Abstract

We have investigated whether the quality of dietary fat and supplementation with coenzyme Q10 (CoQ) modifies expression of genes related with inflammatory response and endoplasmic reticulum stress in elderly persons. Twenty participants received three diets for 4 weeks each: Mediterranean diet + CoQ (Med + CoQ), Mediterranean diet (Med), and saturated fatty acid–rich diet (SFA). After 12-hour fast, volunteers consumed a breakfast with a fat composition similar to that consumed in each of the diets. Med and Med + CoQ diets produced a lower fasting calreticulin, IL-1b, and JNK-1 gene expression; a lower postprandial p65, IKK-b, MMP-9, IL-1b, JNK-1, sXBP-1, and BiP/Grp78 gene expression; and a higher postprandial IkB-a gene expression compared with the SFA diet. Med + CoQ diet produced a lower postprandial decrease p65 and IKK-b gene expression compared with the other diets. Our results support the anti-inflammatory effect of Med diet and that exogenous CoQ supplementation in synergy with a Med diet modulates the inflammatory response and endoplasmic reticulum stress.

AGING is a biological process characterized by time-dependent, progressive physiological declines accompanied by the increased incidence of age-related diseases (1). A growing body of evidence points toward the oxidative damage caused by reactive oxygen species (ROS) as one of the primary determinant of aging (2). However, recent scientific studies have advanced the notion of chronic inflammation as other risk factor underlying aging and age-related diseases as neurodegenerative disorders, type 2 diabetes, atherosclerosis, and cardiovascular diseases (3–5). Addressing the central mechanisms underlying these pathologies will have implications for aging and should lead to new therapeutic approaches for treating these conditions (6).

One potential emerging mechanism involves the endoplasmic reticulum (ER), the organelle responsible for protein folding, maturation, quality control, and trafficking. When the ER becomes stressed due to the accumulation of newly synthesized unfolded proteins, the unfolded protein response is activated. A close examination of ER stress and unfolded protein response pathways has demonstrated many links to major inflammatory and stress signaling networks, including the activation of the JNK-AP1 and NF-κB-IKK pathways (7,8), as well as production of ROS and nitric oxide (9,10).

Nutritional intervention may influence the intrinsic rate of aging as well as the incidence of these age-associated diseases. Increasing evidence suggests that the quality of diet may also be important in modulating inflammation (11,12). Thus, consumption of a meal high in carbohydrates and fat results in multiple metabolic changes including oxidative and inflammatory stress and increase in insulin resistance, transient endothelial dysfunction, and platelet activation with impaired homeostasis. However, extensive scientific evidence shows that the Mediterranean diet (Med diet) prevents the onset and progression of coronary heart disease (13), metabolic disorders, and other aging-related diseases (14,15) and has beneficial effects on selected cancers that are potentially more diet related (16,17). Common components of this diet include monounsaturated fatty acids (MUFA), α-tocopherol, phenolic compounds, phytoesterols, and other antioxidants so the leading hypothesis on the mechanism of this association is a decrease of oxidative stress due to the antioxidant capacity of this diet (16,18).

On the other hand, fasting is not the typical physiological state of the modern human being, which spends most the time in the postprandial state. For all these reasons, it is essential to know what changes are produced during the postprandial phase that is influenced by the quantity and quality of the fat ingested. With regard to the postprandial state, we have recently demonstrated the antioxidant effect of Med diet rich in olive oil and that exogenous coenzyme Q10 (CoQ: 2,3-dimethoxy-5-methy-6-decaprenyl-1,4-benzoquinone) supplementation in synergy with a Med diet (Med + CoQ diet) has an additive effect, improving the postprandial oxidative stress in elderly men and women, with a higher increase in capillary flow, a lower plasma biomarker of oxidative stress levels, and a greater postprandial decrease in plasma antioxidant enzymatic activities with respect to a Western diet rich in saturated fat (SFA diet (19)).

According to these premises, the aim of this study was to determine whether diets with different fat quality influence on the postprandial expression of proinflammatory genes and genes related with ER stress and that this hypothetical improvement could be boosted by supplementation with a natural antioxidant, like CoQ, to a Mediterranean diet in peripheral blood mononuclear cells (PBMCs) of aged persons.

MATERIALS AND METHODS

Participants and Recruitment

Volunteers were recruited using various methods including the use of general practitioner databases and poster and newspaper advertisements. A total of 63 persons were contacted among those willing to enter the study. All participants underwent a comprehensive medical history, physical examination, and clinical chemistry analysis before enrollment and gave their informed consent before joining the study. Inclusion and exclusion criteria were fulfilled by 20 patients (age ≥ 65 years; 10 men and 10 women). Clinical inclusion criteria were age 65 years or older, body mass index 20–40 kg/m2, total cholesterol concentration less than or equal to 8.0 mmol/L, and nonsmokers. Clinical exclusion criteria were age less than 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 greater than or equal to 3 kg within the last 3 months, smokers, and alcohol or drug abuse (based on clinical judgment). 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.

Study Design

Participants were randomly assigned to receive, in a crossover design, three isocaloric diets for 4-week periods each. Three dietary periods were administered continuously (see Supplementary Figure 1). The three diets were as follows: (1) Mediterranean diet supplemented with coenzyme Q (Med + CoQ diet; 200 mg/day 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 [saturated fatty acid], and 4% PUFA [polyunsaturated fatty acid]); (2) Mediterranean diet not supplemented with CoQ (Med diet), with the same composition of the first diet, but supplemented by placebo capsules; and (3) 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, and 4% PUFA).

The cholesterol intake was kept constant (<300 mg/day) 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 aspect. Patients taking capsules were unaware whether they were in the Med + CoQ or Med dietary period (see Supplementary Table 1). The composition of the experimental diets was calculated by using the U.S. Department of Agriculture (20) food tables and Spanish food composition tables for local foodstuffs (21).

Before the start of the intervention period, volunteers completed a 3-day weighed food diary and an extensive Food Frequency Questionnaires, which allowed identification of foods to be modified. At the start of the intervention period, each patient was provided with a handbook for the diet to which they had been randomized. Advice was given on foods to choose and those to avoid if eating outside home. They were also instructed to write down in the diary about any menu eaten out of the home and to call the monitoring study nurse reporting such event. At baseline, volunteers were provided with a supply of study foods to last for 2 weeks. They collected additional study foods every fortnight or when required. At these times, a 24-hour recall of the previous day’s food intake and a short food-use questionnaire based on the study foods were completed to monitor and motivate volunteers to adhere to the dietary advice. A point system was used to assess the number of food exchanges achieved in the 24-hour recall, and additional advice was given if either the 24-hour recall or food-use questionnaire showed inadequate intake of food exchange options. Volunteers were asked to complete 3-day weighed food diaries at baseline, Weeks 2, and 4. Weighed food intake over two weekdays and one weekend day was obtained using scales provided by the investigators. Fat foods were administered by dietitians in the intervention study. The dietary analysis software Dietsource version 2.0 was used. (Novartis S.A., Barcelona, Spain).

At the end of the dietary intervention period, the participants were given a fatty breakfast with the same fat composition as consumed in each of the diets. Patients presented at the clinical centers at 8 hours following a 12-hour fast (Time 0), abstained from alcohol intake during the preceding 7 days. After cannulation of a blood vessel, a fasting blood sample was taken before the test meal, which was then ingested within 20 minutes under supervision. The test meal reflected fatty acid composition of each subject after the chronic dietary intervention. Subsequent blood samples were drawn at 2 and 4 hours. Test meals provided an equal amount of fat (0.7 g/kg 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 follow: Med with CoQ (400 mg in capsules) breakfast (12% SFA, 43% MUFA, and 10% PUFA), Med with placebo capsules breakfast (12% SFA, 43% MUFA, and 10% PUFA), and SFA-rich breakfast (38% SFA, 21% MUFA, and 6% PUFA).

Biochemical Determinations

Plasma samples.—

Venous blood samples were obtained at the end of the each dietary intervention period on fasting state, after a 12-hour fast, before to breakfast ingest, and at 2 and 4 hours after the ingestion of the breakfast. Samples from the fasting and postprandial states were collected in tubes containing 1 g EDTA/L and were stored in containers with ice and kept in the dark. Particular care was taken to avoid exposure to air, light, and ambient temperature. Plasma was separated from whole blood by low-speed centrifugation at 1,500g for 15 minutes at 4°C within 1 hour of extraction.

Isolation of PBMCs.—

PBMCs were isolated from 20 mL of venous blood in tubes containing 1 mg/mL of EDTA. The blood samples were diluted 1:1 in phosphate-buffered saline, and cells were separated in Ficoll gradient 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 Buffer A. This buffer contained 10 mM HEPES, 15 mM KCl, 2 mM MgCl2, and 1 mM EDTA, and at the time of use, 1 mM phenylmethylsulfonyl fluoride and 1 mM Dithiothreitol were added. The cells thus obtained were stored at −80°C for further analysis.

RNA Extraction and Quantitative Real Time-Polymerase Chain Reaction Analysis

RNA extraction.—

Total RNA from PBMCs was extracted using the Trizol method according to the recommendations of the manufacturer (Tri Reagent; Sigma, St Louis, MO) and quantified in a NanoDrop 1000A Spectrophotometer. RNA integrity was verified on agarose gel electrophoresis and stored at −80°C. Next, because polymerase chain reaction can detect even a single molecule of DNA, RNA samples were digested with DNAse I (AMPD-1 KT; Sigma) before RT-PCR.

Biotrove OpenArray real-time PCR.—

Each reaction was performed with 1 μL of a 1:5 (v/v) dilution of the first complementary DNA strand, synthesized from 1 μg of total RNA using the commercial kit iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s instruction.

The reaction of real-time polymerase chain reaction was carried out using the platform OpenArray NT Cycler (Applied Biosystems employing Taqman probes). OpenArray subarrays were preloaded by Biotrove with the selected primer pairs. The individual primer pairs (synthesized by Sigma-Aldrich, St Louis, MO) were preloaded into BioTrove OpenArray plates. Each primer pair was spotted in duplicate. The primers that amplify genes of interest were selected from the database TaqMan Gene Expression Assays (Applied Biosystems), https://products.appliedbiosystems.com/ab/en/US/adirect/ab? Cmd = catNavigate2 & catID = 601267 in Assays search tab taking as search criteria: selection and homo sapiens Gene Expression Assays for each of the genes of interest.

Samples were loaded into OpenArray plates with the OpenArray NT Autoloader according to the manufacturer's protocols. Each subarray was loaded with 5.0 μL of master mix consisted of 1× LighCycler FastStart DNA Master SYBR Green Kit (Roche Applied Sciences, Indianapolis, IN), 1× SYBR Green I 80×, 0.5% glycerol, 0.2% Pluronic F-68, 1 mg/mL bovine serum albumin (New England Biolaboratories, Beverly, MA), 1 mM MgCl2, 400 nM FP, 400 nM RP, 8% Formamide, 0.25× Rox, 1× TfR amplicon, and complementary DNA samples. The PCR OpenArray thermal cycling protocol consisted of 95°C for 10 minutes, followed by cycles of 10 seconds at 95°C, 10 seconds at 53°C, and 10 seconds at 72°C. All samples were tested in duplicate. The Biotrove OpenArray NT Cycler System Software (version 1.0.2) uses a proprietary calling algorithm that estimates the quality of each individual CT value by calculating a CT confidence value for the amplification reaction.

In our assay, CT values with CT confidence values below 700 were regarded as background signals. The remaining positive amplification reactions were analyzed for amplicon specificity by studying the individual melting curves.

The same program allowed the selection of the most stable housekeeping gene in the samples processed for the relativization of the expression of genes of interest. Following this methodology, we analyzed the relative gene expression of these genes: p65 (RelA), IkB-α (inhibitor of kB-subunit α), IKK-β (IkB kinase-subunit β), MMP-9 (metalloproteinase-9), IL-1β (interleukin-1β), JNK-1 (c-Jun N-terminal kinase-1), sXBP-1 (x-box–binding protein-1), CRT (calreticulin), and BiP/Grp78 (glucose-regulated protein 78 kDa).

Statistical Analysis

The Statistical Package for the Social Sciences (SPSS 17.0 for Windows Inc., Chicago, IL) was used for the statistical comparisons. 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 an analysis of variance for repeated measures were performed, followed by Bonferroni’s correction for multiple comparisons. 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, indicative of the degree of the postprandial response in each group of participants with each fat meal (represented by p3). Differences were considered to be significant when p < .05. All data presented in text and tables are expressed as means ± SE.

RESULTS

Metabolic Parameters Levels

The baseline characteristics of the 20 participants who completed the three dietary intervention periods showed that males had higher height, waist circumference, triglycerides, and apolipoprotein B than females. We did not find any other differences by gender (see Supplementary Table 2).

Moreover, we previously observed higher fasting plasma CoQ concentration (p < .001) after the intake of the Med + CoQ diet compared with the Med and SFA diets. At 2 and 4 hours after consumption of the Med + CoQ diet, we observed a greater postprandial increase in plasma CoQ levels compared with the Med and SFA diets (p = .018 and p = .032, respectively; see Supplementary Figure 2 (19)).

Diet Intake and Genes Related With Activation of Nuclear Factor Kappa B in PBMCs

The nuclear factor kappa B (NF-κB) transcription factor functions as homo- or heterodimers of the Rel family of proteins, which includes p50, p65, c-Rel, p52, and RelB. The most common combination of subunits is a heterodimer of the p50 and p65 proteins. Activation of NF-κB dimers is the result of IKK-mediated phosphorylation-induced degradation of the IkB, which enables the NF-κB dimers to enter the nucleus and activate specific target gene expression (22).

At 2 and 4 hours after intake of the Med + CoQ diet, we found lower postprandial p65 (RelA) messenger RNA (mRNA) levels compared with the other diets (p = .008 and p = .012, respectively; Figure 1A). Furthermore, p65 (RelA) mRNA levels were lower after consumption of the Med diet compared with the SFA diet (p = .033).

Figure 1.

Fasting and postprandial levels of p65 (RelA) mRNA (A), IKK-β mRNA (B), and IkB-α mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

Figure 1.

Fasting and postprandial levels of p65 (RelA) mRNA (A), IKK-β mRNA (B), and IkB-α mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

At 2 hours after intake of the Med + CoQ diet, we observed a greater postprandial decrease in IKK-β mRNA levels compared with the other diets (p = .010; Figure 1B). Furthermore, IKK-β mRNA levels were lower after consumption of the Med diet compared with the SFA diet (p = .034). At 4 hours after intake of the Med and Med + CoQ diets, we observed a greater postprandial decrease in IKK-β mRNA levels with respect to the SFA diet (p = .011).

At 2 hours after intake of the Med and Med + CoQ diets, we observed a greater postprandial increase in IkB-α mRNA levels compared with the SFA diet (p = .028). At 4 hours after the Med diet, we found higher postprandial IkB-α mRNA levels compared with the SFA diet (p = .018; Figure 1C).

No significant differences were detected in p65 (RelA), IKK-β, and IkB-α mRNA levels in fasting after intake of any of the three diets (Figure 1A, B, and C, respectively).

Diet Intake and Genes Related With the Inflammatory Response in PBMCs

MMP-9 is a metalloproteinase, which is involved in several stages of atherosclerosis through remodeling of the extracellular matrix (23). We observed a decrease in fasting MMP-9 mRNA levels after intake of the Med diet compared with the SFA diet (p = .034; Figure 2A). At 2 and 4 hours after the SFA diet, we found higher postprandial MMP-9 mRNA levels compared with the Med and Med + CoQ diets (p = .008 and p = .032, respectively; Figure 2A).

Figure 2.

Fasting and postprandial levels of MMP-9 mRNA (A), IL1-β mRNA (B), and JNK-1 mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

Figure 2.

Fasting and postprandial levels of MMP-9 mRNA (A), IL1-β mRNA (B), and JNK-1 mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

IL-1 is an inflammatory cytokine that consists of two distinct ligands (IL-1α and IL-1β), and both subunits appear to play an essential role in many inflammatory response (24).

Fasting IL-1β mRNA levels were lower after participants consumed the Med and Med + CoQ diets than when they consumed the SFA diet (p = .017; Figure 2B). At 2 hours after intake of the Med + CoQ diet, we observed a greater postprandial decrease in IL-1β mRNA levels compared with the other diets (p = .011; Figure 1B). Furthermore, IL-1β mRNA levels were lower after consumption of the Med diet compared with the SFA diet (p = .029). At 4 hours after intake of the Med and Med + CoQ diets, we observed a greater postprandial decrease in IL-1β mRNA levels with respect to the SFA diet (p = .015; Figure 2B).

JNK is involved in inflammatory signals, changes in levels of ROS, and a variety of stress stimuli and consist of 10 isoforms derived from three genes: JNK-1 (4 isoforms), JNK-2 (4 isoforms), and JNK-3 (2 isoforms (25)).

Fasting JNK-1 mRNA levels were lower after participants consumed the Med and Med + CoQ diets than when they consumed the SFA diet (p = .037; Figure 2C). At 2 and 4 hours after the SFA diet, we found higher postprandial JNK-1 mRNA levels compared with the other diets (p = .009 and p = .011, respectively; Figure 2C).

Diet Intake and Genes Related With ER Stress in PBMCs

The transcription factor sXBP-1 has been identified as a key regulator of the mammalian unfolded protein response or ER stress response, which is activated by environmental stressors such as protein overload that require increased ER capacity (26). CRT, BiP/Grp78, calnexin, and other ER Ca2+ binding chaperons and folding enzymes are important component of protein folding and quality control (27).

At 2 and 4 hours after the Med and Med + CoQ diets, we found lower postprandial sXBP-1 mRNA levels compared with the SFA diet (p = .033 and p = .008; Figure 3A). No significant differences were detected in sXBP-1 mRNA levels in fasting after intake of any of the three diets (Figure 3A). In addition, fasting CRT mRNA levels were lower after participants consumed the Med and Med + CoQ diets than when they consumed the SFA diet (p = .031; Figure 3B).

Figure 3.

Fasting and postprandial levels of sXBP-1 mRNA (A), CRT mRNA (B), and BiP/Grp78 mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

Figure 3.

Fasting and postprandial levels of sXBP-1 mRNA (A), CRT mRNA (B), and BiP/Grp78 mRNA (C) in peripheral mononuclear cells according to the type of fat consumed. Data were analyzed using analysis of variance for repeated measures. All values represent the mean ± SEs. Bars with different superscript letters depict statistically significantly differences (p < .05). p1: diet effect, p2: time effect, and p3: Diet × Time interaction.

At 2 hours after intake of the SFA diet, we found a greater postprandial increase in BiP/Grp78 mRNA levels compared with the other diets (p = .021). No significant differences were detected in BiP/Grp78 mRNA levels in fasting and at 4 hours after intake of the three diets (Figure 3C).

DISCUSSION

The present study demonstrates that the consumption of a Med diet reduces the postprandial expression of genes related to both the activation of NF-κB as the inflammatory response such as p65 (RelA) and IKK-β and MMP-9, IL1-β and JNK-1, respectively, and increases the expression of IkB-α mRNA levels in PBMCs. Additionally, the consumption of a Med diet reduces the expression of genes related with ER stress (sXBP-1, CRT, and BiP/Grp78). Moreover, the addition of CoQ had an additive effect on the Med diet because the participants who consumed this diet showed a greater postprandial decrease in gene expression of p65 (RelA), IKK-β, and IL1-β in PBMCs with respect to the other diets.

In the same population of this study, we previously demonstrated that the Med diet improves the postprandial oxidative stress with a higher increase in capillary flow and plasma nitric oxide levels, a lower plasma lipid peroxidation products, nitrotyrosine and protein carbonyl levels, lower plasma antioxidant enzyme activities (GPx, CAT, and SOD (19)), and lower DNA damage in PBMCs (28). Addition of exogenous CoQ in synergy with a Med diet had an additive effect, reducing the postprandial oxidative stress in elderly men and women.

The process of aging has been attributed to cellular free radical damage as well as a decrease in exogenous antioxidants. With the current understanding from human and animal studies, evidence supports that vitamins A and E supplementation may only provide life-span benefits when initiated early in life, and they may accumulate within our body, increasing their risk of toxicity (29–32). In addition, vitamins E and C have in combination shown long-term antiatherogenic effects, but their combined effect on clinical end points has been inconsistent (33).

Thus, interest in CoQ comes from the fact that it has a pivotal role as a redox link between flavoproteins and cytochromes in the mitochondrial respiratory chain (34) where additionally it plays very important antioxidant properties (35). CoQ is the only known bodily-synthesized lipophilic antioxidant, and as an endogenous compound, its toxicity risk may be lesser (36). Not only can reduced CoQ prevent lipid peroxidation chain reaction by itself it can also act by reducing (regenerating) other antioxidants such as α-tocopherol and ascorbate (37,38,39). Previous studies shown that lifelong supplementation of polyunsaturated fatty acid (PUFA) together with dietary CoQ increased life span and may also protect against the deleterious effects of PUFA diet when taken together in rats (29,35). Besides, CoQ can modulate proteins, decrease oxidative stress and cardiovascular risk, as well as control inflammation during aging in rats (40). In healthy humans, plasma oxidative damage may be partially prevented by CoQ supplementation (41), which has been replicated in other populations, like psoriasis (42) or coronary heart disease patients (43). However, whether CoQ added to a Mediterranean has an antioxidant additive effect has not been tested at present.

Accumulating evidence indicates that unresolved, low-grade chronic systemic inflammation plays a significant role in modulating the aging process and age-related diseases, such as metabolic syndrome, atherosclerosis, cancer, and osteoporosis (3–5). In fact, the NF-κB transcription factor can be viewed as the master regulator of the inflammatory process and can be activated by oxidative stimuli. In most cells, NF-κB (p50/p65) is present in an inactive form in the cytoplasm, bound to an inhibitor IkB. Oxidative and other stimuli such as the cytokines (IL-1β, IL-8, and others) result in the phosphorylation of IkB proteins by IKK, ubiquitination, followed by degradation via the 26 S proteasome. This allows active NF-κB to translocate to the nucleus, where it binds to its consensus sequences within the promoter regions of genes, thus activating transcription (44).

Aljada and colleagues (45) have demonstrated that consumption of a hypercaloric breakfast increased the nuclear NF-κB activity, accompanied by a reduction in the cytoplasmic IkB-α expression in healthy participants. Furthermore, our group has already demonstrated that the Med diet decreases NF-κB activation in PBMCs when compared with butter- and walnut-enriched diets or a typical Western diet in healthy young people (46,47).

Consistently, our results shown a greater postprandial decrease in p65 (RelA) and IKK-β mRNA levels after intake of the Med + CoQ diet compared with the SFA diet, with an intermediate effect for the Med diet and greater postprandial increase in IkB-α mRNA levels after intake of the Med and Med + CoQ diets compared with the SFA diet.

The transcription factor NF-κB has been shown to regulate IL-1β, tumor necrosis factor-alpha, interleukin-6 (IL-6), and COX-2 expression (48,49). Thus, we observed, consistently, a greater increase in IL-1β mRNA levels after intake of the SFA diet compared with the Med and Med + CoQ diets.

Another interesting factor is MMP-9, which is involved in several stages of atherosclerosis through remodeling of the extracellular matrix. The expression of MMP-9 in atherosclerotic plaques coincides with the production of free radicals (50). Moreover, MMP-9 might be a potential mediator of the NADPH oxidase–dependent ROS production in the atherosclerotic process, and previous studies suggested that NADPH oxidase activity increase significantly after consumption of a hypercaloric breakfast (45). In our study, we observed a reduction in MMP-9 mRNA levels after the Med and Med + CoQ diets compared with the SFA diet, which indicate that this diet may influence not only the rate of atherosclerosis development but also promote the stability of the plaque (23). The mechanism responsible for rises in the levels of MMP-9 with SFA diet could be the increase in ROS production, inducing the activation of the inflammatory response when this type of fat is consumed.

Activation of stress-kinase signaling has recently been recognized as an important pathophysiological mechanism in the development of diet-induced obesity, type 2 diabetes mellitus, and other aging-related pathologies. Hotamisligil and colleagues (51) revealed that mice deficient for the stress mediator JNK-1 are protected from the development of high-fat diet-induced obesity and glucose intolerance, as well as insulin resistance. The family of JNK kinases can not only be activated by cytokines but also by ER stress and hyperlipidemia (52). In our study, Med and Med + CoQ diets were associated with a lower expression of the JNK-1 and genes related with ER stress as sXBP-1, CRT, and BiP/Grp78, which act to increase ROS levels that may elicit inflammatory responses, thereby providing yet another potential link between ER stress and inflammation (6).

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 or her own control and strengthening the fact that the effects observed are due to the influence of the type of diet. We acknowledge that 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 show that the anti-inflammatory effect of Med diet rich in olive oil and that exogenous CoQ supplementation, in synergy with a Med diet, has an additive effect modulating the inflammatory response and ER stress in elderly men and women and support that the consumption of a Med diet supplemented with CoQ is beneficial for healthy aging of individuals. We can conclude that specific dietary intervention might be a new, interesting, and promising challenge in the treatment (and mainly prevention) of processes that lead to a rise in chronic inflammation and oxidative stress, such as cardiovascular, neurodegenerative diseases, and aging.

FUNDING

This study was supported in part by research grants from the Ministerio de Ciencia e Innovación (AGL 2004-07907, AGL2006-01979, and AGL2009-12270 to J.L-M. and FIS PI10/01041 to P.P-M.), Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P06-CTS-01425 to J.L-M. and CTS5015 to F.P-J.), Consejería de Salud, Junta de Andalucía (06/128, 07/43, PI0193/2009 to J.L-M., 06/129 to F.P-J., PI-0252/09 to J.D-L., and PI-0058/10 to P.P-M.), and Kaneka Corporation (Japan) by the production of CoQ and placebo capsules. The CIBEROBN is an initiative of the Instituto de Salud Carlos III, Madrid, Spain.

SUPPLEMENTARY MATERIAL

Supplementary material can be found at: http://biomedgerontology.oxfordjournals.org/

J.L-M. and F.P-J designed research and conducted research; J.D-L., P.P-M., F.P-J., and J.L-M. provided materials or participants; J.D-L., N.D-C., L.G-G., O.R-Z., F.M.G-M., C.C-T., and E.M.Y-S collected and assembled the data; E.M.Y-S., L.G-G., F.M.G-M., and J.M.V. analyzed the data; E.M.Y-S. wrote the paper; J.M.V., F.M.T., J.D-L., and P.P-M provided significant advice and support in reviewing the drafting of the paper; J.L-M. and F.P-J. had primary responsibility for final content. All authors read an approved the final manuscript.

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Author notes

*
These authors contributed equally to this work
Decision Editor: Rafael de Cabo, PhD