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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 5 2178-2180
Copyright © 2001 by The Endocrine Society

Original Studies

G-308A Polymorphism of the Tumor Necrosis Factor {alpha} Gene Promoter and Salivary Cortisol Secretion1

Roland Rosmond, Monique Chagnon, Claude Bouchard and Per Björntorp

Department of Heart and Lung Diseases (R.R., P.B.), Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden; and Pennington Biomedical Research Center (R.R., M.C., C.B.), Baton Rouge, Louisiana 70808

Address all correspondence and requests for reprints to: Roland Rosmond, M.D., Ph.D., Department of Heart and Lung Diseases, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The objective of the current study was to examine the potential impact of the G->A substitution at position -308 of the tumor necrosis factor {alpha} (TNF-{alpha}) gene promoter on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones including salivary cortisol in 284 unrelated Swedish men born in 1944. The subjects were genotyped by using PCR amplification of the 5' untranslated region of the TNF-{alpha} gene followed by digestion with the restriction enzyme NcoI. The frequencies were 0.77 for allele G and 0.23 for allele A. Tests for differences in salivary cortisol levels between the TNF-{alpha} genotypes revealed that there were significantly higher cortisol levels in the morning, before as well as 30 and 60 min after stimulation by a standardized lunch in homozygotes for the rare allele in comparison with the other genotypes. In addition, homozygotes for the rare allele had a tendency toward higher mean values of body mass index, waist to hip ratio, and abdominal sagittal diameter compared with the other genotype groups. The results also indicated a weak trend toward elevated insulin and glucose levels among men with the A/A genotype. In conclusion, a G->A polymorphism in the 5' untranslated region of the TNF-{alpha} gene is associated with elevated morning cortisol levels as well as elevated postprandial cortisol secretion. This increase in cortisol secretion might be the endocrine mechanism underlying the previously observed associations between the NcoI TNF-{alpha} polymorphism and obesity as well as insulin resistance. However, to what extent this polymorphism is associated with these conditions is uncertain from the present data.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE GENE FOR human tumor necrosis factor (TNF-{alpha}) is located on the short arm of chromosome 6 (1), and a G->A substitution at position -308 upstream from the transcription initiation site in the promoter region of the gene has been identified (2). In vitro experiments have demonstrated that this DNA NcoI restriction fragment length polymorphism increases transcriptional activation of the TNF-{alpha} gene (3). Although controversial, the majority of the data support a direct role for this biallelic polymorphism in the elevated TNF-{alpha} levels observed in homozygotes for the -308 A allele (4).

Some, but not all studies, have indicated a key role for the -308 variant of the TNF-{alpha} gene in the pathogenesis of obesity and obesity-associated insulin resistance (5, 6, 7, 8, 9, 10, 11, 12). Interestingly, recent data suggest that the direct paracrine effect of adipose-derived TNF-{alpha} seems to be inhibition of the leptin production (13, 14). TNF-{alpha} has now been shown to affect more centrally located events that regulate lipid and insulin metabolism, and in recent years a considerable body of knowledge has emerged regarding the reciprocal interaction between the hypothalamic-pituitary-adrenal (HPA) axis and the immune system (15). Several circulating cytokines and mediators including TNF-{alpha} have a major role in the increased HPA axis activity in the response to the stress of inflammation (15). Hence, the objective of the present study was to evaluate the potential impact of the G-308A variant of the TNF-{alpha} gene on obesity and estimates of insulin, glucose, and lipid metabolism as well as circulating hormones including salivary cortisol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
For the present study, the subjects (n = 284) were randomly selected from a larger geographically defined total population cohort of men born in Gothenburg, Sweden, in 1944. The design has been described elsewhere (16, 17). All subjects gave written informed consent before participating in the study, which was approved by the local ethics committee.

Body mass index (BMI; kg/m2), waist to hip ratio (WHR), and abdominal sagittal diameter were measured as described previously (16, 17). Salivary cortisol was measured repeatedly over a random working day. Endocrine measurements, beside cortisol, included testosterone, insulin-like growth factor I, and leptin as described previously (16, 17). Insulin, glucose, triglycerides, and total, high-, and low-density lipoprotein cholesterol, were measured in the overnight fasting state as described previously (16, 17). The serum lipids were determined by an enzymatic procedure in a Roche Molecular Biochemicals Cobas Fara II (Roche Molecular Biochemicals, Mannheim, Germany).

Genotyping was performed on genomic DNA from white blood cells. PCR amplification of the 5' untranslated region of the TNF-{alpha} gene was carried using primers described previously (2). The PCR products were digested with the restriction enzyme NcoI, which generates three genotypes G-308G, G-308A, and A-308A.

All statistical analyses were performed using the SAS System for Windows, release 6.12 (SAS Institute, Inc., Cary, NC). Differences between genotypes were assessed by an analysis of covariance model (18).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The frequency of allele G was 0.77 and 0.23 for allele A. The observed genotype frequencies were 56.5, 40.5, and 3.0% for G/G, G/A, and A/A, respectively. Genotype frequencies were in a Hardy-Weinberg equilibrium. The allelic frequency of the substitution is in accordance with allelic frequencies observed in several other studies in Caucasian populations (8, 9, 11, 12).

The assessment of cortisol in saliva provides several advantages over blood cortisol measurements, as the collection procedure is noninvasive and stress free (19, 20). Because salivary cortisol sampling is laboratory independent, it can be applied under a variety of field settings. The concentrations of cortisol in saliva is independent of the saliva flow and represents the unbound (free) hormone fraction, which reflects accurately the free fraction of cortisol in plasma, despite the conversion of cortisol to cortisone in saliva by 11ß-hydroxysteroid dehydrogenase (21). There are strong correlations with serum-free cortisol (20), which has also been confirmed in our laboratory (r >0.90, our unpublished observations). Moreover, because cortisol, leptin, insulin, and metabolic variables are highly dependent on BMI and WHR (22), these measurements were adjusted for the influence of the anthropometric factors.

In the present study, tests for differences in salivary cortisol levels between the TNF-{alpha} genotypes revealed that before a standardized lunch, as well as 30 and 60 min after, there were higher cortisol levels in the morning in homozygotes for the rare allele in comparison with the other genotypes (Table 1Go). These results indicate that there is an association between the NcoI TNF-{alpha} polymorphism and the regulation of the HPA axis activity. In turn, this biallelic polymorphism in the TNF-{alpha} promoter is known to be associated with elevated TNF-{alpha} levels (4). Such elevated levels of TNF-{alpha} increase basal cortisol levels and potentiate the response to stimuli that normally induce cortisol release (15). This increase in cortisol secretion due to elevated TNF-{alpha} levels might be the endocrine mechanism underlying the observed association between the NcoI TNF-{alpha} polymorphism and obesity (5, 6, 7, 8, 12).


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  		Table 1. Differences in salivary cortisol measurements by the genotype of the G/A polymorphism at position -308 of the TNF-{alpha} gene promoter

 
There were no significant differences between the genotype groups with respect to estimates of obesity (BMI) and body fat distribution (WHR and abdominal sagittal diameter). However, homozygotes for the rare allele had higher mean values of BMI, WHR, and abdominal sagittal diameter compared with the other genotype groups (Table 2Go). The reason why these differences did not reach statistical significance might be due to statistical type II error as the statistical power is less than 50%. Nonetheless, these results tend to confirm some of the previous studies which suggest that homozygotes for the -308 variant are more obese compared with the other genotype groups (7, 8, 12). The impact of this polymorphism seems stronger in women than men (12). However, because discrepant results have been obtained, it seems premature to ascribe the G->A polymorphism at position -308 of the TNF-{alpha} gene a key role in the pathogenesis of obesity and the metabolic syndrome (23).


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  		Table 2. Differences in anthropometric, endocrine, and metabolic measurements by the genotype of the G/A polymorphism at position -308 of the TNF-{alpha} gene promoter

 
Our results indicate a nonsignificant trend toward elevated insulin and glucose levels among men with the A/A genotype. The administration of TNF-{alpha} in humans is associated with an early and sustained rise in plasma glucose concentrations (24). TNF-{alpha} administration also causes an increase in levels of counter-regulatory hormones, such as cortisol, which regulate glucose metabolism (25). The neutralization of TNF-{alpha} in vivo in the Zucker fa/fa rat model results in a marked increase in insulin-stimulated autophosphorylation of the insulin receptor tyrosine kinase, specifically in muscle and fat tissues (26). Taken together, the results of a variety of experimental and clinical studies suggest that TNF-{alpha} is a mediator of obesity-linked insulin resistance (10, 25). However, this concept is mainly based on animal data and is so far only partially supported by studies in humans (27, 28).

In summary, we have shown that a G->A polymorphism in the 5' untranslated region of the TNF-{alpha} gene is associated with elevated morning cortisol levels as well as elevated postprandial cortisol secretion. However, the results from the present population study of middle-aged Swedish men does not support the notion that the -308 nucleotide variation of the TNF-{alpha} gene is associated with either obesity or insulin resistance.


   Footnotes
 
1 This study was supported by grants from the Swedish Medical Research Council (K97-19X-00251-35A) and the Pennington Biomedical Research Center. R.R. also acknowledges the Henning and Johan Throne-Holst Foundation for the support of a postdoctoral fellowship at the Pennington Biomedical Research Center. Back

Received September 27, 2000.

Revised December 1, 2000.

Revised January 22, 2001.

Accepted January 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Nedwin GE, Naylor SL, Sakaguchi AY, et al. 1985 Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Res. 13:6361–6373.[Abstract/Free Full Text]
  2. Wilson AG, di Giovine FS, Blakemore AI, Duff GW. 1992 Single base polymorphism in the human tumour necrosis factor {alpha} (TNF {alpha}) gene detectable by NcoI restriction of PCR product. Hum Mol Genet. 1:353.[Free Full Text]
  3. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. 1997 Effects of a polymorphism in the human tumor necrosis factor {alpha} promoter on transcriptional activation. Proc Natl Acad Sci USA. 94:3195–3199.[Abstract/Free Full Text]
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  12. Hoffstedt J, Eriksson P, Hellström L, Rössner S, Ryden M, Arner P. 2000 Excessive fat accumulation is associated with the TNF {alpha}-308 G/A promoter polymorphism in women but not in men. Diabetologia. 43:117–120.[CrossRef][Medline]
  13. Fawcett RL, Waechter AS, Williams LB, et al. 2000 Tumor necrosis factor-{alpha} inhibits leptin production in subcutaneous and omental adipocytes from morbidly obese humans. J Clin Endocrinol Metab. 85:530–535.[Abstract/Free Full Text]
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This Article
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