Life Sciences 139 (2015) 91–96
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Exercise training and taurine supplementation reduce oxidative stress and 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 b c
Laboratory of Nutrition and Metabolism, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, SP, Brazil School of Physical Education and Sport of Ribeirão Preto, University of São Paulo, SP, Brazil Department of Structural and Functional Biology, University of Campinas, SP, Brazil
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Article history: Received 18 May 2015 Received in revised form 17 August 2015 Accepted 18 August 2015 Available online 24 August 2015 Keywords: Exercise Taurine Endothelial function Oxidative stress Obesity
a b s t r a c t Aim: Few studies have analysed, from a nutritional point of view, the influence of exercise in minimizing detrimental diet-related health effects. This study evaluated the effectiveness of exercise and taurine supplementation in 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 carried out for 7 weeks. Key findings: Exercise effectively reduced insulin (61% and 68%), glucose (30% and 7%) and leptin levels (75% and 67%) in TR and TRTAU groups, respectively. All groups showed a reduction in hepatic 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 treated groups. Exercise, in the absence of taurine, increased Cu–Zn SOD (44%) and reduced gp91phox (34%). Superoxide formation in the aorta was reduced in supplemented (75% in SDTAU) and in trained groups (64% and 77% for TR and TRTAU, respectively). Significance: Exercise and taurine supplementation were effective in preventing endothelial dysfunction induced by 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 related to hypertension and insulin resistance [23,33]. Insulin resistance plays a crucial role in the development of obesity co-morbidities such as type II diabetes mellitus, cardiovascular complications and non-alcoholic fat liver disease [13,17]. Moreover, there is a cross-talk between an increase in fat-mass and obesity-related health complications due to inflammation and oxidative stress [31]. Environmental and cultural behaviours, such as reduced levels of daily 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 insulin signalling and reduce oxidative stress [8]. Nutritional strategies can also minimize these detrimental effects. Taurine (2-aminoethanesulfonic acid), an amino acid involved in bile production, osmoregulation, immune system modulation and a potential antioxidant, has been considered to be ⁎ Corresponding author at: Av. Bandeirantes, 3900, Ribeirão Preto, SP 14026-340, Brazil. E-mail address: [email protected] (C. de Moraes).
http://dx.doi.org/10.1016/j.lfs.2015.08.015 0024-3205/© 2015 Elsevier Inc. All rights reserved.
a good asset in nutritional therapies [2,18]. The beneficial effects of exercise on vascular and metabolic disorders have been reported; however, very few studies have analysed the effects of exercise, from a nutritional point of view, in minimizing diet-related detrimental health effects. The aim of this study was to analyse the effectiveness of exercise and taurine supplementation in preventing vascular and metabolic disorders caused by highly palatable diet intake. 2. Material and methods 2.1. Animals and experimental protocol All procedures were reviewed and approved by the Ethics Committee on Animal Use in Research (CEUA/PUSP-RP protocol number 10.1.1290.53.5) in compliance with the “Principles of laboratory animal care” (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 4 groups: sedentary (SD); sedentary supplemented with taurine solution (2%) in drinking water (SDTAU); trained (TR) and trained
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Table 1 Standard AIN-93 and High-palatable diet ingredients. Ingredient (g/kg)
AIN-93
High-palatable diet
AIN-93 Whole Condensed Milk (Nestlé™) Casein Cornstarch Dextrinized cornstarch Sucrose
– – 200 397 131 100 3
330 330 92 – – 156 –
70 35 10 50 2.5
72 23 7 – –
L-Cystine Soybean oil Mineral mix (AIN-93)a Vitamin mix (AIN-93)a Fibre Choline bitartrate a
[28].
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 to Nandhini and Anuradha [24]. Animals were housed in polypropylene cages (41 × 34 × 30 cm) containing three animals in each and kept on a 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 11 weeks. Animals were fed for 4 weeks prior to exercise training and/or taurine supplementation. Exercise training consisted of treadmill run at 60% maximum speed (ms) for 60 continuous minutes, 5 days a week, for 7 weeks. Training speed was determined after a maximum incremental exercise test, which began at 11.6 m/min and 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 five times within 1 min). The speed at which exhaustion occurred was considered as the maximum speed. Training intensity progressively increased from 40%ms on the first week, 50–55% from the 2nd to 4th week and 60%ms from the 5th to 7th week.
displacement transducer (UgoBasile, Varese, Italy) connected to a PowerLab 400™ data acquisition system (ADInstruments Pty Ltd., Castle Hill, Australia). After 1 h of equilibration, intact aorta rings were pre-contracted with phenylephrine (2 μM) and endothelium-dependent relaxation was assessed by cumulative concentration–response curves to acetylcholine (ACh, 10 nM–100 μM). Cumulative concentration–response curve to sodium nitroprusside (SNP, 100 pM–100 nM) was also calculated in pre-contracted rings. The following equation was used to determine whether the concentration–response data fit into a logistic function: E = Emax / ((1 + (10c / 10x)n) + Φ), where E corresponds to the response; Emax to the maximum response that the agonist can produce; c to the logarithm of the EC50, the concentration of agonist that produces half-maximum response; x to the logarithm of the concentration of agonist; the exponential term, n, to a curve fitting parameter that defines the slope of the concentration–response line and Φ to the response observed in the absence of added agonist. Nonlinear regression analysis to determine Emax, log EC50 and n was 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 dihydroethidium was used to evaluate in situ superoxide production [6]. Transverse aorta sections (10 μm) obtained using a cryostat were incubated in phosphate buffer at 37 °C, for 10 min. Subsequently, fresh phosphate buffer containing hydroethidine (2 μM) was applied to each tissue section and incubated in a lightprotected humidified chamber at 37 °C, for 30 min. Negative control sections received the same volume of phosphate buffer without hydroethidine. Images were obtained with an optical microscope (Olympus BX60, Olympus, Center Valley, PA, USA) equipped with rhodamine filter and camera (Olympus DP-72) using a 20× objective. The fluorescence was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.2. Concentration–response curves in isolated aorta
2.4. Western blot analysis
The thoracic aorta was carefully removed and placed in a freshly prepared 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 tissue was removed and the arteries were cut into 3 mm rings. Each ring was suspended between two wire hooks and mounted in 5 ml organ chambers containing Krebs solution (pH 7.4) at 37 °C and continuously aerated with a mixture of 95% O2 and 5% CO2 under a resting tension of 1.5 g. The tissues isometric tension was recorded by a force
The aortic tissue was homogenized in RIPA lysing buffer (Upstate, Temecula, CA, USA) and protein concentration determined using the Bradford method [3]. Samples containing 50 μg protein were loaded into gels prior to electrophoresis and proteins subsequently transferred by electroblotting onto polyvinylidene difluoride membranes and incubated 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,
Fig. 1. Body weight gain (A) and epididymal fat mass (B) 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, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
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Table 2 Serum and hepatic parameters after 11 weeks.
Serum
Liver
Taurine (mg/mL) Leptin (pg/mL) Insulin (ng/mL) Glucose (mg/mL) Triglyceride (mg/g tissue) TBARS (μg/g protein)
SD
SDTAU
TR
TRTAU
26.8 ± 4.0 12.44 ± 1.13 2.06 ± 0.33 1.87 ± 0.15 149.0 ± 18.3 3.45 ± 0.27
47.3 ± 2.9a 11.26 ± 2.16 1.82 ± 0.32 1.41 ± 0.12a 38.4 ± 7.0a 3.23 ± 0.36
23.2 ± 2.0 2.99 ± 0.71b 0.81 ± 0.08b 1.30 ± 0.04b 27.2 ± 3.8b 1.72 ± 0.32b
46.1 ± 2.3a 3.72 ± 0.73b 0.59 ± 0.08b 1.33 ± 0.03b 21.7 ± 6.0b 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.
Piscataway, NJ, USA) and scanning densitometry used to quantify the immunoblot signals. 2.5. Serum leptin, insulin and glucose concentration Following overnight fasting; the animals were euthanized by decapitation, without previous anaesthetic, 48 h after the last exercise session. Blood samples were taken and plasma/serum immediately separated by centrifugation (8000 g). Blood glucose was assessed by a colorimetric method (Laborlab, São Paulo, Brazil). Leptin and insulin concentrations were determined by fluorescence-labelled microsphere beads using Milliplex™ Map RADPK-81 K (Merck Millipore, Billerica, MA, USA). 2.6. Thiobarbituric acid reactive substances (TBARS) and hepatic triglyceride concentration 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 the decomposition of unstable peroxides from polyunsaturated fatty acids, and the reaction between MDA and thiobarbituric acid results in TBARS. TBARS determination is a well-established method for screening and monitoring lipid peroxidation. For this analysis, liver samples were mixed with 1 mL of a solution containing 15% (w/v) trichloroacetic acid, 0.38% (w/v) thiobarbituric acid and 0.25 N HCl. The mixture was heated for 30 min at 100 °C. The TBARS concentration was measured by absorbance at 535 nm; using 1,1,3,3-tetramethoxypropane (Sigma, St. Louis, MO, USA) as an external standard and the results were expressed as μmol/g protein. Triglyceride and total protein concentration were determined by a colorimetric method using commercial kits (Labtest Diagnóstica, Lagoa Santa, MG, Brazil).
2.7. Statistical analysis Data are presented as means ± standard error of the mean (SEM). Normality test (Kolmogorov and Smirnov) was followed by analysis of variance (two-way ANOVA) and Tukey post-hoc test using SPSS Statistics for Windows, version 17.0 (Chicago, IL, USA) software. Significance was considered at 5% (P b 0.05).
3. Results Seven weeks of exercise was effective in preventing body weight gain but taurine had no additional effect. On the other hand, both taurine and exercise reduced epididymal fat content, as show in Fig. 1. The reduction in epididymal fat mass seen in SDTAU group was not sufficient to prevent an increase in leptin and insulin levels; however, serum glucose was lower than in the SD group. Trained rats had a better metabolic profile; with lower levels of leptin, insulin and glucose (Table 2). In regards to non-alcoholic fatty liver disease markers, both interventions were effective in preventing triglyceride infiltration in the 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 diet intake did not influence maximum response or potency to sodium nitroprusside, the endothelium-dependent response to acetylcholine was affected. A maximum response of 67% was observed in the SD group, which improved not only by taurine supplementation (86% for SDTAU group) but also by exercise training, reaching nearly 90% of maximum relaxation in TR and TRTAU groups. No significant change was observed in acetylcholine potency (Fig. 2). An increase of 32% on extra cellular superoxide dismutase expression was seen in the aortas from all treated groups (Fig. 3C). Exercise training, in the absence of taurine supplementation, was able to increase Cu–Zn superoxide dismutase expression by 44% and reduce NADPH
Fig. 2. Concentration–response curve to acetylcholine (A) and sodium nitroprusside (B) in aorta rings from sedentary (SD), sedentary supplemented with taurine (SDTAU), trained (TR) and trained supplemented with taurine (TRTAU) Wistar rats. Rings were pre-contracted with phenylephrine (2 μM) and relaxation responses expressed as a percentage of contraction. Data are expressed as 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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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 was performed in aortas from 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 = 5–6 per group. Two-way ANOVA, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
oxidase subunit gp91phox by 34% (Fig. 3B and E). Superoxide formation in the aorta was lower in supplemented and trained groups (Fig. 4) but no additional effect was seen in TRTAU group. 4. Discussion
Fig. 4. Representative/quantitative analysis for dihydroethidium-fluorescence obtained from 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, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
Exercise training for seven weeks was effective in preventing excessive body weight gain and epididymal fat increase in trained groups. Similar results have been reported in rats that were fed a highly caloric diet, after six or twelve weeks of treadmill running [8,20]. In the current study, the rats had similar baseline body mass and were randomly distributed into the treatment groups. Thus, the reason for the lower body weight gain observed in TRTAU before 5th week is unclear. Furthermore, trained rats had lower hepatic triglyceride and TBARS. The lipolytic effect of exercise training was mediated by the increase in circulating catecholamines aimed at maintaining energy availability to the skeletal muscle [30]. Taurine supplementation prevented hepatic triglyceride accumulation, probably through an increase in hepatic β-oxidation due to an increase in mitochondrial carnitine or in the expression of PPAR-α and UCP2 [5,11]. Furthermore, SDTAU had lower blood glucose than SD. Similar results have been observed in fructosefed rats supplemented with 2% taurine in drinking water and in vitro experiments demonstrated that taurine stimulates glucose utilization by the skeletal muscle [25]. Taurine modulates insulin signalling at receptor level, increasing insulin-stimulated tyrosine phosphorylation in skeletal muscle and liver and thus enhancing insulin sensitivity [4]. Leptin and insulin affect lipid metabolism by reducing VLDL-TG secretion. Insulin acts peripherally, suppressing adipose tissue lipolysis and diminishing FFA delivery to the liver. Leptin stimulates hepatic oxidative metabolism, depleting the TG pool available for incorporation by VLDL particles [16]. When fat intake overrides the rate of fat oxidation, adipocytes overloaded with triglycerides are less capable of buffering the fatty acid flow. This increase in circulating fatty acids leads to lipid accumulation 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 of resistance, with impairment in the signalling of both hormones. It is important to note that SD rats also had high plasma glucose levels and higher triglyceride accumulation and hepatic lipid peroxidation, a process which initiates when adipocytes become resistant to the anti-lipolytic effect of insulin, leading to an increase in fatty acid release by the adipose tissue [1]. In this state, the inability of the liver to oxidize fatty acids results in lipid droplet accumulation, inflammation and increase in oxidative stress, resulting in non-alcoholic steatohepatitis [32]. Exercise training, but not taurine supplementation, prevented the development of insulin and leptin resistance induced by a highly palatable diet. It has been demonstrated that exercise can improve the peripheral insulin signalling and action through a mechanism that increases insulin-stimulated glucose uptake, due to an increase in total GLUT-4 protein content and activation of hexokinase and glucose 6-phosphate enzymes [19]. In addition, exercise training could modulate leptin concentration through different mechanisms, as well as by reducing fat pad mass and increasing leptin receptors, which leads to improvement on leptin sensitivity in target tissues such as hepatocytes and vascular smooth muscle cells [26,34]. Leptin also has a cardiovascular action; activating the sympathetic nervous system, increasing endothelium-derived nitric oxide and promoting angiogenesis [22]. However, it has been observed that hyperleptinaemia increases vascular tone, which is mediated by sympathetic overactivity and impairment of agonist-induced vasodilation [9]. Furthermore, leptin stimulates macrophages and monocytes to secrete atherogenic 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-dependent vasodilation, as superoxide anion minimizes nitric oxide-induced dilation [15]. The improvement in endothelium-dependent vasodilation observed in the aorta of supplemented and trained rats could be due to an increase in nitric oxide bioavailability. Taurine supplemented rats had higher EC-SOD expression and lower superoxide formation in their aorta. Similar results have been found in the aorta of proteinrestricted rats supplemented with 2.5% taurine in drinking water [21]. Previous studies have shown a protective effect of taurine against excessive mitochondrial superoxide generation, by enhancing mitochondrial electron transport chain activity [18]. During exercise, pulsatile shear stress activates mechanoreceptors in endothelial cells, triggering a signalling pathway that generates nitric oxide followed by vasodilation [15]. Chronic exposure of endothelial cells to exercise-induced shear stress increases nitric oxide synthase protein expression and consequently nitric oxide production. After long-term exercise training, structural adaptation due to nitric oxidemediated remodelling results in a chronic increase in vessel calibre, which normalizes shear stress [14]. Previous studies have shown an increase in nitric oxide metabolites (nitrate/nitrite) in Wistar rats fed a high fat diet after 4 weeks of treadmill exercise but not after 12 weeks [7,8]. In the present study, no changes were observed in nitric oxide metabolites after 7 weeks of exercise (data not show). The improvement in vasodilatation in trained rats fed a highly palatable diet seems to be due to a decrease in superoxide production in the aorta. Accordingly, it has been demonstrated that exercise improves anti-oxidant apparatus, increasing arterial superoxide dismutase and reducing p67phox protein expression [8,29]. 5. Conclusion In summary, moderate-intensity exercise and taurine supplementation were effective approaches in preventing endothelial dysfunction induced by a highly palatable diet. In agreement with the results, the mechanism underlying this response was a decrease in vascular oxidative stress, which may have promoted an increase in nitric oxide bioavailability. It is important to note that taurine supplementation
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Exercise training and taurine supplementation reduce oxidative stress and 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 b c
Laboratory of Nutrition and Metabolism, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, SP, Brazil School of Physical Education and Sport of Ribeirão Preto, University of São Paulo, SP, Brazil Department of Structural and Functional Biology, University of Campinas, SP, Brazil
a r t i c l e
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Article history: Received 18 May 2015 Received in revised form 17 August 2015 Accepted 18 August 2015 Available online 24 August 2015 Keywords: Exercise Taurine Endothelial function Oxidative stress Obesity
a b s t r a c t Aim: Few studies have analysed, from a nutritional point of view, the influence of exercise in minimizing detrimental diet-related health effects. This study evaluated the effectiveness of exercise and taurine supplementation in 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 carried out for 7 weeks. Key findings: Exercise effectively reduced insulin (61% and 68%), glucose (30% and 7%) and leptin levels (75% and 67%) in TR and TRTAU groups, respectively. All groups showed a reduction in hepatic 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 treated groups. Exercise, in the absence of taurine, increased Cu–Zn SOD (44%) and reduced gp91phox (34%). Superoxide formation in the aorta was reduced in supplemented (75% in SDTAU) and in trained groups (64% and 77% for TR and TRTAU, respectively). Significance: Exercise and taurine supplementation were effective in preventing endothelial dysfunction induced by 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 related to hypertension and insulin resistance [23,33]. Insulin resistance plays a crucial role in the development of obesity co-morbidities such as type II diabetes mellitus, cardiovascular complications and non-alcoholic fat liver disease [13,17]. Moreover, there is a cross-talk between an increase in fat-mass and obesity-related health complications due to inflammation and oxidative stress [31]. Environmental and cultural behaviours, such as reduced levels of daily 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 insulin signalling and reduce oxidative stress [8]. Nutritional strategies can also minimize these detrimental effects. Taurine (2-aminoethanesulfonic acid), an amino acid involved in bile production, osmoregulation, immune system modulation and a potential antioxidant, has been considered to be ⁎ Corresponding author at: Av. Bandeirantes, 3900, Ribeirão Preto, SP 14026-340, Brazil. E-mail address: [email protected] (C. de Moraes).
http://dx.doi.org/10.1016/j.lfs.2015.08.015 0024-3205/© 2015 Elsevier Inc. All rights reserved.
a good asset in nutritional therapies [2,18]. The beneficial effects of exercise on vascular and metabolic disorders have been reported; however, very few studies have analysed the effects of exercise, from a nutritional point of view, in minimizing diet-related detrimental health effects. The aim of this study was to analyse the effectiveness of exercise and taurine supplementation in preventing vascular and metabolic disorders caused by highly palatable diet intake. 2. Material and methods 2.1. Animals and experimental protocol All procedures were reviewed and approved by the Ethics Committee on Animal Use in Research (CEUA/PUSP-RP protocol number 10.1.1290.53.5) in compliance with the “Principles of laboratory animal care” (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 4 groups: sedentary (SD); sedentary supplemented with taurine solution (2%) in drinking water (SDTAU); trained (TR) and trained
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Table 1 Standard AIN-93 and High-palatable diet ingredients. Ingredient (g/kg)
AIN-93
High-palatable diet
AIN-93 Whole Condensed Milk (Nestlé™) Casein Cornstarch Dextrinized cornstarch Sucrose
– – 200 397 131 100 3
330 330 92 – – 156 –
70 35 10 50 2.5
72 23 7 – –
L-Cystine Soybean oil Mineral mix (AIN-93)a Vitamin mix (AIN-93)a Fibre Choline bitartrate a
[28].
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 to Nandhini and Anuradha [24]. Animals were housed in polypropylene cages (41 × 34 × 30 cm) containing three animals in each and kept on a 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 11 weeks. Animals were fed for 4 weeks prior to exercise training and/or taurine supplementation. Exercise training consisted of treadmill run at 60% maximum speed (ms) for 60 continuous minutes, 5 days a week, for 7 weeks. Training speed was determined after a maximum incremental exercise test, which began at 11.6 m/min and 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 five times within 1 min). The speed at which exhaustion occurred was considered as the maximum speed. Training intensity progressively increased from 40%ms on the first week, 50–55% from the 2nd to 4th week and 60%ms from the 5th to 7th week.
displacement transducer (UgoBasile, Varese, Italy) connected to a PowerLab 400™ data acquisition system (ADInstruments Pty Ltd., Castle Hill, Australia). After 1 h of equilibration, intact aorta rings were pre-contracted with phenylephrine (2 μM) and endothelium-dependent relaxation was assessed by cumulative concentration–response curves to acetylcholine (ACh, 10 nM–100 μM). Cumulative concentration–response curve to sodium nitroprusside (SNP, 100 pM–100 nM) was also calculated in pre-contracted rings. The following equation was used to determine whether the concentration–response data fit into a logistic function: E = Emax / ((1 + (10c / 10x)n) + Φ), where E corresponds to the response; Emax to the maximum response that the agonist can produce; c to the logarithm of the EC50, the concentration of agonist that produces half-maximum response; x to the logarithm of the concentration of agonist; the exponential term, n, to a curve fitting parameter that defines the slope of the concentration–response line and Φ to the response observed in the absence of added agonist. Nonlinear regression analysis to determine Emax, log EC50 and n was 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 dihydroethidium was used to evaluate in situ superoxide production [6]. Transverse aorta sections (10 μm) obtained using a cryostat were incubated in phosphate buffer at 37 °C, for 10 min. Subsequently, fresh phosphate buffer containing hydroethidine (2 μM) was applied to each tissue section and incubated in a lightprotected humidified chamber at 37 °C, for 30 min. Negative control sections received the same volume of phosphate buffer without hydroethidine. Images were obtained with an optical microscope (Olympus BX60, Olympus, Center Valley, PA, USA) equipped with rhodamine filter and camera (Olympus DP-72) using a 20× objective. The fluorescence was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.2. Concentration–response curves in isolated aorta
2.4. Western blot analysis
The thoracic aorta was carefully removed and placed in a freshly prepared 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 tissue was removed and the arteries were cut into 3 mm rings. Each ring was suspended between two wire hooks and mounted in 5 ml organ chambers containing Krebs solution (pH 7.4) at 37 °C and continuously aerated with a mixture of 95% O2 and 5% CO2 under a resting tension of 1.5 g. The tissues isometric tension was recorded by a force
The aortic tissue was homogenized in RIPA lysing buffer (Upstate, Temecula, CA, USA) and protein concentration determined using the Bradford method [3]. Samples containing 50 μg protein were loaded into gels prior to electrophoresis and proteins subsequently transferred by electroblotting onto polyvinylidene difluoride membranes and incubated 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,
Fig. 1. Body weight gain (A) and epididymal fat mass (B) 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, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
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Table 2 Serum and hepatic parameters after 11 weeks.
Serum
Liver
Taurine (mg/mL) Leptin (pg/mL) Insulin (ng/mL) Glucose (mg/mL) Triglyceride (mg/g tissue) TBARS (μg/g protein)
SD
SDTAU
TR
TRTAU
26.8 ± 4.0 12.44 ± 1.13 2.06 ± 0.33 1.87 ± 0.15 149.0 ± 18.3 3.45 ± 0.27
47.3 ± 2.9a 11.26 ± 2.16 1.82 ± 0.32 1.41 ± 0.12a 38.4 ± 7.0a 3.23 ± 0.36
23.2 ± 2.0 2.99 ± 0.71b 0.81 ± 0.08b 1.30 ± 0.04b 27.2 ± 3.8b 1.72 ± 0.32b
46.1 ± 2.3a 3.72 ± 0.73b 0.59 ± 0.08b 1.33 ± 0.03b 21.7 ± 6.0b 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.
Piscataway, NJ, USA) and scanning densitometry used to quantify the immunoblot signals. 2.5. Serum leptin, insulin and glucose concentration Following overnight fasting; the animals were euthanized by decapitation, without previous anaesthetic, 48 h after the last exercise session. Blood samples were taken and plasma/serum immediately separated by centrifugation (8000 g). Blood glucose was assessed by a colorimetric method (Laborlab, São Paulo, Brazil). Leptin and insulin concentrations were determined by fluorescence-labelled microsphere beads using Milliplex™ Map RADPK-81 K (Merck Millipore, Billerica, MA, USA). 2.6. Thiobarbituric acid reactive substances (TBARS) and hepatic triglyceride concentration 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 the decomposition of unstable peroxides from polyunsaturated fatty acids, and the reaction between MDA and thiobarbituric acid results in TBARS. TBARS determination is a well-established method for screening and monitoring lipid peroxidation. For this analysis, liver samples were mixed with 1 mL of a solution containing 15% (w/v) trichloroacetic acid, 0.38% (w/v) thiobarbituric acid and 0.25 N HCl. The mixture was heated for 30 min at 100 °C. The TBARS concentration was measured by absorbance at 535 nm; using 1,1,3,3-tetramethoxypropane (Sigma, St. Louis, MO, USA) as an external standard and the results were expressed as μmol/g protein. Triglyceride and total protein concentration were determined by a colorimetric method using commercial kits (Labtest Diagnóstica, Lagoa Santa, MG, Brazil).
2.7. Statistical analysis Data are presented as means ± standard error of the mean (SEM). Normality test (Kolmogorov and Smirnov) was followed by analysis of variance (two-way ANOVA) and Tukey post-hoc test using SPSS Statistics for Windows, version 17.0 (Chicago, IL, USA) software. Significance was considered at 5% (P b 0.05).
3. Results Seven weeks of exercise was effective in preventing body weight gain but taurine had no additional effect. On the other hand, both taurine and exercise reduced epididymal fat content, as show in Fig. 1. The reduction in epididymal fat mass seen in SDTAU group was not sufficient to prevent an increase in leptin and insulin levels; however, serum glucose was lower than in the SD group. Trained rats had a better metabolic profile; with lower levels of leptin, insulin and glucose (Table 2). In regards to non-alcoholic fatty liver disease markers, both interventions were effective in preventing triglyceride infiltration in the 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 diet intake did not influence maximum response or potency to sodium nitroprusside, the endothelium-dependent response to acetylcholine was affected. A maximum response of 67% was observed in the SD group, which improved not only by taurine supplementation (86% for SDTAU group) but also by exercise training, reaching nearly 90% of maximum relaxation in TR and TRTAU groups. No significant change was observed in acetylcholine potency (Fig. 2). An increase of 32% on extra cellular superoxide dismutase expression was seen in the aortas from all treated groups (Fig. 3C). Exercise training, in the absence of taurine supplementation, was able to increase Cu–Zn superoxide dismutase expression by 44% and reduce NADPH
Fig. 2. Concentration–response curve to acetylcholine (A) and sodium nitroprusside (B) in aorta rings from sedentary (SD), sedentary supplemented with taurine (SDTAU), trained (TR) and trained supplemented with taurine (TRTAU) Wistar rats. Rings were pre-contracted with phenylephrine (2 μM) and relaxation responses expressed as a percentage of contraction. Data are expressed as 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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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 was performed in aortas from 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 = 5–6 per group. Two-way ANOVA, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
oxidase subunit gp91phox by 34% (Fig. 3B and E). Superoxide formation in the aorta was lower in supplemented and trained groups (Fig. 4) but no additional effect was seen in TRTAU group. 4. Discussion
Fig. 4. Representative/quantitative analysis for dihydroethidium-fluorescence obtained from 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, Tukey post-test (P b 0.05). #Taurine effect, ⁎exercise effect.
Exercise training for seven weeks was effective in preventing excessive body weight gain and epididymal fat increase in trained groups. Similar results have been reported in rats that were fed a highly caloric diet, after six or twelve weeks of treadmill running [8,20]. In the current study, the rats had similar baseline body mass and were randomly distributed into the treatment groups. Thus, the reason for the lower body weight gain observed in TRTAU before 5th week is unclear. Furthermore, trained rats had lower hepatic triglyceride and TBARS. The lipolytic effect of exercise training was mediated by the increase in circulating catecholamines aimed at maintaining energy availability to the skeletal muscle [30]. Taurine supplementation prevented hepatic triglyceride accumulation, probably through an increase in hepatic β-oxidation due to an increase in mitochondrial carnitine or in the expression of PPAR-α and UCP2 [5,11]. Furthermore, SDTAU had lower blood glucose than SD. Similar results have been observed in fructosefed rats supplemented with 2% taurine in drinking water and in vitro experiments demonstrated that taurine stimulates glucose utilization by the skeletal muscle [25]. Taurine modulates insulin signalling at receptor level, increasing insulin-stimulated tyrosine phosphorylation in skeletal muscle and liver and thus enhancing insulin sensitivity [4]. Leptin and insulin affect lipid metabolism by reducing VLDL-TG secretion. Insulin acts peripherally, suppressing adipose tissue lipolysis and diminishing FFA delivery to the liver. Leptin stimulates hepatic oxidative metabolism, depleting the TG pool available for incorporation by VLDL particles [16]. When fat intake overrides the rate of fat oxidation, adipocytes overloaded with triglycerides are less capable of buffering the fatty acid flow. This increase in circulating fatty acids leads to lipid accumulation 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 of resistance, with impairment in the signalling of both hormones. It is important to note that SD rats also had high plasma glucose levels and higher triglyceride accumulation and hepatic lipid peroxidation, a process which initiates when adipocytes become resistant to the anti-lipolytic effect of insulin, leading to an increase in fatty acid release by the adipose tissue [1]. In this state, the inability of the liver to oxidize fatty acids results in lipid droplet accumulation, inflammation and increase in oxidative stress, resulting in non-alcoholic steatohepatitis [32]. Exercise training, but not taurine supplementation, prevented the development of insulin and leptin resistance induced by a highly palatable diet. It has been demonstrated that exercise can improve the peripheral insulin signalling and action through a mechanism that increases insulin-stimulated glucose uptake, due to an increase in total GLUT-4 protein content and activation of hexokinase and glucose 6-phosphate enzymes [19]. In addition, exercise training could modulate leptin concentration through different mechanisms, as well as by reducing fat pad mass and increasing leptin receptors, which leads to improvement on leptin sensitivity in target tissues such as hepatocytes and vascular smooth muscle cells [26,34]. Leptin also has a cardiovascular action; activating the sympathetic nervous system, increasing endothelium-derived nitric oxide and promoting angiogenesis [22]. However, it has been observed that hyperleptinaemia increases vascular tone, which is mediated by sympathetic overactivity and impairment of agonist-induced vasodilation [9]. Furthermore, leptin stimulates macrophages and monocytes to secrete atherogenic 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-dependent vasodilation, as superoxide anion minimizes nitric oxide-induced dilation [15]. The improvement in endothelium-dependent vasodilation observed in the aorta of supplemented and trained rats could be due to an increase in nitric oxide bioavailability. Taurine supplemented rats had higher EC-SOD expression and lower superoxide formation in their aorta. Similar results have been found in the aorta of proteinrestricted rats supplemented with 2.5% taurine in drinking water [21]. Previous studies have shown a protective effect of taurine against excessive mitochondrial superoxide generation, by enhancing mitochondrial electron transport chain activity [18]. During exercise, pulsatile shear stress activates mechanoreceptors in endothelial cells, triggering a signalling pathway that generates nitric oxide followed by vasodilation [15]. Chronic exposure of endothelial cells to exercise-induced shear stress increases nitric oxide synthase protein expression and consequently nitric oxide production. After long-term exercise training, structural adaptation due to nitric oxidemediated remodelling results in a chronic increase in vessel calibre, which normalizes shear stress [14]. Previous studies have shown an increase in nitric oxide metabolites (nitrate/nitrite) in Wistar rats fed a high fat diet after 4 weeks of treadmill exercise but not after 12 weeks [7,8]. In the present study, no changes were observed in nitric oxide metabolites after 7 weeks of exercise (data not show). The improvement in vasodilatation in trained rats fed a highly palatable diet seems to be due to a decrease in superoxide production in the aorta. Accordingly, it has been demonstrated that exercise improves anti-oxidant apparatus, increasing arterial superoxide dismutase and reducing p67phox protein expression [8,29]. 5. Conclusion In summary, moderate-intensity exercise and taurine supplementation were effective approaches in preventing endothelial dysfunction induced by a highly palatable diet. In agreement with the results, the mechanism underlying this response was a decrease in vascular oxidative stress, which may have promoted an increase in nitric oxide bioavailability. It is important to note that taurine supplementation
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