Life Sciences 139 (2015) 91–96

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Exercise training and taurine supplementation reduce oxidative stressand prevent endothelium dysfunction in rats fed a highly palatable diet

Leandro Kansuke Oharomari a, Nádia Fagundes Garcia b, Ellen Cristini de Freitas b, Alceu Afonso Jordão Júnior a,Paula Payão Ovídio a, Aline Rosa Maia c, Ana Paula Davel c, Camila de Moraes b,⁎a Laboratory of Nutrition and Metabolism, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, SP, Brazilb School of Physical Education and Sport of Ribeirão Preto, University of São Paulo, SP, Brazilc Department of Structural and Functional Biology, University of Campinas, SP, Brazil

⁎ Corresponding author at: Av. Bandeirantes, 3900, RibeE-mail address: camimoraes@usp.br (C. de Moraes).

http://dx.doi.org/10.1016/j.lfs.2015.08.0150024-3205/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 18 May 2015Received in revised form 17 August 2015Accepted 18 August 2015Available online 24 August 2015

Keywords:ExerciseTaurineEndothelial functionOxidative stressObesity

Aim: Few studies have analysed, from a nutritional point of view, the influence of exercise in minimizing detri-mental diet-relatedhealth effects. This study evaluated the effectiveness of exercise and taurine supplementationin preventing vascular and metabolic disorders caused by highly palatable diet intake.Main methods: Thirty-two male Wistar rats (255–265 g) were divided into 4 groups: Sedentary (SD);Sedentary + 2% taurine (SDTAU), Trained (TR) and Trained + 2% taurine (TRTAU). Exercise (treadmill, 60%maximum speed, 60 min, 5 days/week) started after 4 weeks of highly palatable diet feeding and was carriedout for 7 weeks.Key findings: Exercise effectively reduced insulin (61% and 68%), glucose (30% and 7%) and leptin levels (75% and67%) in TR and TRTAU groups, respectively. All groups showed a reduction inhepatic triglyceride infiltration (74%for SDTAU, 82% for TR and 85% for TRTAU) but only exercise reduced TBARS (50% for TR and 41% for TRTAU).Impaired relaxation was seen in SD (Emax = 67%) and improved with taurine (Emax = 86%) and exercise(Emax = 90% for TR and TRTAU). Increased expression of EC-SOD (32%) was seen in the aortas from all treatedgroups. Exercise, in the absence of taurine, increased Cu–Zn SOD (44%) and reduced gp91phox (34%). Superoxideformation in the aorta was reduced in supplemented (75% in SDTAU) and in trained groups (64% and 77% for TRand TRTAU, respectively).Significance: Exercise and taurine supplementation were effective in preventing endothelial dysfunction inducedby highly palatable diet intake, through a decrease in vascular oxidative stress.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Excessive fat-mass accumulation, a feature of obesity, is closely relat-ed to hypertension and insulin resistance [23,33]. Insulin resistanceplays a crucial role in the development of obesity co-morbiditiessuch as type II diabetes mellitus, cardiovascular complications andnon-alcoholic fat liver disease [13,17]. Moreover, there is a cross-talkbetween an increase in fat-mass and obesity-related health complica-tions due to inflammation and oxidative stress [31].

Environmental and cultural behaviours, such as reduced levels ofdaily physical activity and overconsumption of energy-dense foods,can contribute to an increase in body fat mass and metabolic disorders.On the other hand, exercise can prevent weight gain, improve insulinsignalling and reduce oxidative stress [8]. Nutritional strategies canalso minimize these detrimental effects. Taurine (2-aminoethanesulfonicacid), an amino acid involved inbile production, osmoregulation, immunesystemmodulation and a potential antioxidant, has been considered to be

irão Preto, SP 14026-340, Brazil.

a good asset in nutritional therapies [2,18]. The beneficial effects of exer-cise on vascular and metabolic disorders have been reported; however,very few studies have analysed the effects of exercise, from a nutritionalpoint of view, in minimizing diet-related detrimental health effects. Theaim of this study was to analyse the effectiveness of exercise and taurinesupplementation in preventing vascular and metabolic disorders causedby highly palatable diet intake.

2. Material and methods

2.1. Animals and experimental protocol

All procedures were reviewed and approved by the Ethics Committeeon Animal Use in Research (CEUA/PUSP-RP protocol number10.1.1290.53.5) in compliance with the “Principles of laboratory animalcare” (NIH publication No. 86-23, revised 1985) and the national law(CONCEA publication No. 11.794, 2008).

Thirty-two male Wistar rats (255–265 g) were divided into 4groups: sedentary (SD); sedentary supplemented with taurine solution(2%) in drinking water (SDTAU); trained (TR) and trained

Table 1Standard AIN-93 and High-palatable diet ingredients.

Ingredient (g/kg) AIN-93 High-palatable diet

AIN-93 – 330Whole Condensed Milk (Nestlé™) – 330Casein 200 92Cornstarch 397 –Dextrinized cornstarch 131 –Sucrose 100 156L-Cystine 3 –

Soybean oil 70 72Mineral mix (AIN-93)a 35 23Vitamin mix (AIN-93)a 10 7Fibre 50 –Choline bitartrate 2.5 –

a [28].

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supplemented with taurine solution (2%) in drinking water (TRTAU).Taurine was purchased from Ajinomoto Food Ingredients (Chicago, IL,USA) and the concentration used was determined according toNandhini and Anuradha [24]. Animals were housed in polypropylenecages (41 × 34 × 30 cm) containing three animals in each and kept ona 12 h light/dark cycle with unlimited access to a highly palatable diet[27,28]. The energy content of the diet derived from 56% carbohydrate,18% protein and 26% fat. A full list of ingredients can be found in Table 1.

The experiment lasted 11weeks. Animals were fed for 4weeks priorto exercise training and/or taurine supplementation. Exercise trainingconsisted of treadmill run at 60%maximum speed (ms) for 60 continu-ousminutes, 5 days aweek, for 7weeks. Training speedwasdeterminedafter a maximum incremental exercise test, which began at 11.6 m/minand increased by 1.6 m/min every 2 min until 20 m/min. Subsequently,the speed was increased by 3.2 m/min and rats ran until exhaustion(determined when the animal touched the bottom of the bay fivetimes within 1 min). The speed at which exhaustion occurred wasconsidered as the maximum speed. Training intensity progressively in-creased from40%ms on the first week, 50–55% from the 2nd to 4thweekand 60%ms from the 5th to 7th week.

2.2. Concentration–response curves in isolated aorta

The thoracic aortawas carefully removed andplaced in a freshly pre-pared Krebs solution containing (mM): NaCl, 118; NaHCO3, 25; glucose,5.6; KCl, 4.7; KH2PO4, 1.2;MgSO4, 1.17 and CaCl2, 2.5. All adherent tissuewas removed and the arteries were cut into 3 mm rings. Each ring wassuspended between two wire hooks and mounted in 5 ml organchambers containing Krebs solution (pH 7.4) at 37 °C and continuouslyaerated with a mixture of 95% O2 and 5% CO2 under a resting tension of1.5 g. The tissues isometric tension was recorded by a force

Fig. 1. Bodyweight gain (A) and epididymal fat mass (B) of sedentary (SD), sedentary supplemeWistar rats. Data are expressed as mean ± SEM of n = 6–7 per group. Two-way ANOVA, Tuke

displacement transducer (UgoBasile, Varese, Italy) connected to aPowerLab 400™data acquisition system(ADInstruments Pty Ltd., CastleHill, Australia).

After 1 h of equilibration, intact aorta ringswere pre-contractedwithphenylephrine (2 μM) and endothelium-dependent relaxation wasassessed by cumulative concentration–response curves to acetylcholine(ACh, 10 nM–100 μM). Cumulative concentration–response curve tosodium nitroprusside (SNP, 100 pM–100 nM) was also calculated inpre-contracted rings. The following equation was used to determinewhether the concentration–response data fit into a logistic function:E = Emax / ((1 + (10c / 10x)n) + Φ), where E corresponds to theresponse; Emax to themaximum response that the agonist can produce;c to the logarithmof the EC50, the concentration of agonist that produceshalf-maximum response; x to the logarithm of the concentration ofagonist; the exponential term, n, to a curve fitting parameter that de-fines the slope of the concentration–response line andΦ to the responseobserved in the absence of added agonist. Nonlinear regression analysisto determine Emax, log EC50 and nwas performed using GraphPad Prism(GraphPad Software, San Diego, CA, USA) with the constraint that Φ =zero.

2.3. Reactive oxygen species (ROS) detection

The oxidative fluorescent dye dihydroethidiumwas used to evaluatein situ superoxide production [6]. Transverse aorta sections (10 μm) ob-tained using a cryostat were incubated in phosphate buffer at 37 °C, for10 min. Subsequently, fresh phosphate buffer containing hydroethidine(2 μM) was applied to each tissue section and incubated in a light-protected humidified chamber at 37 °C, for 30 min. Negative controlsections received the same volume of phosphate buffer withouthydroethidine. Images were obtained with an optical microscope(Olympus BX60, Olympus, Center Valley, PA, USA) equipped with rho-damine filter and camera (Olympus DP-72) using a 20× objective. Thefluorescence was quantified using Image J software (National Institutesof Health, Bethesda, MD, USA).

2.4. Western blot analysis

The aortic tissue was homogenized in RIPA lysing buffer (Upstate,Temecula, CA, USA) and protein concentration determined using theBradford method [3]. Samples containing 50 μg protein were loadedinto gels prior to electrophoresis and proteins subsequently transferredby electroblotting onto polyvinylidene difluoride membranes andincubated in mouse anti Cu/Zn SOD (1:1500, SIGMA, St. Louis, MO, USA)primary antibody. Chemiluminescent signals (ECL plus Amersham,Piscataway, NJ, USA)were captured on X-ray film (Hyperfilm Amersham,

ntedwith taurine (SDTAU), trained (TR) and trained supplementedwith taurine (TRTAU)y post-test (P b 0.05). #Taurine effect, ⁎exercise effect.

Table 2Serum and hepatic parameters after 11 weeks.

SD SDTAU TR TRTAU

Serum Taurine (mg/mL) 26.8 ± 4.0 47.3 ± 2.9a 23.2 ± 2.0 46.1 ± 2.3a

Leptin (pg/mL) 12.44 ± 1.13 11.26 ± 2.16 2.99 ± 0.71b 3.72 ± 0.73b

Insulin (ng/mL) 2.06 ± 0.33 1.82 ± 0.32 0.81 ± 0.08b 0.59 ± 0.08b

Glucose (mg/mL) 1.87 ± 0.15 1.41 ± 0.12a 1.30 ± 0.04b 1.33 ± 0.03b

Liver Triglyceride (mg/g tissue) 149.0 ± 18.3 38.4 ± 7.0a 27.2 ± 3.8b 21.7 ± 6.0b

TBARS (μg/g protein) 3.45 ± 0.27 3.23 ± 0.36 1.72 ± 0.32b 2.04 ± 0.25b

SD: sedentary, SDTAU: sedentary + taurine, TR: trained, TRTAU: trained + taurine. Data are mean ± S.E.M. of n = 5–6 in each group. 2-way ANOVA, Tukey post-test (P b 0.05).a Taurine effect.b Exercise effect.

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Piscataway, NJ, USA) and scanning densitometry used to quantify theimmunoblot signals.

2.5. Serum leptin, insulin and glucose concentration

Following overnight fasting; the animals were euthanized by decap-itation,without previous anaesthetic, 48 h after the last exercise session.Blood sampleswere taken and plasma/serum immediately separated bycentrifugation (8000 g). Blood glucose was assessed by a colorimetricmethod (Laborlab, São Paulo, Brazil). Leptin and insulin concentrationswere determined by fluorescence-labelled microsphere beads usingMilliplex™ Map RADPK-81 K (Merck Millipore, Billerica, MA, USA).

2.6. Thiobarbituric acid reactive substances (TBARS) and hepatic triglycerideconcentration

Liver samples (200 mg) were homogenized in phosphate buffer(0.1 mol/L, pH 7.4) using a Turrax dispenser (MA 102 MINI E —Marconi Model), centrifuged for 15 min at 6500 ×g (3000 rpm)and the supernatant used to quantify TBARS and triglycerides.

Malondialdehyde (MDA) results from thedecomposition of unstableperoxides from polyunsaturated fatty acids, and the reaction betweenMDA and thiobarbituric acid results in TBARS. TBARS determination isa well-establishedmethod for screening andmonitoring lipid peroxida-tion. For this analysis, liver samples were mixed with 1 mL of a solutioncontaining 15% (w/v) trichloroacetic acid, 0.38% (w/v) thiobarbituricacid and 0.25 N HCl. The mixture was heated for 30 min at 100 °C. TheTBARS concentration was measured by absorbance at 535 nm; using1,1,3,3-tetramethoxypropane (Sigma, St. Louis, MO, USA) as an externalstandard and the results were expressed as μmol/g protein. Triglycerideand total protein concentration were determined by a colorimetricmethod using commercial kits (Labtest Diagnóstica, Lagoa Santa, MG,Brazil).

Fig. 2. Concentration–response curve to acetylcholine (A) and sodium nitroprusside (B) in aortand trained supplemented with taurine (TRTAU) Wistar rats. Rings were pre-contracted withData are expressed as mean ± SEM of n = 5–6 per group. Two-way ANOVA, Tukey post-test (

2.7. Statistical analysis

Data are presented as means ± standard error of the mean (SEM).Normality test (Kolmogorov and Smirnov) was followed by analysis ofvariance (two-way ANOVA) and Tukey post-hoc test using SPSS Statis-tics for Windows, version 17.0 (Chicago, IL, USA) software. Significancewas considered at 5% (P b 0.05).

3. Results

Seven weeks of exercise was effective in preventing body weightgain but taurine had no additional effect. On the other hand, both tau-rine and exercise reduced epididymal fat content, as show in Fig. 1.The reduction in epididymal fat mass seen in SDTAU group was not suf-ficient to prevent an increase in leptin and insulin levels; however,serum glucosewas lower than in the SD group. Trained rats had a bettermetabolic profile; with lower levels of leptin, insulin and glucose(Table 2). In regards to non-alcoholic fatty liver disease markers, bothinterventions were effective in preventing triglyceride infiltration inthe hepatic tissue but only physical training significantly (P b 0.05)reduced the marker for lipid peroxidation TBARS (Table 2).

Although metabolic disruption induced by highly palatable dietintake did not influence maximum response or potency to sodium ni-troprusside, the endothelium-dependent response to acetylcholinewas affected. A maximum response of 67% was observed in the SDgroup, which improved not only by taurine supplementation (86% forSDTAU group) but also by exercise training, reaching nearly 90% ofmaximum relaxation in TR and TRTAU groups. No significant changewas observed in acetylcholine potency (Fig. 2).

An increase of 32% on extra cellular superoxide dismutase expres-sion was seen in the aortas from all treated groups (Fig. 3C). Exercisetraining, in the absence of taurine supplementation,was able to increaseCu–Zn superoxide dismutase expression by 44% and reduce NADPH

a rings from sedentary (SD), sedentary supplemented with taurine (SDTAU), trained (TR)phenylephrine (2 μM) and relaxation responses expressed as a percentage of contraction.P b 0.05). #Taurine effect, ⁎exercise effect.

Fig. 4. Representative/quantitative analysis for dihydroethidium-fluorescence obtainedfrom thoracic aortas sections of sedentary (SD), sedentary supplemented with taurine(SDTAU), trained (TR) and trained supplemented with taurine (TRTAU) Wistar rats.Data are expressed as mean ± SEM of n = 6–7 per group. Two-way ANOVA, Tukeypost-test (P b 0.05). #Taurine effect, ⁎exercise effect.

Fig. 3. Aortic protein expression of antioxidant Mn-SOD (A), CuZn-SOD (B) and EC-SOD (C) and pro oxidant NADPH subunits p46phox (D) and gp91phox (E). Western blot analysis wasperformed in aortas from sedentary (SD), sedentary supplemented with taurine (SDTAU), trained (TR) and trained supplemented with taurine (TRTAU)Wistar rats. Data are expressedas mean ± SEM of n = 5–6 per group. Two-way ANOVA, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.

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oxidase subunit gp91phox by 34% (Fig. 3B and E). Superoxide formationin the aorta was lower in supplemented and trained groups (Fig. 4) butno additional effect was seen in TRTAU group.

4. Discussion

Exercise training for seven weeks was effective in preventing exces-sive body weight gain and epididymal fat increase in trained groups.Similar results have been reported in rats that were fed a highly caloricdiet, after six or twelve weeks of treadmill running [8,20]. In the currentstudy, the rats had similar baseline body mass and were randomly dis-tributed into the treatment groups. Thus, the reason for the lowerbody weight gain observed in TRTAU before 5th week is unclear. Fur-thermore, trained rats had lower hepatic triglyceride and TBARS. The li-polytic effect of exercise training was mediated by the increase incirculating catecholamines aimed at maintaining energy availability tothe skeletal muscle [30]. Taurine supplementation prevented hepatictriglyceride accumulation, probably through an increase in hepaticβ-oxidation due to an increase in mitochondrial carnitine or in the ex-pression of PPAR-α and UCP2 [5,11]. Furthermore, SDTAU had lowerblood glucose than SD. Similar results have been observed in fructose-fed rats supplementedwith 2% taurine in drinkingwater and in vitro ex-periments demonstrated that taurine stimulates glucose utilization bythe skeletal muscle [25]. Taurine modulates insulin signalling at recep-tor level, increasing insulin-stimulated tyrosine phosphorylation inskeletal muscle and liver and thus enhancing insulin sensitivity [4].

Leptin and insulin affect lipid metabolism by reducing VLDL-TG se-cretion. Insulin acts peripherally, suppressing adipose tissue lipolysisand diminishing FFA delivery to the liver. Leptin stimulates hepaticoxidative metabolism, depleting the TG pool available for incorporationby VLDL particles [16].When fat intake overrides the rate of fat oxidation,adipocytes overloaded with triglycerides are less capable of buffering thefatty acid flow. This increase in circulating fatty acids leads to lipid accu-mulation in tissues such as the skeletal muscle, blood vessels and liver;contributing to insulin resistance [12]. The hyperleptinaemia and

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hyperinsulinaemia observed in sedentary rats strongly suggest a state ofresistance, with impairment in the signalling of both hormones. It is im-portant to note that SD rats also had high plasma glucose levels andhigher triglyceride accumulation and hepatic lipid peroxidation, a processwhich initiates when adipocytes become resistant to the anti-lipolytic ef-fect of insulin, leading to an increase in fatty acid release by the adiposetissue [1]. In this state, the inability of the liver to oxidize fatty acids resultsin lipid droplet accumulation, inflammation and increase in oxidativestress, resulting in non-alcoholic steatohepatitis [32].

Exercise training, but not taurine supplementation, prevented thedevelopment of insulin and leptin resistance induced by a highly palat-able diet. It has been demonstrated that exercise can improve theperipheral insulin signalling and action through a mechanism thatincreases insulin-stimulated glucose uptake, due to an increase in totalGLUT-4 protein content and activation of hexokinase and glucose6-phosphate enzymes [19]. In addition, exercise training could modu-late leptin concentration through different mechanisms, as well as byreducing fat pad mass and increasing leptin receptors, which leads toimprovement on leptin sensitivity in target tissues such as hepatocytesand vascular smooth muscle cells [26,34].

Leptin also has a cardiovascular action; activating the sympatheticnervous system, increasing endothelium-derived nitric oxide andpromoting angiogenesis [22]. However, it has been observed thathyperleptinaemia increases vascular tone, which ismediated by sympa-thetic overactivity and impairment of agonist-induced vasodilation [9].Furthermore, leptin stimulates macrophages and monocytes to secreteatherogenic cytokines and increase oxidative stress in the vessel wall[10]. In fact, aortas from SD rats had higher superoxide production,which could be related to an impairment in endothelium-dependentvasodilation, as superoxide anion minimizes nitric oxide-induceddilation [15].

The improvement in endothelium-dependent vasodilation observedin the aorta of supplemented and trained rats could be due to an in-crease in nitric oxide bioavailability. Taurine supplemented rats hadhigher EC-SOD expression and lower superoxide formation in theiraorta. Similar results have been found in the aorta of protein-restricted rats supplemented with 2.5% taurine in drinking water [21].Previous studies have shown a protective effect of taurine against exces-sive mitochondrial superoxide generation, by enhancing mitochondrialelectron transport chain activity [18].

During exercise, pulsatile shear stress activatesmechanoreceptors inendothelial cells, triggering a signalling pathway that generates nitricoxide followed by vasodilation [15]. Chronic exposure of endothelialcells to exercise-induced shear stress increases nitric oxide synthaseprotein expression and consequently nitric oxide production. Afterlong-term exercise training, structural adaptation due to nitric oxide-mediated remodelling results in a chronic increase in vessel calibre,which normalizes shear stress [14]. Previous studies have shown an in-crease in nitric oxide metabolites (nitrate/nitrite) in Wistar rats fed ahigh fat diet after 4 weeks of treadmill exercise but not after 12 weeks[7,8]. In the present study, no changeswere observed in nitric oxideme-tabolites after 7weeks of exercise (data not show). The improvement invasodilatation in trained rats fed a highly palatable diet seems to be dueto a decrease in superoxide production in the aorta. Accordingly, it hasbeen demonstrated that exercise improves anti-oxidant apparatus,increasing arterial superoxide dismutase and reducing p67phox proteinexpression [8,29].

5. Conclusion

In summary, moderate-intensity exercise and taurine supplementa-tion were effective approaches in preventing endothelial dysfunctioninduced by a highly palatable diet. In agreement with the results, themechanism underlying this response was a decrease in vascularoxidative stress, which may have promoted an increase in nitric oxidebioavailability. It is important to note that taurine supplementation

had a synergistic effect with exercise training as no added effect wasobserved.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgment

This study was supported by a grant from the São Paulo ResearchFoundation (grant# 2010/12733-4).

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