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ORIGINAL ARTICLES

Is there a relationship between the increase in leptin, CRP, TNF-α, and NO and the degree of obesity in obese Egyptian adolescents?

El-Wakkad, Amanya; Hassan, Nayera Elmorsib; Sibaii, Hibac; El Zayat, Salwac

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doi: 10.1097/01.MJX.0000407614.32015.7a
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Abstract

Introduction

Obesity is considered as a low-grade chronic inflammatory condition 1 characterized by elevated circulating levels of leptin 2, C-reactive protein (CRP) 3, and TNF-α 4; these proinflammatory cytokines are also well-known inducers of nitric oxide (NO) overproduction 5. Leptin, the product of ob gene, is an anorexic hormone secreted by adipose tissue, which is primarily known for its role as a central regulator of food intake, body weight, and adipose stores. Circulating leptin concentrations are proportional to the total adipose mass and it decreases after weight loss 6. It may provide a possible connection between body fat and immune function 6. Obese individuals typically have relatively elevated serum leptin levels, suggesting that, in most humans, obesity may be associated with leptin insensitivity rather than with leptin deficiency 7. The study of Jang et al.8 revealed that NO is considered as a factor with a role in the modulation of food intake and obesity, and that NO is a potential regulator of leptin-induced lipolysis 9. Furthermore, a high-fat diet increased the expression of inducible NO synthase (NOS) in various peripheral tissues 5, and the inhibition of NOS in adipose tissue leads to increased lipolysis 10. Also, the study of Choi et al.11 has demonstrated elevated serum levels of NO metabolites (nitrate and nitrite) in obese adolescents, and this increase begins in participants with a BMI greater than 25 kg/m2. Ntyintyane et al.12 stated that leptin is a hormone secreted by the adipose tissue, and has been positively correlated with obesity. This hormone controls body weight through the central regulation of food intake and energy expenditure 13. In addition, Bruno et al.14 showed that the adipocyte-derived hormone leptin may provide a possible connection with the proinflammatory CRP, TNF-α, and NO 15,16. Also, the study of Dixit et al.17 revealed that NO might be crucial in mediating the endocrine effects of leptin in the pituitary gland and the mononuclear cells.

Dube et al.18 have demonstrated that hyperleptinemia in obesity is associated with increased CRP; a positive correlation between CRP and leptin has also been shown previously 19,20. Recent studies have demonstrated that serum concentrations of CRP 21 and leptin 22 were positively associated with BMI. Bastard et al.23 reported that CRP is regulated by multiple cytokines including TNF-α. The aim of this study was to investigate the relation between the degree of obesity and the serum level of leptin, CRP, TNF-α, and NO.

Methods

The current study was carried out on obese Egyptian adolescents of both sexes, with an age range from 13 to 18 years, in the National Research Centre, Egypt, to estimate the prevalence of obesity among school children and adolescents. It was a cross-sectional survey. Four local public schools situated in the Giza governorate were enrolled in this study regarding adolescents (two primary schools and two secondary schools). The study included boys and girls during the period of October 2007–April 2009. Permission to perform the study was granted by the Ministry of Education, and the directors of the school were included in the research.

The protocol was approved by the ‘Ethical Committee’ of the ‘National Research Centre’. Of the total sample, 103 adolescents (22 boys and 81 girls) with the complaint of obesity were included in the current research after obtaining written informed consent from their parents. Student assent was also obtained. The adolescents were required to meet the following inclusion criteria: age 13–18 years, and BMI greater than 95 percentile for age and sex on the basis of Center of Disease Control Charts 24. Adolescents were excluded if they had a prior major illness, including type I or II diabetes, took medications, or had a condition known to influence body composition, insulin action, or insulin secretion (e.g. glucocorticoids therapy, hypothyroidism, Cushing's disease).

Each adolescent underwent a complete physical examination, including anthropometric measures. The height and the weight were recorded. The height was measured to the nearest 0.5 cm on a Holtain portable anthropometer, and the weight was determined to the nearest 0.1 kg on a Seca scale Balance with the participant dressed in minimum clothes and no shoes. The BMI was calculated as the weight (in kilograms) divided by the height (in meters) squared. According to their BMI results, the participating adolescents were divided into two groups according to the BMI percentile for age and sex: group I with BMI greater than 95 percentile and less than 97 percentile; group II with BMI greater than 97 percentile 24. Blood samples was drawn from the obese Egyptian adolescents, and the serum was separated and kept frozen at −70°C until analysis.

Biochemical assays

Serum CRP levels were determined with an enzyme-linked immunosorbant assay (ELISA) technique according to the method of Roberts et al.25, using commercial kits (BioCheck Inc., Foster City, California, USA), and the sensitivity of the detection level was 0.1 mg\l. Serum concentrations of cytokines TNF-α were measured using commercially available ELISA kits (Ani BiotechOy Orgenium Laboratories Business Unit Finland; Bender MedSystems GmbH Campus Vienna Biocenter, Vienna, Austria), and the sensitivity of detection for TNF was 2.3 pg/ml according to the method described by Adolf and Apfler 26. Leptin levels were determined with an ELISA procedure using commercial kits (Diagnostics Biochem Canada Inc., Ottawa, Canada), and the sensitivity of the detection level was 0.5 ng/ml according to the method of Maffei et al.27. Serum levels of NO were measured using a colorimetric nonenzymatic assay for NO product no. NB88 (Oxford Biomedical Research, Oxford, USA, Superior Science Reliable Results); the kit can be used to accurately measure as little as 1 pmol/ml following the method described by manufacturer.

Statistical analysis

All values are expressed as mean±SD and the differences among the two groups were calculated by Student t-test. Correlations were performed between different parameters using Pearson correlation, and P-value <0.05 was considered significant. All analyses were carried out using SPSS version 9.0 (IBM, Chicago, IL, USA) statistical software.

Results

The data for the two groups are shown in Table 1.

T1-8
Table 1:
Showing the comparison between group I and II for age, sex and BMI percentile

As shown in Table 2, the BMI percentile for age and sex (Center of Disease Control BMI for age growth charts) 24 was used to classify obese adolescents into two groups. The level of proinflammatory cytokine TNF-α was significantly increased (P<0.0005) in the very obese participants in group II (BMI>97 percentile) as compared with group I (BMI>95 percentile and <97 percentile). Also, CRP has shown a very high significant increase (P<0.0005) in group II as compared with group I, whereas leptin has shown a significant increase at P<0.0005 in group II in comparison with group I. NO has shown a significant increase at P<0.005 in the very obese participants in group II as compared with group I.

T2-8
Table 2:
Comparison of different anthropmetrical and biochemical parameters between obese Egyptian adolescents in group I and group II

Correlations between BMI and NO, leptin, CRP, and TNF-α in group II are shown in Table 3. A positive correlation between BMI and NO at r=0.824, P<0.0005 was recorded, as shown in Table 3; also, a highly positive correlation between BMI and leptin at r=0.944, P<0.0005 and a positive correlation of BMI with CRP at r=0.981, P<0.0005 were observed. Similarly, a positive correlation between BMI and TNF-α at r=0.953, P<0.0005 was detected. In addition, there was a positive correlation between NO and leptin, CRP, and TNF-α at r=0.816, P<0.0005; r=0.816, P<0.0005; r=0.822, P<0.0005; and r=0.793, P<0.0005, respectively, and leptin with CRP and TNF-α at r=0.970, P<0.0005 and r=0.915, P<0.0005, respectively. Also, a positive correlation between CRP and TNF-α at r=0.944, P<0.0005 has been observed.

T3-8
Table 3:
Correlation Between different parameters in group II

Discussion

The data in the present study revealed that CRP, NO, TNF-α, and leptin were positively correlated with BMI, which was in agreement with Wu et al.28, Choi et al.11, Weichhaus et al.29, and Buettner et al.22, respectively; in addition, participants with BMI greater than 97 percentile had higher serum concentrations of TNF-α, leptin, CRP, and NO than participants with BMI lesser than 97 percentile, which led us to determine whether this increase is associated with the degree of obesity. No similar study has been reported in this concern. However, it has been shown by the study of Chen et al.20 that leptin and high-sensitivity CRP, and TNF-α, are tightly linked, and this was also true in the current study in group II. Leptin is secreted from adipocytes including production of other adipocytokines such as TNF-α that promote high-sensitivity CRP synthesis by the liver 20. In addition, there was an increase in the serum concentration of NO in obese Egyptian adolescents, and there was a significant correlation between BMI and NO, and this was proved by Choi et al.11. These investigators showed that this increase begins in participants with a BMI greater than 95 percentile 11. Therefore, it seems that increased serum concentrations of NO may be a result of its production in adipose tissue. There was also an increase in TNF-α and NO because TNF-α is a mediator of inflammation and it enhances the production of reactive oxygen species including inducible NO 30. Our results showed increased serum concentrations of NO in obese adolescents and a significant positive correlation between serum NO concentration and BMI of obese Egyptian adolescents .These findings were in agreement with the study of Magdalena et al.31 and Choi et al.11. In addition, there was a significant positive correlation between NO and leptin, and this was in agreement with Konukoglu et al.32. This increase was explained by Mehebik et al.16, who demonstrated that NO produced in response to leptin by adipocytes could also be a mediator of some leptin effects on adipose tissue metabolism, adipogenesis, and the physiological concentrations of leptin stimulate NOS activity in adipocytes. NOS activity is a novel target for leptin in adipocytes and leptin-induced NOS activity is at least in part the result of NOS phosphorylations through both protein kinase A and p42/p44 MAPK activation. This led them to hypothesize that NO is a potentially important factor for leptin signaling in adipocytes. However, it has been shown that the increase in CRP concentration has been reported to be significantly related to the degree of obesity 33.

In the present study, CRP was increased significantly in obese adolescents, and this was consistent with the results of Cook et al.34. Das 35 explained this increase in CRP by activating a complement that binds to Fc receptors and leads to the generation of proinflammatory cytokines. These findings together with the correlation between serum concentration of CRP, leptin, NO, and TNF-α with the BMI in the group of obese participants whose BMI was greater than 30 kg/m2 support the hypothesis that higher concentrations of these parameters in group II may be the result of increased synthesis in the adipose tissue and fat cells, which was in agreement with Magdalena et al.31, and that the opposite effects of these substances on the development of adipose tissue may constitute a mechanism regulating further body mass gain 32. In general, the present study showed that there was an increase in the four parameters measured in the study, and also, there was a correlation between CRP, leptin, TNF-α, and NO, and BMI, which led us to conclude that the degree of obesity is responsible for this increase.

F1-8
Figure

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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