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Original Research

Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats Rafael A. Casuso a,⁎, Antonio Martínez-Amat a , Fidel Hita-Contreras a , Daniel Camiletti-Moirón b , Pilar Aranda b , Emilio Martínez-López c a

Department of Health Sciences, University of Jaén, E-23071 Jaén, Spain Department of Physiology, Faculty of Pharmacy and Institute of Nutrition and Food Technology, Faculty of Sport Sciences, and Institute of Nutrition and Food Technology, University of Granada, Campus Universitario de Cartuja s/n, Granada 18071, Spain c Department of Music, Plastic Expression and Body Language, University of Jaén, E-23071 Jaén, Spain b

ARTI CLE I NFO

A BS TRACT

Article history:

The present study tested the hypothesis that quercetin may inhibit the mitochondrial and

Received 29 December 2014

antioxidant adaptations induced by exercise in cerebellar tissue. Thirty-five 6-week-old Wistar

Revised 29 April 2015

rats were randomly allocated into the following groups: quercetin, exercised (Q-Ex; n = 9);

Accepted 14 May 2015

quercetin, sedentary (Q-Sed; n = 9); no quercetin, exercised (NQ-Ex; n = 9); and no quercetin, sedentary (NQ-Sed; n = 8). After 6 weeks of quercetin supplementation and/or exercise

Keywords:

training, cerebellums were collected. Protein carbonyl content (PCC), sirtuin 1, peroxisome

Flavonoids

proliferator-activated receptor γ coactivator 1α (PGC-1α), messenger RNA levels, citrate

Training

synthase (CS), and mitochondrial DNA were measured. When Q-Sed was compared with

Oxidative stress

NQ-Sed, PCC (P < .005) showed decreased levels, whereas PGC-1α, sirtuin 1 (both, P < .01),

PGC-1α

mitochondrial DNA (P < .001), and CS (P < .01) increased. However, when Q-Ex was compared

SIRT1

with Q-Sed, PCC showed increased levels (P < .001), whereas CS decreased (P < .01).

Wistar rats

Furthermore, the NQ-Ex group experienced an increase in PGC-1α messenger RNA levels in comparison with NQ-Sed (P > .01). This effect, however, did not appear in Q-Ex (P < .05). Therefore, we must hypothesize that either the dose (25 mg/kg) or the length of the quercetin supplementation period that was used in the present study (or perhaps both) may impair exercise-induced adaptations in cerebellar tissue. © 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Quercetin (3,3,4,5,7-pentahydroxyflavone) is a natural polyphenolic flavonoid that is present in large amounts in onions,

garlic, leeks, cabbages, apples, blueberries, tea, and red wine [1]. Despite having quite slow absorption rates [2], quercetin is thought to be significantly accumulated in the lungs, the liver, and the kidneys but can be found in most body tissues [3,4].

Abbreviations: CAT, catalase activity; CS, citrate synthase; mtDNA, mitochondrial DNA; PCC, protein carbonyl content; PGC-1α, peroxisome proliferator–activated receptor γ coactivator 1α; ROS, reactive oxygen species; SIRT1, sirtuin 1; SOD, superoxide dismutase activity; TBARS, thiobarbituric acid reactive substances. ⁎ Corresponding author. Tel.: +34 953 212970; fax: +34 953 012141. E-mail addresses: [email protected] (R.A. Casuso), [email protected] (A. Martínez-Amat), [email protected] (F. Hita-Contreras), [email protected] (D. Camiletti-Moirón), [email protected] (P. Aranda), [email protected] (E. Martínez-López). http://dx.doi.org/10.1016/j.nutres.2015.05.007 0271-5317/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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In the tissues, quercetin exerts some of its biological properties by increasing the transcription of sirtuin 1 (SIRT1) and peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) [5]. Currently, PGC-1α is described in the literature as the master regulator of mitochondrial biogenesis. It coactivates and augments the expression and activity of several transcription factors, which in turn bind the promoters of distinct sets of nuclear-encoded mitochondrial genes [6–8]. In terms of energy/nutrient stress, such as exercise, posttranscriptional activation of PGC-1α is mainly induced by SIRT1, a metabolic sensor that is regulated by NAD+, which in turn induces PGC-1α activation by deacetylation [9–11]. Polyphenolic compounds activate mitochondrial biogenesis by transcriptional regulation of SIRT1 and PGC-1α, which occurs in the brain and in skeletal muscle [5,12,13]. These effects seem to mimic those brought about by exercise in the mitochondrial content of skeletal muscle [8]. However, after a 6-week test of quercetin supplementation on exercised rats, mitochondrial content in skeletal muscle was compromised in response to lower SIRT1 transcription [14]. Furthermore, the brains of rats that consumed quercetin while exercising showed lower mitochondrial content as a result of the ablation of the SIRT1–PGC-1α axis [15]. Furthermore, oxidative damage affected protein structures in the tissues of exercised rats [14,15]. Boots et al [16] described that metabolites (as a result of their antioxidant activity) changed to pro-oxidant agents, which in turn attacked protein structures [16,17]. This paradox might be responsible for cellular and systemic inadaptation to exercise that is observed when quercetin is supplemented during exercise [18]. Exercise improves oxidative status [19] and mitochondrial content [20] in cerebellar tissue. However, quercetin can exert powerful oxidative damage in the cerebellum [21]. Mitochondrial dysfunction and oxidative stress are present in ataxia [22], and the cerebellum plays a primary role in ataxia [23]. It is therefore crucial to determine whether people should be discouraged from consuming isolated quercetin during exercise. Therefore, our research hypothesis is that quercetin may inhibit mitochondrial and antioxidant adaptations caused by exercise in cerebellar tissue. For that purpose, we analyzed mitochondrial content, oxidative stress, and the transcription of SIRT1 and PGC-1α in the cerebellum of rats that were subjected to an exercise regime and concomitantly supplemented with quercetin.

2.

Methods and materials

2.1.

Animals

This study was performed on male Wistar rats (6 weeks old). The animals were maintained for 8 weeks in individual cages under standard light and temperature conditions. They were allowed ad libitum access to food (Harlan, Indianapolis, IN, USA 2014; maintenance chow) and water. All animals were cared for in accordance with the Guide for the Care and Use of Experimental Animals, and all experiments were approved by the committee of ethics of the University of Jaén (Spain).

2.2.

Exercise and supplementation

Rats were randomly assigned to quercetin (Q; n = 17) and nonquercetin (NQ; n = 16) supplementation groups. Both groups were

further divided into Q-exercised (Q-EX; n = 9), Q-sedentary (Q-Sed; n = 8), NQ-exercised (NQ-EX; n = 8), and NQ-sedentary (NQ-Sed; n = 8). The animals were acclimated to the experimental conditions, as well as to the treadmill, for 2 weeks. Treadmill training took place 5 days a week for 6 weeks (Panlab Treadmills, Hollistion, MA, USA for 5 rats LE 8710R). The rats ran at a constant speed of 44 cm/s and at a 10° angle. The rats ran for 20 minutes on each of the first 2 days and for 25 minutes on the third day. The duration of the training was increased by 5 minutes every 2 days. The animals ran for 80 minutes on the last day of the fifth week and also throughout the last week of training [14]. The rats in the quercetin groups were supplemented via gavage (QU995; Quercegen Pharma, Newton, MA, USA) on alternate days during the experimental period. Quercetin was administered at 8:00 AM in the morning, when the 12-hour dark cycle began and also 2 hours before exercise. A dose of 25 mg/kg of quercetin was diluted in a 1% solution of methylcellulose. This dose has been described in the literature as able to promote mitochondrial biogenesis in the brain [5]. Twice a week, rats were weighed to adjust the quercetin dosage. The noquercetin groups were also supplemented using the gavage procedure with the vehicle (1% solution of methylcellulose).

2.3.

Tissue collection

All rats were anesthetized with pentobarbital and were then bled by cannulation of the aorta 48 hours after the final exercise. We opted for a fresh dissection of the cerebellum. Once collected, the cerebellum was rinsed in saline solution, frozen in liquid nitrogen, and stored at −80°C for later analysis.

2.4.

Quantitative real-time polymerase chain reaction

Gene expression of different genes, PGC-1α and NAD(+)-dependent histone deacetylases (SIRT1), was quantitatively assessed by realtime polymerase chain reaction (PCR) using β-actin as the normalizing gene. Total RNA was isolated from cell extracts using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After treatment with DNase, complementary DNA was synthesized from 1.5 μg of total RNA using reverse transcriptase (SuperscriptTM III RT; Invitrogen) with oligo(dT) 15 primers (Promega, Fitchburg, WI, USA). Real-time PCR was performed on the Stratagene MxPro 3005P qPCR system using the Brilliant II SyBR Green QPCR Master Mix (Stratagene, La Jolla, CA, USA). The following primer pairs were used: PGC-1α, 5′-GCGGACAGAA CTGAGAGACC-3′ and 5′-CGACCTGCGTAAAGTATATCCA-3′; SIRT1, 5′-CCTGACTTCAGATCAAGAGATGGTA-3′ and 5′-CTGATTAAAAATA TCTCCACGAACAG-3′; and β-actin, 5′-CTTAGAAGCATTTGCGGTG CCGATG-3′ and 5′-TCATGAAGTGTGACGTTGCATCCGT-3′. Experiments were performed in triplicate, and relative quantities of the target genes corrected with the normalizing gene, β-actin, were calculated using Stratagene MxProTM QPCR Software. Quantification of messenger RNA (mRNA) expression of PGC-1α and SIRT1 was calculated using the ΔΔCT method as previously described [24].

2.5.

Mitochondrial DNA quantification

DNA (mitochondrial and nuclear) was extracted from cerebellar samples using a QIAamp DNA minikit (Qiagen, Chatworth, CA, USA), and the concentration of each sample was spectrophotometrically determined at 260 nm. Real-time PCR was performed

Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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using the Stratagene MxPro 3005P qPCR system using the Brilliant II SyBR Green QPCR Master Mix (Stratagene). Mitochondrial content was estimated as the ratio between copy numbers of mitochondrial DNA (mtDNA; cytochrome b; forward, 5′-AAA GCCACCTTGACCCGATT-3′; reverse, 5′-GATTCGTAGGGCCGC GATA-3′; probe, 5′-CGCTTTCCACTTCATCTTACCATT-3′) vs nuclear DNA (β-actin).

2.6.

Protein carbonyls

Determination of the protein carbonyl content (PCC), an indicator of oxidative stress, was carried out following the 2,4dinitrophenylhydrazine method as described by Levine et al [25]. The results are expressed as millimoles per milligrams protein.

2.8.

and with/without exercise). If this analysis revealed any significant interaction (P < .05), specific differences between mean values were located using Student t test for independent samples. The level of significance was considered at P < .05. Both effect size (η2) and power analysis (1 − β) were performed to determine sample size for all measurements [29,30]. All analyses were performed using the Statistical Package for Social Sciences (IBM-SPSS, version 22.0 for Windows; SPSS, Chicago, IL, USA).

Thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS), major indicators of oxidative stress, were determined in mice cerebellums following the manufacturer's instructions for the Oxikek TBARS Assay Kit (ZeptoMetrix Corp, Buffalo, NY, USA). The final values refer to total protein concentration in the initial extracts (in nanomoles per milligrams of protein).

2.7.

Enzyme assays

For catalase (CAT) activity, cerebellums were rinsed in saline solution and stored at −80°C until used. Cerebellums were homogenized on ice in 5 to 10 mL of cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) per gram of tissue. After centrifugation at 10 000 × g for 15 minutes, the supernatant was collected for protein determination [26] and subsequent analysis. All procedures were performed at 4°C. Catalase activity was studied by monitoring the decomposition of H2O2 at 240 nm, according to the method described by Beers and Sizer [27]. For superoxide dismutase (SOD) activity, cerebellums were homogenized on ice in 5 to 10 mL of cold buffer (20 mM HEPES, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per gram of tissue. After centrifugation at 15 000 × g for 5 minutes, the supernatant was collected for protein determination [26] and subsequent analysis. All procedures were performed at 4°C. Superoxide dismutase activity was assayed by measuring the rate of inhibition of cytochrome c reduction by superoxide anions generated by a xanthine/ xanthine oxidase system [28]. Total citrate synthase (CS) activity was measured in Tris · HCl buffer (50 mM Tris · HCl, 2 mM EDTA, and 250 μM NADH, pH 7.0) and 0.04% Triton-X. The CS reaction was started by the addition of 10 mM oxaloacetate, and activity was measured spectrophotometrically by measuring the disappearance of NADH at 412 nm. The total protein content of all homogenates was determined by the method of Bradford [26].

3.

Results

Fig. 1 shows the effect of exercise training and quercetin on oxidative damage in the cerebellum. A main effect appeared for both quercetin and exercise, indicating a decrease in TBARS content (both P < .001). However, no interactions were found in TBARS (P = .156). Exercise decreased (P = .001, main effect) PCC. When quercetin was supplemented during exercise, cerebellar PCC increased (P < .001, η2 = 0.464, 1 − β = 0.998) in comparison with NQ-Ex and Q-Sed. Q-Sed (P < .05, η2 = 0.051, 1 − β = 0.226) showed a reduced PCC in comparison with NQ-Sed. Total SOD and CAT antioxidant activities are represented in Fig. 2, but the interaction analysis was nonsignificant. Transcription of PGC-1α and SIRT1 is shown in Fig. 3. Quercetin increased SIRT1 transcription (P = .002, main effect for quercetin). The interaction analysis showed that the mRNA level of SIRT1 was higher in Q-Sed (P = .001, η2 = 0.286, 1 − β = 0.909) than in NQ-Sed. In the Q-Ex group, there was a trend toward reduction (P = .052, η2 = 0.222, 1 − β = 0.793) of the transcription of SIRT1 in comparison with Q-Sed. Exercise training increased the mRNA level of PGC-1α (P < .05, main effect for exercise). The interaction analysis showed that NQEx (P = .009, η2 = 0.185, 1 − β = 0.699) and Q-Sed (P = .001, η2 = 0.01, 1 − β = 0.083) increased PGC-1α transcription in rat cerebellums. The increased transcription of PGC-1α that appeared in NQ-Ex was abolished in the Q-Ex group (P = .043, η2 = 0.322, 1 − β = 0.948). Markers of mitochondrial content are displayed in Fig. 4. Quercetin supplementation increased mtDNA content (P = .007 main effect for quercetin). The interaction analysis revealed that NQ-Ex increased (P = .002, η2 = 0.019, 1 − β = 0.111) mtDNA content in comparison with NQ-Sed. Apparently, quercetin mimics this effect because Q-Sed (P < .001, η2 = 0.222, 1 − β = 0.794) showed increased mtDNA content in comparison with NQ-Sed. With regard to CS quercetin, it also displayed increased activity (P < .001; main effect for quercetin). The interaction analysis showed an increased CS activity in both Q-Sed (P < .001, η2 = 0.357, 1 − β = 0.972) and NQ-Ex (P < .001, η2 = 0.106, 1 − β = 0.433) compared with NQ-Sed. Nevertheless, quercetin-induced increases in CS activity were prevented in the Q-Ex group (P < .01, η2 = 0.582, 1 − β = 1.000).

4. 2.9.

3

Discussion

Statistical analyses

The results are presented as means ± SD. Homoscedasticity and normality were tested using the Levene and KolmogorovSmirnov tests, respectively. The results were analyzed using a 2-factor analysis of variance (ANOVA; with/without quercetin

Our results suggest that 25-mg/kg quercetin supplementation during exercise acts as an oxidative agent to the proteins of the rat's cerebellum. Basically, quercetin supplementation increases the transcription of some key coactivators of the mitochondrial biogenesis machinery such as SIRT1 and PGC-1α. Similarly,

Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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Fig. 1 – Effects of quercetin supplementation and exercise on TBARS (A) and PCC (B) in the cerebellum. Non-Q, non–quercetinsupplemented groups: exercised (NQ-Ex; n = 9) and sedentary (NQ-Sed; n = 8). Q, quercetin groups: exercised (Q-Ex; n = 9) and sedentary (Q-Sed; n = 9). Values are means ± SD. The results of a 2-way ANOVA (with/without quercetin and with/without exercise) and Student t test for independent samples when an interaction was significant. †††P < .001, main effect for quercetin; ***P < .001, main effect for exercise; #P < .05 and ###P < .001 for quercetin × exercise interaction.

Fig. 2 – Effects of quercetin supplementation and exercise on CAT (A) and total SOD (B) activity in cerebellum. Non-Q, non–quercetin-supplemented groups: exercised (NQ-Ex; n = 9) and sedentary (NQ-Sed; n = 8). Q, quercetin groups: exercised (Q-Ex; n = 9) and sedentary (Q-Sed; n = 9). Values are means ± SD. The results of a 2-way ANOVA (with/without quercetin and with/without exercise) and Student t test for independent samples when an interaction was significant. ††P < .01, main effect for quercetin; *P < .05 and **P < .01, main effect for exercise.

exercise induces the transcription of PGC-1α, although this effect was hampered in the Q-Ex group. Both exercise and quercetin increase mitochondrial content in cerebellar tissue, but the increased CS activity shown in response to quercetin supplementation is compromised if supplementation occurs concomitantly with exercise. Quercetin seems to regulate cerebellar mitochondrial content by increasing the transcription of both SIRT1 and PGC-1α, whereas exercise only targets PGC-1α. Quercetin is present at relatively low concentrations of 15 to 30 mg/kg fresh weight. Its richest sources are onions (up to 1.2 g/kg fresh weight), curly kale, leeks, broccoli, and blueberries. It is present, however, in all plant foods [1]. Quercetin has quite an extensive metabolism because it can be absorbed by the stomach [31]. Nevertheless, most is absorbed in the small intestine. Conjugation of quercetin occurs in the small intestine and then in the liver, after being incorporated into portal circulation [31]. Quercetin's major circulating compounds in plasma were identified as quercetin 3-O-glucuronide, 3′-Omethylquercetin 3-O-glucuronide, and quercetin 3′-O-sulfate [32]. Once quercetin metabolites reach the blood flow, they are able to transverse the blood-brain barrier and are subsequently accumulated in the brain [33], where they exert their biological effects on the cerebellum [21,34,35].

Exercise training is thought to increase mitochondrial biogenesis in the cerebellum as it does in skeletal muscle [20]. Eight weeks of exercise induced a more powerful relative induction of PGC-1α than SIRT1 within cerebellar tissue [20]. It can therefore be assumed that exercise has a greater effect on the transcription of PGC-1α than on that of SIRT1. Our data support this view because SIRT1 remains unchanged when NQ-Sed and NQ-Ex were compared. However, mitochondrial content is increased after exercise training, which might be explained because exercise targets PGC-1α and not its upstream regulator SIRT1, and PGC-1α is considered to be the main regulator of mitochondrial biogenesis [6–8]. PGC-1α is thought to be regulated by metabolic energy sensors such as AMP-activated protein kinase, whose activation is thought to occur in an SIRT1-dependent manner [11]. It is possible, however, that the preexercise SIRT1 content is enough to deacetylate PGC-1α and trigger the mitochondrial biogenesis process because mitochondrial content is increased after heightened SIRT1 activity despite a decrease in SIRT1 protein content [36]. Similarly to exercise, quercetin seems to increase the mitochondrial content in the brain through activation of the SIRT1–PGC-1α pathway [5,15]. The present study extends this

Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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Fig. 3 – Effects of quercetin supplementation and exercise on PGC-1α and SIRT1 expression in the cerebellum. Non-Q, non– quercetin-supplemented groups: exercised (NQ-Ex; n = 9) and sedentary (NQ-Sed; n = 8). Q, quercetin groups: exercised (Q-Ex; n = 9) and sedentary (Q-Sed; n = 9). Values are means ± SD. The results for a 2-way ANOVA (with/without quercetin and with/without exercise) and Student t test for independent samples when an interaction was significant. ††P < .01, main effect for quercetin; *P < .05, main effect for exercise; #P < .05 and ## P < .01 for quercetin × exercise interaction.

effect to the cerebellar tissue. Furthermore, polyphenols such as isoflavones, resveratrol, and quercetin target SIRT1 at the transcriptional level, which in turn increases PGC-1α mRNA levels [5,12,13]. In a previous study, we found that despite an increased PGC-1α mRNA level in skeletal muscle, mitochondrial content was not increased in exercised rats that were supplemented with quercetin, which can be explained by the unchanged SIRT1 expression [14]. It should be noted that similarly to data regarding the whole brain [15], quercetin supplementation during exercise also inhibits cellular adaptations related to mitochondrial biogenesis in rat cerebellums. The underlying mechanisms may differ between regions of the brain because only PGC-1α is ablated in cerebellar tissue, whereas the entire SIRT1–PGC-1α axis was knocked out in the brain [15]. Thus, as previously reported, the cerebellum may not respond to exercise in the same manner as other areas of the brain [20]. Regarding redox status, quercetin decreased PCC in the sedentary condition regardless of antioxidant activity. This differs from what has previously been shown for the brain as a whole [15]. However, when supplemented during exercise, a pro-oxidant shift occurred by increasing PCC, as has already been described for brain and skeletal muscle tissue [14,15]. This can be explained by the “quercetin paradox,” which suggests that the antioxidant effect of quercetin occurs at the

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Fig. 4 – Effects of quercetin supplementation and exercise on mtDNA content and CS activity. Non-Q, non–quercetinsupplemented groups: exercised (NQ-Ex; n = 9) and sedentary (NQ-Sed; n = 8). Q, quercetin groups: exercised (Q-Ex; n = 9) and sedentary (Q-Sed; n = 9). Values are means ± SD. The results for a 2-way ANOVA (with/without quercetin and with/without exercise) and Student t test for independent samples when an interaction was significant. ††P < .01 and ††† P < .001, main effect for quercetin; ##P < .01 and ###P < .001 for quercetin × exercise interaction.

first stage of supplementation; later on, quercetin metabolites become pro-oxidant agents [16,17]. As a by-product of its antioxidant activity, quercetin is oxidized; if the glutathione concentration is not high enough, the potential oxidative reactions induced by quercetin will be focused on the protein thiols [16]. Martins et al [21] showed that quercetin administration potentiates the oxidative damage induced by methylmercury in mouse cerebellum. This effect was found together with unchanged levels of cerebellar glutathione and glutathione reductase activity induced by quercetin and methylmercury separately and when both treatments were combined. This issue indicates that the glutathione concentration may not be high enough within the cerebellum to face the prooxidant effects derived from the quercetin paradox. Nevertheless, such a hypothesis remains to be further studied because the potential mechanisms remain unclear. Quercetin-induced adaptations have been the subject of several tests on mice in which no metabolic effects mediated by PGC-1α occurred after 3 weeks of supplementation but were achieved instead after 8 weeks of supplementation. Significantly, such effects only appeared with low (0.05 mg) quercetin doses [37]. Regarding the cerebellum, our results depict a scenario in which quercetin prevents exercise-induced mitochondrial

Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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adaptations through PGC-1α ablation in conjunction with increased oxidative damage. The brain is particularly vulnerable to reactive oxygen species (ROS) production because it only accounts for ~2% of body weight but metabolizes 20% of the body's total oxygen and has limited antioxidant capacity [38]. Lipids can be oxidized by numerous ROS and radicals. The resulting lipid peroxidation is a complex process that produces a variety of products (eg, lipid hydroxides and aldehydes). Such aldehydes link the oxidation of lipids and proteins because proteins can be damaged as a result of it binding with them. Nevertheless, the lack of lipid peroxidation evidence suggests that aldehydes may not be triggering the protein damage; hence, such damage might be a result of the direct oxidation of the side chain of amino acids by ROS or by protein glycation with sugars [39]. However, caution should be taken regarding the underlying mechanisms; therefore, further studies are needed to clarify this issue. The only fact is that we have found a disturbance in the redox balance in cells of the cerebellum in favor of oxidants. This is of valuable importance because ataxia is a metabolic disease mainly caused by high oxidative stress together with diminished mitochondrial function in the cerebellum [22]. Moreover, in the early steps of exercise training, an increased oxidative challenge takes place, which in turn triggers most of the adaptations induced by exercise in the brain [40]. This can be attributed to the pro-oxidant effect of quercetin metabolites exceeding the oxidative stress that is tolerated by the cell and may also explain why the Q-Ex group does not achieve mitochondrial adaptations induced by exercise. This requires further study, as little is known about the molecular and physiological effects of polyphenols supplemented during exercise. Nevertheless, our results are relevant for human nutrition because the dosage used in the present study (25 mg/kg) is the one commonly recommended for athletes to enhance exercise performance [41]. It should be considered whether the dose used in the present study is too high for enhancing mitochondrial and oxidative adaptations. As a matter of fact, a dose of 0.05 mg/d for 8 weeks improved the metabolic status of mice fed a high-fat diet through PGC-1α activation, although such effects did not become apparent, neither after 3 weeks of supplementation nor when quercetin was supplemented at a higher dose [37]. Furthermore, a dosage of 17 mg kg−1 d−1 improved mitochondrial content and function in mice fed both high- and low-fat diets for 9 weeks [42]. Thus, to reach a reliable conclusion regarding quercetin supplementation as an ergogenic agent, further studies must be performed, with a focus on matching length and dose with oxidant/antioxidant status and physiological adaptations. The present study has some limitations worth mentioning. First, stomach gavage can be a stressful procedure, which is why we chose to supplement rats every other day. Second, SIRT1 and PGC-1α protein content was not measured and may not correlate with mRNA levels, and therefore, our results can only be applied at the transcriptional level. Third, TBARS is not the best marker to assess the impact of antioxidant supplementation and redox status [39]. Overall, we accept our initial hypothesis that quercetin supplementation during exercise decreases cerebellar mitochondrial biogenesis as a result of the joint effect of lower PGC-1α transcription and increased oxidative stress.

To summarize, both exercise and quercetin increase mitochondrial content in the cerebellum of rats. However, a quercetin dosage of 25 mg/kg may impair exercise-induced adaptations within the cerebellum. More in-depth studies are required to assess the exact dosage and length of supplementation before a consensus can be reached regarding quercetin supplementation during exercise.

Acknowledgment The authors would like to thank all members of the Department of Physiology (University of Granada) for their collaboration and Quercegen Pharma for kindly providing the quercetin used in the study. The present study was funded by the Master's Program in Physical Activity and Health Sciences (University of Jaén, Spain). The authors declare no conflict of interest.

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Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

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Please cite this article as: Casuso RA, et al, Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats, Nutr Res (2015), http://dx.doi.org/10.1016/j.nutres.2015.05.007

Quercetin supplementation does not enhance cerebellar mitochondrial biogenesis and oxidative status in exercised rats.

The present study tested the hypothesis that quercetin may inhibit the mitochondrial and antioxidant adaptations induced by exercise in cerebellar tis...
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