Interaction of the -308G/A promoter polymorphism of the tumor necrosis factor-[alpha] gene with single-nucleotide polymorphism 45 of the adiponectin gene: effect on serum adiponectin concentrations in a Spanish population.
Serum adiponectin concentrations have been shown to be decreased in the presence of obesity, type 2 diabetes, insulin resistance, and cardiovascular disease (8-10). The adiponectin gene is located on chromosome 3q27, on which a diabetes susceptibility locus has recently been mapped (11). Several single-nucleotide polymorphisms (SNPs) in the adiponectin gene have been reported (12). Among those, SNPs 45T>G and 276G>T were originally selected for association studies because of their high frequencies in all populations tested, whereas other reported polymorphisms were rare. Both SNPs 45T>G and 276G>T have been associated with obesity, insulin resistance, and type 2 diabetes (13-15) and with the development of hyperglycemia (16). Most recently, these 2 SNPs have also been reported to be predictors of the conversion from impaired glucose tolerance to type 2 diabetes (17). The exact mechanisms through which SNPs 45 and 276 influence insulin resistance or impaired glucose tolerance are not known at present. SNP 45 is located in exon 2 and is a synonymous mutation, and SNP 276 is located in intron 2. It is also possible, however, that the presence of unknown functional SNPs or other functional genetic loci in linkage disequilibrium with SNPs 45 and 276 are responsible for alterations in insulin sensitivity and glucose homeostasis.
The crystal structures of the trimeric globular head domains of adiponectin and trimeric TNF-[alpha] are very similar (18), and a close inverse functional relationship between TNF-[alpha] and adiponectin gene expression has been reported (19, 20). The TNF-[alpha] gene is therefore a potential candidate gene responsible for the regulation of adiponectin expression and production by adipocytes. Because adiponectin and TNF-[alpha] inhibit each other's synthesis in adipocytes (19), it appears that both adipocytokines interact on different metabolic pathways. On the basis of these considerations, we investigated whether the -308G/A polymorphism in the promoter region of the TNF-[alpha] gene and SNPs 45 and 276 of the adiponectin gene are associated with circulating adiponectin and sTNFR2 concentrations.
Materials and Methods
We studied 809 unrelated Caucasian men (n = 383; 47.3%) and women (n = 426; 52.7%), ages 35-74 years, recruited by a simple random sampling approach from a crosssectional population-based epidemiologic survey in the province of Segovia in central Spain (Castille). The aim of the study was to investigate the prevalence of anthropometric and physiologic variables related to obesity and other components of the metabolic syndrome. From the whole population of Segovia (147 028 inhabitants), our sample included 2992 men and nonpregnant women. A total of 1033 individuals agreed to participate; 91 were not genotyped because they did not give consent to obtain a DNA sample. Individuals with a previous diagnosis of type 1 diabetes (n = 133) were excluded from the study.
All study participants gave written consent to participate. The study protocol was approved by the Ethics Committee of the Hospital Clinico San Carlos of Madrid.
Anthropometric measurements included BMI (kg/[m.sup.2]), WHR, and sagittal abdominal diameter (SAD; cm). Obesity was defined as BMI [greater than or equal to] kg/[m.sup.2] (n = 208; 88 men and 120 women). Systolic and diastolic blood pressures were measured to the nearest even digit by use of a randomzero sphygmomanometer. Each blood pressure measurement was performed 3 times with the participant in the sitting position and resting for 10 min before the measurement.
After participants had fasted overnight, 20 mL of blood was obtained from an antecubital vein without compression. Plasma glucose was determined in duplicate by a glucose oxidase method adapted to an automated analyzer (Hitachi 704; Boehringer Mannheim). Total cholesterol, triglycerides, and HDL-cholesterol were determined by enzymatic methods with commercial reagents sets (Boehringer Mannheim). LDL-cholesterol was calculated by the Friedewald formula. Serum insulin and adiponectin concentrations were determined by RIA (Human Insulin Specific RIA Kit and Human Adiponectin Specific RIA Kit, respectively; Linco Research Inc.). Plasma concentrations of the soluble fraction of TNFR2 were measured by ELISA (HyCult Biotechnology).
In the total study population, 543 participants (71.2%) had unimpaired glucose tolerance, 49 (6.4%) had impaired fasting glucose, 102 (13.4%) had impaired glucose tolerance, and 69 (9.0%) had type 2 diabetes according to the criteria of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (21). These cases of type 2 diabetes had not been diagnosed previously and were discovered during the survey. Blood samples were obtained for the determination of glucose and insulin concentrations in the fasting state and 1 and 2 h after glucose administration. Insulin resistance was estimated by the homeostasis model assessment (HOMA-IR) method according to the formula: insulin ([mu]IU/mL) x glucose (mmol/L)/22.5 (22).
Genomic DNA was extracted from EDTA-whole blood samples with a QIAamp DNA Blood Mini Kit (Qiagen). The -308G/A polymorphism of the TNF-[alpha] gene was screened by restriction fragment length polymorphism analysis after digestion with NcoI restriction enzyme. The PCR primers used were as follows: 5'-AGGCAATAGGTTGAGGGCCAT3' and 5'-TCCTCCCTGCTCCGATTCCG3'. The reaction was carried out in a final volume of 50 [micro]L containing 3 mM Mg[Cl.sub.2], 0.5 mM each of the deoxynucleotide triphosphates (Boehringer Mannheim), 0.2 pmol of each primer, 2.5 U of Taq DNA polymerase (Boehringer Mannheim), and 200 ng of DNA. The reaction mixture was first subjected to 1 cycle of 3 min of denaturation at 94[degrees]C, 1 min of annealing at 60[degrees]C, and 1 min of extension at 72[degrees]C, after which the DNA was amplified for 35 cycles, each consisting of 1 min of denaturation at 94[degrees]C, 1 min of annealing at 60[degrees]C, and 1 min of extension at 72[degrees]C. After amplification, the reaction mixture was subjected to a final cycle of 1 min of denaturation at 94[degrees]C, 1 min of annealing at 60[degrees]C, and 5 min of extension at 72[degrees]C. PCR products were digested at 37[degrees]C overnight with a 10-fold excess of NcoI restriction enzyme and electrophoresed on a 3% agarose gel. NcoI restriction fragment length polymorphisms were detected by ethidium bromide staining, which revealed a 2-allele polymorphism that produced 3 bands of different sizes: a 107-bp fragment corresponding to the -308A allele (restriction site absent) and 87- and 20-bp fragments corresponding to the -308G allele (restriction site present).
We used the SNaPshot ddNTP Primer Extension Kit to genotype SNP 45 (T[alpha]G) and SNP 276 (G[alpha]T) of the adiponectin gene. Forward (5'-GGCTCAGGATGCTGTTGCTGG-3') and reverse (5'-GCTTTGCTTTCTCCCTGTGTCT-3') primers were used to amplify a 328-bp DNA fragment. The following PCR cycling conditions were used: 94[degrees]C for 4 min; 35 cycles of 94[degrees]C, 57[degrees]C, and 72[degrees]C for 30 s each; and 72[degrees]C for 4 min. The PCR product was purified by addition of 1 U of shrimp alkaline phosphatase and 2 U of ExoI and incubation at 37[degrees]C for 60 min and in 75[degrees]C for 15 min.
To determine the genotypes, we used the following primers: 5'-CTGCTATTAGCTCTGCCCGG-3' for SNP 45 and 5'-ACCTCCTACACTGATATAAACTAT-3' for SNP 276. The SNaPshot reaction was performed with a mixture containing 3.75 [micro]L of Tris-HCl, 1.25 [micro]L of SNaPshot Multiplex Ready Reaction Mix (ABI Prism; Applied Biosystems), 0.15 [micro]L of the primer for SNP 45, 0.075 [micro]L of the primer for SNP 276, and 0.775 [micro]L of distilled [H.sub.2]O. The mixture was incubated for 10 s at 94[degrees]C and for 45 cycles of 96[degrees]C for 10 s, 50[degrees]C for 5 s, and 60[degrees]C for 30 s. The reaction mixture was purified by addition of 1 U of shrimp alkaline phosphatase at 37[degrees]C for 60 min and at 75[degrees]C for 15 min. Before samples were loaded on the ABI Prism 3100 Genetic Analyser (Applied Biosystems), 9 [micro]L of formamide and 0.25 [micro]L of size markers were added to 0.5 [micro]L of the reaction mixture and samples were heated at 95[degrees]C for 5 min.
Genotype and allele frequency distributions were compared with the [chi square] test. Student t-test and ANOVA were used to compare continuous variables [expressed as means (SD)], and categorical variables were compared using the [chi square] test. Continuous variables that did not have a gaussian distribution were log-transformed; they are presented in their original scales. Multiple linear regression analysis was carried out to investigate the effect of SNP 45 of the adiponectin gene and the -308G/A polymorphism of the TNF-[alpha] gene on serum adiponectin concentrations (log-transformed). Variables included in the multivariate model were those with biological relevance or an impact on circulating plasma adiponectin concentrations demonstrated in the simple correlation analysis. Adjusted odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated, and the existence of interactions was evaluated. Linear relationships between key variables were tested by Pearson's correlation coefficient. Multiple linear analyses were performed to evaluate the independent relationships of the variables studied. Linkage disequilibrium (D') between the SNPs studied and haplotype frequencies were calculated using Thesias software (23). The null hypothesis was rejected in each statistical test when P was <0.05. Analysis was performed with SPSS for Windows (Ver. 11.0) software (SPSS, Inc.).
In the total study population, the genotype distributions were 73.1% -308G/G, 24.8% -308G/A, and 2.1% -308A/A for the TNF-[alpha] gene (Table 1). Allele frequencies of the -308G and the -308A alleles were 85.4% and 14.6%, respectively, and they were in Hardy-Weinberg equilibrium. Because only a few participants were homozygous for -308A/A (n = 17) and homozygotes and heterozygotes had similar serum adiponectin concentrations [9.7 (5.3) vs 10.3 (5.8) mg/L; P = 0.482] and prevalence of impaired glucose tolerance (17.6% vs 15.0%; P = 0.414), we combined both in all statistical analyses. The genotype distributions (Table 1) and allele frequencies and characteristics of the participants categorized by SNPs 45 and 276 of the adiponectin gene have been reported previously (24).
ASSOCIATION BETWEEN TNF-[alpha] GENE POLYMORPHISM AND METABOLIC VARIABLES
In the overall population, the -308A allele of the TNF-[alpha] gene was associated with higher 2-h postload glucose [6.7 (2.6) vs 6.3 (2.6) mmol/L; P = 0.053] and insulin concentrations [535 (499) vs 455 (368) pmol/L; P = 0.023] compared with -308G/G homozygotes. We found no associations between individual TNF-[alpha] and SNP 45 polymorphisms and HOMA-IR obesity-related variables (BMI and WHR), age, and sex (data not shown). Persons with type 2 diabetes who carried the -308A allele had significantly higher circulating concentrations of sTNFR2 and lower adiponectin concentrations than those with the -308G/G genotype, independent of BMI and HOMA-IR (adjusted OR, 1.52; 95% CI, 1.08 -2.14; P = 0.018) for sTNFR2 concentrations and independent of sex, age, WHR, and HOMA-IR (adjusted OR, 0.13; 95% CI, 0.02-0.97; P = 0.047) for adiponectin concentrations.
We found no association between total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, the metabolic syndrome, and the studied polymorphisms (data not shown).
RELATIONSHIP BETWEEN sTNFR2 CONCENTRATIONS AND ANTHTOPOMETRIC VARIABLES IN PERSONS WITH TYPE 2 DIABETES
Among participants with type 2 diabetes, we found a significantly positive correlation between sTNFR2 concentrations and measures of obesity such as BMI (r = 0.285; P = 0.029), WHR (r = 0.261; P = 0.047), and SAD (r = 0.282; P = 0.030).
COMBINED EFFECTS OF GENES ON METABOLIC VARIABLES
We investigated the effects of genetic interactions between adiponectin SNPs 45 and 276 and TNF-[alpha]-308G/A polymorphisms. The combined effects of these 2 genes on biochemical variables were examined by subgroup analyses. Among carriers of the SNP 45 G allele in the adiponectin gene, those with the -308A allele of the TNF-[alpha] gene had higher 2-h postload glucose [7.1 (2.8) vs 6.2 (2.3) mmol/L; P = 0.014] and insulin concentrations [612 (582) vs 432 (355) pmol/L; P = 0.005] as well as lower circulating adiponectin concentrations [9.7 (5.3) vs 11.3 (5.6) mg/L; P = 0.028] than -308G/G homozygotes. This effect of TNF-[alpha] -308G/A genotypes was not observed among the participants carrying the TT genotype of SNP 45 in the adiponectin gene (Table 2).
To further investigate whether the adverse combination of both genotypes decreases serum adiponectin concentrations, we performed a multiple regression analysis in the overall population with adiponectin concentrations as the dependent variable (Table 3). For each separate polymorphism, we found no statistically significant differences in serum adiponectin concentrations. This model showed, however, that individuals simultaneously having the -308A allele of the TNF-[alpha]gene and the G allele of SNP 45 in the adiponectin gene (n = 71) had lower adiponectin concentrations than those having either gene polymorphism alone (P = 0.005), independent of sex, age, BMI, WHR, HOMA-IR, and glucose tolerance status. Furthermore, participants having these risk genotypes also had the highest incidence of impaired glucose tolerance (21.1%) compared with those having the other genotypes (11.9%; P = 0.037), even after adjustment for sex, age, WHR, and BMI (adjusted OR, 1.26; 95% CI, 1.01-1.56; P = 0.038). We also found a trend toward increased incidence of type 2 diabetes among carriers of these risk genotypes (14.1% vs 8.0%; P = 0.083), but we found no interactive effect between the TNF-[alpha] gene polymorphism and SNP 276 in the adiponectin gene (data not shown).
The main findings of this study are that there may be an interaction between the -308G/A promoter polymorphism of the TNF-[alpha] gene and SNP 45 in the adiponectin gene and that this interaction may be related to a higher incidence of impaired glucose tolerance and low circulating adiponectin concentrations.
We have previously shown an association between Gallele carrier status for SNPs 45 and 276 of the adiponectin gene and impaired glucose tolerance and decreased serum adiponectin concentrations (24). Other reports (13-16) have also shown that several SNPs of the adiponectin gene may, indeed, contribute to obesity, insulin resistance, dyslipidemia, and the risk of type 2 diabetes. The G allele of SNP 45 was not associated with HOMA-IR, BMI, WHR, age, or sex in our population (24), but our results are in agreement with those reported by Zacharova et al. (17) showing that the G allele of SNP 45 is a predictor for the conversion from impaired glucose tolerance to type 2 diabetes. Our findings also agree with reports from studies of Japanese (25) and German (26) populations in which no association of the G allele of SNP 45 with obesity was observed. At variance with our results, Menzaghi et al. (14) showed association of insulin resistance with the T allele. These discrepancies among association studies may be explained, at least partly, by differences in the sample sizes or even in the linkage disequilibrium structure at this locus in different populations.
Moreover, individuals carrying both the TNF-[alpha] and adiponectin risk genotypes (the -308A allele of the TNF-[alpha] gene and the G allele of SNP 45 of the adiponectin gene) had a higher prevalence of impaired glucose tolerance and lower circulating adiponectin concentrations than those carrying individual gene polymorphisms. This result suggests that these 2 genes could participate either in the same pathway or in 2 independent pathways related to the regulation of serum adiponectin concentrations and its impact on glucose homeostasis. Moreover, we observed a trend toward increased incidence of type 2 diabetes among carriers of these risk genotypes. This difference, however, did not reach statistical significance (P = 0.083), perhaps because of the small number of individuals with type 2 diabetes (n = 69) in our study population. The exact molecular mechanisms through which these 2 gene polymorphisms may influence adiponectin gene expression or impaired glucose tolerance are not known. It is possible, however, that the product of one of these genes interacts with the activity of the other gene. Thus, it has been reported recently that TNF-[alpha] inhibits adiponectin production by activating c-Jun Nterminal kinase (JNK), a pathway implicated in obesity and diabetes (27). Furthermore, the precise biological mechanism of this interaction is unknown, and it has not been revealed by genotype-phenotype association studies such as ours. Consistent with our findings, previous studies in cell culture, in animals, and in humans have shown that adiponectin expression at either the mRNA or protein level could be down-regulated by TNF-[alpha] activation (19, 27).
The TNF-[alpha] and adiponectin gene polymorphisms could interact with each other in several ways. For example, TNF-[alpha] might enhance the flux of free fatty acids by stimulating lipolysis in adipose tissue, leading to insulin resistance. Other mechanisms by which TNF-[alpha] impairs insulin sensitivity might be the down-regulation of protein concentrations of IRS-1, GLUT-4, and peroxisome proliferator-activated receptor-[gamma] and adiponectin gene expression in adipocytes. The cross-talk between adiponectin and TNF-[alpha] has also been demonstrated by the suppressive effect of TNF-[alpha] on adiponectin gene expression in vitro (28). In fact, adiponectin and TNF-[alpha] inhibit each other's synthesis in adipocytes (19). In addition, TNF-[alpha]-induced nuclear factor-[alpha] activation is associated with the repression of many adipocyte-abundant genes involved in the uptake and storage of glucose. Importantly, it has recently been reported that adiponectin-null mice have increased concentrations of TNF-[alpha] in adipose tissue and plasma, suggesting interrelated adiponectin and TNF-[alpha] metabolic pathways (29).
In light of our findings, we suggest that both genes could jointly predispose to low adiponectin concentrations, potentially favoring the development of impaired glucose tolerance or type 2 diabetes (30, 31). Our study also provides some evidence of the potential involvement of the -308G/A polymorphism in the TNF-[alpha] gene in the glucose and insulin response to an oral glucose tolerance test. This finding might be of relevance to the natural history of type 2 diabetes because glucose toxicity attributable to chronic hyperglycemia per se impairs both insulin action and insulin secretion (32). As for the association of the -308G/A polymorphism with components of the metabolic syndrome, the available reports have given conflicting results (3, 4, 33-35), likely because of differences in study features such as design, sample size, and sex and age stratification. An important finding of our study is that the -308A allele of the TNF-[alpha] gene was associated with decreased circulating adiponectin concentrations and increased sTNFR2 concentrations in persons with type 2 diabetes, independent of insulin
resistance, BMI, and WHR. Indeed, we found that sTNFR2 concentrations were positively correlated with BMI, WHR, and SAD.
To our knowledge, this is the first study showing that the -308G/A promoter polymorphism of the TNF-[alpha]gene has a gene-gene interaction with SNP 45 of the adiponectin gene, further increasing the risk of impaired glucose tolerance and of low circulating adiponectin concentrations, which might contribute to the metabolic effects of hypoadiponectinemia and its impact on the development of insulin resistance.
We acknowledge the members of the Segovia Insulin Resistance Study Group. We thank Dr. Paul W. Franks (Phoenix Epidemiology & Clinical Research Branch, NIDDK, NIH, Phoenix, AZ) and Dr. Jose Carlos Florez (Massachusetts General Hospital, Boston, MA) for critical reading of the manuscript. We acknowledge Milagros Perez Barba for dedicated and careful technical assistance. This work was supported by Grant FEDER 2FD1997-2309 from Fondo Europeo de Desarrollo Regional, FISS 03/ 1618 from Fondo de Investigaciones Sanitarias, and a grant from Red de Centros RCMN (C03/08), Madrid, Spain. Partial support also came from Educational Grants from Eli Lilly Laboratories (Spain) and Bayer Pharmaceutical Co. (Spain).
Received February 11, 2005; accepted October 6, 2005.
Previously published online at DOI: 10.1373/clinchem.2005.049452
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(3) Nonstandard abbreviations: TNF-[alpha], tumor necrosis factor-[alpha]; sTNFR2, soluble TNF-[alpha] receptor 2; BMI, body mass index; WHR, waist-to-hip ratio; SNP, single-nucleotide polymorphism; SAD, sagittal abdominal diameter; HOMA-IR, homeostasis model assessment for insulin resistance; OR, odds ratio; and CI, confidence interval.
JOSE L. GONZALEZ-SANCHEZ,  MARIA J. MARTINEZ-CALATRAVA,  MARIA T. MARTINEZ-LARRAD,  CARINA ZABENA,  CRISTINA FERNANDEZ-PEREZ,  MARKKU LAAKSO,  and MANUEL SERRANO-RIOS  *
 Department of Internal Medicine II, Hospital Clinico San Carlos, Madrid, Spain.
 Department of Medicine, University of Kuopio, Kuopio, Finland.
* Address correspondence to this author at: Department of Internal Medicine II, Hospital Clinico San Carlos, Cea Bermu dez 66, 5[degrees] G, 28003 Madrid, Spain. Fax 34-91-4429705; e-mail email@example.com.
Table 1. Genotype distributions of the -308G/A TNF-[alpha] polymorphism and SNPs 45 and 276 of the adiponectin gene. No. of individuals (%) Genotype Males Females All P TNF-[alpha] -308 G/A 0.617 GG 277 (72.3) 314 (73.8) 591 (73.1) GA 96 (25.1) 105 (24.6) 201 (24.8) AA 10 (2.6) 7 (1.6) 17 (2.1) Adiponectin SNP 45T>G 0.179 TT 268 (66.1) 286 (62.6) 554 (64.3) TG 129 (31.9) 153 (33.5) 282 (32.7) GG 8 (2.0) 18 (3.9) 26 (3.0) Adiponectin SNP 276G>T 0.592 GG 210 (51.8) 224 (49.0) 434 (50.3) GT 172 (42.5) 201 (44.0) 373 (43.3) TT 23 (5.7) 32 (7.0) 55 (6.4) Table 2. Serum adiponectin concentrations by age group, sex, and genotypes of TNF-[alpha] and SNP 45 of the adiponectin genes. Mean (SD) serum adiponectin, mg/L P Age group, years [less than or equal to]44 9.4 (4.3) >44-54 10.4 (5.7) >54-64 10.5 (4.6) >64 11.8 (5.7) <0.001 Sex Male 8.1 (3.9) Female 12.9 (5.2) <0.001 TNF-[alpha] polymorphism -308G/A -308A/X 10.3 (5.4) -308G/G 10.8 (5.3) 0.268 Combined effects Adiponectin SNP 45GX (a) and TNF-[alpha] -308A/X 9.7 (5.3) TNF-[alpha] -308G/G 11.3 (5.6) 0.028 Adiponectin SNP 45TT (b) and TNF-[alpha] -308A/X 10.6 (5.4) TNF-[alpha] -308G/G 10.5 (5.2) 0.724 (a) Effect of the TNF-[alpha] -308G/A genotype on serum adiponectin concentrations among persons carrying the SNP 45G allele of the adiponectin gene. (b) Effect of the TNF-[alpha] -308G/A genotype on serum adiponectin concentrations among persons carrying SNP 45TT genotype of the adiponectin gene. Table 3. Multiple linear regression analysis with adiponectin concentrations (log-transformed) as the dependent variable in the whole population. Independent [beta] SE 95% CI P variables (a) Sex (F/M) 0.143 0.019 0.106-0.181 <0.001 Age (years) 0.005 0.001 0.003-0.006 <0.001 WHR -0.519 0.103 -0.722 to -0.316 <0.001 HOMA-IR -0.009 0.003 -0.015 to -0.003 0.004 TNF-[alpha] -0.020 0.019 -0.017 to 0.057 0.296 -308G/A (AX vs GG) (b) Adiponectin -0.030 0.017 -0.005 to 0.060 0.092 SNP 45 (GX vs TT) (c) TNF-/Adiponectin -0.090 0.032 -0.153 to -0.026 0.005 SNP 45 (-308AX/ SNP 45GX) (d) (a) Regression coefficient. (b) TNF-[alpha] -308G/A (AX vs GG), persons carrying the A allele compared with GG homozygotes. (c) Adiponectin SNP 45 (GX vs TT), persons carrying the G allele compared with TT homozygotes. (d) TNF-[alpha]/Adiponectin SNP 45 (-308AX/SNP 45GX), persons having both the -308A allele of the TNF-[alpha] gene and the G allele of SNP 45 of the adiponectin gene compared with persons having only one of these gene polymorphisms.
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|Title Annotation:||Endocrinology and Metabolism|
|Author:||Gonzalez-Sanchez, Jose L.; Martinez-Calatrava, Maria J.; Martinez-Larrad, Maria T.; Zabena, Carina;|
|Date:||Jan 1, 2006|
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