Am J Physiol Endocrinol Metab 300: E761–E770, 2011.
First published February 15, 2011; doi:10.1152/ajpendo.00207.2010.
Effect of antioxidant supplementation on insulin sensitivity in response
to endurance exercise training
Christina Yfanti,1 Anders R. Nielsen,1 Thorbjörn Åkerström,1 Søren Nielsen,1 Adam J. Rose,2
Erik A. Richter,2 Jens Lykkesfeldt,3 Christian P. Fischer,1 and Bente K. Pedersen1
1
Centre of Inflammation and Metabolism, Department of Infectious Diseases and CMRC, Rigshospitalet, Faculty of Health
Sciences; 2Molecular Physiology Group, Department of Exercise and Sport Sciences, Section of Human Physiology; and
3
Section of Biomedicine, Department of Disease Biology, Faculty of Life Sciences, University of Copenhagen,
Copenhagen, Denmark
Submitted 8 April 2010; accepted in final form 21 September 2010
Yfanti C, Nielsen AR, Åkerström T, Nielsen S, Rose AJ, Richter
EA, Lykkesfeldt J, Fischer CP, Pedersen BK. Effect of antioxidant
supplementation on insulin sensitivity in response to endurance exercise
training. Am J Physiol Endocrinol Metab 300: E761–E770, 2011. First
published February 15, 2011; doi:10.1152/ajpendo.00207.2010.—While
production of reactive oxygen and nitrogen species (RONS) is asso-
ciated with some of the beneficial adaptations to regular physical
exercise, it is not established whether RONS play a role in the
improved insulin-stimulated glucose uptake in skeletal muscle ob-
tained by endurance training. To assess the effect of antioxidant
supplementation during endurance training on insulin-stimulated glu-
cose uptake, 21 young healthy (age 29 ⫾ 1 y, BMI 25 ⫾ 3 kg/m2) men
were randomly assigned to either an antioxidant [AO; 500 mg vitamin
C and 400 IU vitamin E (␣-tocopherol) daily] or a placebo (PL) group
that both underwent a supervised intense endurance-training program
5 times/wk for 12 wk. A 3-h euglycemic-hyperinsulinemic clamp, a
maximal oxygen consumption (V̇O2max) and maximal power output
(Pmax) test, and body composition measurements (fat mass, fat-free
mass) were performed before and after the training. Muscle biopsies
were obtained for determination of the concentration and activity of
proteins regulating glucose metabolism. Although plasma levels of
vitamin C (P ⬍ 0.05) and ␣-tocopherol (P ⬍ 0.05) increased mark-
edly in the AO group, insulin-stimulated glucose uptake increased
similarly in both the AO (17.2%, P ⬍ 0.05) and the PL (18.9%, P ⬍
0.05) group in response to training. V̇O2max and Pmax also increased
similarly in both groups (time effect, P ⬍ 0.0001 for both) as well as
protein content of GLUT4, hexokinase II, and total Akt (time effect,
P ⱕ 0.05 for all). Our results indicate that administration of antiox-
idants during strenuous endurance training has no effect on the
training-induced increase in insulin sensitivity in healthy individuals.
vitamin C; vitamin E; glucose uptake
RECENTLY, IT WAS SHOWN that a high-fat diet can induce insulin
resistance by increasing the skeletal muscle mitochondrial
production of hydrogen peroxide (1). Conversely, shifting the
balance back toward antioxidant defense by dietary adminis-
tration of antioxidants partially improves health indexes in-
cluding insulin-stimulated glucose disposal in individuals with
type 2 diabetes (20), and, in particular, combined vitamin E
and C supplementation may improve insulin-stimulated glu-
cose metabolism in insulin-resistant, elderly individuals (41).
On the other hand, endurance exercise is also associated
with the generation of reactive oxygen and nitrogen species
(RONS) by the mitochondria as by-products of oxidative
Address for reprint requests and other correspondence: C. Yfanti, Rigshospita-
let, Centre of Inflammation and Metabolism, 7641, Blegdamsvej 9, DK-2100
Copenhagen, Denmark (e-mail: christinayfanti@inflammation-metabolism.dk).
http://www.ajpendo.org 0193-1849/11 Copyright © 2011 the American Physiological Society
metabolism. Despite this, regular physical exercise promotes
numerous health benefits, protects against all caused mortality
(4), and plays a critical role in the treatment of a number of
chronic diseases, such as type 2 diabetes (34). Accordingly,
endurance exercise training has been shown to improve insulin
resistance in patients with type 2 diabetes (15, 28) and en-
hances both insulin-stimulated and non-insulin mediated glu-
cose uptake in skeletal muscle (15, 16). The enhanced insulin-
stimulated glucose disposal by skeletal muscle occurs as a
result of increased protein expression of hexokinase II (HKII),
glucose transporter 4 (GLUT4) (21, 39, 52) and increased
expression or activity of several other molecules involved in
the insulin cascade (21, 39, 52).
There has been considerable evidence that exercise-induced
RONS production plays an important role in the regulation of
signaling pathways (13, 37). In particular, both in vitro (2) and
in vivo (11) experiments have shown that nitric oxide (NO),
essential for the formation of RNS, acts as a stimulus for
exercise-mediated skeletal muscle glucose uptake, indicating a
possible mechanism of the enhanced insulin sensitivity in
response to exercise training.
The RONS-dependent expression (44) of the transcription
factor peroxisome proliferator-activated receptor-␥ (PPAR␥)
and its coactivator-1␣ (PGC-1␣), which increase the expres-
sion of genes involved in mitochondrial biogenesis and oxida-
tive phosphorylation (10), has been shown to be reduced in
type 2 diabetes patients (33). In contrast, endurance training
has been suggested to increase the expression of both PPAR␥
(45) and PGC-1␣ (42, 48), although there have been some
controversial results (35, 49). Furthermore, overexpression of
skeletal muscle PGC-1␣ increases insulin-stimulated glucose
disposal both in insulin-resistant (6) and in healthy (7) rats.
Recently, the hypothesis that antioxidant supplementation
during endurance training may attenuate some of the bene-
ficial effects of endurance exercise has been suggested. For
instance, Gomez-Cabrera et al. (23) found that vitamin C
supplementation in rodents attenuates training-induced mi-
tochondrial biogenesis and endurance capacity. Another
recent study showed that antioxidant supplementation with
vitamins C and E inhibits the beneficial effects of 4 wk of
training on insulin sensitivity and other markers of meta-
bolic training adaptation (40). Moreover, when a 6-wk
aerobic exercise training program is applied in patients with
hypertension, supplementation with antioxidants (vitamins
C and E, and ␣-lipoic acid) is associated with elevated blood
pressure and inhibition of exercise-induced flow-mediated
vasodilatation (50). In support of this hypothesis, experi-
E761
E762 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
ments in mice have shown that when the antioxidant enzyme
glutathione peroxidase (GPX) is overexpressed, the animals
become obese and develop insulin resistance (32), whereas
in case of lacking this enzyme, they are protected from
high-fat diet-induced insulin resistance (45), both indicating
the importance of RONS on insulin signaling and the dele-
terious effects of blocking their action. Thus, even though
antioxidant treatment may improve glucose metabolism in
patients with type 2 diabetes, it may also inhibit other
positive health benefits of regular physical activity.
To examine whether antioxidant treatment affects the adap-
tive increase in insulin sensitivity with regular physical activ-
ity, we conducted a training study using a highly demanding
training protocol in order to achieve the maximum possible
increase in insulin sensitivity. We hypothesized that adminis-
tration of vitamins C and E during training would attenuate the
expected increases in insulin sensitivity in healthy young
individuals.
MATERIALS AND METHODS
Ethical approval. The study was approved by the local Ethics
Committee of Copenhagen and Frederiksberg (KF 01 289434) and
was in accordance with the Declaration of Helsinki. The purpose of
the study and possible risks and discomforts were explained to the
participants before written consent was obtained.
Participants. Twenty-one young, healthy, physically active men
(age 18 – 40 yr) participated in the study. Participants’ characteristics
are listed in Table 1. Before inclusion in the study, participants
underwent a medical examination with blood test screening, an oral
glucose tolerance test (OGTT), and a maximal power (Pmax) test using
an electrically braked cadence-independent cycle ergometer (Monark
839E; Monark, Varberg, Sweden). Exclusion criteria included phys-
ical exercise more than 2–3 times/wk, body mass index (BMI) ⬎ 30
kg/m2, smoking, impaired glucose tolerance, use of medication, and
supplementation with antioxidants.
Supplementation. A double-blinded design was employed, and the
participants were allocated to either the Antioxidant (AO) (n ⫽ 11) or
the Placebo (PL) (n ⫽ 10) group by using a minimization model (47)
according to age, BMI, and Pmax. Participants in the AO group
received oral supplementation with vitamin C (ascorbic acid, 500 mg
daily) and vitamin E (RRR-␣-tocopheryl succinate, 400 IU daily) for
16 wk, while the PL group received placebo tablets. The employed
supplement dosages of vitamin C and vitamin E were ⬃5 and ⬃15
times higher, respectively, than the recommended dietary allowances.
Of note, this combination of vitamins C and E has previously been
shown to attenuate oxidative stress following acute exercise (19). All
participants were instructed to take the supplementation once a day
with breakfast. The supplementation started 4 wk before onset of the
training and continued through the whole training period.
Pmax and V̇O2max. V̇O2max was measured during an incremental
exercise test on an electrically braked cadence-independent cycle
ergometer (Monark 839E) using an indirect calorimetry system
Table 1. Subject baseline characteristics
Antioxidant Group Placebo Group
(n ⫽ 11) (n ⫽ 10)
Age, yr 29 (25–32) 31 (27–34)
Weight, kg 80 (72–88) 81 (75–87)
Height, cm 179 (174–183) 180 (177–184)
BMI, kg/m2 25 (23–27) 25 (24–26)
V̇O2max, ml kg⫺1 min⫺1 50 (46–55) 52 (46–59)
Values are presented as means ⫾ 95% confidence interval (CI). No statis-
tically significant difference between the groups.
AJP-Endocrinol Metab • VOL
(Quark b2; CosMed, Rome, Italy). After a 5-min warm-up at 40% of
V̇O2max, the workload increased every minute by 1/10 of 60% of
expected V̇O2max (12). The expected V̇O2max was calculated on the
basis of the Pmax obtained from the screening test. The test was
terminated when the participant was unable to maintain a cadence of
60 rpm for more than 15 s despite verbal encouragement. Expired
oxygen and carbon dioxide were recorded online. V̇O2max tests were
performed before and after the training period.
The Pmax test was performed in the same way as the V̇O2max test
without recording of expired gasses. A Pmax test was performed at the
prescreening for allocation of the subjects to the two groups and at the
beginning of each training week to determine the intensity of the training
for the following days of the week.
Training protocol. The mode of exercise selected for training was
cycling, and the training frequency was five times a week for 12 wk.
A Pmax test was performed every Monday to determine the intensity
of the training for the following days of the week. Every Tuesday and
Thursday, the training consisted of intervals (intensity 75–91% Pmax
and duration 60 – 80 min) and every Wednesday and Friday of
continuous biking (intensity 55– 66% Pmax and duration 85–155 min).
The participants were allowed to miss only 5% of the total amount of
training, which was equal to 3 training days. In case they had to
refrain from training for more than 3 days, the missing training was
performed during the weekend.
Euglycemic-hyperinsulinemic clamp. The participants reported at
the laboratory at 8:00 AM after an overnight fast. An intravenous
catheter was placed in an antecubital vein of one arm for infusion of
insulin and glucose. A second intravenous catheter was placed in a
dorsal hand vein of the contralateral arm for blood sampling. The
catheterized hand was wrapped in a heating blanket to obtain arteri-
alized venous blood samples for measurement of glucose and potas-
sium concentrations during the clamp. After baseline blood samples
had been obtained, infusion of insulin (100 IU/ml Actrapid; Novo
Nordisk Insulin, Copenhagen, Denmark) started at a constant rate of
80.0 mU·min⫺1·m⫺2 body surface area. Euglycemia (5 mM) was
achieved by coinfusion of glucose (200 g/1,000 ml) at a variable rate.
To maintain potassium at normal value, isotonic saline with potassium
(51 meq/l) was coinfused if needed. Arterialized blood was analyzed
for glucose and potassium concentrations every 10 min. Euglycemic-
hyperinsulinemic clamps were performed before and ⬎72 h after the
last training bout of the training period.
Blood samples. Blood samples were drawn at baseline and at time
points 60, 120, and 180 min after initiation of the clamp. The blood
samples were drawn into EDTA-containing glass tubes, and they were
immediately centrifuged at 3,500 g for 15 min at 4°C. Plasma samples
were then stored in ⫺80°C until analyzed.
Muscle biopsies. Muscle biopsies from vastus lateralis were taken
at time points 0 and 180 min during the insulin clamp, before and after
the training period. All tissue samples were obtained using the
percutaneous needle method with suction (8) under local anesthesia of
the skin and fascia, using 3–5 ml of 20 mg/ml Lidocaine (SAD
Denmark). Muscle tissue was immediately frozen in liquid nitrogen
and stored at ⫺80°C until further analysis.
Body composition. Whole body fat and fat-free tissue mass were
estimated using a dual-energy X-ray absorptiometry (DEXA) scanner
with software version 8.8 (Lunar Prodigy; GE Medical Systems).
Laboratory analysis: plasma. Plasma insulin was measured with a
singleplex assay for human insulin using electrochemiluminescence
(product no. K151BZC; Meso Scale Discovery) according to the
manufacturer’s instructions. The lower limit of detection is 6.0 pg/ml.
The mean coefficient of variance was ⬍10% between duplicates.
Plasma concentration of free fatty acids (FFA) was determined in
duplicates using an automatic analyzer (Cobas fara, Roche; FFA:
NEFA C; Wako Chemicals, Neuss, Germany). High-density lipopro-
tein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol,
and triglycerides (TG) were determined using standard laboratory
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 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY E763
Table 2. Changes in vitamin plasma concentration over time
Antioxidant Group (n ⫽ 11) Placebo Group (n ⫽ 10)

Presupplementation Pretraining Posttraining Presupplementation Pretraining Posttraining

Vitamin C 53.8 (42.2–65.4) 82.0 (68.6–95.4)* 75.0 (60.7–89.2)* 64.7 (56.3–73.2) 66.8 (54.2–79.4) 59.4 (43.1–75.7)
Vitamin E 17.0 (15.1–19.1) 25.4 (22.0–28.0)* 22.5 (20.2–25.0)* 20.8 (17.8–24.2) 21.3 (18.5–24.5) 19.8 (17.0–23.0)
Values are presented as means ⫾ 95% CI in ␮mol/l. *P ⬍ 0.05 vs. presupplementation values.

procedures (Department of Clinical Biochemistry, Rigshospitalet Uni-
versity Hospital, Copenhagen, Denmark).
RNA isolation and quantitative real-time PCR. Skeletal muscle
biopsies were homogenized in TRIzol Reagent (Invitrogen, Carlsbad,
CA) using a motor-driven homogenizer (Polytron, Kinematica, New-
ark, NJ), and total RNA was isolated according to the manufacturer’s
protocol. Total RNA was dissolved in RNase-free water, quantified
spectrophotometrically at 260 nm, and reverse-transcribed using re-
verse transcription reagents (Applied Biosystems, Foster City, CA)
according to the manufacturer’s protocol. Random hexamers were
used for first-strand cDNA synthesis. The mRNA levels of of PPAR␥
and PGC-1␣, and the endogenous control 18S rRNA were determined
by real-time RT-PCR using an ABI PRISM 7900HT sequence detec-
tor (Applied Biosystems).
Primers and MGB probes were designed using Primer Express
software (Applied Biosystems) or obtained using the Universal Probe
Library (Roche Applied Science).
Primers and probes were premixed with TaqMan Universal Master
Mix or SYBR Green PCR Master Mix (Applied Biosystems) and
distributed into 384-well MicroAmp optical plates (Applied Biosys-
tems). cDNA aliquots of 3 ␮l were added in triplicates. The amplifi-
cation of genomic DNA typically amounted to a maximum of ⬍1% of
the target gene when the TRIzol protocol was used. A twofold dilution
series was made from a pooled sample of a small part of all the
samples. This was run on each plate together with the samples and
used to construct a standard curve from which the mRNA content of
the target genes was calculated from the cycle threshold (CT) values
of the unknown samples. The relative expressions of PPAR␥ and
PGC-1␣ were determined after normalization to the endogenous
control 18S.
Muscle lysate preparation. Twenty-five milligrams of skeletal
muscle was used for protein extraction. Muscle lysates were prepared
adding 500 ␮l of ice-cold homogenization buffer [50 mM Tris, 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 5
mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM
PMSF, 1 mM dithiotreitol, 0.5% (vol/vol) protease inhibitor cocktail
and 1% (vol/vol) Nonidet P-40] to muscle tissue. The muscle tissue
was then homogenized using cooled racks in a TissueLyser (Qiagen)
for 1 min at 20 Hz followed by a 15-min incubation on ice. The
procedure was repeated twice and, if there were still visible particles
of the tissue, a third time. Homogenates were then rotated end over
end for 1 h at 4°C and centrifuged at 13,000 g at 4°C for 15 min. The
supernatant was taken and stored at ⫺80°C. An aliquot of 10 ␮l of
each lysate was taken and diluted for protein concentration determi-
nation prior to storage.
Protein concentration of tissue extracts was determined in triplicate
using the bicinchoninic acid (BCA) method using bovine serum
albumin standards (Pierce, Rockford, IL) and BCA assay reagents
(Pierce). A maximal coefficient of variance of 5% was accepted
between replicates.
Electrochemiluminescence. Total and phosphorylated (Tyr1150/1151)
insulin receptor (IR) protein content was measured using a multiplex
electrochemiluminescence assay for insulin signaling (product nos.
K15152C for total protein and K15151C for phosphorylated protein,
MSD), according to the manufacturer’s instructions (5).
Western blotting. Equal amounts of denatured proteins (27) from
the tissue homogenates were separated by gel electrophoresis using a
NuPage 8% Bis-Tris gel (Invitrogen, Taastrup, Denmark), followed
by immunoblotting to PVDF membranes (Hybond-P, GE Healthcare).
Membranes were then incubated in blocking buffer (skimmed milk)
for 1 h at room temperature to reduce background signal. Subse-
quently, the membranes were incubated with primary and secondary
antibodies for optimized times and concentrations and washed with
Tris-buffered saline containing 0.1% Tween-20. Primary antibodies
used were anti-Akt substrate of 160 kDa (AS160; catalog no. 2447,
Cell Signaling Technology), anti-phospho-Ser/Thr-AS160 (catalog
no. 9611, Cell Signaling Technology), anti-Akt (catalog no. 9272,
Cell Signaling Technology), anti-phospho-Ser473-Akt (catalog no.
9271, Cell Signaling Technology), anti-GLUT4 (catalog no. PA1-
1065, Affinity Bioreagents), and anti-HKII (catalog no. 6521, Santa
Cruz Biotechnology). Secondary antibodies were from DakoCytoma-
tion (Denmark). Protein bands were detected using Supersignal West
Femto (Pierce) and quantified using a CCD image sensor (Chemi-
DocXRS, Bio-Rad) and software (Quantity One, Bio-Rad).
Preliminary experiments demonstrated that the amounts of protein
loaded were within the dynamic range for the conditions used and the

Table 3. Changes before and after 12 wk of endurance training

Antioxidant Group (n ⫽ 11) Placebo Group (n ⫽ 10)

Pre Post Pre Post

Fitness
V̇O2max, ml kg⫺1 min⫺1 50.4 (45.8–55.0) 58.9 (53.8–64.0)† 52.1 (45.7–58.5) 62.4 (55.3–69.5)†
Body composition
Fat mass, kg 15.2 (9.1–21.3) 13.9 (8.8–18.9) 16.1 (11.1–21.1) 12.7 (7.8–17.6)
Fat-free mass, kg 60.2 (55.0–65.3) 61.5 (57.4–65.7) 61.9 (58.1–65.6) 63.7 (60.0–67.4)
Body weight, kg 79.7 (71.5–87.9) 78.2 (70.8–85.7) 80.9 (74.8–87.1) 79.2 (73.1–85.3)
Metabolic parameters
HDL, mmol/l 1.4 (1.3–1.6) 1.7 (1.5–1.9)* 1.5 (1.3–1.8) 1.8 (1.6–1.9)*
LDL, mmol/l 2.5 (2.1–2.9) 2.6 (2.3–3.0) 2.8 (2.3–3.4) 2.9 (2.3–3.6)
TG, mmol/l 0.9 (0.7–1.2) 1.0 (0.7–1.3) 0.9 (0.7–1.2) 1.3 (0.9–1.9)
FFA, ␮mol/l 414.7 (288.0–541.5) 339.4 (204.3–474.5) 452.0 (331.6–572.4) 398.4 (334.6–462.2)
Glucose, mmol/l 5.3 (5.0–5.7) 5.4 (5.1–5.7) 5.2 (5.0–5.5) 5.2 (5.0–5.4)
Values are presented as means ⫾ 95% CI. TG, triglycerides; FFA, free fatty acids. *P ⬍ 0.05, †P ⬍ 0.0001 vs. pre training values.

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E764 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY

results obtained (data not shown), and immunoreactive bands mi-
grated at expected relative mobilities. Reactive Brown (RB) protein
stain (51) was used as a loading and transfer control.
Analysis of vitamin C and ␣-tocopherol. Plasma samples for
vitamin C measurements were mixed with an equal amount of 10%
metaphosphoric acid containing 2 mM disodium-EDTA and frozen
immediately until analysis. The concentration of ascorbate was mea-
sured by reversed-phase HPLC with colorimetric detection (31). The
concentration of ␣-tocopherol in plasma samples was measured by
HPLC with amperometric detection (18).
Statistical analysis. All data were tested for normality of distribu-
tion before further analysis by use of histograms and probability plots.
The data that were not normally distributed a priori were subjected to
logarithmic transformation to obtain normal distribution.
A general linear mixed model was used to analyze the effect of
time and supplementation. An interaction between the two was also
tested. A random subject-specific component was introduced to adjust
for interindividual variations. The fit of the model was evaluated by
testing the residuals for normal distribution and variance homogene-
ity. In the post hoc analysis, possible effects of time and group were
tested by Student’s paired t-tests. The level of significance was set at
P ⬍ 0.05.
The statistical analysis was performed using SAS statistical software
(9.1, SAS Institute, Cary, NC). Results are presented as means ⫾ SE or
geometric means ⫾ 95% confidence interval (CI) for the log-transformed
data.
RESULTS
The two groups did not differ at baseline regarding V̇O2max
and anthropometrical measurements (Table 1). Plasma insulin
concentrations during the glucose clamp did not differ between
groups and did not change with exercise training (969 ⫾ 57 to
990 ⫾ 85 pmol/l pre- vs. posttraining for AO, 1,004 ⫾ 76 to
982 ⫾ 82 pmol/l pre- vs. posttraining for PL).
Vitamins C and E concentration in plasma. Vitamin con-
centrations were measured at baseline, after 4 wk of supple-

Fig. 1. Glucose infusion rate (GIR) measured during a 180-min hyperinsulinemic-euglycemic clamp for the Antioxidant group (A) and the Placebo group (B).
GIR calculated as area under the curve (AUC) for the time 60 –180 min during hyperinsulinemic-euglycemic clamp for both groups before and after training.
GIR AUC is expressed as mg/kg body wt. Open bars, Antioxidant group (n ⫽ 11); filled bars, Placebo group (n ⫽ 10). Values are means ⫾ SE. *P ⬍ 0.05 vs.
pretraining values.

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 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
mentation without training, and at the end of 12 wk of training.
The two groups did not differ at baseline. Plasma ascorbic acid
and vitamin E (␣-tocopherol) concentrations increased (P ⬍
0.05) in the AO group after 4 wk of supplementation and
remained elevated (P ⬍ 0.05) during the whole training period
(Table 2).
V̇O2max and Pmax. V̇O2max (Table 3) increased by ⬃17% (P ⬍
0.0001) in the AO group and 20% in the PL (P ⬍ 0.0001)
group. Pmax (see Fig. 4) increased gradually every week in a
AJP-Endocrinol Metab • VOL
E765
similar way for both groups (time effect: P ⬍ 0.0001). Pmax
was 24% higher the AO (P ⬍ 0.0001) and 20% higher in the
PL (P ⬍ 0.0001) group after the training period. V̇O2max and
Pmax were similar when AO and PL were compared before as
well as after training.
Insulin-stimulated glucose uptake. Insulin-stimulated glucose
uptake was increased ⬃15% after the 12-wk training period (Fig.
1) in both the AO (P ⬍ 0.05) and the PL groups (P ⬍ 0.05). There
were no differences between the two groups (P ⫽ 0.68).
Fig. 2. Total protein content (A) and phosphor-
ylation (B) of insulin-signaling molecules be-
fore and after 12 wk of training. Values are
expressed as arbitrary units. Values for phos-
phorylated proteins are presented as ⌬ changes
between pre- and post-insulin levels. Open
bars, Antioxidant group (n ⫽ 11); filled bars,
Placebo group (n ⫽ 10). Values are means ⫾
95% CI for Akt and Akt Ser473 and as geo-
metric means ⫾ 95% CI for all other proteins.
NIS, non-insulin-stimulated; IS, insulin-stim-
ulated. *P ⬍ 0.05 vs. pretraining values.
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E766 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
Protein content of insulin signaling molecules. The protein
contents of the insulin signaling molecules IR, Akt, and
AS160, total and phosphorylated, are presented in Fig. 2.
Protein content of the total IR, Akt, and AS160 was measured
before and after the training period. Phosphorylation level of
the same proteins was measured during insulin stimulation and
before and after the training period. Both the total and the
phosphorylated proteins were normalized to the loading control
Reactive Brown protein stain. Noteworthy, using normaliza-
tion of the phosphorylated proteins to the total proteins rather
than the protein stain did not change the outcome of the data
analysis.
Total protein content of Akt was significantly increased after
12 wk of training in both groups (P ⬍ 0.05 for both groups).
There was also a tendency for increase after training of IR
(time effect: P ⫽ 0.08). There was no difference between
groups for total protein content of AS160 (P ⫽ 0.45) or for any
of the phosphorylated proteins (p-IR: P ⫽ 0.40; p-Akt: P ⫽
0.10; p-AS160: P ⫽ 0.68).
Protein contents of GLUT4 and HKII. Protein content of
HKII increased significantly in response to training in both the
AO group (P ⬍ 0.05) and the PL group (P ⬍ 0.005; Fig. 3).
With regard to GLUT4 (Fig. 3), the increase in response to
training was, however, borderline significant (P ⫽ 0.05). There
was no difference between groups for either HKII (P ⫽ 0.70)
or GLUT4 (P ⫽ 0.11).
Skeletal muscle PPAR␥ and PGC-1␣ mRNA expression.
There was an overall increase in PPAR␥ basal mRNA expres-
sion (overall training effect: P ⬍ 0.001) in response to 12 wk
of endurance training that was localized in the AO group. More
specifically, PPAR␥ basal mRNA expression increased by
77% in the AO group (P ⬍ 0.005) and by 26% in the PL group
(P ⬎ 0.05) but with no difference between the groups (P ⫽
0.87). There was no effect of either the training or the supple-
mentation on the basal mRNA expression of PGC-1␣ (Fig. 4).
Body composition and metabolic parameters. Total body fat
mass decreased and total body fat-free mass increased after 12
wk of training; however, the post-values were not significantly
different from the pre-values. There were no changes in total
body weight (Table 3).
Plasma HDL increased in response to training by ⬃21%
(P ⬍ 0.05) in the AO group and 17% in the PL (P ⬍ 0.05)
Fig. 3. Protein content of GLUT4 and hexoki-
nase II (HXK2) before and after 12 wk of
training. Values are expressed as arbitrary units.
Open bars, Antioxidant group (n ⫽ 11); filled
bars, Placebo group (n ⫽ 10). Values are means ⫾
95% CI for GLUT4 and as geometric means ⫾
95% CI for HXK2. *P ⬍ 0.05, **P ⬍ 0.005 vs.
pretraining values.
AJP-Endocrinol Metab • VOL
group. There were no changes in plasma LDL, plasma TG,
plasma FFA, and plasma glucose concentration before and
after the training period (Table 3).
DISCUSSION
The aim of the present study was to test whether antioxidant
supplementation in healthy individuals would reduce training-
induced insulin sensitivity. In contrast to our hypothesis, the
present study showed that combined supplementation with
vitamins C and E before and during 12 wk of supervised
strenuous bicycle exercise training had no effect on insulin-
stimulated glucose uptake in skeletal muscle.
An intense endurance training protocol containing progres-
sive increases in both duration and intensity of the training
sessions was employed in the present study to obtain a robust
training effect whereby possible effects of the supplementation
would be easier to detect. There was, however, no indication
that the supplementation with antioxidants had any effect on
performance or insulin-stimulated glucose uptake. Despite the
limited number of participants in the present study, the highly
controlled design (e.g., supervised training sessions 5 times/
wk) and absence of P values anywhere near statistical signif-
icance when the two groups were compared indicate that the
risk of a type 2 error is low.
In the present study, we observed a very clear increase in
V̇O2max and insulin-stimulated glucose uptake in response to
the training. Clearly, there are currently very few studies that
have examined the interaction of antioxidant supplementation
and exercise training on insulin sensitivity. Supplementation
with the antioxidant ␣-lipoic acid during endurance training
has no effect on insulin action in rodents (43), however, a
recent study in humans reports that antioxidant supplementa-
tion with 400 IU of vitamin E and 1,000 mg of vitamin C daily
may inhibit the insulin-sensitizing effect of training (40).
Accordingly, our results are in contrast to previous findings.
Importantly, we also found that the increased insulin sensi-
tivity was associated with corresponding increases in total Akt,
GLUT4, and HKII in skeletal muscle, which is in agreement
with previous training studies (16, 21). Previous studies have
demonstrated a significant increase of GLUT4 protein content
in response to endurance training (15, 21, 26, 29), although the
300 • MAY 2011 • www.ajpendo.org
 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
Fig. 4. Skeletal muscle basal mRNA content of PPAR␥ and PGC-1␣ before
and after 12 wk of training. Open bars, Antioxidant group (n ⫽ 11); filled bars,
Placebo group (n ⫽ 10). Values are means ⫾ SE for PPAR␥ and as geometric
means ⫾ 95% CI for PGC-1␣. **P ⬍ 0.005 vs. pretraining values.
increase just failed to reach statistical significance (P ⫽ 0.05)
in the present study. However, it should be noted that the
participants in our study were young, healthy, and physically
active men with mean V̇O2max ⬎ 50 ml·kg⫺1·min⫺1 even
before training, thus a priori well above normal average V̇O2max
for young, healthy, Scandinavian men (17). Therefore, it could
be that the protein content of GLUT4 was already high before
the participants started training, whereby a further increase in
response to the employed training may have been limited.
Basal expression of the transcription factor PPAR␥ was
increased after the training period, which is in accord with
results from previous studies (40, 45). The increase, however,
reached statistical significance in the post hoc analysis only in
the AO group. The biological relevance of this finding appears,
however, to be limited, since the two groups otherwise pro-
duced very similar responses to the training. Nevertheless, the
result is in contrast with the results from a previous study
that has examined the effect of vitamins C and E supple-
mentation during 4 wk of training on PPAR␥ basal mRNA
AJP-Endocrinol Metab • VOL
E767
expression (40), showing that the antioxidant supplementa-
tion blunts the expression. Recent studies have suggested a
mechanistic link between oxidative stress and PPAR␥.
Thus, PPAR␥ activation reduces oxidative stress in adi-
pocytes (30), whereas in a skeletal muscle cell model of
insulin resistance PPAR␥ activation exerts an antioxidant
effect that prevents insulin resistance (14). On the other
hand, oxidative stress may reduce PPAR␥ mRNA expres-
sion and activity in endothelial cells (9), whereas the use of
antioxidant vitamins prevents this reduction. Considering
the above, the increased levels on PPAR␥ mRNA content in
the AO group in our study could be the result of the additive
effect of the exercise training and the antioxidant supple-
mentation. However, since the exact mechanisms of the
relation among oxidative stress, exercise, and PPAR␥ acti-
vation are not clarified yet, it is difficult to draw further
conclusions.
Regarding PGC-1␣, basal mRNA expression did not change
in response to either endurance training or antioxidant supple-
mentation. Results from a rodent study (24) have demonstrated
a 163% increase in mRNA content after 7 days of swimming
training, but results from human studies are controversial:
Pilegaard et al. (35) found no change in PGC-1␣ mRNA
content after 4 wk of knee extensor training. Tunstall et al. (49)
demonstrated the same result after 9 days of bicycling training,
whereas in another human study (42), PGC-1␣ mRNA content
increased after 6 wk of endurance running training. The reason
for the disparate results among the above studies, including
ours, may be related to differences in training mode, exercise
intensity, and duration.
Of note, the number of studies investigating the effect of
antioxidant supplementation during endurance training on the
expression of the transcription factor PPAR␥ and its coactiva-
tor PGC-1␣ is limited. The contraction-induced RONS may act
as a signal for the upregulation of PGC-1␣ (25, 44), and
experiments in muscle cells (44) have demonstrated that anti-
oxidant supplementation suppresses this effect. Supplementa-
tion with 1,000 mg of vitamin C daily during endurance
training in rodents blunted the upregulation of PGC-1␣ protein
Fig. 5. Maximal power output per week expressed as watts. Œ, Antioxidant
group (n ⫽ 11);
, Placebo group (n ⫽ 10). Values are means ⫾ SE. Overall
training effect: P ⬍ 0.0001.
300 • MAY 2011 • www.ajpendo.org
E768 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
content (23). Human data (40) demonstrate that the combina-
tion of 1,000 mg of vitamin C and 400 IU of vitamin E daily
during 4 wk of training also prevents the increase in PPAR␥
and PGC-1␣ mRNA content.
The discrepancy between our results and previous findings
may have different explanations. It could be speculated that the
continuous increases in the duration and intensity of the train-
ing sessions resulted in a progressively higher amount of
RONS produced during each exercise session. In support of
this hypothesis, it has been shown both in animals (22) and in
humans (46) that the higher the exercise intensity the higher the
amount of RONS produced. Consequently, even if the antiox-
idant vitamins prevented some of the RONS produced, it could
be speculated that the exercise-induced RONS might exceed
the antioxidative capacity of the vitamins and thus still be
capable of activating the various redox-sensitive signaling
pathways (46). In addition, some oxidants have insulin-mim-
icking effects, which activate insulin-signaling pathways (3).
Given the above, it could be speculated that amount of RONS
generated during each exercise session in the AO group could
have been adequate to trigger the insulin-signaling pathways
despite increased antioxidative capacity obtained by the sup-
plementation. On the other hand, regular exercise seems to
lower both the basal levels of RONS and the levels of RONS
produced during exercise of the same initial intensity (38).
Accordingly, if the amount of antioxidant supplements remains
the same, the latter would probably be more effective in
blunting the small amount of RONS produced during exercise
at the end of a training program, thereby preventing RONS-
required adaptations.
Taking into account the long duration of the training period
in the present study, it could also be speculated that the
vitamins C and E supplementation might have had an effect
during the first week of the training period, at the initial phase
of the training adaptation. Weekly measurements of Pmax did
not, however, indicate such transient effects of the antioxidants
as Pmax was increasing every week in a similar way in both
groups (Fig. 5).
It is worth noticing that in the human study by Ristow et al.
(40) the duration of the individual training sessions was con-
stant during the training period, yet there is no information
about the intensity of the exercise. Furthermore, the duration of
the training period was only 4 wk compared with 12 wk in our
training study. Taken together, the two training protocols differ
with regard to duration, intensity, and total amount of training.
Each of these factors may explain the different results between
the two studies.
We investigated the effect of vitamins C and E supplemen-
tation on insulin sensitivity, assessed by differences in the
glucose infusion rate during insulin clamps, and differences in
the protein content of the insulin signaling cascade molecules.
We did not find any differences in the phosphorylation level of
the proteins p-IR, Akt Ser473, or p-AS160 during the insulin
clamps before or after training. However, we do not know
whether the antioxidant supplementation perhaps changed the
kinetics of the phosphorylation level of the proteins between
the PL and the AO groups. It has been shown, for example, that
TNF-␣ infusion decreases insulin-induced phosphorylation of
AS160 after 1 h, whereas this effect is lost after 3 h (36). A
marked change in phosphorylation kinetics is, however, un-
AJP-Endocrinol Metab • VOL
likely, since the glucose infusion rate profiles were similar in
the two groups (Fig. 1, A and B).
In conclusion, the present study has demonstrated a marked
effect of 12 wk of strenuous endurance training on insulin
sensitivity in healthy young males and thereby underscores the
fact that there is room for further improvement in glucose
metabolism, even in healthy, moderately- to well-trained peo-
ple. In addition, supplementation with vitamins C and E during
strenuous endurance training did not affect the training-in-
duced improvement in insulin sensitivity in healthy, young
individuals. Although the results from the present study do not
indicate a harmful effect of the antioxidant supplementation
during training, it is clear that, at least in this population, the
antioxidant vitamins do not offer any additional beneficial
effect to the endurance training. Considering the extent of
usage of antioxidant supplements within the sports world,
individuals who are healthy, follow a balanced diet, and are
engaged in a regular training program to improve their fitness,
should probably be more critical toward antioxidant vitamin
supplementation.
ACKNOWLEDGMENTS
Ruth Rousing and Hanne Villumsen are acknowledged for their excellent
technical assistance.
GRANTS
The Center of Inflammation and Metabolism (CIM) is supported by a grant
from the Danish National Research Foundation (# 02-512-55). This study was
further supported by the Danish Medical Research Council, the Commission of
the European Communities (Grant Agreement no. 223576, MYOAGE), and by
grants from the Greek State Scholarships Foundation and the Danish Ministry
of Culture Committee on Sports Research. CIM is part of the UNIK Project:
Food, Fitness & Pharma for Health and Disease, supported by the Danish
Ministry of Science, Technology and Innovation.
DISCLOSURES
No conflicts of interest are declared by the author(s).
REFERENCES
1. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin CT,
Price JW, III, Kang L, Rabinovitch PS, Szeto HH, Houmard JA,
Cortright RN, Wasserman DH, Neufer PD. Mitochondrial H2O2 emis-
sion and cellular redox state link excess fat intake to insulin resistance in
both rodents and humans. J Clin Invest 2009.
2. Balon TW, Nadler JL. Evidence that nitric oxide increases glucose
transport in skeletal muscle. J Appl Physiol 82: 359 –363, 1997.
3. Bashan N, Kovsan J, Kachko I, Ovadia H, Rudich A. Positive and
negative regulation of insulin signaling by reactive oxygen and nitrogen
species. Physiol Rev 89: 27–71, 2009.
4. Bauman AE. Updating the evidence that physical activity is good for
health: an epidemiological review 2000 –2003. J Sci Med Sport 7: 6 –19,
2004.
5. Beltran PJ, Mitchell P, Chung YA, Cajulis E, Lu J, Belmontes B, Ho
J, Tsai MM, Zhu M, Vonderfecht S, Baserga R, Kendall R, Radinsky
R, Calzone FJ. AMG 479, a fully human anti-insulin-like growth factor
receptor type I monoclonal antibody, inhibits the growth and survival of
pancreatic carcinoma cells. Mol Cancer Ther, 2009.
6. Benton CR, Holloway GP, Han XX, Yoshida Y, Snook LA, Lally J,
Glatz JF, Luiken JJ, Chabowski A, Bonen A. Increased levels of
peroxisome proliferator-activated receptor gamma, coactivator 1 alpha
(PGC-1alpha) improve lipid utilisation, insulin signalling and glucose
transport in skeletal muscle of lean and insulin-resistant obese Zucker rats.
Diabetologia 53: 2008 –2019, 2010.
7. Benton CR, Nickerson JG, Lally J, Han XX, Holloway GP, Glatz JF,
Luiken JJ, Graham TE, Heikkila JJ, Bonen A. Modest PGC-1alpha
overexpression in muscle in vivo is sufficient to increase insulin sensitivity
300 • MAY 2011 • www.ajpendo.org
 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY
and palmitate oxidation in subsarcolemmal, not intermyofibrillar, mito-
chondria. J Biol Chem 283: 4228 –4240, 2008.
8. Bergstrom J. Percutaneous needle biopsy of skeletal muscle in physio-
logical and clinical research. Scand J Clin Lab Invest 35: 609 –616, 1975.
9. Blanquicett C, Kang BY, Ritzenthaler JD, Jones DP, Hart CM.
Oxidative stress modulates PPAR gamma in vascular endothelial cells.
Free Radic Biol Med 48: 1618 –1625, 2010.
10. Bonen A. PGC-1alpha-induced improvements in skeletal muscle metab-
olism and insulin sensitivity. Appl Physiol Nutr Metab 34: 307–314, 2009.
11. Bradley SJ, Kingwell BA, McConell GK. Nitric oxide synthase inhibi-
tion reduces leg glucose uptake but not blood flow during dynamic
exercise in humans. Diabetes 48: 1815–1821, 1999.
12. Buchfuhrer MJ, Hansen JE, Robinson TE, Sue DY, Wasserman K,
Whipp BJ. Optimizing the exercise protocol for cardiopulmonary assess-
ment. J Appl Physiol 55: 1558 –1564, 1983.
13. Burgoyne JR, Madhani M, Cuello F, Charles RL, Brennan JP,
Schroder E, Browning DD, Eaton P. Cysteine redox sensor in PKGIa
enables oxidant-induced activation. Science 317: 1393–1397, 2007.
14. Chung SS, Kim M, Youn BS, Lee NS, Park JW, Lee IK, Lee YS, Kim
JB, Cho YM, Lee HK, Park KS. Glutathione peroxidase 3 mediates the
antioxidant effect of peroxisome proliferator-activated receptor gamma in
human skeletal muscle cells. Mol Cell Biol 29: 20 –30, 2009.
15. Dela F, Larsen JJ, Mikines KJ, Ploug T, Petersen LN, Galbo H.
Insulin-stimulated muscle glucose clearance in patients with NIDDM.
Effects of one-legged physical training. Diabetes 44: 1010 –1020, 1995.
16. Dela F, Mikines KJ, von Linstow M, Secher NH, Galbo H. Effect of
training on insulin-mediated glucose uptake in human muscle. Am J
Physiol Endocrinol Metab 263: E1134 –E1143, 1992.
17. Dyrstad SM, Aandstad A, Hallen J. Aerobic fitness in young Norwegian
men: a comparison between 1980 and 2002. Scand J Med Sci Sports 15:
298 –303, 2005.
18. Finckh B, Kontush A, Commentz J, Hubner C, Burdelski M,
Kohlschutter A. High-performance liquid chromatography-coulometric
electrochemical detection of ubiquinol 10, ubiquinone 10, carotenoids, and
tocopherols in neonatal plasma. Methods Enzymol 299: 341–348, 1999.
19. Fischer CP, Hiscock NJ, Penkowa M, Basu S, Vessby B, Kallner A,
Sjoberg LB, Pedersen BK. Supplementation with vitamins C and E
inhibits the release of interleukin-6 from contracting human skeletal
muscle. J Physiol 558: 633–645, 2004.
20. Franzini L, Ardigo D, Zavaroni I. Dietary antioxidants and glucose
metabolism. Curr Opin Clin Nutr Metab Care 11: 471–476, 2008.
21. Frosig C, Rose AJ, Treebak JT, Kiens B, Richter EA, Wojtaszewski
JF. Effects of endurance exercise training on insulin signaling in human
skeletal muscle: interactions at the level of phosphatidylinositol 3-kinase,
Akt, and AS160. Diabetes 56: 2093–2102, 2007.
22. Gambelunghe C, Rossi R, Micheletti A, Mariucci G, Rufini S. Physical
exercise intensity can be related to plasma glutathione levels. J Physiol
Biochem 57: 9 –14, 2001.
23. Gomez-Cabrera MC, Domenech E, Romagnoli M, Arduini A, Borras
C, Pallardo FV, Sastre J, Vina J. Oral administration of vitamin C
decreases muscle mitochondrial biogenesis and hampers training-induced
adaptations in endurance performance. Am J Clin Nutr 87: 142–149, 2008.
24. Goto M, Terada S, Kato M, Katoh M, Yokozeki T, Tabata I,
Shimokawa T. cDNA Cloning and mRNA analysis of PGC-1 in epitroch-
learis muscle in swimming-exercised rats. Biochem Biophys Res Commun
274: 350 –354, 2000.
25. Irrcher I, Ljubicic V, Hood DA. Interactions between ROS and AMP
kinase activity in the regulation of PGC-1␣ transcription in skeletal muscle
cells. Am J Physiol Cell Physiol 296: C116 –C123, 2009.
26. Kim HJ, Lee JS, Kim CK. Effect of exercise training on muscle glucose
transporter 4 protein and intramuscular lipid content in elderly men with
impaired glucose tolerance. Eur J Appl Physiol 93: 353–358, 2004.
27. Laemmli UK. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227: 680 –685, 1970.
28. Lazarevic G, Antic S, Cvetkovic T, Vlahovic P, Tasic I, Stefanovic V.
A physical activity programme and its effects on insulin resistance and
oxidative defense in obese male patients with type 2 diabetes mellitus.
Diabetes Metab 32: 583–590, 2006.
29. Little JP, Safdar A, Wilkin GP, Tarnopolsky MA, Gibala MJ. A
practical model of low-volume high-intensity interval training induces
mitochondrial biogenesis in human skeletal muscle: potential mechanisms.
J Physiol 588: 1011–1022, 2010.
AJP-Endocrinol Metab • VOL 300 • MAY 2011 •
E769
30. Luo W, Cao J, Li J, He W. Adipose tissue-specific PPARgamma
deficiency increases resistance to oxidative stress. Exp Gerontol 43:
154 –163, 2008.
31. Lykkesfeldt J. Determination of ascorbic acid and dehydroascorbic acid
in biological samples by high-performance liquid chromatography using
subtraction methods: reliable reduction with tris[2-carboxyethyl]phos-
phine hydrochloride. Anal Biochem 282: 89 –93, 2000.
32. McClung JP, Roneker CA, Mu W, Lisk DJ, Langlais P, Liu F, Lei XG.
Development of insulin resistance and obesity in mice overexpressing
cellular glutathione peroxidase. Proc Natl Acad Sci USA 101: 8852–8857,
2004.
33. Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S,
Miyazaki Y, Kohane I, Costello M, Saccone R, Landaker EJ, Goldfine
AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, Mandarino LJ.
Coordinated reduction of genes of oxidative metabolism in humans with
insulin resistance and diabetes: Potential role of PGC1 and NRF1. Proc
Natl Acad Sci USA 100: 8466 –8471, 2003.
34. Pedersen BK, Saltin B. Evidence for prescribing exercise as therapy in
chronic disease. Scand J Med Sci Sports 16, Suppl 1: 3–63, 2006.
35. Pilegaard H, Saltin B, Neufer PD. Exercise induces transient transcrip-
tional activation of the PGC-1alpha gene in human skeletal muscle. J
Physiol 546: 851–858, 2003.
36. Plomgaard P, Bouzakri K, Krogh-Madsen R, Mittendorfer B, Zierath
JR, Pedersen BK. Tumor necrosis factor-alpha induces skeletal muscle
insulin resistance in healthy human subjects via inhibition of Akt substrate
160 phosphorylation. Diabetes 54: 2939 –2945, 2005.
37. Powers SK, Duarte J, Kavazis AN, Talbert EE. Reactive oxygen
species are signalling molecules for skeletal muscle adaptation. Exp
Physiol 95: 1–9, 2010.
38. Radak Z, Chung HY, Goto S. Exercise and hormesis: oxidative stress-
related adaptation for successful aging. Biogerontology 6: 71–75, 2005.
39. Ren JM, Semenkovich CF, Gulve EA, Gao J, Holloszy JO. Exercise
induces rapid increases in GLUT4 expression, glucose transport capacity,
and insulin-stimulated glycogen storage in muscle. J Biol Chem 269:
14396 –14401, 1994.
40. Ristow M, Zarse K, Oberbach A, Kloting N, Birringer M, Kiehntopf
M, Stumvoll M, Kahn CR, Bluher M. Antioxidants prevent health-
promoting effects of physical exercise in humans. Proc Natl Acad Sci USA
106: 8665–8670, 2009.
41. Rizzo MR, Abbatecola AM, Barbieri M, Vietri MT, Cioffi M, Grella
R, Molinari A, Forsey R, Powell J, Paolisso G. Evidence for anti-
inflammatory effects of combined administration of vitamin E and C in
older persons with impaired fasting glucose: impact on insulin action. J
Am Coll Nutr 27: 505–511, 2008.
42. Russell AP, Feilchenfeldt J, Schreiber S, Praz M, Crettenand A,
Gobelet C, Meier CA, Bell DR, Kralli A, Giacobino JP, Deriaz O.
Endurance training in humans leads to fiber type-specific increases in
levels of peroxisome proliferator-activated receptor-gamma coactivator-1
and peroxisome proliferator-activated receptor-alpha in skeletal muscle.
Diabetes 52: 2874 –2881, 2003.
43. Saengsirisuwan V, Perez FR, Kinnick TR, Henriksen EJ. Effects of
exercise training and antioxidant R-ALA on glucose transport in insulin-
sensitive rat skeletal muscle. J Appl Physiol 92: 50 –58, 2002.
44. Silveira LR, Pilegaard H, Kusuhara K, Curi R, Hellsten Y. The
contraction induced increase in gene expression of peroxisome prolifera-
tor-activated receptor (PPAR)-gamma coactivator 1alpha (PGC-1alpha),
mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in
primary rat skeletal muscle cells is dependent on reactive oxygen species.
Biochim Biophys Acta 1763: 969 –976, 2006.
45. Spangenburg EE, Brown DA, Johnson MS, Moore RL. Alterations in
peroxisome proliferator-activated receptor mRNA expression in skeletal
muscle after acute and repeated bouts of exercise. Mol Cell Biochem 332:
225–231, 2009.
46. Sureda A, Ferrer MD, Tauler P, Romaguera D, Drobnic F, Pujol P,
Tur JA, Pons A. Effects of exercise intensity on lymphocyte H2O2
production and antioxidant defences in soccer players. Br J Sports Med 43:
186 –190, 2009.
47. Taves DR. Minimization: a new method of assigning patients to treatment
and control groups. Clin Pharmacol Ther 15: 443–453, 1974.
48. Taylor EB, Lamb JD, Hurst RW, Chesser DG, Ellingson WJ, Green-
wood LJ, Porter BB, Herway ST, Winder WW. Endurance training
increases skeletal muscle LKB1 and PGC-1␣ protein abundance: effects of
time and intensity. Am J Physiol Endocrinol Metab 289: E960 –E968,
2005.
www.ajpendo.org
E770 ANTIOXIDANTS, TRAINING, AND INSULIN SENSITIVITY

49. Tunstall RJ, Mehan KA, Wadley GD, Collier GR, Bonen A, Har-
greaves M, Cameron-Smith D. Exercise training increases lipid metab-
olism gene expression in human skeletal muscle. Am J Physiol Endocrinol
Metab 283: E66 –E72, 2002.
50. Wray DW, Uberoi A, Lawrenson L, Bailey DM, Richardson RS. Oral
antioxidants and cardiovascular health in the exercise-trained and untrained
elderly: a radically different outcome. Clin Sci (Lond) 116: 433–441, 2009.
51. Yonan CR, Duong PT, Chang FN. High-efficiency staining of pro-
teins on different blot membranes. Anal Biochem 338: 159 –161,
2005.
52. Yu M, Blomstrand E, Chibalin AV, Wallberg-Henriksson H, Zierath
JR, Krook A. Exercise-associated differences in an array of proteins
involved in signal transduction and glucose transport. J Appl Physiol 90:
29 –34, 2001.

AJP-Endocrinol Metab • VOL 300 • MAY 2011 • www.ajpendo.org