Plasma Interleukin-8 Concentrations Are Increased in Obese Subjects and Related to Fat Mass and Tumor Necrosis Factor-{alpha}

System
Marek Straczkowski, Stella Dzienis-Straczkowska, Agnieszka Stpie, Irina Kowalska, Malgorzata Szelachowska and Ida Kinalska J. Clin. Endocrinol. Metab. 2002 87: 4602-4606, doi: 10.1210/jc.2002-020135

To subscribe to Journal of Clinical Endocrinology & Metabolism or any of the other journals published by The Endocrine Society please go to: http://jcem.endojournals.org//subscriptions/

Copyright The Endocrine Society. All rights reserved. Print ISSN: 0021-972X. Online

0013-7227/02/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 87(10):4602 4606 Copyright 2002 by The Endocrine Society doi: 10.1210/jc.2002-020135

Plasma Interleukin-8 Concentrations Are Increased in Obese Subjects and Related to Fat Mass and Tumor Necrosis Factor- System
MAREK STRACZKOWSKI, STELLA DZIENIS-STRACZKOWSKA, AGNIESZKA STEPIEN, IRINA KOWALSKA, MALGORZATA SZELACHOWSKA, AND IDA KINALSKA
Department of Endocrinology, Diabetology and Internal Medicine, Medical Academy, Bialystok 15-276, Poland
Obesity is associated with the increased risk of cardiovascular disease; however, mechanisms responsible for such an increase are not fully understood. IL-8 is a cytokine that might have atherogenic properties. Recent in vitro studies revealed that IL-8 is produced and secreted by human adipocytes. The aim of the present study was to evaluate plasma IL-8 concentrations in obese subjects and the relationships between circulating IL-8 and anthropometric and biochemical parameters and TNF- system. A total of 75 subjects with normal glucose tolerance, 35 lean and 40 obese, were recruited for this study. Plasma IL-8 levels were measured in fasting state, after an oral glucose tolerance test and after the euglycemic hyperinsulinemic clamp. A significant increase in plasma IL-8 was observed in the obese group. In simple regression analysis, performed for the initial evaluation of relationships, plasma IL-8 was related to body mass index, percentage of body fat, fat mass (FM), and soluble TNF- receptor 2 (sTNFR2) in both groups and with waist-to-hip ratio and sTNFR1 in the obese. In multiple regression analysis, FM, waist-to-hip ratio, gender, sTNFR2, and low density lipoprotein cholesterol were responsible for 44% of IL-8 variability. During oral glucose tolerance testing, mean plasma IL-8 concentrations increased in both groups, whereas clamp resulted in a significant increase in plasma IL-8 only in the obese. We conclude that plasma IL-8 levels are increased in obese subjects, and are related to FM and TNF- system. Increase in circulating IL-8 might be one of the factors linking obesity with greater cardiovascular risk. (J Clin Endocrinol Metab 87: 4602 4606, 2002)

BESITY IS ASSOCIATED with the increased prevalence of type 2 diabetes mellitus (1) and is also an independent risk factor for cardiovascular disease (2). Mechanisms linking obesity with atherosclerosis and cardiovascular disease are not completely understood. In recent years, many studies have focused on endocrine and paracrine function of fat cells (3). Adipocytes synthesize and secrete substances involved in regulation of food intake, energy expenditure, and local and whole-body carbohydrate and lipid metabolism (3). These substances include leptin, TNF- , IL-6, resistin, and many others (3, 4). It is proposed that one of the adipocyte-derived cytokines is also IL-8 (5, 6). IL-8 is produced mainly by macrophages and monocytes and plays a role in modulating an inflammatory response (7). Inflammation might contribute to the pathogenesis of atherosclerosis (8), and IL-8 is probably also an atherogenic factor. Oxidized low-density lipoprotein (LDL) particles are able to stimulate production and secretion of IL-8 by macrophages from human atherosclerotic plaques and high levels of IL-8 in macrophage-derived human foam cells were found (9). IL-8 is a potent chemoattractant and may be responsible for the recruitment of neutrophils and T lymphocytes into the subendothelial space. It also induces adhesion of monocytes to endothelium (10) and migration of vascular smooth muscle cells (11). All those processes lead to intimal thickening and atherosclerosis (10).
Abbreviations: CV, Coefficients of variation; EASIA, enzyme amplified sensitivity immunoassay; FM, fat mass; LDL, low-density lipoprotein; OGTT, oral glucose tolerance test; sTNFR1 or 2, soluble TNFreceptor 1 or 2; WHR, waist-to-hip ratio.

Another function of IL-8 is inhibition of tissue inhibitor of metalloproteinase expression, which results in the increased release of matrix-degrading metalloproteinases and in consequence the instability of atherosclerotic plaque (12). Elevated serum IL-8 levels were found in type 1 and type 2 diabetic subjects, and it is suggested that this cytokine might also contribute to the development of diabetic macroangiopathy (13). Recently, two elegant studies of Bruun et al. (5, 6) revealed, through the in vitro experiments, that IL-8 is produced and secreted by human adipocytes. IL-8 was released from adipose tissue fragments into the medium, and IL-8 mRNA was found in isolated adipocytes (5, 6). Therefore, the authors suggested that the correlation found between the severity of obesity and the development of atherosclerosis and cardiovascular disease might be related to the ability of human adipose tissue to produce and release IL-8 (6). However, there are no in vivo data about plasma IL-8 levels in obesity. Additionally, it was found that adipocyte IL-8 mRNA expression and protein synthesis and release is stimulated by IL-1 and TNF- (6) and inhibited by the insulin-sensitizing agents thiazolidinedione ciglitazone and biguanide metformin (5). Both insulin resistance and TNF- overexpression in adipose tissue and skeletal muscle are important features of human obesity, related to each other, as TNF- induces insulin resistance by acting via autocrine-paracrine pathway (14). It is well known that insulin resistance plays a role in the pathogenesis of obesity-related complications (15). TNF- may also increase cardiovascular risk in obesity via other mechanisms (16). This cytokine has two cell surface

4602

Straczkowski et al. IL-8 in Obesity

J Clin Endocrinol Metab, October 2002, 87(10):4602 4606 4603

receptors, TNFR1 and TNFR2, that are also present in plasma in soluble forms (sTNFR1 and 2). sTNFR2 is more stable protein than TNF- and is increased in obese subjects (17). It is proposed that sTNFR2 might serve as the best predictor of local TNF- system activity in obesity (17). The relationship between these parameters and plasma IL-8 in vivo in humans has not been determined so far. Therefore, in the present study we evaluate plasma IL-8 levels in lean and obese subjects with normal glucose tolerance and the relationships of plasma IL-8 with anthropometric measurements, insulin action, and TNF- system. We also examine the effect of oral glucose load and hyperinsulinemia on circulating IL-8 concentrations.
Subjects and Methods
A total of 75 subjects, 35 lean [body mass index (BMI) 27.8, 14 males, and 21 females] and 40 with marked overweight or obesity (BMI 27.8, 18 males, and 22 females, obese group), were recruited for this study. All the participants had no cardiovascular disease, hypertension, infections, or any other serious medical problems. Before entering the study, physical examination and resting electrocardiography were performed. Subjects underwent an oral glucose tolerance test (OGTT), and they all had normal glucose tolerance according to WHO criteria. The study protocol was approved by the Ethics Committee of Medical Academy, Bialystok. All the subjects gave written informed consent before entering the study. Analyses were performed after an overnight fast. The BMI was calculated as body weight height2 and expressed in kg/m2. The waistto-hip ratio (WHR) was estimated. The waist circumference was measured at the smallest circumference between the rib cage and the iliac crest, with the subject in the standing position. The hip circumference was measured at the widest circumference between the waist and the thighs. Percentage of body fat was assessed by bioelectric impedance analysis using the Tanita TBF-511 Body Fat Analyzer (Tanita Corp., Tokyo, Japan), fat mass (FM), and fat-free mass were calculated. Insulin sensitivity was evaluated by the euglycemic hyperinsulinemic clamp technique as described by DeFronzo et al. (18). On the morning of the study, two venous catheters were inserted into antecubital veins, one for the infusion of insulin and glucose and the other in the contralateral hand for blood sampling; that hand was heated to approximately 60 C. Insulin (Actrapid HM, Novo Nordisk, Copenhagen, Denmark) was given as a primed-continuous iv infusion for 2 h at 50 mU kg 1 h 1, resulting in constant hyperinsulinemia of approximately 550 pmol/liter. Arterialized blood glucose was obtained every 5 min and 40% dextrose (2.22 mol/liter) infusion was adjusted to maintain plasma glucose levels at 5.0 mmol/liter. The glucose infusion rate approached stable values during final 40 min of the study and the rate of whole-body glucose uptake (M value) was calculated as the mean glucose infusion rate from 80 120 min, corrected for glucose space, and normalized per kilogram of fat-free mass (Mffm). Fasting blood samples were also taken from the antecubital vein before the beginning of the clamp for the determination of glycated hemoglobin (HbA1c), plasma lipids, TNF- , sTNFR1, sTNFR2, and IL-8. Plasma IL-8 concentration were also estimated at the end (at 120 min) of the OGTT and clamp. For the determination of plasma TNF system and IL-8, samples were frozen at 70 C. Plasma glucose was measured immediately by the enzymatic method using glucose analyzer. Plasma insulin was measured with the Medgenix enzyme amplified sensitivity immunoassay (EASIA) test (Biosource Technologies Europe, Nivelles, Belgium). The minimum detectable concentration was 1.05 pg/liter, and the intraassay and interassay coefficients of variation (CV) were less than 5.5% and 10%, respectively. In that method, human and animal proinsulins present no crossreaction. HbA1c were measured by the HPLC method (Bio-Rad Laboratories, Inc., Muenchen, Germany). Plasma cholesterol and triglycerides were assessed by the enzymatic methods (Cormay, Warsaw, Poland). Plasma TNF- concentrations were measured by the Immunoassay Kit (Biosource Technologies International, Camarillo, CA) with the minimum detectable concentration 1.7 pg/ml and with the intraassay and

interassay CV less than 5.2% and 8.5%, respectively. Plasma sTNFR1 and sTNFR2 were determined with the EASIA kits (Biosource Technologies Europe). The minimum detectable concentration was 0.05 ng/ml for sTNFR1 and 0.1 ng/ml for sTNFR2. The intraassay and interassay CV for both receptors were less than 6.5% and 9%, respectively. sTNFR1 EASIA does not cross-react with sTNFR2 and TNF- does not interfere with the assay. Plasma IL-8 concentration was assessed with the human ultrasensitive ELISA kit (Biosource Technologies International) with the standard curve range of 0.39 25.0 pg/ml and the detection limit of the method less than 100 fg/ml. The intraassay and interassay CV were less than 7.5% and 9.0%, respectively. The statistics were performed with the STATISTICA 5.0 program (StatSoft, Krakow, Poland). Differences between the groups were evaluated with the unpaired Students t test. Differences between fasting and postload IL-8 levels in respective groups were assessed using the paired Students t test. Relationships between variables were estimated with the simple and multiple regression analysis. The level of significance was accepted at P value less than 0.05.

Results

Anthropometric and biochemical characteristics of the studied groups are presented in Table 1. Obese subjects had markedly higher levels of fasting glucose (P 0.0002), insulin (P 0.005), HbA1c (P 0.002), total and LDL cholesterol, and triglycerides (all P 0.002) and were more insulin resistant (P 0.001) in comparison to the lean controls. The values of soluble forms of both TNF- receptors were also significantly increased in the obese group (sTNFR1, P 0.00005, sTNFR2, P 0.005), whereas TNF- did not differ between the studied groups. The mean plasma IL-8 concentration was 3.81 1.37 pg/ml and ranged from 1.57.7 pg/ml. We observed an increased IL-8 levels in the obese subjects (P 0.0005). Next we divided the obese subjects into the subgroup with visceral (men, WHR 0.95, n 8; women, WHR 0.85, n 12) and peripheral obesity (men, WHR 0.95, n 10; women, WHR 0.85, n 10). We found significantly higher plasma IL-8 in the subjects with visceral (4.89 1.35 pg/ml) in
TABLE 1. Clinical characteristics of the studied groups
Lean subjects (n 24) Obese subjects (n 30)

Age (yr) BMI (kg/m2) WHR Percentage of body fat FM (kg) Fat-free mass (kg) HbA1c (%) Plasma glucose (mmol/liter) Plasma insulin (pmol/liter) Plasma cholesterol (mmol/ liter) Plasma triglycerides (mmol/ liter) High-density lipoprotein cholesterol (mmol/liter) LDL cholesterol (mmol/liter) Mffm ( mol kg 1 min 1) TNF (pg/ml) sTNFR1 (ng/ml) sTNFR2 (ng/ml) IL-8 (pg/ml)

36.00 22.85 0.79 17.33 12.01 54.86 5.40 4.82 72.65 4.66 1.16 1.38 2.74 44.50 5.34 1.96 4.18 3.24

8.37 2.48 0.06 5.71 5.53 8.57 0.36 0.62 34.56 1.14 0.67 0.36 1.09 16.33 1.38 0.36 0.85 1.07

39.85 32.70 0.89 38.51 38.20 58.18 5.89 5.38 127.85 5.53 1.83 1.22 3.53 31.69 5.76 2.40 4.97 4.31

11.03 3.29a 0.07a 9.25a 15.48a 7.59 0.83a 0.59a 96.91a 1.17a 1.00a 0.38 1.03a 14.58a 2.99 0.46a 1.36a 1.43a

Data are means SD. a P 0.05 in obese vs. lean group.

4604

J Clin Endocrinol Metab, October 2002, 87(10):4602 4606

Straczkowski et al. IL-8 in Obesity

comparison to peripheral type of obesity (3.74 1.28 pg/ml, P 0.01). Similar observation was made separately in women (4.94 1.23 vs. 3.59 1.26 pg/ml, P 0.02), whereas in men the difference did not reach the level of significance (4.81 1.61 vs. 3.89 1.35 pg/ml, P 0.20). No significant difference in plasma IL-8 between males and females was detected in the whole group and within the subgroups of lean and obese subjects. Table 2 shows the correlations between plasma IL-8 and other examined parameters in lean and obese subjects. IL-8 levels were positively related to BMI, percentage of body fat, FM, and sTNFR2 in both groups. IL-8 concentrations were also associated with WHR and sTNFR1 in the obese group, whereas in lean individuals correlation with WHR was of borderline significance. Figure 1 shows the relationships between plasma IL-8 and WHR, FM, and sTNFR2 in obese subjects. When we analyzed both groups together, there were also marked correlations between circulating IL-8 and fasting insulin (r 0.23, P 0.05), total (r 0.29, P 0.02) and LDL cholesterol (r 0.28, P 0.02), whereas the relationship between IL-8 and insulin sensitivity was of borderline significance (r 0.22, P 0.058). In multiple regression analysis with plasma IL-8 as the 0.64, P dependent variable, we found that WHR ( 0.34, P 0.01) were independent 0.0001) and gender ( predictors of IL-8 levels. Although, as already mentioned, plasma IL-8 did not differ between men and women, an effect of female gender on determining an increase of IL-8 was present in this regression model. Coefficients for FM and 0.24, P 0.09 sTNFR2 were of borderline significance ( 0.22, P 0.06, respectively). Such a result is proband ably the effect of close colinearity between FM and sTNFR2 (r 0.59, P 0.001). When each of those parameters where included in the analysis without the other, it was also an 0.37; sTNFR2, independent predictor of IL-8 (FM, 0.32; both P 0.005). In the stepwise regression analysis, FM and WHR were responsible for 37% of IL-8 variability (P 0.000005) with gender increasing R2 value to 0.40 (P 0.000005) and also with sTNFR2 and LDL cholesterol, slightly improving correlation coefficient (R2 0.44; P 0.000005). Age, plasma glucose, insulin, and Mffm did not

enter the regression model. We chose sTNFR2 as the best predictor of TNF- activity because it was most strongly related to IL-8. During an OGTT, mean plasma IL-8 concentrations increased both in lean (from 3.24 1.07 pg/ml to 4.05 1.61 pg/ml, P 0.01) and in obese subjects (from 4.31 1.43 pg/ml to 4.83 1.61 pg/ml, P 0.02). Although the observed change in plasma IL-8 ( IL-8OGTT) was slightly higher in the lean group, this difference was not statistically significant (P 0.38). IL-8OGTT was related neither to the factors determining fasting concentrations of the cytokine, nor to glucose and insulin responses during an OGTT. Plasma IL-8 levels after glucose load were still higher in the obese in comparison to lean subjects (P 0.05). In contrast, the euglycemic hyperinsulinemic clamp resulted in a significant increase in plasma IL-8 levels in obese (from 4.31 1.43 pg/ml to 6.51 3.48 pg/ml, P 0.00005), but not in lean subjects (3.24 1.07 pg/ml vs. 3.41 1.03 pg/ml, P 0.20). The difference in IL-8 between lean and obese individuals increased after the clamp (IL-8 levels about 90% higher in the obese, P 0.000005). The change in plasma IL-8 during the clamp ( IL-8clamp) in the obese group was related to fasting IL-8 levels (r 0.32, P 0.05), FM (r 0.33, P 0.05) and steady-state insulin concentrations (r 0.42, P 0.01), but not to WHR (r 0.06, P 0.69). The correlation between IL-8clamp and baseline sTNFR2 approached the level of significance (r 0.30, P 0.061).
Discussion

Mean values of plasma IL-8 found in our study are similar to those reported by other investigators (19). We observed, for the first time to our knowledge, an increase in plasma IL-8 in obese individuals. As already mentioned, this might be one of the factors responsible for the increased cardiovascular risk in obesity. We do not provide a direct evidence about the source of the elevated plasma IL-8. However, previous studies of Bruun et al. (5, 6) showed IL-8 production by the adipocytes and in our in vivo observation IL-8 levels were related to fat mass. Therefore, we hypothesize that the main source of the

TABLE 2. Correlations between plasma IL-8 and other clinical parameters in lean and in obese subjects
Lean subjects (n r 35) P Obese subjects (n r 40) P

Age (yr) BMI (kg/m2) WHR Percentage of body fat FM (kg) Fat-free mass (kg) HbA1c (%) Plasma glucose (mmol/liter) Plasma insulin (pmol/liter) Plasma cholesterol (mmol/liter) Plasma triglycerides (mmol/liter) High-density lipoprotein cholesterol (mmol/liter) LDL cholesterol (mmol/liter) Mffm ( mol kg 1 min 1) TNF (pg/ml) sTNFR1 (ng/ml) sTNFR2 (ng/ml)

0.10 0.39 0.32 0.34 0.35 0.15 0.11 0.11 0.04 0.29 0.28 0.06 0.22 0.01 0.02 0.25 0.37

0.57 0.02 0.06 0.042 0.039 0.39 0.51 0.51 0.80 0.09 0.10 0.73 0.20 0.97 0.92 0.15 0.028

0.01 0.31 0.42 0.46 0.43 0.12 0.06 0.10 0.13 0.11 0.08 0.17 0.14 0.15 0.24 0.31 0.32

0.97 0.049 0.006 0.003 0.005 0.42 0.69 0.51 0.42 0.48 0.58 0.29 0.37 0.37 0.12 0.049 0.045

Straczkowski et al. IL-8 in Obesity

J Clin Endocrinol Metab, October 2002, 87(10):4602 4606 4605

FIG. 1. Correlations between IL-8 and WHR (A), FM (B), and sTNFR2 (C) in the obese group.

increased plasma IL-8 in obese individuals is adipose tissue. Additionally, our data suggest, although indirectly, that there may be regional difference of fat depots in ability to synthesize IL-8. Subjects with visceral obesity had higher plasma IL-8 and there was a relationship between IL-8 concentrations and WHR. These findings indicate that abdominal (visceral or sc) adipose tissue might produce and secrete more IL-8 than peripheral fat and give another, among others, possible explanation of the accelerated atherogenesis in individuals with visceral type of obesity. The limitation of those findings is that WHR is, at best, a surrogate measure of body fat distribution; the association between IL-8 and different fat compartments should be further explored. The increase in IL-8 levels was about 30% in obese individuals and about 50% when subjects with visceral obesity where analyzed separately. The observed elevation is much

smaller than that reported in inflammation (20) or diabetes (13), where the main source of IL-8 are macrophages and endothelial cells. Similar difference concerns other cytokines elevated in obesity, like TNF- and its receptors or IL-6 (21). Small prolonged increase in circulating IL-8 might be sufficient to elicit atherogenic function. Another interesting finding is correlation between TNFsystem and IL-8. TNF- stimulates IL-8 synthesis and release by different cell types, including adipocytes (6). TNF- is overexpressed in adipose tissue of obese subjects (22), and it might be one of the factors responsible for the IL-8 production in obesity. Plasma sTNFR2 levels are suggested to be the best indicator of TNF- system activity (17), which was observed also in our previous studies (23). In the present study, sTNFR2 was the parameter of TNF- system, which was most strongly related to IL-8 and predicted plasma values of this cytokine independently of other variables, except FM. The main limitation of our report is that it does not reveal any cause-effect relationship. On the basis of our findings, we cannot exclude the possibility that both TNF- and IL-8 are up-regulated in obesity in similar way by other, as yet undetermined factors. However, in the context of in vitro observations (6) and our results, we can hypothesize that higher circulating IL-8 levels in obesity are partly the result of TNFaction. TNF- increases cardiovascular risk via different mechanisms, mostly taking place in endothelium, like inhibition of insulin signaling leading to nitric oxide synthesis in the endothelial cells (24), stimulation of adhesion molecules expression (16), and many others. It is also possible that TNFmight have an influence on atherogenic process through its local action in adipose tissue, not only by inducing insulin resistance, but also by modulating adipocyte endocrine and paracrine function, for instance by increasing IL-8 synthesis and secretion. Insulin resistance is also associated with accelerated atherogenesis (15). In vitro IL-8 was down-regulated by the insulin-sensitizing agents thiazolidinedione ciglitazone and biguanide metformin (5). Therefore, it was tempting to speculate that insulin resistance might itself up-regulate IL-8 expression and secretion and that this might be an additional link with atherosclerosis. However, we were unable to detect a correlation between plasma IL-8 and insulin sensitivity in lean and obese subjects. Although the observed correlation values were of borderline significance when all the individuals were analyzed together, we suppose, on the basis of our findings, that such a tend would rather reflect an association of both parameters with body fat. In the study of Makino et al. (25), no relationship between insulin resistance and circulating IL-8, in contrast to IL-6, in patients with cancer was reported. Inhibitory effect of ciglitazone may be therefore associated with the fact, that thiazolidinediones are also inhibitors of TNF- expression (26). Bruun et al. (6) reported no effect of incubation with insulin on the IL-8 production by adipose tissue fragments. In the present study, fasting IL-8 was not related to fasting insulin within the groups of lean and obese subjects. However, we demonstrated an effect of acute hyperinsulinemia on plasma IL-8 levels. To the best of our knowledge, this is the first report about the effect of insulin on circulating IL-8. Our

4606

J Clin Endocrinol Metab, October 2002, 87(10):4602 4606

Straczkowski et al. IL-8 in Obesity 6. Bruun JM, Pedersen SB, Richelsen B 2001 Regulation of interleukin 8 production and gene expression in human adipose tissue in vitro. J Clin Endocrinol Metab 86:12671273 7. Baggiolini M, Loetscher P, Moser B 1995 Interleukin-8 and the chemokine family. Int J Immunopharmacol 17:103108 8. Ross R 1999 Atherosclerosisan inflammatory disease. N Engl J Med 340: 115126 9. Liu Y, Hulten LM, Wiklund O 1997 Macrophages isolated from human atherosclerotic plaques produce IL-8, and oxysterols may have a regulatory function for IL-8 production. Arterioscler Thromb Vasc Biol 17:317323 10. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone Jr MA, Luster AD, Luscinskas FW, Rosenzweig A 1999 MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 398:718 723 11. Yue TL, Mckenna PJ, Gu JL, Feuerstein GZ 1993 Interleukin-8 is chemotactic for vascular smooth muscle cells. Eur J Pharmacol 240:81 84 12. Moreau M, Brocheriou I, Petit L, Ninio E, Chapman MJ, Rouis M 1999 Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterol-loaded human macrophages: relevance to stability of atherosclerotic plaque. Circulation 99:420 426 13. Zozulinska D, Majchrzak A, Sobieska M, Wiktorowicz K, Wierusz-Wysocka B 1999 Serum interleukin-8 level is increased in diabetic patients. Diabetologia 42:117118 14. Hotamisligil GS, Spiegelman BM 1994 Tumor necrosis factor : a key component of the obesity-diabetes link. Diabetes 43:12711278 15. Reaven GM 1988 Role of insulin resistance in human disease. Diabetes 37: 15951607 16. Straczkowski M, Lewczuk P, Dzienis-Straczkowska S, Kowalska I, Stepien A, Kinalska I 2002 Elevated soluble intercellular adhesion molecule-1 levels in obesity: relationship to insulin resistance and tumor necrosis factor- system activity. Metabolism 51:7578 17. Hotamisligil GS, Arner P, Atkinson RL, Spiegelman BM 1997 Differential regulation of the p80 tumor necrosis factor receptor in human obesity and insulin resistance. Diabetes 46:451 455 18. DeFronzo RA, Tobin JD, Andres R 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 237:E214 E223 19. Gonzalez C, Cava F, Ayllon A, Guevara P, Navajo JA, Gonzalez-Bultrago JM 2001 Biological variation of interleukin-1 , interleukin-8 and tumor necrosis factor- in serum of healthy individuals. Clin Chem Lab Med 39:836 841 20. Santana C, Guindeo MC, Gonzalez G, Garcia-Munoz F, Saavedra P, Domenech E 2001 Cord blood levels of cytokines as predictors of early neonatal sepsis. Acta Paediatr 90:1176 1181 21. Dandona P, Weinstock R, Thusu K, Abdel-Rahman E, Aljada A, Wadden T 1998 Tumor necrosis factor- in sera of obese patients: fall with weight loss. J Clin Endocrinol Metab 83:29072910 22. Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM 1995 Increased adipose tissue expression of tumor necrosis factor- in human obesity and insulin resistance. J Clin Invest 95:2409 2415 23. Straczkowski M, Kowalska I, Dzienis-Straczkowska S, Stepien A, Skibinska E, Szelachowska M, Kinalska I 2001 Changes in tumor necrosis factorsystem and insulin sensitivity during exercise training program in obese women with normal and impaired glucose tolerance. Eur J Endocrinol 145: 273280 24. Kim F, Gallis B, Corson MA 2001 TNF inhibits flow and insulin signaling leading to NO production in aortic endothelial cells. Am J Physiol 280:C1057 C1065 25. Makino T, Noguchi Y, Yoshikawa T, Doi C, Nomura K 1998 Circulating interleukin 6 concentrations and insulin resistance in patients with cancer. Br J Surg 85:1658 1662 26. Hube F, Hauner H 1999 The role of TNF in human adipose tissue: prevention of weight gain at the expense of insulin resistance? Horm Metab Res 31:626 631 27. Urakaze M, Temaru R, Satou A, Yamazaki K, Hamazaki T, Kobayashi M 1996 The IL-8 production in endothelial cells is stimulated by high glucose. Horm Metab Res 28:400 401

study also suggests that this effect occurs at supraphysiological insulin concentrations and is dependent on obesity. One may hypothesize that adipose tissue is the probable site of insulin action regulating IL-8 levels. It should be noted that clamp conditions are nonphysiological and the question whether our findings might constitute an additional possible link between hyperinsulinemia and atherogenesis requires further investigations. It was demonstrated that high glucose concentrations are able to stimulate IL-8 production from cultured endothelial cells (27), and in diabetic subjects circulating IL-8 is related to a degree of metabolic control (13). Although fasting IL-8 levels were not related to fasting glucose concentrations, our results show that oral glucose load might have an effect on circulating IL-8, even in normoglycemic individuals. The effect of glucose ingestion is probably independent of obesity; the difference between lean and obese subjects after the OGTT reflects the difference in basal conditions. There also might be other factors responsible for the IL-8 increase in obesity, which remain to be determined. We conclude that plasma IL-8 levels are increased in normoglycemic obese subjects, and are related to fat mass and TNF- system. Circulating IL-8 is also acutely up-regulated by hyperinsulinemia in the obese. In the context of previously known actions of IL-8, it is possible that an increase in circulating IL-8 might be one of the factors linking obesity with greater cardiovascular risk.
Acknowledgments
Received January 30, 2002. Accepted June 26, 2002. Address all correspondence and requests for reprints to: Marek Straczkowski, M.D., Department of Endocrinology, Diabetology and Internal Medicine, Medical Academy, Bialystok, M.C. Sklodowskiej 24a, 15-276 Bialystok. E-mail: mstraczkowski@poczta.onet.pl.

References
1. Chan JM, Stampfer MJ, Ribb EB, Willett WC, Colditz GA 1994 Obesity, fat distribution and weight gain as risk factors for clinical diabetes in man. Diabetes Care 17:961969 2. Hubert H, Feinleb M, McNamara P, Castelli W 1983 Obesity as an independent risk factor for cardiovascular disease: a 26-year follow-up of participants in the Framingham study. Circulation 67:968 977 3. Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA 2001 The adipocyte: a model for integration of endocrine and metabolic signalling in energy metabolism regulation. Am J Physiol 280:E827E847 4. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA 2001 The hormone resistin links obesity to diabetes. Nature 409:307312 5. Bruun JM, Pedersen SB, Richelsen B 2000 Interleukin-8 production in human adipose tissue. Inhibitory effects of anti-diabetic compounds, the thiazolidinedione ciglitazone and the biguanide metformin. Horm Metab Res 32: 537541