Mediterranean Diet Supplemented With Coenzyme Q10 Modulates the Postprandial Metabolism of Advanced Glycation End Products in Elderly Men and Women

Mediterranean Diet Supplemented With Coenzyme Q10 Modulates the Postprandial Metabolism of... Abstract Advanced glycation end products (AGEs) and oxidative stress are elevated with aging and dysmetabolic conditions. Because a Mediterranean (Med) diet reduces oxidative stress, serum AGEs levels, and gene expression related to AGEs metabolism in healthy elderly people, we studied whether supplementation with coenzyme Q10 (CoQ) was of further benefit. Twenty participants aged ≥ 65 (10 men and 10 women) were randomly assigned to each of three isocaloric diets for successive periods of 4 weeks in a crossover design: Med diet, Med + CoQ, and a Western high-saturated-fat diet (SFA diet). After a 12-hour fast, volunteers consumed a breakfast with a fat composition similar to the previous diet period. Analyses included dietary AGEs consumed, serum AGEs and AGE receptor-1 (AGER1), receptor for AGEs (RAGE), glyoxalase I (GloxI), and estrogen receptor α (ERα) mRNA levels. Med diet modulated redox-state parameters, reducing AGEs levels and increasing AGER1 and GloxI mRNA levels compared with the SFA diet. This benefit was accentuated by adding CoQ, in particular, in the postprandial state. Because elevated oxidative stress/inflammation and AGEs are associated with clinical disease in aging, the enhanced protection of a Med diet supplemented with CoQ should be assessed in a larger clinical trial in which clinical conditions in aging are measured. Oxidative stress, Coenzyme Q10, Mediterranean diet, Age-related diseases, Advanced glycation end products Aging is a biological process characterized by a time-dependent, progressive, and physiological decline, accompanied by an increased incidence of age-related diseases (1). Both reactive oxygen species (ROS) and oxidative stress have been pointed out as primary determinants of aging (2). A certain amount of oxidative damage takes place even under normal conditions, but the rate of this damage increases during pathological conditions such as diabetes, cardiovascular diseases, cancer, or other aging-related diseases (3). The contribution of advanced glycation end products (AGEs) in the origin and progression of age-associated diseases has been reported, increasing oxidative stress and inflammation, which underlies their pathology (4–6). AGEs constitute a heterogeneous group of compounds derived from the nonenzymatic glycation of proteins, lipids, and nuclear acids via a complex sequence of reactions referred as the Maillard reaction (7). Glyoxal, methylglyoxal (MG), (8) and N-carboxymethyllysine (CML) are the most well-studied AGEs and serve as markers of AGE accumulation in several tissues (9). Small amounts of AGEs are generated in vivo as a normal consequence of metabolism, and they gradually accumulate during aging, especially in the context of associated chronic diseases (10). However, dietary AGEs (dAGEs) are one of the most important exogenous AGEs sources that contribute to the body AGEs pool (11). In this sense, studies in healthy participants on self-selected diets with low or high dAGEs content showed a strong correlation of AGEs intake and serum AGE levels (sAGEs) (12). Consumption of an AGE-restricted diet is an effective method to reduce the body’s total AGE concentration and that, in turn, decreases oxidative stress and inflammation in many populations (13,14). AGEs content of a diet depends on the nutrient composition and on the way food is processed (15,16). In fact, there is growing evidence that the Western-style diet, rich in saturated fatty acids (SFA), is a plentiful source of exogenous AGEs (17). When evaluating the influence of the diet on oxidative stress, most studies analyzed several factors in the fasting state after different dietary regimens. However, fasting is not the typical physiological state of the modern human being, who spends most of their time in a postprandial state (18). Postprandial oxidative stress is characterized by an increase in oxidative stress biomarkers after the intake of a meal (19). In line with this, the Mediterranean diet (Med diet), which is rich in monounsaturated fatty acids (MUFA) and minimally processed natural foods, was found to reduce postprandial oxidative stress and inflammation (20). Interest in Coenzyme Q10 (CoQ; 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone) was stimulated by the fact that it may be an additional antioxidant source based on its key role in mitochondrial bioenergetics and its widely studied antioxidant capacity under lipophilic conditions (21). Aging, poor eating habits, and stress affect the organism’s ability to provide adequate amounts of CoQ. Many researchers suggest that using CoQ supplements may help to maintain the health status of elderly people and may also treat some of the age-related diseases. For instance, it might be used as adjunctive therapy in the treatment of congestive heart failure (22) as well as for slowing the progression of Parkinson’s disease in its early stages (23). We previously showed that postprandial oxidative stress was reduced after a Mediterranean diet supplemented with CoQ (Med + CoQ diet) when compared with a SFA-rich diet (24–26). However, the effect of Mediterranean diet and CoQ in the metabolism of AGEs associated with aging has not been established. In this sense, our main aim was to determine whether the Med + CoQ diet may have a favorable effect on the oxidative stress by modifying the levels of sAGEs and the expression of genes related to AGEs metabolism in healthy elderly people. Methods Participants and Recruitment Volunteers were recruited using various methods including general practitioner databases and advertisements by posters and newspapers. Among those willing to consider entering the study, a total of 63 persons were contacted and showed their willingness to enter the study. Inclusion and exclusion criteria were fulfilled by 20 patients (age ≥ 65 years; 10 men and 10 women). Clinical inclusion criteria were as follows: age ≥ 65 years, body mass index = 20–40 kg/m2, total cholesterol concentration ≤ 8.0 mmol/L, and nonsmokers. The clinical exclusion criteria were as follows: age < 65 years, diabetes or other endocrine disorders, chronic inflammatory conditions, kidney or liver dysfunction, iron deficiency anemia (hemoglobin < 12 g/dL men, <11 g/dL women), prescribed hypolipidemic and anti-inflammatory medication, fatty acid supplements including fish oil, consumers of high doses of antioxidant vitamins (A, C, E, β-carotene), highly trained or endurance athletes or those who participate in more than three periods of intense exercise per week, weight change ≥ 3 kg within the last 3 months, smokers, alcohol, or drug abuse (based on clinical judgment). Forty-three patients were excluded after reviewing the inclusion and exclusion criteria, being the main cause of exclusion the existence of chronic diseases. Before joining the study, all participants underwent a comprehensive medical history, physical examination, and clinical chemistry analysis and gave informed consent. The study protocol was approved by the Human Investigation Review Committee of the Reina Sofia University Hospital, according to institutional and Good Clinical Practice guidelines (protocol number 772). Study Design Participants were randomly assigned to receive, in a crossover design, three sequential isocaloric diets for 4-week periods each. The experimental design of the study was described previously (Supplementary Figure 2) (24–26). Because all participants consumed the three diets, the final sample size was considered to be 60. The three diets were as follows: (a) Mediterranean diet supplemented with CoQ (Med + CoQ diet; ubiquinone: 200 mg/d in capsules), containing 15% of energy as protein, 47% of energy as carbohydrate, and 38% of total energy as fat (24% MUFA [provided by virgin olive oil], 10% SFA, 4% polyunsaturated fatty acid [PUFA]); (b) Mediterranean diet not supplemented with CoQ (Med diet), with the same composition of the first diet but supplemented by placebo capsules; and (c) Western diet rich in saturated fat (SFA diet), with 15% of energy as protein, 47% of energy as carbohydrate, and 38% of total energy as fat (12% MUFA, 22% SFA, 4% PUFA). From the Med/Med + CoQ diets, 80% of the MUFA was provided by virgin olive oil, which was used for cooking, salad dressing, and as a spread. Butter was used as the main source of SFA during the SFA dietary period (Supplementary Table 3). The cholesterol intake was kept constant (<300 mg/d) during the three periods. Both the CoQ and the placebo capsules were specially produced by the same company (Kaneka Corporation, Osaka, Japan) and were identical in weight and external aspects. Patients taking the capsules were unaware whether they were in the Med + CoQ or Med dietary period (Supplementary Table 1). The composition of the experimental diets was calculated by using the U.S. Department of Agriculture (1987) food tables and Spanish food-composition tables for local foodstuffs (27,28). At the end of each dietary intervention period, participants were given a fatty breakfast with the same fat composition as consumed in each of the assigned diets. Patients presented at the clinical centers 8-hours following a 12-hour fast (time 0) and abstained from alcohol intake during the preceding 7 days. A fasting blood sample was taken in the morning, following which the test meal, was ingested within 20 minutes under supervision. Subsequent blood samples were drawn at 4 hours. Test meals provided an equal amount of fat (0.7 g/kg of body weight), cholesterol (5 mg/kg of body weight), and vitamin A (60,000 IU/m2 body surface area). The test meal provided 65% of energy as fat, 10% as protein, and 25% as carbohydrates. The composition of the breakfasts was as follows: Med with CoQ breakfast (400 mg in capsules; 12% SFA, 43% MUFA, 10% PUFA); Med with placebo capsules breakfast (12% SFA, 43% MUFA, 10% PUFA); and SFA-rich breakfast (38% SFA, 21% MUFA, 6% PUFA). Biochemical Determinations Serum samples Samples from each dietary intervention were collected in the fasting state and 4 hours after the meal intake (postprandial state) and stored with particular care to avoid exposure to air and light. Serum was separated from whole blood by centrifugation at 1,500g for 20 minutes at 20°C within 1 hour of extraction. dAGEs intake Assessment of dAGEs content was performed using 3-day weighed food diaries emphasizing cooking methods that were completed by the participants, at baseline, Week 2, and Week 4 for each diet. Similarly, dAGEs content was also determined after each test breakfast. Then, dAGEs content was estimated from a database of approximately 560 foods that lists AGE values (16) and was expressed as AGE kilounits per 100-gram food. Measurement of AGEs Serum CML and MG (sCML and sMG) were determined by well-validated competitive ELISAs (OxiSelect Methylglyoxal Competitive Elisa Kit and OxiSelect Nε-Carboxymethyllysine Competitive Elisa Kit [Cell Biolabs, San Diego], respectively), which are based on noncross-reactive monoclonal antibodies (mabs) for protein-bound CML (4G9 mab) and protein-bound MG derivatives. The immunogens used to generate the mabs (ie, lysine-MG-H1 [3D11 mab] and protein-bound CML) were characterized by high-performance liquid chromatography (29). The resulting values reflect relatively stable protein- or peptide-associated CML and MG and not the free compounds. The interassay coefficients of variation were 2.8% and 5.2% for CML and MG, respectively; the intra-assay coefficients of variation were 2.6% and 4.1% for CML and MG, respectively (13). Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were isolated from 20 mL of venous blood, at fasting and postprandial states, in tubes containing 1 mg/mL of ethylenediaminetetraacetic acid. The blood samples were diluted 1:1 in phosphate-buffered saline, and cells were separated in Ficoll gradient (Ficoll-Paque PLUS; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) by centrifugation at 800g for 25 minutes at 20°C. The cells were collected and washed with cold phosphate-buffered saline two times and finally resuspended in a buffer. The buffer contained 10 mM HEPES, 15 mM KCl, 2 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid (at the time of use, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol were added; Sigma–Aldrich, St. Louis, MO). The cells thus obtained were stored at −80°C for further analysis. RNA extraction Total RNA from PBMCs was extracted using the trizol method according to the recommendations of the manufacturer (Tri Reagent; Sigma–Aldrich) and quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK). RNA integrity was verified on agarose gel electrophoresis and stored at −80°C. Samples were digested with DNAse I (AMPD-1 KT; Sigma–Aldrich) before real-time PCR. Quantitative Real-Time PCR for Gene Expression Analysis Real-time PCR was carried out using the OpenArray NT Cycler system (Applied Biosystems, Carlsbad, CA), following the manufacturer’s instructions. Each reaction was performed with 1 μL of a 1:5 (v/v) dilution of the first cDNA synthesized from 1 μg 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 20 participants at fasting and at 4 hours after intake of the fat overload. Primer pairs were selected from the database TaqMan Gene Expression assays (Applied Biosystems; https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601267), for the following genes: AGE receptor-1 (AGER1), receptor for AGEs (RAGE), glyoxalase I (GloxI), and estrogen receptor α (ERα). 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 OpenArray Real-Time qPCR Analysis Software (Applied Biosystems). Statistical Analysis PASW Statistics, Version a8, SPSS statistical software (Chicago, IL) was used for statistical analysis. The Kolmogorov–Smirnov test did not show a significant departure from normality in the distribution of variance values. In order to evaluate data variation, Student’s t test and analysis of variance for repeated measures was performed, followed by Bonferroni’s correction for multiple comparisons, depending on the existence of two or more groups in each comparison. We studied the statistical effects of the type of fat meal ingested, independent of time (represented by p1), the effect of time (represented by p2), and the interaction of both factors (represented by p3). Pearson’s correlation analyses were performed to examine the correlations between AGER1 and RAGE mRNA levels and sCML and sMG and other parameters related to inflammation and redox state described in our previous reports (nuclear factor [erythroid-derived 2]-like 2 [Nrf2], thioredoxin [Trx], NADPH oxidase subunits [p40phox and p67phox], interleukin-8 [IL-8] mRNA levels, and plasmatic nitric oxide, 8-isoprostanes, and protein carbonyls levels) (24,26). Differences were considered to be significant when p < .05. All data presented in text and tables are expressed as means ± SE. Results Baseline Characteristics of the Study Population All participants completed the three dietary intervention periods. Men had higher height, waist circumference, triglyceride, and Apo B than women. There were no other differences by gender (Supplementary Table 2). dAGEs Consumed in Each Dietary Intervention Med diet (with or without CoQ supplementation) and SFA diet differed in quality fat composition, and this was mirrored by differences in dAGEs levels (Figure 1A and B). In both the fasting and postprandial phase after SFA diet, participants consumed a higher amount of dAGEs compared with Med or Med + CoQ diets (all p < .05; Figure 1A and B). There were no differences in the amount of food-related AGEs between Med and Med + CoQ diets (Figure 1A). Figure 1. View largeDownload slide dAGEs levels consumed after 4 weeks of each dietary intervention (A) and after 4-hour postprandial state (B). Fasting and postprandial levels of MG (C) and CML (D) in serum samples according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGEs = advanced glycation end products; ANOVA = analysis of variance; CML = N-carboxymethyllysine; CoQ = coenzyme Q10; dAGEs = dietary advanced glycation end products; Med = Mediterranean; MG = methylglyoxal; SFA = saturated fat. Figure 1. View largeDownload slide dAGEs levels consumed after 4 weeks of each dietary intervention (A) and after 4-hour postprandial state (B). Fasting and postprandial levels of MG (C) and CML (D) in serum samples according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGEs = advanced glycation end products; ANOVA = analysis of variance; CML = N-carboxymethyllysine; CoQ = coenzyme Q10; dAGEs = dietary advanced glycation end products; Med = Mediterranean; MG = methylglyoxal; SFA = saturated fat. Plasma CoQ and Serum AGEs Levels After Each Dietary Intervention Fasting and postprandial plasma CoQ levels were higher after Med + CoQ diet compared with the other diets (all p < .05). Moreover, Med + CoQ diet produced an increase in plasma CoQ levels during the postprandial phase compared with fasting state (p = .019; Supplementary Figure 1). Conversely, fasting sMG and sCML levels were higher after SFA diet compared with both Med and Med + CoQ diets (all p < .05; Figure 1C and D). Furthermore, Med + CoQ diet produced lower postprandial sMG and sCML levels compared with Med diet (p = .013 and .019, respectively). We also observed lower postprandial levels of both sAGEs forms after Med diet compared with SFA diet (p = .005 and .009, respectively; Figure 1C and D). Interestingly, sMG levels decreased significantly during the postprandial phase when compared with the fasting state in the Med + CoQ diet group (p = .023) but were unchanged in the Med group and were even increased in SFA group (Figure 1C). Dietary Intake and Expression of Genes Related to Receptors for AGEs in PBMCs The antioxidant properties of AGER1 are due to the fact that it reduces AGE levels in the intracellular and extracellular spaces (13,14). Conversely, cell surface RAGE binds AGEs and induces the generation of ROS and promotes an inflammatory response (30). Med and Med + CoQ diets produced higher fasting and postprandial AGER1 mRNA levels and lower fasting and postprandial RAGE mRNA levels compared with SFA diet. However, we observed higher postprandial AGER1 mRNA levels after Med + CoQ diet compared with Med diet (all p < .05; Figure 2A and B). We did not find differences on fasting and postprandial RAGE mRNA levels between Med + CoQ and Med diets (Figure 2B). Conversely, Med and Med + CoQ diets produced an increase in AGER1 mRNA levels, during the postprandial phase compared with the fasting state (p = .025 and .006, respectively), whereas consumption of the SFA diet resulted in a decrease of postprandial AGER1 mRNA levels in comparison with the fasting state (Figure 2A). Figure 2. View largeDownload slide Fasting and postprandial levels of AGER1 mRNA (A), RAGE mRNA (B), GloxI mRNA (C), and ERα mRNA (D) in PBMCs according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGER1 = advanced glycation end product receptor-1; ANOVA = analysis of variance; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs. Figure 2. View largeDownload slide Fasting and postprandial levels of AGER1 mRNA (A), RAGE mRNA (B), GloxI mRNA (C), and ERα mRNA (D) in PBMCs according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGER1 = advanced glycation end product receptor-1; ANOVA = analysis of variance; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs. Dietary Intake and Expression of Genes Related to AGE Metabolism and Oxidative Stress in PBMCs GloxI detoxifies reactive α-oxoaldehyde, thereby removing AGEs, particularly MG (31). In addition, AGEs have been implicated in the reduction in number and function of estrogen receptors (ERs) (32). Med and Med + CoQ diets produced higher fasting and postprandial GloxI mRNA levels and higher postprandial ERα mRNA levels compared with SFA diet (all p < .05; Figure 2C and D). There were no differences in fasting ERα mRNA levels among the three diets. Conversely, Med + CoQ diet produced higher postprandial GloxI mRNA levels compared with Med diet (p = .018; Figure 2C). Conversely, Med diet produced an increase in ERα mRNA levels, and both Med and Med + CoQ diets produced an increase in GloxI mRNA levels, during the postprandial phase compared with fasting state (all p < .05) but not after SFA diet (Figure 2C and D). Relationship Between AGEs and Inflammatory and Redox-State-Related Parameters Figures 3 and 4 show the correlation results for the relationship between AGER1 and RAGE mRNA levels and parameters related to redox state and inflammation. A univariate analysis showed a moderate positive correlation between AGER1 mRNA levels and Nrf2 mRNA levels (r = .396; p = .018), nitric oxide levels (r = .401; p = .010) and Trx mRNA levels (r = .444; p = .002), a moderate negative correlation with protein carbonyl levels (r = −.305; p = .032), and a weak negative correlation with 8-isoprostanes levels (r = −.299; p = .024). As anticipated, RAGE mRNA levels showed a strong positive correlation with IL-8 mRNA levels (r = .511; p = .003) and 8-isoprostanes levels (r = .613; p = .011), and a moderate positive correlation with sMG content (r = .301; p = .007) and gene expression of a subunit of NADPH oxidase such as p67phox mRNA levels (r = .361; p = .001). Figure 3. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and expression of genes related to oxidative stress/inflammation, in PBMCs. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; p67phox = subunit of NADPH oxidase; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs; Trx = thioredoxin. Figure 3. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and expression of genes related to oxidative stress/inflammation, in PBMCs. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; p67phox = subunit of NADPH oxidase; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs; Trx = thioredoxin. Figure 4. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and plasma and serum oxidative stress/inflammation-related variables. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; RAGE = receptor for AGEs; sMG = serum methylglyoxal. Figure 4. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and plasma and serum oxidative stress/inflammation-related variables. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; RAGE = receptor for AGEs; sMG = serum methylglyoxal. Discussion Recent knowledge points toward high levels of ROS and oxidative stress as the primary determinants of aging, together with low levels of CoQ present during the aging process, explain the fact of selecting an elderly population in our study. Conversely, current dietary habits would predict that modern-day humans spend most of the time in the postprandial state (18), which is known to be a stressful condition because this is the time in which an oxidative stress situation normally occurs (19). Therefore, the postprandial phase is probably one of the most essential periods to reveal the healthy or detrimental effects of dietary interventions by studying variations in oxidative stress. In this sense, our results corroborated the antioxidant and anti-inflammatory effects of the consumption of a Med diet, both in fasting and postprandial states (20). Moreover, the addition of the CoQ to a Med-style diet provides an additional beneficial effect of reducing the oxidative and inflammatory state, particularly in the postprandial phase (24,26). Here, we have shown for the first time that CoQ supplementation resulted in a greater decrease in endogenous AGEs levels, sMG and sCML, during postprandial state, which is likely a consequence of an increase in gene expression related with AGEs metabolism, AGER1 and GloxI. Although other clinical studies have been focused on the antioxidant effects of CoQ, the current study is the first cross-randomized controlled dietary trial that analyzes the effect of supplementation with CoQ on AGEs during the postprandial phase in an aged population. Another novel feature of this study is the correlations between the diets and the metabolism of AGEs. Several studies have shown that a low-AGE diet reduced circulating AGE levels and chronic inflammation/oxidative stress in patients with age-related diseases (4,14). The current study extends these previous data and shows that consumption of Med diet, with or without supplementation with CoQ, provided lower dAGEs content compared with SFA diet. Med diet components (MUFA, α-tocopherol, phenolic compounds, or phytoesterols) could provide the antioxidant capacity of this diet, suggesting its direct association with a decrease of oxidative stress (33). Previous studies showed that sAGEs levels are directly related to AGEs consumed via the diet (34), a finding that is corroborated by the current study. In contrast, consumption of SFA diet, with higher content in dAGEs, was associated with increased sMG and sCML levels. Dietary intervention studies demonstrated that SFA-rich diets contain elevated quantities of AGEs with adverse consequences on health (4,35). In response to high AGEs levels, the host defense system employs different mechanisms to restrict their toxicity. The glyoxalase system (mainly GloxI and GloxII) (11) and AGER1/RAGE receptors have been shown to be a part of the innate and adaptive defense that serve to respond to AGEs. GloxI is an important regulator of the control of intracellular AGEs because it binds and detoxifies AGEs formed during normal metabolism. AGER1 binds extracellular AGEs and delivers them to the lysosomes where they are detoxified (30), thus reducing AGE-related ROS and inflammation (13,36). Another means of reducing extracellular AGEs levels is provided by circulating RAGE because it binds AGEs. However, after this binding, RAGE induces oxidative stress/inflammation, and thus, it can be considered as a deleterious response (37). These defense mechanisms are reduced in aging and other age-related diseases, resulting in suppression of the antioxidant defense system and increased levels of pro-oxidant mechanisms, which results in a further increase in AGEs levels, ROS, and finally leads to tissue injury (29). The current study supports these findings and other previous studies showing that sAGEs serve as stimuli for changes in gene expression. Namely, higher AGER1 and GloxI mRNA levels and the lower RAGE mRNA levels observed in participants with the Med diet intake could also explain the reduction in sMG and sCML levels compared with the SFA diet. Although both Med and Med + CoQ diets contained similar levels of dAGEs, supplementation of a Med diet with CoQ led to further reductions in the postprandial levels of sMG and sCML accompanied by increased AGER1 and GloxI mRNA levels. Besides being part of the mammalian mitochondrial electron transport chain, CoQ is related with protection and elimination of oxidative damage due its antioxidant properties in all cell membranes preventing lipid peroxidation and participating in the regeneration of other antioxidants, such as α-tocopherol (38). Although the reduced form of this molecule, ubiquinol, is the one that acts as a potent lipid-soluble antioxidant scavenging free-radical species and regenerating the active form of other antioxidants (21), our study showed a high protection of the redox state when a Med diet was supplemented with ubiquinone, the oxidized form. In accordance, efficient mechanisms for reduction of CoQ seem to be present in the blood, and the majority of this compound is in the reduced state even after a fourfold elevation of concentration on dietary supplementation with its oxidized form (39). CoQ deficiency results in an impairment of the assembly and/or stability of the respiratory chain enzymes, which leads to imbalanced oxidative phosphorylation and enhanced ROS production (40), and a reduced cell capacity against oxidative damage (38). Therefore, the addition of CoQ to the diet could be expected to enhance respiratory chain activity, reduce ROS production, and protect from protein oxidation and lipid peroxidation (41), outcomes that could contribute to a reduction in the formation of endogenous AGEs. This ultimately might lead to preservation of anti-AGE defenses (GloxI and AGER1). Although estrogen levels are decreased in aging, they remain sufficient to stimulate ERs, suggesting that the insensitivity to estrogen in the postmenopausal period is due to the decrease in number and function of ERs (32). Several studies have implicated AGEs in ERα inhibition (6,42). ERα mRNA levels were increased in those participants consuming the Med and Med + CoQ diets. Moreover, Med and Med + CoQ diets contained the lowest levels of AGEs and contributed to a reduction of sAGEs levels, which may have led to the increased postprandial ERα mRNA levels in these participants. The oxidant and inflammatory action of circulating AGEs is normally controlled by AGER1, considered as the main host defense against glycoxidants, whereas the AGEs binding to RAGE produces the activation of inflammatory and immune responses, which finally leads to cell damage (29,36). In this way, AGER1 mRNA levels were mainly associated with genes/markers related to antioxidant defenses (a moderate correlation with Nrf2 and Trx mRNA and plasma nitric oxide levels), whereas RAGE mRNA levels showed associations with inflammatory/oxidative markers (a strong correlation with IL-8 mRNA and 8-isoprostanes levels, and a moderate correlation with sMG and p67phox mRNA levels, a subunit of NAPDH oxidase enzyme, related to ROS production in the cell). The present study has the advantage of a randomized crossover design in which all the participants have experienced the three diet periods, each individual acting as his/her own control and strengthening the fact that the effects observed are indeed due to the influence of the type of diet. Our study has certain limitations because ensuring adherence to dietary instructions is difficult in a feeding trial. However, adherence to the recommended dietary patterns was satisfactory, as can be judged by the measurements of compliance. In conclusion, our results support the protective effect of a Med diet against oxidative stress/inflammation, and this protection is enhanced by supplementing with CoQ, thus reducing postprandial circulating AGEs levels and modulating the expression of genes related to AGEs metabolism in elderly men and women. This fact may be a good starting point for assessing the benefits of both consuming low-AGE diets and the use of CoQ on clinical features associated to age. Supplementary Material Please visit the article online at http://gerontologist.oxfordjournals.org/ to view supplementary material. Funding This work was supported in part by research grants from the Ministerio de Ciencia e Innovación (AGL2004-07907, AGL2006-01979, and AGL2009-12270), Ministerio de Ciencia y Competitividad (AGL2012-39615 to J.L.-M., FIS PI10/01041 to P.P.-M., and FIS PI13/00023 to J.D.-L.), Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P06-CTS-01425 and PI0193/09 to J.L.-M., PI-0252/09 to J.D.-L., and PI-0058/10 to P.P.-M.), Proyecto de Excelencia, Consejería de Economía, Innovación, Ciencia y Empleo (CVI-7450 to J.L.-M.); Merck Serono and Fundacion 2000 (Clinical Research in Cardiometabolic to P.P.-M.). Conflict of Interest The authors have no conflicts of interest to disclose. Acknowledgments The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. The authors’ responsibilities were as follows: J.L.-M. and F.P.-J. designed research and conducted research; J.D.-L., A.G.-R., J.F.A.-D., F.G.-D., P.P.-M., and J.L.-M. provided materials or participants; J.L.-M., A.C., J.F.A.-D., G.M.Q.-N., and E.M.Y.-S. collected and assembled data; J.L.-M. and E.M.Y.-S. analyzed data; J.L.-M. and E.M.Y.-S. wrote the manuscript; G.E.S., J.D.-L., F.J.-T., and P.P.-M. provided significant advice and support in reviewing the drafting of the manuscript; J.L.-M. and E.M.Y.-S. had primary responsibility for final content. All authors read an approved the final manuscript. References 1. Harman D. Origin and evolution of the free radical theory of aging: a brief personal history, 1954–2009. Biogerontology . 2009; 10: 773– 781. doi: 10.1007/s10522-009-9234-2 Google Scholar CrossRef Search ADS PubMed  2. Dröge W. The plasma redox state and ageing. Ageing Res Rev . 2002; 1: 257– 278. Google Scholar CrossRef Search ADS PubMed  3. Elahi MM Kong YX Matata BM. 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RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes. J Am Soc Nephrol . 2009; 20: 742– 752. doi: 10.1681/ASN.2008050514 Google Scholar CrossRef Search ADS PubMed  38. López-Lluch G Rodríguez-Aguilera JC Santos-Ocaña C Navas P. Is coenzyme Q a key factor in aging? Mech Ageing Dev . 2010; 131: 225– 235. doi: 10.1016/j.mad.2010.02.003. Google Scholar CrossRef Search ADS PubMed  39. Dallner G Sindelar PJ. Regulation of ubiquinone metabolism. Free Radic Biol Med . 2000; 29: 285– 294. Google Scholar CrossRef Search ADS PubMed  40. Quinzii CM López LC Von-Moltke Jet al.  . Respiratory chain dysfunction and oxidative stress correlate with severity of primary CoQ10 deficiency. FASEB J . 2008; 22: 1874– 1885. doi: 10.1096/fj.07-100149 Google Scholar CrossRef Search ADS PubMed  41. Imada I Sato EF Kira Y Inoue M. Effect of CoQ homologues on reactive oxygen generation by mitochondria. Biofactors . 2008; 32: 41– 48. Google Scholar CrossRef Search ADS PubMed  42. Mukherjee TK Reynolds PR Hoidal JR. Differential effect of estrogen receptor alpha and beta agonists on the receptor for advanced glycation end product expression in human microvascular endothelial cells. Biochim Biophys Acta . 2005; 1745: 300– 309. doi: 10.1016/j.bbamcr.2005.03.012 Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences Oxford University Press

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Abstract

Abstract Advanced glycation end products (AGEs) and oxidative stress are elevated with aging and dysmetabolic conditions. Because a Mediterranean (Med) diet reduces oxidative stress, serum AGEs levels, and gene expression related to AGEs metabolism in healthy elderly people, we studied whether supplementation with coenzyme Q10 (CoQ) was of further benefit. Twenty participants aged ≥ 65 (10 men and 10 women) were randomly assigned to each of three isocaloric diets for successive periods of 4 weeks in a crossover design: Med diet, Med + CoQ, and a Western high-saturated-fat diet (SFA diet). After a 12-hour fast, volunteers consumed a breakfast with a fat composition similar to the previous diet period. Analyses included dietary AGEs consumed, serum AGEs and AGE receptor-1 (AGER1), receptor for AGEs (RAGE), glyoxalase I (GloxI), and estrogen receptor α (ERα) mRNA levels. Med diet modulated redox-state parameters, reducing AGEs levels and increasing AGER1 and GloxI mRNA levels compared with the SFA diet. This benefit was accentuated by adding CoQ, in particular, in the postprandial state. Because elevated oxidative stress/inflammation and AGEs are associated with clinical disease in aging, the enhanced protection of a Med diet supplemented with CoQ should be assessed in a larger clinical trial in which clinical conditions in aging are measured. Oxidative stress, Coenzyme Q10, Mediterranean diet, Age-related diseases, Advanced glycation end products Aging is a biological process characterized by a time-dependent, progressive, and physiological decline, accompanied by an increased incidence of age-related diseases (1). Both reactive oxygen species (ROS) and oxidative stress have been pointed out as primary determinants of aging (2). A certain amount of oxidative damage takes place even under normal conditions, but the rate of this damage increases during pathological conditions such as diabetes, cardiovascular diseases, cancer, or other aging-related diseases (3). The contribution of advanced glycation end products (AGEs) in the origin and progression of age-associated diseases has been reported, increasing oxidative stress and inflammation, which underlies their pathology (4–6). AGEs constitute a heterogeneous group of compounds derived from the nonenzymatic glycation of proteins, lipids, and nuclear acids via a complex sequence of reactions referred as the Maillard reaction (7). Glyoxal, methylglyoxal (MG), (8) and N-carboxymethyllysine (CML) are the most well-studied AGEs and serve as markers of AGE accumulation in several tissues (9). Small amounts of AGEs are generated in vivo as a normal consequence of metabolism, and they gradually accumulate during aging, especially in the context of associated chronic diseases (10). However, dietary AGEs (dAGEs) are one of the most important exogenous AGEs sources that contribute to the body AGEs pool (11). In this sense, studies in healthy participants on self-selected diets with low or high dAGEs content showed a strong correlation of AGEs intake and serum AGE levels (sAGEs) (12). Consumption of an AGE-restricted diet is an effective method to reduce the body’s total AGE concentration and that, in turn, decreases oxidative stress and inflammation in many populations (13,14). AGEs content of a diet depends on the nutrient composition and on the way food is processed (15,16). In fact, there is growing evidence that the Western-style diet, rich in saturated fatty acids (SFA), is a plentiful source of exogenous AGEs (17). When evaluating the influence of the diet on oxidative stress, most studies analyzed several factors in the fasting state after different dietary regimens. However, fasting is not the typical physiological state of the modern human being, who spends most of their time in a postprandial state (18). Postprandial oxidative stress is characterized by an increase in oxidative stress biomarkers after the intake of a meal (19). In line with this, the Mediterranean diet (Med diet), which is rich in monounsaturated fatty acids (MUFA) and minimally processed natural foods, was found to reduce postprandial oxidative stress and inflammation (20). Interest in Coenzyme Q10 (CoQ; 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone) was stimulated by the fact that it may be an additional antioxidant source based on its key role in mitochondrial bioenergetics and its widely studied antioxidant capacity under lipophilic conditions (21). Aging, poor eating habits, and stress affect the organism’s ability to provide adequate amounts of CoQ. Many researchers suggest that using CoQ supplements may help to maintain the health status of elderly people and may also treat some of the age-related diseases. For instance, it might be used as adjunctive therapy in the treatment of congestive heart failure (22) as well as for slowing the progression of Parkinson’s disease in its early stages (23). We previously showed that postprandial oxidative stress was reduced after a Mediterranean diet supplemented with CoQ (Med + CoQ diet) when compared with a SFA-rich diet (24–26). However, the effect of Mediterranean diet and CoQ in the metabolism of AGEs associated with aging has not been established. In this sense, our main aim was to determine whether the Med + CoQ diet may have a favorable effect on the oxidative stress by modifying the levels of sAGEs and the expression of genes related to AGEs metabolism in healthy elderly people. Methods Participants and Recruitment Volunteers were recruited using various methods including general practitioner databases and advertisements by posters and newspapers. Among those willing to consider entering the study, a total of 63 persons were contacted and showed their willingness to enter the study. Inclusion and exclusion criteria were fulfilled by 20 patients (age ≥ 65 years; 10 men and 10 women). Clinical inclusion criteria were as follows: age ≥ 65 years, body mass index = 20–40 kg/m2, total cholesterol concentration ≤ 8.0 mmol/L, and nonsmokers. The clinical exclusion criteria were as follows: age < 65 years, diabetes or other endocrine disorders, chronic inflammatory conditions, kidney or liver dysfunction, iron deficiency anemia (hemoglobin < 12 g/dL men, <11 g/dL women), prescribed hypolipidemic and anti-inflammatory medication, fatty acid supplements including fish oil, consumers of high doses of antioxidant vitamins (A, C, E, β-carotene), highly trained or endurance athletes or those who participate in more than three periods of intense exercise per week, weight change ≥ 3 kg within the last 3 months, smokers, alcohol, or drug abuse (based on clinical judgment). Forty-three patients were excluded after reviewing the inclusion and exclusion criteria, being the main cause of exclusion the existence of chronic diseases. Before joining the study, all participants underwent a comprehensive medical history, physical examination, and clinical chemistry analysis and gave informed consent. The study protocol was approved by the Human Investigation Review Committee of the Reina Sofia University Hospital, according to institutional and Good Clinical Practice guidelines (protocol number 772). Study Design Participants were randomly assigned to receive, in a crossover design, three sequential isocaloric diets for 4-week periods each. The experimental design of the study was described previously (Supplementary Figure 2) (24–26). Because all participants consumed the three diets, the final sample size was considered to be 60. The three diets were as follows: (a) Mediterranean diet supplemented with CoQ (Med + CoQ diet; ubiquinone: 200 mg/d in capsules), containing 15% of energy as protein, 47% of energy as carbohydrate, and 38% of total energy as fat (24% MUFA [provided by virgin olive oil], 10% SFA, 4% polyunsaturated fatty acid [PUFA]); (b) Mediterranean diet not supplemented with CoQ (Med diet), with the same composition of the first diet but supplemented by placebo capsules; and (c) Western diet rich in saturated fat (SFA diet), with 15% of energy as protein, 47% of energy as carbohydrate, and 38% of total energy as fat (12% MUFA, 22% SFA, 4% PUFA). From the Med/Med + CoQ diets, 80% of the MUFA was provided by virgin olive oil, which was used for cooking, salad dressing, and as a spread. Butter was used as the main source of SFA during the SFA dietary period (Supplementary Table 3). The cholesterol intake was kept constant (<300 mg/d) during the three periods. Both the CoQ and the placebo capsules were specially produced by the same company (Kaneka Corporation, Osaka, Japan) and were identical in weight and external aspects. Patients taking the capsules were unaware whether they were in the Med + CoQ or Med dietary period (Supplementary Table 1). The composition of the experimental diets was calculated by using the U.S. Department of Agriculture (1987) food tables and Spanish food-composition tables for local foodstuffs (27,28). At the end of each dietary intervention period, participants were given a fatty breakfast with the same fat composition as consumed in each of the assigned diets. Patients presented at the clinical centers 8-hours following a 12-hour fast (time 0) and abstained from alcohol intake during the preceding 7 days. A fasting blood sample was taken in the morning, following which the test meal, was ingested within 20 minutes under supervision. Subsequent blood samples were drawn at 4 hours. Test meals provided an equal amount of fat (0.7 g/kg of body weight), cholesterol (5 mg/kg of body weight), and vitamin A (60,000 IU/m2 body surface area). The test meal provided 65% of energy as fat, 10% as protein, and 25% as carbohydrates. The composition of the breakfasts was as follows: Med with CoQ breakfast (400 mg in capsules; 12% SFA, 43% MUFA, 10% PUFA); Med with placebo capsules breakfast (12% SFA, 43% MUFA, 10% PUFA); and SFA-rich breakfast (38% SFA, 21% MUFA, 6% PUFA). Biochemical Determinations Serum samples Samples from each dietary intervention were collected in the fasting state and 4 hours after the meal intake (postprandial state) and stored with particular care to avoid exposure to air and light. Serum was separated from whole blood by centrifugation at 1,500g for 20 minutes at 20°C within 1 hour of extraction. dAGEs intake Assessment of dAGEs content was performed using 3-day weighed food diaries emphasizing cooking methods that were completed by the participants, at baseline, Week 2, and Week 4 for each diet. Similarly, dAGEs content was also determined after each test breakfast. Then, dAGEs content was estimated from a database of approximately 560 foods that lists AGE values (16) and was expressed as AGE kilounits per 100-gram food. Measurement of AGEs Serum CML and MG (sCML and sMG) were determined by well-validated competitive ELISAs (OxiSelect Methylglyoxal Competitive Elisa Kit and OxiSelect Nε-Carboxymethyllysine Competitive Elisa Kit [Cell Biolabs, San Diego], respectively), which are based on noncross-reactive monoclonal antibodies (mabs) for protein-bound CML (4G9 mab) and protein-bound MG derivatives. The immunogens used to generate the mabs (ie, lysine-MG-H1 [3D11 mab] and protein-bound CML) were characterized by high-performance liquid chromatography (29). The resulting values reflect relatively stable protein- or peptide-associated CML and MG and not the free compounds. The interassay coefficients of variation were 2.8% and 5.2% for CML and MG, respectively; the intra-assay coefficients of variation were 2.6% and 4.1% for CML and MG, respectively (13). Isolation of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were isolated from 20 mL of venous blood, at fasting and postprandial states, in tubes containing 1 mg/mL of ethylenediaminetetraacetic acid. The blood samples were diluted 1:1 in phosphate-buffered saline, and cells were separated in Ficoll gradient (Ficoll-Paque PLUS; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) by centrifugation at 800g for 25 minutes at 20°C. The cells were collected and washed with cold phosphate-buffered saline two times and finally resuspended in a buffer. The buffer contained 10 mM HEPES, 15 mM KCl, 2 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid (at the time of use, 1 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol were added; Sigma–Aldrich, St. Louis, MO). The cells thus obtained were stored at −80°C for further analysis. RNA extraction Total RNA from PBMCs was extracted using the trizol method according to the recommendations of the manufacturer (Tri Reagent; Sigma–Aldrich) and quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology, Cambridge, UK). RNA integrity was verified on agarose gel electrophoresis and stored at −80°C. Samples were digested with DNAse I (AMPD-1 KT; Sigma–Aldrich) before real-time PCR. Quantitative Real-Time PCR for Gene Expression Analysis Real-time PCR was carried out using the OpenArray NT Cycler system (Applied Biosystems, Carlsbad, CA), following the manufacturer’s instructions. Each reaction was performed with 1 μL of a 1:5 (v/v) dilution of the first cDNA synthesized from 1 μg 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 20 participants at fasting and at 4 hours after intake of the fat overload. Primer pairs were selected from the database TaqMan Gene Expression assays (Applied Biosystems; https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601267), for the following genes: AGE receptor-1 (AGER1), receptor for AGEs (RAGE), glyoxalase I (GloxI), and estrogen receptor α (ERα). 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 OpenArray Real-Time qPCR Analysis Software (Applied Biosystems). Statistical Analysis PASW Statistics, Version a8, SPSS statistical software (Chicago, IL) was used for statistical analysis. The Kolmogorov–Smirnov test did not show a significant departure from normality in the distribution of variance values. In order to evaluate data variation, Student’s t test and analysis of variance for repeated measures was performed, followed by Bonferroni’s correction for multiple comparisons, depending on the existence of two or more groups in each comparison. We studied the statistical effects of the type of fat meal ingested, independent of time (represented by p1), the effect of time (represented by p2), and the interaction of both factors (represented by p3). Pearson’s correlation analyses were performed to examine the correlations between AGER1 and RAGE mRNA levels and sCML and sMG and other parameters related to inflammation and redox state described in our previous reports (nuclear factor [erythroid-derived 2]-like 2 [Nrf2], thioredoxin [Trx], NADPH oxidase subunits [p40phox and p67phox], interleukin-8 [IL-8] mRNA levels, and plasmatic nitric oxide, 8-isoprostanes, and protein carbonyls levels) (24,26). Differences were considered to be significant when p < .05. All data presented in text and tables are expressed as means ± SE. Results Baseline Characteristics of the Study Population All participants completed the three dietary intervention periods. Men had higher height, waist circumference, triglyceride, and Apo B than women. There were no other differences by gender (Supplementary Table 2). dAGEs Consumed in Each Dietary Intervention Med diet (with or without CoQ supplementation) and SFA diet differed in quality fat composition, and this was mirrored by differences in dAGEs levels (Figure 1A and B). In both the fasting and postprandial phase after SFA diet, participants consumed a higher amount of dAGEs compared with Med or Med + CoQ diets (all p < .05; Figure 1A and B). There were no differences in the amount of food-related AGEs between Med and Med + CoQ diets (Figure 1A). Figure 1. View largeDownload slide dAGEs levels consumed after 4 weeks of each dietary intervention (A) and after 4-hour postprandial state (B). Fasting and postprandial levels of MG (C) and CML (D) in serum samples according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGEs = advanced glycation end products; ANOVA = analysis of variance; CML = N-carboxymethyllysine; CoQ = coenzyme Q10; dAGEs = dietary advanced glycation end products; Med = Mediterranean; MG = methylglyoxal; SFA = saturated fat. Figure 1. View largeDownload slide dAGEs levels consumed after 4 weeks of each dietary intervention (A) and after 4-hour postprandial state (B). Fasting and postprandial levels of MG (C) and CML (D) in serum samples according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGEs = advanced glycation end products; ANOVA = analysis of variance; CML = N-carboxymethyllysine; CoQ = coenzyme Q10; dAGEs = dietary advanced glycation end products; Med = Mediterranean; MG = methylglyoxal; SFA = saturated fat. Plasma CoQ and Serum AGEs Levels After Each Dietary Intervention Fasting and postprandial plasma CoQ levels were higher after Med + CoQ diet compared with the other diets (all p < .05). Moreover, Med + CoQ diet produced an increase in plasma CoQ levels during the postprandial phase compared with fasting state (p = .019; Supplementary Figure 1). Conversely, fasting sMG and sCML levels were higher after SFA diet compared with both Med and Med + CoQ diets (all p < .05; Figure 1C and D). Furthermore, Med + CoQ diet produced lower postprandial sMG and sCML levels compared with Med diet (p = .013 and .019, respectively). We also observed lower postprandial levels of both sAGEs forms after Med diet compared with SFA diet (p = .005 and .009, respectively; Figure 1C and D). Interestingly, sMG levels decreased significantly during the postprandial phase when compared with the fasting state in the Med + CoQ diet group (p = .023) but were unchanged in the Med group and were even increased in SFA group (Figure 1C). Dietary Intake and Expression of Genes Related to Receptors for AGEs in PBMCs The antioxidant properties of AGER1 are due to the fact that it reduces AGE levels in the intracellular and extracellular spaces (13,14). Conversely, cell surface RAGE binds AGEs and induces the generation of ROS and promotes an inflammatory response (30). Med and Med + CoQ diets produced higher fasting and postprandial AGER1 mRNA levels and lower fasting and postprandial RAGE mRNA levels compared with SFA diet. However, we observed higher postprandial AGER1 mRNA levels after Med + CoQ diet compared with Med diet (all p < .05; Figure 2A and B). We did not find differences on fasting and postprandial RAGE mRNA levels between Med + CoQ and Med diets (Figure 2B). Conversely, Med and Med + CoQ diets produced an increase in AGER1 mRNA levels, during the postprandial phase compared with the fasting state (p = .025 and .006, respectively), whereas consumption of the SFA diet resulted in a decrease of postprandial AGER1 mRNA levels in comparison with the fasting state (Figure 2A). Figure 2. View largeDownload slide Fasting and postprandial levels of AGER1 mRNA (A), RAGE mRNA (B), GloxI mRNA (C), and ERα mRNA (D) in PBMCs according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGER1 = advanced glycation end product receptor-1; ANOVA = analysis of variance; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs. Figure 2. View largeDownload slide Fasting and postprandial levels of AGER1 mRNA (A), RAGE mRNA (B), GloxI mRNA (C), and ERα mRNA (D) in PBMCs according to the different diets consumed. Data were analyzed using ANOVA for repeated measures. All values represent the mean ± SE. Bars with different superscript letters depict statistically significantly differences. *Differences between fasting and postprandial phase (p < .05). p1: diet effect, p2: time effect, p3: diet × time interaction. AGER1 = advanced glycation end product receptor-1; ANOVA = analysis of variance; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs. Dietary Intake and Expression of Genes Related to AGE Metabolism and Oxidative Stress in PBMCs GloxI detoxifies reactive α-oxoaldehyde, thereby removing AGEs, particularly MG (31). In addition, AGEs have been implicated in the reduction in number and function of estrogen receptors (ERs) (32). Med and Med + CoQ diets produced higher fasting and postprandial GloxI mRNA levels and higher postprandial ERα mRNA levels compared with SFA diet (all p < .05; Figure 2C and D). There were no differences in fasting ERα mRNA levels among the three diets. Conversely, Med + CoQ diet produced higher postprandial GloxI mRNA levels compared with Med diet (p = .018; Figure 2C). Conversely, Med diet produced an increase in ERα mRNA levels, and both Med and Med + CoQ diets produced an increase in GloxI mRNA levels, during the postprandial phase compared with fasting state (all p < .05) but not after SFA diet (Figure 2C and D). Relationship Between AGEs and Inflammatory and Redox-State-Related Parameters Figures 3 and 4 show the correlation results for the relationship between AGER1 and RAGE mRNA levels and parameters related to redox state and inflammation. A univariate analysis showed a moderate positive correlation between AGER1 mRNA levels and Nrf2 mRNA levels (r = .396; p = .018), nitric oxide levels (r = .401; p = .010) and Trx mRNA levels (r = .444; p = .002), a moderate negative correlation with protein carbonyl levels (r = −.305; p = .032), and a weak negative correlation with 8-isoprostanes levels (r = −.299; p = .024). As anticipated, RAGE mRNA levels showed a strong positive correlation with IL-8 mRNA levels (r = .511; p = .003) and 8-isoprostanes levels (r = .613; p = .011), and a moderate positive correlation with sMG content (r = .301; p = .007) and gene expression of a subunit of NADPH oxidase such as p67phox mRNA levels (r = .361; p = .001). Figure 3. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and expression of genes related to oxidative stress/inflammation, in PBMCs. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; p67phox = subunit of NADPH oxidase; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs; Trx = thioredoxin. Figure 3. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and expression of genes related to oxidative stress/inflammation, in PBMCs. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; p67phox = subunit of NADPH oxidase; PBMCs = peripheral blood mononuclear cells; RAGE = receptor for AGEs; Trx = thioredoxin. Figure 4. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and plasma and serum oxidative stress/inflammation-related variables. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; RAGE = receptor for AGEs; sMG = serum methylglyoxal. Figure 4. View largeDownload slide Correlation analysis between AGER1 and RAGE mRNA levels and plasma and serum oxidative stress/inflammation-related variables. r (correlation coefficient) has been determined by Pearson’s method; p < .05 is considered significant. AGER1 = advanced glycation end product receptor-1; RAGE = receptor for AGEs; sMG = serum methylglyoxal. Discussion Recent knowledge points toward high levels of ROS and oxidative stress as the primary determinants of aging, together with low levels of CoQ present during the aging process, explain the fact of selecting an elderly population in our study. Conversely, current dietary habits would predict that modern-day humans spend most of the time in the postprandial state (18), which is known to be a stressful condition because this is the time in which an oxidative stress situation normally occurs (19). Therefore, the postprandial phase is probably one of the most essential periods to reveal the healthy or detrimental effects of dietary interventions by studying variations in oxidative stress. In this sense, our results corroborated the antioxidant and anti-inflammatory effects of the consumption of a Med diet, both in fasting and postprandial states (20). Moreover, the addition of the CoQ to a Med-style diet provides an additional beneficial effect of reducing the oxidative and inflammatory state, particularly in the postprandial phase (24,26). Here, we have shown for the first time that CoQ supplementation resulted in a greater decrease in endogenous AGEs levels, sMG and sCML, during postprandial state, which is likely a consequence of an increase in gene expression related with AGEs metabolism, AGER1 and GloxI. Although other clinical studies have been focused on the antioxidant effects of CoQ, the current study is the first cross-randomized controlled dietary trial that analyzes the effect of supplementation with CoQ on AGEs during the postprandial phase in an aged population. Another novel feature of this study is the correlations between the diets and the metabolism of AGEs. Several studies have shown that a low-AGE diet reduced circulating AGE levels and chronic inflammation/oxidative stress in patients with age-related diseases (4,14). The current study extends these previous data and shows that consumption of Med diet, with or without supplementation with CoQ, provided lower dAGEs content compared with SFA diet. Med diet components (MUFA, α-tocopherol, phenolic compounds, or phytoesterols) could provide the antioxidant capacity of this diet, suggesting its direct association with a decrease of oxidative stress (33). Previous studies showed that sAGEs levels are directly related to AGEs consumed via the diet (34), a finding that is corroborated by the current study. In contrast, consumption of SFA diet, with higher content in dAGEs, was associated with increased sMG and sCML levels. Dietary intervention studies demonstrated that SFA-rich diets contain elevated quantities of AGEs with adverse consequences on health (4,35). In response to high AGEs levels, the host defense system employs different mechanisms to restrict their toxicity. The glyoxalase system (mainly GloxI and GloxII) (11) and AGER1/RAGE receptors have been shown to be a part of the innate and adaptive defense that serve to respond to AGEs. GloxI is an important regulator of the control of intracellular AGEs because it binds and detoxifies AGEs formed during normal metabolism. AGER1 binds extracellular AGEs and delivers them to the lysosomes where they are detoxified (30), thus reducing AGE-related ROS and inflammation (13,36). Another means of reducing extracellular AGEs levels is provided by circulating RAGE because it binds AGEs. However, after this binding, RAGE induces oxidative stress/inflammation, and thus, it can be considered as a deleterious response (37). These defense mechanisms are reduced in aging and other age-related diseases, resulting in suppression of the antioxidant defense system and increased levels of pro-oxidant mechanisms, which results in a further increase in AGEs levels, ROS, and finally leads to tissue injury (29). The current study supports these findings and other previous studies showing that sAGEs serve as stimuli for changes in gene expression. Namely, higher AGER1 and GloxI mRNA levels and the lower RAGE mRNA levels observed in participants with the Med diet intake could also explain the reduction in sMG and sCML levels compared with the SFA diet. Although both Med and Med + CoQ diets contained similar levels of dAGEs, supplementation of a Med diet with CoQ led to further reductions in the postprandial levels of sMG and sCML accompanied by increased AGER1 and GloxI mRNA levels. Besides being part of the mammalian mitochondrial electron transport chain, CoQ is related with protection and elimination of oxidative damage due its antioxidant properties in all cell membranes preventing lipid peroxidation and participating in the regeneration of other antioxidants, such as α-tocopherol (38). Although the reduced form of this molecule, ubiquinol, is the one that acts as a potent lipid-soluble antioxidant scavenging free-radical species and regenerating the active form of other antioxidants (21), our study showed a high protection of the redox state when a Med diet was supplemented with ubiquinone, the oxidized form. In accordance, efficient mechanisms for reduction of CoQ seem to be present in the blood, and the majority of this compound is in the reduced state even after a fourfold elevation of concentration on dietary supplementation with its oxidized form (39). CoQ deficiency results in an impairment of the assembly and/or stability of the respiratory chain enzymes, which leads to imbalanced oxidative phosphorylation and enhanced ROS production (40), and a reduced cell capacity against oxidative damage (38). Therefore, the addition of CoQ to the diet could be expected to enhance respiratory chain activity, reduce ROS production, and protect from protein oxidation and lipid peroxidation (41), outcomes that could contribute to a reduction in the formation of endogenous AGEs. This ultimately might lead to preservation of anti-AGE defenses (GloxI and AGER1). Although estrogen levels are decreased in aging, they remain sufficient to stimulate ERs, suggesting that the insensitivity to estrogen in the postmenopausal period is due to the decrease in number and function of ERs (32). Several studies have implicated AGEs in ERα inhibition (6,42). ERα mRNA levels were increased in those participants consuming the Med and Med + CoQ diets. Moreover, Med and Med + CoQ diets contained the lowest levels of AGEs and contributed to a reduction of sAGEs levels, which may have led to the increased postprandial ERα mRNA levels in these participants. The oxidant and inflammatory action of circulating AGEs is normally controlled by AGER1, considered as the main host defense against glycoxidants, whereas the AGEs binding to RAGE produces the activation of inflammatory and immune responses, which finally leads to cell damage (29,36). In this way, AGER1 mRNA levels were mainly associated with genes/markers related to antioxidant defenses (a moderate correlation with Nrf2 and Trx mRNA and plasma nitric oxide levels), whereas RAGE mRNA levels showed associations with inflammatory/oxidative markers (a strong correlation with IL-8 mRNA and 8-isoprostanes levels, and a moderate correlation with sMG and p67phox mRNA levels, a subunit of NAPDH oxidase enzyme, related to ROS production in the cell). The present study has the advantage of a randomized crossover design in which all the participants have experienced the three diet periods, each individual acting as his/her own control and strengthening the fact that the effects observed are indeed due to the influence of the type of diet. Our study has certain limitations because ensuring adherence to dietary instructions is difficult in a feeding trial. However, adherence to the recommended dietary patterns was satisfactory, as can be judged by the measurements of compliance. In conclusion, our results support the protective effect of a Med diet against oxidative stress/inflammation, and this protection is enhanced by supplementing with CoQ, thus reducing postprandial circulating AGEs levels and modulating the expression of genes related to AGEs metabolism in elderly men and women. This fact may be a good starting point for assessing the benefits of both consuming low-AGE diets and the use of CoQ on clinical features associated to age. Supplementary Material Please visit the article online at http://gerontologist.oxfordjournals.org/ to view supplementary material. Funding This work was supported in part by research grants from the Ministerio de Ciencia e Innovación (AGL2004-07907, AGL2006-01979, and AGL2009-12270), Ministerio de Ciencia y Competitividad (AGL2012-39615 to J.L.-M., FIS PI10/01041 to P.P.-M., and FIS PI13/00023 to J.D.-L.), Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P06-CTS-01425 and PI0193/09 to J.L.-M., PI-0252/09 to J.D.-L., and PI-0058/10 to P.P.-M.), Proyecto de Excelencia, Consejería de Economía, Innovación, Ciencia y Empleo (CVI-7450 to J.L.-M.); Merck Serono and Fundacion 2000 (Clinical Research in Cardiometabolic to P.P.-M.). Conflict of Interest The authors have no conflicts of interest to disclose. Acknowledgments The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain. 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The Journals of Gerontology Series A: Biomedical Sciences and Medical SciencesOxford University Press

Published: Mar 1, 2018

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