Mol. Cells, Vol. 18, No. 3, pp. 340-345
Molecules and Cells KSMCB 2004
Polymorphism of the Tumor Necrosis Factor Alpha Gene and Waist-Hip Ratio in Obese Korean Women Jae-Young Um1,3, Byung-Ku Kang2, Si-Hyeong Lee2, Jo-Young Shin2, Seung-Heon Hong3, and Hyung-Min Kim1,* 1
Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, Seoul 130-701, Korea; College of Oriental Medicine, Wonkwang University, Iksan 570-749, Korea; 3 Department of Oriental Pharmacy, College of Pharmacy, Wonkwang University, Iksan 570-749, Korea. 2
(Received July 5, 2004; Accepted August 13, 2004)
A number of candidate genes have been in implicated in the pathogenesis of obesity in humans. Tumor necrosis factor-alpha (TNFα) is expressed primarily in adipocytes, and elevated levels of this cytokine have been linked to obesity and insulin resistance. Recently, the A allele of a polymorphism at position –308 in the promoter region of TNFα (G-308A) has been shown to increase transcription of the gene in adipocytes. We therefore designed this study to test whether obese and nonobese subjects differ in terms of TNFα genotype distribution. We also investigated whether the genotypes affect anthropometric parameters, such as body mass index (BMI). The study included 153 obese healthy women and 82 non-obese women. Total fat mass and percent body fat were determined by dual-energy X-ray absorptiometry. Genomic DNA was extracted and used for NcoI restriction fragment length polymorphismbased genotyping of TNFα. No differences were observed in allele and genotype frequencies between obese and non-obese women, and no association of TNFα polymorphism with BMI was observed for genotype in the obese women. In addition, age, percent body fat, BMI, and cholesterol levels did not vary with TNFα genotype. However, waist-to-hip ratio (WHR) was significantly lower in subjects with TNFα GA or AA genotypes (0.94 ± 0.07 vs. 0.92 ± 0.03, P < 0.005). These results indicate that polymorphism at position –308 of the TNFα promoter is not a significant factor for BMI, but affects WHR in obese healthy Korean women. Keywords: Koreans; Obese Women; Polymorphism; Tumor Necrosis Factor-Alpha; Waist-Hip Ratio. * To whom correspondence should be addressed. Tel: 82-2-961-9448; Fax: 82-2-968-1085. E-mail:
[email protected]
Introduction Obesity is increasing in prevalence and is associated with several adverse health problems, including type 2 diabetes, hyperlipidemia, and hypertension. The influence of obesity on the development of obesity-related disorders is complex and probably involves interactions between genetic, nutritional, and metabolic factors (Comuzzie and Allison, 1998; Perusse and Bouchard, 1999). The cytokine, tumor necrosis factor (TNF)α, acting as a modulator of gene expression in adipocytes, has been implicated in the development of insulin resistance and obesity (Hotamisligil and Spiegelman, 1994). Fat tissue is a significant source of endogeneous TNFα production, and the expression of this cytokine is elevated in human obesity in both adipose (Hotamisligil et al., 1995; Kern et al., 1995) and muscle tissues (Saghizadeh et al., 1996). Increased expression of TNFα strongly correlates with the level of hyperinsulinemia (Hotamisligil et al., 1993) and glucose disposal rate during application of the euglycemic clamp technique (Saghizadeh et al., 1996). The gene for human TNFα is located on the short arm of chromosome 6 (Nedwin et al., 1985), and a G → A substitution has been identified at position –308 upstream from the transcription initiation site in the promoter region of the gene (Wilson et al., 1992). In vitro experiments have demonstrated that this NcoI restriction fragment length polymorphism (RFLP) increases transcriptional activation of the TNFα gene (Wilson et al., 1997). Although controversial, the majority of the data support a direct role for this biallelic polymorphism in the elevation of TNFα levels observed in homozygotes for the −308 A allele (Abraham and Kroeger, 1999). Recent studies of the relationship between this polymorphism and insulin resistance have given inconsistent results (Deng et al., 1996; Fernandez-Real et al., 1997; Hamann et al., 1995; Wal-
Jae-Young Um et al.
ston et al., 1999). Fernandez-Real et al. (1997) showed that the polymorphism influenced insulin sensitivity via an increase in body fat in a group of non-diabetic normotensive Spanish subjects. A recent study showed no difference between type 2 diabetes mellitus patients and healthy control subjects in the frequencies of alleles at – 308 (Hamann et al., 1995) and Walston et al. reported that TNFα polymorphism at –308 site did not relate to any traits of obesity and insulin resistance in a group of non-diabetic subjects (Walston et al., 1999). Because the etiology of human obesity is complex and multifactorial, we excluded obese subjects with metabolic disorders including diabetes and dyslipidemia, in order to investigate only the relationship between TNF genotype and obesity. We designed this study to test whether obese and non-obese subjects differ in TNFα genotype distribution, and whether the genotypes affect the anthropometric parameters of obese healthy women.
Materials and Methods Subjects Subjects were healthy obese (body mass index, BMI ≥ 25 kg/m2, range 25−39.1) women between the ages of 15−40 years. All subjects were nonsmokers and had no evidence of cancer, liver, renal, hematological disease or metabolic disorder other than obesity. A total of 153 women met all study criteria and were enrolled. All subjects were normotensive, and subjects with dyslipidemia (total cholesterol > 250 mg/dl, HDLcholesterol < 35 mg/dl) and/or diabetes were excluded. All methods and procedures for the study were approved by the institutional review board of our hospital. Each participant provided written informed consent.
Phenotypic measurements Anthropometry Height (in cm) and weight (in kg) were measured to calculate BMI as weight (kg)/height (m) squared. Waist circumference (measured at the narrowest point superior to the hip) was divided by the circumference of the hip (measured at its greatest gluteal protuberance) to obtain the waist-to-hip ratio (WHR). Dual-energy X-ray absorptiometry Fat mass was determined by dual-energy X-ray absorptiometry (DEXA). Genotyping Genomic DNA was extracted from whole blood by an inorganic procedure (Miller et al., 1988). The single base pair polymorphism at position –308 in the promoter region of the TNFα gene was examined by the NcoI (Takara, Japan) RFLP method (Cabrera et al., 1995). The primers used were 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and 5′-TCCTCCCTGCTCCGA-TTCCG-3′.
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Statistical analysis The mean levels of all numerical values were tested by Student’s t-test or ANOVA. Comparisons of genotypes and allele frequencies of the TNFα genotypes between groups were carried out using Pearson’s chi-squared test. Odds ratios were calculated with 95% confidence intervals, and all statistical analyses were performed with SPSS v10.00 (SPSS Inc.) statistical analysis software. A P-value of less than 0.05 was considered statistically significant.
Results Clinical characteristics of obese women The clinical characteristics of the obese women and controls are presented in Table 1. 27.5% of the obese women were classified as BMI 25−26 (n = 42), 35.9% as BMI 27−29 (n = 55), and 36.6% as BMI 30−40 (n = 56). As expected, weight, fat mass, percent body fat (PBF), and WHR differed between the three BMI groups (Table 1). Frequencies of alleles and genotypes Genotype frequencies in all groups were in accordance with the HardyWeinberg equilibrium. The distribution of TNFα genotype in the obese women (BMI ≥ 25) was as follows; GG, 129 (84.3%); GA, 22 (14.4%); and AA, 2 (1.3%). This was not significantly different from the distribution in the non-obese women (BMI < 25): GG, 68 (82.9%); GA, 12 (14.6%); and AA, 2 (2.4%). The allele frequencies in the obese women were: G, 280 (91.5%); and A, 26 (8.5%). This also was not significantly different from the distribution in non-obese women: G, 1480 (90.2%); and A, 16 (9.8%) (Table 2). In addition, TNFα –308 G > A did not show any association with obesity by logistic regression analysis controlling for confounding factors. Characteristics of obese women according to TNFα genotype Table 3 describes the relationships between the TNFα genotype and anthropometric parameters in obese women. Interestingly, WHR was significantly lower in women with GA + AA genotypes than in those with the GG genotype (0.92 ± 0.03 vs. 0.94 ± 0.07, P = 0.004). The remaining variables were similar in the GG and GA + AA genotypes as well as among the three genotypes. We also investigated the relationships between TNFα genotype and anthropometric parameters in non-obese women. However, we did not find any differences between GG and GA + AA genotypes in non-obese women (data not shown). TNFα polymorphism and BMI We failed to detect any statistically significant association between TNFα polymorphism and BMI (Table 4). We note that the frequency of the A allele was lower in the groups with BMI 27−40 kg/m2 than in the group with BMI 25−26 kg/m2, although
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Table 1. Characteristics of obese women according to BMI (n = 153). Non-obese Controls 82 025.6 ± 7.5 062.2 ± 19.5 161.3 ± 5.2 163.7 ± 25.4 077.3 ± 38.8 019.5 ± 2.1 033.3 ± 2.6 00.85 ± 0.04 112.9 ± 9.5 072.9 ± 9.5
N (%) Age (year) Weight (kg) Height (cm) Total cholesterol (mg/dl) Triglyceride (mg/dl) Fat mass (kg) PBF (%) WHR SBP (mm Hg) DBP (mm Hg)
~26 42(27.5) 028.0 ± 9.8 064.4 ± 4.9 158.7 ± 6.5 182.9 ± 45.3 116.7 ± 112.0 023.2 ± 2.6 035.9 ± 2.9 00.89 ± 0.03 110.8 ± 11.6 069.1 ± 7.9
BMI (kg/m2) 27~29 55(35.9) 031.5 ± 10.0 070.8 ± 6.0 158.2 ± 6.0 174.6 ± 34.7 122.2 ± 79.5 026.1 ± 3.2 036.1 ± 6.2 00.93 ± 0.04 116.7 ± 17.2 071.3 ± 11.3
30~ 56(36.6) 030.2 ± 11.3 085.7 ± 14.6 163.5 ± 7.0 196.2 ± 41.5 163.2 ± 140.6 034.4 ± 8.4 139.6 ± 6.0 10.98 ± 0.07 121.7 ± 11.5 077.4 ± 7.5
P*
0.258 < 0.001 < 0.001 0.027 0.086 < 0.001 0.001 < 0.001 0.083 0.023
Values are means ± SD; BMI, body mass index; PBF, percentage body fat; WHR, ratio of waist-to-hip circumstance; SBP, systolic blood pressure; DBP, diastolic blood pressure. * By one-way ANOVA (among BMI groups).
Table 2. Genotype and allele frequencies of TNFα in non-obese and obese women. Non-obese, n (%) n = 82 Genotype GG GA AA Allele G A
Obese, n (%) n = 153
P*
068 (82.9) 012 (14.6) 002 (02.4)
129 (84.3) 022 (14.4) 002 (01.3)
x 2 = 0.416, P = 0.812
148 (90.2) 016 (09.8)
280 (91.5) 026 (08.5)
x 2 = 0.208, P = 0.649
OR (95% CI)
0.859 (0.447-1.652)
OR, odds ratio; CI, confidence interval. * By x 2 test (two-sided).
the difference was not statistically significant. In obese women with BMI ≥ 30 kg/m2, carriers of the GA + AA genotypes tended to exhibit a lower frequency of the A allele than did women with BMI < 30 kg/m2 (14.3% vs. 16.5%).
Discussion The purpose of the present study was to determine whether the G/A polymorphism of the TNFα gene is associated with obesity and anthropometric parameters in women. Obesity is a complex metabolic disorder with a strong genetic component, and there are many candidate genes for obesity and its related phenotypes. Most of these genes are candidates because mutations in them cause rare genetic syndromes affecting adipocyte differentiation (Bouchard et al., 1998). Recent interest has focused on the role of the TNFα gene in insulin-resistance and obesity. We therefore examined the association between the polymorphism of TNFα and obesity without metabolic disease.
However, we did not find significant differences between obese women and non-obese women in the frequencies of TNFα polymorphism. A number of polymorphisms have been identified in TNFα including the TNFα –308G > A promoter polymorphism (Shin et al., 2004). However, our study focused on the latter, which is particularly interesting because of its known involvement in differential transcriptional activation (Wilson et al., 1997), elevated plasma TNFα levels, and higher levels of TNF on stimulation in vivo and ex vivo (Louis et al., 1998). Nevertheless, there should be further studies of other polymorphisms of TNF genes since all the single-nucleotide polymorphisms (SNP) in TNF genes are in very strong linkage disequilibrium. Interestingly, we found that WHR was lower in women with GA + AA genotypes than in those with the GG genotype. This finding differs from those reported by Rosmond et al. (2001), who found no association between TNFα and obesity (BMI) and body fat distribution (WHR and abdominal sagittal diameter). Contrary to their findings, some studies have suggested an association of TNFα
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Table 3. Characteristics of obese women according to TNFα genotype (n = 153). Genotypes GG 129 (84.3) 030.1 ± 11.3 074.8 ± 13.7 160.3 ± 6.5 186.2 ± 43.2 135.6 ± 115.3 028.7 ± 7.9 029.2 ± 4.1 037.5 ± 5.9 00.94 ± 0.07 119.0 ± 14.6 074.1 ± 9.7
N (%) Age (year) Weight (kg) Height (cm) Total cholesterol (mg/dl) Triglyceride (mg/dl) Fat mass (kg) BMI (kg/m2) PBF (%) WHR SBP (mm Hg) DBP (mm Hg)
GA + AA 24 (15.7) 029.4 ± 08.20 072.4 ± 11.50 159.6 ± 9.10 178.9 ± 26.70 125.0 ± 84.22 026.6 ± 4.60 028.3 ± 2.20 036.8 ± 3.60 00.92 ± 0.03 112.2 ± 6.70 072.2 ± 6.7
P* 0.522 0.422 0.654 0.477 0.694 0.212 0.265 0.605 0.004 0.182 0.577
Values are means ± SD; BMI, body mass index; PBF, percentage body fat; WHR, ratio of waist-to-hip circumstance; SBP, systolic blood pressure; DBP, diastolic blood pressure. * By t-test (GG genotype vs. the other genotypes).
Table 4. Frequencies of TNFα genotypes according to BMI in obese women (n = 153). BMI (kg/m2)
Genotypes
P*
25−26, n (%) 27−29, n (%) 30−40, n (%)
GG 34 (81.0) 47 (85.5) 48 (85.7)
GA + AA 08 (19.0) 08 (14.5) 08 (14.3)
x2 = 0.496, P = 0.780
< 30, n (%) ≥ 30, n (%)
81 (83.5) 48 (85.7)
16 (16.5) 08 (14.3)
x2 = 0.131, P = 0.717
OR (95% CI)
0.84 (0.34-2.12)
BMI, body mass index; OR, odds ratio; CI, confidence interval. * By x 2 test (two-sided).
polymorphism with obesity in several European populations (Fernandez-Real et al., 1997; Herrmann et al., 1998). However, the design of those studies and the ethnic populations examined were quite different from the present study, and the results are hardly comparable. Moreover, obesity in women (BMI ≥ 25) was not the main focus of those studies. It is well known that body fat distribution generally differs between men and women. Men often have upperbody obesity, whereas peripheral obesity is the most common form among women. In fact, several of the polymorphisms have a different impact on body fat in men and women. Many studies suggest that Trp64Arg polymorphism in the β3-adrenoceptor is only associated with obesity in women (Arner and Hoffstedt, 1999). Gln27Glu polymorphism in the β2-adrenoceptor has been linked to obesity in women but not in men in a Swedish population (Hellstrom et al., 1999). These data indicate that men and women have different obesity genes. Therefore, further studies are needed to investigate the association of TNFα gene polymorphism with male obesity.
We selected TNFα, a candidate gene for obesity in Pima Indian, mainly because of the reported overexpression of TNFα mRNA in the adipose tissue of rodents, which are genetic models for insulin resistance and associated obesity (Hotamisligil et al., 1993). Levels of TNFα mRNA and protein in the adipose tissue of obese human subjects were found to be 2.5 times higher than in lean controls and highly correlated with fasting insulin concentration (Hotamisligil et al., 1995). In addition, it was reported that substitution of adenine (A) for guanine (G) at position –308 in the promoter region of TNFα enhanced the transcription of this cytokine in cultured cells (Wilson et al., 1997). Indeed, some of the previous studies suggest that homozygotes for the –308 variant are more obese than groups with other genotypes (Fernandez-Real et al., 1997; Hoffstedt et al., 2000; Norman et al., 1995). We therefore expected to find higher WHR in GA + AA subjects. Instead we observed lower WHR in women with GA + AA genotypes. Nonetheless, these results tend to confirm that several cytokines (e.g., TNFα, IL-1α, IL-6, and IFN-γ) suppress food intake in animals and humans in
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the same manner as leptin (Langstein and Norton, 1991). It may be premature to view the polymorphism at position −308 of the TNFα gene as having a key role in the pathogenesis of obesity since it is probably related to differences in TNFα synthesis, secretion, and activity. However, we believe that the increased basal expression of TNFα as a consequence of the polymorphism could be a protective factor against increases WHR. Further studies are needed to test this idea. In summary, we observed no differences in allele and genotype frequencies between obese women and nonobese women, and no association of TNFα polymorphism with BMI for genotype in obese women. However, WHR was significantly lower in subjects with TNFα GA and AA genotype. These results suggest that TNFα promoter polymorphism at position –308 does not have a significant influence on BMI, but affects WHR in obese healthy Korean women.
Acknowledgment This research was supported by Wonkwang University in 2003.
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