Central obesity

Pro-inflammatory profiles of Indonesian adult men with central obesity: a preliminary study on TNF-α, sTNFR-2 and IL-1β

ABSTRACT

BACKGROUND:

Central obesity is closely associated with chronic inflammation, characterized by abnormal cytokine production such as IL-1β, tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor-2 (TNFR-2). Central obesity and chronic inflammation form a complex link with insulin resistance, leading to the development of type-2 diabetes mellitus (T2DM) and cardio-vascular disease (CVD). Knowing the coinciding occurrence of chronic inflammation and central obesity, this study aimed to examine pro-inflammatory cytokine profiles of Indonesian adult men with central obesity.

METHODS:

This cross-sectional study recruited 80 apparently healthy Indonesian adult men, aged 23-53 years old with waist circumference ranging from 64-125 cm. This study was done in Jakarta

MEASUREMENTS:

Measurement included clinical parameters like systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), aspartate amino transferase (AST), alanine amino transferase (ALT), creatinine, high sensitivity C-reactive protein (hsCRP); anthropometric parameters, namely weight, height and waist circumference; and pro-inflammatory cytokines TNF-α, soluble TNFR-2 (sTNFR-2) and IL-1β.

RESULTS:

Basic characteristics of the subjects showed linear increase in values of SBP, DBP and the serum concentrations of AST, ALT, FBG, GFR, hsCRP (p < 0,005 respectively) with the degree of obesity. sTNFR-2 and IL-1 β positively correlated with hsCRP (rs= 0.277, p= 0.013 and rs= 0.257, p= 0.022, respectively), WC (rs= 0.380, p= 0.001 and rs= 0.400, p < 0.001, respectively) and body mass index (BMI) (rs= 0. 364, p= 0.001 and rs= 0.399, p < 0.001, respectively). Moreover, TNF-α did not show correlations with hsCRP, WC, and BMI.

CONCLUSION:

There was linear increased in the serum concentrations of sTNFR-2 and IL-1 β in subjects with central obesity. Both pro-inflammatory markers were correlated with hsCRP, WC and BMI, but this did not occur for TNF-α. sTNFR-2 and IL-1 β are, therefore, considered as valid biomarkers to indicate chronic inflammation in Indonesian adult men with central obesity.

INTRODUCTION

The worldwide epidemic of obesity, fostered by the modern lifestyle characterized by the lack of physical activity and energy-dense diet, has increased incrementally in many developed and developing countries. Indonesia is not an exceptional country. The prevalence of overweight and obesity amongst Indonesian adults, using the body mass index (BMI) cut-off > 25kg/m2, was 48.97% in men and 40.65% in women.1 The concomitant occurrence of central/abdominal obesity has become a great concern, given it is the major risk leading to cardio-vascular disease (CVD), type 2 diabetes (T2DM), and other degenerative diseases like cancer.2,3.

It should be pointed out that different fat depots have distinct metabolic characteristics, leading to individual differences in the impact of obesity on cardio-metabolic risks. Obesity is known to be associated with low-grade chronic inflammation, marked by increased production of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1 β (IL-1β) and C-reactive protein (CRP)5 due to increasing number of infiltrating macrophages population in adipose tissue. Pro-inflammatory cytokines produced in adipose tissue interfere with signals and activation of insulin on adipose tissue, liver and muscle, leading to insulin resistance8, evidenced by consistent links between inflammation with insulin resistance, T2DM and CVD.2,4-7

The roles of TNF-α associated with insulin resistance have been well documented in both animal and human studies.9 TNF-α can induce disruption of tyrosine phosphorylation in insulin receptor and insulin receptor substrate-1 (IRS-1), and activate serine/threonine phosphorylation in IRS-1.10 Experimentally, inhibition of TNF-α in rodent models improved insulin sensitivity significantly.11 Concentrations of TNF-α were reported to be associated positively with BMI and central obesity. In a reciprocal pattern, TNF-α concentrations decline in a linear manner with weight loss, and the decline of TNF-α improved insulin sensitivity.10.12,13

As a consequence of increasing production of TNF-α, adipose tissue of obese individuals expressed soluble tumor necrosis factor receptor-2 (sTNFR-2). Hotamisligil et al.11 showed that the expression of TNFR-2 mRNA in fat tissue was two-fold and the sTNFR-2 concentrations in the blood circulation was six-fold in obese women compared to non-obese woman. The bound TNF-α and TNFR-2 complex directly binds to the TNF receptor associated factor (TRAF), and then go downstream on the path to activate intra-cellular nuclear factor kappa B (NFҡB) and c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). 11

IL-1β, produced by monocytes and macrophages, exerts its biological function by binding to IL-1 type I receptor, which then activates the inhibitor-ҡB kinase/nuclear factor-ҡB (IKK/NF-ҡB) pathway and the three types of mitogen-activated protein kinase mitogen-activated protein kinase (MAPKs): extracellular-regulated kinase (ERK), JNK, and p38MAPK. Previous research showed that the expression of IL-1 β and its receptor increased in obese adipose tissue, and increased the risk of T2DM.8

TNF-α, sTNFR-2 and IL-1 β could play an important role in insulin resistance and CVD related obesity. But until now there has been no comprehensive information on TNF-α, sTNFR-2 and IL-1β in Indonesia. Therefore this study is aimed to examine the proinflammatory cytokines profile and their interaction in Indonesian adult men with central obesity.

SUBJECTS AND METHOD

Subjects

Subjects were recruited from medical checkup patients visiting Prodia Clinical Laboratory in Jakarta, employees of some companies and members of nearby communities who were interested in participating in this study. These preliminary results were part of the big project on Inflammation and Angiogenesis in Indonesian Adult Men with Central Obesity undertaken by the Principal Investigator (SCR) in Jakarta. Eighty apparently healthy Indonesian male adults, aged 25-55 were eligible for the study. No subjects had renal, hepatic, endocrine, and oncological diseases; fever, received anti-diabetic drugs, or other drugs known to affect lipoprotein metabolism for at least 3 weeks prior to the study.

Subjects were divided into 3 groups, they were non-obese group with waist circumference (WC) < 90 cm (n= 25), obes I group with WC 90 - 100 cm (n= 27) and obese II with WC > 100 cm (n=28). This subject classification was based on the Asia Pacific criteria for central obesity cut-offs recommended by the International Diabetes Federation (IDF)14 and World Health Organization (WHO).15

All subjects agreed to sign informed consent prior to the commencement of the study. The proposal and clinical protocol of this study were approved by the Medical Ethics Committee of the Faculty of Medicine, Hasanuddin University.

Clinical assessments

Blood pressure was measured using a standardized method as described in the previous study.16

Anthropometric assessments

The anthropometric indicators were measured twice. The mean values were used in the analyses. Body weight (BW), height and waist circumference were measured using a standardized method as described in the previous study.16

Biochemical assessments

Fasting blood specimens were collected in the morning between 07:00 - 10:00 am. Plasma and serum were separated immediately by centrifugation and aliquots were frozen at - 20 0C for batched analysis for TNF-α, sTNFR-2 and IL-1β. Routine biochemical parameters like serum high sensitivity C-reactive protein (hsCRP), fasting blood glucose (FBG), aspartate amino transferase (ALT), alanine aminotransferase (ALT), creatinine, total bilirubin concentrations were determined immediately soon after the blood separation process. All biochemical analyses were performed at Prodia Clinical Laboratory.

Serum glucose, creatinine, ALT, AST, total bilirubin and hsCRP concentrations were measured using the Prodia Clinical Laboratory's routine chemistry procedures. Serum AST, ALT, plasma glucose concentrations were determined using an automated glucose oxidase method. Serum creatinine concentrations were determined using the International Federation of Clinical Chemistry (IFCC) method and reagents manufactured by Roche diagnostic ® Mannheim, Germany). Subjects were defined suffering from chronic kidney disease if the value of glomerular filtration rate (GFR) was < 60 ml/minute/1,73m2 in line with the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) guidelines 2000.17 GFR was calculated using the Cockroft and Gault formula.

[(140-age) X body weight/kg]

GFR (mL/min)=

[72 X creatinine serum (mg/dL)]

Plasma hsCRP was determined using chemiluminescent immunometric asssay. Commercially available kits and reagents were purchased to measure serum hsCRP (for use on Immulite® 2000 systems produced by Siemens).

With respect to pro-inflammatory cytokines, commercially available kits were purchased to measure serum TNF-α (Quantikine® HS High Sensitivity ELISAs Human TNF-α/TNFSF1A produced by R&D Systems, Inc., Minneapolis, MN, USA), TNFR-2 (Quantikine® Human sTNF RII/TNFRSF1B produced by R&D Systems, Inc., Minneapolis, MN, USA), and IL-1β (Quantikine® HS High Sensitivity ELISAs Human IL-1β/IL-1F2 produced by R&D Systems, Inc., Minneapolis, MN, USA),

Statistical analysis

Data analysis was done using SPSS 13.0 statistical analysis software for Windows (SPSS Inc., Chicago, IL, USA). Distributions of continuous variables were assessed for normality using the Kolmogorov-Smirnov. For continuous variables with normal distributions, such as anthropometric and biochemical measures, descriptive statistics were presented as mean ± SD. Statistical analysis was performed using a one-way analysis of variance for a quantitative dependent variable by a single factor (independent) variable. Associations between continuous variables with approximately normal distributions were described using Spearman's correlation coefficients. All tests were two-sided and considered significant at p < 0.05.

RESULTS

Table 1 describes the clinical and biochemical characteristics of the subjects, by central obesity categories, with mean, median, minimum and maximum values. Subjects' age ranged from 26 to 56 years old, with WC ranged from 65 to 125 cm. Other clinical and biochemical indicators were in the range of the study inclusion criteria.

Table 2 shows the mean value of clinical and biochemical variables of non-obese, obese I and II subjects, indicating significant differences between the groups. There was linear increase in the values of SBP and DBP, and almost all biomarker concentrations with the degree of central obesity, except for serum total bilirubin concentrations. Serum TNF-α concentrations did not show significant differences between the groups. Mean values of sTNFR-2 and IL-1β showed significant differences between the groups of obese I and II (both at p < 0.05).

Tabel 1 : Clinical and biochemical characteristics of the subjects

Variable

Units

Mean

s.d

Median

Min

Max

Age

year

37.1

5.7

38.0

26.0

51.0

Clinical and anthropometric indicators

94.0

11.8

94.0

65.0

125.0

BMI

kg/m2

27.7

4.5

27.4

18.1

45.1

SBP

mmHg

111.7

9.0

110.0

90.0

130.0

DBP

mmHg

76.2

6.8

80.0

60.0

89.0

Biochemical indicators

AST

U/L

23.5

7.5

22.0

13.0

52.0

ALT

U/L

31.2

18.3

26.0

6.0

97.0

Total Bilirubin

mg/dL

0.8

0.3

0.7

0.4

2.3

FBG

mg/dL

85.8

7.4

86.0

69.0

104.0

GFR

ml/mnt

121.7

26.8

117.9

74.2

191.8

hsCRP

µg/dL

2.1

2.0

1.4

0.2

9.3

Inflammatory markers

TNF-α

pg/mL

3.31

3.13

2.53

1.23

26.92

sTNFR-2

µg/mL

2.18

0.44

2.09

1.41

3.49

IL-1β

pg/mL

0.38

0.68

0.14

0.01

3.55

Tabel 2. Differences in clinical and biochemical indicators of the subjects, by central obesity categories.

Variables

Units

Degree of central obesity

Mean ± s.d

Non-obese

Obese I

Obese II

(n= 25)

(n= 27)

(n= 28)

Age

Year

35.4

7.6

37.2

5.2

38.4

4.9

Clinical indicators

SBP

mmHg

107.9

8.0b

113.1

9.4

113.8

8.6b

DBP

mmHg

72.8

7.1ab

77.7

6.1a

77.7

6.2b

Biochemicals indicators

AST

U/L

20.8

3.8b

21.8

5.8c

27.5

9.6bc

ALT

U/L

21.7

9.6ab

28.6

12.1ac

42.2

23.3bc

Tot Bil

mg/dL

0.8

0.2

0.8

0.4

0.8

0.2

FBG

mg/dL

84.2

6.0b

84.6

6.5c

88.4

8.8bc

GFR

mL/mnt

105.7

21.8b

115.8

19.8c

142.4

23.7bc

hsCRP

µg/dL

1.02

1.06ab

2.17

2.11a

3.14

2.04b

Inflammatory Markers

TNF-α

pg/mL

3.78

5.08

2.15

1.56

3.23

1.75

sTNFR-2

µg/mL

2.02

0.36b

2.10

0.29c

2.40

0.53bc

IL-1β

pg/mL

0.16

0.23b

0.19

0.23c

0.76

1.01bc

Data are expressed as means ± s.d. Abbreviations: WC, waist circumference; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; AST, aspartate amino transferase; ALT, alanine amino transferase; Tot bil, total bilirubin; GFR, glomerular filtration rate; hsCRP, high sensitivity C-reactive protein; TNF-α, tumor necrotic factor-α; sTNFR-2, soluble tumor necrotic factor receptor-2; IL-1β, interleukin-1β; s.d., standard deviation. Differences among groups were determined by one-way analysis of variance; asignificantly different between non-obese and obese I subjects (p < 0.05); bsignificantly different between non-obese and obese II subjects (p < 0.05); csignificantly different between obese I and obese II subjects (p < 0.05).

Table 3 shows that sTNFR-2 and IL-1β positively correlated with hsCRP (rs= 0.277, p= 0.013 and rs= 0.257, p= 0.022, respectively), WC (rs= 0.380, p= 0.001 and rs= 0.400, p < 0.001, respectively) and BMI (rs= 0.364, p= 0.001 and rs= 0.399, p < 0.001, respectively). However, TNF-α did not show any correlation with hsCRP, WC, and BMI.

Tabel 3. Relationships between inflammatory cytokines with hsCRP, waist circumference and body mass index.

Variable

Units

TNF-α

sTNFR-2

IL-1β

hsCRP

µg/dL

0.056

0.277

0.013

0.257

0.022

0.173

0.380

0.001

0.400

0.000

BMI

kg/m2

0.183

0.364

0.001

0.399

0.000

Description:

WC, waist circumference; hsCRP, high sensitivity C-reactive protein; TNF-α, tumor necrotic factor-α; sTNFR-2, soluble tumor necrotic factor receptor-2; IL-1β, interleukin-1 β; rs, Spearman correlation (2-tailed); p, value of significant; NS, non significant

DISCUSSION

Prospective study had shown that central obesity is a major risk factor for type 2 diabetes and cardiovascular disease, and WC is the best predictors of intra peritoneal and posterior subcutaneous abdominal adipose tissue mass.18 The increased in ALT, GFR and hsCRP concentrations and SBP and DBP value were accompanied by increasing WC. An increasing value of physical examination and laboratory test in obesity due to metabolism disorders.19

Table 2 shows the concentration of pro-inflammatory cytokine, sTNFR-2 and IL-1β decreased from non-obese group to the obese group I, and then it increased significantly to obese II. The possibility is due to body's compensatory mechanisms against obesity, in which the concentration of pro-inflammatory factors, in the early occurrence of obesity, has not been expressed in the circulation. When the state of obesity continues, the expression of proinflammatory cytokines released into the circulation. Mechanism of biological variation in the body is unknown, since there is currently no published data about the body compensatory mechanism of pro-inflammatory cytokines to obesity.

Several studied have published, that the state of obesity associated with low levels of inflammation.4 Both sTNFR-2 and IL-1β concentration showed increased in the circulation consistent with the development of adipose tissue and increased of hsCRP which is a biochemical marker for systemic low grade inflammation.20 Infiltration of monocytes and macrophages were found in the growing adipose tissue.8 sTNFR-2 concentration correlated positively with TNF-α in the circulation, since the release of sTNFR-2 stimulated by TNF-α.21 sTNFR-2 plays as modulators of biological function of TNF-α.22 TNF-α in obesity did not show the difference between groups, it may that TNF-α in the circulation quickly bound and neutralized by TNF-α receptor that becomes very low in concentration.23 mTNR-2 expression was higher in obese than non-obese. Hence the concentrations of sTNFR-2 were found to increased in the serum obese individuals.23

Examination of TNF-α activity is more reliable using concentrations of sTNFR-2 compared with TNF-α concentration of the total, because the sTNFR-2 protein is more easily detected in the plasma and the concentration remained elevated at a longer period.24

Concentration of IL-1β were higher in obese II groups. IL-1β decreases insulin induced glucose transport in adipocytes mainly by inhibiting IRS-1 expression through a reduction in IRS-1 mRNA that is dependent on ERK pathway activation and by a posttranscriptional mechanism that is independent of ERK. IL-1 β that is produced by resident macrophages in adipose tissue could act in synergy with TNF- β and IL-6 to impair adipocytes biology that could be an important event in the development of the insulin resistance.8

CONCLUSION

References:

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