94 Original Basic
TNF-α Gene Expression in Subcutaneous Adipose Tissue Associated with HOMA in Asian Indian Postmenopausal Women
Affiliations
Key words ▶ adipokines ● ▶ metabolic syndrome ● ▶ adipose tissue ● ▶ hyperinsulinemia ● ▶ leptin ● ▶ obesity ● ▶ postmenopause ●
received 30.03.2013 accepted after second revision 14.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358706 Published online: December 2, 2013 Horm Metab Res 2014; 46: 94–99 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. S. Tiwari Professor and Head Department of Physiology King George’s Medical University Uttar Pradesh Lucknow 226003 India Tel.: + 91/522/2257 542 Fax: + 91/522/2257 539 research.physiology@gmail. com
S. Tiwari1, Sadashiv1, B. N. Paul2, S. Kumar3, A. Chandra4, S. Dhananjai1, M. P. S. Negi5 1
Department of Physiology, King George’s Medical University, Lucknow, India Immunobiology Division, Indian Institute of Toxicological Research, Lucknow, India 3 Director AIIMS, Bhopal, Madhya Pradesh, India 4 Department of Gastroenterology, King George’s Medical University, Lucknow, India 5 Institute for Data Computing and Training, Lucknow, India 2
Abstract
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The present study determines the fat depotspecific expression of leptin and TNF-α and its association with biochemical parameters in postmenopausal women. A total of 108 postmenopausal women were recruited prospectively; 54 were with metabolic syndrome (cases) and 54 were without metabolic syndrome (controls). Leptin and TNF-α mRNA expression in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were done by Real TimeRT PCR. In cases, the mean ( ± SD) serum estrogen was significantly lower (41.33 ± 24.90 vs. 23.95 ± 14.45, p < 0.001) while leptin (12.85 ± 4.51 vs. 10.34 ± 3.89, p = 0.002) and TNF-α (13.81 ± 7.13 vs. 8.00 ± 4.38, p < 0.001) were significantly higher as compared to controls. Further, the mean relative VAT mRNA expression of both leptin
Introduction
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Adipocytes are highly specialized cells that maintain whole body energy homeostasis by regulating glucose and lipid metabolism. Adipose tissue is not only an energy storage depot, but is also regarded nowadays as an extremely active endocrine organ [1], which secretes adipocyte-derived molecules, including lipid metabolites and adipocytokines [adiponectin, leptin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, etc.] [2]. It is well documented that accumulation of visceral adipose tissue (VAT) rather than subcutaneous adipose tissue (SAT) is a stronger risk factor for adverse health profile [3]. Abnormal production of adipose tissue derived proteins is suggested to play a role in the pathogenesis of insulin resistance and the metabolic syndrome seen in relation to obesity [4–7]. TNF-α, a cytokine produced in adipose tissue, has been shown to be increased in obesity, type 2 diabetes, and atherosclerosis [8, 9]. Increased
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
(0.33 ± 0.29 vs. 0.05 ± 0.09, p < 0.001) and TNF-α (0.32 ± 0.31 vs. 0.13 ± 0.09, p < 0.001) and expression of SAT leptin (4.91 ± 4.01 vs. 0.50 ± 0.92, p < 0.001) also lowered significantly in cases as compared to controls. Further, the relative VAT expression of both leptin (r = − 0.32, p < 0.001) and TNF-α (r = − 0.23, p < 0.01) showed significant and negative correlation with glucose; expression of SAT leptin showed significant and positive correlation with HDL (r = 0.20, p < 0.05) and serum estrogen (r = 0.30, p < 0.01) while negative correlation with glucose (r = − 0.26, p < 0.01) and serum TNF-α (r = − 0.29, p < 0.01); and expression of SAT TNF-α showed significant and positive correlation with insulin (r = 0.21, p < 0.05) and HOMA (r = 0.20, p < 0.05). In conclusion, the VAT and SAT leptin mRNA expressions may have a modulatory role in metabolic syndrome.
circulating TNF-α level is positively associated with obesity [9]. In 1993, Hotamisligil et al. not only showed that TNF-α was elevated in plasma and in adipose tissue of obese rodents but also discovered that neutralization of TNF-α in obese fa/fa rats caused a significant increase in the peripheral uptake of glucose in response to insulin. These data clearly indicated a role for TNF-α in insulin resistance and type 2 diabetes that often accompany obesity [10]. A number of studies have demonstrated that TNF-α can impair insulin signaling in hepatocytes and adipose tissue [11, 12]. Although TNF-α is an established link between insulin resistance and inflammation in obese rodent models, whether such a link exists in humans is still debated. Yet, the few TNF-α neutralization studies, which addressed in vivo whether TNF-α is involved in insulin resistance of obese subjects yielded negative results possibly because of their limited power [13–16]. Leptin is a 16-kDa hormone and also secreted by adipocytes. Obesity is also associated with
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Authors
Original Basic 95
triglyceride, TC: total cholesterol, and HDL: high density lipoprotein) were determined using a semi-automated analyzer (Microlab 300, Merck). Serum leptin and TNF-α level were measured by enzyme-linked immunosorbent assay (Human TNF-α ELISA 950 090 096/192, Diaclone and Diagnostics Biochem Canada Inc., Cat. No. CAN-L-4260, Version 6.0, London, Ontario, Canada).
RNA extraction Total RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO, USA) RNA was measured spectrophotometrically at 260 and 280 nm while RNA integrity was checked by visual inspection of the 2 ribosomal RNAs 18 S and 28 S on agarose gel.
Real-time PCR measurement of leptin and TNF-α mRNA One-step RT-PCR was carried out using Quanti Tect SYBR Green RT-PCR master mix kit (Qiagen). PCR amplification was carried out in Light Cycler 480 (Roche, Real time thermal cycler) with 96-well PCR plates using following temperature profile: 50 °C, 30 min, (reverse transcription); 95 °C, 15 min (initial denaturation); followed by 40 cycles of 94 °C for 15 s, 59 °C for 30 s, and 72 °C for 30 s for denaturation, annealing and extension steps, respectively. Primer sequence of human leptin and TNF-α were 5′-GCTGTGCCCATCCAAAAAGT-3′ (forward) 5′-ACTGCCAGTGTCTGGTCCAT3′ (reverse) and 5′-CAGAGGGAAGAGTTCCCCAG-3′ (forward) 5′-CCTTGGTCTGGTAGGAGACG-3′ (reverse). The following primer sequence of β-actin as internal control with following sequence was 5′-GTGGCATCCACGAAACTACCTT-3′ (forward) and 5′-GGACTCCTGATACTCCTGCTTG-3′ (reverse). The PCR primers were synthesized by (Agile Life Science Technologies, India). Expression of β-actin or glyceraldehyde-3-phosphate dehydrogenase was used to normalize leptin and TNF-α expression values. There was no difference in glyceraldehyde-3-phosphate dehydrogenase or β-actin expression between adipocytes from controls and cases or between the visceral and subcutaneous depots.
Subjects and Methods
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Calculation Insulin resistance
Total 170 postmenopausal female subjects were enrolled at K.G.’s Medical University, Lucknow, India who underwent elective abdominal surgery for gall bladder stone or hysterectomy. A total of 130 subjects who gave their written consent were enrolled for the study. Among these, 54 women (45–70 years) were classified as presenting metabolic syndrome (case) according to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) [29] criteria for metabolic syndrome and 76 were without metabolic syndrome. From 76 women without metabolic syndrome, 54 age-matched, unrelated to metabolic syndrome were selected and considered as control. Thus, in total 108 postmenopausal women (control = 54 and case = 54) were recruited for the study. Abdominal visceral and subcutaneous adipose tissues were obtained during surgery. No specific standard diet and hormonal therapy were given to the patients during the surgery. All tissue samples were stored in RNAlatar (Sigma Chemical Co., St. Louis, MO, USA) for RNA extraction. The study was approved by the Institutional Ethics Committee. Informed consent was obtained from each patient.
HOMA an index of insulin resistance (IR) was calculated using the homeostasis model assessment [HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mM)/22.5] [30].
Subjects
Relative gene expression Relative leptin and TNF-α mRNA expression levels of both visceral and subcutaneous were calculated using [(1/2)∆Ct].
Statistical analysis Continuous data were summarized as mean ± SD. Two independent groups were compared by parametric independent Student’s t-test while dependent by paired t-test. The significance of both parametric independent t-test and dependent t-test was also confirmed by nonparametric alternatives Mann-Whitney U-test and Wilcoxon matched pairs test, respectively. Association between variables was done by Pearson correlation analysis and simple linear regression analysis. A 2-tailed (α = 2) p < 0.05 was considered statistically significant.
Biochemical estimation Blood samples were taken the next morning after admission to the hospital for surgery (day of surgery). Plasma insulin and estrogen concentrations were determined using immune radiometric assay (IRMA), radioimmunoassay (RIA, Immunotech). Plasma glucose and lipid profile (Merck kit) concentrations (TG:
Results
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Basic characteristics The basic characteristics of 2 groups (controls and cases) at pres▶ Table 1. The mean entation (enrollment) are summarized in ●
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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increased plasma leptin level [17]. The secretion is also dependent on the fat depot, being higher in the SAT than in the VAT [18]. Leptin plasma concentration [19] and mRNA expression in adipose tissue [20] are directly related to obesity severity, as an increase of fat mass is associated with an increase of leptin, which makes leptin an indicator of total fat mass. In fact, in generalized lipodystrophy, where adipose tissue is nearly absent, leptin administration improves insulin sensitivity [21]. This highlights the influence of this adipocyte hormone on whole-body glucose homoeostasis. However, in common human obesity, there are high circulating leptin levels, suggesting leptin resistance, and leptin administration has little or no effect on insulin resistance [22]. Therefore, while leptin deficiency very likely contributes to insulin resistance when adipose tissue is lacking, leptin resistance is a main feature of human obesity. So far, the precise role of leptin in insulin resistance remains unclear. SAT and VAT show functional differences. For example, genes for angiotensinogen (blood pressure regulation), complement factors [23], and fatty acid-binding protein 4 (involved in fatty acid trapping in adipocytes) are expressed at higher levels in VAT than in SAT [24]. Leptin, however, is mainly produced by human SAT while TNF-α is equally produced by both fat depots [25], although others report differently from in vitro studies [26]. Therefore, the results are conflicting [27, 28]. Thus, the question of regional differences in TNF-α and leptin expression and secretion in visceral vs. subcutaneous adipose tissue remains unanswered at the present time. Most importantly, no study has yet clearly examined how these adipokines released by adipocytes from these 2 fat depots is affected in overall and visceral obese women. The purpose of this study was to examine the fat depot-specific expression of leptin and TNF-α and its association with biochemical parameters in postmenopausal women.
96 Original Basic
Variables
Controls (n = 54)
Cases (n = 54)
t-Value (DF = 106)
p-Value
Age (years) Weight (kg) Height (cm) BMI (kg/m2) WC (cm) HC (cm) WHR SBP (mm Hg) DBP (mm Hg)
54.56 ± 6.80 69.78 ± 18.39 156.91 ± 7.24 28.13 ± 6.16 95.02 ± 15.28 104.24 ± 10.33 0.91 ± 0.11 124.91 ± 8.94 83.65 ± 8.84
56.06 ± 7.32 68.35 ± 13.10 154.58 ± 7.99 28.70 ± 5.71 95.94 ± 14.55 103.76 ± 13.03 0.92 ± 0.08 131.78 ± 14.43 84.22 ± 8.68
1.10 0.47 1.59 0.50 0.32 0.21 0.76 2.97 0.34
0.272 0.642 0.115 0.618 0.748 0.832 0.450 0.004 0.734
Table 1 Basic characteristics of 2 groups at enrollment.
Values are mean ± SD BMI: Body mass index; WC: Waist circumference; HC: Hip circumference; WHR: Waist to hip ratio; SBP: Systolic blood pressure;
Variables
Controls (n = 54)
Cases (n = 54)
t-Value (DF = 106)
p-Value
Glucose (mg/dl) Insulin (μU/ml) HOMA TG (mg/dl) TC (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Serum leptin (ng/ml) Serum TNF-α (pg/ml) Serum estrogen (pg/ml)
96.41 ± 13.80 9.66 ± 4.84 2.31 ± 1.25 140.13 ± 71.63 160.32 ± 35.12 43.91 ± 8.42 88.38 ± 35.48 28.03 ± 14.33 10.34 ± 3.89 8.00 ± 4.38 41.33 ± 24.90
109.48 ± 7.89 13.51 ± 4.59 3.65 ± 1.28 163.26 ± 32.90 169.36 ± 32.53 36.97 ± 9.05 99.74 ± 32.58 32.65 ± 6.58 12.85 ± 4.51 13.81 ± 7.13 23.95 ± 14.45
6.04 4.24 5.51 2.16 1.39 4.13 1.73 2.16 3.10 5.09 4.44
< 0.001 < 0.001 < 0.001 0.033 0.168 < 0.001 0.086 0.033 0.002 < 0.001 < 0.001
Table 2 Biochemical parameter levels of 2 groups at enrollment.
Values are mean ± SD HOMA: Homeostasis model assessment; TG: Triglyceride; TC: Total cholesterol; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TNF: Tumor necrosis factor
Biochemical profile The biochemical parameter levels of 2 groups at presentation are ▶ Table 2. The mean levels of all biochemical summarized in ● parameter were higher in cases as compared to controls, except HDL and estrogen. Comparing the mean level of each biochemical parameter between the 2 groups, t-test revealed significantly different (p < 0.05 or p < 0.01, or p < 0.001) and higher glucose (96.41 ± 13.80 vs. 109.48 ± 7.89; p < 0.001), insulin (9.66 ± 4.84 vs. 13.51 ± 4.59; p < 0.001), HOMA (2.31 ± 1.25 vs. 3.65 ± 1.28; p < 0.001), TG (140.13 ± 71.63 vs. 163.26 ± 32.90; p = 0.033), VLDL (28.03 ± 14.33 vs. 32.65 ± 6.58 p = 0.033), leptin (10.34 ± 3.89 vs. 12.85 ± 4.51; p = 0.002), and TNF-α (8.00 ± 4.38 vs. 13.81 ± 7.13; p < 0.001) while significantly (p < 0.001) lower HDL (43.91 ± 8.42 vs. 36.97 ± 9.05; p < 0.001) and estrogen (41.33 ± 24.90 vs. 23.95 ± 14.45; p < 0.001) in cases as compared to controls. However, at presentation, mean TC and LDL were found similar (p > 0.05) between the 2 groups.
Leptin and TNF-α mRNA expression in visceral adipose tissue We have made comparison of relative leptin and TNF-α mRNA expression in visceral adipose tissue (VAT) of controls and cases
Mean ± SD
a
0.75
Leptin
0.50 0.25
***
Fig. 1 Relative VAT leptin a and TNF-α b mRNA expressions of 2 groups. *** p < 0.001 as compared to controls.
0.00 Controls Cases Groups
b 0.75 Mean ± SD
age, BMI (body mass index), WC (waist circumference), WHR (waist to hip ratio), SBP (systolic blood pressure), and DBP (diastolic blood pressure) were slightly higher in cases as compared to controls while weight, height, and HC (hip circumference) were higher in controls as compared to cases. Comparing the mean basic characteristics of 2 groups, t-test revealed similar (p > 0.05) basic characteristics between the 2 groups except SBP. At presentation, the mean SBP of cases was significantly higher than controls (124.91 ± 8.94 vs. 131.78 ± 14.43; p = 0.004).
TNF-α
0.50 0.25
***
0.00 Controls Cases Groups
▶ Fig. 1). In visceral adipose tissue, the mean expressions of (● both leptin (0.33 ± 0.29 vs. 0.05 ± 0.09, p < 0.001) and TNF-α (0.32 ± 0.31 vs. 0.13 ± 0.09, p < 0.001) lowered significantly in cases as compared to controls.
Leptin and TNF-α mRNA expression in subcutaneous adipose tissue We also compared relative leptin and TNF-α mRNA expression ▶ Fig. 2). in subcutaneous adipose tissue (SAT) in both groups (● In subcutaneous adipose tissue, the mean expression of leptin (4.91 ± 4.01 vs. 0.50 ± 0.92, p < 0.001) also lowered significantly in
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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DBP: Diastolic blood pressure
Original Basic 97
Fig. 2 Relative SAT leptin a and TNF-α b mRNA expressions of 2 groups. ns p > 0.05 or *** p < 0.001 as compared to controls.
7.5 5.0 2.5
a
***
b
TNF-α ns
Mean ± SD
Mean ± SD
1
VAT
Controls Cases Groups 0.75
**
0.50 0.25
Mean ± SD
***
0.50 0.25
VAT
Leptin
10.0
***
7.5 5.0 2.5
Fig. 3 Relative VAT and SAT leptin a and TNF-α b mRNA expressions of controls. ns p > 0.05 or *** p < 0.001 as compared to VAT.
0.0 VAT
Mean ± SD
SAT
TNF-α
0.75
Controls Cases Groups
b
Groups
0.00
0.00
a
Fig. 4 Relative VAT and SAT leptin a and TNF-α b mRNA expressions of cases. ** p < 0.01 or *** p < 0.001 as compared to VAT.
0
0.0
b
Leptin
2
Groups
SAT
TNF-α
0.75
ns
0.50 0.25 0.00 VAT
Groups
SAT
Groups
SAT
Correlation The correlation of relative VAT and SAT mRNA expressions of both leptin and TNF-α with biochemical profiles (glucose, insulin, HOMA, TG, TC, HDL, LDL, VLDL, serum leptin, serum TNF-α, ▶ Table 3. and serum estrogen) of all subjects are summarized in ● The relative VAT leptin mRNA expression showed significant and negative (inverse) correlation with both glucose (r = − 0.32, p < 0.001) and serum TNF-α (r = − 0.21, p < 0.05). Further, relative VAT TNF-α mRNA expression also showed significant and negative correlation with both glucose (r = − 0.23, p < 0.01). In contrast, relative SAT leptin mRNA expression showed significant and positive (direct) correlation with both HDL (r = 0.20, p < 0.05) and serum estrogen (r = 0.30, p < 0.01) while significant and negative correlation with both glucose (r = − 0.26, p < 0.01) and serum TNF-α (r = − 0.29, p < 0.01). Conversely, relative SAT TNF-α mRNA expression showed significant and direct association (correlation) with both insulin (r = 0.21, p < 0.05) and HOMA (r = 0.20, p < 0.05).
Discussion cases as compared to controls. However, the relative mean mRNA expression of TNF-α did not differ significantly between the groups, that is, it was found to be statistically the same (0.36 ± 0.28 vs. 0.34 ± 0.28, p = 0.667).
Leptin and TNF-α mRNA expression in visceral and subcutaneous adipose tissue The comparatively relative VAT and SAT mRNA expressions of both ▶ Fig. 3, 4 leptin and TNF-α in controls and cases are shown in ● respectively. In controls, the relative mean mRNA expression of leptin was significantly higher in subcutaneous adipose tissue as compared to visceral adipose tissue (0.33 ± 0.29 vs. 4.91 ± 4.01; p < 0.001); however, the expression of TNF-α was found similar between the 2 tissues (0.32 ± 0.31 vs. 0.36 ± 0.28; p = 0.368). Conversely, in cases, the relative mean mRNA expressions of both leptin (0.05 ± 0.09 vs. 0.50 ± 0.92; p = 0.001) and TNF-α (0.13 ± 0.09 vs. 0.34 ± 0.28; p < 0.001) was found significantly different and higher in subcutaneous adipose tissue as compared to visceral adipose tissue.
▼
Previous studies from several groups [31–33] have provided the evidence for a regional difference in leptin expression, as they demonstrated higher leptin mRNA levels in SAT than in VAT samples from centrally obese and non obese subjects. In the present study we have made the comparison of VAT leptin and TNF-α mRNA expression and SAT leptin and TNF-α mRNA expression including regional difference between controls and cases. Our finding demonstrates that SAT leptin mRNA expression was significantly lower in cases as compared to controls. This finding is consistent with the recent study, which indicates that SAT leptin expression was lower in women with increased visceral fat volume [34]. However, our results also demonstrate that leptin mRNA expression was higher in SAT as compared to VAT in both the groups. This is in support of the study of Montague et al. who also found higher expression of leptin gene in subcutaneous adipose tissue compared to visceral adipose tissue [18]. However, subcutaneous fat is responsible of 80 % of total leptin production. This was shown in cultures ex vivo where the production of leptin was higher in subcutaneous adipocytes than visceral adi-
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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Leptin
10.0
Mean ± SD
Mean ± SD
a
98 Original Basic
Variables Glucose (mg/dl) Insulin (μU/ml) HOMA TG (mg/dl) TC (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Serum leptin (ng/ml) Serum TNF-α (pg/ml) Serum estrogen (pg/ml)
Relative VAT leptin
Relative VAT TNF-α mRNA
Relative SAT leptin mRNA
Relative SAT TNF-α mRNA
mRNA expression
expression
expression
expression
− 0.26** − 0.11 ns − 0.17 ns − 0.03 ns − 0.06 ns 0.20* − 0.11 ns − 0.03 ns − 0.13 ns − 0.29** 0.30**
− 0.01 ns 0.21* 0.20* 0.08 ns 0.10 ns 0.05 ns 0.05 ns* 0.08 ns − 0.11ns − 0.08 ns − 0.01 ns
− 0.32*** − 0.06 ns − 0.15 ns 0.01ns 0.01ns 0.11ns − 0.02 ns 0.01ns − 0.03 ns − 0.21* 0.12 ns
− 0.23** 0.06ns − 0.02 ns − 0.14 ns 0.03ns − 0.04 ns 0.08 ns − 0.14 ns − 0.05 ns − 0.17 ns 0.09 ns
VAT: Visceral adipose tissue; SAT: Subcutaneous adipose tissue ns
p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001
pocytes [35]. This could be a possible explanation for higher leptin expression in SAT in humans and stimulation of leptin by estrogen in females [36]. Interestingly the present study finds the significant and positive correlation between leptin mRNA expression and estrogen. The present finding suggests that estrogens may be modulators of leptin’s catabolic action in the brain. Higher levels of estrogen have been associated with increased central leptin sensitivity in rodents [37]. Differences in central leptin sensitivity caused by the presence or absence of estrogens may occur downstream of leptin receptor transcription and translation [38]. Leptin plasma concentration and mRNA expression in adipose tissue are directly related to obesity severity, as an increase of fat mass is associated with an increase of leptin, which makes leptin an indicator of total fat mass [20]. In the present study, we have found higher serum leptin concentration in cases as compared to controls. The finding is in well agreement with previous findings, which shows significant effect of serum leptin on insulin action, potentially linking obesity with insulin resistance [39]. Leptin is important in the regulation of appetite and energy balance, as shown by animal studies [40]. In humans and rodents, plasma leptin concentrations are highly correlated with BMI [41]. It appears that with increasing leptin concentrations, the hormone induces target cells to become resistant to its actions. In mice that became obese after being fed a high-fat diet, leptin concentrations increased, and this increase was accompanied by an increased expression of SOCS-3 (suppressor-of-cytokine-signaling), a potent inhibitor of leptin signaling [38]. One study reported that leptin impairs insulin signaling by increasing IRS-1 phosphorylation at the serine 318 site [42]. Leptin may task through insulin receptor substrate IRS-1 and IRS-2 suggesting that there are cross-talks of insulin and leptin signaling pathways [43]. However, the role of elevated leptin in obesity-associated insulin resistance is still a debate. In the present study, like leptin, VAT TNF-α mRNA expression was significantly lower in cases as compare to controls. The present study did not find any significant difference of SAT TNF-α mRNA expression between cases and controls. This finding corroborates with Koistinen et al. who showed similar TNF-α mRNA expression in 7 lean and 10 obese nondiabetic and 9 type-2 diabetic men [44]. However, a recent study by Selcuk Gormez et al. shows higher expression of TNF-α in subcutaneous adipose tissue of metabolic syndrome’s subjects [45]. Our results show that TNF-α mRNA expression was higher in SAT as compared to VAT of case group. Our finding is consistent
with Bullo et al. who reported that TNF-α mRNA expressions of the adipose tissue in obese and morbid obese patients were significantly higher than in controls. [46]. Increased expression of TNF-α mRNA in the subcutaneous abdominal adipose tissue depot has been documented in obese rodents and humans [10, 27, 28], leading to the hypothesis that TNF-α may play a crucial role in obesity-related insulin resistance [46] although clinical studies have shown that visceral adipose tissue is closely linked to insulin resistance. Moreover, TNF-α is weakly expressed either in subcutaneous or in deep human adipose tissue depots and this expression is not always modified in obesity [45]. This corresponds with the evaluation of in vivo secretion, which showed that TNF-α production by subcutaneous abdominal adipose tissue was quantitatively negligible in lean and obese subjects [47]. The results of the present study show that subcutaneous adipose tissue TNF-α mRNA expression is positively associated with insulin and HOMA. The findings are in well agreement with previous findings, which show HOMA index correlated positively with TNF-α gene expression [34]. Our results confirm and extend the findings of Hotamisligil et al. in a small sample of 19 obese females [10]. These authors reported that the adipose-tissue expression of TNF-α was positively related to BMI and decreased in proportion to the loss in body weight. This suggests that TNF-α has an important role in the pathophysiology of obesity. Previous studies support the role of VAT in disease development but we cannot ignore the role of subcutaneous adipose tissue. Thus, the present study concludes that the SAT leptin and TNF-α mRNA expressions may have a modulatory role in metabolic syndrome and leptin expression may be under the control of estrogen. Further study is needed to understand the regulatory mechanism of leptin and TNF-α expression.
Acknowledgements
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We thank Indian Council of Medical Research, New Delhi for support of this study.
Conflict of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
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Table 3 Correlation of relative VAT and SAT leptin and TNF-α mRNA expressions with biochemical profiles (n = 108).
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Original Basic 99
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TNF-α Gene Expression in Subcutaneous Adipose Tissue Associated with HOMA in Asian Indian Postmenopausal Women
Affiliations
Key words ▶ adipokines ● ▶ metabolic syndrome ● ▶ adipose tissue ● ▶ hyperinsulinemia ● ▶ leptin ● ▶ obesity ● ▶ postmenopause ●
received 30.03.2013 accepted after second revision 14.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1358706 Published online: December 2, 2013 Horm Metab Res 2014; 46: 94–99 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. S. Tiwari Professor and Head Department of Physiology King George’s Medical University Uttar Pradesh Lucknow 226003 India Tel.: + 91/522/2257 542 Fax: + 91/522/2257 539 research.physiology@gmail. com
S. Tiwari1, Sadashiv1, B. N. Paul2, S. Kumar3, A. Chandra4, S. Dhananjai1, M. P. S. Negi5 1
Department of Physiology, King George’s Medical University, Lucknow, India Immunobiology Division, Indian Institute of Toxicological Research, Lucknow, India 3 Director AIIMS, Bhopal, Madhya Pradesh, India 4 Department of Gastroenterology, King George’s Medical University, Lucknow, India 5 Institute for Data Computing and Training, Lucknow, India 2
Abstract
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The present study determines the fat depotspecific expression of leptin and TNF-α and its association with biochemical parameters in postmenopausal women. A total of 108 postmenopausal women were recruited prospectively; 54 were with metabolic syndrome (cases) and 54 were without metabolic syndrome (controls). Leptin and TNF-α mRNA expression in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were done by Real TimeRT PCR. In cases, the mean ( ± SD) serum estrogen was significantly lower (41.33 ± 24.90 vs. 23.95 ± 14.45, p < 0.001) while leptin (12.85 ± 4.51 vs. 10.34 ± 3.89, p = 0.002) and TNF-α (13.81 ± 7.13 vs. 8.00 ± 4.38, p < 0.001) were significantly higher as compared to controls. Further, the mean relative VAT mRNA expression of both leptin
Introduction
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Adipocytes are highly specialized cells that maintain whole body energy homeostasis by regulating glucose and lipid metabolism. Adipose tissue is not only an energy storage depot, but is also regarded nowadays as an extremely active endocrine organ [1], which secretes adipocyte-derived molecules, including lipid metabolites and adipocytokines [adiponectin, leptin, tumor necrosis factor (TNF)-α, interleukin (IL)-6, etc.] [2]. It is well documented that accumulation of visceral adipose tissue (VAT) rather than subcutaneous adipose tissue (SAT) is a stronger risk factor for adverse health profile [3]. Abnormal production of adipose tissue derived proteins is suggested to play a role in the pathogenesis of insulin resistance and the metabolic syndrome seen in relation to obesity [4–7]. TNF-α, a cytokine produced in adipose tissue, has been shown to be increased in obesity, type 2 diabetes, and atherosclerosis [8, 9]. Increased
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
(0.33 ± 0.29 vs. 0.05 ± 0.09, p < 0.001) and TNF-α (0.32 ± 0.31 vs. 0.13 ± 0.09, p < 0.001) and expression of SAT leptin (4.91 ± 4.01 vs. 0.50 ± 0.92, p < 0.001) also lowered significantly in cases as compared to controls. Further, the relative VAT expression of both leptin (r = − 0.32, p < 0.001) and TNF-α (r = − 0.23, p < 0.01) showed significant and negative correlation with glucose; expression of SAT leptin showed significant and positive correlation with HDL (r = 0.20, p < 0.05) and serum estrogen (r = 0.30, p < 0.01) while negative correlation with glucose (r = − 0.26, p < 0.01) and serum TNF-α (r = − 0.29, p < 0.01); and expression of SAT TNF-α showed significant and positive correlation with insulin (r = 0.21, p < 0.05) and HOMA (r = 0.20, p < 0.05). In conclusion, the VAT and SAT leptin mRNA expressions may have a modulatory role in metabolic syndrome.
circulating TNF-α level is positively associated with obesity [9]. In 1993, Hotamisligil et al. not only showed that TNF-α was elevated in plasma and in adipose tissue of obese rodents but also discovered that neutralization of TNF-α in obese fa/fa rats caused a significant increase in the peripheral uptake of glucose in response to insulin. These data clearly indicated a role for TNF-α in insulin resistance and type 2 diabetes that often accompany obesity [10]. A number of studies have demonstrated that TNF-α can impair insulin signaling in hepatocytes and adipose tissue [11, 12]. Although TNF-α is an established link between insulin resistance and inflammation in obese rodent models, whether such a link exists in humans is still debated. Yet, the few TNF-α neutralization studies, which addressed in vivo whether TNF-α is involved in insulin resistance of obese subjects yielded negative results possibly because of their limited power [13–16]. Leptin is a 16-kDa hormone and also secreted by adipocytes. Obesity is also associated with
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Authors
Original Basic 95
triglyceride, TC: total cholesterol, and HDL: high density lipoprotein) were determined using a semi-automated analyzer (Microlab 300, Merck). Serum leptin and TNF-α level were measured by enzyme-linked immunosorbent assay (Human TNF-α ELISA 950 090 096/192, Diaclone and Diagnostics Biochem Canada Inc., Cat. No. CAN-L-4260, Version 6.0, London, Ontario, Canada).
RNA extraction Total RNA was isolated using Tri-Reagent (Sigma Chemical Co., St. Louis, MO, USA) RNA was measured spectrophotometrically at 260 and 280 nm while RNA integrity was checked by visual inspection of the 2 ribosomal RNAs 18 S and 28 S on agarose gel.
Real-time PCR measurement of leptin and TNF-α mRNA One-step RT-PCR was carried out using Quanti Tect SYBR Green RT-PCR master mix kit (Qiagen). PCR amplification was carried out in Light Cycler 480 (Roche, Real time thermal cycler) with 96-well PCR plates using following temperature profile: 50 °C, 30 min, (reverse transcription); 95 °C, 15 min (initial denaturation); followed by 40 cycles of 94 °C for 15 s, 59 °C for 30 s, and 72 °C for 30 s for denaturation, annealing and extension steps, respectively. Primer sequence of human leptin and TNF-α were 5′-GCTGTGCCCATCCAAAAAGT-3′ (forward) 5′-ACTGCCAGTGTCTGGTCCAT3′ (reverse) and 5′-CAGAGGGAAGAGTTCCCCAG-3′ (forward) 5′-CCTTGGTCTGGTAGGAGACG-3′ (reverse). The following primer sequence of β-actin as internal control with following sequence was 5′-GTGGCATCCACGAAACTACCTT-3′ (forward) and 5′-GGACTCCTGATACTCCTGCTTG-3′ (reverse). The PCR primers were synthesized by (Agile Life Science Technologies, India). Expression of β-actin or glyceraldehyde-3-phosphate dehydrogenase was used to normalize leptin and TNF-α expression values. There was no difference in glyceraldehyde-3-phosphate dehydrogenase or β-actin expression between adipocytes from controls and cases or between the visceral and subcutaneous depots.
Subjects and Methods
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Calculation Insulin resistance
Total 170 postmenopausal female subjects were enrolled at K.G.’s Medical University, Lucknow, India who underwent elective abdominal surgery for gall bladder stone or hysterectomy. A total of 130 subjects who gave their written consent were enrolled for the study. Among these, 54 women (45–70 years) were classified as presenting metabolic syndrome (case) according to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) [29] criteria for metabolic syndrome and 76 were without metabolic syndrome. From 76 women without metabolic syndrome, 54 age-matched, unrelated to metabolic syndrome were selected and considered as control. Thus, in total 108 postmenopausal women (control = 54 and case = 54) were recruited for the study. Abdominal visceral and subcutaneous adipose tissues were obtained during surgery. No specific standard diet and hormonal therapy were given to the patients during the surgery. All tissue samples were stored in RNAlatar (Sigma Chemical Co., St. Louis, MO, USA) for RNA extraction. The study was approved by the Institutional Ethics Committee. Informed consent was obtained from each patient.
HOMA an index of insulin resistance (IR) was calculated using the homeostasis model assessment [HOMA-IR = fasting insulin (μU/ml) × fasting glucose (mM)/22.5] [30].
Subjects
Relative gene expression Relative leptin and TNF-α mRNA expression levels of both visceral and subcutaneous were calculated using [(1/2)∆Ct].
Statistical analysis Continuous data were summarized as mean ± SD. Two independent groups were compared by parametric independent Student’s t-test while dependent by paired t-test. The significance of both parametric independent t-test and dependent t-test was also confirmed by nonparametric alternatives Mann-Whitney U-test and Wilcoxon matched pairs test, respectively. Association between variables was done by Pearson correlation analysis and simple linear regression analysis. A 2-tailed (α = 2) p < 0.05 was considered statistically significant.
Biochemical estimation Blood samples were taken the next morning after admission to the hospital for surgery (day of surgery). Plasma insulin and estrogen concentrations were determined using immune radiometric assay (IRMA), radioimmunoassay (RIA, Immunotech). Plasma glucose and lipid profile (Merck kit) concentrations (TG:
Results
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Basic characteristics The basic characteristics of 2 groups (controls and cases) at pres▶ Table 1. The mean entation (enrollment) are summarized in ●
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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increased plasma leptin level [17]. The secretion is also dependent on the fat depot, being higher in the SAT than in the VAT [18]. Leptin plasma concentration [19] and mRNA expression in adipose tissue [20] are directly related to obesity severity, as an increase of fat mass is associated with an increase of leptin, which makes leptin an indicator of total fat mass. In fact, in generalized lipodystrophy, where adipose tissue is nearly absent, leptin administration improves insulin sensitivity [21]. This highlights the influence of this adipocyte hormone on whole-body glucose homoeostasis. However, in common human obesity, there are high circulating leptin levels, suggesting leptin resistance, and leptin administration has little or no effect on insulin resistance [22]. Therefore, while leptin deficiency very likely contributes to insulin resistance when adipose tissue is lacking, leptin resistance is a main feature of human obesity. So far, the precise role of leptin in insulin resistance remains unclear. SAT and VAT show functional differences. For example, genes for angiotensinogen (blood pressure regulation), complement factors [23], and fatty acid-binding protein 4 (involved in fatty acid trapping in adipocytes) are expressed at higher levels in VAT than in SAT [24]. Leptin, however, is mainly produced by human SAT while TNF-α is equally produced by both fat depots [25], although others report differently from in vitro studies [26]. Therefore, the results are conflicting [27, 28]. Thus, the question of regional differences in TNF-α and leptin expression and secretion in visceral vs. subcutaneous adipose tissue remains unanswered at the present time. Most importantly, no study has yet clearly examined how these adipokines released by adipocytes from these 2 fat depots is affected in overall and visceral obese women. The purpose of this study was to examine the fat depot-specific expression of leptin and TNF-α and its association with biochemical parameters in postmenopausal women.
96 Original Basic
Variables
Controls (n = 54)
Cases (n = 54)
t-Value (DF = 106)
p-Value
Age (years) Weight (kg) Height (cm) BMI (kg/m2) WC (cm) HC (cm) WHR SBP (mm Hg) DBP (mm Hg)
54.56 ± 6.80 69.78 ± 18.39 156.91 ± 7.24 28.13 ± 6.16 95.02 ± 15.28 104.24 ± 10.33 0.91 ± 0.11 124.91 ± 8.94 83.65 ± 8.84
56.06 ± 7.32 68.35 ± 13.10 154.58 ± 7.99 28.70 ± 5.71 95.94 ± 14.55 103.76 ± 13.03 0.92 ± 0.08 131.78 ± 14.43 84.22 ± 8.68
1.10 0.47 1.59 0.50 0.32 0.21 0.76 2.97 0.34
0.272 0.642 0.115 0.618 0.748 0.832 0.450 0.004 0.734
Table 1 Basic characteristics of 2 groups at enrollment.
Values are mean ± SD BMI: Body mass index; WC: Waist circumference; HC: Hip circumference; WHR: Waist to hip ratio; SBP: Systolic blood pressure;
Variables
Controls (n = 54)
Cases (n = 54)
t-Value (DF = 106)
p-Value
Glucose (mg/dl) Insulin (μU/ml) HOMA TG (mg/dl) TC (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Serum leptin (ng/ml) Serum TNF-α (pg/ml) Serum estrogen (pg/ml)
96.41 ± 13.80 9.66 ± 4.84 2.31 ± 1.25 140.13 ± 71.63 160.32 ± 35.12 43.91 ± 8.42 88.38 ± 35.48 28.03 ± 14.33 10.34 ± 3.89 8.00 ± 4.38 41.33 ± 24.90
109.48 ± 7.89 13.51 ± 4.59 3.65 ± 1.28 163.26 ± 32.90 169.36 ± 32.53 36.97 ± 9.05 99.74 ± 32.58 32.65 ± 6.58 12.85 ± 4.51 13.81 ± 7.13 23.95 ± 14.45
6.04 4.24 5.51 2.16 1.39 4.13 1.73 2.16 3.10 5.09 4.44
< 0.001 < 0.001 < 0.001 0.033 0.168 < 0.001 0.086 0.033 0.002 < 0.001 < 0.001
Table 2 Biochemical parameter levels of 2 groups at enrollment.
Values are mean ± SD HOMA: Homeostasis model assessment; TG: Triglyceride; TC: Total cholesterol; HDL: High density lipoprotein; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein; TNF: Tumor necrosis factor
Biochemical profile The biochemical parameter levels of 2 groups at presentation are ▶ Table 2. The mean levels of all biochemical summarized in ● parameter were higher in cases as compared to controls, except HDL and estrogen. Comparing the mean level of each biochemical parameter between the 2 groups, t-test revealed significantly different (p < 0.05 or p < 0.01, or p < 0.001) and higher glucose (96.41 ± 13.80 vs. 109.48 ± 7.89; p < 0.001), insulin (9.66 ± 4.84 vs. 13.51 ± 4.59; p < 0.001), HOMA (2.31 ± 1.25 vs. 3.65 ± 1.28; p < 0.001), TG (140.13 ± 71.63 vs. 163.26 ± 32.90; p = 0.033), VLDL (28.03 ± 14.33 vs. 32.65 ± 6.58 p = 0.033), leptin (10.34 ± 3.89 vs. 12.85 ± 4.51; p = 0.002), and TNF-α (8.00 ± 4.38 vs. 13.81 ± 7.13; p < 0.001) while significantly (p < 0.001) lower HDL (43.91 ± 8.42 vs. 36.97 ± 9.05; p < 0.001) and estrogen (41.33 ± 24.90 vs. 23.95 ± 14.45; p < 0.001) in cases as compared to controls. However, at presentation, mean TC and LDL were found similar (p > 0.05) between the 2 groups.
Leptin and TNF-α mRNA expression in visceral adipose tissue We have made comparison of relative leptin and TNF-α mRNA expression in visceral adipose tissue (VAT) of controls and cases
Mean ± SD
a
0.75
Leptin
0.50 0.25
***
Fig. 1 Relative VAT leptin a and TNF-α b mRNA expressions of 2 groups. *** p < 0.001 as compared to controls.
0.00 Controls Cases Groups
b 0.75 Mean ± SD
age, BMI (body mass index), WC (waist circumference), WHR (waist to hip ratio), SBP (systolic blood pressure), and DBP (diastolic blood pressure) were slightly higher in cases as compared to controls while weight, height, and HC (hip circumference) were higher in controls as compared to cases. Comparing the mean basic characteristics of 2 groups, t-test revealed similar (p > 0.05) basic characteristics between the 2 groups except SBP. At presentation, the mean SBP of cases was significantly higher than controls (124.91 ± 8.94 vs. 131.78 ± 14.43; p = 0.004).
TNF-α
0.50 0.25
***
0.00 Controls Cases Groups
▶ Fig. 1). In visceral adipose tissue, the mean expressions of (● both leptin (0.33 ± 0.29 vs. 0.05 ± 0.09, p < 0.001) and TNF-α (0.32 ± 0.31 vs. 0.13 ± 0.09, p < 0.001) lowered significantly in cases as compared to controls.
Leptin and TNF-α mRNA expression in subcutaneous adipose tissue We also compared relative leptin and TNF-α mRNA expression ▶ Fig. 2). in subcutaneous adipose tissue (SAT) in both groups (● In subcutaneous adipose tissue, the mean expression of leptin (4.91 ± 4.01 vs. 0.50 ± 0.92, p < 0.001) also lowered significantly in
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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DBP: Diastolic blood pressure
Original Basic 97
Fig. 2 Relative SAT leptin a and TNF-α b mRNA expressions of 2 groups. ns p > 0.05 or *** p < 0.001 as compared to controls.
7.5 5.0 2.5
a
***
b
TNF-α ns
Mean ± SD
Mean ± SD
1
VAT
Controls Cases Groups 0.75
**
0.50 0.25
Mean ± SD
***
0.50 0.25
VAT
Leptin
10.0
***
7.5 5.0 2.5
Fig. 3 Relative VAT and SAT leptin a and TNF-α b mRNA expressions of controls. ns p > 0.05 or *** p < 0.001 as compared to VAT.
0.0 VAT
Mean ± SD
SAT
TNF-α
0.75
Controls Cases Groups
b
Groups
0.00
0.00
a
Fig. 4 Relative VAT and SAT leptin a and TNF-α b mRNA expressions of cases. ** p < 0.01 or *** p < 0.001 as compared to VAT.
0
0.0
b
Leptin
2
Groups
SAT
TNF-α
0.75
ns
0.50 0.25 0.00 VAT
Groups
SAT
Groups
SAT
Correlation The correlation of relative VAT and SAT mRNA expressions of both leptin and TNF-α with biochemical profiles (glucose, insulin, HOMA, TG, TC, HDL, LDL, VLDL, serum leptin, serum TNF-α, ▶ Table 3. and serum estrogen) of all subjects are summarized in ● The relative VAT leptin mRNA expression showed significant and negative (inverse) correlation with both glucose (r = − 0.32, p < 0.001) and serum TNF-α (r = − 0.21, p < 0.05). Further, relative VAT TNF-α mRNA expression also showed significant and negative correlation with both glucose (r = − 0.23, p < 0.01). In contrast, relative SAT leptin mRNA expression showed significant and positive (direct) correlation with both HDL (r = 0.20, p < 0.05) and serum estrogen (r = 0.30, p < 0.01) while significant and negative correlation with both glucose (r = − 0.26, p < 0.01) and serum TNF-α (r = − 0.29, p < 0.01). Conversely, relative SAT TNF-α mRNA expression showed significant and direct association (correlation) with both insulin (r = 0.21, p < 0.05) and HOMA (r = 0.20, p < 0.05).
Discussion cases as compared to controls. However, the relative mean mRNA expression of TNF-α did not differ significantly between the groups, that is, it was found to be statistically the same (0.36 ± 0.28 vs. 0.34 ± 0.28, p = 0.667).
Leptin and TNF-α mRNA expression in visceral and subcutaneous adipose tissue The comparatively relative VAT and SAT mRNA expressions of both ▶ Fig. 3, 4 leptin and TNF-α in controls and cases are shown in ● respectively. In controls, the relative mean mRNA expression of leptin was significantly higher in subcutaneous adipose tissue as compared to visceral adipose tissue (0.33 ± 0.29 vs. 4.91 ± 4.01; p < 0.001); however, the expression of TNF-α was found similar between the 2 tissues (0.32 ± 0.31 vs. 0.36 ± 0.28; p = 0.368). Conversely, in cases, the relative mean mRNA expressions of both leptin (0.05 ± 0.09 vs. 0.50 ± 0.92; p = 0.001) and TNF-α (0.13 ± 0.09 vs. 0.34 ± 0.28; p < 0.001) was found significantly different and higher in subcutaneous adipose tissue as compared to visceral adipose tissue.
▼
Previous studies from several groups [31–33] have provided the evidence for a regional difference in leptin expression, as they demonstrated higher leptin mRNA levels in SAT than in VAT samples from centrally obese and non obese subjects. In the present study we have made the comparison of VAT leptin and TNF-α mRNA expression and SAT leptin and TNF-α mRNA expression including regional difference between controls and cases. Our finding demonstrates that SAT leptin mRNA expression was significantly lower in cases as compared to controls. This finding is consistent with the recent study, which indicates that SAT leptin expression was lower in women with increased visceral fat volume [34]. However, our results also demonstrate that leptin mRNA expression was higher in SAT as compared to VAT in both the groups. This is in support of the study of Montague et al. who also found higher expression of leptin gene in subcutaneous adipose tissue compared to visceral adipose tissue [18]. However, subcutaneous fat is responsible of 80 % of total leptin production. This was shown in cultures ex vivo where the production of leptin was higher in subcutaneous adipocytes than visceral adi-
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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Leptin
10.0
Mean ± SD
Mean ± SD
a
98 Original Basic
Variables Glucose (mg/dl) Insulin (μU/ml) HOMA TG (mg/dl) TC (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) Serum leptin (ng/ml) Serum TNF-α (pg/ml) Serum estrogen (pg/ml)
Relative VAT leptin
Relative VAT TNF-α mRNA
Relative SAT leptin mRNA
Relative SAT TNF-α mRNA
mRNA expression
expression
expression
expression
− 0.26** − 0.11 ns − 0.17 ns − 0.03 ns − 0.06 ns 0.20* − 0.11 ns − 0.03 ns − 0.13 ns − 0.29** 0.30**
− 0.01 ns 0.21* 0.20* 0.08 ns 0.10 ns 0.05 ns 0.05 ns* 0.08 ns − 0.11ns − 0.08 ns − 0.01 ns
− 0.32*** − 0.06 ns − 0.15 ns 0.01ns 0.01ns 0.11ns − 0.02 ns 0.01ns − 0.03 ns − 0.21* 0.12 ns
− 0.23** 0.06ns − 0.02 ns − 0.14 ns 0.03ns − 0.04 ns 0.08 ns − 0.14 ns − 0.05 ns − 0.17 ns 0.09 ns
VAT: Visceral adipose tissue; SAT: Subcutaneous adipose tissue ns
p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001
pocytes [35]. This could be a possible explanation for higher leptin expression in SAT in humans and stimulation of leptin by estrogen in females [36]. Interestingly the present study finds the significant and positive correlation between leptin mRNA expression and estrogen. The present finding suggests that estrogens may be modulators of leptin’s catabolic action in the brain. Higher levels of estrogen have been associated with increased central leptin sensitivity in rodents [37]. Differences in central leptin sensitivity caused by the presence or absence of estrogens may occur downstream of leptin receptor transcription and translation [38]. Leptin plasma concentration and mRNA expression in adipose tissue are directly related to obesity severity, as an increase of fat mass is associated with an increase of leptin, which makes leptin an indicator of total fat mass [20]. In the present study, we have found higher serum leptin concentration in cases as compared to controls. The finding is in well agreement with previous findings, which shows significant effect of serum leptin on insulin action, potentially linking obesity with insulin resistance [39]. Leptin is important in the regulation of appetite and energy balance, as shown by animal studies [40]. In humans and rodents, plasma leptin concentrations are highly correlated with BMI [41]. It appears that with increasing leptin concentrations, the hormone induces target cells to become resistant to its actions. In mice that became obese after being fed a high-fat diet, leptin concentrations increased, and this increase was accompanied by an increased expression of SOCS-3 (suppressor-of-cytokine-signaling), a potent inhibitor of leptin signaling [38]. One study reported that leptin impairs insulin signaling by increasing IRS-1 phosphorylation at the serine 318 site [42]. Leptin may task through insulin receptor substrate IRS-1 and IRS-2 suggesting that there are cross-talks of insulin and leptin signaling pathways [43]. However, the role of elevated leptin in obesity-associated insulin resistance is still a debate. In the present study, like leptin, VAT TNF-α mRNA expression was significantly lower in cases as compare to controls. The present study did not find any significant difference of SAT TNF-α mRNA expression between cases and controls. This finding corroborates with Koistinen et al. who showed similar TNF-α mRNA expression in 7 lean and 10 obese nondiabetic and 9 type-2 diabetic men [44]. However, a recent study by Selcuk Gormez et al. shows higher expression of TNF-α in subcutaneous adipose tissue of metabolic syndrome’s subjects [45]. Our results show that TNF-α mRNA expression was higher in SAT as compared to VAT of case group. Our finding is consistent
with Bullo et al. who reported that TNF-α mRNA expressions of the adipose tissue in obese and morbid obese patients were significantly higher than in controls. [46]. Increased expression of TNF-α mRNA in the subcutaneous abdominal adipose tissue depot has been documented in obese rodents and humans [10, 27, 28], leading to the hypothesis that TNF-α may play a crucial role in obesity-related insulin resistance [46] although clinical studies have shown that visceral adipose tissue is closely linked to insulin resistance. Moreover, TNF-α is weakly expressed either in subcutaneous or in deep human adipose tissue depots and this expression is not always modified in obesity [45]. This corresponds with the evaluation of in vivo secretion, which showed that TNF-α production by subcutaneous abdominal adipose tissue was quantitatively negligible in lean and obese subjects [47]. The results of the present study show that subcutaneous adipose tissue TNF-α mRNA expression is positively associated with insulin and HOMA. The findings are in well agreement with previous findings, which show HOMA index correlated positively with TNF-α gene expression [34]. Our results confirm and extend the findings of Hotamisligil et al. in a small sample of 19 obese females [10]. These authors reported that the adipose-tissue expression of TNF-α was positively related to BMI and decreased in proportion to the loss in body weight. This suggests that TNF-α has an important role in the pathophysiology of obesity. Previous studies support the role of VAT in disease development but we cannot ignore the role of subcutaneous adipose tissue. Thus, the present study concludes that the SAT leptin and TNF-α mRNA expressions may have a modulatory role in metabolic syndrome and leptin expression may be under the control of estrogen. Further study is needed to understand the regulatory mechanism of leptin and TNF-α expression.
Acknowledgements
▼
We thank Indian Council of Medical Research, New Delhi for support of this study.
Conflict of Interest
▼
The authors declare that they have no conflicts of interest in the authorship or publication of this contribution.
Tiwari S et al. TNF-α and Subcutaneous Adipose Tissue … Horm Metab Res 2014; 46: 94–99
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Table 3 Correlation of relative VAT and SAT leptin and TNF-α mRNA expressions with biochemical profiles (n = 108).
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