Systemic Inflammation, Adipose Tissue Tumor ... - Wiley Online Library

Systemic Inflammation, Adipose Tissue Tumor Necrosis Factor, and Leptin Expression Monica Bullo´,* Pilar Garcıá-Lorda,* Isabel Megias,* and Jordi Salas-Salvado´*†

Abstract ´ , MONICA, PILAR GARCIÁ-LORDA, ISABEL BULLO MEGIAS, AND JORDI SALAS-SALVADO ´ . Systemic inflammation, adipose tissue tumor necrosis factor, and leptin expression. Obes Res. 2003;11:525-531. Objectives: To explore the links between tumor necrosis factor ␣ (TNF␣) and leptin adipose tissue expression and low-grade systemic inflammation and to determine the relationship between inflammation and the degree of adiposity, the presence of type 2 diabetes, and other cardiovascular risk factors. Research Methods and Procedures: Ninety-one women (BMI 19 to 65 kg/m2) were divided into tertiles of CRP. Insulin resistance was calculated using the HOMA method. Albumin, fibrinogen, C-reactive protein (CRP), interleukin-6, sTNFR1, sTNFR2, and leptin levels were measured in serum and plasma samples. TNF␣ and leptin expression were measured by reverse transcription-polymerase chain reaction in abdominal subcutaneous adipose tissue samples. Results: CRP was positively related to BMI and upper distribution of adiposity. TNF␣ and leptin adipose tissue expression were higher in the upper tertile of CRP. Also, peripheral levels of both soluble TNFRs and leptin were higher in patients with the greatest inflammation degree. Diabetes, dislipidemia, and hypertension were most prevalent in patients in the upper CRP tertile. Inflammatory markers of diabetic women were significantly different from those of nondiabetic women, even after adjusting for differences in body fat. BMI, type 2 diabetes, and adipose TNF␣ mRNA levels were significant predictors of serum CRP levels (r2 ⫽ 0.28, p ⬍ 0.001). Discussion: These results are in agreement with the hypoth-

Received for review July 22, 2002. Accepted in final form January 22, 2003. *Unitat de Nutricio´ Humana, Facultat de Medicina i Cie`ncies de la Salut de Reus, Universitat Rovira i Virgili, Reus, Spain and †Unitat de Nutricio´, Servei de Medicina Interna, Hospital Universitari Sant Joan, Reus, Spain. Address correspondence to Jordi Salas-Salvado´, M.D., Ph.D., Unitat de Nutricio´ Humana Facultat de Medicina i Cie`ncies de la Salut de Reus Universitat Rovira i Virgili C/Sant Llorenç, 21 43201 Reus, Spain. E-mail: [email protected] Copyright © 2003 NAASO

esis that the synthesis of adipose tissue TNF␣ and leptin could induce the production of interleukin-6, CRP, and other acute-phase reactants, thus contributing to the maintenance of chronic low-grade inflammation state involved in the progression of obesity and its associated comorbidities. Key words: C-reactive protein, IL-6, albumin, TNF␣system, leptin

Introduction Obesity is a chronic metabolic condition with important public health implications. It is associated with a high risk of cardiovascular disease, insulin resistance, hypertension, and other comorbidities. Type 2 diabetes is associated with an increased risk of coronary artery disease, especially among women (1,2). In the last few years, there has been considerable evidence to support the inflammatory nature of atherosclerotic coronary heart disease (3–5). Several authors have suggested that inflammation may be the link between obesity, type 2 diabetes, and cardiovascular disease. In fact, in the last few years, increases in several inflammatory markers such as C-reactive protein (CPR)1 or interleukin-6 (IL-6) have been reported in obesity (6 –9). It has been suggested recently that the production of some cytokines such as TNF␣ (10), IL-6 (11), and leptin (12) by adipose tissue may be involved in the low-grade acutephase response associated with obesity and related comorbidities (13,14). However, whether the production of adipose tissue cytokine modulates the production of acutephase reactants has not yet been shown. Only indirect evidences such as the stimulatory effect of TNF␣ on IL-6 production (15), the pro-inflammatory properties of IL-6 in the production of acute-phase proteins by the liver (16,17), and the relationship between the adipose tissue content of IL-6 and serum CRP (18) sustained the possible relationship

1 Nonstandard abbreviations: CRP, C-reactive protein; IL, interleukin; HOMA, homeostasis model assessment; ESR, erythrocyte sedimentation rate.

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Inflammation, Obesity, and Type 2 Diabetes, Bullo´ et al.

between adipose tissue and inflammation. Also, an inhibitory role of adipocyte lipoprotein lipase and insulin receptor activity by TNF␣ and IL-6 (19,20) or impaired insulin actions of leptin (21,22) may be related to the development and maintenance of metabolic disorders associated with obesity. There have been several recent studies, but so far little is known about adipose tissue cytokine production and its relationship to peripheral acute-phase reactants in obesity and related metabolic derangements. Therefore, we have studied the links between several cytokines produced by adipose tissue with peripheral levels of the most important acute-phase inflammation markers and how these cytokines relate both to the degree of adiposity and to the presence of type-2 diabetes or other cardiovascular risk factors in a group of women with several degrees of adiposity.

Research Methods and Procedures Subjects Ninety-six women, 19 to 25 years of age, with a BMI between 19 and 65 kg/m2, were previously recruited for another study (23). Five of the women were excluded from this study because their leukocyte counts were higher than 11 ⫻ 109 cells/L. Criteria for exclusion were the presence of infectious, inflammatory, neoplastic, or systemic diseases, hypothyroidism or endocrine diseases other than diabetes, or the active use of antiobesity drugs. The hospital ethics committee approved the study protocol, and all subjects gave their informed written consent to participate in the study. Plasma and Serum Sample Collection and Laboratory Procedures Body composition was estimated by bioelectrical impedance (Human-In Scan, Dietosystem, Sabadell, Spain) (24). Fat-free mass was estimated using the gender-specific equations validated by Segal (25). Fasting glucose, triglycerides and lipid profile, albumin, fibrinogen, and erythrocyte sedimentation rates (ESRs) were assayed by the hospital’s routine chemistry laboratory. White blood cell count was assessed using a quantitative automated hematology analyzer. Insulin plasma concentrations were measured by radioimmunoassay (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Insulin resistance was measured from fasting glucose and insulin concentrations using the HOMA method as previously described (26). Serum CRP levels were assessed by a commercial enzyme-linked immunosorbent assay kit (Alpha Diagnostic International, San Antonio, TX) with a detection limit of 0.35 ng/mL. The intra- and interassay coefficients of variation were ⬍4.5% and 5%, respectively. Immunoenzymometric assays were used to determine the levels of both sTNFR plasma levels (BioSource, Fleunes, Belgium). Leptin levels were assessed 526


by radioimmunoassay (Linco Research, St. Louis, MO). IL-6 was assessed by a high-sensitivity commercially available enzyme-linked immunosorbent assay (Amersham Pharmacia Biotech) with a detection limit of 0.1 pg/mL. The intra- and interassay coefficients of variation were ⬍10%. Adipose Tissue Measurements Adipose tissue samples were obtained from subcutaneous abdominal depots by surgical procedures in which the anesthetic procedure was standardized. (Epinephrine was avoided.) Quantitative competitive polymerase chain reaction assays were performed to measure TNF␣ and leptin expression as has been described previously (23). TNF␣ and leptin mRNA levels were expressed as specific mRNA divided by beta-actin mRNA. Statistical Methods Statistical analysis was performed using the SPSS/PC statistical program (version 10.0 for Windows; SPSS, Inc., Chicago, IL). Descriptive results of continuous variables are expressed as means and SE. Differences in mean values between groups were assessed by the Student’s t test. Relationships between two quantitative variables were assessed by Pearson’s correlation coefficient. A stepwise multiple regression analysis was performed to identify the independent predictors of CRP serum levels. Covariance analysis was used to adjust variables, and the significance of the differences between groups was assessed by the F statistic. Significance was set at p ⬍ 0.05.

Results Twenty of the women in the overall study population were diabetic (27). Eight of these women were treated by diet alone, and 12 were treated by oral antidiabetic agents. Good glycemic control (hemoglobin A1c ⱕ 7.0%) was observed in eight patients. None of the diabetic women was taking thiazolidenedione during the study. Fifty-two women were dyslipidemic, and 35 women had hypertension. Fortyfive percent of patients were premenopausal. Only nine patients were current smokers. Volunteers were divided into tertiles of CRP serum levels; cut points were 0.196 to 0.97 mg/L. The baseline characteristics for the groups are shown in Table 1. As predicted, patients with higher levels of serum CRP were significantly more obese and showed higher levels of positive acute-phase reactants. Serum albumin was significantly lower in the medium and upper tertile. Although no significant differences were observed between tertiles, plasma IL-6 levels were higher in the medium and upper tertile than in the lower tertile (1.05 ⫾ 0.24 pg/mL, 0.99 ⫾ 0.20 pg/mL, and 0.53 ⫾ 0.11 pg/mL, respectively). Plasma leptin levels and both soluble TNF␣ receptor levels were


Table 1. Baseline characteristics of women in relationship to the tertiles of C-Reactive Protein (CRP) in milligrams per liter

Age (years) BMI (kg/m2) Body fat (%) Waist circumference (cm) Glucose (mM) HOMA IR Total cholesterol (mM) High-density lipoprotein-cholesterol (mM) VLDL-cholesterol (mM) LDL-cholesterol (mM) Triglycerides (mM) Apolipoprotein A (mg/dL) Albumin (g/L) ESR (mm/h) Fibrinogen(g/L) Leukocyte count (⫻10E9/l)

Tertile 1 (CRP < 0.196)

Tertile 2 (0.196 < CRP < 0.97)

Tertile 3 (CRP > 0.97)

48.50 (1.95) 30.70 (1.36) 38.37 (1.86) 95.55 (3.20) 5.66 (0.21) 3.22 (0.64) 5.83 (0.18) 1.84 (0.07) 0.52 (0.04) 3.49 (0.19) 1.16 (0.1) 157.67 (4.29) 45.43 (0.61) 10.99 (1.33) 3.77 (0.20) 6.10 (0.26)

47.87 (1.73) 36.93 (1.60)* 43.31 (1.74)* 107.04 (3.42)* 6.09 (0.30) 5.47 (1.06) 5.57 (0.16) 1.65 (0.04) 0.65 (0.04) 3.36 (0.14) 1.47 (0.1) 150.64 (4.14) 43.42 (0.60)* 11.91 (1.38) 4.40 (0.16)* 6.62 (0.19)

50.07 (1.06) 40.33 (1.29)† 46.42 (1.77)* 111.89 (2.21)† 7.21 (0.51)*‡ 8.69 (1.38)*‡ 5.93 (0.24) 1.54 (0.17)* 0.73 (0.07)* 3.58 (0.22) 1.95 (0.23)†‡ 145.72 (4.76)* 42.72 (0.58)* 19.67 (3.44)*‡ 4.32 (0.25)* 7.11 (0.23)*

LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein. Values are expressed as means (SE). * p ⬍ 0.01. † p ⬍ 0.001 vs. the lowest tertile. ‡ p ⬍ 0.05 vs. medium tertile.

also significantly higher in patients who had high levels of CRP (Figure 1). The differences in peripheral levels of cytokines between CRP tertiles were also maintained after adjusting values for differences in body fat. Subcutaneous adipose TNF␣ and leptin expression were also higher in patients with high degree of inflammation (Figure 1). Also, adipose tissue TNF␣ and leptin expression were significantly higher in the upper and medium tertile than in the lower tertile, even after adjusting for adiposity (1.19 ⫾ 0.13, 0.94 ⫾ 0.10, and 0.79 ⫾ 0.10, respectively, for TNF␣, and 0.91 ⫾ 0.03, 0.85 ⫾ 0.03, and 0.69 ⫾ 0.06, respectively, for leptin, p ⬍ 0.05). In a bivariate correlation analysis, CRP and IL-6 were positively related to the BMI (r ⫽ 0.39, r ⫽ 0.25, and p ⬍ 0.05, respectively) and the upper body distribution of adiposity (r ⫽ 0.32, r ⫽ 0.25, and p ⬍ 0.05 respectively). CRP serum levels were also positively related to VLDL-cholesterol (r ⫽ 0.25, p ⬍ 0.005) and triglycerides (r ⫽ 0.23, p ⬍ 0.005). IL-6 was positively related to total-cholesterol:high-density lipoprotein-cholesterol ratio (r ⫽ 0.25, p ⬍ 0.05) and negatively related to apolipoprotein A (r ⫽ ⫺0.24, p ⬍ 0.05). CRP was also related to both sTNFR and leptin levels (r ⫽ 0.297 for sTNFR1, r ⫽ 0.309 for sTNFR2, and r ⫽ 0.308 for leptin, p ⬍ 0.05), adipose

tissue TNF␣ and leptin expression (Figure 2), and the degree of insulin resistance determined by HOMA (r ⫽ 0.22, p ⫽ 0.039). Diabetes, dyslipidemia, and hypertension were

Figure 1: Plasma levels of both soluble TNF receptors and leptin, and adipose tissue expression of both cytokines in CRP tertile ´ groups. (*, p ⬍ 0.05; **, p ⬍ 0.001 vs. the lowest tertile; and Yp ⬍ 0.05 vs. medium tertile). Values are expressed as means (SE).


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Figure 2: Relationship between CRP serum levels and TNF␣ or leptin adipose tissue expression.

more prevalent in patients in the upper CRP tertile than in patients in the lower or medium tertile (Figure 3). In fact, when the women were classified according to the presence or absence of type 2 diabetes, 36.6% of those in the upper tertile of CRP were diabetic as opposed to 6.6% of those in the lower tertile or 23.3% of those in the medium tertile. Other inflammatory markers, such as sTNFR, leukocyte count, albumin, and serum CRP levels, were significantly different between diabetic and nondiabetic women. These differences remained even after adjusting for differences in body fat (2.16 ⫾ 0.30 mg/L serum CRP levels in diabetic women vs. 1.30 ⫾ 0.20 mg/L in nondiabetic women, p ⫽ 0.018). No significant differences were observed in serum CRP, IL-6, albumin, ESR, plasma leptin levels, or both soluble TNF␣ receptor levels between patients with good glycemic control and patients with poor glycemic control, or between patients treated with diet and patients treated with oral hypoglycemics. We did not observe any significant differences in any of the inflammatory parameters in 528


Figure 3: Inflammatory markers in relation to the number of metabolic disorders associated with obesity (diabetes, dyslipidemia, and hypertension). None or one metabolic disease (0), two metabolic diseases (1), and three metabolic diseases (2). p ⬍ 0.05 vs. group 0 (*).

relation to the type of antihypertensive therapy, smoking status, or menopausal state after adjusting for differences in adiposity (data not shown). Finally, when CRP was considered as a dependent variable in a forward stepwise multiple regression analysis with BMI, age, type 2 diabetes, HOMA, and adipose tissue TNF␣ expression as independent variables, it was found that BMI, type 2 diabetes, and TNF␣ mRNA were predictors of CRP variability (r2 ⫽ 0.28, p ⬍ 0.001).


Discussion In this study, we report that the degree of adiposity, the presence of type 2 diabetes, and adipose tissue TNF␣ expression are predictors of serum CRP concentrations. In general, the relationship between peripheral inflammatory markers, plasma cytokines, and overall adipose tissue cytokine expression supports the dynamic role of adipose tissue in the etiology and maintenance of chronic inflammation associated with obesity, type 2 diabetes, and cardiovascular disease. Low-grade systemic inflammation, mainly characterized by higher levels of serum CRP and plasma IL-6, has recently been observed in obesity, type 2 diabetes, and cardiovascular disease (10,14,28,29). Other inflammatory markers, such as blood cell count, fibrinogen, ESR, inflammation-associated hemostasis variables, and low-serum albumin, have also been associated with obesity and its metabolic derangements (30 –32). These relationships, and the reduction of inflammatory marker levels observed in some longitudinal studies performed in obese people (33–35), suggest that there may be a link between inflammation, obesity, and their comorbidities. Several authors have recently suggested that this lowdegree inflammation observed in obese people is caused by the higher secretion of pro-inflammatory cytokines described in the adipose tissue of these patients (11,23). TNF␣ is a potent cytokine that induces the production of IL-6 (36), which is the major determinant of the acute-phase response (16,17) and is also produced by adipose tissue. Surprisingly, despite the possible role of the TNF␣-system in the lowgrade systemic inflammation associated with obesity, few studies have measured TNF␣ plasma levels to show a positive relationship with CRP serum levels in obesity (9,35). Because circulating TNF␣ is not considered a good measure of this cytokine activity (37,38), in our study we have evaluated the TNF␣-system, including the adipose tissue cytokine expression and the plasma sTNFR1 and sTNFR2 concentrations. We have shown that there is a positive relationship between subcutaneous adipose TNF␣ expression and both sTNFR and CRP serum levels. It is important to note that adipose tissue TNF␣ mRNA was a predictor of CRP levels, regardless of the degree of obesity. Moreover, adipose tissue expression of TNF␣ was also related to other inflammatory markers such as albumin, ESR, leukocyte count, fibrinogen, and alkaline phosphatase. Although it seems that subcutaneous adipose tissue almost does not contribute to the secretion of circulating TNF␣ (11), this cytokine could have an autocrine role in the production of other pro-inflammatory cytokines such as IL-6 or leptin from adipose depots. We also evaluated the adipose tissue expression of leptin, showing a positive relationship with serum CRP levels and plasma IL-6. In fact, several authors have attributed some pro-inflammatory properties to leptin (39,40). Therefore, it is possible that

although controversial data argue against the relationship between TNF␣ and adipocyte leptin production (41,42), the possible overlap described between these two proteins (38) could be a mechanism through which TNF␣ can modulate the inflammation (43). However, the design of our study did not enable a direct cause-effect relationship to be established between cytokine adipose tissue production and peripheral inflammatory processes. Long-term longitudinal studies will be required. Recent data also indicate that there is a relationship between insulin resistance and an increased acute-phase response. It has also been suggested that diabetes is a disease of the innate immune system (13). The relationship observed between CRP and IL-6 levels with insulin resistance measurements sustained this hypothesis (44). However, in our study, CRP serum levels, but not IL-6, were significantly higher in obese diabetic patients than in obese nondiabetic patients, even after we adjusted for differences in adiposity. This supports the notion that the relationship between IL-6 and insulin action may, therefore, be mediated by adiposity. These discrepancies between the two acutephase reactants in relation to diabetes have also been observed in a recent prospective study carried out with a large cohort of women (29). It is important to note that other inflammatory markers, such as lower serum albumin levels or higher leukocyte number, fibrinogen, and ESR, were also associated with insulin resistance and type 2 diabetes independently of obesity. This independent association may be due to the higher levels of soluble TNFR and adipose tissue TNF␣ expression in diabetic patients, which was previously reported by our group (23,38), and to the direct role of TNF␣ in the production of hepatic albumin and fibrinogen described in vitro (45). However, insulin is a positive modulator of hepatic albumin synthesis (46) and has an inhibiting role in fibrinogen production (47). Therefore, the TNF␣ per se or the insulin resistance induced by TNF␣ and leptin may be indirect mechanisms involved in acute-phase reactions. Finally, evidence is growing that low inflammatory status may be crucial in the progression and severity of atherosclerotic disorders. However, whether CRP or IL-6 plays a causative role or simply acts as a marker of acute-phase reactions is unknown. The design of our study does not enable us to determine the role of these inflammatory markers. However, the positive relationship of CRP, IL-6, and other acute-phase reactants with several components of lipid profile supports the hypothesis that inflammation plays a direct role in the etiology of cardiovascular disease. However, this relationship was markedly reduced when differences in fat mass were taken into account. Taken together, these data suggest that adipose tissue TNF␣ and leptin, which are both overexpressed in obesity, can induce the production of IL-6, CRP, and other acutephase reactants through their autocrine or endocrine role. OBESITY RESEARCH Vol. 11 No. 4 April 2003

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This low-peripheral inflammatory status can contribute to an increase in the risk of diabetes and cardiovascular disease. Although the changes in acute-phase reactants are smaller than those in acute infectious diseases, the chronicity of low-grade inflammation may be decisive in the progression of obesity and its comorbidities. Longitudinal studies are needed to confirm the role of adipose tissue in the acute-phase response associated with obesity and its related comorbidities.

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