ORIGINAL ARTICLE

Lipid-peroxidation and peroxiredoxin-overoxidationin the erythrocytes of non-insulin-dependent type 2diabetic men during acute exercise

Christian Brinkmann • Jenny Blossfeld • Martin Pesch • Bastian Krone •

Kathrin Wiesiollek • Dario Capin • Georgina Montiel • Martin Hellmich •

Wilhelm Bloch • Klara Brixius

Received: 1 July 2011 / Accepted: 30 September 2011 / Published online: 18 October 2011

� Springer-Verlag 2011

Abstract Single bouts of exercise induce an acute state of

oxidative stress. It is largely unknown what this means in the

context of diseases which are associated with increased

oxidative stress, e.g., type 2 diabetes mellitus (T2DM). Free

radicals can destroy the structure of erythrocytes and reduce

their deformability. Antioxidative peroxiredoxins are highly

abundant in erythrocytes. Therefore, we immunohisto-

chemically examined whether the free radical-induced

erythrocyte lipid-peroxidation measured by 8-iso-prosta-

glandin-F2a (8-Iso-PGF) as well as the erythrocyte con-

tents of overoxidized peroxiredoxins (PRDX-SO2–3)

differ between overweight/obese T2DM men (n = 15,

years = 59 ± 10 (mean ± SD)) and overweight/obese

non-diabetic control subjects (n = 12, years = 53 ± 4)

during acute exercise (WHO-step test). We further studied

whether physical training affects the oxidative stress

response to acute exercise. Seven men belonging to the

diabetic group took part in a moderate intensity cycling

endurance training. Erythrocyte 8-Iso-PGF significantly

increased during acute exercise and decreased in the 30-min

recovery phase in untrained diabetic and non-diabetic men

(P B 0.05). Increases/decreases in 8-Iso-PGF in relation to

exercise/recovery time were similar in both groups. A sig-

nificant exercise-induced increase in the contents of eryth-

rocyte PRDX-SO2–3 was only observed in T2DM men

(P B 0.05). PRDX-SO2–3 contents were not reduced during

recovery. Following physical training, the magnitude of

exercise-induced increases in 8-Iso-PGF (relative to exercise

time) was significantly lower in the erythrocytes of T2DM

men (P B 0.05), whereas increases in PRDX-SO2–3 were

significantly higher (P B 0.05). Exercise-induced erythro-

cyte lipid-peroxidation is similar in untrained overweight/

obese T2DM patients and overweight/obese control sub-

jects, while antioxidative mechanisms differ. Physical

training might improve oxidative stress in T2DM men’s

erythrocytes during acute exercise.

Keywords Oxidative stress � Exercise � Type 2 diabetes �Erythrocytes � Lipid-peroxidation � Peroxiredoxin

Introduction

In patients suffering from type 2 diabetes mellitus (T2DM),

oxidative stress (the imbalance between free reactive oxygen

species (ROS) and the antioxidative defense capacity) can

induce/potentiate different secondary complications, e.g.,

cardio-vascular illnesses (Rains and Jain 2011).

The increased autoxidation of glucose, an intensified

formation of advanced glycation products, an elevated

activation of the polyol-pathway, mitochondrial ROS-pro-

duction as well as the increased activity of NADPH-oxi-

dases can be responsible for a considerable amount of free

radicals in T2DM subjects (Brownlee 2001; Inoguchi et al.

Communicated by Susan A. Ward.

C. Brinkmann (&) � J. Blossfeld � M. Pesch � B. Krone �K. Wiesiollek � D. Capin � W. Bloch � K. Brixius

Department of Molecular and Cellular Sport Medicine,

Institute of Cardiovascular Research and Sport Medicine,

German Sport University Cologne, Am Sportpark

Mungersdorf 6, 50933 Cologne, Germany

e-mail: ch.brinkmann@gmx.net

G. Montiel

Department of Preventive and Rehabilitative Sport Medicine,

Institute of Cardiovascular Research and Sport Medicine,

German Sport University Cologne, Cologne, Germany

M. Hellmich

Institute of Medical Statistics, Informatics and Epidemiology,

University of Cologne, Cologne, Germany

123

Eur J Appl Physiol (2012) 112:2277–2287

DOI 10.1007/s00421-011-2203-x

2000; Kaneto et al. 2010; Wolff and Dean 1987). In

addition, there is growing evidence that the antioxidative

capacity is weakened in patients exhibiting T2DM, since

reduced activity of antioxidative enzymes, e.g., of super-

oxide dismutase (SOD), catalase (CAT) and glutathione

peroxidase (GPX) has been reported (Bhatia et al. 2003;

Memisogullari et al. 2003).

Thus, numerous publications have demonstrated

increased systemic oxidative stress levels in T2DM

patients in blood plasma compared with non-diabetic

control subjects (Collier et al. 1992; Osuntokl et al. 2007;

Pandey et al. 2010; Sato et al. 1979).

Single exercise bouts can increase oxidative stress,

among others, probably attributable to a rise in mitochon-

drial ROS-production, as well as in the ROS-generation

resulting from the autoxidation of oxyhemoglobin, oxy-

myoglobin, catecholamines or the activity of xanthine- and

NADPH-oxidases (Cooper et al. 2002; Fisher-Wellman and

Bloomer 2009).

Red blood cells are confronted with ROS generated by

the intracellular autoxidation of hemoglobin or coming

from the extracellular plasma. In the situations of high

oxidative stress, the structure of erythrocytes can be neg-

atively affected and their deformability which is essential

to pass through small capillaries can be reduced (Minetti

et al. 2007). Moreover, Minetti et al. argue that red blood

cells could turn into harmful ‘‘pro-oxidant bullets’’

spreading oxidative stress throughout the entire body when

ROS are not sufficiently buffered by the antioxidative

system. To protect themselves from oxidative damage,

erythrocytes are characterized by a strong endogenous

antioxidative capacity. It has, therefore, been asserted that

red blood cells can even help lower systemic oxidative

stress by taking up and metabolizing peroxides from the

extracellular plasma (Cho et al. 2010; Winterbourn and

Stern 1987).

Peroxiredoxins are highly abundant antioxidative pro-

teins in erythrocytes. Most of them are only slowly recy-

cled/reduced by the thioredoxin system (Low et al. 2008).

It has, thus, been demonstrated in erythrocytes in vitro that

peroxiredoxins can be overoxidized in the situations of

high amounts of free radicals (Cho et al. 2010). To date,

little is known about the activity of these antioxidative

enzymes in T2DM patients’ red blood cells.

In accordance with the abovementioned aspects, Villa-

Caballero et al. (2000) hypothesized that exercise with an

incremental workload induces an increase in oxidative

stress in both T2DM and non-diabetic control subjects,

with more rapid and higher increases observable in T2DM

patients. This might be harmful, especially for the exer-

cising diabetic subjects. However, training was thought to

reduce exercise-induced increases in oxidative stress in

T2DM patients especially due to an up-regulation of the

antioxidative capacity in the long-term. This topic has thus

far been researched quite sparsely and no research group, to

our knowledge, has examined oxidative stress levels in red

blood cells of T2DM patients during acute exercise,

although erythrocytes have been assigned a central role for

the redox homeostasis in the cardio-vascular system

(Nikolaidis and Jamurtas 2009).

Therefore, the aim of the present study was to examine

the influence of acute exercise on erythrocyte oxidative

stress in untrained/trained T2DM patients and non-diabetic

control subjects. We performed immunohistochemical

stainings using antibodies for the lipid-peroxidation marker

8-iso-prostaglandin-F2a (8-Iso-PGF) which has been

established as being a valid marker for oxidative stress

and free radical-dependent cell damages (Basu 1998; Pra-

tico et al. 2004; Roberts and Morrow 2000). In addition,

exercise-induced changes in the contents of overoxidized

peroxiredoxins (PRDX-SO2–3) were quantified immuno-

histochemically to better understand the functioning of

peroxiredoxins as very important antioxidants in red blood

cells.

Methods

The protocol for the research project was approved by a

suitably constituted Ethics Committee of the German Sport

University before the investigation. It conformed to the

provisions of the Declaration of Helsinki. Written informed

consent was obtained from all subjects.

Subjects

Male subjects were recruited via a newspaper ad. The

inclusion criteria required the subjects to belong to an age

group of around 50 years and be overweight/obese. The

diabetic patients were to exhibit non-insulin-dependent

type 2 diabetes (diagnosed by the family physician (crite-

ria: fasting glucose values C126 mg/dl, HbA1c value C6%

and/or 2 h oral glucose tolerance test indicating glucose

levels C200 mg/dl)). Finally, a total of 15 diabetic men

took part in the study. They declared that neither diabetic

complications nor any cardio-vascular diseases (apart from

well-controlled hypertension, n = 11) had ever been

diagnosed. The duration of the diabetic disease had been

6 ± 7 years (self-report). Within the control group, 12

non-diabetic men were studied for comparison. The sub-

jects’ characteristics are presented in Table 1. Diabetic and

non-diabetic control subjects did not significantly differ in

age, body mass index (BMI) or physical fitness in order to

exclude the influence of age, overweightness, physical fit-

ness when analyzing oxidative stress between the two

groups and to focus on the influence of type 2 diabetes.

2278 Eur J Appl Physiol (2012) 112:2277–2287

123

Fasting glucose values in blood serum indicating the cur-

rent glycemic status were significantly higher in the dia-

betic than in the control group. Most of the subjects were

taking medications during the investigation period. Medi-

cation intake and the subjects’ health condition are shown

in Table 2. It was determined by questionnaire that none of

the subjects had regularly exercised during the last 3 years

before the commencement of the study.

Study design

The erythrocyte oxidative stress response to an acute bout

of exercise was compared between untrained T2DM men

and non-diabetic control subjects. Accordingly, venous

blood was collected before, immediately after the WHO-

step test on a bicycle ergometer and 30-min post-exercise.

In addition, venous blood that was collected under resting

conditions was used to compare basal oxidative stress in

the erythrocytes of T2DM men with that of non-diabetic

control subjects. Furthermore, the effect of regular physical

activity on exercise-induced erythrocyte oxidative stress

was investigated. The subjects of the diabetic group were

asked to participate in a cycling endurance training for 3

months. The training program started 3 days after the

initial performance test. The patients were instructed not to

change their dietary habits and medication-intake during

the training intervention. The WHO-step test was per-

formed once again 3 days after the end of the training

period. Venous blood was collected in accordance with the

same principles as before the training.

Endurance training

Seven diabetic patients (Table 3) took part in the endur-

ance training program. The exercise regimen was designed

based on experiences from previous studies involving

training for diabetic subjects (Sigal et al. 2004). Accord-

ingly, the diabetic subjects were instructed to take part in a

supervised cycling endurance training 3 times a week on

non-consecutive days. Subjects were encouraged to train

on their own (walking or Nordic walking after initial pro-

fessional instruction) when they missed a training session.

We documented that patients participated in at least 90% of

the training sessions. The training intensity was about 75%

of the maximal heart rate (220-age beats/min). The training

bouts (effective time of performance) continuously

increased from a duration of 25 min in the first week to

50 min in the last.

Performance diagnostics

An endurance test was performed on the upright bike

‘‘Ergometrics900’’ (Ergoline, Bitz, Germany) coupled with

an ECG (‘‘Ergoscript EK3012’’, Ergoline). During exer-

cise, respiratory gas measurement was done using the

‘‘ZAN 600 USB’’ system (nSpire Health, Longmont, Col-

orado, USA). Subjects were tested with the following

stopping criteria: muscular exhaustion, angina pectoris,

ischemia, paleness, cyanosis, arrhythmia, respiratory

insufficiency, hypertension (systolic blood pressur-

e [250 mmHg or diastolic blood pressure [115 mmHg),

aberration, dizziness and/or co-ordination problems. Start-

ing at 25 W resistance, the intensity gradually increased by

25 W every 2 min (WHO-step test). VO2peak was deter-

mined as the highest oxygen uptake during performance.

The consumption of caffeine was not permitted within 12 h

before testing. The subjects were always tested at the same

time of day and were instructed not to engage in physically

exhausting activities 24 h before the measuring.

Erythrocyte preparation and immunohistochemistry

Immunohistochemistry is a standard procedure used in cell

biology that has proven successful in the semiquantitative

analysis of proteins in erythrocytes (Fischer et al. 2007; Suhr

et al. 2009). Both the protocol for the fixation of erythrocytes

as well as the standard immunohistochemical protocol were

similar to those used by Suhr et al. (2009). Immunohisto-

chemical stainings were carried out with the following pri-

mary antibodies in the main fields of cell-covered slides:

8-Iso-PGF IgG polyclonal (Acris, Hiddenhausen, Germany)

as the lipid-peroxidation marker and PRDX-SO2–3 IgG

monoclonal (AbFrontier, Seoul, Korea) detecting two forms

of overoxidized peroxiredoxins, those with the cysteine

Table 1 Subjects’ characteristics: comparison of data from non-diabetic male control subjects (CON) and men suffering from type 2 diabetes

mellitus (T2DM)

Age (years) BMI (kg/m2) Fasting glucose (mg/dl) VO2peak (ml/min/kg)

Untrained CON 53 ± 4 30 ± 2 94 ± 9 23.9 ± 5.4

Untrained T2DM 59 ± 10 32 ± 4 163 ± 43* 22 ± 4.9

Values are means ± SD

BMI body mass index, VO2peak highest oxygen consumption

* Significantly different from control subjects (P B 0.05)

Eur J Appl Physiol (2012) 112:2277–2287 2279

123

sulfinic acid (CP-SO2H) and those with the cysteine sulfonic

acid (CP-SO3H). The antibodies were used with a dilution of

1:1,500 (8-Iso-PGF) and 1:200 (PRDX-SO2–3). The primary

antibodies were absent for a negative immunohistochemical

control (IHC-C) which was separated from the main field on

the slides. For the intensity analysis of immunostaining, the

gray values [DU = density unit] of at least 40 erythrocytes

from at least 5 randomly selected areas of each slide were

measured in the main field. At least 10 erythrocytes from at

least 2 randomly selected areas were measured in the IHC-C.

Table 2 Medication intake and chronic diseases (self-report) in non-diabetic male control subjects (CON) and men suffering from type 2

diabetes mellitus (T2DM)

ID Medications Chronic diseases

CON-1 Ramipril Hypertension

CON-2 Euthyrox, Zyprexa Depression, thyroid hypofunction

CON-3 Carmen Hypertension

CON-4 Atacand Hypertension

CON-5 Marcumar, Nebivolol, Thyronajod Hypertension, thyroid hypofunction

CON-6 Lorzaar Hypertension

CON-7 Atacand Hypertension

CON-8 None None

CON-9 None None

CON-10 None None

CON-11 None None

CON-12 None None

ID Medications Chronic diseases apart from type 2 diabetes

T2DM-1 Jutabis, NovoNorm Hypertension

T2DM-2 ASS, Mescorit None

T2DM-3 ASS, Cabaseril, Metformin, Thyronajod Parkinson’s disease, thyroid hypofunction

T2DM-4 Amlodipin, ASS, Enalapril, Metformin, Simvastatin Hyperlipidemia, hypertension

T2DM-5 Metformin, Ramipril Hypertension

T2DM-6 Celebrex, Cymbalta, Glucobay, Metformin, Simvabeta, Tamsulosin Arthrosis, benign prostatic hyperplasia,

depression, hyperlipidemia

T2DM-7 Allopurinol Gout

T2DM-8 Allopurinol, Amlodipin, Metformin, Simvastatin, Torasemid Gout, hyperlipidemia, hypertension

T2DM-9 ASS, Carmen, Competact, Januvia, Natrilix, Nebilet, Simvabeta Hyperlipidemia, hypertension

T2DM-10 Amaryl, Metformin, Metoprolol, Ramipril, Simvastatin Hyperlipidemia, hypertension

T2DM-11 ASS, Glibenclamid, Metformin, Ramipril, Simvastatin Hyperlipidemia, hypertension

T2DM-12 Metformin, Ramipril, HCT Hypertension

T2DM-13 Bisoprolol, Enalapril, L-Thyroxin, Metformin, Simvastatin Hyperlipidemia, hypertension, thyroid hypofunction

T2DM-14 Pantozol, Ramipril Hypertension

T2DM-15 Delmuno, Dytide, Metformin Hypertension

ID identification number

Table 3 Subjects characteristics: comparison of data from men suffering from type 2 diabetes mellitus (T2DM) before physical training (BT)

and after physical training (AT)

Age (years) BMI (kg/m2) Fasting glucose (mg/dl) VO2peak (ml/min/kg)

T2DM BT 57 ± 10 32 ± 4 166 ± 33 21.8 ± 6.1

T2DM AT 57 ± 10 31 ± 4* 132 ± 21* 26.2 ± 4.5*

Values are means ± SD

BMI body mass index, VO2peak highest oxygen consumption

* Significantly different from the T2DM patients’ pre-training value (P B 0.05)

2280 Eur J Appl Physiol (2012) 112:2277–2287

123

Finally, the intensity of immunostaining (for all erythrocytes

of an individual) was determined as follows:

Arbitrary gray value¼ 1

40

X40

i¼1

ðerythrocyte gray valuemain field i

� background gray valuemain field iÞ

� 1

10

X10

j¼1

ðerythrocyte gray valueIHC�Cj

� background gray valueIHC�CjÞ

The background gray value was measured in a cell-free

area of the slide. For comparative analyses, only values of

erythrocytes incubated in the same immunostaining

procedure (under identical conditions) were chosen. For

staining intensity detection, a light microscope (‘‘Axiophot’’,

Zeiss, Jena, Germany) coupled to a video camera (‘‘3CCD’’,

Sony, Tokio, Japan) was used together with the ‘‘KS300’’

software (Zeiss). The analysis was conducted using the

software ‘‘Image J’’ (National Institutes of Health, Bethesda,

Maryland, USA). The magnification was 4009.

Statistical analyses

Data are presented as mean values ± standard deviations

(SD). Non-parametric (rank-based) hypotheses tests

were used throughout as normality of continuous data

distributions seemed questionable (skewness, outliers).

Specifically, the Friedman ANOVA was performed for

repeated measures. If found statistically significant,

implemented post-hoc tests for multiple pairwise compar-

isons were conducted (Bonferroni corrected). Moreover,

unpaired samples of diabetic and non-diabetic men were

compared by the Mann–Whitney U test, paired data (pre-

vs. post-training values) using the Wilcoxon signed rank

test. P values B0.05 were found to indicate statistical

significance. All statistical analyses were carried out using

the ‘‘SPSS 18.0’’ program (PASW Statistics, SPSS Inc.,

Chicago, Illinois, USA).

Results

Exercise-induced lipid-peroxidation and peroxiredoxin-

overoxidation in the erythrocytes of untrained non-

insulin-dependent T2DM patients and

untrained non-diabetic control subjects

We first examined how lipid-peroxidation (as measured by

8-Iso-PGF) as well as the peroxiredoxin-overoxidation

(PRDX-SO2–3) in the erythrocytes of T2DM men change

during acute exercise. In both type 2 diabetic and non-

diabetic men, statistical analysis revealed significant

changes in erythrocyte 8-Iso-PGF contents over time

(Fig. 1): the density of 8-Iso-PGF significantly increased

during acute exercise and significantly decreased in the

Fig. 1 a Density of 8-Iso-PGF and PRDX-SO2–3 at rest, immedi-

ately after the WHO-step test and at 30-min recovery in non-diabetic

male control subjects (CON) and men suffering from non-insulin-

dependent type 2 diabetes mellitus (T2DM). Values are means ± SD.

�Significantly different from at rest (P B 0.05). �Significantly differ-

ent from immediately after exercise (P B 0.05). b Original photos of

the 8-Iso-PGF and PRDX-SO2–3 immunohistochemical stainings

Eur J Appl Physiol (2012) 112:2277–2287 2281

123

following 30-min recovery nearly to the level before

exercise. The density of overoxidized peroxiredoxins

(PRDX-SO2–3) significantly changed over time in T2DM

patients’ erythrocytes, but not in non-diabetic control

subjects’ red blood cells (Fig. 1). We observed a significant

increase in PRDX-SO2–3 contents during acute exercise in

T2DM men. Following a 30-min recovery period, PRDX-

SO2–3 levels were still increased compared with the levels

at resting conditions. Neither increases in erythrocyte

8-Iso-PGF levels during the WHO-step test (in relation to

the individual exercise time during the WHO-step test) nor

decreases in 8-Iso-PGF levels during recovery significantly

differed between T2DM patients and control subjects

(Fig. 2).

We were interested in determining whether the detected

similarities/differences in the oxidative stress response

during acute exercise between T2DM men and non-dia-

betic control subjects were possibly associated with simi-

larities/differences in the basal oxidative stress status.

Therefore, we examined 8-Iso-PGF levels in the erythro-

cytes and found no significant differences for erythrocyte

8-Iso-PGF in T2DM patients and control subjects at resting

conditions (Fig. 3).

Exercise-induced lipid-peroxidation and peroxiredoxin-

overoxidation in the erythrocytes of non-insulin-

dependent T2DM patients before and after

physical training

To determine whether regular physical training can affect

acute exercise-induced lipid-peroxidation and peroxir-

edoxin-overoxidation in the erythrocytes of T2DM

patients, we immunohistochemically analyzed erythrocytes

from blood taken after 3 months of endurance training. As

presented in Table 3, physical fitness improved after the

training period.

8-Iso-PGF increased in the erythrocytes of T2DM men

during an acute bout of exercise and decreased in the

recovery period similar to the observations before training

(Fig. 4). Furthermore, we found significant changes in

erythrocyte PRDX-SO2–3 levels during acute exercise

which were also similar to changes before training: PRDX-

SO2–3 contents increased from at rest to immediately after

exercise and were not reduced in the 30-min recovery

period (Fig. 4).

Nevertheless, there were some changes in the oxidative

stress response to acute exercise after the training period

(Fig. 5). Following physical training, increases in erythro-

cyte 8-Iso-PGF levels (in relation to exercise time) were

significantly decreased during the WHO-step test.

Decreases in 8-Iso-PGF in the recovery phase did not differ

between untrained and trained men. Increases in PRDX-

SO2–3 contents (in relation to exercise time) were signifi-

cantly higher in the erythrocytes of T2DM men following

physical training.

To discuss changes in the oxidative stress response to an

acute bout of exercise pre- versus post-training, we wanted

to determine whether oxidative stress at rest was also

affected by physical training. We observed a significant

reduction in 8-Iso-PGF in the erythrocytes of T2DM men

following physical training based on immunohistochemical

analyses (Fig. 3).

Discussion

The present study revealed a similar exercise-induced

oxidative stress response in the erythrocytes of T2DM

patients relative to non-diabetic control subjects as mea-

sured by 8-Iso-PGF. However, we observed increased

contents of overoxidized peroxiredoxins only in T2DM

patients during strenuous exercise. Physical training low-

ered oxidative stress (8-Iso-PGF) at rest and reduced

exercise-induced increases in 8-Iso-PGF contents (in rela-

tion to exercise time) in the erythrocytes of T2DM patients.

Furthermore, peroxiredoxin-overoxidation was increased

during acute exercise following physical training in the

erythrocytes of T2DM men.

Fig. 2 Increases/decreases in

8-Iso-PGF density during acute

exercise and at 30-min recovery

(in relation to exercise/recovery

time) in non-diabetic male

control subjects (CON) and men

suffering from non-insulin-

dependent type 2 diabetes

mellitus (T2DM). Values are

means ± SD

2282 Eur J Appl Physiol (2012) 112:2277–2287

123

Exercise-induced lipid-peroxidation and peroxiredoxin-

overoxidation in the erythrocytes of untrained non-

insulin-dependent T2DM patients and untrained non-

diabetic control subjects

Single bouts of exercise can increase the amount of free

radicals in the short-term. This has already been demon-

strated by Ashton et al. (1998) using electron spin reso-

nance spectroscopy as early as in 1998 (the signal was

fourfold increased post- vs. pre-exercise).

Erythrocytes are confronted with ROS from blood plasma

as well as with ROS resulting from the autoxidation of

hemoglobin. It can be assumed that this cell-internal ROS-

generation is also increased during acute exercise (Cooper

et al. 2002). High amounts of free radicals can reduce the red

blood cell deformability and even lead to their destruction

(Minetti et al. 2007; Dikmenoglu et al. 2008).

The demonstration that the activity of antioxidative

enzymes SOD, CAT and GPX in the erythrocytes of T2DM

subjects can be decreased (Bhatia et al. 2003; Memisogullari

Fig. 3 a Density of 8-Iso-PGF

in the erythrocytes of non-

diabetic male control subjects

(CON) and men suffering from

non-insulin-dependent type 2

diabetes mellitus (T2DM) as

well as of T2DM men before

physical training (BT) and after

physical training (AT) at rest.

Values are means ± SD.

*Significantly different from the

pre-training value (P B 0.05).

b Original photos of the 8-Iso-

PGF immunohistochemical

stainings

Fig. 4 a Density of 8-Iso-PGF and PRDX-SO2–3 at rest, immedi-

ately after the WHO-step test and at 30-min recovery in men suffering

from non-insulin-dependent type 2 diabetes mellitus before physical

training (BT) and after physical training (AT). Values are

means ± SD. �Significantly different from at rest (P B 0.05).�Significantly different from immediately after exercise (P B 0.05).

b Original photos of the 8-Iso-PGF and PRDX-SO2–3 immunohisto-

chemical stainings

Eur J Appl Physiol (2012) 112:2277–2287 2283

123

et al. 2003) raises the question whether T2DM patients’

erythrocytes are more defenceless against exercise-induced

free radicals and whether there are more rapid and higher

increases in oxidative stress during acute exercise than in

non-diabetic subjects.

Our results indicate similar changes in lipid-peroxidation

as measured by 8-Iso-PGF. Erythrocyte 8-Iso-PGF levels

increased during exercise and decreased in the following

30-min recovery phase in T2DM men and non-diabetic

control subjects. Increases and decreases (in relation to

exercise/recovery time) were also similar in both groups.

The data are in line with the findings of Miyazaki et al.

(2001) who found an increased lipid-peroxidation in the

erythrocyte membrane of healthy subjects immediately

after acute exercise compared with values at rest.

The finding that the contents of overoxidized peroxire-

doxins only increased in the diabetic group, but not in non-

diabetic men during acute exercise is particularly interesting.

It has been shown experimentally in different cell types,

e.g., in HeLa and yeast cells, that peroxiredoxins can

overoxidize in the presence of high amounts of hydrogen

peroxides (Lim et al. 2008; Seo et al. 2009). Consequently,

the peroxiredoxin system is ‘‘overloaded’’ and overoxida-

tion occurs (Low et al. 2008). Cho et al. (2010) recently

confirmed this mechanism in human erythrocytes and

found overoxidized peroxiredoxins when exposing red

blood cells to high hydrogen peroxide levels in vitro.

What could be the reason for an increase in the contents

of overoxidized peroxiredoxins in vivo to only occur in

T2DM men and not in non-diabetic control subjects under

increased oxidative stress as is the case during acute

exercise?

It can be suggested that the peroxiredoxin system

becomes more relevant in diabetes to buffer hydrogen per-

oxides in the erythrocytes if the activity of other enzymes

(e.g., of erythrocyte CAT, GPX) is reduced under patho-

logical conditions (Bhatia et al. 2003; Memisogullari et al.

2003). Furthermore, it can be speculated that the thioredoxin

system which is responsible for reducing oxidized perox-

iredoxins is negatively influenced by hyperglycemia (Minn

et al. 2005; Schulze et al. 2004). Moreover, it can be

hypothesized that the regulation of the activity of peroxire-

doxins through posttranslational modification (Seo et al.

2009; Jang et al. 2006) is different between the two groups.

However, increases in lipid-peroxidation, as measured by

8-Iso-PGF (relative to exercise time), were similar between

the diabetic and non-diabetic group. Therefore, the ratio of

exercise-generated free radicals and the total antioxidative

capacity seem to be similar in the patients of both our groups,

whereas antioxidative mechanisms obviously differ.

Finally, we examined whether T2DM men have

increased erythrocyte oxidative stress levels at rest relative

to non-diabetic subjects. In this case, exercise-induced

increases in oxidative stress would probably sum up to a

higher level of oxidative stress during acute exercise

compared with control subjects.

Different processes including hyperglycemia-dependent

mechanisms can be responsible for an elevated amount of

free radicals in T2DM subjects (Brownlee 2001; Inoguchi

et al. 2000; Kaneto et al. 2010; Wolff and Dean 1987).

Fig. 5 Increases/decreases in

8-Iso-PGF and PRDX-SO2–3

density during acute exercise

and at 30-min recovery (in

relation to exercise/recovery

time) in men suffering from

non-insulin-dependent type 2

diabetes mellitus before

physical training (BT) and after

physical training (AT). Values

are means ± SD. *Significantly

different from the pre-training

value (P B 0.05)

2284 Eur J Appl Physiol (2012) 112:2277–2287

123

In this context, Memisogullari et al. (2003) identified

increased oxidative stress in the erythrocytes of T2DM

men indicated by an increased concentration of malondi-

aldehyde (MDA). In our study, 8-Iso-PGF levels did not

significantly differ between overweight/obese T2DM and

overweight/obese non-diabetic men. It must be noted that

most of the diabetic subjects in our study took medications,

which, in part, are known to reduce ROS-production

(Ouslimani et al. 2005). Furthermore, the control subjects

were overweight/obese (BMI-matched) and it is accepted

that overnutrition increases systemic oxidative stress

(Urakawa et al. 2003). Therefore, it might be plausible that

oxidative stress is already increased in the erythrocytes of

overweight/obese men, and it is possible that arising T2DM

does not further increase oxidative stress levels, at least not

considerably.

A 30-min recovery period suffices to reduce the contents

of erythrocyte 8-Iso-PGF close to the basic level in both

diabetic as well as non-diabetic men. Nevertheless, the

contents of overoxidized peroxiredoxins were not signifi-

cantly reduced in the erythrocytes of the diabetic patients.

The peroxidatic cysteine of peroxiredoxins can be over-

oxidized to the sulfinic (CP-SO2H) and further on to the

sulfonic (CP-SO3H) acid form. The sulfinic form is

reversible and can be reduced by sulfiredoxin that is

expressed in red blood cells (Cho et al. 2010), whereas the

sulfonic form is probably irreversible (Seo et al. 2009).

Whether the sulfiredoxin system in the erythrocytes was

negatively affected by diabetes or whether peroxiredoxins

in the erythrocytes of the T2DM men in our study were

primarily overoxidized to the irreversible form remains

open. Further research on these questions is necessary.

Exercise-induced lipid-peroxidation and peroxiredoxin-

overoxidation in the erythrocytes of non-insulin-

dependent T2DM patients before and after

physical training

Several human studies have revealed a positive effect of

physical training on the activity/contents of antioxidative

enzymes, for example, of SOD, CAT, GPX in erythrocytes

or in skeletal muscle in the long-term (Ennezat et al. 2001;

Linke et al. 2005; Miyazaki et al. 2001; Ohno et al. 1988).

An up-regulation of antioxidative enzymes/proteins can

also be expected in the erythrocytes of the T2DM men in

our study, since we have revealed a decrease in basal

erythrocyte 8-Iso-PGF contents at rest as well as lower

increases in exercise-induced erythrocyte oxidative stress

(8-Iso-PGF in relation to exercise time) after training

compared with before training.

In line with our study, Miyazaki et al. (2001) examined

the influence of physical training on the oxidative stress

response in the erythrocytes of healthy men. They reported

a decreased magnitude of the increases in lipid-peroxida-

tion (thiobarbituric acid reactive substances (TBARS)) in

the erythrocyte membrane after acute exercise following

physical training. Miyazaki et al. also found an increase of

antioxidative enzyme activities at rest as well as a

decreased free radical production by neutrophils during

acute exercise following training. Consequently, it can be

deduced that regular physical activity can contribute to the

protection of erythrocytes from oxidative damage at rest

and during acute exercise.

Looking at our 8-Iso-PGF data, it could be possible that

the documented decreases in 8-Iso-PGF during the WHO-

step test following training are largely due to an increased

exercise time following physical training. When analyzing

absolute rather than relative increases (i.e., not in relation

to exercise time), no significant change is in fact obser-

vable pre- versus post-training. However, our data indicate

that less oxidative stress arises at the same workload pre-

versus post-training, i.e., oxidative stress increases more

slowly during the WHO-step test after the training

intervention.

Surprisingly, the magnitude of increases in overoxidized

peroxiredoxins was higher after than before training.

As a possible explanation it can be assumed that training

increases PRDX contents in the erythrocytes and, further-

more, the ratio of PRDX contents relative to other anti-

oxidative components. As a result, the probability of

peroxiredoxin oxidation and overoxidation increases in the

presence of high amounts of free radicals. This hypothesis

may be accepted, especially when the thioredoxin system is

weakened and/or sulfiredoxin activity/contents are reduced

in T2DM.

Of course the question is whether a high concentration

of overoxidized peroxiredoxins can be useful for the

erythrocytes of T2DM patients in any way. It has been

described in the literature that overoxidized peroxiredoxins

can convert into high-molecular-weight complexes (no

longer scavenging hydrogen peroxides) and act as chap-

erones which might protect proteins from (oxygen radical-

mediated) denaturation (Rhee et al. 2007).

Limitations

The use of only one measure (8-Iso-PGF) to determine the

presence of oxidative stress can be considered a limitation

of the study. The ratio of oxidized/reduced glutathione,

TBARS or other indicators for the redox state of the

erythrocytes should be examined in future studies (also

using other methods as well). It would also be particularly

interesting to compare the effect of physical training

between overweight/obese type 2 diabetic men and over-

weight/obese non-diabetic subjects to better elucidate the

effect of training on the type 2 diabetic organism,

Eur J Appl Physiol (2012) 112:2277–2287 2285

123

independent of obesity. It is, however, not too easy to

clearly determine the extent of the influence of diabetes

(before and after training) on oxidative stress variables,

because most of the subjects in our study also exhibited

other risk factors such as hyperlipidemia or hypertension,

which can affect the oxidative stress situation (Alexander

1995; Araujo et al. 1995).

Conclusions

The oxidative stress response to an acute bout of exercise,

measured as 8-Iso-PGF in the erythrocytes, is similar in

untrained overweight/obese non-diabetic control subjects

and T2DM patients, while antioxidative mechanisms

obviously differ. Regular physical activity can contribute to

the lowering of erythrocyte lipid-peroxidation (8-Iso-PGF)

at resting conditions as well as during acute exercise in

T2DM men. Therefore, cycling endurance training as

performed in our study can be considered a beneficial

strategy in diabetes to efficiently counteract ROS in

erythrocytes and prevent oxidative damage.

Acknowledgments The study was funded by the German Sport

University Cologne. The authors would like to thank A. Voss,

M. Ghilav and B. Collins for technical assistance.

Conflict of interest There is no conflict of interest for any of the

authors.

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