1
Journal of Exercise Physiologyonline
August 2013
Volume 16 Number 4
Editor-in-Chief
Official Research Journal
Tommy
of the American
Boone, PhD,
Society
MBA
of
Review
Board
Exercise
Physiologists
Todd Astorino, PhD
JulienISSN
Baker,
1097-9751
PhD
Steve Brock, PhD
Lance Dalleck, PhD
Eric Goulet, PhD
Robert Gotshall, PhD
Alexander Hutchison, PhD
M. Knight-Maloney, PhD
Len Kravitz, PhD
James Laskin, PhD
Yit Aun Lim, PhD
Lonnie Lowery, PhD
Derek Marks, PhD
Cristine Mermier, PhD
Robert Robergs, PhD
Chantal Vella, PhD
Dale Wagner, PhD
Frank Wyatt, PhD
Ben Zhou, PhD
Official Research Journal
of the American Society of
Exercise Physiologists
ISSN 1097-9751
JEPonline
Muscle Glycogen Restoration in Females and Males
Following Moderate Intensity Cycling Exercise in
Differing Ambient Temperatures
Michael J. Carper1, Scott R. Richmond2, Samantha A. Whitman 3, Luke
S. Acree4, and Michael P. Godard5
1Human
Performance Laboratory, Pittsburg State University, Pittsburg,
KS, 2Exercise Science Laboratory, Missouri State University, Springfield,
MO, 3Department of Pharmacology and Toxicology, University of
Arizona, Tucson, AZ, 4Abbvie, Metabolic Division, North Chicago, IL,
5Department of Nutrition and Kinesiology, University of Central Missouri,
Warrensburg, MO
ABSTRACT
Carper MJ, Richmond SR, Whitman SA, Acree LD, Godard MP.
Muscle Glycogen Restoration in Females and Males Following Moderate
Intensity Cycling Exercise in Differing Ambient Temperatures.
JEPonline 2013;16(4):1-18. The purpose of this study was to determine
differences in muscle glycogen restoration between males and females
following 90 min of cycle ergometry at varying ambient temperatures. A
total of 8 females (n=4) and males (n=4) performed a 90 min cycle
ergometry trial (70% VO2 max) followed by 8, 1-min sprints (125% VO2
max) at 5C, 25C, and 35C ambient temperature. The subjects were
placed in a 20ºC environment and received a carbohydrate supplement
(1 g CHO·kg body mass-1) at immediate- and 60 min post-exercise.
Muscle biopsies were obtained before and at immediate- and 180 min
post-exercise for the determination of muscle glycogen. Blood samples
were obtained before exercise, every 0.5 hr during exercise, and every
0.5 h for 180 min following exercise for determination of serum glucose,
insulin, and free fatty acids. There was a significant difference in muscle
glycogen resynthesis between groups following cycling exercise at 5ºC,
25ºC, and 35ºC. Males significantly increased muscle glycogen to near
or above baseline levels. There was no significant difference between
groups for muscle glycogen immediately post-exercise. The primary
finding from this study indicates that males restore significantly more
muscle glycogen than females in the 180 min immediately following
moderate intensity cycling exercise at 5ºC, 25ºC, and 35ºC.
Key Words: Carbohydrate, VO2 max, Metabolism
2
INTRODUCTION
Muscle glycogen restoration following exercise bouts has been studied extensively, beginning with
ground-breaking work in the mid 1960s (2). Although research of muscle glycogen utilization and
restoration has been of importance over the last few decades, the effects of differing ambient
temperatures on restoration has received little attention. The majority of the previous reports have
been conducted in ambient room temperature settings (22,29,30) with the purpose of measuring
muscle glycogen restoration and/or accumulation rates. However, there were no alterations of the
surrounding ambient temperature. Thus, the influence of different ambient temperatures during
exercise on muscle glycogen restoration in male and/or female athletes has yet to be discerned.
Other factors have been recognized to influence glycogen restoration. For instance, it has been
demonstrated that post-exercise supplementation with carbohydrate can increase muscle glycogen
resynthesis rates whether provided alone or in combination with proteins and fats (29). Additionally,
the resynthesis of muscle glycogen following a bout of exercise has been shown to be affected by
pre-exercise muscle glycogen content (22). Consequently, muscle glycogen resynthesis rates
previously reported (29) may have been confounded and misinterpreted due to the absence of preexercise muscle biopsies to determine pre-exercise muscle glycogen content.
Exercise fuel metabolism has been well characterized in males, but is very misunderstood in females.
There are data that suggest males and females place different priorities on lipid and carbohydrate
metabolism during exercise. With the same relative intensity of submaximal exercise, females have
been reported to oxidize relatively more lipids and less carbohydrate than males (27). Concurrently, a
lesser decline in muscle glycogen was observed in response to endurance exercise in females (28).
It is still not clear how gender-based differences in the pattern of fuel oxidation during endurance
exercise and restoration following exercise are mediated metabolically, especially when subjects are
exposed to differing environmental conditions.
In addition to the literature on carbohydrate and lipid oxidation in males and females, there exists
discrepancies with regards to muscle glycogen restoration following exercise at differing ambient
temperatures. Numerous investigations have studied the effects of heat and cold stress and ambient
conditions on muscle glycogen utilization in males but not females (5,3,15,18,31). Heat stress has
been shown to increase glycogenolysis (6), muscle lactate accrual (8), and blood glucose
concentrations during endurance exercise in males. Following heat acclimation muscle
glycogenolysis and muscle lactate accumulation have been shown to decrease (6). Moreover, no
increase in muscle glycogen metabolism during exercise in the heat is observed in well-trained,
acclimatized females and males (23). There is some controversy as to which substrate, carbohydrate
or fat, demonstrates the most significant role in energy production during cold stress or exposure.
Some suggest there is an increase in the amount of muscle glycogen utilized (16,32) and others
suggest there is an increased amount of lipid utilized (12).
To date, the influence of varying ambient temperatures on muscle glycogen restoration in males and
females after exercise remains equivocal. In addition, no published studies have compared muscle
glycogen restoration between males and females following cycling exercise at varying ambient
temperatures. Therefore, the primary purpose of this investigation was to determine the effects of
varying ambient temperatures (5C, 25C, and 35C) on muscle glycogen restoration following 90 min
of moderate intensity cycling exercise.
3
METHODS
Subjects
A total of 18 subjects were recruited and completed preliminary testing for this study. Ten subjects
withdrew from the study due to time commitment, muscle biopsies, core temperature measurement,
and blood collection. Eight male (n=4) and female (n=4) trained cyclists completed this investigation
after giving written informed consent in accordance with guidelines established by the Human
Subjects Committee-Lawrence at the University of Kansas. Physical characteristics of the subjects
are presented in Table 1.
Table 1. Descriptive Characteristics of Subjects.
Characteristics
Males
Age (yr)
Height (cm)
Females
34.0  2.9
30.3  2.3
175.7  4.8
141.8  51.4
Weight (kg)
78.5  8.4*
64.2  4.4
VO2 max (mL·kg-1·min-1)
57.9  11.4
45.8  2.6
4.4  1.0*
3.0  0.2
VO2 max (L·min-1)
Body Fat (%)
15.2  5.2
19.3  7.2
Values are mean ± SD for males (n=4) and females (n=4). *Significantly different from females (P≤0.05).
Subject testing consisted of four preliminary testing days followed by one primary testing day for each
of the three exercise conditions (5C, 25C, and 35C). All female subjects were taking triphasic oral
contraceptives and had been for the previous 6 months. Triphasic oral contraceptives are suggested
to provide better menstrual cycle control and decrease any androgenic side effects such as altered
carbohydrate and lipid metabolism (25). Female subjects were tested in what would normally be the
mid-follicular phase of the menstrual cycle (determined as days 7-11 from the onset of menstruation)
to ensure lower levels of estrogen (E2) and progesterone (P4), thus reducing the unpredictability of
ovarian hormone effects on substrate utilization.
Procedures
Preliminary Testing
Aerobic Capacity Test. Prior to the experimental trial, subjects performed a graded exercise test on
an electronically braked cycle ergometer (Lode, Groningen, The Netherlands) to determine maximal
oxygen uptake (VO2 max). Expired gases were collected using a metabolic measurement cart
(Sensormedics 2900, Yorba Linda, CA) for determination of respiratory exchange ratio (RER =
VCO2/VO2) and oxygen consumption (VO2) every 20 sec. Maximal oxygen uptake was determined as
reported previously (4).
Dietary Control. The subjects were instructed to complete a three day food record using two week
days and at least one weekend day. All subjects were educated on appropriate measurement and
recording techniques for food records. Subjects were presented nutritional hand-outs representing
traditional measurements to use as references. Subject diets were analyzed, using a commercially
available dietary analysis program (Food Processor v. 7.04, ESHA Research, Salem, OH) for percent
4
carbohydrate (%CHO), percent protein (%PRO), percent fat (%FAT), and total calories (Tot Cal)
(Table 2). Using foods that were listed on the three day food records, diets consisting of 60% CHO,
15% PRO, and 25% FAT were prescribed to the subjects 24 hrs prior to each of the three primary
testing days (described below). Similar types of diets have been reported to maintain normal muscle
glycogen stores (4,20,26,27). Subjects adhered to these specific diets 24 hrs prior to each primary
testing day.
Table 2. Three Day Food Record Average Calories and Macronutrient Composition 24 hrs
Prior to Depletion Ride.
Males
Females
Three Day Food Record
Average Calories
2969.5  1715
1690.3  470.2
3038.1  1532.8
1713.3  543.4
1730.8  715.4*
1046.9  4.4
Macronutrient composition
Average calories
Carbohydrate calories
515.4  180.2
231.8  74.2
Fat calories
800.6  561*
361.3  139.4
% carbohydrate
60.7  16.6
62.1  8.2
18.3  5.2
13.7  1.8
26.1  10.6
21.1  4.2
Protein calories
% protein
% fat
Values are mean  for males (n=4) and females (n=4). Significance (P≤0.05). *Significant between
group differences
Oral Glucose Tolerance Test. The laboratory methods used to perform the oral glucose tolerance
tests (OGTT) have been previously described (21). Briefly, each subject consumed a glucose solution
that consisted of 1 g glucosekg body mass-1 derived from a commercially available carbohydrate
drink. Subjects were required to have a fasting serum glucose concentration of 115 mgdL-1, a peak
value of 200 mgdL-1, and a 2-hr concentration of 140 mgdL-1 (10).
Pre-glycogen Depletion Ride. Twenty-four hours prior to each primary testing day subjects consumed
a diet based on the average of their three day food records. That evening, subjects reported to the
laboratory for a 45-min pre-glycogen depletion ride at an intensity of 70% VO2 max. Prior to the ride, a
resting needle muscle biopsy of the vastus lateralis was collected with the aid of suction (1). This
biopsy was considered the pre-depletion ride muscle biopsy, as subjects did not consume any type of
calorie containing foods for the next 15 hrs. Following the pre-depletion ride, subjects fasted for 10
hrs, during which only water was appropriate to consume. This overnight fast was to ensure that
minimal muscle glycogen resynthesis occurred prior to the actual 90-min depletion ride and to also
ensure that the subjects would be glycogen depleted following the ride (described below).
5
Primary Testing
Glycogen Depletion Ride. During each of the three separate trials, the subjects reported to the
laboratory in a 10-hr fasted state to perform the glycogen depletion ride at 5C, 25C, or 35C. The
temperature at which the subjects would perform the 90-min cycling exercise was determined via
random sampling prior to the subject arriving to the laboratory. Following the insertion of a 1” Teflon
catheter into an antecubital vein, kept patent with 0.9% saline, a resting blood sample was collected.
The subjects cycled for 90 min at 70% VO2 max followed by 8, 1-min sprints at 125% VO2 max. It has
been demonstrated that high intensity sprints following moderate intensity exercise facilitates muscle
glycogen degradation (4). Every 30 min throughout the exercise a 5 mL blood sample was collected
to determine serum glucose (GLU), serum insulin (INS), and serum free fatty acids (FFA).The
subjects were allowed and encouraged to consume water ad libitum throughout the cycling protocol.
Recovery from Exercise. Immediately post-exercise, a blood sample was collected. In addition, a
muscle biopsy of the vastus lateralis was obtained on the opposite leg from the pre-depletion ride
biopsy. Following blood collection and muscle biopsy, the subject was given a carbohydrate drink (1 g
CHOkg body mass-1) which was brought to a final volume of 0.5 L. The subject remained in the
laboratory in a sitting or supine resting position at room temperature for 180 min following the 90-min
ride. Activity was limited to restroom breaks only. A second carbohydrate drink consisting of the same
concentration, carbohydrate content, and volume as the first drink was given to the subject at 60 min
post-exercise. At 30, 60, 90, 120, 150, and 180 min post-exercise, blood samples were again
collected. At 180 min post-exercise, a third muscle biopsy was obtained through the same incision as,
and proximal to, the immediate post-depletion ride biopsy. This has been shown to be an acceptable
manner in which to obtain post-exercise muscle biopsies with no disturbance in glycogen formation
(33).
Muscle Biopsy
As stated, muscle biopsies were obtained pre-depletion ride as well as immediately post-exercise and
180 min post-exercise. All muscle samples were dissected free of blood and any visible connective
tissue and directly frozen in liquid nitrogen. All muscle samples were stored in liquid nitrogen until
analysis of muscle glycogen was completed.
Blood Sampling and Analysis
All blood samples were collected from a 1” Teflon catheter, placed in an antecubital vein, via a
Vacutainer vial containing no additive (Vacutainer, Franklin Lakes, NJ). All blood samples were
centrifuged at ~1300 x g, 4C for 20 min. The serum was transferred to appropriate tubes and stored
at −80°C until analyzed for GLU, INS, and FFA. For all assays, measurements were made in
triplicate. Serum GLU was determined using a reagent kit (Sigma Chemical #315, St. Louis, MO)
prepared according to manufacturer’s guidelines. The quinoneimine dye formed in the reaction was
measured using a spectrophotometer (SpectronicGenesys2, Spectronic Instrument, Inc.,
Rochester, NY) set at a wavelength of 505 nm and the absorbance (A) reading set to zero. Serum
INS was determined via immunoassay (Access Immunoassay System, Beckman Coulter, Fullerton,
CA). Serum FFA were assayed using a non-esterified fatty acid (NEFA)-C test kit (Wako Chemical
#994-75409, Wako Chemicals USA, Inc., Richmond, VA) and was prepared according to
manufacturer’s guidelines. The concentration of NEFA’s was determined using a 96 well microplate
reader (Ceres UV 900 HDi, Bio-Tek Instruments, Inc., Winooski, VT) with the spectrophotometer set
at a wavelength of 550 nm.
Muscle Glycogen Determination
Muscle glycogen analysis was based on previously described procedures (7,19). Briefly, for each
muscle sample a 2 mL cryotube was labelled to which 2 N HCl was added. The muscle samples were
6
placed in a freezer and allowed to reach approximately −15C. The muscle was then dissected and
weight recorded to the nearest mg. The sample was then placed in 2N HCl, boiled in water for 30
min, and neutralized with 0.67 N NaOH. Reagent cocktail (ThermoTrace #TR-15421, Melbourne,
Australia) was transferred to the appropriate tube. Muscle sample extract, standard, or distilled water
was transferred to the reagent cocktail for the samples, standards, and blanks, respectively. Samples
were then allowed to stand at room temperature for approximately 3 min to let the reaction proceed to
its endpoint. At the end of the 3-min incubation, the absorbance was recorded spectrophotometrically
at a wavelength of 340 nm (SpectronicGenesys2, Spectronic Instrument, Inc., Rochester, NY) and
muscle glycogen content calculated.
Rectal (Tre) Core Temperature
Rectal core temperature was measured as previously described (6). Briefly, prior to each of the three
rides all subjects inserted a rectal thermistor probe (Yellow Springs Instruments) 10 cm beyond the
anal sphincter and subsequently entered the environmental chamber, previously set to the
designated environmental temperature. Core temperatures were recorded every 15 min during
exercise (Table 3). For safety reasons, core temperatures were measured every 15 min following the
exercise bout at 35ºC to ensure that the subjects were properly thermoregulating.
Statistical Analyses
Descriptive statistics were computed for all subjects and independent t-tests were employed to detect
between group differences in age, height, body mass, VO 2 max, and percent body fat (%BF). A twoway repeated measures analysis of variance (ANOVA) was used to detect if there were any
interactions or main effects for the dependent variables. If any significant interactions were detected
Bonferroni post-hoc analysis was used for further statistical examination. All results are mean ± SD.
The level of significance was set at P0.05. All statistical analyses were performed with Statistical
Package for the Social Sciences (SPSS 20, Inc., Chicago, IL).
RESULTS
Subject Characteristics
There were no significant differences between or within groups with regard to age, height, and
percent body fat (refer to Table 1). However, there were significant differences between groups for
body mass and maximal oxygen consumption (VO2 max; Lmin-1).
Dietary Records. Nutrient analysis of 24-hr food records prior to the testing session revealed no
significant difference between or within groups with regard to the mean % carbohydrate, protein, and
fat consumption (60.7 ± 16.6 vs. 62.1 ± 8.2%; 18.3 ± 5.2 vs. 13.7 ± 1.8%; 26.1 ± 10.6 vs. 21.1 ± 4.2%;
males vs. females, respectively). There was no significant difference in caloric consumption between
rides within each group (refer to Table 2).
OGTT. The measurements for the oral glucose tolerance test revealed no significant between group
differences for the serum GLU (Figure 1).
7
Figure 1. Oral glucose tolerance tests (OGTT). --■--, Males; ▲ , Females. OGTT using 1 g CHO•kg body
mass-1 commercially available CHO drink. Values are means ± SD for males (n=4) and females (n=4).
*Significant between group differences (P≤0.05).
Depletion Ride
Rectal Core Temperature. Core temperature did not differ between males and females for any of the
rides. However, there were significant differences within the groups for each of the rides as shown in
Table 3.
Table 3. Depletion Ride Rectal Core Temperatures (ºC).
Baseline
30 min
60 min
90 min
25ºC Ride
Males
36.9  0.22
37.5  1.2
38.2  1.1
38.8  0.58†
Females
37.4  0.82
38.3  0.58†
38.3  0.38†
38.6  0.60†
Males
37.5  0.52
38.1  0.96
40.0  1.32†
39.5  0.46†
Females
37.1  0.22
38.7  0.60†
39.3  0.42†
39.4  0.30†
Males
37.0  0.48
38.4  0.52†
38.8  0.42†
38.6  0.78†
Females
37.3  0.20
38.4  0.08†
38.1  0.58†
39.3  0.24†
35ºC Ride
5ºC Ride
Values are means  SD for males (n=4) and females (n=4). Significance (P≤0.05). *Significant between
group differences. †Significant difference from Baseline.
8
Average Watts. All subjects performed the depletion rides at the same relative intensity (70% VO 2
max) and the sprints following the depletion ride (125% VO 2 max). Although there were significant
differences between groups for average watts sustained throughout the 90-min cycling protocol and
the sprints following the 90-min ride, the power outputs were adjusted so that all subjects exercised at
70% and 125% VO2 max.
Muscle Glycogen Content
Males and females had similar muscle glycogen content before each of the three cycling trials at
25C (71.6 vs. 88.8 mmol·kg wet wt-1; P = 0.17; males and females, respectively; Figure 2A), 35C
(106.7 vs. 84.5 mmol·kg wet wt-1; P = 0.27; males and females, respectively; Figure 2B), and 5C
(85.6 vs. 82.5 mmol·kg wet wt-1; P = 0.21; males and females, respectively; Figure 2C).
Figure 2.
A.
Glycogen Resynthesis
(mmol/kg wet wt/hr)
Muscle glycogen (mmol/kg wet wt)
120
100
80
30
25
20
15
10
‡
5
0
Males
60
†
40
Females
‡#
†
20
0
Before
Im m ed pst
180 m in pst
Tim e (m in)
Glycogen Resynthesis
(mmol/kg wet wt/hr)
B.
Muscle glycogen (mmol/kg wet wt)
120
100
30
‡
25
20
15
10
5
0
Males
80
†
60
Females
*#
†
40
20
0
Before
Im m ed pst
Tim e (m in)
180 m in pst
9
C.
Glycogen Resynthesis
(mmol/kg wet wt/hr)
Muscle glycogen (mmol/kg wet wt)
120
100
80
30
25
‡
20
15
10
5
0
Males
60
Females
*#
†
†
40
20
0
Before
Im m ed pst
Tim e (m in)
180 pst
Figure 2. A. Depletion and post-depletion ride muscle glycogen (mmol·kg wet wt-1) at (A) 25ºC, (B) 35ºC, and
(C) 5ºC. Inserts are muscle glycogen resynthesis rates. ░, Males. ▓, Females. Values are means ± SD for
males (n=4) and females (n=4). Significance (P≤0.05). *Significant between group differences. †Significant
difference from before- to immediate post-exercise. ‡Significant difference from immediate post- to 180 min
post-exercise. #Significant difference from before- to 180 min post-exercise (P≤0.05).
During the 90-min ride at 25ºC (Figure 2A), males significantly decreased muscle glycogen by 70%
(71.6 to 21.4 mmol·kg wet wt-1) and females by 68% (88.8 to 27.8 mmol·kg wet wt-1). From immediate
post- to 180 min post-exercise, males and females significantly increased muscle glycogen by 64%
and 36%, (21.4 to 59.5 mmol·kg wet wt-1 and 27.8 to 43.7 mmol·kg wet wt-1, males and females,
respectively). However, males increased muscle glycogen at 180 min post-exercise to within 16% of
resting levels while females’ muscle glycogen content remained 51% below and significantly different
from resting levels. Rates of muscle glycogen resynthesis were 12.7 and 5.3 mmol·kg wet wt-1·hr-1 for
males and females, respectively (Figure 2A).
During the ride at 35C (Figure 2B), males significantly decreased muscle glycogen by 57% (106.7 to
45.8 mmol·kg wet wt-1) and females by 37% (84.4 to 52.4 mmol·kg wet wt-1). From immediate post- to
180 min post-exercise males significantly increased muscle glycogen by 54% (45.8 to 99.4 mmol·kg
wet wt-1), thus increasing glycogen to 6% below resting levels. At the same time point, the muscle
glycogen content of the females increased by 4% (52.4 to 54.8 mmol·kg wet wt-1) and remained 35%
below, and significantly different from resting levels. Rates of muscle glycogen resynthesis were 22.9
and 1.0 mmol·kg wet wt-1·hr-1 for males and females, respectively (Figure 2B).
During the ride at 5C (Figure 2C), males significantly decreased muscle glycogen by 77% (85.6 to
19.4 mmol·kg wet wt-1) and females by 54% (82.4 to 37.8 mmol·kg wet wt-1). From immediate post- to
180 min post-exercise, males significantly increased muscle glycogen by 76% (19.4 to 83.5 mmol·kg
wet wt-1). The increase was within 2% of resting levels. Females increased muscle glycogen levels by
31% (37.8 to 55.3 mmol·kg wet wt-1), and remained 33% below, and significantly different from
resting levels. Rates of muscle glycogen resynthesis were 24.6 and 5.8 mmol·kg wet wt-1·hr-1, for
males and females, respectively (Figure 2C).
10
Blood Parameters
Glucose
There were no significant differences between groups for serum GLU at any time point during or
through 180 min following the 90-min depletion rides at 25ºC and 35ºC (Figure 3A and Figure 3B).
However, there was a significant difference between groups at 180 min post-exercise at 5ºC (Figure
3C). Serum GLU peaked during the 25ºC and 35ºC rides at 30 min and decreased to below baseline
by 90 min of exercise for both groups. Following the cessation of exercise and the consumption of the
carbohydrate drink, serum GLU significantly increased at 30 min post-exercise for both groups
following all rides. Serum GLU levels began to steadily decreased for both males and females at 30
min post-CHO consumption for the 25ºC ride (Figure 3A). Interestingly, following the ride at 35ºC
(Figure 3B) serum GLU did not begin to steadily decrease until 150 min post-CHO consumption for
the males and 120 min post-CHO consumption for the females. Following the consumption of CHO
after the ride at 5ºC (Figure 3C), serum GLU levels began to steadily decrease at 120 min postexercise for the females, where it did not for the males.
Figure 3.
A.
Post Exercise
[Glucose] (mmol/L)
14
12
Exercise
10
8
6
4
2
0
Base
30
60
90
30
60
90
120
150
180
150
180
Time
B.
Post Exercise
[Glucose] (mmol/L)
12
10
Exercise
8
6
4
2
0
Base
30
60
90
30
60
Time
90
120
11
C.
Post Exercise
[Glucose] (mmol/L)
12
10
Exercise
*
8
6
4
2
0
Base
30
60
90
30
60
90
120
150
180
Time
Figure 3. Depletion and post-depletion ride serum glucose (mmol·L-1) at (A) 25ºC, (B) 35ºC, and (C) 5ºC. --■-, Males. ▲ , Females. Values are means ± SD for males (n=4) and females (n=4). *Significant between
group differences (P≤0.05).
Insulin
There were no significant differences between groups with respect to serum insulin during or through
180 min following the depletion ride at 25ºC (Figure 4A). There were significant differences between
groups in serum insulin at 150 min post-exercise at 35ºC (Figure 4B) and at 150 min post- and 180
min post-exercise at 5ºC (Figure 4C). Serum insulin levels decreased during exercise during each
ride and reached the lowest levels at 90 min of exercise for both males and females. Following the
exercise bouts, insulin levels peaked at 90 min post-exercise and 30 min post-exercise at 25ºC and
35ºC, respectively. Serum insulin peaked at 90 min post-exercise for the females and 120 min postexercise for the males at 5ºC. Insulin levels did not reach baseline values for either group at 180 min
post-exercise following any of the exercise bouts.
Figure 4.
A.
Post Exercise
[Insulin] (pmol/L)
350
300
Exercise
250
200
150
100
50
0
Base
30
60
90
30
60
Time
90
120
150
180
12
B.
Post Exercise
[Insulin] (pmol/L)
350
300
*
Exercise
250
200
150
100
50
0
Base
30
60
90
30
60
90
120
150
180
Time
C.
Post Exercise
[Insulin] (pmol/L)
350
300
Exercise
250
200
*
150
*
100
50
0
Base
30
60
90
30
60
90
120
150
180
Time
Figure 4. Depletion and post-depletion ride serum insulin (pmol·L-1) (A) 25ºC, (B) 35ºC, and (C) 5ºC. --■--,
Males, ▲ Females. Values are means ± SD for males (n=4) and females (n=4). *Significant between group
differences (P≤0.05).
Free Fatty Acids
Depletion and post-depletion ride serum FFA values are expressed in Figures 5 A-C. There were
significant between group differences for serum FFA during exercise at 25ºC and at baseline through
90 min of exercise at 35ºC and 5ºC. There were significant differences between groups in serum FFA
at 30, 60, 120, and 150 min post-exercise at 25ºC, at 90 through 180 min post-exercise at 35ºC, and
at 60, 120, and 150 min post-exercise at 5ºC. Serum FFA values decreased steadily from 30 to 180
min post-exercise for both groups following each exercise bout.
13
Figure 5.
A.
Post Exercise
Exercise
1.2
[FFA] (mE/L)
1.0
* *
0.8
0.6
*
*
*
* *
0.4
0.2
0.0
Base
30
60
90
30
60
90
120
150
180
Time (min)
B.
Post Exercise
Exercise
1.2
*
[FFA] (mE/L)
1.0
0.8
0.6
* *
*
* * * *
0.4
0.2
0.0
Base
30
60
90
30
60
90
120
150
180
Time
C.
Post Exercise
Exercise
1.2
[FFA] (mE/L)
1.0
0.8
0.6
* * * *
*
* *
0.4
0.2
0.0
Base
30
60
90
30
60
Time
90
120
150
180
14
Figure 5. Depletion and post-depletion ride serum FFA (mE·L-1) at (A) 25ºC, (B) 35ºC, and (C) 5ºC. --■--,
Males, ▲ , Females. Values are means ± SD for males (n=4) and females (n=4). *Significant between group
differences (P ≤0.05).
DISCUSSION
The purpose of this investigation was to determine the effects of temperature on muscle glycogen
restoration between males and females following 90 min of moderate intensity cycling exercise
followed by 8, 1-min sprints at varying ambient temperatures (25ºC, 35ºC, and 5ºC). Specifically,
muscle glycogen restoration was examined, following an exhaustive cycling protocol, for 180 min
employing two liquid carbohydrate feedings (1 g CHO·kg body mass-1). The primary findings of this
investigation are that males and females do not restore muscle glycogen to the same extent following
90 min of cycling exercise at differing ambient temperatures when consuming 1 g CHO·kg body
mass-1 at immediate and 60 min post-exercise. Females received the same amount of CHO·kg body
mass-1 even though they had less relative lean mass. Therefore, females received more grams of
CHO·kg body mass-1 but muscle glycogen restoration was still lower despite the higher relative load
of CHO.
Previously, males and females have been shown to have similar glycogen synthesis rates with postexercise supplementation containing CHO alone (1 g CHO·kg body mass-1) (30). Conversely, our
findings show the rates of post-exercise muscle glycogen resynthesis were different between males
and female following each of the rides (Figure 2 A-C). At baseline muscle glycogen content can
influence the amount of muscle glycogen metabolized during exercise and the rate of muscle
glycogen resynthesis. More importantly, the current study, which controlled for nutritional and
exercise habits 24 hrs prior to the preliminary testing day, demonstrates that males and females had
similar muscle glycogen contents prior to each of the three trials.
Muscle glycogen content following exercise can also influence the rate of muscle glycogen
resynthesis (22). Moreover, overall muscle glycogen resynthesis occurs at a faster rate when there is
a greater decrease in muscle glycogen levels following exercise (22). While we demonstrate that
males and females utilized muscle glycogen similarly during each of the three trials, the trial at 35C
(Figure 2B) did not elicit as great of a muscle glycogen depletion in either group as did the 25C
(Figure 2A) or 5C (Figure 2C) trials. This conflicts with the previous notion that the utilization of
muscle glycogen is increased during exercise in a hot environment (~35.4ºC) compared to a cool
environment (~16.4ºC) (13).
The elevated core temperatures of our subjects during exercise at 35C (Table 3) may explain why
glycogen depletion was delayed in this trial, as hotter temperatures have been shown to instigate
central nervous system stress and accelerate the onset of fatigue (9,17). The majority of the subjects
had symptoms of central nervous system stress (e.g., fatigue and dizziness) upon completing the
exercise, particularly at the 35C trial. Furthermore, the possibility exists that our subjects were
acclimated to exercising at elevated temperatures, which has been shown to reduce carbohydrate
dependency as an energy source (6). Unfortunately, we are unable to address this with our subjects
as the recorded training histories did not include environmental training conditions.
Substrate metabolism during exercise has been shown to differ between males and females (24,28).
The study by Tarnopolsky et al. (28) demonstrated that females oxidized significantly more lipid and
less carbohydrate and protein when compared to males during exercise at 75% VO2 peak. This
increase in lipid oxidation may have contributed to the decrease in muscle glycogen utilization in the
15
females. In the current study, we show that females have significantly more serum FFA during and
post-exercise than males during all rides. This increased post-exercise serum FFA may have
contributed to the decreased muscle glycogen resynthesis observed in females. However, the lack of
RER data during exercise does not allow us to fully conclude that females were in fact oxidizing more
lipid than males, which may help to explain the decrease in post-exercise muscle glycogen
resynthesis observed in this group.
Increasing rates of muscle glycogen resynthesis after exercise can also occur from utilizing greater
amounts of triglycerides to supply lipid fuel for oxidative muscle metabolism (14). However, we
speculate that FFA was not the primary source of energy for the females during these particular
exercise bouts, as we found that males and females used similar amounts of muscle glycogen.
Additionally, previous reports have evidenced a decrease in interstitial glycerol concentrations
following a constant insulin infusion, suggesting that interference from FFA on muscle glycogen
resynthesis is unlikely (11). During post-exercise recovery, we found an increase in serum INS from
immediate post- to 120-150 min post-exercise and a concomitant decrease in serum FFA. These
results may suggest the 180 min post-exercise recovery period for females was not long enough in
duration to significantly increase muscle glycogen concentrations to resting levels.
CONCLUSIONS
This study shows that 90 min of moderate intensity exercise at varying ambient temperatures
decreases muscle glycogen concentrations to the same extent in both males and females, but muscle
glycogen restoration in the first 180 min after exercise was significantly faster in males. Therefore, it
appears that males and females do not restore muscle glycogen at the same rate during a 3-hr
restoration period utilizing two oral carbohydrate bolus’ consisting of 1 g CHO·kg body mass-1. These
findings suggest that although males and females utilize muscle glycogen to the same extent during
moderate intensity exercise at differing temperatures, the mechanisms utilized to restore this valuable
energy source may be dissimilar. While this study presents pertinent insight to potential mechanisms
regarding muscle glycogen restoration in trained males and females, many discrepancies continue to
surface in the growing body of scientific literature. Therefore, future investigations are encouraged to
discern these inconsistencies and clarify the process(es) of muscle glycogen restoration between
trained male and female athletes.
ACKNOWLEDGMENTS
The authors would like to thank John P. Thyfault, PhD, University of Missouri for assistance in data
analysis. This project was supported in part by a grant from the Gatorade Sports Science Institute.
Address for correspondence: Michael J. Carper, PhD, Department of Health, Human Performance,
and Recreation, Pittsburg, KS 66762. Phone: 1-620-235-6155; Email: mcarper@pittstate.edu
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