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A Polymorphisms of the Tumor Necrosis Factor 
Gene Promotor Are Not Associated with Features of the Insulin Resistance Syndrome or Altered Birth Weight in Danish Caucasians1
Steno Diabetes Center and Hagedorn Research Institute (S.K.R., S.A.U., J.N.J., T.H., O.P.), DK-2820, Gentofte, Denmark; and Center of Preventive Medicine (K.B.-J.), Glostrup University Hospital, DK-2600, Glostrup, Denmark
Address correspondence and requests for reprints to: Søren K. Rasmussen, M.Sc., Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark.
Abstract
Recently, two G
A polymorphisms at positions -308 and -238, in the
promoter of the tumor necrosis factor 
(TNF-) gene, have been
identified. These variants have, in different ethnic groups, been
linked to estimates of insulin resistance and obesity. The objective of
the present study was to investigate whether these genetic variants of
TNF- were associated with features of the insulin resistance
syndrome or alterations in birth weight in two Danish study populations
comprising 380 unrelated young healthy subjects and 249
glucose-tolerant relatives of type 2 diabetic patients, respectively.
All study participants underwent an iv glucose tolerance test with the
addition of tolbutamide after 20 min. In addition, a number of
biochemical and anthropometric measures were performed on each subject.
The subjects were genotyped for the polymorphisms by applying PCR
restriction fragment length polymorphism. Neither of the variants was
related to altered insulin sensitivity index or other features of the
insulin resistance syndrome (body mass index, waist to hip ratio, fat
mass, fasting serum lipids or fasting serum insulin or C-peptide).
Birth weight and the ponderal index were also not associated with the
polymorphisms. In conclusion, although the study was carried out on
sufficiently large study samples, the study does not support a major
role of the -308 or -238 substitutions of the TNF- gene in the
pathogenesis of insulin resistance or altered birth weight among Danish
Caucasian subjects.
TUMOR NECROSIS FACTOR (TNF-) is a
potent cytokine that originally was identified as a factor implicated
in inflammatory and immunoregulatory actions. Later on, research has
indicated a key role for the TNF- protein in the pathogenesis of
obesity-associated insulin resistance. TNF- messenger RNA levels in
skeletal muscle and adipose tissue of humans are positively correlated
with body mass index (BMI), fat mass, and insulinemia and inversely
correlated with lipoprotein lipase activity (1, 2, 3), and analysis of
sib-pairs has demonstrated linkage between the TNF- locus and body
fat content among Pima Indians (4). Moreover, mice lacking the TNF-
gene are less insulin resistant than wild-type mice (5). These studies
strongly suggest that TNF- impairs insulin action, and the mechanism
seems to involve impairments of the proximal insulin signaling pathway
(6, 7). However, the molecular mechanism and regions within the TNF-
gene that are responsible for the alterations in TNF- expression in
insulin-resistant states have not been clarified. Recently, attention
has been focused on two nucleotide polymorphisms identified in the
promoter region of the TNF- gene: a GA substitution at position
-308 and a GA substitution at position -238. Interestingly,
in vitro experiments have demonstrated that the -308
variant increases transcriptional activation of the TNF- gene (8, 9). This finding is consistent with recent clinical studies showing a
relationship between the -308 variant and obesity (10, 11) and insulin
resistance (10). The potential functional impact of the polymorphism at
position -238 of the TNF- gene has not yet been investigated in
in vitro expression studies. However, the variant is located
within a putative Y box, which is a regulatory motif typical of the
promoter region of major histocompatibility complex class II genes
(12). Furthermore, a TNF- repressor site has been identified between
-254 and -230 of the gene (13), suggesting that the variant might
affect the transcription of the gene. The potential functional
significance of this variant was further indicated by a recent report
that demonstrates an association between the -238 variant and
increased insulin sensitivity (14). Hence, the objectives of the
present study were to evaluate the potential impact of the two
polymorphisms of the TNF- gene in a population-based sample of 380
young Danish Caucasians and in 249 glucose-tolerant relatives of type 2
diabetes patients on estimates of obesity, fasting values of serum
insulin and serum lipids, and the insulin sensitivity index as
estimated from an iv glucose tolerance test (IVGTT) using Bergman’s
Minimal Model. Because insulin resistance has been associated with low
birth weight (15), we also evaluated the potential impact of the
variants on birth weight and the ponderal index in the same study
groups.
Materials and Methods
The study participants, consisting of 380 healthy subjects, were randomly recruited from a population of young individuals aged 18–32 yr. Physiological characteristics of this population sample have been reported previously (16). The other study population, consisting of 249 glucose-tolerant relatives of type 2 diabetic patients from 62 families, were recruited from the Danish family resource bank at the Department of Human Genetics, University of Copenhagen, or from the outpatient clinic at Steno Diabetes Center. Physiological characterization of this population has been reported previously (17). All were Danish Caucasians by self-identification. Data of birth length and birth weight were obtained from the midwife records stored in the Danish Provencial Archives for Zealand, Lolland Falster, and Bornholm. Ponderal index was calculated as (birth weight)/(birth length)3.
BMI, waist to hip ratio, fat mass, plasma concentration of glucose, serum levels of triglyceride, total cholesterol, and high-density lipoprotein (HDL) cholesterol were analyzed as described (16). Serum insulin and C-peptide were analyzed using the routine procedures of Steno Diabetes Center. Fat mass was measured with an impedance technique (16). Insulin sensitivity index (Si) was estimated from an IVGTT in combination with injection of iv tolbutamide, as described previously (16). Prior to the participation in the study, informed consent was obtained from all subjects. The study was approved by the Ethical Committee of Copenhagen and was in accordance with the principles of the Declaration of Helsinki II.
The presence of the variant was determined by PCR-restriction fragment length polymorphism, as described (14). Differences between wild-type, heterozygous, and homozygous carriers of the variants among the young healthy subjects were tested with a generalized linear model including gender and genotype as fixed factors and BMI and age as covariate factors, using Statistical Package of Social Science for Windows (SPSS, Inc., Chicago, IL), version 9. Age and BMI were excluded from the model for analysis of birth weight and ponderal index, and BMI was excluded from the model for analysis of waist to hip ratio, fat mass, and fat percentage. For analysis of data obtained from relatives of diabetic patients a variance component model was used, as described (17).
Data are presented as means (SD). A P value less than 0.05 was considered significant.
Results and Discussion
In the population-based sample of young Danish Caucasians, the
allelic frequency of the substitution at position -308 was 18.9%
(95% confidence interval, 15.9–21.9%), which is in accordance with
allelic frequencies observed in Spanish (10), British (14), White
American (18, 19), African American (19), French (11), and Australian
subjects (20) but lower than the frequency observed in the Irish
population (11). The allelic frequency of the substitution at position
-238 was 6.3% (4.4–8.2%), comparable with frequencies found in
other study populations (11, 14, 18). The observed genotype frequencies
in the population-based sample of young Danes were in Hardy-Weinberg
equilibrium, and the polymorphisms were not in linkage disequilibrium
(
2 = 2.2, P > 0.05). However,
in the 62 families examined we observed only three haplotypes:
-308G/-238G, -308A/-238G and -308G/-238A. There were no
significant differences between the genotype groups with respect to
estimates of obesity (BMI, fat mass, fat percentage), fasting serum
lipids, fasting serum insulin or C-peptide or the insulin sensitivity
index, regarding the -238 or the -308 polymorphism (Tables 1
and 2).
Neither were the variants related to alterations in birth weight or the
ponderal index (Tables 1 and 2). Heterozygous carriers of the -308
variant among the young healthy subjects had a higher waist to hip
ratio (P = 0.007) compared to wild-type and homozygous
carriers, which we consider as an incidental finding because we could
not replicate this finding among glucose-tolerant relatives of type 2
diabetic patients.
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Regarding, the -238 variant, previous results also are inconsistent.
In the study of British nondiabetic relatives and control subjects, the
-238 variant was associated with increased insulin sensitivity as
estimated by two independent methods (14), whereas this variant did not
show any relationship with type 2 diabetes in the New England study
(18) or associations with estimates of obesity in the study of Irish
and French subjects (11) or in the study of Caucasian and African
American subjects (19), which coincides with our findings. The reasons
for these conflicting results are not clear. Some of the previous
studies are based on a small number of study participants, and,
therefore, the results might represent statistical type I and type II
errors. The power of the present study (population-based sample of
young Danish Caucasians) to detect an effect of the variants of about
40% on the insulin sensitivity index, as reported previously (10), is
more than 99% and 
95%, regarding the -308 and -238 variant,
respectively, indicating a low risk of false negative results. These
estimations consider the variants in their heterozygous forms, whereas
the corresponding calculated power is 55% with respect to A/A genotype
of the -308 variant. Power estimations concerning the other variables
examined in the study are comparable. Because only two homozygous
carriers of the -238 polymorphism were identified we cannot exclude an
effect of this variant in homozygous form. In this respect, it should
be noted that conclusions drawn from previous studies are based on
analyses of carriers (combined heterozygous and homozygous)
vs. noncarriers of the variants.
Alternatively, the disparity between our and previous studies
might reflect differences in genetic background, although, as noted,
allele frequencies of both variants are comparable in most ethnic
groups, besides the higher frequency of the -308A allele in the Irish
population. Another explanation could be that the -308 and -238
polymorphisms serve as markers for a yet unidentified functional
variant, and, therefore, the disparity might be due to different degree
of linkage in the study populations. Also, differences in study design
should be considered. Our study populations are young (mean age, 25
yr), healthy, and randomly recruited and middle-aged (mean age, 42 yr)
glucose-tolerant relatives of type 2 diabetic patients, whereas
previous studies have examined older and selected groups of
individuals. It might be hypothesized that the polymorphisms show
phenotypically expression only in combination with other yet
unidentified genetic risk factors or at a later stage of age. In
conclusion, the present study based on sufficiently large study samples
of young healthy subjects and middle-aged glucose-tolerant relatives of
type 2 diabetic patients does not support the hypothesis that the -308
or the -238 nucleotide variants of the TNF- gene play a major role
in the pathogenesis of insulin resistance or low birth weight in the
Danish population.
Acknowledgments
We thank Dorte Gøth-Johansen, Sandra Urioste, Annemette Forman, Lene Aabo, Bente Mottlau, Susanne Kjellberg, Lis Ølholm, Maja Lis Halkjær, and Nis Pedersen for dedicated and careful technical assistance and Grete Lademann for secretarial support.
Footnotes
1 Supported by grants from the Danish Research Academy, the
Danish Medical Research Council, the University of Copenhagen, the
Velux Foundation, the Danish Diabetes Association, and European
Economic Community (BMH4-CT-98-3084). 
Received October 18, 1999.
Revised January 10, 2000.
Accepted January 10, 2000.
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