Mediterranean tomato-based sofrito protects against vascular alterations in obese zucker rats by preserving no bioavailability

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Molecular nutrition & food research (2017): March 2017 DOI: https://doi.org/10.1002/mnfr.201601010 Copyright: 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. This is the peer reviewed version of the following article: Rodriguez-Rodriguez R, Jiménez-Altayó F, Alsina L, Onetti Y, Rinaldi de Alvarenga JF, Claro C, Ogalla E, Casals N, Lamuela-Raventos RM. Mediterranean tomato-based sofrito protects against vascular alterations in obese Zucker rats by preserving NO bioavailability. Mol Nutr Food Res. 2017 Sep;61(9). doi: 10.1002/mnfr.201601010. Epub 2017 Apr 5. PMID: 28319651. t https://doi.org/10.1002/mnfr.201601010 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 Mediterranean tomato-based sofrito protects against vascular disturbances in obese Zucker rats by preserving NO bioavailability Rosalia Rodriguez-Rodriguez1, Francesc Jiménez-Altayó2, Laia Alsina2, Yara Onetti2, Carmen Claro3, Elena Ogalla3, Nuria Casals1,5, Rosa M. Lamuela-Raventos4,5 1 Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Barcelona, Spain. 2 Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain. 3 Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain. 4 Nutrition, Food Science Department and Gastronomy, XaRTA, INSA-UB Pharmacy, University of Barcelona, Barcelona, Spain. 5 CIBER Fisiopatología de la Obesidad y la Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain 2 Abstract Scope: Sofrito, a key component of the Mediterranean diet, provides nutritional interest due to its high content in bioactive compounds from tomato, onion and olive oil, and especially to the lipid matrix in which these compounds are found. In this study, the potential beneficial effects of dietary intake of sofrito on obesity-related vascular disturbances were explored in obese Zucker rats. Methods and results: Obese and lean rats were fed a control diet supplemented or not with 2% sofrito for 8 weeks. Vascular function was evaluated in aortic rings in organ baths. Dihydroethidium staining and immunofluorescence was used to determine aortic superoxide anion and peroxynitrite production, respectively. Despite food intake was higher in sofrito-fed obese rats, no differences were appreciated on body weight gain, organ weights and lipid profile compared to obese control rats. In aorta, sofrito intake attenuated phenylephrineinduced vasoconstriction in obese and lean rats. This effect was associated with recovery of the nitric oxide (NO) influence on vasoconstriction in obese animals. Furthermore, vasoprotection was accompanied by normalization of serum NO metabolites and vascular superoxide anion and peroxynitrite levels in obese rats. Conclusion: This study demonstrates for the first time that tomato-based sofrito protects against vascular alterations that could precede major cardiometabolic complications associated to obesity by restoring physiological vascular and circulating NO levels. These results could contribute to explain the therapeutic properties against obesity-related disorders attributable to the Mediterranean diet. Therefore, sofrito is an attractive dietary approach against obesity and its cardiovascular health risks, at least in the early stages of obesity. Keywords: sofrito; Mediterranean diet; obesity; vascular function; nitric oxide List of abbreviations: 3 1. Introduction Obesity is one of the leading public health problems in developing countries. Besides its direct impact on life quality, obesity is a major risk factor for highly lethal chronic complications such as insulin resistance and cardiovascular disease [1]. Particularly, the pathophysiology of obesity-related vascular disorders is an important target for developing new therapeutic strategies aimed to prevent or ameliorate cardiovascular complications in obesity [2]. Although traditional dietary approaches have proven successful as part of the treatment of obesity and cardiovascular pathologies in clinical trials, the exact role and importance of the individual components of the dietary pattern on specific aspects of these diseases need further evaluation [3, 4]. Research has therefore turned its attention to functional foods and nutraceutics as main components of a healthy prototype diet that could have the potential ability to modulate physiological and pathophysiological molecular mechanisms, thus resulting in favorable health outcomes [4]. In this sense, the Mediterranean dietary pattern has been shown to reduce the burden or even prevent the development of cardiovascular disease, cancer and obesity, among other pathologies [3, 5, 6]. Within the Mediterranean diet, tomato and tomato sauces are typical. In fact, tomato sauce is the most commonly consumed tomato product worldwide and particularly in Spain, where it represents almost 40.8 % of all sauce consumption [7]. In addition, consumption of tomato products has been consistently associated with a lower risk of several types of cancer and coronary heart disease, an effect mainly attributable to its high content in bioactive compounds such as polyphenols, carotenoids and vitamin C [8–11]. Tomato-based sofrito, a key component of the Mediterranean diet, is particularly interesting for its high content in bioactive compounds, not only from tomato but also from onion and virgin olive oil, and especially for the lipid matrix in which these compounds are found [12]. Regarding the latter, recent investigations have demonstrated that mechanical and thermal Comentado [RR1]: Rosa, sólo he encontrado este dato en la ref.7, no encuentro dato de consumo mundial comparando con otras sauces consumption. Solo tengo el dato de consumo de tomate mundial per capita. ¿Tienes algún dato adicional? 4 treatments, as well as oil matrix addition during tomato and sofrito sauce processing, may increase the bioaccessibility, extractability and bioavailability of phenolic compounds from tomato [12, 13]. Once they reach the adequate bioavailability, predominant phytochemicals present in sofrito, especially polyphenols and carotenoids, can exert their health-promoting effects [13–15]. Epidemiological and preclinical studies have demonstrated that fruits, vegetables and beverages rich in carotenoids (e.g. lycopene and beta-carotene) and polyphenols (e.g. rutin, quercetin and naringenin) delay the onset of atherosclerosis and exhibit anti-inflammatory and antiplatelet activities [16, 17]. Additionally, these phytochemicals are able to reduce neointimal thickening by inhibiting proliferation of vascular smooth muscle cells, and can also prevent vascular dysfunction by improving nitric oxide (NO) availability [16]. In particular, recent evidence suggests that carotenoids and polyphenols are key players not only in the treatment of inflammation and vascular complications, but also in restoring normal adipocyte function in obese subjects [4, 18, 19]. Interestingly, an inverse association has been established between plasmatic levels of carotenoids and inflammatory and oxidative stress markers in obese patients [20]. The fact that obesity is an inflammatory state that contributes to oxidative stress and vascular complications [2], indicates that a dietary source of carotenoids and polyphenols could represent an attractive therapeutic strategy to prevent or ameliorate the obesity-related complications. In the present study, the potential beneficial effects of a shortto middle-term dietary intake of Mediterranean sofrito were explored for the first time in an animal model of obesity. Concretely, we used the Zucker rat that is an obesity model extremely useful to explore vascular and metabolic complications associated to obesity [21]. Thus, this study is an attempt to evidence the potential contribution of sofrito consumption in the healthy properties of the Mediterranean diet. 2. Materials and methods 2.1. Animals and diets 5 Six week-old male obese Zucker rats and their lean littermate controls were purchased from Charles River (Charles River Laboratories, Barcelona, Spain). At 8 weeks of age, obese and lean rats were randomly assigned to the following groups (n=8): Lean rats fed control chow diet (LC), obese rats fed control chow diet (OC), lean rats fed chow diet supplemented in 2 % sofrito (LS), and obese rats fed chow diet supplemented in 2 % sofrito (OS). Control (standard) chow diet (Global Diet 2014) was provided by Harlan Laboratories (Milan, Italy). Sofrito (Gallina Blanca-Star, Barcelona, Spain) was previously characterized [12] and content of polyphenols and carotenoids is shown in Table I. 2% Sofrito supplement was calculated according to previous studies in humans in which 600-900 g sofrito/week is administered. Body weight and food intake was weekly evaluated. After 8 weeks of administration of the experimental diets, animals were killed by decapitation. Blood samples were immediately collected and thoracic aorta, liver, visceral and epididymal adipose tissues (VAT and EAT, respectively) were dissected. The protocol for animal handling and experimentation agreed with the European Union guidelines for the ethical management of animals and was approved by the Committee of Ethical Experimentation of the Universitat of Barcelona. 2.2. Blood biochemical assays Serum samples were obtained from blood by centrifugation for 5 minutes at 2000 g at room temperature. Glucose, total cholesterol and triglycerides levels in serum were determined using enzymatic-based assays following manufacturer's instructions (CatBio Laboratories, Barcelona, Spain). Serum levels of nitric oxide metabolites (NOx) were determined by using a methodology based on the colorimetric Griess reaction. Absorbance was measured spectrophotometrically at 540 nm [22]. 2.3. Tissue preparation Comentado [RR2]: He incluido la ingesta calórica en la tabla I 6 Thoracic aorta was cleaned of adhering fat and connective tissue and placed in cold KrebsHenseleit solution (KHS) (in mmol/L: NaCl 112; KCl 4.7; NaHCO3 25; MgSO4 1.2; CaCl2 2.5; KH2PO4 1.2 and glucose 11.1; pH 7.4). For detection of superoxide anion (O2˙−), aortic rings were maintained in 30 % sucrose-KHS overnight, placed into cryomolds containing Tissue-Tek OCT embedding medium (Sakura Finetek Europe, the Netherlands), and immediately frozen in liquid nitrogen for storage at −80°C [23]. For immunofluorescence studies, aortas were fixed in 4 % phosphate-buffered paraformaldehyde (pH 7.4) for 1 h and then washed in phosphatebuffered saline (PBS). Afterwards, arterial segments were placed in 30% sucrose PBS overnight, transferred to cryomolds with embedding medium, frozen in liquid nitrogen, and kept at −80°C until analysis [23]. 2.4. Reactivity experiments Aortic rings were disposed in organ baths, and vascular function was measured as previously described [23]. Contractile capacity of the vessels was assessed with KCl 100 mmol/L solution. After 30 minutes of stabilization, endothelial-dependent vasodilatations were studied by evaluating the relaxation induced by acetylcholine (ACh; 0.001–10 μmol/L) performed in vessels contracted to 60-80% of KCl contraction with phenylephrine (Phe). To investigate contractile responses mediated by α1-adrenoceptor stimulation, concentration–response curves to Phe (0.001–100 μmol/L) were performed. Concentration–response curves to Phe were constructed in the absence or presence of the nonselective NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME, 300 μmol/L). 2.5. In situ detection of superoxide anion The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate production of arterial O2˙− in situ, as previously described [23]. Briefly, 14-µm thick aortic sections placed on gelatin-coated slides were incubated with DHE (2 mmol/L) in Krebs-HEPES buffer. Preparations were viewed by laser scanning confocal microscope (TCS SP2; Leica, Heidelberg, Germany; 20x 7 objective). DHE fluorescence was visualized by excitation at 546 nm and emission at 610 nm. In these experiments, DHE fluorescence was abolished by the O2˙− scavenger, Mn(III)tetrakis(1methyl-4-pyridyl)porphyrin (0.1434 mg/ml, 30 min, 37°C), indicating the specificity of this reaction. Integrated optical densities were quantified using MetaMorph Image Analysis Software (Molecular Devices, Sunnyvale, CA, USA). The fluorescence signal per area was measured in at least two rings of each animal, and the results were expressed as arbitrary units. 2.6. Immunofluorescence The in situ production of peroxynitrite (ONOO-) was measured indirectly by analyzing nitrotyrosine levels, as previously described [24]. Frozen transverse sections (14-µm thick) cut onto gelatin-coated slides were incubated with rabbit polyclonal antibody against nitrotyrosine (1:100; Merck Millipore, Darmstadt, Germany) in PBS containing 2% bovine serum albumin for 1 h at 37°C in a humidified chamber. After washing, arterial preparations were incubated with the secondary antibody IgG conjugated to cyanine 3 (1:200; Jackson ImmunoResearch Laboratories Inc, West Grove, PA, USA) for 1 h at 37°C in a humidified chamber. Signals were viewed using a laser scanning confocal microscope (TCS SP2; Leica, Heidelberg, Germany; 20x objective). Cyanine 3-labeled antibody was visualized by excitation at 561 nm and detection at 600 to 700 nm. The specificity of the immunostaining was evaluated by omission of the primary antibody and processed as described above. Under these conditions, no staining was observed in the arterial wall in any experimental situation. Optical density of immunofluorescence was assayed with MetaMorph Image Analysis Software (Molecular Devices, Sunnyvale, CA, USA). The fluorescence signal per area was measured in at least two rings of each animal, and the results were expressed as arbitrary units. 2.7. Data analysis and statistics Results were shown as mean ± SEM of the number of rats (n) indicated in the figure legends. Vasoconstrictor responses were expressed as a percentage of tone generated by KCl 100 8 mmol/L. Vasodilator responses to ACh were expressed as a percentage of the previous tone generated by Phe. Area under the curve (AUC) was calculated from each individual concentration-response curve to ACh and Phe, and illustrated as arbitrary units. The dependence of vasoconstrictor and vasodilator response on strain and diet or on diet and vessel treatment was assessed by a two-way ANOVA with repeated measurements on the concentration factor. For NOx, superoxide and nitrotyrosine fluorescence levels, differences between groups were analyzed by unpaired t-test. Differences were considered significant when P< 0.05. Data analysis was carried out using GraphPad Prism Software 5.0 (San Diego, CA, USA). 3. Results and discussion 3.1. Effect of sofrito-supplemented diet on body weight gain, food intake, organ weights and serum biochemical determinations As illustrated in Table II, obese rats gained more body weight, and liver and white adipose tissue weights were higher compared to lean rats. Eight-weeks administration of sofritosupplemented diet was not able to modify body weight gain and organ weights in obese rats. Moreover, it is important to mention that, despite food and caloric intake in OS group was significantly higher than in OC rats, this hyperphagia did not imply higher body weight gain or higher values of liver and white adipose tissue weights in sofrito-fed rats (Table II). 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Concentration-response curves to phenylephrine (Phe) in the absence or presence of the NO synthesis inhibitor L-NAME in aortic rings from lean (A-B) and obese Zucker (C-D) rats. LC: lean rats fed a control diet, LS: lean rats fed a sofrito-supplemented diet, OC: obese rats fed a control diet, OS: obese rats fed a sofrito-supplemented diet. Data are mean ± SEM (n=5-6). *P<0.05 vs LC; +P<0.05 vs LS; #P<0.05 vs OS. Figure 3. Serum NO metabolites (nitrates and nitrites, NOx) levels after sofrito-supplemented diet administration in lean and obese Zucker rats. LC: lean rats fed a control diet, LS: lean rats fed a sofrito-supplemented diet, OC: obese rats fed a control diet, OS: obese rats fed a sofritosupplemented diet. Data are mean ± SEM (n=5-6). *P<0.05 vs LC. Figure 4. Representative photomicrographs and quantification of dihydroethidium-derived (an indirect indicator of superoxide anion) fluorescence (A) and nitrotyrosine (an indirect indicator of peroxynitrite) immunofluorescence (B) levels in confocal microscopic aortic sections from lean and obese Zucker rats. LC: lean rats fed a control diet, LS: lean rats fed a sofritosupplemented diet, OC: obese rats fed a control diet, OS: obese rats fed a sofritosupplemented diet. Data are mean ± SEM (n=5-7). *P<0.05; **P<0.01 vs LC. 22 Table I. Characterization of polyphenol and carotenoid content of the Mediterranean sofrito used in the study. The sofrito is mainly composed by tomato (50%) (pulp and concentrated), onion (37%), virgin olive oil (12%) and salt. Quantification of individual polyphenols or carotenoids (µg/g FW) and total polyphenols by Folin-Ciocalteau (µg GAE/g FW) is expressed as mean ± SD. Polyphenols Carotenoids Ferulic acid Chlorogenic acid Caffeic acid Sinapic acid Protocatechuic acid p-Coumaric acid p-Hydroxybenzoic acid Rutin Quercetin Quercetin-3-O-glucoside Naringenin Naringenin-7-O-glucòsid Total polyphenols (µg GAE/g FW) 03,89 ± 0,16 05,50 ± 0,21 04,72 ± 0,12 03,54 ± 0,10 02,26 ± 0,07 04,01 ± 0,07 03,01 ± 0,08 16,66 ± 0,67 13,77 ± 0,25 04,20 ± 0,09 10,33 ± 0,44 04,21 ± 0,05 202 ± 2 trans-α-Carotene trans-β-Carotene trans-Lutein trans-Lycopene 5-cis-Lycopene 9-cisLycopene 13-cisLycopene Total Carotenoids 01,10 ± 0,03 29,69 ± 0,11 03,42 ± 0,04 40,50 ± 0,33 02,81 ± 0,07 01,88 ± 0,10 02,09 ± 0,07 81,49 Adapted from [12]. SD: standard deviation; GAE: gallic acid equivalent; FW: fresh weight. 23 Table II. Body weight, food intake, organ weights and serum biochemical determinations LC LS OC OS Body weight (g) 383.6 ± 8.7 378.4 ± 6.5 518.3 ± 15.6* 517.3 ± 14.5+ Food intake (g/day/rat) 20.00 ± 1.24 23.61 ± 1.48 28.64 ± 1.77* 34.44 ± 1.13+ Caloric intake (Kcal) 61.99 ± 3.86 73.71 ± 4.61 88.79 ± 5.50* 107.55 ± 3.52+ # Organ weight (g/ 100g b.w.) Liver 3.78 ± 0.16 3.46 ± 0.08 5.11 ± 0.35* 5.44 ± 0.35+ VAT 0.78 ± 0.10 0.91 ± 0.10 1.58 ± 0.24* 1.35 ± 0.19+ EAT 0.75 ± 0.10 0.77 ± 0.15 1.94 ± 0.10* 0.89 ± 0.10+ Serum parameters Glucose (mmol/L) 7.1 ± 0.15 7.633 ± 0.12 8.433 ± 0.47* 9.4 ± 1.134+ Triglycerides (mmol/L) 1.496 ± 0.15 1.327 ± 0.12 5.684 ± 0.54* 4.76 ± 0.28+ Cholesterol (mmol/L) 3.494 ± 0.10 3.523 ± 0.12 6.09 ± 0.47* 7.07 ± 0.39+ Data are mean ± SEM. *P<0.05 vs LC; +P<0.05 vs LS; #P<0.05 vs OC. LC: lean rats fed a control diet, LS: lean rats fed a sofrito-supplemented diet, OC: obese rats fed a control diet, OS: obese rats fed a sofrito-supplemented diet. VAT: visceral adipose tissue, EAT: epididymal adipose tissue.