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Increase in intranuclear nuclear factor B and decrease in inhibitor B in mononuclear cells after a mixed meal: evidence for a proinflammatory effect^1,2,3

¹ From the Division of Endocrinology, Diabetes and Metabolism, State University of New York at Buffalo and Kaleida Health, Buffalo, NY.

² Supported by the William G McGowan Charitable Fund, Washington, DC.

³ Address reprint requests to P Dandona, Diabetes–Endocrinology Center of Western New York, 3 Gates Circle, Buffalo, NY 14209. E-mail: pdandona{at}kaleidahealth.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: In view of the stimulatory effect of glucose on reactive oxygen species (ROS) generation, we investigated the possibility that a mixed meal stimulates ROS generation and possibly induces concomitant proinflammatory changes.

Objective: The objective was to determine whether the intake of a 900-kcal mixed meal induces an increase in ROS generation by leukocytes and an inflammatory response at the cellular level.

Design: Nine normal-weight subjects were given a 900-kcal mixed meal, and 8 normal-weight subjects were given 300 mL water after an overnight fast. Blood samples were collected at 0, 1, 2, and 3 h. ROS generation by mononuclear cells and polymorphonuclear leukocytes and the expression of p47^phox subunit were measured. Intranuclear nuclear factor B (NF-B) binding and the expression of inhibitor B (IB), IB kinase (IKK), and IB kinase ß (IKKß) were measured. Plasma concentrations of C-reactive protein (CRP) and soluble intercellular adhesion molecule were also measured.

Results: ROS generation by mononuclear cells and polymorphonuclear leukocytes and p47^phox expression increased significantly. The expression of IKK and IKKß and DNA-binding activity of NF-B increased significantly, whereas IB expression decreased. Plasma CRP concentrations increased. The intake of 300 mL water did not induce a change in any of the above indexes.

Conclusions: These data show that the intake of a mixed meal results in significant inflammatory changes characterized by a decrease in IB and an increase in NF-B binding, plasma CRP, and the expression of IKK, IKKß, and p47^phox subunit. These proinflammatory changes are probably relevant to the state of chronic hypertension and obesity and to its association with atherosclerosis.

Key Words: Nuclear transcription factor B • inhibitor B • mononuclear cells • mixed meal • inflammation • reactive oxygen species

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
We recently showed that glucose (1) and lipid and protein (2) intakes cause an increase in the generation of reactive oxygen species (ROS) by leukocytes and that each of these macronutrients induces a distinct pattern of increase in ROS generation. Thus, glucose induces a peak in ROS generation by both mononuclear cells (MNCs) and polymorphonuclear leukocytes (PMNLs) at 2 h, whereas cream (lipid) produces a peak at 1 h. The peak increase in ROS generation is the greatest with glucose, whereas it is the least with casein (protein). On the other hand, cream intake causes a prolonged increase in lipid peroxidation. Consistent with these observations, we also showed that a 48-h fast results in a marked decrease in ROS generation by leukocytes and oxidative damage of amino acids (3). Thus, we suggest that nutritional intake may be the major modulator of ROS generation. Indeed, we showed recently that the state of obesity reflecting chronic hypernutrition is associated with marked oxidative stress, as reflected in an increase in indexes of lipid peroxidation, protein carbonylation, and oxidative damage of amino acids, which diminishes after dietary restriction over a relatively short period of time, eg, 4 wk (4).

Because our studies to date have not dealt with real food, but instead with pure macronutrients, we have now undertaken our first study with food items. We chose to study the effect of a mixed meal composed of carbohydrates, protein, and fats. Such a mix was previously shown to impair endothelium-mediated vasodilatation in the brachial artery (5, 6). In addition, Ceriello et al (7) showed an increase in oxidative stress and LDL oxidation in diabetes after a meal challenge.

We hypothesized that the intake of a mixed meal causes an increase in oxidative stress and proinflammatory activity in circulating MNCs. In this study we investigated the effect of the intake of this mixed meal on ROS generation by PMNLs and MNCs. Because we previously showed that glucose intake results in an increase in the p47^phox subunit, the key protein of NADPH oxidase in leukocytes, an increase in intranuclear nuclear factor B (NF-B), and a decrease in IB (8), we decided to also investigate the effect of the mixed meal on intranuclear NF-B binding activity and the expression of inhibitor B (IB), IB kinase (IKK), IB kinase ß (IKKß), and p47^phox subunit in MNC. Plasma C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), and -tocopherol were also measured. These indexes would allow us to explore a possible link between nutrition, oxidative stress, and inflammation.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
Nine nondiabetic subjects aged 29–38 y ( ± SEM: 32 ± 3 y) of normal weight [body mass index (in kg/m²): 25.4 ± 2.7; ± SEM] were included in the study. The subjects came to the Clinical Research Center of the Diabetes–Endocrinology Center of Western New York after an overnight fast between 0800 and 0900. A fasting blood sample was obtained, and the subjects were asked to eat a mixed meal containing 910 kcal (egg-muffin and sausage-muffin sandwiches and 2 hash browns, which contained 81 g carbohydrate, 51 g fat, and 32 g protein) over 15 min. Additional blood samples were obtained 1, 2, and 3 h after the meal was eaten. The control subjects (age: range, 26–50 y; ± SD, 35 ± 8 y; body mass index: 24.3 ± 2.0) were 8 volunteers who were given 300 mL water to drink in the fasting state. Blood samples were obtained before and 1, 2, and 3 h after the drink. The protocol was approved by the Institutional Review Board of the State University of New York at Buffalo based at The Millard Fillmore Hospital. All participants gave their written informed consent.

Isolation of PMNLs and MNCs
Blood samples were collected in tubes containing Na-EDTA as an anticoagulant; 3.5 mL of the anticoagulated blood sample was carefully layered over 3.5 mL of the PMNL isolation medium (Robbins Scientific Corp, Sunnyvale, CA). Samples were centrifuged at 450 x g in a swing outrotor for 30 min at 22 °C. At the end of centrifugation, 2 bands separate out at the top of the red blood cell pellet. The top band consists of MNCs, whereas the bottom band consists of PMNLs. The MNC and PMNL bands were harvested with a Pasteur pipette, repeatedly washed with Hank’s balanced salt solution, and reconstituted to a concentration of 4 x 10⁵ cells/mL in Hank’s balanced salt solution.

NF-B electrophoretic mobility shift assay
DNA-binding protein extracts were prepared from MNCs with the method described by Andrews and Faller (9). Total protein concentrations were determined with the bicinchoninic acid protein assay (Pierce, Rockland, IL). The NF-B gel retardation assay was performed with the use of a NFB binding protein detection kit (Life Technologies, Long Island, NY). Briefly, the double-stranded oligonucleotide containing a tandem repeat of the consensus sequence for the NF-B binding site was radiolabeled with -P³² by T4 kinase. Then, 5 µg of the nuclear extract was mixed with the incubation buffer, and the mixture was preincubated at 4 °C for 15 min. Labeled oligonucleotide (60 000 cpm) was added, and the mixture was incubated at room temperature for 20 min. Samples were then applied to wells of 6% nondenaturing polyacrylamide gel. The gel was dried under vacuum and exposed to X-ray film. Densitometry was performed with the use of Bio-Rad molecular analyst software (Hercules, CA).

p47^phox Subunit, IB, IKK, and IKKß Western blotting
Western blotting was carried out as previously described. Briefly, total protein concentrations were determined with the use of a bicinchoninic acid protein assay (Pierce, Rockland, IL); 20 µg MNC homogenate was electrophoresed on sodium dodecyl sulfate polyacrylamide gels for the p47 ^phox subunit. Forty micrograms of total homogenates was used for IB (Cell Signaling Technology Inc, Beverly, MA), IKK (PharMingen, San Diego), and IKKß (Transduction Labs, Lexington, KY) Western blotting.

Measurement of reactive oxygen species generation
Five hundred microliters of PMNLs or MNCs (2 x 10⁵ cells) were delivered into a Chronolog Lumi-Aggregometer cuvette. Fifteen microliters of 10 mmol luminol/L was then added, followed by 1.0 µL of 10 mmol formylmethionyl leucinyl phenylalanine/L. Chemiluminescence was recorded for 15 min (a protracted record after 15 min did not alter the relative amounts of chemiluminescence produced by various blood samples). Our method, developed independently (10, 11), is similar to that published by Tosi and Hamedani (12). In this assay system, the release of superoxide radical as measured by chemiluminescence has been shown to be linearly correlated with that measured by the ferricytochrome C method (12). We further established that, in our assay system, there is a dose-dependent inhibition of chemiluminescence by superoxide dismutase and catalase as well as diphenylene iodonium (data not shown), which is a specific inhibitor of NADPH oxidase—the enzyme responsible for the production of superoxide radicals. The specific inhibitory effect of diphenylene iodonium on NADPH oxidase was established by Hancock and Jones (13).

Measurement of plasma vitamin E, sICAM-1, CRP, and insulin
-Tocopherol was measured in plasma by HPLC as described previously (1, 4). Plasma sICAM-1 was assayed with an enzyme-linked immunosorbent assay kit from R&D systems (Minneapolis). The intraassay CV for sICAM-1 is 4.6%, whereas the interassay CV is 6.1%. The CRP (intraassay CV: 4%; interassay CV: 5%) and insulin (intraassay CV: 2.6%; interassay CV: 6.2%) enzyme-linked immunosorbent assay kits were purchased from Diagnostic Systems Laboratories Inc (Webster, TX).

Statistical analysis
The statistical analysis was carried out by using SIGMASTAT software (version 2.03; Jandel Scientific, San Rafael, CA). The analysis was carried out with one-factor repeated-measures analysis of variance (ANOVA) with the use of Dunnett’s test for comparisons against the baseline (0 h) for normally distributed data. Dunn’s test was used for the nonparametric data. Two-factor ANOVA was used to evaluate the interaction between treatment (water compared with the mixed meal) and time. Student’s t test was used to compare the area under the curves (AUCs). For the comparisons of ROS generation by PMNLs and MNCs before and after the meal challenge, the data were normalized to a baseline value of 100% because the baseline ROS generation varied markedly from one person to another. We previously showed this same level of variability while showing a consistency (reproducibility) in ROS generation within individuals (10). The AUC for all ROS generation was then measured because the peak of ROS generation for both PMNLs and MNCs varied from one subject to another, whereas an increase occurred in all subjects. The AUC was calculated for NF-B binding activity, IB, IKK, IKKß, and p47^phox and then compared between the meal-fed and control subjects by using Student’s t test for unpaired data. CRP and sICAM-1 concentrations were also normalized to a baseline value of 100% because the baseline concentrations varied markedly from one subjects to another. A P value < 0.05 was used to assess the significance for all statistical analyses. The results are presented as means ± SEMs.

	RESULTS

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Glucose, lipid, and insulin concentrations
Plasma glucose concentrations did not change significantly after the meal challenge (Table 1). Plasma insulin concentration increased significantly at 1 h and remained elevated at 2 and 3 h when compared with baseline. Plasma triacylglycerol concentrations increased significantly 2 and 3 h after the meal challenge. Plasma HDL, LDL, and cholesterol did not change significantly after food intake.

View this table: [in this window] [in a new window]   TABLE 1. Glucose, insulin, triacylglycerol, -tocopherol, and total, HDL-, and LDL-cholesterol concentrations after a 900-kcal mixed-meal challenge1

NF-B binding activity

Figure 1

View larger version (31K): [in this window] [in a new window]   FIGURE 1.. A: Representative electrophoretic mobility shift assay showing the relative nuclear transcription factor B (NF-B) binding to the double-stranded oligonucleotide containing the NF-B DNA-binding site after a 900-kcal mixed-meal challenge or water intake. Band-shift assays were performed with the use of 5 µg mononuclear cell extract for each time point. Lane 1 represents 0 h, lane 2 represents 1 h, lane 3 represents 2 h, and lane 4 represents 3 h after meal intake. Two complexes were detected in the gels (I and II). The super shift assay showed that complex I is composed of p50:p65 subunits, whereas complex II is composed of p50:p50 subunits. Lane 5: super shift assay with the use of an antibody against the p65 subunit. Lane 6: super shift assay with the use of an antibody against the p50 subunit. Lane 7: antibody against c-fos showing no effect with the electrophoretic mobility shift assay. Lane 8: inhibition of NF-B binding with the use of 2 µL competitor NF-B oligonucleotide. Lane 9: nonspecific oligonucleotide (AP-1) that shows no effect on binding. This gel clearly shows an increase in complex I formation after the 900-kcal meal challenge and a decrease in the p50:p50 complex (II); however, the decrease in the p50:p50 complex was not consistent in other subjects and did not change significantly (data not shown). B: Mean (± SEM) change in NF-B binding activity for complex I after intake of the mixed meal by densitometric quantitative analysis. There was a significant interaction between treatment (water compared with meal) and time (P < 0.005, two-factor ANOVA). *Significantly different from baseline (0 h), P < 0.05 (one-factor ANOVA for repeated measures followed by Dunnett’s test). n = 9 for meal intake and n = 8 for water intake. C: Area under the curve (AUC) for NF-B binding activity after water and meal intakes. *Significantly different from water intake, P < 0.05 (Student’s t test).

IB, IKK, and IKKß concentrations

Figure 2

Figure 3

Figure 4

View larger version (25K): [in this window] [in a new window]   FIGURE 2.. A: Representative Western blot showing the relative expression of inhibitor B (IB) in mononuclear cell homogenates 0, 1, 2, and 3 h after a 900-kcal mixed-meal challenge or water intake. B: Mean (± SEM) change in IB protein concentrations in mononuclear cells by densitometric quantitative analysis. There was a significant interaction between treatment (water compared with meal) and time (P < 0.05, two-factor ANOVA). *Significantly different from baseline (0 h), P < 0.05 (one-factor ANOVA for repeated measures followed by Dunnett’s test). n = 9 for meal intake and n = 8 for water intake. C: Area under the curve (AUC) for IB protein expression after water and meal intakes. *Significantly different from water intake, P < 0.05 (Student’s t test).

View larger version (29K): [in this window] [in a new window]   FIGURE 3.. A: Representative Western blot showing the relative expression of inhibitor B kinase (IKK) in mononuclear cell homogenates 0, 1, 2, and 3 h after a 900-kcal mixed-meal challenge or water intake. B: Mean (± SEM) change in IKK concentrations in mononuclear cells by densitometric quantitative analysis. There was a significant interaction between treatment (water compared with meal) and time (P < 0.005, two-factor ANOVA). *Significantly different from baseline (0 h), P < 0.05 (one-factor ANOVA for repeated measures followed by Dunnett’s test). n = 9 for meal intake and n = 8 for water intake. C: Area under the curve (AUC) for IKK protein expression after water and meal intakes. *Significantly different from water intake, P < 0.05 (Student’s t test).

View larger version (21K): [in this window] [in a new window]   FIGURE 4.. A: Representative Western blot showing the relative expression of inhibitor B kinase ß (IKKß) in mononuclear cell homogenates 0, 1, 2, and 3 h after a 900-kcal mixed-meal challenge or water intake. Note that IKKß concentrations did not increase significantly, largely because of variation in the magnitude of change after the meal. B: Mean (± SEM) change in IKKß concentrations in mononuclear cells by densitometric quantitative analysis. n = 9 for meal intake and n = 8 for water intake. There was no significant interaction between treatment (water compared with meal) and time (two-factor ANOVA). C: Area under the curve (AUC) for IKKß protein expression after water and meal intakes. *Significantly different from water intake, P < 0.05 (Student’s t test).

Figure 5

Figure 6

View larger version (26K): [in this window] [in a new window]   FIGURE 5.. A: Representative Western blot showing the relative expression of p47phox subunit in mononuclear cell homogenates 0, 1, 2, and 3 h after a 900-kcal mixed-meal challenge or water intake. B: Mean (± SEM) change in p47phox subunit protein expression in mononuclear cell homogenates by densitometric quantitative analysis. There was a significant interaction between treatment (water compared with meal) and time (P < 0.05, two-factor ANOVA). *Significantly different from baseline (0 h), P < 0.05 (one-factor ANOVA for repeated measures followed by Dunnett’s test). n = 9 for meal intake and n = 8 for water intake. C: Area under the curve (AUC) for p47phox subunit protein expression in mononuclear cell homogenates after water and meal intakes. *Significantly different from water intake, P < 0.05 (Student’s t test).

View larger version (28K): [in this window] [in a new window]   FIGURE 6.. Mean (± SEM) reactive oxygen species (ROS) generation by mononuclear cells (MNCs) and polymorphonuclear leukocytes (PMNLs) after a 900-kcal mixed-meal challenge or water intake. *Significantly different from water intake, P < 0.05 (Student’s t test for unpaired data). n = 9 for meal intake and n = 8 for water intake.

Plasma CRP, sICAM-1, and -tocopherol concentrations

Table 2

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

The increase in IKK and IKKß after the caloric challenge of the mixed meal is of interest because this enzyme is induced by endotoxin challenge and is responsible for the phosphorylation, ubiquitination, and eventual proteasomal degradation of IB. This decrease in IB releases NF-B to translocate into the nucleus. There are recent data that show that IKK may have an additional function: it may translocate into the nucleus and act as a coactivator of NF-B–induced tumor necrosis factor and other proinflammatory gene expression (19).

p47^phox Subunit expression increased significantly at 1 h and persisted at an elevated concentration for 3 h. This suggests that NADPH oxidase activity increased significantly after the meal, which is consistent with our previous data on the effects of glucose intake (8). Our data are thus consistent with the NF-B–mediated modulation of the p47^phox subunit. We previously showed that the suppression of intranuclear NF-B is associated with a decrease in p47^phox subunit expression, as observed after hydrocortisone (20), rosiglitazone (P Mohanty, A Aljada, H Ghanim et al, unpublished observations, 2001), troglitazone (21), and insulin (22, 23) injections, whereas an increase in NF-B is associated with a parallel increase in p47^phox subunit expression as observed after glucose intake (8). The essential role of NADPH oxidase and superoxide radical in the pathogenesis of atherosclerosis was recently shown. ApoE -/- mice, in whom the p47^phox gene has been deleted, do not develop atherosclerotic lesions even when fed a high-cholesterol diet (24). Thus, it is of interest that p47^phox and superoxide radical get induced with each meal and that obesity is characterized by a marked increase in oxidative stress (4).

The magnitude of the peak increase in the p47^phox subunit after the intake of the mixed meal was similar to that observed after glucose intake, but the peak increase in ROS generation was at different times in the subjects studied. The lack of a significant increase in ROS generation at a single time point contrasts with the increases we previously showed after glucose (1), cream, and casein (8) challenges. This may be the result of the variability caused by the variable digestion of food before absorption and the peak of ROS generation being blunted by the innate antioxidants in the mixed meal, including vitamins C and E (25). However, when the AUC of an increase from baseline in ROS generation at 1, 2, and 3 h was combined and comparisons were made between the mixed-meal group and the control subjects, there was a significant increase in ROS generation by both MNCs and PMNLs in the mixed-meal group compared with the control subjects, who drank 300 mL water.

Our observations have relevance to the pathogenesis of atherosclerosis, in which inflammation may play a role through leukocyte-endothelial interactions. Such damage is of special relevance to diabetic patients because glycated proteins in these patients may trigger inflammation through advanced glycation end product receptors (26, 27). The proinflammatory changes induced by a meal may act in combination with the increase in oxidative stress, which is known to be increased in diabetes. Similarly, macrovascular disease in obesity may be related to increased oxidative stress, which we recently showed in obese subjects (4), and to the proinflammatory changes induced by meal intake. Thus, these observations may be relevant to the pathogenesis of atherosclerosis, which is a chronic inflammatory state of the arterial wall (28, 29), in both diabetes and obesity. It is also relevant to the reversal of atherosclerosis and the improvement in myocardial perfusion, which was shown after intense lifestyle changes by Ornish et al (30).

These observations may also help explain the abnormalities in brachial artery reactivity observed after consumption of the 900-kcal meal in the current study. Vogel et al (6) showed that such a meal results in a significant reduction in postischemic flow-mediated vasodilation of the brachial artery. Furthermore, they showed that prior intake of vitamins E and C prevents this abnormality (31). They concluded that the abnormality in vascular reactivity induced by the meal was mediated through an increase in ROS generation and a possible reduction in the bioavailability of endothelial nitric oxide. Our observations provide actual evidence that confirms their hypothesis in terms of increased ROS generation and altered endothelium-mediated vasodilatation probably through reduced nitric oxide bioavailability.

The concomitant increase in IKK and IKKß expression, decrease in IB, and increase in intranuclear NF-B are consistent with leukocytic activation and a proinflammatory effect of the intake of a moderate-sized meal. It is of interest that these processes get triggered within 1 h of a meal and remain active for 3 h and that CRP also increases after such a meal. The duration of this proinflammatory effect is relevant because the time for another meal is usually 4–5 h after a meal, with the potential for further NF-B activation, ROS load, and potential proinflammatory changes. It is possible that chronic overeating may result in permanent increases in NF-B binding activity and in total expression as a protein in the cell. This area needs further investigation because the state of obesity is known to be associated with an increase in oxidative stress and an increase in plasma concentrations of proinflammatory mediators such as tumor necrosis factor , interleukin 6, and CRP (32–34).

The fact that insulin is secreted in response to the meal raises the theoretical possibility that it may mediate this proinflammatory effect. However, we already showed that insulin has a potent and rapid antiinflammatory effect both in vivo (22, 23) and in vitro (35, 36). Insulin suppresses intranuclear NF-B, induces IB, and suppresses the p47^phox subunit of NADPH oxidase, ICAM-1, MCP-1, and CRP, all of which are proinflammatory.

Our data are relevant to the concept that, whereas insulin and insulin sensitizers of the thiazolidinediones class have an ROS-suppressive and a profound antiinflammatory effect, macronutrient intake induces oxidative stress and proinflammatory changes. Thus, our current understanding of the relation between macronutrient intake and insulin, which contains metabolic implications only, has to incorporate the respective inflammatory and antiinflammatory effects of the macronutrient intake and insulin action.

In conclusion, a moderate-sized mixed meal results in a significant increase in the proinflammatory transcription factor NF-B in the nucleus, a decrease in IB (which is probably a consequence of the increase in IKK and IKKß), and a parallel increase in the cellular p47^phox subunit, which is the key protein component of NADPH oxidase and in ROS generation. These changes occur within 1 h of meal consumption and last for 3 h. These observations are consistent with potent inflammatory changes and raise fundamental issues about the relation between food intake, oxidative damage, inflammation, and atherosclerosis and about the ideal way to eat and modify lifestyle.

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