Systemic low-grade inflammation is related to both circulating and adipose tissue TNFα, leptin and IL-6 levels in obese women

Abstract

OBJECTIVE: We assessed the relationships between four circulating acute phase proteins and the circulating and adipose tissue levels of three adipocytokines.

SUBJECTS: In all, 15 nondiabetic obese women with a body mass index (BMI) above 32 kg/m2 were investigated.

METHOD: Circulating concentrations of C-reactive protein (CRP), alpha 1 acid glycoprotein (AAG), fibrinogen, alpha 1 antitrypsin and both circulating and adipose tissue levels of interleukin (IL)-6, tumor necrosis factor (TNF)α and leptin were measured by either nephelometry or enzyme-linked immunosorbent assay.

RESULTS: We found a strong positive correlation between both circulating and adipose tissue levels of IL-6, TNFα and leptin and serum CRP levels. All these adipose tissue adipocytokines were also positively correlated with serum AAG levels. These correlations disappeared when adjusted for fat mass, suggesting that the relationship observed was dependent on fat amount.

CONCLUSION: Our results indicate a strong relationship between adipocytokines and inflammatory markers, and suggest that cytokines secreted by adipose tissue in obese subjects could play a role in increased inflammatory proteins secretion by the liver.

Introduction

Low-grade systemic inflammation, mainly characterized by increased levels of circulating C-reactive protein (CRP), is associated with an increased risk of cardiovascular disease (CVD).1 However, the mechanisms responsible for increased CRP levels are not well elucidated. Obese individuals have significantly higher CRP levels than nonobese subjects. This relationship remains after restricting the analysis to healthy, nonsmoking subjects.2, 3 One possible link between CRP and obesity assessed by body mass index (BMI) could be the cytokine production by adipose tissue. Among these cytokines, IL-6 is known as the major regulator of acute phase protein synthesis as clearly demonstrated in human hepatocytes.4 Indeed, we have previously shown that IL-6 from adipose tissue could be one of the links between circulating CRP and obesity in human,5 which is in accordance with another study.6 However, adipose tissue IL-6 secretion only partly explains liver production of CRP. Other adipocytokines, synthesized either almost exclusively (leptin) or partially (tumor necrosis factor (TNF)α) by adipocytes, could also be involved in this process.7, 8 In the past few years, many studies have been focussed on high-sensitivity (hs) CRP as a marker of low-grade inflammation.1, 2, 3, 4, 5 However, it is not known whether adipocytokines are related specifically to CRP or to a whole set of acute phase proteins produced by the liver in obesity. Indeed, other inflammation markers such alpha 1 acid glycoprotein (AAG) and alpha 1 anti-trypsin (AAT) have been found to be increased in obesity.9, 10, 11 We therefore assessed the relationships between several circulating acute phase proteins, that is, CRP, AAG, fibrinogen (Fg) and AAT levels and both circulating and adipose tissue leptin, IL-6, TNFα and plasminogen activator inhibitor (PAI)-1 content in obese women.

Subjects and methods

In all, 15 Caucasian nondiabetic obese women (age: 48±4 y; BMI: 40.2±1.9 kg/m2; waist/hip ratio: 0.98±0.02) were included in this study (Table 1). Of these, 10 patients were postmenopausal and five premenopausal as attested by the results from circulating FSH and oestradiol levels. Except obesity, all the subjects were in good health. All the patients reported a stable body weight, defined as a variation of less than 2 kg, for the 12-month period preceding the study. None was engaged in any type of exercise program or was excessively sedentary. All these subjects were involved in clinical investigations that were approved by the ethical committee of the Assistance Publique – Hôpitaux de Paris and that were performed according to the French legislation. Venous blood samples were taken between 08:00 and 09:00, after an overnight fast and stored at −80°C before immunoassays analysis. Subcutaneous abdominal adipose tissue biopsies were taken after an overnight fast as described previously.12, 13 Adipose tissue samples were immediately frozen in liquid nitrogen and stored at −80°C until protein analysis.

Table 1 Characteristics of the nondiabetic obese subjects (n=15)

Fasting plasma glucose was assayed enzymatically (hexokinase) using a multiparametric analyser (Hitachi 911, Roche-Boehringer, Meylan, France). Fasting plasma insulin was measured using commercial radioimmunoassay kits (Bi-Insulin IRMA, ERIA-Pasteur, Paris, France). Insulin resistance was assessed by the calculation of the HOMA index (fasting plasma insulin × fasting plasma glucose)/22.5. The serum levels of leptin, TNFα and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA, Quantikine leptin; Quantikine High Sensitivity TNFα and Quantikine IL-6; R&D Systems, Oxford, UK). Plasma PAI-1 was determined by ELISA (Stago, Asnières, France) as described previously.12 The same ELISA assay kits were used to determine the immunoreactive cytokines from adipose tissue after homogenization of 200 mg of frozen tissue in 400 μl of a buffer (pH 7.4) containing 10 mmol/l of Tris-HCl, 250 mmol/l of sucrose and a cocktail of protease inhibitors (Complete™, Boehringer Mannheim) as reported previously.12, 13 hs-CRP, AAG, Fg and AAT were assessed by immunonephelometry on IMMAGE (Beckman-Coulter, Villepinte, France). A body composition analysis by dual X-ray absorptiometry was performed, using the QDR 1000 from Hologic (Watham, MA, USA).

Statistical analysis

All results are presented as mean±s.e. The univariate associations between parameters were assessed by Pearson's correlation coefficient. Bivariate regression analysis was used for adjustment for age and fat mass. The threshold for significance was set at P=0.05.

Results

Relationship between anthropometric parameters, circulating adipocytokines and acute phase proteins

BMI was significantly correlated with circulating IL-6 (r=0.600, P<0.02), leptin (r=0.679, P<0.005) with a tendency for TNFα (r=0.503, P=0.06). BMI was also significantly correlated with hs-CRP (r=0.617, P<0.02) and AAT (r=0.684, P<0.005) but not with AAG and Fg (r=0.332, P: NS and r=0.287, P: NS respectively). We found significant positive correlations between the percentage of fat mass and the circulating levels of hs-CRP (r=0.732, P<0.005), AAT (r=0.845, P<0.0001), AAG (r=0.529, P<0.05) and leptin (r=0.831, P<0.0001). In contrast, no significant correlation was observed between the percentage of fat mass and the level of Fg, IL-6 or TNFα (r=0.408, P: NS; r=0.488, P: NS and r=0.381, P: NS, respectively). Finally, no correlation was observed with waist-to-hip ratio.

Relationship between the level of adipose tissue content in adipocytokine, circulating adipocytokines and acute phase proteins

We found a significant correlation between hs-CRP and the adipose tissue content in IL-6, TNFα and leptin (Figure 1a). In contrast, no significant correlation was found between hs-CRP and adipose tissue PAI-1 levels (r=0.317, P: NS), suggesting that hs-CRP was specifically related to adipocytokines. hs-CRP was also significantly correlated with circulating IL-6 (r=0.532, P<0.05), TNFα (r=0.571, P<0.05) and leptin (r=0.720, P<0.005). Interestingly, as observed for hs-CRP, we found a significant correlation between AAG and the adipose tissue content in IL-6, TNFα and leptin (Figure 1b). However, no significant correlations were found between AAG and circulating levels of IL-6, TNFα and leptin (r=0.262, P: NS; r=−0.192, P: NS and r=0.435, P: NS, respectively). Finally, no significant correlation was observed between AAG and both circulating and adipose tissue PAI-1 levels (r=−0.003, P: NS and r=0.486, P: NS, respectively). Fg was only correlated with adipose tissue TNFα content (r=0.587, P<0.05), whereas AAT correlated with both circulating IL-6 and leptin levels (r=0.546, P<0.05) and (r=0.773, P<0.0005). Interestingly, adipose tissue IL-6 was significantly correlated with adipose tissue leptin and TNFα (r=0.549, P<0.05 and r=0.600, P<0.05,) while adipose tissue leptin and TNFα were also correlated (r=0.594, P<0.05). Finally, adipose tissue PAI-1 was only correlated with adipose tissue TNFα level (r=0.655, P<0.01).

Figure 1
figure1

Correlation between (a) circulating CRP levels and total adipose tissue IL-6, TNFα and leptin content and (b) between circulating α1 glycoprotein acid and total adipose tissue IL-6, TNFα and leptin content in 15 nondiabetic obese women.

All the correlations observed between circulating and adipose tissue IL-6, leptin and TNFα levels and CRP remained significant after adjustment for age, but they disappeared after adjustment for fat mass, suggesting that adipose tissue is involved in low-grade inflammation in obese women. When adjusted for age, the correlations remained significant between adipose tissue IL-6, leptin and TNFα. Finally, when adjusted for fat mass, these correlations remained significant for IL-6 and leptin but not for TNFα.

Discussion

Over the past few years, it has become increasingly clear that inflammation could represent a mechanism responsible for CVD and that increased circulating inflammation markers could be predictive of cardiovascular events.14, 15 Among the inflammation markers available, hs-CRP appeared as one of the most powerful independent predictors of cardiovascular events.1, 6, 14, 15 The mechanisms involved in increased CRP levels are not well elucidated. CRP has been linked to the degree of obesity and it has been proposed that cytokines such as IL-6 and TNFα could be a link between BMI, CRP and CVD.5, 6 More recently, leptin has also been linked to inflammation in human.7, 8, 16 However, it is not clear whether cytokines originating from adipose tissue represent an important message for overall acute phase protein synthesis by the liver. These cytokines could be secreted by adipocytes and by inflammatory cells such as macrophages present in adipose tissue from obese subjects.17 In the present study, we found a significant correlation between CRP and circulating levels of adipocytokines, that is, IL-6, TNFα and leptin in obese women as reported previously.6, 7, 16 When adjusted for fat mass, these correlations disappeared, which suggest that the relationship observed was related to increased fat amount. In order to assess whether adipose tissue was involved in low-grade inflammation, we directly measured the adipocytokines content in adipose tissue. Interestingly, we report for the first time a positive association between the adipose tissue content of IL-6, TNFα and leptin and the level of circulating inflammation proteins in nondiabetic obese women, suggesting that adipose tissue secretion products could be involved in the regulation of acute phase protein synthesis. These results are in line with two recent papers showing that obesity-induced chronic inflammation in adipose tissue could play a crucial role in obesity-related insulin resistance.17, 18 These studies clearly demonstrated that fat macrophages infiltration is enhanced in obesity in both mice and human. This could partly explain the increased adipose tissue IL-6 and TNFα levels, which in turn could induce insulin resistance in adipose cells as recently suggested.19 Indeed, both IL-6 and TNFα are able to induce insulin resistance in adipose cells by inhibiting insulin signalling.20, 21

In addition to adipose tissue IL-6, TNFα and leptin content, we analysed PAI-1, another protein mainly released from human adipose tissue,22 not considered as a stimulatory mediator of CRP synthesis by the liver. We found no correlation between both plasma and adipose tissue PAI-1 and circulating CRP levels. This suggests that some but not all adipose tissue products are related to the systemic inflammatory process. Our results are in line with a recent published study reporting a positive correlation between hs-CRP and both leptin and TNFα adipose tissue mRNA expression in a population including women with diabetes and metabolic alterations with a wide range of BMI (19–65 kg/m2).8 We selected only nondiabetic obese women in the present work to get rid of the alterations linked to hyperglycaemia. Our results indicate a strong relationship between adipocytokines and inflammatory markers, and suggest that cytokines secreted by adipose tissue could play a role in increased inflammatory proteins secretion by the liver in obesity. This could explain increased CRP and AAG levels observed in obese patients.2, 3, 9 CRP could be deleterious on the arterial wall since it has been found to promote directly endothelial cell inflammation and atherosclerotic processes.23, 24, 25, 26 Therefore, to reduce CRP levels could help to prevent vascular damage. This emphasizes the importance of weight loss programs in obese subjects since previous studies showed a significant reduction in proinflammatory adipocytokines and circulating hs-CRP levels after weight loss.13, 27, 28, 29, 30, 31

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Acknowledgements

This work was supported by grants from Le Ministère de la Santé (Programme Hospitalier de Recherche Clinique No. AOA94042) and INSERM.

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Correspondence to J-P Bastard.

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Maachi, M., Piéroni, L., Bruckert, E. et al. Systemic low-grade inflammation is related to both circulating and adipose tissue TNFα, leptin and IL-6 levels in obese women. Int J Obes 28, 993–997 (2004). https://doi.org/10.1038/sj.ijo.0802718

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Keywords

  • C-reactive protein
  • inflammation
  • cytokine
  • adipocytokine
  • adipose tissue

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