Effect of moderate exercise immediately
followed by induced hyperglycemia on gene
expression and content of the glucose
transporter-4 protein in skeletal muscles of horses
Yvette S. Nout, DVM; Kenneth W. Hinchcliff, BVSc, PhD; Edward Jose-Cunilleras, DVM;
Lawrence R. Dearth, PhD; Gloria S. Sivko, DVM; James W. DeWille, PhD
ObjectiveTo determine the effect of a single bout
of exercise and increased substrate availability after
exercise on gene expression and content of the glu-
cose transporter-4 (GLUT-4) protein in equine skeletal
muscle.
Animals6 healthy adult Thoroughbreds.
ProceduresThe study was designed in a balanced,
randomized, 3-way crossover fashion. During 2 trials,
horses were exercised at 45% of their maximal rate
of oxygen consumption for 60 minutes after which 1
group received water (10 mL/kg), and the other group
received glucose (2 g/kg, 20% solution) by nasogas-
tric intubation. During 1 trial, horses stood on the
treadmill (sham exercise) and then received water (10
mL/kg) by nasogastric intubation. Muscle glycogen
concentration and muscle GLUT-4 protein and mRNA
content were determined before exercise and at 5
minutes and 4, 8, and 24 hours after exercise.
ResultsAlthough exercise resulted in a 30% reduc-
tion in muscle glycogen concentration, no significant
difference was detected in muscle GLUT-4 protein or
mRNA content before and after exercise. Glycogen
replenishment was similar in both exercised groups
and was not complete at 24 hours after exercise.
Horses that received glucose had significantly higher
plasma glucose and insulin concentrations for 3 hours
after exercise, but no effect of hyperglycemia was
detected on muscle GLUT-4 protein or mRNA con-
tent.
ConclusionUnder the conditions of this study, nei-
ther exercise nor the combination of exercise fol-
lowed by hyperglycemia induced translation or tran-
scription of the GLUT-4 protein in horses. (Am J Vet
Res 2003;64:14011408)
S keletal muscle glucose metabolism is enhanced
during high-intensity and moderate-intensity exer-
cise, primarily through muscle glycogen and blood-
borne glucose utilization. Glucose utilization in mus-
cle is a function of the supply of glucose to muscle
from blood, the glucose transport capacity of the sur-
Received February 20, 2003.
Accepted April 30, 2003.
From the Departments of Veterinary Clinical Sciences (Nout,
Hinchcliff, Jose-Cunilleras) and Veterinary Biosciences (Dearth,
Sivko, DeWille), College of Veterinary Medicine, The Ohio State
University, Columbus, Ohio 43210.
Funded by Equine Research Funds of The Ohio State University,
Columbus, Ohio.
The authors thank Lori Avila and Leia Hill for technical assistance.
Address correspondence to Dr. Hinchcliff.
AJVR, Vol 64, No. 11, November 2003
face membrane of the myocyte, and the rate of glucose
metabolism within the cell. Transport of glucose
through the sarcolemma appears to be the primary
rate-limiting step for glucose metabolism1,2 and glyco-
genesis3 in striated muscle. Glucose transport into the
muscle cell is a saturable process, mediated by glucose
transporter (GLUT) proteins, of which GLUT-4 is the
major isoform expressed in skeletal muscle. Transport
through GLUT-4 is stimulated by at least 2 separate
pathways. The chief acute physiologic stimulus of glu-
cose disposal is mediated through insulin. However,
other stimuli such as muscle contractions and hypoxia
can independently activate glucose uptake through
GLUT-4.4-6 In horses, the importance of this glucose
transport mechanism has yet to be determined.
Exercise training has been shown to increase
skeletal muscle GLUT-4 protein content in humans,7,8
rats,9-16 and, recently, horses.17 Furthermore, exercise
has been shown to increase GLUT-4 gene expression in
humans18 and rats2,13,15,16,19; however, this has not been
studied in horses. In rats, it has been shown that this
increase in GLUT-4 gene expression is an early adap-
tive response to exercise, and it is speculated that this
adaptation plays an important role in enhancing mus-
cle glycogen replenishment.2,20 Glucose transporter-4 is
the major transporter responsible for glucose transport
after exercise; however, it is not essential for glycogen
synthesis.21 Although GLUT-4-deficient mice were able
to replenish their glycogen stores at 24 hours after
exercise,21 replenishment of glycogen in the presence of
GLUT-4 is enhanced in mice21 and humans.8
Furthermore, in exercised mice, increased GLUT-4
gene expression occurs fast enough to affect glycogen
resynthesis.22
Restoration of glucose homeostasis and replenish-
ment of glycogen stores are critical for optimal perfor-
mance during subsequent exercise. Fatigue is associat-
ed with depletion of muscle glycogen concentra-
tions,23,24 and reduced concentrations of muscle glyco-
gen before exercise are associated with impaired athlet-
ic performance in humans25,26 and horses.27 Restoration
of muscle glycogen concentration is achieved by glyco-
gen synthesis principally from blood-borne glucose
and is dependent on substrate availability, intracellular
transport capacity, and enzyme concentrations. Muscle
glycogen synthesis in horses not provided supplemen-
tal glucose is slow, with up to 3 days required for
restoration of muscle glycogen concentration to values
before exercise.28,29 It has been shown that induction of
hyperglycemia, and hence hyperinsulinemia, by inges-
1401
tion or IV administration of glucose or glucose poly-
mers enhances the rate of muscle glycogen resynthesis
in humans30 and horses.31,32 However, in horses, the role
of GLUT-4 in muscle glycogen replenishment after
exercise has not been investigated.
The purpose of the study reported here was to
examine the effect of a single bout of exercise on
equine skeletal muscle GLUT-4 protein and GLUT-4
gene expression and determine whether a period of
hyperglycemia after exercise would enhance the
GLUT-4 protein content with or without an increase in
gene expression. More specifically, horses underwent a
period of moderate-intensity exercise, and hyper-
glycemia was induced by intragastric administration of
a glucose solution. Muscle glycogen concentration and
muscle GLUT-4 protein and mRNA content were deter-
mined to examine the effect of exercise and hyper-
glycemia on these variables.
Materials and Methods
Experimental protocolThe effect of exercise and
hyperglycemia on muscle GLUT-4 protein content and
GLUT-4 gene expression was examined in a balanced, ran-
domized, 3-way crossover design in which 6 horses per-
formed 3 trials separated by 7-day intervals. The order in
which the horses performed the trials was randomized, but
all horses performed all 3 trials. Sedentary horses (SED-
group horses) stood on the treadmill for 60 minutes (sham
exercise) and received water (10 mL/kg) via nasogastric tube
immediately after the sham exercise. During the other 2 tri-
als, horses ran on a treadmill set at a 4o incline for 60 minutes
at approximately
45% of their
maximal rate of oxygen con-
sumption (VO2max). The VO2max was determined during an
incremental exercise test 6 days before the start of the study.
Immediately after exercise, exercised horses received water
(WATER-group horses) at a dose of 10 mL/kg, or exercised
horses received a 20% glucose solution (GLUC-group hors-
es) at a dose of 2 g/kg via a nasogastric tube. Horses were not
exercised for 72 hours before the study, and food was with-
held for 18 hours prior to all trials. After collection of blood
samples and muscle specimens 8 hours after exercise, horses
were fed only hay (ad libitum) until collection of the 24-hour
samples. All experiments were conducted after approval by
the Institutional Laboratory Animal Care and Use Committee
of The Ohio State University and were performed in compli-
ance with their guidelines and recommendations.
HorsesSix clinically normal Thoroughbreds (4 mares
and 2 geldings) were used for this study. Their mean ( SE)
age was 7 1 years, and their mean body mass was 478
15 kg. Horses were weighed once every week during the
training period and before each trial during the study period.
Horses were housed in box stalls (3 X 4 m) and fed grass hay
and cracked corn according to National Research Council
guidelines to maintain an ideal body weight (body condition
score of 5 to 6 out of 9). They had ad libitum access to fresh
water and a salt and mineral block.
Prior to onset of the study, the horses had been turned
out in pasture for at least 2 months. Horses were then housed
in box stalls and underwent a 1-month conditioning period
during which they were accustomed to running on a high-
speed treadmill.a Horses were run on the treadmill 3 to
4 d/wk at 4 to 5 m/s for 40 minutes. During this time, the
horses were accustomed to wearing the facemask used to col-
lect respiratory gases during the VO2max test. At the end of the
training period, the horses underwent 1 bout of exercise at 5
to 6 m/s for 60 minutes. During the 7-day interval between
the trials, horses were maintained in an exercise program
1402
similar to the one used during days 2, 3, and 4 of the train-
ing period. The treadmill was set at a 4o incline throughout
the conditioning and experimental period.
Maximal rate of oxygen consumptionAll horses
underwent an incremental exercise test prior to the study to
determine their VO2max. During this test, the horses were run
at 4 m/s for 90 seconds followed by 7 m/s for 90 seconds with
subsequent increases
of 1 m/s every 90 seconds. During the
exercise test, VO2 was measured every 10 seconds by indirect
calorimetryb as previously described.33,34 The VO2max was
defined as the value at which oxygen consumption reached a
plateau (change in VO2 < 4 mLkg1min1 despite further
increases in speed). Regression analysis of the running speed
to VO2 relationship was used to determine the speed that
elicited 45% of VO2max.
Sample collectionPrior to each trial, a catheter was
placed aseptically in a jugular vein, through which blood was
collected before exercise, immediately after completion of
exercise, every 30 minutes following water or glucose admin-
istration for 8 hours, and at 24 hours after completion of the
exercise. Blood was collected in evacuated tubes containing
potassium oxalate and sodium fluoride for determination of
plasma glucose concentrations and plasma lactate concentra-
tions and in evacuated tubes containing no additive for deter-
mination of serum immunoreactive insulin (IRI) concentra-
tions. Plasma or serum was harvested within 30 minutes of
blood collection by centrifugation at 1,500 X g for 20 minutes
at 4oC. Serum was stored at 80oC until analysis.
Muscle biopsy specimens (approx 1 g) were obtained by
the same person from the semimembranosus or semitendi-
nosus muscles by cut-down incisions. Biopsy specimens were
collected at a depth of approximately 1 cm from the skin after
induction of local anesthesia by SC infiltration with 2%
mepivacaine hydrochloride. Horses were not sedated for this
procedure. Either the right or left hind limb was used to col-
lect 5 biopsy specimens during 1 trial. The right and left hind
limbs were alternated for the 3 trials. Biopsy specimens were
used for determination of glycogen concentration, GLUT-4
protein, and GLUT-4 mRNA content. Muscle was collected
prior to the trial (before exercise), within 5 minutes of com-
pleting the exercise, and at 4, 8, and 24 hours after exercise.
Muscle specimens were divided into 3 samples, flash frozen
in liquid nitrogen immediately after collection, and stored at
80oC until analysis. Horses received phenylbutazone (1 g
orally twice daily for 3 days) after the fifth muscle biopsy
specimen was collected.
Plasma glucose and lactate and serum immunoreac-
tive insulin concentrationsPlasma was incubated with a
glucose reagentc in a microplated according to manufactur-
ers recommendations to determine plasma glucose concen-
trations. Absorbance of NADH at 340 nm was measured
spectrophotometricallye by accompanying software.e Plasma
was incubated with a lactate reagentc to determine plasma
lactate concentrations. Absorbance of a chromogen at 540
nm was measured spectrophotometricallye by accompany-
ing software.e Serum IRI concentrations were determined by
use of a commercially available radioimmunoassayf that has
been validated for horses.35 All analyses were performed in
duplicate.
Muscle glycogen concentrationFrozen muscle speci-
mens were freeze-dried, and 20 mg (dry weight) was
processed as previously described.36 Muscle was hydrolyzed
with 500 L of 2.0M HCl for 2 hours at 100oC. The solution
was neutralized by adding 1.5 mL of 0.66M NaOH, and the
glycogen concentration (glucosyl u/kg of dry weight) of the
hydrolysate was subsequently determined as described for
plasma glucose concentrations. The acid hydrolysis was per-
AJVR, Vol 64, No. 11, November 2003
formed in duplicate, and for each sample, the absorbance of
NADH at 340 nm was measured in duplicate spectrophoto-
metricallye by accompanying software. Twenty microliters of
a 1M glycogen solution prepared from bovine liverc was
processed similarly (ie, acid hydrolysis and spectrophoto-
metric glucose concentration determination) and used for
glycogen standards.
Muscle GLUT-4 proteinFrozen muscle specimens (20
mg) were pulverized, and whole-cell lysates were obtained by
homogenization for 30 minutes at 4oC in a buffer containing
20mM Tris (pH, 8.0), 137mM NaCl, 10% glycerol, 1%
IGEPAL, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxy-
cholate, 2mM EDTA, 1mM phenylmethylsulfonyl fluoride,
and 1X protease inhibitors.g The homogenate was centrifuged
(4oC) for 10 minutes at 3,000 X g and the supernatant col-
lected. The protein concentration of the supernatant was
determined spectrophotometrically by use of a protein assay
reagent kit.h Aliquots containing 25 g of protein were dilut-
ed (1:1) in a protein loading buffer and boiled for 5 minutes
prior to separation by electrophoresis on a 12% Tris-HCl geli
(15 wells, 15 L each well) at 100 V for 90 minutes. The pro-
teins were transferred electrophoretically to a polyvinylidene
diflouride membranej at 150 V for 120 minutes. The
polyvinylidene diflouride membrane was blocked in PBS
solution with Tween (PBST; 1X PBS solution and 0.05%
Tween) containing 10% nonfat dry milk solution for 60 min-
utes. The blots were washed in PBST and subsequently incu-
bated in PBST containing 5% nonfat dry milk solution and
primary polyclonal anti-rat GLUT-4 antisera (1:2,000)k for 60
minutes. After the blot was washed in PBST, the membrane
was incubated in PBST containing 5% nonfat dry milk solu-
tion with anti-rabbit IgG horseradish peroxidase-conjugated
secondary antibodiesl (1:2,000) for 60 minutes. The blot was
washed in PBST (3 times for 10 minutes) prior to viewing of
the antibody-bound GLUT-4 protein by use of enhanced-
chemiluminescence western blot technique detection
reagents.m Quantification of resulting enhanced-chemilumi-
nescence western blot technique films was performed by use
of a software program.n Values obtained from densitometric
scans were converted to ratios for comparison to the density
of the before-exercise sample. Evenness of loading was veri-
fied by examination of the gels stained with Coomassie Blue
and by reprobing the blots with actin monoclonal IgG.o
Prior to analyzing these samples, this western blot tech-
nique was performed on horse muscle, rat cardiac muscle
(positive control), and horse spleen (negative control) speci-
mens in order to validate the presence of the GLUT-4 protein
at the correct site (approx 43 kd). A broad-range protein
markerl and a full-range markerl were used for size determi-
nation of the GLUT-4 protein.
Muscle GLUT-4 mRNAFrozen muscle specimens
(500 mg) were pulverized, and total RNA was isolated by use
of a commercial kitp and a separation reagent.q After centrifu-
gation, isopropanol was added to the aqueous part, and the
product was frozen (20oC) for 12 hours. After centrifuga-
tion, the total RNA was washed with 70% ethanol, and for-
masol was added to stabilize the RNA. The amount of
retrieved total RNA was quantified by measuring the
absorbance at 260 and 280 nm.r An equal amount of total
RNA (30 to 45 g) was diluted (1:1) in an RNA running
buffer containing formaldehyde and a dye and incubated at
65oC for 15 minutes prior to separation by electrophoresis on
a 1.2% agar gel containing formaldehyde and ethidium bro-
mide at 100 V for 4 hours. The 5S, 18S, and 28S ribosomal
bands were viewed and photographed by ultraviolet transil-
lumination to ensure that the RNA was intact and evenly
loaded. The RNA was transferred by osmosis from the agar to
an ultraviolet membranes for 24 hours at room temperature
AJVR, Vol 64, No. 11, November 2003
(approx 20oC). Cross-linking was performed with an ultravi-
olet linkers at 1,200 J.
A northern blot technique was performed by incubation
of the membrane in hybridization bufferc and background
quencherl at 42oC for 12 hours and incubation with a probe
that contained human GLUT-4 DNA at 42oC for 12 hours.
The probe was synthesized by use of a labeling systemt and
-32P-dCTP. Detection of the GLUT-4-labeled mRNA was per-
formed by phosphorimaging.u Quantification was performed
by computer-integrated densitometry.v Presence of equal
amounts of RNA was confirmed by use of a northern blot
technique with cyclophilin receptor protein partial cDNA
that was used as a constitutive probe.
Statistical analysisData are presented as mean ( SE)
values. Data (glucose, insulin, glycogen, GLUT-4 protein,
and GLUT-4 mRNA) were analyzed by use of 2-way repeated
measures ANOVA, with treatment (SED, WATER, and
GLUC) and time as independent factors. The plasma lactate
concentrations were analyzed by use of a 1-way repeated
measures ANOVA. The null hypothesis was rejected at
= 0.05 for the main effects (time and treatment). Significant
differences identified by ANOVA were isolated by use of the
Student-Newman-Keuls method for pairwise multiple com-
parison post hoc test. The statistical computations were per-
formed with a software package.w Values of P < 0.05 were
considered significant
Results
Experimental protocolHorses were run at 5.5
0.1 m/s to achieve 45% of VO2max. Volumes of water and
glucose solution used in each trial were identical and
were 4.7 0.2 L.
Plasma glucoseA significant effect of treatment
on plasma glucose concentrations was found (Fig 1).
Plasma glucose concentrations for SED-group horses
remained constant during the study period (5.47
0.05 mmol/L). A significant increase in the plasma glu-
cose concentration after exercise was found in both
GLUC-group and WATER-group horses. Plasma glu-
cose concentrations after exercise were 8.79 1.37 and
9.59 1.57 mmol/L for GLUC-group and WATER-
group horses, respectively. In the WATER-group hors-
es, plasma glucose concentrations declined rapidly and
were not significantly different from those of the SED-
group horses 30 minutes after exercise. In contrast,
hyperglycemia persisted in the GLUC-group horses,
with plasma glucose concentrations remaining signifi-
cantly higher than in the SED-group and WATER-
group horses until 3 hours after exercise.
Serum immunoreactive insulinA significant
effect of treatment on serum IRI concentrations was
found (Fig 1). Serum IRI concentrations for the SED-
group horses remained constant during the study time
(7.57 1.31 IU/mL). A slight increase of the serum
IRI concentration after exercise was found in the
GLUC-group and WATER-group horses; however, this
increase was not significant. Serum IRI concentrations
after exercise were 10.14 2.17 and 9.51
2.25 IU/mL for GLUC-group and WATER-group
horses, respectively. In the WATER-group horses,
serum IRI concentrations remained similar to those of
the SED-group horses. In contrast, serum IRI concen-
trations in the GLUC-group horses increased signifi-
cantly from before exercise (6.68 0.93 IU/mL) to
1403

Figure 1Mean ( SE) plasma glucose (A) and serum
immunoreactive insulin (B) concentrations before (pre) and after
(post) exercise and sham exercise. Treatments were either sham
exercise followed by water administration (Sedentary) or 60
minutes of exercise
at 45% of their maximal rate of oxygen con-
sumption (V O2max) followed by water (Water) or glucose
(Glucose) administration. *Significantly (P < 0.05) different from
sedentary-group horses. $Significant (P < 0.05) difference
between glucose-group horses and water-group horses.
#Significant (P < 0.05) difference between sedentary-group
horses and water-group horses.
Figure 2Mean ( SE) muscle glycogen concentration before
(pre) and after (post) exercise. Treatments were either sham
exercise followed by water administration
(Sedentary) or 60
minutes of exercise at 45% of V O2max followed by water (Water)
or glucose (Glucose) administration. *Significantly (P < 0.05) dif-
ferent from immediately before exercise for glucose-group hors-
es. #Significantly (P < 0.05) different from immediately before
exercise for water-group horses.
23.31 5.81 IU/mL at 1 hour, 26.32 7.24 IU/mL points, the muscle glycogen concentration remained sig-
at 2 hours, and 23.26 9.02 IU/mL at 3 hours after
exercise and glucose administration. This hyperinsu-
linemic response was significant from 1 to 3 hours after
exercise and glucose administration, compared with
the SED-group and WATER-group horses.
Plasma lactatePlasma lactate concentration of the
SED-group horses taken immediately after the sham exer-
cise was 0.74 0.03 mmol/L. In the GLUC-group and
1404
Figure 3Western immunoblot of rat heart (RH), equine skeletal
muscle (EM), and equine spleen (ES). Notice the presence of glu-
cose transporter-4 (GLUT-4) protein in RH and EM, but not in ES.
Figure 4Representative western immunoblot demonstrating
detection of GLUT-4 protein. Analysis of EM before (pre) exer-
cise and at 5 minutes (post) and 4, 8, and 24 hours after exer-
cise from 1 horse after undergoing sedentary, water, and glu-
cose treatments. Notice the evenness of loading in the
Coomassie Blue (CB) stained gel.
WATER-group horses, the plasma lactate concentrations
immediately after exercise were significantly higher at
3.99 0.51 mmol/L (P = 0.016) and 3.44 0.35 mmol/L
(P = 0.015), respectively, compared with SED-group hors-
es.
Muscle glycogenMean ( SE) muscle glycogen
concentration before exercise in the SED-group horses
was 600 47 mmol/kg of dry weight and did not change
significantly during the trial (Fig 2). The before-exercise
muscle glycogen concentration in the GLUC-group hors-
es was 616 38 mmol/kg of dry weight, and this
decreased significantly to 434 32 mmol/kg of dry weight
immediately after exercise. At 4 hours after exercise, the
muscle glycogen concentration remained decreased at
434 30 mmol/kg of dry weight. At 8 and 24 hours after
exercise, muscle glycogen concentration increased from
values immediately after exercise; however, at both time
points, the muscle glycogen concentration remained sig-
nificantly lower than in the before-exercise sample. The
before-exercise muscle glycogen concentration in the
WATER-group horses was 700 102 mmol/kg of dry
weight, and this decreased significantly to 490 59
mmol/kg of dry weight immediately after exercise. At 8
and 24 hours after exercise, an increase in muscle glyco-
gen concentration was found; however, at both time
nificantly lower than in the before-exercise sample.
Muscle GLUT-4 proteinThe GLUT-4 protein
was readily detected at approximately 43 kd in rat
heart and equine muscle, but not in equine spleen
(Fig 3). No detectable differences in density of the
bands were found at the 5 time points (before exercise
to 24 hours after exercise) nor between the 3 treatment
groups (Fig 4 and 5).
AJVR, Vol 64, No. 11, November 2003

Figure 5Mean ( SE) arbitrary units of muscle GLUT-4 protein
content pre- and post-exercise. Treatments were either sham
exercise followed by water administration
(Sedentary) or 60
minutes of exercise at 45% of VO2max followed by water (Water)
or glucose (Glucose) administration. Arbitrary units are defined
as the ratio of density of GLUT-4 in the after-treatment muscle
specimen to the density of GLUT-4 in the before-treatment mus-
cle specimen.
Figure 6Northern blot of GLUT-4 mRNA and cyclophilin (CP)
as detected at 2.7 and 0.7 kilobases (kb), respectively.
Analysis of EM (pre) exercise and at 5 minutes and 4, 8, and
24 hours after (post) exercise from 1 horse after undergoing
sedentary, water, and glucose treatments. *Nonspecific bind-
ing.
Figure 7Mean ( SE) arbitrary units of muscle GLUT-4 mRNA
before (pre) and after (post) exercise. Treatments were either
sham exercise followed by water administration
(Sedentary) or
60 minutes of exercise at 45% of VO2max followed by water
(Water) or glucose (Glucose) administration. Arbitrary units are
defined as the ratio of density of GLUT-4 mRNA to the density
of the housekeeping gene, CP.
Muscle GLUT-4 mRNAThe GLUT-4 mRNA was
detected by use of a northern blot technique between
the 18S and 28S bands at approximately 2.7 kilobases
(kb). Results of northern blot analysis did not reveal
significant differences between the 5 time points
(before exercise to 24 hours after exercise) nor
between the 3 treatment groups (Fig 6 and 7). This
AJVR, Vol 64, No. 11, November 2003
was confirmed by densitometry on the GLUT-4 mRNA
bands normalized to the cyclophilin bands that were
detected by northern blot analysis at approximately 0.7
kb.
Discussion
No detectable effect of a single bout of exercise by
moderately trained horses was found on muscle GLUT-
4 protein or mRNA content. Horses that received glu-
cose had significantly higher plasma glucose and
serum IRI concentrations for 3 hours after exercise, but
this did not affect muscle GLUT-4 protein or mRNA
content. Exercise in our study resulted in a 30% reduc-
tion of muscle glycogen concentration; however,
increased substrate availability did not enhance muscle
glycogen resynthesis. Glycogen replenishment was
similar in both exercised groups and was not complete
at 24 hours after exercise.
Glucose transporter-4 is the main insulin-respon-
sive glucose transporter and is recycled between intra-
cellular storage pools and the plasma membrane, trans-
verse tubules, and sarcolemmal vesicles.37,38 In the
absence of stimuli such as insulin or exercise, approxi-
mately 90% of GLUT-4 is sequestered intracellularly.39
The exact mechanism for the translocation of GLUT-4
from the storage vesicles to the plasma membrane that
occurs after exercise is unclear but may involve calcium,
5-AMP-activated kinase, nitric oxide, and mitogen-acti-
vated protein kinase.40 Results of our study indicate that
a single bout of moderate to intense exercise has no
effect on muscle GLUT-4 protein content, which is sim-
ilar to what has been reported by McCutcheon et al.17 In
rat muscle experiments performed in vitro, a single bout
of exhaustive exercise did not result in immediate
change of GLUT-4 protein or mRNA content.41 Similarly,
Kuo et al20 did not find an increase in rat skeletal muscle
GLUT-4 protein content immediately after exercise;
however, a significant increase was found between 1 and
16 hours after exercise.19,20 A single bout of exercise in
rats also increased muscle GLUT-4 content 18 hours
after exercise.42 Results of another study revealed no
increase of GLUT-4 protein content after exercise, but
did indicate that a single bout of exercise induced
translocation of GLUT-4 to the cell membrane.38
Furthermore, the immediate increase of GLUT-4 protein
in the cell membrane appears to be associated with an
increase in insulin-mediated membrane glucose trans-
port, both of which return back to baseline rapidly after
exercise has ceased.43 Although findings in our study
and a study17 performed earlier in horses did not provide
evidence for an increase in muscle GLUT-4 protein con-
tent after exercise, the degree of translocation of GLUT-
4 from the intracellular vesicles to the plasma membrane
was not determined. In both studies, whole-cell GLUT-
4 protein was determined, rather than the membrane-
bound fraction of GLUT-4. In order to study transloca-
tion of GLUT-4 within the cell, separation of cellular
components is required so that intracellular and sar-
colemmal GLUT-4 protein content can be determined.
This procedure was not performed in our study.
Whereas increases in muscle GLUT-4 protein content
immediately after a single bout of exercise are small, if
present at all, exercise training of at least 7 days dura-
1405
tion has been shown to result in a significant increase in
muscle GLUT-4 protein content in humans,7,8 rats,2,9-11,13-16
and horses.17 However, this increase in muscle GLUT-4
protein content following endurance training is associ-
ated with decreased translocation of GLUT-4 to the sar-
colemmal membrane and decreased glucose utiliza-
tion.44 Trained animals have an increased muscle glyco-
gen concentration at rest, and an increased muscle
glycogen concentration decreases glucose utilization.45 It
may be speculated that the increased GLUT-4 content in
trained animals allows a rapid increase in glucose uti-
lization during high-intensity exercise.
No significant effect was detected, under the con-
ditions of our study, of exercise on GLUT-4 gene
expression. This is in contrast to what has been found
previously in humans18 and rats.2,16,20 In rats, GLUT-4
mRNA was increased immediately after exercise, but
declined rapidly back to baseline when measured at 1.5
and 5.0 hours after exercise.20 Similarly, Neufer et al16
revealed that exercise in trained and untrained rats
results in an increase in GLUT-4 mRNA content at 3
hours after exercise, but no change is found at 30 min-
utes and 24 hours after exercise. Interestingly, further
studies2,19 have revealed significant increases in GLUT-
4 mRNA and protein content at 16 hours after exercise.
Similar to what has been shown for GLUT-4 protein
content, exercise training has been shown to increase
muscle GLUT-4 mRNA content.13 However, this
increase in GLUT-4 mRNA appears to be short-lived, as
in 1 study15 GLUT-4 mRNA was increased at 24 hours
after the last bout of exercise in an exercise training
program, but returned to baseline at 48 hours after
exercise and was not different from that before the
training program.
Other factors that may have played a role in the
lack of GLUT-4 mRNA change in our study are related
to the exercise protocol and the analytic technique
used. First, the stimulus in our study may not have
been sufficient to induce increased gene transcription.
Future studies may need to examine GLUT-4 gene
expression in horses in which a greater degree of
glycogen depletion has been achieved; muscle glyco-
gen depletion was 50% in rats in which an effect of
exercise on GLUT-4 gene expression was detected.
Second, detection of subtle differences in horse mus-
cle mRNA may be more accurate when the northern
blot technique is performed with a species-specific
probe. The sequence of the equine GLUT-4 gene has
been determined, but results were not available for our
study.46
The amount of muscle glycogen depletion that was
achieved in our study was similar to what has been
reported earlier.47 Furthermore, similar to what has
been reported by other investigators, glycogen replen-
ishment was not complete at 24 hours after exer-
cise.29,48-50 In our study, no effect of increased substrate
availability was observed on glycogen resynthesis,
which has also been shown in other equine studies.48-50
More recently, investigators have been able to demon-
strate increased glycogen replenishment following IV
administration of large amounts of glucose.31,32 In
humans and rats, however, it has been well established
that increased substrate availability provided orally or
1406
IV enhances muscle glycogen resynthesis.30 Moreover,
in rats51 and humans,8 the increased muscle glycogen
resynthesis following exercise and carbohydrate feed-
ing has been associated with increased GLUT-4 protein
content. The lack of effect of carbohydrate supplemen-
tation on GLUT-4 protein and mRNA content and
glycogen resynthesis in our study is interesting and
may be the result of species differences in metabolism
and work intensity.
The GLUT-4 is the major transporter responsible
for glucose transport after exercise, but it is not essen-
tial for glycogen resynthesis. In mice deficient of
GLUT-4, glycogen resynthesis is delayed, and glycogen
super compensation does not occur; however, glyco-
gen replenishment is achieved at 24 hours after exer-
cise.21 Furthermore, in rats, glucose transport and cell
surface GLUT-4 content are dependent on muscle
glycogen content, with decreased GLUT-4 transloca-
tion to the plasma membrane present in muscle with a
high glycogen concentration.45,52 Also, glucose adminis-
tration following exercise lessened the exercise-
induced increase of GLUT-4 mRNA in rats.19 Moreover,
hyperglycemia in rats has been shown to result in a sig-
nificant decrease in glucose transport and in GLUT-4
protein and mRNA content.53,54 This provides evidence
for translational rather than pretranslational events
that predominantly regulate GLUT-4 gene expression.
Carbohydrate administration, therefore, has a modify-
ing rather than stimulating effect on exercise-induced
GLUT-4 gene expression. This may indicate that unless
muscle glycogen is considerably depleted, no substan-
tial effects from enhanced substrate delivery on muscle
glycogen replenishment should be expected. Other
potential causes for the lack of effect of hyperglycemia
on glycogen replenishment include saturation of
GLUT-4, other rate-limiting factors, and replenishment
of other glycogen stores, such as the liver glycogen
stores, first. In humans, the Michaelis-Menten constant
of GLUT-4 is 2 to 10 mmol/L, which suggests that this
transport system can be saturated during physiologic
conditions55 and may be fully effective in horses after
exercise. Although few reports exist on liver glycogen
resynthesis following exercise, it appears that muscle
glycogen replenishment occurs prior to liver glycogen
replenishment.56,57
Although in our study we did not find increases in
either GLUT-4 protein or GLUT-4 mRNA after a single
bout of exercise, we did find a substantial hyper-
glycemic and hyperinsulinemic response following
glucose administration after exercise that resulted in a
30% reduction of muscle glycogen stores. We speculate
that the lack of GLUT-4 transcription and translation
under these conditions is the result of species differ-
ences in glucose metabolism and glycogen synthesis.
Further studies in horses that have performed exercise
that induces a greater reduction in muscle glycogen
concentration or is repeated over several days are
required to determine the significance of GLUT-4 in
equine skeletal muscle.
a
Sato, BIAB Industrial, Uppsala, Sweden.
b
Oxymax-XL, Columbus Instruments, Columbus, Ohio.
c
Sigma Chemical Co, St Louis, Mo.
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Powerwave X select and KC4v.3.0 with Power Reports, Bio-Tek
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f 15. Reynolds TH IV, Brozinick JT Jr, Larkin LM, et al. Transient
Coat-A-Count Insulin, Diagnostics Products Corp, Los Angeles,
Calif. enhancement of GLUT-4 levels in rat epitrochlearis muscle after
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Boehringer Mannheim, Indianapolis, Ind.
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Pierce, Rockford, Ill.
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Immobilon-P transfer membrane, Milipore Corp, Bedford, Mass.
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Donated by Dr. Samuel W. Cushman, Experimental Diabetes,
Metabolism, and Nutrition Section Chief, Diabetes Branch, tal muscle GLUT4 content and muscle membrane glucose transport
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