 1 weekend day, and 2 weekdays with normal training) to determine the amount of energy and the nutrient composition of their habitual diet. Food records were analyzed using a computer program (Dankost 2000, Danish Catering Center, Copenhagen, Denmark). On the basis of these individual food records, subjects consumed a controlled, isocaloric diet during the 8 days preceding the exercise experiment. The diet consisted of ~15 energy percent (E%) protein, ~20 E% fat, and ~65 E% carbohydrate. This nutrient composition was similar to the habitual diet of the subjects. Food items with a high ratio of 13C to 12C were avoided in the controlled diet to keep the background enrichment of 13CO2 in breath and blood as low as possible during the exercise experiment. During the 8 days, all food intake was strictly controlled, and all food was weighed to an accuracy of 1 g and registered in a dietary record. Throughout the 8 days, adherence to the diet was evidenced by the close resemblence between the prescribed dietary content and the actual intake.
Exercise experimental protocol.
The exercise test consisted of bicycling on a Krogh bicycle ergometer
for 90 min at 58% O2 peak.
Methods
Breath samples. Expired volumes of air from the Douglas bags were measured with a chain-suspended Collins spirometer, and a small sample of mixed expiratory air was analyzed for O2 (Servomex S-3A) and CO2 (Beckman LB2). Furthermore, samples of mixed expiratory air from the Douglas bags were collected into 10-ml evacuated glass tubes (Vacutainer, Becton-Dickinson, Meylan, France) for subsequent determination of the enrichment of 13CO2 in the expiratory air.
Muscle biopsies.
The biopsy was freed from all visible blood and fat, rapidly frozen in
liquid nitrogen, and stored at 80°C for subsequent biochemical
analysis. Before analysis, the muscle tissue was freeze-dried and
dissected free of all visible adipose tissue, connective tissue, and
blood under a microscope. The dissected muscle fibers were pooled and
then divided into subpools for determination of MCTG and glycogen
concentrations. The glycogen concentration was determined by a
fluorometric method (15), and the concentration of
MCTG was determined according to Kiens and Richter (13).
Blood samples. Blood hemoglobin, O2 saturation, PO2, PCO2, and pH were determined by means of a blood gas analyzer (ABL510, Radiometer Medical, Copenhagen, Denmark). From these values, the hematocrit (Hct) and the blood O2 and CO2 concentrations were calculated according to equations provided by Siggaard-Andersen et al. (30). Blood glucose and lactate concentrations were measured on a glucose analyzer (YSI model 2300 Stat Plus, Yellow Springs Instrument, Yellow Springs, OH). Plasma FA concentration was measured by a colorimetric commercial assay kit (Wako Chemicals, Neuss, Germany) which was performed by a COBAS FARA autoanalyzer (COBAS FARA 2, Roche Diagnostic, Basel, Switzerland). Concentrations of insulin (Pharmacia Insulin Radioimmunoassay 100, Pharmacia & Upjohn Diagnostics, Uppsala, Sweden) as well as epinephrine and norepinephrine (KatCombi Radioimmunoassay, Immuno-Biological Laboratories, Hamburg, Germany) in plasma were determined by radioimmunoassay.
Isotope analyses. [U-13C]palmitate (98% enriched), NaH[13C]O3 (99% enriched), and [6,6-2H2]D-glucose (99% enriched) were purchased from Cambridge Isotope Laboratories, Andover, MA. On the day of the exercise experiment, the palmitate tracer in solution was added to methanolic potassium hydroxide to form the potassium salt, dried under nitrogen, redissolved in sterile water at 60°C, passed through a 0.22-µm sterile filter (Millex-Or), and added, and thereby complexed, to sterile 20% (wt/vol) human albumin (State Serum Institute, Copenhagen, Denmark).
Enrichments of 13CO2 in expiratory air samples and blood samples were determined by gas chromatography-isotope ratio mass spectrometry (GC-IRMS; Deltaplus, Finnigan MAT, Bremen, Germany). Before injection into the GC, samples were brought to pressure with pure helium, and phosphoric acid was added to the blood samples to release CO2 in gaseous form. Each injection introduced 20 µl of samples into the column in split mode (split ratio 1:2 for breath and 1:5 for blood samples). A fused silica 25 m × 0.32 mm CP-Poraplot Q column (Chrompack, VARIAN, Analytical Instruments, Værløse, Denmark) was used for chromatography. A deactivated, fused silica 5 m × 0.32 mm column coated with cyanophenylmethyl (Chrompack, VARIAN) was used as a postcolumn. Helium was the carrier gas at 1.8 ml/min. Injector and oven temperatures were set at 35 and 30°C, respectively. Derivatization of palmitate to its methyl ester was modified from the method described by Patterson et al. (21). Briefly, heptadecanoic acid was added to plasma samples or blanks and mixed for 10 min. Precipitation of proteins was carried out by adding ice-cold acetone, mixing for 1 min, placing atCalculations
Plasma FA and glucose kinetics.
Enrichments are reported in units of tracer-to-tracee ratio (TTR),
which is defined as TTR = ratiosa ratiobk, where sa is sample and bk is background sample. At
rest, systemic rates of appearance (Ra) and disappearance
(Rd) were calculated using steady-state equations
(39). Non-steady-state equations modified for use with
stable isotopes were applied during exercise (25). The effective volume of distribution was assumed to be 40 ml/kg BM for
palmitate and 165 ml/kg BM for glucose (25). For glucose, however, it did not affect the results whether a distribution volume of
40, 100, or 165 ml/kg BM was applied. Across the exercising leg,
palmitate uptake and release were calculated using the following equations provided by Wolfe (39)
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Background correction. Enrichments of 13CO2 in breath and blood during exercise were corrected for the increase in background enrichment after initiation of exercise observed during background experiments in three subjects. No increase in background enrichment at initiation of exercise was observed for [U-13C]palmitate and [6,6-2H2]glucose.
Acetate correction factor.
The acetate correction factor c was applied when calculating plasma FA
oxidation rates systemically as well as across the exercising leg to
account for the amount of 13CO2 label from
[U-13C]palmitate lost by fixation at any step between the
entrance of labeled acetyl-CoA into the tricarboxylic acid cycle and
recovery in the breath or venous blood, respectively, despite complete oxidation of [U-13C]palmitate (37). A
correction factor of 0.23 at rest and 0.91 during exercise was applied
in the calculation of palmitate oxidation rates. The values of this
factor were based on studies by Schrauwen and colleagues (27,
28) and a study by Van Loon et al. (38), where the
acetate correction factor was determined in endurance-trained males
bicycling at the same relative and absolute workload as the males in
the present study. In one of the studies by Schrauwen et al.
(28), it was shown that 13CO2
label recovery was practically similar in females and males at rest,
whereas males had a slightly higher recovery of
13CO2 label than females during bicycle
exercise at 40-50% O2 peak. However, this gender difference disappeared when correction was made
for oxygen uptake/LBM and respiratory exchange ratio (RER) (28). In the present study, oxygen uptake/LBM and RER were
similar in females and males during exercise, indicating that,
presumably, no gender difference existed in
13CO2 label recovery during exercise.
Therefore, we applied identical acetate correction factors in females
and males.
Indirect calorimetry.
The relative contributions from fat and carbohydrate to the systemic
oxidative metabolism (in percentages of total oxygen uptake) were
estimated from the RER, on the assumption that RER reflected the
systemic nonprotein respiratory quotient (RQ). The following equations
were used, modified from Ref. 6
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Statistics
Data are presented as means ± SE unless otherwise stated. For variables independent of time, an unpaired t-test was performed to test for differences between genders. In a few cases, data suitable for an unpaired t-test were not distributed normally, and a Mann-Whitney rank-sum test was run instead. For variables measured before and after exercise as well as variables measured before and during exercise, a two-way analysis of variance (ANOVA), with repeated measures for the time factor, was performed to test for differences between genders or changes due to time. When a significant main effect of time was found, significant pairwise differences were performed using Tukey's post hoc test. In all cases, a probability of 0.05 was used as the level of significance. ![]() |
RESULTS |
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Workload
Females as well as males completed the 90-min bicycle exercise test at a workload averaging 58 ± 1%Respiratory and Cardiovascular Parameters
Resting femoral venous blood flow was 0.22 ± 0.02 and 0.32 ± 0.05 l/min in females and males, respectively (NS). During exercise, a constant blood flow was observed in the female subjects (averaging 5.6 ± 0.2 l/min). On the other hand, in the male subjects, femoral venous blood flow increased (P < 0.01) continuously from 15 min (5.9 ± 0.2 l/min) to 60 min (6.5 ± 0.3), after which it leveled off (averaging 6.3 ± 0.3 l/min during the last 30 min of exercise). During exercise, a main effect of gender was observed (P < 0.05).No measurable differences in the Hct of femoral arterial and venous blood were observed at rest or during exercise (P > 0.05). At rest, the Hct was 39 ± 1 and 43 ± 1% in females and males, respectively. After 15 min of exercise, an increase (P < 0.001) in Hct was observed to 42 ± 1% in females and 47 ± 1% in males, whereafter Hct decreased slightly but significantly (P < 0.001) to 40 ± 1 and 44 ± 1% at 90 min of exercise in females and males, respectively. A main effect of gender was observed in Hct (P < 0.05).
Resting oxygen uptake across the leg was 13 ± 2 and 20 ± 4 ml/min in females and males, respectively (NS). During exercise, leg oxygen uptake was constant in both groups, averaging 838 ± 48 ml/min and 999 ± 39 ml/min in females and males, respectively (P < 0.05).
Leg RQ increased at initiation of exercise from 0.79 ± 0.01 and 0.80 ± 0.01 at rest to 0.91 ± 0.02 and 0.89 ± 0.02 at 15 min of exercise in females and males, respectively. During the 1st h of exercise, leg RQ was constant, averaging 0.90 ± 0.02 and 0.88 ± 0.02 in females and males, respectively, but then decreased (P < 0.01) to 0.87 ± 0.02 and 0.82 ± 0.01 at 90 min in females and males, respectively. No gender differences were observed in leg RQ (NS).
At rest, RER was similar in females (0.79 ± 0.02) and males
(0.79 ± 0.02) (NS) (Fig. 1). At
initiation of exercise, RER increased (P < 0.001) in
females as well as in males and did not change during the 1st h of
exercise (averaging 0.89 ± 0.02 and 0.91 ± 0.01 in
females and males, respectively). However, from 60 to 90 min, RER
decreased significantly to 0.87 ± 0.02 in females and 0.88 ± 0.01 in males. No gender difference was observed in RER during
exercise at any time point (NS).
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Metabolite Concentrations
The arterial concentrations of blood glucose, plasma FA, and glycerol are shown in Fig. 2. After an increase (P < 0.001) in arterial blood glucose concentration (Fig. 2A) at initiation of exercise, it did not change significantly from 15 to 30 min. During the next 30 min, arterial glucose concentration decreased (P < 0.05) to a lower level, which was maintained throughout exercise.
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During the first 15 min of exercise, arterial plasma FA concentration decreased in males (P < 0.05) but not in females (NS) (Fig. 2B). Then, it increased (P < 0.01) continuously until the end of exercise in males, whereas a borderline significant increase (P = 0.06) was observed in females.
At initiation of exercise, an increase (P < 0.001) was observed in arterial plasma glycerol concentration (Fig. 2C), followed by a further significant increase throughout exercise.
The arterial blood lactate concentration averaged 0.5 ± 0.1 mM at rest in both groups. During exercise, small increases to a maximum value of 1.1 ± 0.2 mM after 15 min were observed in both groups.
There were no significant gender differences in arterial glucose, plasma FA, glycerol, or lactate concentrations during exercise.
Glucose Kinetics
The enrichment of [6,6-2H2]glucose in arterial plasma is shown in Fig. 3A. The [6,6-2H2]glucose enrichment did not change significantly during the first 75 min of exercise, whereafter it decreased from 75 to 90 min (P < 0.01). Furthermore, a main effect of gender was observed (P < 0.05).
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The systemic glucose Ra and Rd are provided in
Table 2 and were similar in females and
males at rest (NS), but lower in females than in males during exercise
(P < 0.05).
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At initiation of exercise, the glucose net uptake across the leg (Fig.
4A) increased
(P < 0.001). However, throughout exercise, it
did not change significantly, averaging 114 ± 24 and 110 ± 21 µmol · kg lean leg mass
(LLM)1 · min
1 during the last hour
of exercise in females and males, respectively. No significant gender
differences were observed at rest or during exercise.
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Plasma FA Kinetics
The enrichments of [U-13C]palmitate in plasma and 13CO2 in the expiratory air and blood at rest and during exercise are shown in Fig. 3. The only significant changes in the enrichments during the last 60 min of the exercise period occurred in the femoral venous enrichment of [U-13C]palmitate, which decreased slightly from 30 min (P < 0.001) and 60 min (P < 0.05) to 90 min; the enrichment of 13CO2 in the expiratory air, which increased slightly from 30 to 60 min (P < 0.01), 75 min (P < 0.01), and 90 min (P < 0.001); and the femoral arterial enrichment of 13CO2, which increased slightly from 30 to 90 min (P < 0.05). No significant gender differences were observed in the enrichments of [U-13C]palmitate and 13CO2.The contribution of palmitate to the total plasma FA concentration was similar in arterial and femoral venous blood, averaging 27.8 ± 2.6% in females and 34.5 ± 5.2% in males. The gender difference was not statistically significant.
The systemic plasma FA turnover and oxidation at rest and during the last hour of exercise are shown in Table 2. These kinetic parameters were not significantly different between females and males.
Across the leg, a net release of FA to plasma was observed at rest in
females as well as in males (Fig. 4B). From the initiation of exercise, a net uptake of plasma FA was observed across the leg,
which did not change significantly throughout exercise, averaging 79 ± 19 µmol/min in females and 158 ± 40 µmol/min in
males during the last hour of exercise (P < 0.05).
However, when expressed relative to LLM, no significant gender
difference was observed in leg plasma FA net uptake (averaging 9.1 ± 2.6 and 14.2 ± 3.8 µmol · kg
LLM1 · min
1 during the last hour of
exercise in females and males, respectively, Fig. 4B).
The plasma FA total uptake and oxidation across the active muscles were
similar in females and males (NS), whereas females had a higher release
of FA to plasma (P < 0.01) (Table
3).
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Muscle Samples
Muscle tissue samples were obtained from only six of the female subjects. Glycogen concentrations in vastus lateralis muscle before exercise were not significantly different between females and males (Fig. 5A). At termination of exercise, a significant decrease (P < 0.001) in glycogen concentration was observed in both groups. No significant gender difference was observed after exercise, and the glycogen utilization was similar in the two groups (NS).
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Concentrations of MCTG in vastus lateralis before exercise were higher in females than in males (P < 0.05) (Fig. 5B). At termination of exercise, a decrease (P < 0.01) in MCTG concentration was observed in females; however, no significant change occurred in the males. Thus the change in MCTG concentration during exercise was significantly different between females and males.
Substrate Utilization Across the Leg
On the basis of the substrate utilizations across the leg provided above, relative contributions to the oxidative metabolism across the leg during 90 min of exercise derived from blood glucose, plasma FA, muscle glycogen, and MCTG were estimated (Fig. 6).
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The active muscle mass in one of the exercising legs was estimated from the decrease in glycogen concentration in vastus lateralis during exercise and the glycogen oxidation during exercise. The glycogen oxidation was calculated as total leg carbohydrate oxidation estimated from indirect calorimetry minus glucose uptake across the leg. The active muscle mass per leg was 3.3 ± 0.3 and 4.3 ± 0.2 kg in females and males, respectively (P < 0.05).
Total oxygen uptake across the leg during exercise was 72.3 ± 4.5 and 87.9 ± 2.9 l O2 in females and males, respectively (P < 0.01). The relative contribution from blood-borne glucose and muscle glycogen, respectively, averaged 13.6 ± 1.5 and 48.5 ± 4.9% in females and 14.3 ± 1.5 and 42.8 ± 2.1% in males. Thus, in females, carbohydrates accounted for 62.1 ± 4.7% of the oxidative metabolism, whereas in males carbohydrates accounted for 57.1 ± 3.3%. Plasma FA (isotopically determined oxidation) and MCTG, respectively, contributed 12.0 ± 2.5 and 25.0 ± 6.0% to the oxidative metabolism in females and 9.6 ± 1.5 and 5.0 ± 7.3% in males. Thus measurable lipid utilization contributed 37.0 ± 6.4 and 14.6 ± 6.7% to the oxidative metabolism in females and males, respectively. The relative contribution from MCTG to the oxidative metabolism as well as the total measurable lipid utilization differed between females and males (P < 0.05 for both). None of the other energy sources differed significantly between females and males in their relative contribution to the oxidative metabolism during the bicycle exercise test.
In females, 0.9 ± 6.1% of the total oxygen consumption across the leg could not be accounted for as measurable substrate utilization. In males, the amount of oxygen that could not be accounted for amounted to 28.3 ± 6.5% of the total oxygen consumption across the leg. Thus the amount of oxygen not accountable for differed between females and males (P < 0.01).
Hormones
At initiation of exercise, the arterial insulin concentration did not change significantly, but it decreased (P < 0.001) continuously from 30 to 90 min of exercise in females as well as in males (Fig. 7A). The arterial epinephrine concentration did not change significantly from rest to exercise, but it increased (P < 0.01) continuously from 30 to 90 min of exercise in both females and males (Fig. 7B). The norepinephrine concentration increased (P < 0.001) from rest to exercise and did not change significantly during the exercise period (Fig. 7C). No gender differences were observed at any of the time points in plasma concentrations of insulin, epinephrine, and norepinephrine (NS).
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DISCUSSION |
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The present study revealed an equal relative contribution from carbohydrates and lipids as fuel across the leg (as estimated from leg RQ) during submaximal prolonged bicycle exercise at the same relative workload in endurance-trained females and males. However, a marked gender difference in the utilization of the different lipid sources was observed. Thus females degraded MCTG during exercise, whereas males did not. Furthermore, in females, the oxygen uptake across the leg for lipid oxidation was accounted for solely by MCTG and plasma FA oxidation. On the other hand, in males, MCTG and plasma FA oxidation did not cover the total amount of oxygen uptake for lipid oxidation, indicating that males oxidized additional lipid sources. Finally, we observed that females released a significantly higher amount of FA to plasma across the leg during exercise than did the male subjects.
The finding that RER was also similar in females and males is in line with some previous studies (3, 17, 26) but in contrast to others (7, 11, 19, 34, 36), where females utilized more lipids than males during exercise. In the present study, females were all tested in the midfollicular phase of their menstrual cycle, and all subjects ingested a well controlled experimental diet for 8 days before the main exercise experiment. Besides the menstrual status of the females and the dietary status of the subjects, a possible gender difference in the relative oxidation of carbohydrates and lipids might be influenced by the type and intensity of exercise, which differ markedly among some of the previous studies (3, 7, 17, 36).
It might be argued that the lack of a gender difference in the relative contribution from carbohydrates and lipids to the oxidative metabolism in the present study might be ascribed to the relatively small number of subjects. However, we have recently found similar RER during exercise in a larger number of females (n = 20) and males (n = 21), including both trained and untrained subjects (33). Moreover, we have also recently observed that breakdown of MCTG in females is independent of training status (33). Thus the conclusions reached in the present study are not to any major extent dependent on the training status of the subjects.
Lipid Sources Utilized
In the present study, males had a significantly higher net uptake of plasma FA across the leg during exercise than females despite an equal arterial concentration and delivery of plasma FA in females and males. However, when expressed relative to LLM, no gender difference was observed in net plasma FA uptake, indicating that the amount of muscle mass involved during exercise is responsible for plasma FA net uptake. Moreover, we observed that the systemic turnover of plasma FA, determined by tracer technique, was similar in females and males when expressed relative to LBM. The arterial glycerol and FA concentrations, where no gender difference was observed, are also in line with the finding of similar systemic plasma FA turnover in females and males. Expressing systemic plasma FA turnover relative to BM in the present study (data not shown) did not result in a gender difference either, supporting previous studies (3, 7). However, it is probably more correct to express plasma FA turnover relative to LBM to correct for the possible influence of the apparent gender difference in body composition on the turnover of plasma FA. Across the exercising leg, the total uptake and oxidation of plasma FA were similar in females and males, whereas the release of FA to plasma was higher in females than in males. The observation of a partial gender difference in plasma FA kinetics across the exercising leg is in immediate contrast to findings in a recent study by Burguera et al. (3), where the plasma FA total uptake and release across the leg were similar in females and males during exercise. However, there might be no contradiction between the two findings, because in the study by Burguera et al., subjects were untrained and bicycle exercise was performed at 45%Even though the amount of plasma FA taken up systemically as well as
across the leg was in the same range as in previous studies (3,
26), only 32-34% of the systemic plasma FA total uptake occurred across the two legs. This is in accord with previous observations in our laboratory, where 40-50% of the systemic
plasma FA total uptake occurred across the legs during the last
30 min of 1-h bicycle exercise at 70%
O2 peak in moderately trained
males (10). That such a relatively small fraction of the
systemic plasma FA total uptake occurs in the active muscles implies
that more than one-half of the systemic plasma FA total uptake would
presumably occur in adipose tissue, liver, heart, and accessory as well
as in inactive muscles during exercise, which may seem unlikely. At
present, we are not able to explain these observations. A possibility
to consider is contamination of the sampled femoral venous blood with
blood draining tissues other than skeletal muscle, e.g., subcutaneous
adipose tissue, which would have a high concentration of FA. Therefore,
even if the contamination in terms of blood volume were small compared with the large flow perfusing the active muscles, the femoral venous
concentration of FA might be artificially increased. Still, blood from
adipose tissue would be expected to have a rather low level of
enrichment with [13C]palmitate, since the FA would
be derived from intracellular, mainly unlabeled triacylglycerols.
Thus the arteriovenous difference of [13C]palmitate would
be only slightly underestimated if contamination with blood draining
adipose tissue was significant. The effect of such possible
contamination on the calculation of plasma FA total uptake is therefore
expected to be minor. Furthermore, even if contamination were an issue,
it would not to any major extent influence the calculation of leg
plasma FA oxidation and consequently the estimation of relative
utilization of the different energy sources, because plasma FA
oxidation rates across the leg depend primarily on femoral arterial and
venous 13CO2 concentrations. The femoral venous
13CO2 concentration would not be expected to be
changed much by contamination of small volumes of blood from adipose tissue.
At rest only ~40% of systemic plasma FA total uptake was oxidized in
the present study irrespective of gender. This supports previous
studies in resting isolated rat muscle that used the pulse-chase
technique, where a significant incorporation of exogenous palmitate
into MCTG occurred (4, 22). During exercise, however, 86-96% of systemic plasma FA total uptake was oxidized in both females and males according to plasma FA total uptake and oxidation calculated from the tracer technique. This was slightly higher than in
previous studies calculating this percentage in moderately trained
females (5) and males (18). Across the leg in
the present study, 100 and 84% of the plasma FA total uptake was
oxidized in females and males, respectively. In the study by Guo et al. (8), the pulse-chase technique was applied to evaluate the kinetics of plasma palmitate after its entrance into the muscle cell.
The authors showed that a significant incorporation of plasma palmitate
into MCTG in vastus lateralis muscle occurred during exercise at 45%
O2 peak in untrained females and males. The finding of a 100 and 84% oxidation of plasma FA total uptake in
the present study, in contrast to the study by Guo et al., might,
however, be expected, due to the higher workload in the present study.
Furthermore, the higher training level and, consequently, the higher
skeletal muscle oxidative capacity of the subjects in the present study
compared with that in the study by Guo et al., presumably increase
their ability to oxidize a large fraction of the plasma FA taken up
into skeletal muscle rather than incorporate plasma FA into MCTG.
Therefore, it is not at all unexpected that, during exercise at 58%
O2 peak, a very high fraction of the
plasma FA taken up across the leg was oxidized irrespective of gender.
Finally, during submaximal exercise, parts of the motor neurons in the
leg are inactive; therefore, part of the incorporation of plasma FA
into MCTG may occur in nonactive muscle fibers. Obviously, the lower
the relative exercise intensity, the higher the proportion of inactive fibers.
In the present study, females had a significantly higher resting
concentration of MCTG in vastus lateralis muscle than males, and females utilized significant amounts of MCTG during exercise, whereas males did not. This observation has recently been shown in our
laboratory on a larger number of females and males, including both
trained and untrained subjects (33). Other studies where the muscle biopsy technique was applied have also shown that males do
not utilize MCTG during prolonged submaximal exercise (2, 14,
32). Furthermore, Guo et al. (8) observed no change in MCTG palmitate concentration in vastus lateralis during 90-min bicycle exercise at 45% O2 peak in
untrained females and males. A gender comparison was not made in their
study. On the other hand, in a recent study by Romijn et al.
(26), the difference between systemic lipid oxidation
(determined by indirect calorimetry) and systemic plasma FA total
uptake (determined by tracer technique) was similar in females and
males during exercise, leading the authors in that study to conclude
that, during exercise, no gender difference in whole body MCTG
oxidation exists. However, in the present study, the difference between
systemic lipid oxidation (averaging 28.7 ± 4.8 and 23.8 ± 2.5 µmol FA equivalents · kg LBM
1 · min
1 during exercise in
females and males, respectively) and systemic plasma FA oxidation
(averaging 15.1 ± 1.8 and 12.4 ± 3.0 µmol FA
equivalents · kg
LBM
1 · min
1 during exercise in
females and males, respectively) was also similar in females and males
[i.e., a difference of 13.6 ± 4.2 µmol FA
equivalents · kg
LBM
1 · min
1 in females and
11.4 ± 4.7 µmol FA equivalents · kg
LBM
1 · min
1 in males (NS)],
despite the obvious gender difference in MCTG utilization in vastus
lateralis muscle. Therefore, a methodological problem must exist when
indirectly estimating whole body MCTG oxidation as the difference
between systemic lipid oxidation and systemic plasma FA total uptake or
oxidation. As mentioned before, the fact that only one-third of the
systemic plasma FA uptake occurred in the legs during exercise further
indicates that systemic plasma FA uptake is a poor measure of leg
plasma FA metabolism during exercise. Therefore, subtraction of
systemic plasma FA turnover from total lipid oxidation cannot be
expected to produce a reasonable estimate of MCTG breakdown, in accord
with our findings that whole body nonplasma FA oxidation is not
equivalent to MCTG utilization. This point of view has been voiced
before (2, 10). Thus increasing evidence suggests that the
indirect estimation of MCTG utilization by subtraction of systemic
plasma FA turnover from total lipid oxidation is not valid.
Carbohydrate Sources
In contrast to previous studies (17, 26), we observed a higher systemic glucose turnover during exercise in endurance-trained males than in endurance-trained females. Furthermore, we observed a generally higher systemic glucose turnover than Romijn et al. (26), despite comparable workloads and training status of the subjects. These discrepancies are difficult to explain. However, from the data presented it is possible to calculate that 93 and 75% of the systemic glucose total uptake occurred across the two legs during exercise in females and males, respectively, indicating that our determination of systemic glucose turnover was not too high compared with the glucose uptake across the legs. Despite the observed gender difference in systemic glucose turnover expressed relative to LBM, we found a similar glucose net uptake across the leg in females and males expressed in absolute terms (data not shown) as well as relative to LLM. This suggests that males might have a higher total uptake of glucose in adipose tissue, liver, heart, brain, or inactive muscles during exercise compared with females.In the present study, the content of glycogen in vastus lateralis was
similar in females and males before exercise. During exercise, the
utilization of glycogen in vastus lateralis was also similar in females
and males. This is in contrast to the study by Tarnopolsky et al.
(36), where the utilization of muscle glycogen in vastus
lateralis was higher in males than in females. It should, however, be
noted that the study by Tarnopolsky et al., where subjects
completed 95 min of treadmill running at 63% O2 peak, revealed a significantly
higher systemic oxidation of total carbohydrate in males than in
females, thus differing from the conditions in the present study.
Substrate Utilization Across the Leg
The estimated active muscle mass plays an important role when the total utilization of muscle glycogen and MCTG is calculated. The estimation of active muscle mass is based on the assumptions that the difference in glycogen concentration between the two muscle tissue samples obtained before and after exercise, respectively, is representative of all active leg muscle tissue and that all of the glucose taken up across the leg during exercise is oxidized (12). Therefore, the estimation of active muscle mass is subject to potentially large variation, which might influence the variability especially of the total MCTG utilization and therefore also the amount of oxygen not accountable for as utilized substrate. For instance, an active muscle mass 0.5 kg (12%) higher than originally estimated in the male subjects would change the contributions from blood glucose, glycogen, plasma FA, and MCTG to the oxidative metabolism across the leg to 14, 53, 10, and 6%, respectively, decreasing the percentage of oxygen uptake across the leg not accountable for as substrate utilized from 28 to 17%. Despite these considerations, the MCTG utilization (25.0 ± 6.0 and 5.0 ± 7.3% of total oxygen uptake in females and males, respectively) and the sum of plasma FA and MCTG utilization (37.0 ± 6.4 and 14.6 ± 6.7%) differed significantly between females and males. On the other hand, the utilization of blood glucose (13.6 ± 1.5 and 14.3 ± 1.5%) and glycogen (48.5 ± 4.9 and 42.8 ± 2.1%) was similar in females and males. Finally, a significant gender difference was also observed in the percentage of oxygen uptake across the leg not accountable for as substrate utilized (0.9 ± 6.1 and 28.3 ± 6.5%). It might be speculated that the unaccountable oxygen consumption in males originated from oxidation of VLDL-TG or TG located between the muscle fibers, as has been suggested earlier (9, 12). Furthermore, a contribution from protein to the oxidative metabolism during exercise should not be disregarded (12) and seems to be larger in males than in females (23).In conclusion, in the present study, the oxidative metabolism during prolonged submaximal exercise was evaluated in endurance-trained males compared with endurance-trained females in their midfollicular phase of the menstrual cycle. Despite similar relative contributions from carbohydrates and lipids to the oxidative metabolism during exercise at the same relative workload in endurance-trained females and males, we observed a gender difference in the relative utilization of the different lipid sources. Thus, although the plasma FA oxidation across the exercising leg was similar in females and males, MCTG was significantly degraded in females but not in males. In females, measured substrate oxidation accounted for 99% of the leg oxygen uptake, whereas in males, 28% of leg oxygen uptake was unaccounted for in terms of measured oxidized lipid substrates. These findings thus may indicate that males utilized a third lipid source, presumably VLDL-TG or TG located between muscle fibers.
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ACKNOWLEDGEMENTS |
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We are thankful to Gerrit van Hall for support and advice on analysis and calculation of stable isotope tracer data. Furthermore, we acknowledge the skilled technical assistance of Irene Bech Nielsen, Nina Pluszek, Betina Bolmgren, and Winnie Taagerup.
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FOOTNOTES |
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This study was supported by the Danish National Research Foundation, Grant 504-12, and by the Danish Sports Research Council.
Address for reprint requests and other correspondence: Carsten Roepstorff, Copenhagen Muscle Research Centre, Dept. of Human Physiology, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark (E-mail: croepstorff{at}ifi.ku.dk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00266.2001
Received 20 June 2001; accepted in final form 5 October 2001.
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