Peripheral blood mononuclear cells as in vivo model for dietary intervention induced systemic oxidative stress

Food and Chemical Toxicology 72 (2014) 178–186

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Peripheral blood mononuclear cells as in vivo model for dietary intervention induced systemic oxidative stress Antonio Camargo a,b,1, Patricia Peña-Orihuela a,b,1, Oriol Alberto Rangel-Zúñiga a,b, Pablo Pérez-Martínez a,b, Javier Delgado-Lista a,b, Cristina Cruz-Teno a,b, Carmen Marín a,b, Francisco Tinahones b,c, María M. Malagón b,d, Helen M. Roche e, Francisco Pérez-Jiménez a,b, José López-Miranda a,b,⇑ a

Lipids and Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital, University of Cordoba, Cordoba, Spain CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Spain Endocrinology and Nutrition Service, Hospital Virgen de la Victoria, Málaga, Spain d Department of Cell Biology, Physiology, and Immunology, IMIBIC/Reina Sofia University Hospital/University of Córdoba, Cordoba, Spain e UCD Institute of Food & Health/UCD Conway Institute, School of Public Health and Population Sciences, University College Dublin, Ireland b c

a r t i c l e

i n f o

Article history: Received 19 March 2014 Accepted 15 July 2014 Available online 22 July 2014 Keywords: Mononuclear cells Oxidative stress Diet Metabolic syndrome Postprandial state LIPGENE study

a b s t r a c t Our aim was to assess the use of peripheral blood mononuclear cells (PBMC) as an in vivo cellular model to evaluate diet-induced changes in the oxidative stress status by analyzing the gene expression pattern of NADPH-oxidase subunits and antioxidant genes. A randomized, controlled trial assigned metabolic syndrome patients to 4 diets for 12 weeks each: (i) high-saturated fatty acid (HSFA), (ii) highmonounsaturated fatty acid, and (iii), (iv) two low-fat, high-complex carbohydrate diets supplemented with n-3 polyunsaturated fatty acids or placebo. A fat challenge reflecting the fatty acid composition as the original diets was conducted post-intervention. The mRNA levels of gp91phox (P < 0.001), p22phox (P = 0.005), p47phox (P = 0.001) and p40phox (P < 0.001) increased at 2 h after the intake of the HSFA meal. The expression of SOD1, SOD2, GSR, GPx1, GPX4, TXN, TXNRD1 and Nrf2 increased after the HSFA meal (p < 0.05). In contrast, the expression of these genes remained unaltered in response to the other dietary interventions. Our results suggest that the increased expression of antioxidant genes in PBMC seems to be due to the response to the postprandial oxidative stress generated mainly in adipose tissue after the consumption of an HSFA diet. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Metabolic syndrome (MetS), as a multi-component disorder characterized by hypertriglyceridemia, low HDL cholesterol, hyperglycaemia, abdominal obesity and hypertension, is closely linked to cardiovascular disease (CVD) and type 2 diabetes (Grundy et al., 2004). MetS etiology is the result of a complex interaction between Abbreviations: MetS, metabolic syndrome; CVD, cardiovascular disease; ROS, reactive oxygen species; HSFA, high-saturated fatty acid; HMUFA, high-monounsaturated fatty acid; LFHCC, low-fat, high-complex carbohydrate diet; LFHCC n-3, low-fat, high-complex carbohydrate diet supplemented with n-3 polyunsaturated fatty acids; PBMC, peripheral blood mononuclear cells; E, energy; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; LC, long chain; TG, triglycerides; HDL-c, high-density lipoprotein-cholesterol; LDL-c, low-density lipoprotein-cholesterol; LPO, lipid peroxides. ⇑ Corresponding author. Address: Servicio de Medicina Interna, Unidad de Lípidos y Arteriosclerosis, Hospital Universitario Reina Sofía, Avda. Menéndez Pidal, s/n, 14004 Córdoba, Spain. Tel.: +34 957 012882; fax: +34 957 204763. E-mail address: [email protected] (J. López-Miranda). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.fct.2014.07.024 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.

genetic, metabolic and environmental factors including dietary habits and, especially, the quality of dietary fat (Phillips et al., 2006). Along with the pro-inflammatory and pro-thrombotic state, characteristic in MetS patients (Grundy et al., 2004; Gustafson, 2010), oxidative stress plays a key role in the pathogenesis of the syndrome. Oxidative stress, which results from an imbalance between the production and detoxification of reactive oxygen species (ROS) (Durackova, 2010), is also involved in the pathogenesis and etiology of several diseases, including impaired glucose tolerance and insulin resistance, diabetes and cardiovascular disease (Ceriello and Motz, 2004). Moreover, oxidative stress induces inflammation through the generation of inflammatory mediators, such as interleukins and adhesion molecules (Roebuck, 1999). It has also been suggested that oxidative stress in adipose tissue is a major contributor for the development of MetS, through dysregulation of the release of adipocytokines and by generating systemic oxidative stress (Furukawa et al., 2004; Palmieri et al., 2006). Research evidences increasingly reinforce the notion that diet, in particular the quality of dietary fat, can modulate oxidative

A. Camargo et al. / Food and Chemical Toxicology 72 (2014) 178–186

stress. Previous dietary intervention studies have demonstrated that altering fat composition can significantly reduce oxidative stress in MetS patients (Roberts et al., 2006, 2002). However, these studies assessed oxidative stress markers in the fasting state, yet humans spend most of the day in a continuous postprandial state (de Koning and Rabelink, 2002). In fact, postprandial oxidative stress is characterized by an imbalance between the production of ROS and their elimination by the antioxidant system (Durackova, 2010; Scandalios, 2002), with an increase in oxidative stress biomarkers occurring after meals (Cardona et al., 2008; Devaraj et al., 2008; Ursini and Sevanian, 2002). We have previously demonstrated that the HMUFA diet improves plasma postprandial oxidative stress parameters, and that the LFHCC and LFHCC n-3 diets have an intermediate effect relative to the HMUFA and HSFA diets in a MetS population in the Spanish cohort of MetS included in the LIPGENE study (Perez-Martinez et al., 2010). Further, we demonstrated that consumption of the HSFA diet increased oxidative stress due to an imbalance between ROS generation and removal, as a consequence of the increased expression of NADPH-oxidase and the decreased expression of antioxidant enzymes in adipose tissue (Pena-Orihuela et al., 2013). On the basis of these previous findings, herein we hypothesized that diet, specifically the quantity and quality of dietary fat, could also modify the expression of pro-oxidant and anti-oxidant genes in the peripheral blood mononuclear cells (PBMC) of MetS patients. These cells are a sub-set of white blood cells, which include monocytes and lymphocytes, which play a critical role in the immune system and are involved in the initiation and progression of atherosclerotic lesions (Bouloumie et al., 2005; Osterud and Bjorklid, 2003), the pathogenic substrate responsible for CVD in MetS patients (Grundy et al., 2004). Moreover, these cells are now being used increasingly for gene expression studies because they are easy to collect repeatedly in sufficient quantities (de Mello et al., 2008; Ghanim et al., 2004). Moreover, it has been shown that PBMC modify their gene expression profile in response to stimuli such as dietary fat, metabolic factors such as dyslipidemia, and inflammatory molecules produced by other organs and tissues (Bories et al., 2012; de Mello et al., 2012; Ziegler-Heitbrock, 2000). The aim of the present work was to assess the use of PBMC as an in vivo cellular model to evaluate the systemic oxidative response to dietary intervention. For this purpose, we studied the effect of four diets differing in quantity and quality of fat on the postprandial expression of the pro-oxidant gp91phox, p22phox, p67phox, p47phox, p40phox and the anti-oxidant, SOD1 SOD2, GSR, GPx1, GPx4, TXN, TXNRD1, and Nrf2 genes in PBMC of patients with MetS. 2. Materials and methods 2.1. Participants and recruitment The current study was conducted within the framework of the LIPGENE study (‘‘Diet, genomics and the metabolic syndrome: an integrated nutrition, agro-food, social and economic analysis’’), a Framework 6 Integrated Project funded by the European Union. A subgroup of 75 subjects with the MetS from the LIPGENE cohort was accepted for the postprandial study and successfully concluded the dietary intervention and the post-intervention postprandial lipaemia studies. All the participants gave written informed consent and underwent a comprehensive study of their medical history, physical examination and clinical chemistry analysis before enrolment. Clinical Trial Registration Number: NCT00429195. This study was carried out in the Lipids and Atherosclerosis Unit at Reina Sofia University Hospital, from February 2005 to April 2006. The experimental protocol was approved by the local Ethics Committee, in line with the Helsinki Declaration.

2.2. Design The patients were randomly stratified into 1 of 4 dietary interventions for 12 weeks. The MetS was defined by published criteria (Grundy et al., 2004), which conformed to the LIPGENE inclusion and exclusion criteria (Shaw et al., 2009). A post-intervention high-fat meal was administered, providing the same amount of

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fat (0.7 g/kg body weight) contained in the intervention period. The intervention study design and intervention protocol, which also provides information about pre, mild, and post-intervention food consumption and dietary compliance have been previously described (Shaw et al. (2009)).

2.3. Randomization and intervention Randomization was completed centrally, according to age, gender, and fasting plasma glucose concentration, using the Minimisation Program for Allocating patients to Clinical Trials (Department of Clinical Epidemiology, London Hospital Medical College, UK) randomization program. The four diets differed in fat quantity and quality, while remaining isoenergetic. Composition of diets in the pre- and post-intervention period and dietary targets have been previously described (Cruz-Teno et al., 2012). Two diets were designed to provide 38% energy (E) from fat: (i) a high-fat, saturated fatty acid-rich diet (HSFA), which was designed to provide 16% E as SFA, and (ii) a high monounsaturated fatty acid rich diet (HMUFA) designed to provide 20% E from MUFA. The other two diets were low-fat, high-complex carbohydrate-rich diets [(iii) LFHCC and (iv) LFHCC (n-3); both 28% E from fat]. The LFHCC (n-3) diet included a 1.24-g/d supplement of long chain (n-3) PUFA [ratio of 1.4 eicosapentaenoic acid (EPA):1 docosahexaenoic acid (DHA)] and the LFHCC diet included a 1.2-g/d supplement of control high-oleic sunflower seed oil capsules (placebo). The composition of fatty acid capsules has been previously described (Jimenez-Gomez et al., 2010). The test meal, which represents a fat overload providing the same amount of fat (65%), allowed us to study the postprandial responses that allowed us to study the postprandial responses after long-term adaptation to the corresponding diet. The patients arrived at the clinical center at 8:00 a.m. after a 12 h. fast, refrained from smoking during the fasting period and abstained from alcohol intake during the preceding 7 days. After cannulation, a fasting blood sample was taken before the test meal, which then was ingested within 20 min. under supervision. Then, the blood samples were drawn at 4 h. The test meals were prepared in the center, reflecting the fatty acid composition of each subject’s chronic dietary intervention. These test meals provided an equal amount of fat (0.7 g/kg body weight), E content (40.2 kJ/kg body weight), cholesterol (5 mg/kg of body weight), fiber, and vitamin A [62.9 lmol vitamin A (retinol)/m2 body surface area]. The test meal provided 65% of E as fat, 10% as protein, and 25% as carbohydrates. During the postprandial assessment, the participants rested and did not consume any other food, but were allowed to drink water. The composition of the test meal has been previously described (Meneses et al., 2011). Briefly, HSFA, 38% E from SFA, based on butter, whole milk, white bread and eggs; HMUFA, 43% E from MUFA, based on olive oil, skimmed milk, white bread, eggs, egg yolks, and tomatoes; LFHCC with placebo capsules, a6% E as PUFA; LFHCC with LC (n-3) PUFA, 16% E as PUFA [1.24 g/d of LC (n-3) PUFA (ratio 1.4 EPA:1 DHA)]. Meals after LFHCC diets were based on butter, olive oil, skimmed milk, white bread, eggs, egg yolks, and walnuts.

2.4. Monitoring for adverse effects The volunteers were visited every 2 weeks for study. The clinical investigators assessed adverse events by using physical examinations, used a checklist of diet-related symptoms, and gave advice on how to remedy them.

2.5. Measurements Blood was collected in tubes containing EDTA to give a final concentration of 0.a% EDTA. Plasma was separated from red cells by centrifugation at a 500g for 15 min at 4 °C. Biomarkers determined in frozen samples were analyzed by laboratory researchers who were unaware of the interventions. Lipid parameter determinations were previously described (Jimenez-Gomez et al., 2010). Briefly, plasma triglycerides (TG) and cholesterol concentrations were assayed using enzymatic procedures (Allain et al., 1974; Bucolo and David, 1973). High-density lipoprotein-cholesterol (HDL-c) was measured by precipitation of an aliquot of plasma with dextran sulfate–Mg2+, as previously described (Warnick et al., 1982). Low-density lipoprotein-cholesterol (LDL-c) was calculated using the following formula: LDL-c = plasma cholesterol – HDL-c + large TRL-c + small TRL-c, where TRL-C is TG-rich lipoprotein-cholesterol. Determinations of the oxidative stress biomarkers H2O2, LPO (lipid peroxides), and protein carbonyl levels in plasma were previously described (Perez-Martinez et al., 2010).

2.6. Isolation of RNA from PBMC and quantification of the gene expression by Real-time PCR PBMC were isolated at the beginning and at the end of the period of dietary intervention at fasting state, as well as at 2 and 4 h after the meal intake. Blood was processed and PBMC were isolated as previously described (Cruz-Teno et al., 2012). Total RNA from PBMC was extracted using the Trizol method and quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop TechnologyÒ, Cambridge, UK). RNA integrity was measured by loading on agarose gels in 1X TBE 1.6%.

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Real-time PCR reactions were carried out using the OpenArray™ NT Cycler system (Applied Biosystems, Carlsbad, CA, USA), following the manufacturer’s instructions. Each reaction was performed with a ll of a 1:5 (v/v) dilution of the first cDNA synthesized from 1 lg of total RNA using the commercial kit High Capacity cDNA Reverse Transcription Kit with RNAse inhibitor (Applied Biosystems), following the manufacturer’s instructions. The gene expression analysis was performed on duplicated samples from 75 subjects at fasting and at 2 and 4 h after intake of the fat overload. Primer pairs were selected from the database TaqMan Gene Expression assays (Applied Biosystems, Carlsbad, CA, USA; https://products. appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601267), for the following genes: nuclear factor erythroid-derived 2-like 2 (Nrf2), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), catalase (CAT), glutathione peroxidase 1 (GPx1), glutathione peroxidase 4 (GPx4), glutathione reductase (GSR), thioredoxin (TXN) and thioredoxin reductase a (TXNRD1), NADPH oxidase subunits (gp91phox, p22phox, p47phox, p67phox and p40phox), and the transcription factor PU.1. The relative expression for each analyzed gene was calculated with GADPH (glyceraldehyde-3-phosphate dehydrogenase) as a housekeeping gene. The data set was analyzed by OpenArrayÒ Real-Time qPCR Analysis Software (Applied Biosystems, Carlsbad, CA, USA). 2.7. Western blotting Cytoplasmic and nuclear lysates were prepared from PBMC following the method described previously (Hernandez-Presa et al., 1997). The extracted proteins were quantified using the Bradford method. Electrophoretic separation was carried out with 40 lg of protein for both cytoplasmic and nuclear fractions in sodium dodecyl sulfate polyacrylamide electrophoresis gels. The following proteins were detected using their corresponding antibodies: Nrf2 (C-20: rabbit polyclonal, Santa Cruz Biotechnology, Inc.), TFIIB (C-a8: rabbit polyclonal, Santa Cruz Biotechnology, Inc.), and b-actin (C-2: mouse monoclonal, Santa Cruz Biotechnology, Inc.). Immunocomplexes were detected with an appropriate peroxidase conjugated secondary antibody (Sigma Aldrich) and detected with a chemiluminescent kit (Immun-Star™ WesternC™). Values for cytoplasmic Nrf2 were normalized with the signal for b-actin and values for nuclear Nrf2 were normalized with the signal for TFIIB. Protein levels were quantified using the Image Lab™ version 3.0 image analysis software (Bio-Rad Laboratories, Inc.). Results were expressed in arbitrary units (de Mello et al.). 2.8. Analysis of data PASW Statistics, Version a8, SPSS statistical software (Chicago, IL, USA) was used for statistical analysis. The normal distribution of variables to characterize the postprandial response was assessed using the Kolmogorov–Smirnov test. The gene expression values of gp91phox, p67phox, p47phox, p40phox, PU.1, CAT, SOD2, GPx4, GSR and TXN were log transformed to get a normal distribution. We performed ANOVA for repeated measurements to determine the postprandial effect of the fat meal composition, with dietary intervention as the inter-subject factor. The global p-values indicate: Pa: the effect of the diet and fat meal composition ingested (between-subject effect); P2: the time effect (within-subject effect); and P3: the interaction of both factors (diet by time interaction). Post hoc statistical analysis was completed using Bonferroni’s multiple comparisons test. Values of nuclear Nrf2 protein levels were log transformed before statistical analyses. Postprandial protein levels were analyzed by one-way ANOVA, global p-values P: diet effect. The relationship among parameters was also analyzed using Pearson’s linear correlation coefficient. The data presented are expressed as mean ± SEM.

3. Results 3.1. Effect of diet on NADPH-oxidase gene expression in the postprandial state

Moreover, we found that p40phox mRNA levels were also enhanced after the HSFA (P = 0.022) or the LFHCC (P = 0.038) meals with respect to the HMUFA meal (Fig. 1E). In addition, when all the post-intervention measurements were evaluated together (0, 2, and 4 h), we found higher p47phox mRNA levels after the intake of an HSFA meal when compared to the HMUFA (P = 0.007), LFHCC (P = 0.007) and LFHCC n-3 meals (P < 0.001) (Fig. 1D). Moreover, we also found higher p40phox mRNA levels after the intake of an HSFA meal compared with the HMUFA (P = 0.008) and the LFHCC n-3 meals (P < 0.001) (Fig. 1E). Finally, p67phox mRNA levels also increased after the HSFA meal as compared with the LFHCC meal (P = 0.016) (Fig. 1C). 3.2. Effects of diet on antioxidant markers in the postprandial state We observed a postprandial increase in GSR and GPx1 mRNA levels (both P < 0.001) at 2 h after the intake of the HSFA meal, while the intake of the other meals had no effect on the expression of these genes (Fig. 2D and E). In addition, the mRNA levels of GSR, GPx1, TXNRD1 and Nrf2 were higher at 2 h after the intake of the HSFA meal than after the intake of the HMUFA (P = 0.001, P = 0.019, P = 0.021, and P = 0.001, respectively), LFHCC (P = 0.001, P = 0.038, P = 0.002, and P = 0.001, respectively), and LFHCC n-3 meals (P < 0.001, P = 0.001, P = 0.001, and P < 0.001, respectively) meals (Figs. 2D, E, H, and 3). The intake of the HSFA meal also increased SOD2 and TXN mRNA postprandial levels as compared to the LFHCC (P = 0.046 and P = 0.002, respectively), and LFHCC n-3 meals (both P < 0.001) (Fig. 2B and G). We also found that the SOD1 and GPx4 mRNA levels were higher at 2 h after the intake of an HSFA meal than 2 h after the intake of the LFHCC n-3 meal (P = 0.029 and P = 0.005) (Fig. 2A and F). Taking all the post-intervention measurements (0, 2 and 4 h) together, we observed higher GSR mRNA levels after the HSFA meal than after the intake of HMUFA (P = 0.013) and LFHCC (P = 0.006) meals (Fig. 2D), higher CAT and TXNRD1 mRNA levels after the intake of the HSFA meal than after the LFHCC (P = 0.039 and P = 0.013) and LFHCC n-3 meals (P = 0.011 and 0.033) (Fig. 2C and H), and higher GPx1 (P = 0.044) and Nrf2 (P < 0.001) mRNA levels after the intake of the HSFA meal than after the LFHCC n-3 meal (Figs. 2E and 3). 3.3. Meal intake and Nrf2 protein levels in PBMC We observed higher nuclear Nrf2 protein levels after the intake of an HSFA meal as compared with the intake of the LFHCC and LFHCC n-3 meals (P = 0.018 and P = 0.011, respectively), while the cytoplasmic Nrf2 protein levels were lower after the intake of the HSFA and LFHCC n-3 meals as compared with those observed in response to the HMUFA meal (P = 0.047 and P = 0.003, respectively) (Fig. 4). 3.4. Correlation analysis

We observed a postprandial increase in gp91phox (P < 0.001), p22phox (P = 0.005), p47phox (P = 0.001), and p40phox (P < 0.001) mRNA levels post-intervention, at 2 h after the intake of an HSFA meal (Fig. 1), while the expression of these genes remained unchanged after the intake of the other meals tested. Post hoc statistical analysis also showed that gp91phox, p47phox, p40phox and P.U.1 mRNA levels were higher at 2 h after the intake of the HSFA meal than at 2 h after the intake of the HMUFA (P = 0.020, P = 0.001, P = 0.002, and P = 0.027, respectively), LFHCC (P = 0.034, P < 0.001, P = 0.010, and P = 0.042, respectively), and LFHCC n-3 (P = 0.001, P < 0.001, P < 0.001 and P = 0.007, respectively) meals. The mRNA levels of p47phox remained higher at 4 h in MetS fed the HSFA meal as compared to that observed after the intake of HMUFA (P = 0.050) and LFHCC n-3 (P = 0.024) meals (Fig. 1D).

We analyzed the relationship between the expression of the gp91phox, p22phox, p67phox, p47phox, p40phox, PU.1, Nrf2, SOD1, SOD2, CAT, GPx1, GPx4, GSR, TXN, and TXNRD1 genes in PBMC and the plasma concentrations of total cholesterol, triglycerides, HDL-c, LDL-c, lipid peroxides, H2O2 and protein carbonyls in the postprandial state. We found a positive correlation between plasma protein carbonyl levels and the gene expression of the NADPH-oxidase subunits and all the antioxidant genes in PBMC at 2 h after the meal intake (Table 1). On the other hand, a negative correlation was observed between plasma protein carbonyls and the mRNA levels of p22phox (r: 0.342; P = 0.003) and Nrf2 (r: 0.275; P = 0.020) at 4 h after the meal intake (Table 1). Likewise, plasma

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Fig. 1. Gene expression of NADPH-oxidase subunits in PBMC. Mean (±S.E.M.) of postprandial gene expression of (A) gp91 , (B) p22 , (C) p67phox, (D) p47phox, (E) p40phox, (F) PU.1. ANOVA for repeated measures (n = 75). P1: diet effect; P2: time effect; P3: diet by time interaction. HSFA: high-saturated fatty acid; HMUFA: highmonounsaturated fatty acid; LFHCC n-3: low-fat, high-complex carbohydrate diet supplemented with n-3 fatty acids; LFHCC: low-fat, high-complex carbohydrate diet supplemented with placebo. à means P < 0.05 for the HSFA meal compared with HMUFA, LFHCC and LFHCC n-3 meals; § means P < 0.05 for the HSFA meal compared with LFHCC meal;   means P < 0.05 2 h after the HSFA meal compared with HMUFA, LFHCC and LFHCC n-3 meals; ¥ means P < 0.05 4 h after the HSFA meal compared with HMUFA and LFHCC n-3 meals; O means P < 0.05 4 h after the HMUFA meal compared with HSFA and LFHCC meals; ⁄ means P < 0.05 2 h after HSFA meal versus fasting state; a means P < 0.05 4 h after HSFA meal versus 2 h.

H2O2 concentration correlated positively with the mRNA levels of genes in PBMC at 2 h after the meal intake, as shown in Table 1. Finally, we found a positive correlation between plasma lipid peroxides concentrations and PU.1 mRNA levels at 2 h after the meal intake (r: 0.241; P = 0.041). and with the mRNA levels of gp91phox (r: 0.312; P = 0.008), p67phox (r: 0.287; P = 0.014), p40phox (r: 0.275; P = 0.019), SOD2 (r: 0.317; P = 0.007), GSR (r: 0.303; P = 0.010) and TXN (r: 0.257; P = 0.029) at 4 h after the meal intake (Table 1).

4. Discussion Our data showed that the consumption of an HSFA meal increased the postprandial expression in PBMC of the antioxidant defence-related genes SOD1, SOD2, CAT, GPx1, GPx4, GSR, TXN, and TXNRD1 as compared to the consumption of an HMUFA meal and LFHCC meal, either supplemented or not with LC3, which caused no effects on the expression of these genes. Additionally, the consumption of an HSFA meal induced a higher postprandial gene expression of different NADPH-oxidase subunits (gp91phox, p22phox, p47phox and p40phox), an enzyme that generates ROS (Inoguchi and Nawata, 2005).

These data are consistent with previous results obtained in the same MetS cohort on oxidative stress parameters in plasma. To be more specific, the consumption of an HSFA diet increased the postprandial oxidative stress as compared to the other three diets (Perez-Martinez et al., 2010). Interestingly, the MetS cohort also exhibited enhanced expression levels of NADPH-oxidase and decreased mRNA content of antioxidant enzymes in subcutaneous adipose tissue after the consumption of the HSFA diet, which is suggestive of enhanced oxidative stress in this tissue. When viewed together, these observations support the notion that changes in oxidative parameters in PBMC reflect those occurring in adipose tissue in MetS patients (Pena-Orihuela et al., 2013). This is also in agreement with the mechanism proposed by Furukawa et al., who showed that systemic oxidative stress in obesity is caused by ROS production in the adipose tissue but not in other tissues, as a consequence of an increase in the expression of NADPHoxidase, and by a decrease in the expression of antioxidant enzymes (Furukawa et al., 2004). Humans nowadays spend most of the time in the postprandial state (Lairon et al., 2007). This condition is characterized by increased oxidative stress (Cardona et al., 2008; Devaraj et al., 2008; Ursini and Sevanian, 2002), caused by an imbalance between ROS production as metabolic by-products or generated by enzymes

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GPx1 mRNA levels

1.2

HMUFA

2

D

me (h) HSFA

p1: 0.051 p2: 0.654 p3: 0.026

B

me (h)

GSR mRNA levels

0.45

SOD2 mRNA levels

A. Camargo et al. / Food and Chemical Toxicology 72 (2014) 178–186

SOD1 mRNA levels

182

2

4

me (h) LFHCC

LFHCC-n3

HSFA

HMUFA

LFHCC

LFHCC-n3

Fig. 2. Antioxidant response in PBMC. Mean (±S.E.M.) of postprandial gene expression in (A) SOD1, (B) SOD2, (C) CAT, (D) GSR, (E) GPx1, (F) GPx4, (G) TXN, (H) TXNRD1. ANOVA for repeated measures (n = 75). P1: diet effect; P2: time effect; P3: diet by time interaction. HSFA: high-saturated fatty acid; HMUFA: high-monounsaturated fatty acid; LFHCC n-3: low-fat, high-complex carbohydrate diet supplemented with n-3 polyunsaturated fatty acids; LFHCC: low-fat, high-complex carbohydrate diet supplemented with placebo. à means P < 0.05 for the HSFA meal compared with HMUFA, LFHCC and LFHCC n-3 meals; £ means P < 0.05 for the HSFA meal compared with LFHCC and LFHCC n-3 meals; – means P < 0.05 for the HSFA meal compared with HMUFA and LFHCC meals; D means P < 0.05 for the HSFA meal compared with LFHCC n-3 meal;   means P < 0.05 2 h after the HSFA meal compared with HMUFA, LFHCC and LFHCC n-3 meals; # means P < 0.05 2 h after the HSFA meal compared with LFHCC and LFHCC n-3 meals; ¤ means P < 0.05 2 h after the HSFA meal compared with LFHCC n-3 meal; ⁄ means P < 0.05 2 h after HSFA meal versus fasting state; a means P < 0.05 4 h after HSFA meal versus 2 h.

such as NADPH-oxidase, involved in the production of ROS (Inoguchi and Nawata, 2005) and ROS detoxification (Durackova, 2010; Scandalios, 2002). However, postprandial oxidative stress can be modulated by diet. In fact, several studies based on the measurement of oxidative markers in plasma demonstrated that the consumption of saturated fatty acids causes harmful effects related to increased oxidative stress (Perez-Martinez et al., 2010; Roberts et al., 2006, 2002; Yubero-Serrano et al., 2011). In line with this, our study showed that the consumption of an HSFA meal increased the postprandial expression of the antioxidant genes SOD1, SOD2, GPx1, GPx4, GSR,

TXN and TXNRD1, at 2 h after the intake of a meal reflecting the fatty acid composition of the previous HSFA diet, while they remained unchanged after the intake of the other meals in PBMC, a sub-set of white blood cells whose gene expression has been proved to be useful in distinguishing between ill and healthy states (Burczynski and Dorner, 2006; de Mello et al., 2008; Ghanim et al., 2004) which has been widely used for gene expression studies (Bories et al., 2012; Camargo et al., 2012, 2010; Cruz-Teno et al., 2012; de Mello et al., 2012; Perez-Herrera et al., 2013; YuberoSerrano et al., 2013). In contrast, we previously observed a down-regulation of several antioxidant genes, including CAT,

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A. Camargo et al. / Food and Chemical Toxicology 72 (2014) 178–186 Δ †

Nrf2 mRNA levels

0.8 0.7 0.6 0.5

Δ

Δ

0.4

blood mononuclear cells seem to reinforce their antioxidant defences by increasing the expression of antioxidant genes. Along with the changes in gene expression indicated above, the HSFA meal also enhanced the expression levels of GSR and TXNRD1 as compared with the HMUFA, LFHCC and the LFHCC n-3 meals. GSR catalyzes the reduction of oxidized glutathione disulfide (GSSG) to the sulphydryl form GSH, which is used as a substrate for GPx activity in the presence of oxidative stress (Mustacich and Powis, 2000). TXNRD1 has the capacity to reduce oxidized thioredoxin and to carry out the reduction of substrates by TXN (Mustacich and Powis, 2000). Thus, it is likely that GSH and TXN turnover is increased to produce the reduced and active antioxidant forms in response to the HSFA meal, These results the occurrence of an adaptive cell response to reinforce the antioxidant systems in order to cope with the higher oxidative stress induced by the HSFA diet, along with oxidized macromolecules or proteins (Taguchi et al., 2011), which are presumably more abundant after an HSFA meal intake than after the intake of the other meals, on the basis of our previous study (Pena-Orihuela et al., 2013; Perez-Martinez et al., 2010). In oxidative stress conditions, the Keap1-Nrf2 complex located in the cytoplasm is disrupted, and Nrf2 migrates to the nucleus to bind specifically to the antioxidant-response element (ARE) and promote the expression of genes encoding an antioxidant response such as CAT, SOD and GPx (Zhang, 2006). Thus, and consistent with the gene expression changes observed after the HSFA meal, we also observed an increase in the nuclear protein levels of Nrf2 and a concomitant decrease in the cytoplasmic fraction after the HSFA meal intake, which may have occurred as a response to the rise in ROS after the HSFA meal. These results coincide with a previous study performed in an elderly population (Inoguchi and Nawata, 2005). The ROS are generated as by-products of metabolism, but several enzymes, including NADPH-oxidase, are also involved in ROS production (Coate and Huggins, 2010; Du et al., 2012). Our group has shown that the quality of the dietary fat is also a factor that may modulate the expression of this enzymatic complex (PerezHerrera et al., 2013; Yubero-Serrano et al., 2013). In fact, it has been shown that the consumption of saturated fat induces an increase in the postprandial expression of p22phox and p47phox NADPH-oxidase subunits as compared to the consumption of monounsaturated fat (Suzuki et al., 1998). In line with this, the current study showed that in metabolic syndrome patients, the

p1 <0.001 p2: 0.115 p3 <0.001

0.3 0.2

0

2

4

me (h) HSFA

HMUFA

LFHCC

LFHCC-n3

Fig. 3. Postprandial Nrf2 gene expression in PBMC. Mean (±S.E.M.) of postprandial gene expression. ANOVA for repeated measures (n = 75). P1: diet effect; P2: time effect; P3: diet by time interaction. HSFA: high-saturated fatty acid; HMUFA: high-monounsaturated fatty acid; LFHCC n-3: low-fat, high-complex carbohydrate diet supplemented with n-3 polyunsaturated fatty acids; LFHCC: low-fat, highcomplex carbohydrate diet supplemented with placebo. D means P < 0.05 for the HSFA meal compared with LFHCC n-3 meal;   means P < 0.05 2 h after the HSFA meal compared with HMUFA, LFHCC and LFHCC n-3 meals.

GPx1, GPx3 and TXNRD1 after the HSFA meal in the adipose tissue of a sub-group of the population analyzed in this study (Pena-Orihuela et al., 2013), which cause, at least partially, an imbalance between ROS generation and detoxification after HSFA diet consumption, leading to an increased oxidative stress state (Pena-Orihuela et al., 2013; Perez-Martinez et al., 2010). The PBMC have been extensively used to study the changes in the inflammatory and oxidative stress status evoked by dietary interventions (Cruz-Teno et al., 2012; de Mello et al., 2012; Yubero-Serrano et al., 2013). However, in terms of oxidative stress, the results conflicted with the Furukawa hypothesis about increased systemic oxidative stress generated by the adipose tissue as a consequence of the down-regulation of antioxidant genes (Perez-Herrera et al., 2013; Yubero-Serrano et al., 2013), since the studies in PBMC showed the increase of their antioxidant defence system when oxidative stress increased (Kodydkova et al., 2009). Based on previous studies in the population in study, the higher mRNA levels of the antioxidant genes SOD1, SOD2, CAT, GPx1 and GPx4 at 2 h after the intake of a meal reflecting the fatty acid composition of the HSFA diet is suggestive of a higher postprandial oxidative stress generated in adipose tissue after HSFA diet consumption (Pena-Orihuela et al., 2013; Perez-Martinez et al., 2010). In response to this increased oxidative stress, peripheral

2.5

Nuclear Nrf2

2.0

B

a

P=0.004

Cytoplasmic Nrf2

A

1.5 1.0 0.5

abc

0.0

bc

c

2.0

P=0.007

b

1.5 1.0 0.5 0.0

abc

a

ac

-0.5 -1.0 -1.5 -2.0 -2.5

-0.5 HSFA

HMUFA

C

LFHCC

0h

LFHCC-n3

4h 0h 4h

HSFA

0h 4h

HMUFA

LFHCC

LFHCC-n3

0h 4h

57kDa

Nuclear Nrf2

57kDa

Cytoplasmic Nrf2 HSFA

HUMFA

LFHCC

LFHCC-n3

Fig. 4. Postprandial Nrf2 protein levels in nuclear and cytoplasmic fraction of PBMC after the intake of the four meals. Values are the mean ± S.E.M. of postprandial protein level (n = 36). Values represent the increment between fasting state and 4 h after meals intake. One-way ANOVA statistical analysis. Bars with different superscript letters depict statistically significantly differences between diets (p < 0.05). Postprandial levels of nuclear Nrf2 (A) and cytoplasmic Nrf2 (B) in peripheral mononuclear cells, according to the meal consumed. Representative immunoblot of nuclear and cytoplasmic Nrf2 (C).

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Table 1 Correlations between the postprandial mRNA levels for gp91phox, p22phox, p67phox, p47phox, p40phox, PU.1, Nrf2, SOD1, SOD2, CAT, GPx1, GPx4, GSR, TXN and TXNRD1 with plasma oxidative stress biomarkers such as lipid peroxides (nmol/ml), H2O2 (lM) and protein carbonyls (nmol/ml) in the MetS patients, 4 h after the administration of the fatty meal with a fat composition similar to that consumed in each of the HSFA, HMUFA, LFHCC, or LFHCC n-3 diets. n = 75, r: Pearson’s linear correlation coefficient. 2 h after the meal intake

4 h after the meal intake

P r

H2O2

Protein carbonyls

Lipid peroxides

H2O2

Lipid peroxides

Protein carbonyls

gp91phox mRNA

0.002 0.358 n.s.

0.003 0.341 0.037 0.247 0.001 0.389 <0.001 0.493 <0.001 0.428 0.001 0.389 <0.001 0.458 0.014 0.29 <0.001 0.42 0.010 0.303 0.003 0.345 0.001 0.394 <0.001 0.484 0.027 0.26 0.002 0.365

n.s.

n.s.

n.s.

n.s.

n.s.

0.008 0.312 n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

0.041 0.241 n.s.

n.s.

0.019 0.275 n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

0.007 0.317 n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

0.010 0.303 0.029 0.257 n.s.

p22phox mRNA p67phox mRNA p47phox mRNA p40phox mRNA PU.1 mRNA Nrf2 mRNA SOD1 mRNA SOD2 mRNA CAT mRNA GPx1 mRNA GPx4 mRNA GSR mRNA TXN mRNA TXNRD1 mRNA

0.001 0.400 n.s. 0.002 0.364 0.001 0.382 0.023 0.268 0.025 0.265 0.001 0.372 <0.001 0.443 0.002 0.358 0.018 0.279 n.s. 0.009 0.307 0.003 0.350

consumption of an HSFA diet increased the postprandial expression of gp91phox, p47phox and p40phox NADPH-oxidase subunit genes, and the expression of the PU.1 gene, a transcription factor involved in the positive regulation of gp91phox transcription, as compared to the other diets. Unlike the antioxidant genes, which are induced in PBMC and down-regulated in adipose tissue after HSFA diet consumption, the expression of the NADPH-oxidase subunits seems to increase in both PBMC and adipose tissue after HSFA diet consumption and remains unchanged after the intake of the other three diets in both PBMC and adipose tissue. It is noteworthy that we observed a close parallelism between the gene expression changes after the HSFA meal intake, where the antioxidant gene expression increased at 2 h after the meal intake and decreased to fasting levels at 4 h in the current study, and the plasma oxidative stress parameter, which increased at 2 h after the meal intake and decreased to fasting levels at 4 h (Babior, 1999, 2004; Landis and Tower, 2005). Consistent with this, our results also showed a positive correlation between several plasma oxidative stress parameters such as H2O2, protein carbonyls and lipid peroxide with the postprandial expression of NADPH-oxidase subunits and antioxidant genes. These observations support the notion that an increased expression of antioxidant genes occurs in PBMC in response to the increased expression of NADPH-oxidase subunits and a rise in ROS after HSFA consumption, which in turn generates secondary products and oxidative damage products such as H2O2, lipid peroxides and protein carbonyls. One of the limitations of our study lies in the fact that we performed a genomic approach, as we analyzed the gene expression of the pro-oxidant and antioxidant. Further studies providing protein data may shed more light in the effect of the quantity and quality

0.014 0.287 n.s.

0.003 0.342 n.s. n.s. n.s. n.s. 0.020 0.275 n.s.

n.s.

n.s. n.s.

of dietary fat in the modulation of the systemic oxidative stress and their specific cellular and molecular mechanisms. In conclusion, our results suggest that in terms of oxidative stress, PBMC and adipose tissue respond differentially to dietary fat. Although the NADPH-subunits seem to respond in a similar way, up-regulation after HSFA diet consumption in both PBMC and adipose tissue, the antioxidant genes are down-regulated in adipose tissue after HSFA diet consumption, which presumably is the responsible for the higher postprandial oxidative stress after the consumption of this diet, whereas PBMC increased the expression of antioxidant genes in response to the higher postprandial oxidative stress after the consumption of an HSFA diet. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgments The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. This work was supported in part by Research Grants from the European Union [LIPGENE European Integrated Project505944], from the Ministerio de Ciencia e Innovacion [Grant Numbers AGL2004-07907, AGL2006-01979, AGL2009-2270, AGL2012-39615 (to J.L.-M.)]; CIBER Fisiopatologia de la Obesidad

A. Camargo et al. / Food and Chemical Toxicology 72 (2014) 178–186

y Nutricion [Grant Number CB06/03/0047]; Consejeria de Innovacion, Ciencia y Empresa, Junta de Andalucia [Grant Number P06CTS-01425 (to J.L.-M.) CTS-03039 (to M.M.M.)]; and Consejeria de Salud, Junta de Andalucia [Grant Numbers 06/128, 07/43, PI-0193 (to J.L.-M.)].); Fondo Europeo de Desarrollo Regional (FEDER). P.P-O has a fellowship from Programa de Formación de Profesorado Universitario (FPU) del Ministerio de Educación, Gobierno de España. We thank to Mª Jose Gomez-Luna for technical support. A.C., P.P-O., O.A.R-Z., P.P-M., J.D-L., C.C-T. and C.M. conducted research; A.C., P.P-O., P.P-M., J.D-L., analyzed data; A.C., P.P-O., F.J. T., M.M.M., H.M.R., F.P-J. and J.L-M. interpreted the data and wrote the paper; A.C., F.P-J. and J.L-M. had primary responsibility for final content; All authors read and approved the final manuscript. References Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W., Fu, P.C., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470–475. 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