Whey protein increases muscle weight gain through inhibition of oxidative
effects induced by resistance exercise in rats

Kely R. Teixeira, Marcelo E. Silva, Wanderson G. de Lima, Maria L.

Pedrosa, Fabiano K. Haraguchi

PII: S0271-5317(16)30323-2
DOI: doi: 10.1016/j.nutres.2016.08.003
Reference: NTR 7670

To appear in: Nutrition Research

Received date: 28 February 2016

Revised date: 14 August 2016
Accepted date: 16 August 2016

Please cite this article as: Teixeira Kely R., Silva Marcelo E., de Lima Wanderson G.,
Pedrosa Maria L., Haraguchi Fabiano K., Whey protein increases muscle weight gain
through inhibition of oxidative effects induced by resistance exercise in rats, Nutrition
Research (2016), doi: 10.1016/j.nutres.2016.08.003

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 ACCEPTED MANUSCRIPT

Whey protein increases muscle weight gain through inhibition of oxidative effects

induced by resistance exercise in rats

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Kely R. Teixeira a, Marcelo E. Silva a,b, Wanderson G. de Lima b, Maria L. Pedrosa a,b,

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Fabiano K. Haraguchi b,c,*.

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a
Programa de Pós-Graduação em Saúde e Nutrição (PPGSN), School of Nutrition,

University of Ouro Preto (UFOP), Minas Gerais, Brazil.

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b
Núcleo de Pesquisas em Ciências Biológicas (NUPEB), University of Ouro Preto (UFOP),
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Minas Gerais, Brazil.
c
Programa de Pós-Graduação em Nutrição e Saúde (PPGNS), Departamento de Educação
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Integrada em Saúde (DEIS), University of Espírito Santo (UFES), Espírito Santo, Brazil.
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* Corresponding author
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Departamento de Educação Integrada em Saúde (DEIS), University of Espírito Santo, 29040-

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090 Vitória, Espírito Santo, Brazil;

Phone: 55 27 98173-7700

Fax: 55 27 3335-7016

fabianokenji@gmail.com

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Abbreviations

RONS; reactive oxygen and nitrogen species

SOD; superoxide dismutase

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CAT; catalase

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GPx; glutathione peroxidase

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GCS; gamma-glutamylcysteine synthetase

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Nrf2; nuclear factor erythroid 2–related factor 2

WP; whey protein

RE; resistance exercise

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CS; control sedentary

CE; control exercised

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WS; whey protein sedentary

WE; whey protein exercised

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EDL; extensor digitorum longus

HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

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EGTA; ethylene glycol tetraacetic acid

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DTNB; 5,5´-dithio-bis-(2-nitrobenzoic) acid

TNB; 2-nitro-5-thiobenzoic acid

EDTA; ethylenediaminetetraacetic acid

NADPH; nicotinamide adenine dinucleotide phosphate

TBARS; thiobarbituric acid-reactive substances

TBA; thiobarbituric acid

mRNA; messenger ribonucleic acid

cDNA; complementary deoxyribonucleic acid

CuZn SOD; copper zinc superoxide dismutase

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Keap1; Kelch-like ECH-associated protein 1

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Abstract

Whey protein (WP) is known for its nutritional value and antioxidant properties. The aim of

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this study was to evaluate whether the antioxidant properties of WP could contribute to

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muscle weight gain in response to resistance exercise (RE). We hypothesized that WP

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ingestion could increase muscle weight gain in rats subjected to an RE program through

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inhibition of oxidative effects induced by high-intensity RE. Thirty-two male Fischer rats

were randomly assigned to control sedentary, control exercised, WP sedentary, and WP

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exercised groups (n=8/group). RE consisted of inducing the rats to perform sets of jumps for
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8 weeks. Body and muscle weight gains, muscle glutathione content, histopathology, muscle

antioxidant enzyme activities, and gene expression were evaluated. Body and muscle weight
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gains of exercised rats fed WP were higher than those of control exercised rats.

Concomitantly, RE induced an increase in phagocyte infiltration, protein oxidation, and

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downregulation of glutathione peroxidase and gamma-glutamylcysteine synthetase mRNA

expression in gastrocnemius muscle (P<0.05), effects that were inhibited by WP ingestion.

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Cytosolic superoxide dismutase and catalase mRNA expression were reduced only by RE
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(P<0.05), and muscle glutathione content was increased only by WP (P<0.05) with no

significant interaction observed (P>0.05). These findings suggest that differences in body and

muscle weight gain in exercised rats fed control or WP diets were mediated in part by the

antioxidant properties of WP, and indicate that when associated with resistance exercise, WP

represents a nutritional aid to support muscle growth.

Key words: Rat; glutathione peroxidase; gamma-glutamylcysteine synthetase; superoxide

dismutase; catalase; muscle growth.

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1. Introduction

The generation of reactive oxygen and nitrogen species (RONS) by physical exercise can be

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characterized as a dose-response phenomenon, in which a low dose has a stimulatory effect

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and a high dose has an inhibitory effect, resulting in an inverted U-shaped dose–response

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curve, based on the concept of hormesis [1]. Exercise of moderate intensity produces low

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levels of RONS that, in turn, causes adaptation of the antioxidant and repair systems [2]. In

contrast, high-intensity exercise can accelerate the generation of RONS, which frequently

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exceeds the capacity for antioxidant defense and results in an oxidative stress status.
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Consequently, a decrease in muscle strength and force production, muscle soreness, and even

structural muscle damage can occur [3,4]. However, it is not clear whether exercise-induced
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oxidative stress affects muscle growth.

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The antioxidant defense systems include enzymes such as superoxide dismutase (SOD),

catalase (CAT), glutathione peroxidase (GPx), and constitutive glutathione enzymes, such as
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gamma-glutamylcysteine synthetase (GCS). Studies have shown differences in the enzymatic

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antioxidant responses induced by physical exercise [5,6], which are most likely due to

differences between exercise protocols [7]. Acute and chronic exercises generally improve

the antioxidant defense system during the recovery period, although divergent results have

been reported [7,8]. The mechanism that controls antioxidant enzyme gene expression in

different tissues, including skeletal muscle, involves the transcription factor nuclear factor

erythroid 2-related factor 2 (Nrf2), which has been described as playing a central role in

cellular protection against oxidative stress, initiating transcriptional activation of antioxidant

response elements in the promoter region of antioxidant genes [9,10]. Recently, Gounder et al.

[10] demonstrated that moderate exercise training, but not high endurance training,

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modulated Nrf2 levels and antioxidant enzyme gene expression in the hearts of aged mice.

This suggests that the regulation of antioxidant defenses by exercise could also occur in a

dose-dependent manner.

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In recent years, much attention has been given to the possible role of nutritional compounds

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in preventing the deleterious effects of exercise-induced oxidative damage [11,12], although

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their benefits are still a matter of debate [13,14]. In this context, whey protein (WP) deserves

special attention because it is present in a variety of sports supplements and several benefits

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have been reported [15,16], including the combination of WP with physical exercise [17,18].
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We previously showed that WP prevented muscle protein oxidation and maintained low

levels of peroxidized lipids in resistance-exercised rats, suggesting that the higher muscle and
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body weight gains observed in exercised rats fed WP may have been mediated in part through

a reduction in tissue (muscle and liver) oxidation [19]. The primary mechanism by which WP
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exerts its antioxidant properties is based on its capacity to increase liver glutathione levels

[20,21]. In vitro studies also suggest that the antioxidant effects of WP may occur in part
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through the increase of antioxidant enzyme activity, such as CAT, GPx, and SOD, and that
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Nrf2 is also involved in triggering this antioxidant response [22]. However, it is not known if

the antioxidant properties of WP could contribute to muscle weight gain in response to high-

intensity resistance exercise (RE).

In the present study, we hypothesized that WP ingestion could increase muscle weight gain in

rats subjected to an RE program, through inhibition of oxidative effects induced by high

intensity RE. To test this hypothesis, we subjected rats to an 8-week RE program, feeding

control or WP diets, and evaluated body and muscle weight gain, muscle histopathology, and

gene expression and activity of muscle antioxidant enzymes.

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2. Methods and materials

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2.1 Animals and diets

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Thirty-two male Fischer rats (60 days old) were randomly assigned to four experimental

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groups (n = 8 per group): control sedentary (CS), control exercised (CE), whey protein

sedentary (WS), and whey protein exercised (WE). The animals were housed individually in

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galvanized wire cages in a room with controlled temperature (23 ± 1 °C), relative humidity
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(55 ± 10 %), and a 12-h light/dark cycle. CS and CE rats were fed the AIN-93M standard diet

[23], and WS and WE animals received the AIN-93M diet containing WP instead of control
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protein. The composition of the diets is shown in Table 1. Food and water were provided ad

libitum and consumption was measured each week, with correction for spilled losses. The
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animals received care in accordance with the guidelines of the Brazilian College of Animal

Experimentation (CEEA). The study was approved by the Ethics Committee of the Federal
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University of Ouro Preto (Protocol # 036/2008).

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2.2 Exercise training program and experimental procedures

The present study is part of a larger study evaluating the effects of RE, alone or in

combination with WP ingestion, on biochemical and molecular parameters of muscle and

liver protein metabolism in rats [17,19].

Exercised rats were subjected to a high-intensity RE program for 8 weeks, as previously

reported [17,19], to mimic a weightlifting training model for hind limb muscles.

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In the first week, as an adaptation period, rats from each exercised group (CE and WE) were

subjected to swimming without weights for 15 min in a 40-cm-deep swimming pool with a

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water temperature of 32 ± 1 °C. After the adaptation period, the RE consisted of inducing the

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animals to perform sets of jumps in a circular plastic container with a depth corresponding to

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150 % of their body length. Weights were attached to the animal’s chest to promote

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submersion and the resistance to the exercise. When the rats touched the bottom of the

container, they had to jump to emerge from the water to breathe. The RE program consisted

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of inducing the animals to perform four sets of 10 jumps per day, five times per week for 8
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weeks. A one-minute rest interval was included between each set of jumps. Exercise intensity

was increased weekly by changing the maximum weight supported by each animal to
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perform the set of jumps correctly (~25 % of body weight in week 1, ~30 % in week 2, ~ 35 %

in week 3, ~40 % in week 4, ~45 % in week 5, ~50 % in week 6, and ~ 55 % in weeks 7 and
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8); 55 % of body weight was the greatest weight supported by the rats to perform all jumping

sets correctly. A pilot study with six animals in a single group was performed for 10 weeks to
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determine the amount by which the % load could be increased weekly without affecting
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exercise execution, and the highest depth that allowed rats to jump and breathe at the surface

without moving from a vertical to a horizontal position. This protocol aimed to provide high-

intensity RE. During the exercise sets, sedentary animals were kept in a similar aquatic

environment consisting of a 5-cm-deep swimming pool. At the end of the eighth training

week, and within 24 h of the last training session, all rats were fasted for 12 h, anesthetized

by isoflurane inhalation (4 %), and exsanguinated by cardiac puncture. Gastrocnemius and

extensor digitorum longus (EDL) muscles of both legs were immediately excised, washed,

weighed, sliced, and stored at -80 °C until further analysis, or buffered in formaldehyde for

histopathological analysis.

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2.3 Tissue preparation

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Gastrocnemius muscle samples were homogenized using a Potter-Elvehjem glass at 4 ºC with

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specific solutions (10 % w/v): sulfosalicylic acid (5 %) for total muscle glutathione,

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potassium phosphate buffer (pH 7.2) for catalase activity, and 4-(2-hydroxyethyl)-1-

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piperazineethanesulfonic acid (HEPES) (20 mM, pH 7.2), containing 1 mM ethylene glycol

tetraacetic acid (EGTA), 70 mM sucrose, and 210 mM mannitol for total SOD activity. After

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homogenization, samples were centrifuged for 10 min at 3000 ×g at 4 ºC and the supernatant
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was used for analysis.
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2.4 Muscle antioxidant enzyme activities

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Catalase activity was determined as previously described [24]. A 10-µL sample of

supernatant was mixed with 50 µL K2HPO4, 40 µL Milli-Q water (Millipore, Bedford, MA,
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USA), and 900 µL 2.5 mmol/L H2O2. Concentrations of H2O2 and samples were chosen such
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that the degradation rate was linear at 30 s, 1 min, 2 min, and 3 min. The rate of H2O2

decomposition was measured spectrophotometrically at 240 nm at 25 ºC. The decomposition

of hydrogen peroxide was calculated using the molar extinction coefficient 39.4 M-1cm-1. One

U of catalase is equivalent to the hydrolysis of 1 µmol of H2O2 per minute. Total protein

concentrations were determined by the method described by Lowry et al. [25] using bovine

serum albumin as the standard.

Total SOD activity was measured using the Superoxide Dismutase Assay Kit #706002

(Cayman Chemical Company, Ann Arbor, MI, USA), which utilizes tetrazolium salt for the

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detection of superoxide radicals generated by xanthine oxidase and hypoxanthine [26], and

measures CuZn SOD and Mn SOD types. One unit of SOD is defined as the amount of

enzyme needed to exhibit 50 % dismutation of the superoxide radical. Briefly, 10 µL of the

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supernatant was used in the assay. The reaction was initiated by adding 20 µL xanthine

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oxidase. The plate was incubated on a shaker for 20 min at room temperature, and the

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absorbance was measured at 450 nm using a plate reader (Biotek ELx808, Winooski, VT,

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USA). Bovine erythrocyte SOD (CuZn SOD) was used to generate the standard curve.

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2.5 Total muscle glutathione content, and muscle biomarkers of oxidative damage.
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Total glutathione content was determined in gastrocnemius muscle homogenates using the
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Sigma Kit #CS0260 (Saint Louis, MO, USA), as previously described [27]. This assay uses a

kinetic method to measure glutathione based on the reduction of 5,5´-dithio-bis-(2-

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nitrobenzoic) acid (DTNB) to 2-nitro-5-thiobenzoic acid (TNB), which can be

spectrophotometrically determined at 412 nm. Supernatant (10 µl) was mixed and incubated
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for 5 min with 150 µL of a solution containing 95 mM potassium phosphate buffer (pH 7.0),
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0.95 mM ethylenediaminetetraacetic acid (EDTA), 48 mM nicotinamide adenine dinucleotide

phosphate (NADPH), 0.031 mg/mL DTNB, 0.115 units/mL glutathione reductase, and 0.24 %

5-sulfosalicylic acid. Absorbance was measured using a plate reader (Biotek ELx808,

Winooski, VT, USA). Glutathione (reduced powder Sigma #G4251) was used to generate the

standard curve.

Protein carbonyls and peroxidized lipid content values are from our previously published data,

and methods have been described in detail elsewhere [19]. Peroxidized lipids were quantified

through thiobarbituric acid-reactive substance (TBARS) formation. Malondialdehyde (MDA),

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a product of lipid peroxidation, reacts with thiobarbituric acid (TBA) at low pH and high

temperature to form a pink-colored complex. Although TBARS is one of the most widely

used methods to detect MDA [28], TBA may react with several other compounds derived

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from oxidation, including aldehydes other than MDA, which can affect its accuracy and

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specificity [29].

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2.6 Quantitative real-time reverse transcription polymerase chain reaction

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Total RNA was isolated from 40–50 mg freeze-dried gastrocnemius muscle using the
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guanidine thiocyanate method with a lysis buffer solution (Promega Co., Madison, WI, USA).

The amount of RNA obtained was measured in a NanoVue spectrophotometer (GE

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Healthcare, United Kingdom). Two micrograms of RNA was used as a template to synthesize

cDNA, using a reverse transcriptase kit with random primers (Applied Biosystems, Foster
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City, CA, USA) in a total volume of 20 µL. The cDNA was stored at -80 °C until further

analysis.
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Real-time PCR was performed using the ABI 7300 Sequence Detection System (Applied

Biosystems) and the Power SYBR Green PCR Master Mix Kit (Applied Biosystems). The

primers were derived from Rattus norvegicus gene sequences (National Center of

Biotechnology Information GenBank) and were constructed using the Prime-BLAST

program (http://ncbi.nlm.nih.gov/tools/primer-blast/) as follows: rat CuZn SOD forward 5-

GAGCAGAAGGCAAGCGGTGAA-3 and reverse 5-CCACATTGCCCAGGTCTC-3; rat

CAT forward 5-ATTGCCGTCCGATTCTCC-3 and reverse 5-

CCAGTTACCATCTTCAGTGTAG-3; rat GPx forward 5-

CAGTTCGGACATCAGGAGAAT-3 and reverse 5-AGAGCGGGTGAGCCTTCT-3; rat

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GCS forward 5-ATCTGGATGATGCCAACGAGTC-3 and reverse 5-

CCTCCATTGGTCGGAACTCTACT-3. The expression of the 18S ribosomal RNA

housekeeping gene was determined as the endogenous control using the primers18S forward

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5-GTAAGTGCGGGTCATAAG-3 and reverse 5-CCATCCAATCGGTAGTAGC-3. The

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reactions were carried out under the following conditions: 50 °C for 2 min and 95 °C for 10

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min, followed by 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (primer

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annealing and product extension). The specificity of the products obtained was confirmed by

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analysis of the dissociation curves of the amplified products. The data obtained were

analyzed using the comparative 2-∆∆CT method [30]. Target gene expression was determined
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relative to the expression of the endogenous 18S gene. qPCR was conducted under tightly

controlled conditions to reduce variability, as recommended by the minimum information for

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publication of quantitative real-time PCR experiments (MIQE) guidelines [31]. All analyses

were performed in triplicate.

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2.7 Histopathological evaluation

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Muscle fragments not exceeding 4 mm in diameter were fixed in 10 % formaldehyde solution,

dehydrated, cleared, and embedded in paraffin. Paraffin sections of approximately 4 µm were

obtained by sectioning embedded fragments with a rotary microtome. The sections were

mounted on glass slides, previously cleaned and degreased, and then stained with

hematoxylin-eosin for visualization of histological changes. Digital morphometric analysis,

conducted to determine the average size and area of the inflammatory focus, was carried out

using a Leica DM5000 light microscope (Wetzlar, Germany) equipped with the Leica Qwin

Plus software. Inflammatory cells were quantified in the muscle by counting the number of

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cell nuclei present in tissue sections using ImageJ software (NIH, USA). For morphometric

analyses, 16 randomly taken images covering 1.5 × 106 µm2 of area were used.

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2.8 Statistical analyses

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The Shapiro-Wilk test (P>0.05) was used to test all variables for normal distribution and

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homogeneity of variances by Levene´s test. Data were analyzed by two-way ANOVA. The

classification variables were diet (CS+CE × WS+WE), exercise (CS+WS × CE+WE), and the

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interaction between diet and exercise (CS × CE × WS × WE). Tukey’s post hoc test was used
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to determine differences between the four groups when a statistically significant diet ×

exercise interaction was observed (P<0.05). Statistically different values were marked with
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different superscript letters when a significant interaction was observed. Body weight gain

during the experiment was evaluated weekly by one-way ANOVA. Values are means ± SD.
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Power analysis was based on our previous studies, to detect differences in gastrocnemius

muscle weight gain required to reach a power of 0.85 and α = 0.05, and indicated that 8
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rats/group were required [19]. The Statistical Package for the Social Sciences (version 21)
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was used for these analyses.

3. Results

3.1 Initial and final body weight, body weight gain, muscle weights, and food intake

Initial body weight was similar in all groups. After 8 weeks, CE rats exhibited the lowest

body and muscle weights. WE rats exhibited gastrocnemius and EDL weights similar to those

of CS and WS rats, but higher than those of CE rats. Food intake was significantly reduced

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only by exercise, and with no significant interaction (Table 2). Evaluation of body weight

gain showed a lower weight gain in CE rats after 3 weeks of RE, which persisted until the

end of the experiment (Fig. 1).

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3.2 Muscle antioxidant enzyme activities, muscle glutathione content, and biomarkers of

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oxidative damage

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Muscle total SOD and CAT activities were reduced by both RE and the WP diets, but no

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significant interaction was observed. On the other hand, total muscle glutathione content was
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increased only by the WP diets, independently of RE (Table 3). Muscle protein carbonyl

levels were increased significantly by RE in control rats, but not in WP-exercised rats. The
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WP diet was able to reduce muscle TBARS levels significantly, independently of RE, with no

significant interaction observed (data previously published) [19] (Table 3).

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3.3 Real-time PCR

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The results for antioxidant enzyme gene expression are shown in Fig. 2. Cytosolic SOD (Fig.

2A) and CAT (Fig. 2B) mRNA expression were reduced only by the RE program.A

significant interaction between RE and WP ingestion was observed for both GPx and GCS

mRNA expression. RE induced a downregulation of GPx mRNA in control rats, which was

inhibited by WP ingestion (Fig. 2C). GCS gene expression was not reduced significantly by

RE in control rats, but WE rats showed higher GCS mRNA expression than CE rats (Fig. 2D).

3.4 Histopathological evaluation

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Histological analysis of the gastrocnemius muscle after 8 weeks is presented in Fig. 3A. A

normal histological appearance with few inflammatory cells was observed in the

gastrocnemius muscle of CS, WS, and WE rats. The increased number of inflammatory cells

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(inflammatory infiltrate) was more pronounced in CE rats compared with that in the other

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groups. Quantification of inflammatory cells in the gastrocnemius muscle showed a higher

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number of inflammatory cells in the muscle tissue per microscopic field in CE rats, compared

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with all other groups (Fig. 3B).

4. Discussion
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The present study evaluated the effects of WP ingestion on body and muscle weight gain,
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muscle histopathology, gene expression, and muscle antioxidant enzyme activity in rats

subjected to an 8-week RE program. The main findings were that WP ingestion inhibited the
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oxidative effects induced by RE, including the downregulation of gene expression of

glutathione system enzymes and phagocyte infiltration in gastrocnemius muscle cells.

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Concomitantly, exercised rats fed a WP diet showed higher body and muscle weight gain
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than exercised rats fed the control diet.

Considering that the generation of RONS by physical exercise can be characterized as a

particular type of dose-response phenomenon, high-intensity exercise may be deleterious

because high levels of RONS production induced by exercise can cause an oxidative stress

status in muscle cells, increasing the risk of oxidative damage to cellular components [1,3]. It

is well known that oxidative damage induced by exercise can affect muscle contraction, force

production [4], and consequently, exercise performance, but its role in muscle growth is

unclear and the contribution of the antioxidant system to muscle growth in vivo is still an

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untested hypothesis. Schoenfeld [32] reported that muscle damage induced by exercise may

be related to muscle hypertrophy through intracellular signaling pathways that involve

inflammatory cell activation. However, the extent of damage that would be optimal to induce

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maximum muscle growth is unclear, and additional exercise-induced damage beyond this

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hypothetical threshold may be detrimental to muscle growth. The results of the present study

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suggest that this might also occur in control exercised rats.

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Gene expression of antioxidant enzymes is predominantly regulated by the transcription

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factor Nrf2, the master regulator of antioxidant gene transcription [9]. It has been reported
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that exercise can activate the Nrf2 pathway in different animal models and in human subjects

[10,33,34]. In addition, data from the literature suggest that Nrf2 activation could be related
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to physiological (age of subjects) and exercise (acute versus chronic and moderate versus

high intensity) conditions [10,35,36]. Gounder et al. [10] reported differences in Nrf2 levels
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and antioxidant gene expression after moderate and high intensity exercise in aged mice, but

not in young ones. Aged mice were highly susceptible to oxidative stress following high
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endurance exercise stress, but an increase in Nrf2 levels and adaptive redox homeostasis was
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observed after moderate exercise training. Although in the present study mRNA expression

of proteins of the Nrf2 pathway were not evaluated, it seems reasonable to speculate, based

on previous data [10] that the RE program might have interfered with Nrf2 pathway

activation in CE rats. This appears to have been detrimental to muscle growth, since RE

resulted in ineffective adaptation of the antioxidant system, including a downregulation of

antioxidant enzyme mRNA expression and a reduction of muscle antioxidant activities.

Consequently, a disturbance in oxidative balance was generated, increasing protein oxidation

[19] and the presence of inflammatory infiltrate in the gastrocnemius of CE rats, an oxidative

state that appears to have negatively affected the muscle growth. In fact, inflammatory cells

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can generate RONS via NADPH and mitochondrial oxidases, and other enzymes [37], which

are critical in the regulation of inflammatory response. In the present study, data suggest that

WP could reduce RONS generation by inflammatory cells, decreasing phagocyte infiltration

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and tissue inflammation, which might have contributed in part to inhibiting oxidative damage,

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and to the differences in muscle weight gain between CE and WE rats.

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Tissue glutathione synthesis is dependent on the dietary supply of cysteine, the rate-limiting

substrate for intracellular glutathione synthesis [38]. WP, with a cysteine content up to 5-fold

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higher than that of casein (control protein), is an important source of glutamyl-cysteine
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groups [19]. This fact could explain the higher muscle total glutathione content in WP rats,

and partially explain the differences in gene expression of GPx and GCS in control and WP
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rats, because both enzymes are strongly correlated with glutathione content, in contrast to the

other two antioxidant enzymes evaluated, CuZn SOD and CAT. Thus, the results indicate that
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WP protected muscle cells from oxidative damage induced by RE, through its capacity to

stimulate glutathione synthesis. Besides cysteine, alpha-lactalbumin, a protein component of

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whey, has antioxidant activity based on its iron-chelating properties [39]. Molecular
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mechanisms involved in antioxidant enzyme gene transcription induced by WP in vivo are

scarce, and most recent studies have proposed possible mechanisms in cultured cell models

[22,40]. C2C12 myoblast cells exposed to H2O2 and treated with WP showed an upregulation

of Nrf2 mRNA expression and an increase in several antioxidant enzyme activities and

glutathione content, indicating that the cytoprotective effects of WP might be mediated in

part by activation of Nrf2 [22]. In the present study, antioxidant enzyme gene expression may

have been downregulated through RE-induced inhibition of the Nrf2 pathway. It is therefore

possible that WP ingestion might have counteracted this effect, modulating the Nrf2 pathway

and allowing muscle cells to cope with excessive RONS generated by RE. This would

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suggest that activation of Nrf2 pathway by WP contributed, in part, to muscle growth in

response to exercise.

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Although WP inhibited the downregulation of GPx and GCS mRNA expression induced by

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RE, it significantly reduced muscle SOD and CAT activities. One possibility is that an

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increase in reduced glutathione concentration potentially increases the availability of

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reducing equivalents used by GPx to detoxify hydrogen peroxide [41], a fact that could

improve the oxidative balance and partially reduce the activity of both enzymes.

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Differences in food intake cannot entirely explain the differences we observed in body and

muscle weight gain in exercised rats, since the body and muscle weights of WE rats were
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similar to those of the CS and WS groups, but higher than those of CE rats. Oxidative

damage induced by RE may induce alterations in DNA, lipids, and proteins [3,4], reducing
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cellular biological function [42] and possibly the muscle protein synthesis induced by RE

[19]. WP is a high-quality protein source containing large amounts of leucine, a crucial amino
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acid that triggers muscle protein synthesis [43]. WP has been shown to meet more biological
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requirements than other proteins, including casein (control protein), because it contains

different bioactive peptides that can positively affect protein utilization for growth [44]. In

addition, when compared with casein, WP digestion/absorption kinetics promote a blood

leucine peak with different effects on protein turnover [45]. The findings of the present study

and data from the literature suggested that a synergistic effect between the biological quality

of WP and its antioxidant properties were crucial to differences in body and muscle weights

between CE and WE rats.

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Nutritional strategies to improve muscle mass are particularly important in older individuals,

in whom sarcopenia is commonly observed. Excessive RONS production and the oxidative

stress generated during aging contribute to sarcopenia development [46]. Our data

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corroborates previous reports [47], suggesting that WP could represent an effective

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nutritional aid to support muscle growth in this clinical condition [47]. However, some

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limitations of the present study should be highlighted: 1) The limited number of antioxidant

SC
enzymes evaluated. Evaluation of other antioxidant enzymes such as SOD isoforms, heme

oxygenase-1, glutathione S-transferase, and NADPH quinone oxidoreductase should help

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improve knowledge of how WP ingestion can enhance antioxidant defenses, in response to,
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or in the absence of, RE; 2) The use of TBARS as a biomarker of lipid peroxidation.

Although the TBARS assay has been widely adopted to measure MDA content [28], its
ED

application to tissue extracts has produced artifacts, including interference from related

species or overestimation derived from analysis conditions, which can affect its accuracy and
PT

reliability [29]; 3) Nrf2 pathway proteins, including Nrf2 itself, and others including Kelch-

like ECH-associated protein 1 (Keap1), a Nrf2 suppressor, were not evaluated. Binding of
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Keap1 to NrF2 prevents its translocation to the nucleus and therefore, its access to the gene
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promoter regions [48]. Whether the Nrf2 pathway is involved in WP-induced gene expression

of antioxidant enzymes, and muscle growth impaired by a high intensity RE program,

warrants further investigation.

In conclusion, these results support our hypothesis, since WP ingestion increases muscle

weight gain in resistance-exercised rats through inhibition of the oxidative effects induced by

a high intensity RE program, including the downregulation of gene expression of antioxidant

enzymes, mainly those of glutathione system, phagocyte infiltration, and protein oxidation.

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These findings indicate that WP represents an effective nutritional aid to promote muscle

growth when associated with RE, confirming its health-promoting benefits.

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Acknowledgments

P
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We thank the Federal University of Ouro Preto (UFOP, Minas Gerais, Brazil), Fundação de

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Amparo à Pesquisa de Minas Gerais (FAPEMIG, Minas Gerais Research Foundation),

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council

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for Scientific and Technological Development), and the University of Espírito Santo (UFES,
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Espírito Santo, Brazil).
ED

Conflicts of interest
PT

None
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Table 1 - Ingredient composition (g/kg) of the diets fed to rats

Ingredients CS/CE WS/WE

Caseina 140 -

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Whey Proteinb - 150

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Mineral Mixture (AIN-93M) 35 35

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Vitamin Mixture (AIN-93M) 10 10

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Soybean oil 40 40

Sucrose 100 100

Cellulose
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Choline 2.5 2.5

Cornstarch 622.5 612.5

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Total 1.000 1.000

CS, control sedentary; CE, control exercised; WS, whey protein sedentary; WE, whey protein
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exercised
a
Isofar (Rio de Janeiro, Brazil). Crude protein 85%
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b
Probiótica (São Paulo, Brazil). Crude protein 80%
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Table 2 - Initial and final body weight, gastrocnemius, and extensor digitorum longus muscle

weights, and food intake of the experimental groups after 8 weeks.

T
ANOVA
Experimental

P
(P value)

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Groups

CS CE WS WE D E DxE

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Initial body weight
119 ± 5 119 ± 3 118 ± 7 119 ± 4 0.813 0.838 0.994

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(g)

302 ± 264 ± 313 ± 308 ±

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Final body weight (g) <0.001 <0.001 <0.001
13a 10b 15a 20a

3522 ± 2985 ± 3426 ± 3393 ±

Gastrocnemius* (mg) 0.048 0.001 0.003
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a b a a
153 181 263 284

270 ± 246 ± 274 ± 274 ±

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EDL* (mg) 0.003 0.027 0.021

a b a a
14 10 19 15
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 Muscle weight** 3793 ± 3231 ± 3700 ± 3667 ±

0.037 0.001 0.003
a b a a
(mg) 160 190 280 290
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765 ± 671 ± 754 ± 707 ±

Food intake (g) 0.406 <0.001 0.099
30 36 74 44

Values are means ± SD (n=8/group). EDL, extensor digitorum longus; CS, control sedentary;

CE, control exercised; WS, whey protein sedentary; WE, whey protein exercised. *Right +

left muscles; **Gastrocnemius + EDL. Data were analyzed by two-way ANOVA followed

by the Tukey's post hoc test. Within a row, mean values with different letters differ, P<0.05.

D corresponds to diet effect (CS+CE × WS+WE), E to exercise effect (CS+WS × CE+WE),

and D × E is the interaction between the corresponding parameters (CS × CE × WS × WE).

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Table 3 - Total superoxide dismutase activity, catalase activity, total muscle glutathione,

protein carbonyls, and thiobarbituric acid-reactive substance content of the experimental

groups after 8 weeks.

T
Experimental ANOVA (P

P
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Groups value)

CS CE WS WE D E DxE

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2.0 ± 1.9 ± 1.6 ± 1.3 ± <0.00
SOD (U/mg of protein) 0.003 0.498

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0.2 0.2 0.4 0.1 1

<0.00
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CAT (U/mg of protein) 83 ± 17 65 ± 15 76 ± 24 39 ± 11 0.015 0.208
1

Total muscle glutathione 1.95 ± 1.98 ± 2.08 ± 2.38 ±

0.011 0.127 0.188
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(mmol/mg of muscle) 0.34 0.22 0.32 0.22

Protein carbonyls* 6.9 ± 11.9 ± 9.3 ± 7.1 ±

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0.168 0.064 0.001

b a b b
(mmol/mg of protein) 2.3 3.3 1.8 1.8
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TBARS* (mmol/mg of 5.8 ± 6.3 ± 5.7 ± 5.4 ±

0.031 0.464 0.420
protein) 1.3 1.8 1.9 1.6
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Values are means ± SD (n=8/group). SOD, superoxide dismutase; CAT, catalase; TBARS,

thiobarbituric acid-reactive substances; CS, control sedentary; CE, control exercised; WS,

whey protein sedentary; WE, whey protein exercised. Data were analyzed by two-way

ANOVA followed by the Tukey's post hoc test. Within a row, mean values with different

letters differ, P<0.05. D corresponds to diet effect (CS+CE × WS+WE), E to exercise effect

(CS+WS × CE+WE), and D × E is the interaction between the corresponding parameters (CS

× CE × WS × WE). *Data from Haraguchi et al. 2011 [19] with kind permission of Springer

Science + Business Media.

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350

300
Body Weight (g)

250
*

T
*
*

P
200 *
*

CS

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150 * CE
WS
WE

SC
100
0 1 2 3 4 5 6 7 8

Weeks

NU
MA
Figure 1
ED
PT
CE
AC

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A D: P=0.406
150 E: P=0.009
CuZn SOD mRNA expression (% of control)

D x E: P=0.289

125

100

T
P
75

RI
50

SC
25

0
CS CE WS WE

B NU
MA
D: P=0.080
120 E: P<0.001
D x E: P=0.053
CAT mRNA expression (% of control)

100
ED

80

60
PT

40

20
CE

0
CS CE WS WE
AC

C
250
D: P=0.002
225 E: P=0.301
a
GPx mRNA expression (% of control)

D x E: P=0.001
200 a

175

150

125
a
100

75

50 b

25

0
CS CE WS WE

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D
225 D: P=0.025
E: P=0.723 a
200
GCS mRNA expression (% of control)

D x E: P=0.020

175

T
150
a,b

P
125
a,b
100

RI
75
b
50

SC
25

0
CS CE WS WE

NU
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Figure 2
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PT
CE
AC

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 ACCEPTED MANUSCRIPT

T
P
RI
SC
B D: P=0.025
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120 E: P=0.048
D x E: P=0.019 a
Inflammatory cels/microscopic field

100
b b
b
80
ED

60
PT

40

20
CE

0
CS CE WS WE
AC

Figure 3

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Figure Captions

Fig. 1 - Body weight gain throughout 8 weeks of the experiment. Values are means ± SD

P T
(n=8/group). Data were analyzed by one-way ANOVA followed by the Tukey's post hoc test.

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Asterisks indicate differences for CE group, P<0.05. CS, control sedentary; CE, control

SC
exercised; WS, whey protein sedentary; WE, whey protein exercised;

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Fig. 2 - Quantitative real-time PCR assay of antioxidant enzymes in gastrocnemius muscle
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after 8 weeks. Copper/zinc superoxide dismutase (A), Catalase (B), Glutathione peroxidase

(C), and Gamma-glutamylcysteine synthetase (D) of sedentary (black bars) or exercised (grey
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bars) rats fed with control or whey protein diets. Values are means ± SD (n=8/group). Data

were analyzed by two-way ANOVA followed by the Tukey's post hoc test. Bars with
PT

different letters differ, P<0.05. D corresponds to diet effect (CS+CE × WS+WE); E

CE

corresponds to exercise effect (CS+WS × CE+WE); and D × E is the interaction between the

corresponding parameters (CS × CE × WS × WE). CS, control sedentary; CE, control

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exercised; WS, whey protein sedentary; WE, whey protein exercised.

Fig. 3 - Photomicrographs of histological muscle sections (A). Hematoxylin-eosin staining.

Scale bar, 50 μM. Arrows indicate increased numbers of inflammatory cells (inflammatory

infiltrate). Number of inflammatory cells in the muscle tissue per microscopic field (B).

Values are means ± SD of sedentary (black bars) or exercised (grey bars) rats fed with control

or whey protein diets. Data were analyzed by two-way ANOVA followed by the Tukey's post

hoc test. Bars with different letters differ, P<0.05. D corresponds to diet effect (CS+CE ×

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WS+WE); E corresponds to exercise effect (CS+WS × CE+WE); and D × E is the interaction

between the corresponding parameters (CS × CE × WS × WE). CS, control sedentary; CE,

control exercised; WS, whey protein sedentary; WE, whey protein exercised.

P T
RI
SC
NU
MA
ED
PT
CE
AC

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