Substrate metabolism during different exercise intensities in endurance- trained women

Abstract

We have studied eight endurance-trained women at rest and during exercise at 25, 65, and 85% of maximal oxygen uptake. The rate of appearance (R(a)) of free fatty acids (FFA) was determined by infusion of [(2)H(2)]palmitate, and fat oxidation rates were determined by indirect calorimetry. Glucose kinetics were assessed with [6,6-(2)H(2)]glucose. Glucose R(a) increased in relation to exercise intensity. In contrast, whereas FFA R(a) was significantly increased to the same extent in low- and moderate-intensity exercise, during high-intensity exercise, FFA R(a) was reduced compared with the other exercise values. Carbohydrate oxidation increased progressively with exercise intensity, whereas the highest rate of fat oxidation was during exercise at 65% of maximal oxygen uptake. After correction for differences in lean body mass, there were no differences between these results and previously reported data in endurance-trained men studied under the same conditions, except for slight differences in glucose metabolism during low-intensity exercise (Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E, and Wolfe RR. Am J Physiol Endocrinol Metab 265: E380-E391, 1993). We conclude that the patterns of changes in substrate kinetics during moderate- and high-intensity exercise are similar in trained men and women.

Full text

Substrate metabolism during different exercise
intensities in endurance-trained women
J. A. ROMIJN,
1
E. F. COYLE,
2
L. S. SIDOSSIS,
3,4
J. ROSENBLATT,
3,4
AND R. R. WOLFE
3,4
1
Department of Endocrinology and Metabolism, Leiden University Medical Center,
2300 RC Leiden, The Netherlands;
2
Human Performance Laboratory,
Department of Kinesiology and Health, The University of Texas at Austin, Austin 78712;
3
Metabolism Unit, Shriners Burns Institute, and
4
Departments of Anesthesiology
and Surgery, University of Texas Medical Branch, Galveston, Texas 77550
Romijn, J.A., E. F. Coyle, L. S. Sidossis, J. Rosenblatt,
and R. R. Wolfe. Substrate metabolism during different
exercise intensities in endurance-trained women. J Appl
Physiol 88: 1707–1714, 2000.—We have studied eight endur-
ance-trained womenatrestand during exercise at25,65,and
85%ofmaximal oxygenuptake.The rateof appearance(R
a
)of
free fatty acids (FFA) was determined by infusion of
[
2
H
2
]palmitate, and fat oxidation rates were determined by
indirect calorimetry. Glucose kinetics were assessed with
[6,6-
2
H
2
]glucose. Glucose R
a
increased in relation to exercise
intensity. In contrast, whereas FFA R
a
was significantly
increased to the same extent in low- and moderate-intensity
exercise, during high-intensity exercise, FFA R
a
was reduced
comparedwiththeotherexercisevalues.Carbohydrateoxida-
tion increased progressively with exercise intensity, whereas
the highest rate of fat oxidation was during exercise at 65% of
maximal oxygen uptake. After correction for differences in
lean body mass, there were no differences between these
results and previously reported data in endurance-trained
men studied under the same conditions, except for slight
differences in glucose metabolism during low-intensity exer-
cise (Romijn JA, Coyle EF, Sidossis LS, GastaldelliA, Horow-
itz JF, Endert E, and Wolfe RR. Am J Physiol Endocrinol
Metab 265: E380–E391, 1993).We conclude that the patterns
of changes in substrate kinetics during moderate- and high-
intensity exercise are similarin trained men and women.
stable isotopes; body composition; glucose; glycogen; fatty
acids
THERE IS A COMMON PERCEPTION that women may be
relatively more suited to endurance exercise than men
because of a greater ability to use fatty acids as energy
substrates during exercise. This notion is supported by
several studies that have found some index of fat
metabolism to be greater in women than in men at
comparable exercise intensity (e.g., Refs. 7, 22). On the
other hand, several other studies have failed to show a
gender difference in substrate metabolism (3, 23). It
has been suggested that the difference in substrate
metabolism between men and women during exercise
becomes less as training status increases (19), but all
data are not consistent with this explanation. For
example, endurance-trained women were found to de-
rive more energy from lipids than endurance-trained
men did when exercising at 65% of maximal oxygen
consumption (V
˙
O
2max
) (21).
If women are in fact better able to rely on fat during
exercise than men, it may be because women generally
have a higher percentage of body fat than men. This
could theoretically lead to greater availability of fatty
acids during exercise and, inturn, to more fat oxidation
at a given exercise intensity than in a man with less
body fat. In this regard, changes in fatty acid availabil-
ity have been shown to have an effect on fatty acid
oxidation during high-intensity exercise (16). On the
other hand, it may be erroneous to assume that women
have an increased rate of lipolysis because of their
greater amount of body fat. Our laboratory has previ-
ously shown that changes in the percentbody fat result
in altered lipolytic sensitivity that makes individuals
with low body fat better able to mobilize fatty acids
than might be anticipated otherwise. Thus lipolytic
responsiveness is enhanced in subjects with low body
fat (11) and decreased inobese subjects (29).Consistent
with these observations, our laboratory found that
highly trained athletes had higher rates of lipolysis at
rest (18) than untrained controls did. However, it is not
clear whether this reflects a response to training, a
change in body composition, or some other factor. In
trained men, it is difficult to distinguish a training
effect on fat metabolism from a body-composition effect
because body fat is low in all endurance-trained men.
This is not necessarily the case in endurance-trained
women, who may have a relatively large range in body
composition. The purpose of the present study was
therefore twofold: 1) to compare with the previously
reported data from endurance-trained men (15) the
response of glucose and lipid kinetics in endurance-
trained women to three intensities of exercise and 2)to
evaluate the role of bodycomposition on the response of
substrate kinetics to exercise in the trained women. To
accomplish these goals, we have quantified glucose and
fatty acid kinetics by using stable isotopically labeled
tracers at three intensities of exercise in eight endur-
ance-trained women with a range in body composition
from 7.7 to 26.9% body fat.
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 thisfact.
J Appl Physiol
88: 1707–1714, 2000.
8750-7587/00 $5.00 Copyright
r
2000 the American Physiological Society 1707http://www.jap.org

METHODS
Subjects
We studied eight well-trained female cyclists with percent
body fat ranging from 7.7 to 26.9%. Their physical character-
istics are shown in Table 1. All subjects were healthy, as
indicated by medical history and physical examination. They
were consuming a weight-maintaining diet containing at least
300–400 g of carbohydrates daily. They were in energy balance,
documented by their stable weight in the 2 mo preceding the
study. They were studied in the postabsorptive state after a
10- to 12-h fast and did not exercise the day before the
studies.Thesubjects wereeumenorrheicand studied through
a variety of phases of the menstrual cycle. The men we used
for comparison were describedpreviously (15).
Body composition was determined by densitometry by
using the hydrostatic weighing technique with correction for
residual lung volume (25). Relative body fat was estimated
from total body density (26). Assessment of body composition
was performed within 1wk of the isotope-infusion protocol.
V
˙
O
2max
had been determined several weeks before the
present protocol while the subjects cycled on a stationary
ergometer (model 819, Monark). V
˙
O
2max
was determined
during an incremental cycling protocol lasting 7–10 min. The
study was approved by the institutional review boards of the
University of Texas, Galveston andAustin.
Exercise Protocol
The subjects were studied on 2 consecutive days in the
postabsorptive state. On one occasion, the protocol consisted
of at least 120 min of bed rest, 60 min of exercise at 25% of
V
˙
O
2max
on a stationary ergometer (model 819, Monark),
followed by 60 min of bed rest, and, finally, 30 min of exer-
cise at 85% of V
˙
O
2max
. We have previously shown that the
effects of very-low-intensity exercise (120 min at 25% of
V
˙
O
2max
) on glucose and fat metabolism subside within1hof
bed rest (15). On the other day, the protocol consisted of at
least 120minof bed rest followedby60min of exercise at65%
of V
˙
O
2max
. The order of the two protocols was determined by
random allocation.
Indirect Calorimetry
Indirect calorimetry was performed at rest for at least 15
min continuously during the first 30 min of exercise (all 3
levels of exercise) and for 5–10 min at 15-min intervals
throughout the remainder of the exercise periods at 25 and
65% of V
˙
O
2max
. The values obtained from 20 to 30 min of
exercise were used to calculate and compare substrate oxida-
tion rates among the three levels of exercise. Our laboratory
(14) has previously shown that indirect calorimetry is a valid
method to measure substrate oxidation rates in trained
subjects during cycling exercise at 80–85% of V
˙
O
2max
because
ofthe relativephysiological state(i.e., constantlactate concen-
tration, heart rate, and so forth) over this time interval. The
restingvalueswere obtained afterthesubjects hadbeenlying
on a bed for at least 1 h. Inspired volumes of air were
measured with a dry-gas meter (model CD-4, Parkinson-
Cowan). Aliquots of expired gas were sampled from a mixing
chamber for O
2
(model S3A, Applied Electrochemistry) and
CO
2
(model LB-2, Beckman). Analog outputs from the gas
analyses and gas meter were directed to a laboratory com-
puter for calculation of oxygen uptake and carbon dioxide
production.
Isotope Infusion
Teflon catheters were placed percutaneously in an antecu-
bital vein, and a sampling catheter was inserted in a dorsal
hand vein of the contralateral side. The heated hand tech-
nique was used to obtain arterialized blood samples. The
subjects lay on a bed for 1 h after catheter placement. Then,
after a blood sample was drawn to determine background
enrichment, primed, constant infusions of [6,6-
2
H
2
]glucose
(99% enriched, Merck, Rahway, NJ; 0.22 µmol·kg
21
·min
21
;
priming dose 17.6 µmol/kg) and [
2
H
2
]palmitate (0.04 µmol·
kg
21
·min
21
; no priming dose) were started by using cali-
brated syringe pumps (HarvardApparatus, Natick,MA). The
exact infusion rates in each experiment were determined by
measuring the concentrations in the infusates. The palmitate
tracer (99% enriched) was purchased from Tracer Technolo-
gies (Newton, MA). Palmitate was bound to albumin (Cutter
Biological, Berkeley, CA) by following previously described
procedures (27). After2hofinfusion at rest, exercise was
started and the rate of isotope administration was doubled
for palmitate (25 and 65% of V
˙
O
2max
) and glucose (65% of
V
˙
O
2max
) and tripled for glucose (85% of V
˙
O
2max
) to minimize
changes in substrate isotopic enrichment. These changes in
isotope infusion rates were determined from results of a
previousstudyon metaboliceffectsof similarexerciseintensi-
tiesinmen (15).Duringthe intervalbetween theexperiments
with exercise at 25 and 85%, the isotope infusions were
continued at the basalrates.
Blood Sampling
The first blood samples were drawn before the isotope
infusion was started to determine background enrichment.
Blood was also taken 110, 115, and 120 min after the
beginning of infusion to measure resting kinetics. During
high-intensity exercise, blood was drawn after 5, 10, 15, 20,
25, and 30 min of exercise. In moderate- and low-intensity
exercise, samples were drawn every 10 min for 60 min. All
samples were collected in 10-ml vacutainers containing
lithium heparin and were placed on ice. Plasma was sepa-
rated by centrifugation within 5 min of sampling and subse-
quently frozen until furtherprocessing.
Sample Analysis
Plasma glucose concentration was measured on a glucose
analyzer (Beckman Instruments) by use of the glucose oxi-
dase method. The enrichment of [6,6-
2
H
2
]glucose in plasma
was determined as previously described (27). Briefly, plasma
was deproteinated with barium hydroxide and zinc sulfate
solutions. Glucose was extracted by mixed-bed anion-cation
exchange chromatography (AG-1-X8 and AG 5OW-X8, Bio-
Rad Laboratories, Richmond, CA) and reacted with acetic
anhydride and pyridine to form the pentaacetate derivative.
Isotopic enrichment was determined by using gas chromato-
Table 1. Physical characteristics of study subjects
Subject
No.
Age,
yr
Weight,
kg
Height,
m
Weight,
kg
Fat,
%body
weight
V
˙
O
2max
,
l/min
1 31 48 1.65 44 7.7 2.75
2 27 49 1.63 42 13.4 2.75
3 29 55 1.60 47 14.8 3.30
4 26 63 1.66 52 17.8 3.60
5 28 64 1.65 53 18.4 3.83
6 25 69 1.78 55 20.5 3.90
7 29 65 1.65 51 21.6 4.15
8 18 72 1.77 53 26.9 3.65
Mean6SE 276 16163 1.6760.02 5062 17.662 3.496 0.2
V
˙
O
2max
, maximal oxygen uptake.
1708 SUBSTRATE METABOLISM IN EXERCISING WOMEN

graph-mass spectrometry (model 5985B, Hewlett-Packard,
Fullerton, CA) with electronic-impact ionization, selectively
monitoring ions at mass-to-charge ratio (m/z) 202, 201, and
200. Correction was made for the contribution of singly
labeled molecules (m/z 201) to the apparent enrichment at
m/z 202 (27).
FFA were extracted from plasma, isolated by thin-layer
chromatography, and derivatized to their methyl esters.
Palmitate and total free fatty acid (FFA) concentrations were
determined by gas chromatography (model 5890, Hewlett-
Packard)byusing heptadecanoicacidas an internalstandard
(27). Isotopic enrichment of palmitate was measured by gas
chromatograph-mass spectrometry analysis of the methyl
ester derivatives (model 5992, Hewlett-Packard). Ions of m/z
270 and 272 wereselectively monitored.
Calculations
Indirect calorimetry. Carbohydrate and fat oxidation rates
were calculated by using stoichiometric equations (4). Nitro-
gen excretion rate was assumed to be 135 µg·kg
21
·min
21
.
This average value was taken from the measured values
determined in another study performed in our laboratory (1).
A 30% error in this assumed value (which exceeds the total
range of values in the previous study) would have no signifi-
cant effect on the calculated values of fat and carbohydrate
oxidation in exercise in the present study. Fatty acid oxida-
tion was determined by converting the rate of triglyceride
oxidation (g·kg
21
·min
21
) to its molar equivalent, assuming
the average molecular mass of triglyceride is 860 g/mol (4)
and multiplying the molar rate of triglyceride oxidation by
three because each moleculecontains 3 mol of fatty acids.
Rates of appearance. Rate of appearance (R
a
) and, when
appropriate, rate of disappearance (R
d
5 tissue uptake) of
glucose and palmitate at rest were calculated by using the
equation of Steele (20), as modified for use with stable
isotopes (27). During exercise and the first 30 min ofrecovery,
the non-steady-state approximation of Steele was used in
conjunction with a spline-fitting program to smooth the raw
data(24).The effectivevolume of distributionwasassumed to
be165ml/kg forglucose and40ml/kg forpalmitate. Thevalue
for palmitate was chosen because acute changes in palmitate
concentration are essentially restricted to plasma (because
FFA are bound to albumin). The FFA R
a
was calculated by
dividing the palmitate R
a
by the fractional contribution of
palmitate to the total FFA concentration, as determined by
gas chromatography.
Statistical Analysis
The results obtained during 20–30 min of exercise were
used for comparison of the subjects of the three levels of
exercise. The effect of time on the response within each
exercise level was analyzed by two-way analysis of variance
for a randomized block, with the subjects as blocks and time
as treatment. If necessary, the time effects were compared by
Fisher’s least significant difference test. The results of the
three exercise intensifies were compared by two-way analysis
ofvarianceforrandomized block design,inwhich the subjects
are blocks and the three exercise levels are treatments. If
necessary, the analysis of variance wasfollowedbyamultiple
comparison to detect differencesamong groups.
To analyze the effects of body composition and gender on
different metabolic responses (e.g., glucose production) we
used the repeated-measures analysis of covariance. Letting
Y
s,ef
denote the measured metabolic response from subject(s)
[person(s)] exercising at intensity level e, with body composi-
tion parameter f, the model is Y
s,ef
5 S
s
1 E
e
1 f(FE)
e
1
error
s,ef
, in which the error terms are assumed to be indepen-
dent, identically distributed, normal random variables with
mean value 0 and common SD
e
. The body composition factor
was either fat mass (FM), fat free mass (FFM), or the ratio
FM/FFM. Thus the repeated-measures aspect is included via
the subject effect S
s
, and the effect of body composition is
accounted for by the f(FE) term. To ensure unique estimates,
the baseline constraints E
1
5 0 and (FE)
1
5 0 were imposed.
In this case, E
e
and (FE)
e
represent the rise over baseline
levels of intercept and slope, respectively. The effect of body
composition at baseline, along with any other baseline vari-
abilityassociatedwithbodycompositionorotherfixedcharac-
teristics of subjects, is embodied in the S
s
. If the body
composition were the only such factor, the values of the S
s
would specify the average effect of body composition on the
baseline metabolic response. Simultaneously, 0.95level confi-
dence intervals for the parameters E
e
and (FE)
e
were gener-
ated in association with a repeated-measures analysis of
covariance F-test of the hypothesis H
o
:E
e
5 0, (FE)
e
5 0, e 5
2,3,4. Data fromaprevious paper (15)fromour laboratory, in
which the exact same experimental procedures were used in
trained male subjects, were used for evaluation of gender
effect.
RESULTS
Resting State
At rest, there were no differences in concentrations,
R
a
, or oxidation rates of FFAor glucose before the three
levels of exercise. Themeanvalues are showninTable 2.
Exercise
Lactate concentrations. Lactateconcentrationdid not
change during low-intensity exercise and increased
from 0.84 6 0.03 to 1.68 6 0.18 mmol/l (P , 0.05)
during moderate exercise intensity. During high-
intensityexercise,lactateconcentrationswereconsider-
ablyhigher,but,nonetheless,physiologicalsteadystate
was maintained. This was reflected in plasma lactate
concentrations over the last 15 min of exercise (0 min:
0.84 6 0.03, 15 min: 7.92 6 0.17, 30 min: 7.30 6 0.71
mmol/l).
Glucose metabolism. Tracer-to-tracee ratios and sub-
strate kinetics are shown in Figs. 1 and 2, respectively.
During low-intensity exercise, plasma glucose concen-
tration and glucose R
a
did not change from the resting
values. In contrast, during moderate- and high-intensity
exercise, plasma glucose concentration and glucose R
a
increased significantly in relation to exercise intensity
(Tables 2 and 3).
FFAmetabolism.FFAconcentrationsincreasedgradu-
ally during low-intensity exercise, whereas they tran-
siently decreased and subsequently increased during
moderate exercise intensity [not significant (NS) vs.
low-intensity exercise]. During high-intensity exercise,
FFA concentrations were considerably decreased com-
pared with the values obtained at rest and during
low-intensity exercise (P, 0.05; Table 3). Palmitate R
a
,
FFAR
a
, and FFAuptake were significantly increased to
the same extent in low- and moderate-intensity exer-
cise(Fig.2,Tables2and3).Duringhigh-intensityexercise,
palmitate R
a
,FFAR
a
and FFA uptake were significantly
1709
SUBSTRATE METABOLISM IN EXERCISING WOMEN

lower than the values during the two lower exercise
intensities (P, 0.05; Fig. 2, Tables 2 and 3).
Substrate oxidation rates. Substrate oxidation rates
are given in Tables 2 and 3. During moderate-intensity
exercise, carbohydrate oxidation rates increased above
the value during low-intensity exercise, and carbohy-
drate oxidation was the highest during high-intensity
exercise. The highest rate of fat oxidation was during
exercise at 65% of V
˙
O
2max
(Table 3), whereas there was
no significant difference between low- and high-intensity
exercise.
During exercise at 25% of V
˙
O
2max
, FFA uptake and
FFA oxidation rates, expressed in fatty acid equiva-
lents, were similar (Table 2), indicating that plasma
could transport adequate amounts of FFAfor oxidation
from adipose tissue. However, during moderate and
high exercise intensity, fatoxidation ratesexceeded the
maximal amounts that could be obtained from plasma,
the differencebeing the minimal contribution of muscle
triglycerides toenergy requirements (Fig. 3). From Fig.
3,itisalsoevident thatthemaximalcombinedcontribu-
tion of plasma glucose and FFA to oxidation rates
hardly differs between the three levels of exercise.
Apparently, the decrease in FFA R
d
during high-
intensity exercise more or less balances the increase in
glucose R
d
.
Relationship of Results to Gender
Table 2 shows the comparison between the women
and the previously described men. V
˙
O
2max
, expressed
per kilogram of lean body mass, was similar in both
groups. In addition, respiratory exhange ratio (RER)
values obtained during different exercise intensifies
were not different. Despite decreased glucose R
d
and
increased rate of carbohydrate oxidation during low-
intensity exercise in the women, there were no differ-
ences in glucose and fat metabolism between men and
women after correction for differences in lean body
mass. The minimal contribution of muscle triglycerides
to energy requirements were 0 vs. 9% (men vs. women)
at 25% of V
˙
O
2max
(P , 0.05), 23 vs. 25% at 65% ofV
˙
O
2max
(NS) and 11 vs. 17% at 85% of V
˙
O
2max
(NS). Muscle
glycogen contributed minimally, 0 vs. 9% (men vs.
women) at 25% of V
˙
O
2max
(P , 0.05), 41 vs. 34% at 65%
of V
˙
O
2max
(NS), and 63 vs. 58% at 85% of V
˙
O
2max
(NS).
DISCUSSION
The results of this study indicate that substrate
metabolisminendurance-trainedwomen respondssimi-
larly to moderate- and high-intensity exercise, as our
laboratory has previously reported in endurance-
trained men (15). A greater relative amount of body fat
in women is a potential basis for expecting a difference
in substrate metabolism between trained men and
women. In this study, we were able to assess the effect
ofbodycompositionon substratemetabolismbycompar-
ing men and women with comparable training status
but with different body composition. Because we found
no metabolic effect of gender, substrate metabolism
during exercise is determined by energy requirements
of lean body mass, rather than by gender or body
composition.
Therewas a slight differenceinstudydesignbetween
the present study and the previous study in men (15).
In the present study in women, exercise at 85% of
V
˙
O
2max
was performed on the same day as the exercise
study at 25% V
˙
O
2max
. The two exercise studies were
separated by an interval of 1 h. In the study in men, we
performed the three exercise protocols on 3 separate
days. One might argue that this difference in design
affects our conclusion as to the comparison of the
results at 85% of V
˙
O
2max
. However, there was no gender
effect after correction for differences in lean body mass.
Moreover, in the study in men, we documented that all
changes induced by exercise at 25% of V
˙
O
2max
subsided
within the first hour of recovery. Finally, exercise at
25% of V
˙
O
2max
in trained subjects only involves cycling
withoutanyresistance. This very low exerciseintensity
is not reflected in any change in glucose R
a
. Therefore,
Table 2. Comparison of anthropometric values and
substrate metabolism at rest and after 20–30 min of
exercise at different exercise intensities in
endurance-trained men and women
Men
(n5 5)
Women
(n5 8)
P
Value
Weight 75.263.6 60.663.2 ,0.02
Height 1.7860.03 1.6760.02 ,0.05
Lean body mass 68.263.3 49.561.6 ,0.01
Fat mass 8.160.2 11.16 1.7 NS
V
˙
O
2max
l/min 5.0160.3 3.560.2 ,0.05
ml/kg lean body mass 73.66 3.5 70.16 2.0 NS
RER
Rest 0.7860.01 0.7660.01 NS
25% 0.7360.01 0.7560.01 NS
65% 0.8160.02 0.8160.01 NS
85% 0.9160.01 0.9060.01 NS
FFAuptake, µmol·kg lean
body mass
21
·min
21
Rest 17.061.4 15.961.9 NS
25% 29.062.9 26.661.6 NS
65% 25.262.5 29.463.0 NS
85% 19.063.7 15.661.5 NS
Fatty acid oxidation,
µmol·kg lean body
mass
21
·min
21
Rest 6.760.5 5.860.5 NS
25% 26.161.6 30.161.7 NS
65% 47.064.6 53.363.5 NS
85% 33.465.0 36.863.9 NS
R
d
glucose, µmol·kg lean
body mass
21
·min
21
Rest 11.56 0.4 11.66 0.4 NS
25% 14.360.4 11.26 0.6 ,0.01
65% 24.561.2 28.562.7 NS
85% 56.962.1 51.7611.7 NS
Carbohydrate oxidation,
µmol·kg lean body
mass
21
·min
21
Rest 6.460.9 6.261.2 NS
25% 11.56 3.4 22.26 3.7 ,0.01
65% 150.9630.0 130.7612.7 NS
85% 331.1617.5 285615.5 NS
Values are means 6 SE; n, no. of subjects. RER, respiratory
exchange ratio; FFA, free fatty acid; R
d
, rate of disappearance; NS,
not significant.
1710 SUBSTRATE METABOLISM IN EXERCISING WOMEN

it seems unlikely that the slight differences in study
design between the two studies affect our conclusion to
a considerable extent.
Several other studies have evaluated the changes in
substrate metabolism in relation to exercise intensity.
Our data extend the observations of Friedlander et al.
(5), which involved the effects of exercise at 52 and
65% of V
˙
O
2max
in women after 8–12 wk of endurance
training. They observed that there was no significant
difference in FFAR
a
between these exercise intensities,
in line with our observation that FFAR
a
is not different
even between 25 and 65% of V
˙
O
2max
in trained women.
Fig. 1. Tracer-to-tracee ratios of palmitate (A) and glucose
(B) at rest and during exercise at 25, 65, and 85% of
maximal oxygen uptake. Values are means 6 SE for 8
women.
Fig. 2. Rates of appearance (R
a
) ofpalmitate (A)and
glucose (B) at rest and during exercise at 25, 65, and
85% of maximal oxygen uptake. Values are means 6
SE for 8 women.
1711SUBSTRATE METABOLISM IN EXERCISING WOMEN

Moreover, our data show for the first time that, in
women, FFAR
a
at 85% of V
˙
O
2max
is much lower and not
different from the values obtained at rest, as occurs in
men. In a second study with a similar design, Fried-
lander et al. (6) demonstrated that glucose R
d
was
directly related to exercise intensity in trained and
untrained women. Our data arein accordance with this
conclusion over a wider range of exercise intensities.
However, there were also quantitative differences be-
tween the studies of Friedlander et al. and the present
study. For instance, FFA R
a
and whole body fat oxida-
tion rates were considerably lower in women in the
study of Friedlander et al. (5) than in our study.
Conversely, glucose R
d
was higher in women after
training in the study of Friedlander et al. (6) than in
our study. These discrepancies are at least in part
related to differences in subject characteristics. For
instance, the women in our study had a lower amount
of body fat than those in the studies of Friedlander et
al. (18 vs. 24%) and a higher V
˙
O
2max
(70 vs. 54 ml·kg
lean body mass
21
·min
21
). These anthropometric values
suggest that the differences in the absolute values
between the studies of Friedlander et al. and our study
are at least in part explained by a differencein training
status.
Several studies have compared substrate metabo-
lism during exercise between men and women. Fried-
lander et al. (6) compared the training-induced alter-
ations of carbohydrate metabolism in men and women.
They found no gender difference in glucose R
a
during
exercise in trained men and women,in accordancewith
our data. Moreover, we found no differences in FFA R
a
between endurance-trained men and women after cor-
rection for differences in lean body mass. In contrast to
our results, however, Friedlander et al. found a gender
differenceinsubstrateoxidation,inthatwomenshowed
a reduction in RER after training, in contrast to men.
In accordance, other authors observed that women
derived more of the total energy expended from fat
oxidation than men did during exercise at 40 or 65% of
V
˙
O
2max
(9, 22). The reason for this discrepancy in the
resultsderivedfromindirectcalorimetry betweendiffer-
ent studies is unclear. Most studies evaluate exercising
women in the midfollicular phase (e.g., Refs. 6, 22),
whereas our study was not controlled for the effects of
the menstrual phase. There are indications that the
menstrual cycle affects the metabolic response to exer-
cise. For instance, lipid oxidation during moderate
exercise intensities ishigher in the midluteal phase(8).
Gender differences may, therefore, not be as clear in
women studied throughout all phases of the menstrual
cycle. During submaximal exercise intensity, this gen-
der difference in lipid oxidation disappeared (8). There-
fore, at higher intensities, it is likely that substrate
selection is governed by the exercise itself rather than
by mitigating factors like menstrual cycle.
In this study, we compared five men with eight
women. In this respect, it is appropriate to evaluatethe
statistical power of the comparisons between both
groups. We calculated the detectable difference be-
tween the means as a function of the a-value of 0.05, a
b-value of 0.80, the number of subjects, and the SD of
the observations. Although the detectable difference
varied somewhat between the different variables, the
mean difference between both groups that could be
detected was 15%. Therefore, we cannot exclude the
possibility that there may have been smaller differ-
ences between both groups, which could have been
detected in a study with a larger number of subjects.
However, it is unlikely that this explains the absence of
a difference in RER values between men and women,
Table 3. Substrate concentrations, kinetics, and
oxidation in women after 30 min of exercise
25%
V
˙
O
2max
65%
V
˙
O
2max
85%
V
˙
O
2max
FFAconcentration,
µmol/l 6986103* 7716102* 359668
FFAuptake,
µmol·kg
21
·min
21
21.661.7* 25.363.1* 12.661.2
Fat oxidation,
µmol·kg
21
·min
21
21.561.6† 43.163.5* 30.163.6
Glucose concentration,
mg/dl 8362*† 9566* 141617
R
d
glucose,
µmol·kg
21
·min
21
9.160.5*† 23.262.0* 41.769.8
Carbohydrate oxida-
tion,
µmol·kg
21
·min
21
18.063.2*† 106.5611.5* 232.16 15.1
Values are means 6 SE for 8 subjects. *P , 0.05 vs. 85% V
˙
O
2max
.
†P , 0.05 vs.65% V
˙
O
2max
.
Fig. 3. Maximal caloric contribution of plasma
free fatty acids (FFA) and glucose and minimal
contribution of muscle triglyceride and glycogen
stores in relation to exercise intensity. Values
represent mean value of 8 endurance-trained
women.
1712 SUBSTRATE METABOLISM IN EXERCISING WOMEN

because these values were almost identical in both
groups.
In light of the lack of a relationship between relative
fat mass and the lipolytic response, as reflected by FFA
R
a
, to exercise, it is appealing to consider the energy
requirements of performing the exercise as the major
determinant of the rate of FFAR
a
, because it is the rate
of energy utilization that determines the requirement
for substrate oxidation. Nonetheless, several aspects of
our data argue againstthis interpretation.Most impor-
tantly, fatty acid uptake did not increase as the energy
requirement increased. In the absence of a change in
uptake, plasma fatty acid concentrations will not de-
crease, and thus any given peripheral fat cell would
have no direct feedback signal. In any case, there is no
evidence that FFA concentrations influence FFA R
a
in
vivo. For example, in short-term fasting the concentra-
tion of FFAincreases two- to threefold, and FFAR
a
also
increases (28). Thus there is no direct feedback mecha-
nism whereby FFAconcentration affects FFAR
a
.
The absence of a direct link between the energy
utilization of the lean body mass and FFA R
a
requires
the identification of an alternative mechanism to ex-
plain how the change in energy utilization during
exercise could control lipolysis. Insulin, epinephrine,
and adenosine are the most important short-term
regulators of lipolysis (2), but none provides a link
between energy utilization of lipolysisor FFAR
a
during
exercise at different intensities. Thus catecholamines
are only elevated to a great extent during high-
intensity exercise (15), yet in this study [as in previous
studies (10, 15)] fatty acid release was less during
high-intensity exercise than during low intensities.
Insulin concentration is generally suppressed during
exercise (16), and differences in insulin concentration
at different exercise intensities cannot explain the
corresponding rates of lipolysis. Adenosine is a potent
inhibitor of lipolysis in some circumstances (12). How-
ever, because an increase in adenosine will generally
accompany increased ATP turnover, changes in adeno-
sine concentration would not be expected to cause
lipolysis to be positively correlated with energy expen-
diture. In fact, our laboratory (13) has previously
shown that changes in adenosine activity cannot ex-
plain the lipolytic response to exercise. Therefore, it is
impossible to provide a physiological basis for the
notion that energy utilization during exercise controls
lipolysis, either directly or indirectly, through any of
the control mechanisms known to be important in in
vivo regulations of lipolysis.
An implication of the preceding argument is that the
regulation of lipolysis is not necessarily related to the
rate of energy expenditure. This notion is supported by
empirical data from a variety of circumstances. In the
present study, the maximal lipolytic response was
elicited at 25% of V
˙
O
2max
(Fig. 2), yet energy expendi-
ture increased severalfold at higher intensities. In
contrast, the response to fasting elicits a doubling or
more in the rate of lipolysis, yet the rate of energy
utilization does not increase (28). Furthermore, the
extra fatty acids supplied by increased lipolysis in
fasting far exceed the decrease in caloric equivalents
due to decreased hepatic glucose production (17). Thus,
in a wide variety of physiological circumstances, the
rate of lipolysis is not directly related to the rate of
energy expenditure. The major hormones regulating
lipolysis, insulin and catecholamines, are primarily
controlled by factors other than availability of fatty
acids. Therefore, direct feedback control of lipolysis via
hormonal control is unlikely.
Because our data indicate that changes in lipolysis
are not primarily responsible for changes in fat oxida-
tion during exercise, it is not surprising that we found
no effect of body composition on any metabolic param-
eter. Differences in fat mass per se should be expected
to affect substrate metabolism by means of differences
in the availability of fatty acids. Because differences in
body composition would be expected to be one of the
major reasons for differences in substrate metabolism
in exercise between men and women, the lack of an
effectofbodycompositionisconsistentwiththesimilar-
ity inresponses between men and women. On the other
hand,thereisevidencethatsexsteroidsmayplayarole
in substrate metabolism in exercise (19). The present
study was not designed to assess the role ofsex steroids
because the phase of the menstrual cycle during which
our subjects were studied was not controlled. It is
unlikely, however, that this affected our conclusion
because the coefficient of variation of the data in the
female subjects in the present study was generally less
than in the male athletes who were studied previously
(15).Consequently,whereasvariationsin theconcentra-
tions of sex steroidsmay have contributed tovariability
of the results, that variability cannot explain the
inability to find significant differences between men
and women. Rather, it is likely that any possible effects
of sex steroids were overshadowed by the effects of
strenuous endurance training.
This work was supported by Shriners of North America Grant
8940 andby NationalInstitute of Diabetes andDigestive andKidney
Diseases Grant DK-46017. J. A. Romijn was supported by a grant
from the Dutch DiabetesFoundation.
Present address of J. A. Romijn: Dept. of Endocrinology and
Metabolism, Leiden University Medical Center, PO Box 9600, 2300
RC Leiden, The Netherlands(E-mail: jaromijn@endo.azl.nl).
Address for correspondence: R. R. Wolfe, Metabolism Unit, Shri-
ners Burns Institute, 815Market St.,Galveston, TX77550.
Received 5 February 1998;accepted infinal form8 December1999.
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