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Gene in the Danish MONICA Cohort: Homozygosity of the Ala Allele Confers a Decreased Risk of the Insulin Resistance Syndrome
Department of Endocrinology (L.F., K.B., S.M., S.A.U.) and Clinical Biochemistry (M.F.), Hvidovre University Hospital, Hvidovre DK-2650, Denmark; Center of Preventive Medicine (T.J.), Glostrup University Hospital, Glostrup DK-2600, Denmark; and Steno Diabetes Center (K.B.-J.), Copenhagen DK-2820, Denmark
Address all correspondence and requests for reprints to: Søren A. Urhammer, M.D., D.M.Sci, Department of Endocrinology, Hvidovre University Hospital, Kettegård Alle 30, DK-2650 Hvidovre, Copenhagen, Denmark. E-mail: . sau{at}dadlnet.dk
Abstract
The Pro12Ala polymorphism of PPAR-
2 has been shown to influence insulin sensitivity and the risk of type 2 diabetes in various ethnic populations. We examined whether the polymorphism was related to the insulin resistance syndrome (IRS) among nondiabetic Danish subjects. The Pro12Ala variant was examined using PCR-restriction fragment length polymorphism in a phenotypically well characterized population-based sample of 2245 nondiabetic subjects. The study participants were characterized by a number of anthropometric and biochemical measurements and the European Group for the Study of Insulin Resistance criteria enabling a classification of the study population in an IRS group and a non-IRS group. The allelic frequency of the Pro12Ala polymorphism in the total study sample was 14% (95% confidence interval, 13–15%). Two hundred ninety-four subjects fulfilled the European Group for the Study of Insulin Resistance criteria defining the IRS. The frequency of the Ala allele was 12.6% in the IRS group and 14.2% among subjects classified as not having the IRS (P = 0.15). However, the frequency of the variant in the homozygous form was significantly lower in the IRS group [0.7% (0–1.6%)] compared with the frequency in the non-IRS group [2.8% (2.1–3.5%); P = 0.02; odds ratio, 0.24 (0.06–0.99)]. Moreover, in the total study population, homozygous carriers of the variant had lower levels of fasting serum triglyceride [1.1 ± 0.4 mmol/liter (means ± SD) vs. 1.4 ± 0.9 mmol/liter; P = 0.04] and a lower diastolic blood pressure (79 ± 8 mm Hg vs. 82 ± 11 mm Hg; P = 0.01) compared with wild-type carriers. The same tendency was observed with regard to the homeostasis model assessment estimate of insulin resistance (P = 0.16). There were no differences between genotype groups with respect to measures of body composition (BMI and waist circumference). In conclusion, homozygosity of the codon 12 variant of PPAR-2 confers a reduced risk of the IRS among Danish Caucasian subjects.
THE TERMS SYNDROME X, the metabolic syndrome, or the insulin resistance syndrome (IRS) refer to patients who, in the presence of insulin resistance, exhibit a cluster of common abnormalities such as hypertension, dyslipidemia, central obesity, and type 2 diabetes. The syndrome identifies patients with an enhanced propensity to develop premature arteriosclerosis and is associated with an increased cardiovascular morbidity and mortality (1). Multiple mechanisms, including genetic components, are thought to contribute to the pathogenesis of insulin resistance, and in recent years the role of obesity and adipose tissue has been highlighted. The significance of the peroxisome proliferator-activated receptor- (PPAR-), a transcription factor involved in adipogenesis and a functional receptor for a new class of insulin sensitizers, the thiazolidinediones, has recently been recognized. Thus far, several genetic variants have been identified in the second isoform of PPAR-, PPAR-2, and shown to influence the development of several of the components of the IRS, including insulin sensitivity, glucose tolerance, and obesity. The most common variation, a C to A nucleotide substitution in exon 2 resulting in an amino acid change, Pro to Ala at codon 12, is present with an allele frequency of approximately 15% among Caucasians. In vitro experiments have demonstrated that this variant is associated with a reduced transcriptional activity (2). In line with this, clinical studies in humans have demonstrated relationships between the polymorphism and alterations in BMI and insulin sensitivity (2, 3, 4, 5, 6, 7, 8), and recently a meta-analysis showed association of this variant with a decreased risk of type 2 diabetes (3).
The present study addresses the question whether the Pro12Ala polymorphism of PPAR-2 influences the risk of the IRS in a large phenotypically well characterized population- based sample of 2245 nondiabetic Danish subjects.
Subjects and Methods
Subjects
In 1982–84, a random sample of 4581 Danes from the referral area of Glostrup Country Hospital was invited to participate in a health survey. The sample was selected to represent an equal number of men and women aged 30, 40, 50, and 60 yr. The study was a part of an international World Health Organization-coordinated study, MONItoring of trends and determinants in CArdiovascular diseases, (MONICA). In 1993–94, 2656 of these subjects participated in a reexamination. After PCR amplification of DNA samples and exclusion of type 2 diabetic patients (fasting plasma glucose >7.0 or treated diabetes), 2245 subjects were eligible for analysis. All were Danish Caucasians by self-identification. Physiological characteristics of this population sample have been presented previously (9).
Before participation in the study, informed consent was obtained for 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.
Anthropometric and biochemical variables
Data of blood pressure and serum lipids have been described previously (9). BMI was calculated as weight (kilograms) divided by the squared height (meters). Plasma concentration of glucose was analyzed using a hexokinase reagent kit. Serum insulin was determined by enzyme-linked two-site immunoassay specific for intact insulin and without cross-reactivity of proinsulin (10).
Insulin resistance was calculated using homeostasis model assessment (HOMA) analysis: (Fasting serum insulin x fasting plasma glucose)/22.5 (11).
The European Group for the Study of Insulin Resistance (EGIR) criteria (12) were applied to stratify the study population in an IRS group and a non-IRS group. This definition was used for practical purpose, i. e. a hyperinsulinemic-euglycemic clamp was not performed in this epidemiological study. Thus, the IRS group was defined by the presence of insulin resistance (highest quartile of HOMA values) and two of the following: hyperglycemia (fasting plasma glucose 
6.1, but nondiabetic); hypertension (systolic/diastolic >140/90 mm Hg or treated for hypertension); dyslipidemia [triglyceride >2.0 mmol/liter or high-density lipoprotein (HDL) cholesterol <1.0 mmol/liter or treated for dyslipidemia]; and central obesity (waist circumference 94 cm in men and 80 cm in women).
Detection of the Pro12Ala polymorphism in the PPAR- gene
Genomic DNA was obtained from human leukocyte nuclei isolated from whole blood. PCR amplification of the DNA segment containing the variant was carried out in a volume of 25 µl, containing 200 ng DNA, 0.2 mM of each dNTP, 1.5 mM MgCl2, 1.0 µM of each primer (sense, 5'-CAAGCCCAGTCCTTTCTGTG-3'; antisense, 5'-CAGGAAACAGCTATGACCAGTGA AGGAATCGCTTTCCG-3'), 0.5 IU of Taq DNA polymerase, and 10x PCR buffer. PCR conditions were: denaturation at 94C for 15 min, 35 cycles of denaturation (94C, 40 sec), annealing (55C, 40 sec) and extension (72C, 40 sec), and a final extension at 72C for 10 min. Restriction fragment length polymorphism was detected after digestion with MspI, which cuts the wild-type allele at a site introduced by the reverse primer (mismatched nucleotide is indicated by bold letter).
Statistics
Fischer’s exact test was applied to test for significance of differences in allele and genotype frequencies. Differences in continuous variables between groups of subjects were tested with ANOVA or t test when the distribution of the variable or the logarithmically transformed variable approached a normal distribution, and the variances of the variables were equal in the groups compared. Otherwise Kruskall-Wallis test or Mann-Whitney rank sum tests were used. Subsequently, multiple regression analysis including age, BMI, and gender when appropriate were performed. Statistical Package of Social Science for Windows (version 9.0, SPSS, Inc., Chicago, IL) was used. P value less than 0.05 was considered significant.
Results
The allelic frequency of the Pro12Ala polymorphism in the total study population of 2245 subjects was 14.0% (95% confidence interval, 13.0–15.0%). Five hundred eighteen subjects (23%) carried the variant in its heterozygous form, whereas 56 (2.5%) of the population were homozygous for the less common Ala allele. The observed genotype frequencies were in Hardy-Weinberg equilibrium. Using the EGIR criteria (12), the study population was stratified into an IRS group and a group not having this syndrome (non-IRS). Two hundred ninety-four subjects fulfilled the EGIR criteria. The allelic frequency of the Pro12Ala polymorphism was 12.6% in the IRS group and 14.2% among subjects classified as not having the IRS (P = 0.15; Table 1
). However, the genotype frequency of Ala/Ala homozygotes was significantly lower in the IRS group [0.7% (0–1.6%)] compared with the frequency in the non-IRS group [2.8% (2.1–3.5%), P = 0.02; odds ratio, 0.24 (0.06–0.99)]. Within the total study population, age and gender distribution did not differ between carriers of the three genotypes, whereas significant differences were observed with respect to diastolic blood pressure and fasting serum triglyceride (Table 2). Comparing Ala/Ala homozygotes with subjects carrying the wild type, diastolic blood pressure and serum triglyceride were significantly decreased, P = 0.01 and P = 0.04, respectively. When controlling for BMI, age, and gender in a multiple regression analysis, the levels were still significantly lower (P = 0.04 and P = 0.03, respectively). The same tendency was observed with regard to the HOMA estimate of insulin resistance (P = 0.16). Heterozygous carriers did not differ from wild-type carriers regarding these variables. Neither were there any differences between genotype groups with respect to body weight distribution, fasting plasma glucose, or serum insulin. There was no interaction of the variant and gender or degree of obesity on the examined variables (data not shown).
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The primary objective of the present study was to examine whether the Pro12Ala variant of the PPAR-2 gene was associated with IRS as defined by the EGIR criteria (12). We found an allelic frequency of the polymorphism similar to previous studies of the variant in Caucasian subjects (4, 5, 7, 13). Although a direct relationship between the variant allele per se and the IRS was not observed, the present study demonstrates a significant association between the homozygous variant genotype (Ala/Ala) and a reduced risk of the IRS, i.e. homozygosity of the variant protecting against the IRS. Consistent with this finding, the present study also revealed an approximately 30% reduction in serum triglyceride and a 5% reduction in diastolic blood pressure among carriers of the Ala/Ala genotype. The directionality of these significant findings is in accordance with previous reports. Although most single studies among Caucasians failed to show associations of the variant with type 2 diabetes (14, 15, 16, 17, 18), recent meta-analyses using family-based association study approach (3) and case control design (13) demonstrated that the variant confers a decreased risk of type 2 diabetes. The mechanism has been suggested to involve improved insulin sensitivity. In line with this hypothesis, several previous studies in nondiabetic Caucasian study populations, including Scandinavians (2, 8, 13, 19, 20), have demonstrated significant improved insulin sensitivity estimated by hyperinsulinemic-euglycemic clamp techniques associated with the Ala allele. Although a similar trend was observed in the present study group, we were not able to show a significant association of the variant with the HOMA estimate of insulin sensitivity. However, although HOMA values of insulin sensitivity correlate with the estimate of insulin sensitivity obtained by the clamp (21), it is a nondynamic estimate based on fasting values of insulin and glucose. Thus, the HOMA model might be inadequate to detect the effect of the Pro12Ala variant on insulin sensitivity. Accordingly, previous studies in nondiabetic and diabetic patients were unable to detect effects of the variant on insulin sensitivity calculated by this method (22, 23).
In line with the present study, previous investigations found associations of the Pro12Ala variant with alterations in serum lipids. In a group of elderly (70 yr) Finnish subjects, homozygous carriers of the Ala allele exhibited significantly lower levels of serum triglyceride and higher levels of serum HDL cholesterol (2). Whether these effects were independent of a concomitant decrease in BMI is, however, not stated in the paper (2). Similarly, in another Finnish study of type 2 diabetic patients, carriers (heterozygous and homozygous carriers grouped together) had decreased levels of serum triglyceride, in this case independent of BMI (24), which is consistent with our results. In contrast, other studies in Japanese subjects found no effect of the variant on serum lipids (23). Neither in the French WHO-MONICA population of 839 subjects was the variant related to disturbances in serum triglyceride (16). However, in this study data of homozygous Ala carriers were not available, making a direct comparison with our results difficult.
The association between the Pro12Ala variant and blood pressure is of particular interest given the recent report by Barrosa and associates (25) of three type 2 diabetic patients with early onset hypertension and mutations in the PPAR- gene. The mechanism by which PPAR- affects blood pressure is, however, unknown. Because PPAR- is a nuclear receptor expressed in a variety of tissues, including small and large vessels, and it stimulates transcription of multiple genes necessary for adipogenesis, lipid metabolism, and insulin signaling (26, 27), it is possible that a biologically active variant of this protein might affect several of the components of the IRS by other mechanisms than altered insulin sensitivity. In contrast to the present investigation, another Finnish study revealed associations of the variant with higher blood pressure among nondiabetic patients as well as among severely obese type 2 diabetic patients (24). Most previous studies that have examined the impact of the variant on blood pressure, found, however, no significant associations between the variant and alterations in blood pressure (4, 15, 22, 23, 28, 29). Several of these studies were performed in Japanese subjects (22, 23, 28) in whom the allelic frequency of the variant is reported to be considerably lower than in Caucasians. Yet, the disparity between the studies might reflect differences in genetic background. Furthermore, with respect to the impact on blood pressure, conclusions drawn from previous studies are all based on analyses of heterozygous and homozygous carriers grouped together, whereas data of Ala/Ala carriers are missing.
In general, most studies have revealed inconsistent results with respect to several parameters, including those discussed above. It is possible that this specific PPAR- variant, on the basis of variable interaction with other unknown genetic and environmental factors, expresses variable effects in different study subgroups. Thus, the variant has previously, in another Danish study cohort of young men, been shown to exhibit divergent modulating effects on BMI depending on the degree of obesity (7), whereas another study among Scandinavians found no relationship to BMI (13), consistent with our results. Nevertheless, it is also possible that some of the inconsistency reflects insufficient statistical power in many studies. In our study, the power to detect effects of 30% of the homozygous genotype on triglycerides and BMI was estimated to be above 85%, indicating low risks of false negative results.
From the present investigation, we conclude that the common Pro12Ala variant of the PPAR- gene in its homozygous form is associated with reduced levels of serum triglyceride and diastolic blood pressure and a protection against the IRS among nondiabetic Danish subjects.
Acknowledgments
Footnotes
This work was supported by grants from the Danish Medical Research Council, the Danish Hospital Foundation of Medical Research, region of Copenhagen, the Faroe Islands and Greenland, the Danish Insurance Health Foundation, and the Danish Diabetes Association.
Abbreviations: EGIR, European Group for the Study of Insulin Resistance; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; IRS, insulin resistance syndrome.
Received February 8, 2002.
Accepted April 26, 2002.
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