help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0778
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, C.
Right arrow Articles by Dandona, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, C.
Right arrow Articles by Dandona, P.
Related Collections
Right arrow Cardiovascular Endocrinology
Right arrow Obesity
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 11 4476-4479
Copyright © 2007 by The Endocrine Society

BRIEF REPORT

Prolonged Reactive Oxygen Species Generation and Nuclear Factor-{kappa}B Activation after a High-Fat, High-Carbohydrate Meal in the Obese

Chinmay Patel, Husam Ghanim, Shreyas Ravishankar, Chang Ling Sia, Prabhakar Viswanathan, Priya Mohanty and Paresh Dandona

Division of Endocrinology, Diabetes, and Metabolism, State University of New York at Buffalo and Kaleida Health, Buffalo, New York 14209

Address all correspondence and requests for reprints to: Paresh Dandona, M.D., Ph.D., Diabetes-Endocrinology Center of WNY, 3 Gates Circle, Buffalo, New York 14209. E-mail: pdandona{at}kaleidahealth.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: Because obesity is associated with chronic oxidative and inflammatory stress, and high-fat, high-carbohydrate meals induce significant oxidative and inflammatory stress in normal subjects, we have now hypothesized that the intake of a high-fat, high-carbohydrate meal would result in a greater and more prolonged oxidative and inflammatory stress in the obese than in normal subjects.

Methods: Ten normal-weight and eight obese subjects were given a high-fat, high-carbohydrate meal after an overnight fast. Blood samples were collected at baseline and hourly following the meal for 3 h.

Results: Reactive oxygen species generation by mononuclear cells increased significantly by 2 h in both groups but continued to increase significantly at 3 h in the obese subjects, whereas in normal subjects it returned to baseline. Levels of p47phox increased significantly (by 81 ± 26%) at 3 h in obese individuals (P < 0.05), whereas there was no significant change in p47phox in normal subjects. Nuclear factor-{kappa}B DNA binding in mononuclear cells increased significantly (by 48 ± 58%, P < 0.036) at 2 h but not at 3 h in normal subjects, whereas in the obese, nuclear factor-{kappa}B increased significantly at both 2 and 3 h (by 36 ± 57 and 42 ± 63%, respectively, P < 0.004). Matrix metalloproteinase-9 concentrations were significantly higher in the obese at baseline (580 ± 103.9 vs. 373 ± 30.03 ng/ml, P < 0.05) and increased to significantly greater concentrations after the meal than in the lean subjects.

Conclusions: High-fat, high-carbohydrate meals induced a significantly more prolonged and greater oxidative and inflammatory stress in the obese. This may contribute to the increased atherogenic risk in obesity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE INTAKE OF glucose leads to an increase in reactive oxygen species (ROS) generation by mononuclear cells and inflammation as reflected in an increase in nuclear factor-{kappa}ß (NF-{kappa}B), activator protein-1 (AP-1), and early growth response (Egr-1) binding after glucose intake with an increase in the expression of matrix metalloproteinase-2 (MMP-2) and tissue factor in mononuclear cells (1, 2) in normal subjects. There is a concomitant increase in plasma concentrations of MMP-2, MMP-9, and tissue factor. More recently, we demonstrated that a moderately sized high-fat, high-carbohydrate meal (900 kcal) also induced oxidative stress and inflammation in normal subjects (3). Hyperglycemia has been also shown to produce oxidative stress and inflammation in glucose-intolerant and diabetic patients after a meal challenge (4, 5). Clearly, glucose, fat, and macronutrient intake induces oxidative stress and is proinflammatory and prothrombotic (6).

Because the obese are in a chronic state of oxidative stress and low-grade inflammation (7, 8), we have now hypothesized that the magnitude and the duration of ROS generation and inflammation after a high-fat, high-carbohydrate meal is greater and more prolonged in the obese than in normal-weight subjects.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Ten (five males and five females) normal-weight subjects [body mass index (BMI) = 23.3 ± 0.4 kg/m2] aged 23–41 (mean ± SEM, 30.5 ± 6.5) yr and eight (three males) obese subjects (BMI = 35.5 ± 2.3 kg/m2) aged 24–60 (mean ± SEM, 36.9 ± 13.5) yr were asked to eat a high-fat, high-carbohydrate meal containing 1800 kcal (Big Mac, large French fries, a large Coke, and apple pie, which contains 62% carbohydrate, 30% fat, and 8% protein) over 15 min after an overnight fast. Blood samples were obtained before and at 1, 2, and 3 h after the meal. The protocol was approved by the Institutional Review Board of the State University of New York at Buffalo. All participants gave their written and informed consent.

Methods

Mononuclear cell isolation, ROS generation by formyl-Met-Leu-Phe (fMLP)-induced mononuclear cells and Western blotting for p47phox from mononuclear cells was done as previously described (9). NF-{kappa}B EMSA on mononuclear cells nuclear extracts, measurements of plasma insulin, glucose, and free fatty acid (FFA) concentrations, and homeostasis model assessment of insulin resistance (HOMA-IR) calculation were carried out as described before (7). The concentration of MMP-9 in plasma was measured with ELISA kit (R&D Systems, Minneapolis, MN).

Statistical analysis

The statistical analysis was carried out using Sigmastat software (Jandel Scientific, San Rafael, CA). The analysis was carried out with one-factor repeated-measures ANOVA for comparisons of the change from baseline (0 h). Two-factor ANOVA was used to evaluate the interaction between treatment groups (obese compared with normal weight) and time. Paired t test was used to compare data at two different time points in the same groups, and unpaired t test was used to compare the data at a particular time point between two groups. Data were normalized to a baseline value of 100 U and percent change from baseline calculated to allow comparison between two groups and at two different time points in same group. A P value of < 0.05 was used to assess the significance for all statistical analyses. The results are presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Plasma glucose, insulin, HOMA-IR, FFA, and triglyceride concentration

There was a significant increase in plasma glucose concentrations (one-way ANOVA, P = 0.018) in the obese group, whereas it did not change significantly in the normal-weight subjects. There was a significant difference in plasma glucose levels between the groups (P = 0.042) (Table 1Go). The basal and postprandial insulin concentrations were significantly higher in the obese (P < 0.001 by two-way ANOVA). There was no significant difference in plasma triglyceride and FFA concentration between the two groups at baseline (0 h) or after the meal. FFA concentrations fell rapidly after the meal in both groups (P < 0.05), whereas triglycerides increased (P < 0.05). HOMA-IR was significantly higher in the obese (4.3) than that in lean subjects (1.4).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Plasma glucose, insulin, HOMA-IR, triglyceride, free fatty acid (FFA) concentrations

 
ROS generation

Baseline levels of ROS generation by mononuclear cells were not different between the two groups. In normal subjects, ROS generation by mononuclear cells increased significantly by 41 ± 57% over the baseline with the peak increase at 2 h (P = 0.045, ANOVA). In the obese group, ROS generation by mononuclear cells increased significantly over baseline by 88 ± 40% at 3 h (P = 0.017, ANOVA). At 3 h, ROS generation declined toward baseline in normal individuals, whereas it was still significantly elevated in obese individuals, but the difference between the groups was not statistically significant (P = 0.085, t test).

Expression of p47phox protein

Baseline levels of p47phox subunit of NADPH oxidase in mononuclear cell homogenates were relatively higher but not statistically different in obese subjects compared with that in lean subjects. p47phox expression increased significantly by 81 ± 26% over the baseline at 3 h in obese individuals (P < 0.05, ANOVA), whereas it did not change significantly in the lean group. This increase was significantly greater in the obese group (two-way ANOVA, P < 0.05) when compared with the lean group.

NF-{kappa}B binding activity

Baseline levels of NF-{kappa}B DNA binding in mononuclear cells were relatively higher but not statistically different in obese subjects compared with that in lean subjects. NF-{kappa}B DNA binding in mononuclear cells increased significantly by 48% (48 ± 58%, P < 0.036, by paired t test, Fig. 1BGo) over baseline at 2 h in the lean subjects after the meal but returned to baseline by 3 h. In the obese subjects, NF-{kappa}B DNA binding increased significantly at both 2 and 3 h after the meal by 51% (36 ± 57%) and 59% (42 ± 63%) over the baseline, respectively (Fig. 1BGo) (ANOVA, P = 0.004). Furthermore, the change in NF-{kappa}B DNA binding at 3 h compared with 2 h (3 h – 2 h) in lean and obese groups was –33 ± 12 and 6 ± 18, respectively, which was also significantly different when compared by t test (P < 0.017, Fig. 1CGo).


Figure 1
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 1. A, EMSA image of NF-{kappa}B binding activity from nuclear extract on one obese and one lean subject at 0, 1, 2, and 3 h after 1800-kcal mixed meal; B, change from baseline in NF-{kappa}B binding in obese and normal weight groups (notice that NF-{kappa}B binding is decreasing at 3 h in normal-weight individuals while it was still increasing in the obese); C, change in NF-{kappa}B activity between 2 and 3 h (3 h – 2 h) after the mixed meal. *, P < 0.05 by one-way ANOVA in the obese; +, P < 0.05 by paired t test within the group; **, P < 0.0017 by t test.

 
MMP-9 concentrations

Fasting MMP-9 concentrations were significantly higher in obese subjects than in normal-weight subjects (580 ± 103.9 vs. 373 ± 30.3 ng/ml, P < 0.05). In both groups, there was a significant increase in MMP-9 concentrations at 3 h (one-way ANOVA, P < 0.05). The magnitude of increase at 3 h was significantly greater in the obese (702 ± 108.8 ng/ml) compared with normal-weight subjects (473 ± 25 ng/ml) (P < 0.05, paired t test).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our data show clearly that after a large (1800-kcal) high-fat, high-carbohydrate meal in the obese, ROS generation by mononuclear cells, the expression of p47phox, and intranuclear NF-{kappa}B binding increased in the mononuclear cells as did plasma MMP-9 concentrations. The increase in these indices was more prolonged in the obese than in normal subjects. In addition, the magnitude of oxidative stress and inflammation in the obese was greater at the baseline (but not statistically significant for all indices) and throughout the period of 3 h.

Because ROS generation and intranuclear NF-{kappa}B DNA binding by the mononuclear cells of the obese was greater than those in normal-weight subjects at baseline and because the percent increase at peak (2 h) after the meal was similar between the groups, the magnitude of the total increase in these indexes was significantly greater in the obese. Furthermore, there was no decline in these indices in the obese at 3 h, whereas there was a decline in these indices in normal subjects at 3 h. Also, p47phox subunit of NADPH oxidase and plasma MMP-9 levels were significantly higher after the meal in obese subjects compared with those in the lean. This suggests that the ability of obese subjects to handle oxidative and inflammatory stress challenge is less than that in normal subjects.

ROS generation measurements were carried out after fMLP stimulation, consistent with our previous work and that of others using this agonist or endotoxin (10). The addition of agonist enhances the signal as in platelet aggregation studies. The measurements on NF-{kappa}B binding and p47phox were carried out without fMLP stimulation.

We have previously shown that obesity is associated with chronic oxidative and inflammatory stress and that caloric restriction results in a fall in these indices (7, 11). These observations have been confirmed by work of Davi and Patrono and colleagues (12, 13). Furthermore, a 48-h fast results in a marked reduction in ROS generation, p47phox expression, and oxidative damage to amino acids (14). Clearly, macronutrient intake is a key factor in generating oxidative and inflammatory stress. Thus, it is not surprising that a challenge with a large high-fat, high-carbohydrate meal resulted in a further acute increase in oxidative and inflammatory stress, which was significantly greater in its magnitude and duration in the obese than that observed in normal-weight subjects.

Consistent with the increase NF-{kappa}B binding and ROS generation, there was also an increase in plasma MMP-9 concentrations after the meal in both the obese and the normal-weight subjects. The mean basal plasma MMP-9 concentration in the obese was significantly greater than that in normal subjects, whereas the percent increase from the basal was similar in the two groups. Thus, the overall sequential MMP-9 concentrations in the obese were significantly greater than those in normal-weight subjects throughout the experimental period from the fasting state to 3 h.

Because oxidative and inflammatory stress plays a key role in the pathogenesis of atherosclerosis, it is relevant that the obese have enhanced oxidative and inflammatory stress in the fasting state and that any further macronutrient challenge leads to a greater increase in such stress (15, 16, 17). It is likely that such a practice on a regular basis further enhances the cumulative atherogenic risk with an increase in cardiovascular events.

There has been one previous attempt to compare the proinflammatory effect of 1) a hyperglycemic clamp (15 mmol/liter = 270 mg/liter) and 2) hyperglycemic pulses in insulin-resistant and normal subjects. Endogenous insulin secretion was inhibited with an iv infusion of somavastatin in this study (2). This study demonstrated more prolonged increases in TNF{alpha}, IL-6, and IL-18 in insulin-resistant subjects than those in normal subjects, similar to what we observed in our study. However, this study investigated the effect of hyperglycemia, whereas endogenous insulin secretion was totally inhibited, a totally artificial situation. We, on the other hand, have investigated the effect of a large high-fat, high-carbohydrate meal, not uncommon in our daily lives, without the induction of hyperglycemia. This simple experimental plan and these observations are novel. These observations are relevant to our lifestyles.

In conclusion, 1) the intake of a large high-fat, high-carbohydrate meal results in marked oxidative and inflammatory stress, 2) this increase is significantly greater in the obese and lasts for a longer duration of time in the obese population when compared with normal-weight subjects, and 3) the above observations may contribute to the pathogenesis of atherosclerosis in the obese.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online September 4, 2007

Abbreviations: BMI, Body mass index; FFA, free fatty acid; fMLP, formyl-Met-Leu-Phe; HOMA-IR, homeostasis model assessment of insulin resistance; MMP-2, matrix metalloproteinase-2; NF-{kappa}B, nuclear factor-{kappa}B; ROS, reactive oxygen species.

Received April 16, 2007.

Accepted August 29, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Aljada A, Friedman J, Ghanim H, Mohanty P, Hofmeyer D, Chaudhuri A, Dandona P 2006 Glucose ingestion induces an increase in intranuclear nuclear factor {kappa}B, a fall in cellular inhibitor {kappa}B, and an increase in tumor necrosis factor {alpha} messenger RNA by mononuclear cells in healthy human subjects. Metabolism 55:1177–1185[CrossRef][Medline]
  2. Aljada A, Ghanim H, Mohanty P, Syed T, Bandyopadhyay A, Dandona P 2004 Glucose intake induces an increase in activator protein 1 and early growth response 1 binding activities, in the expression of tissue factor and matrix metalloproteinase in mononuclear cells, and in plasma tissue factor and matrix metalloproteinase concentrations. Am J Clin Nutr 80:51–57[Abstract/Free Full Text]
  3. Aljada A, Mohanty P, Ghanim H, Abdo T, Tripathy D, Chaudhuri A, Dandona P 2004 Increase in intranuclear nuclear factor {kappa}B and decrease in inhibitor {kappa}B in mononuclear cells after a mixed meal: evidence for a proinflammatory effect. Am J Clin Nutr 79:682–690[Abstract/Free Full Text]
  4. Ceriello A, Bortolotti N, Motz E, Crescentini A, Lizzio S, Russo A, Tonutti L, Taboga C 1998 Meal-generated oxidative stress in type 2 diabetic patients. Diabetes Care 21:1529–1533[Abstract]
  5. Esposito K, Nappo F, Marfella R, Giugliano G, Giugliano F, Ciotola M, Quagliaro L, Ceriello A, Giugliano D 2002 Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation 106:2067–2072[Abstract/Free Full Text]
  6. Bowen PE, Borthakur G 2004 Postprandial lipid oxidation and cardiovascular disease risk. Curr Atheroscler Rep 6:477–484[Medline]
  7. Ghanim H, Aljada A, Hofmeyer D, Syed T, Mohanty P, Dandona P 2004 Circulating mononuclear cells in the obese are in a proinflammatory state. Circulation 110:1564–1571[Abstract/Free Full Text]
  8. Vincent HK, Taylor AG 2006 Biomarkers and potential mechanisms of obesity-induced oxidant stress in humans. Int J Obes (Lond) 30:400–418[CrossRef][Medline]
  9. Dandona P, Thusu K, Hafeez R, Abdel-Rahman E, Chaudhuri A 1998 Effect of hydrocortisone on oxygen free radical generation by mononuclear cells. Metabolism 47:788–791[CrossRef][Medline]
  10. Kopprasch S, Graessler J, Kohl M, Bergmann S, Schroder HE 1996 Comparison of circulating phagocyte oxidative activity measured by chemiluminescence in whole blood and isolated polymorphonuclear leukocytes. Clin Chim Acta 253:145–157[CrossRef][Medline]
  11. Dandona P, Mohanty P, Ghanim H, Aljada A, Browne R, Hamouda W, Prabhala A, Afzal A, Garg R 2001 The suppressive effect of dietary restriction and weight loss in the obese on the generation of reactive oxygen species by leukocytes, lipid peroxidation, and protein carbonylation. J Clin Endocrinol Metab 86:355–362[Abstract/Free Full Text]
  12. Basili S, Pacini G, Guagnano MT, Manigrasso MR, Santilli F, Pettinella C, Ciabattoni G, Patrono C, Davi G 2006 Insulin resistance as a determinant of platelet activation in obese women. J Am Coll Cardiol 48:2531–2538[Abstract/Free Full Text]
  13. De Cristofaro R, Rocca B, Vitacolonna E, Falco A, Marchesani P, Ciabattoni G, Landolfi R, Patrono C, Davi G 2003 Lipid and protein oxidation contribute to a prothrombotic state in patients with type 2 diabetes mellitus. J Thromb Haemost 1:250–256[CrossRef][Medline]
  14. Dandona P, Mohanty P, Hamouda W, Ghanim H, Aljada A, Garg R, Kumar V 2001 Inhibitory effect of a two day fast on reactive oxygen species (ROS) generation by leucocytes and plasma ortho-tyrosine and meta-tyrosine concentrations. J Clin Endocrinol Metab 86:2899–2902[Abstract/Free Full Text]
  15. Libby P, Ridker PM, Maseri A 2002 Inflammation and atherosclerosis. Circulation 105:1135–1143[Abstract/Free Full Text]
  16. Dandona P 2002 Endothelium, inflammation, and diabetes. Curr Diab Rep 2:311–315[Medline]
  17. Davi G, Falco A 2005 Oxidant stress, inflammation and atherogenesis. Lupus 14:760–764[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Patel, C.
Right arrow Articles by Dandona, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Patel, C.
Right arrow Articles by Dandona, P.
Related Collections
Right arrow Cardiovascular Endocrinology
Right arrow Obesity

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals