Introduction

Obesity is an important predictor and cause of type 2 diabetes and cardiovascular disease. Both lifestyle factors, such as energy expenditure associated with physical activity, and genetic factors play important roles in the development of obesity. The peroxisome proliferator-activated receptor-γ co-activator 1α (PPARGC1A, sometimes referred to as PGC1α), a master regulator of skeletal muscle metabolism, has recently been implicated in obesity and type 2 diabetes by epidemiological and mechanistic studies [1]. The expression of the gene encoding PPARGC1A (PPARGC1A) and a set of genes involved in oxidative phosphorylation is decreased in skeletal muscle from subjects with impaired glucose homeostasis or a family history of type 2 diabetes [2, 3]. These data suggest that PPARGC1A dysfunction is an important early and inherited trait of metabolic disease. A genome scan of obesity has shown a linkage peak in the region that contains PPARGC1A [4]. Importantly, the PPARGC1A Gly482Ser polymorphism has been associated with type 2 diabetes, obesity, and with an age-dependent decrease in PPARGC1A mRNA and levels of the protein [5–8]. We hypothesised that the risk associated with 482Ser is subject to sex- and age-specific effects and may be modulated by physical activity. We tested this hypothesis by investigating the influence of the PPARGC1A Gly482Ser polymorphism on obesity in a population-based study with available data on physical activity.

Subjects and methods

Subjects

This study is based on data from a population-based study in Vara, a small community in a rural area of south-western Sweden, as part of the Skaraborg Project. The parameters assessed have previously been described in detail [9]. Inclusion criteria were attendance at an outpatient department for assessment of clinical parameters, completion of health questionnaires, and successful genotyping of the PPARGC1A Gly482Ser polymorphism. A total of 1,811 subjects were surveyed in this study. Of these, 899 females and 902 males had the full information required for the study (Table 1). The participants answered structured questionnaires concerning ethnicity, demography, socioeconomic status, education, lifestyle and mental health. Weight and height were recorded with subjects in light clothing without shoes to the closest 0.1 kg and 1 cm, respectively, and BMI (kg/m2) calculated. Obesity was defined as a BMI ≥30 kg/m2. Leisure-time physical activity (LTPA) was divided into four different categories, ranging from sedentary to hard exercise. Subjects who participated in regular physical activity for at least 2 h a week were considered to be physically active during leisure time (level 3 or 4), and those reporting less were considered to have a low physical activity (level 1 or 2) [10]. The research ethical committee in Gothenburg approved the study and informed written consent was obtained from the subjects prior to their participation.

Table 1 Characteristics of the study population
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DNA extraction and genotyping

DNA was extracted from whole blood using the QiaGen MiniPrep Kit (Qiagen, Hilden, Germany) at the DNA/RNA Genotyping Laboratory at the SWEGENE Resource Center for Profiling Polygenic Disease (Malmö University Hospital, Lund University, Malmö, Sweden). The PPARGC1A Gly482Ser polymorphism was genotyped using an allelic discrimination assay, which was performed with an ABI 7900 system according to manufacturer’s recommendations (Applied Biosystems, Foster City, CA, USA) using the PCR primers 5′-TGGAGAATTGTTCATTACTGAAATCACTGT-3′ (forward) and 5′-GGTCATCCCAGTCAAGCTGTTTT-3′ (reverse), and the TaqMan MGB probes Fam-5′-ACAAGACCAGTGAACTG-3′ and Vic-5′-CAAGACCGGTGAACTG-3′. Genotyping efficiency was 99.8%, and 100% genotyping accuracy was shown when 5% of the samples were re-run. The frequency distribution was in Hardy–Weinberg equilibrium (data not shown).

Statistical analysis

Standard methods were used for descriptive statistics. All analyses were adjusted for age and stratified by sex. Although age was used as a continuous variable, for stratification, subjects were categorised as being 30 to <50 years or ≥50 years. Proportions were age-standardised by 5-year age groups, using the whole Vara population as standards. Associations between obesity and categorical variables were analysed by logistic regression and expressed as odds ratios (OR) with 95% CIs. All tests were two-sided, and statistical significance was assumed when the p value was less than 0.05. All statistical analyses were performed using SPSS for Windows XP, version 11.5 (SPSS, Chicago, IL, USA).

Results

Clinical characteristics and PPARGC1A Gly482Ser genotype distribution are shown in Table 1. Both the men and women were generally overweight, with a mean BMI (±SD) of 27.0±4.6 and 26.8±4.6 kg/m2, respectively. According to the criteria set out by the World Health Organization, 72% of the men and 60% of the women were overweight or obese, and 20 and 25%, respectively, were obese. Overall, we found no association between 482Ser and obesity in either sex, although a trend for increased risk was noted in the men (OR=1.37, 95% CI 0.97–1.92, p=0.074).

When we stratified the population according to age and sex to test our primary hypothesis we observed a significant association between the Ser allele and obesity (OR=1.99, 95% CI 1.14–3.47, p=0.015) in elderly males (age ≥50 years). When a potential dominant effect of the Ser allele was explored in this subgroup, the calculated odds ratio was 2.09 (95% CI 1.02–4.31) in those who were heterozygous (Gly/Ser) and 3.12 (95% CI 1.14–8.55) in subjects homozygous for the Ser allele (Table 2). Accordingly, upon formal testing, the interaction between PPARGC1A Gly482Ser and age was significant in men (p=0.025) but not in women (p=0.407). The risk was restricted to males with a low LTPA level; however, the association with obesity was significantly weaker for homozygous 482Gly carriers (OR=0.44, 95% CI 0.22–0.87, p=0.018). The association was absent in younger men and in women of all ages. A low level of LTPA was associated with an increased risk of obesity in both men (OR=2.10, 95% CI 1.43–3.08, p<0.001) and women (OR=2.72, 95% CI 1.79–4.15, p<0.001). There was no significant interaction between PPARGC1A Gly482Ser and LTPA either in the study population as a whole, or in men or women when analysed separately (data not shown).

Table 2 The interaction of the PPARGC1A Gly482Ser polymorphism with LTPA and age in the association with obesity in men and women
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Discussion

To our knowledge, this is the first population-based study that has observed an interaction between the PPARGC1A Gly482Ser polymorphism and physical activity with respect to the risk of obesity. Our data confirm that 482Ser is associated with obesity but that this risk is dependent upon age, sex and physical activity. An increased risk was only seen in physically inactive men aged ≥50 years. In other words, elderly male homozygous carriers of 482Gly who are physically inactive exhibit relative protection against obesity. Conversely, male carriers of the risk allele may be able to compensate for this risk by means of increased physical activity.

Based on previous information, our a-priori-defined hypothesis was that the risk of obesity associated with 482Ser is influenced by age, sex and level of physical activity [1–8]. The data were primarily analysed in separate groups according to these criteria. This represents sound hypothesis testing, but caution is still warranted when interpreting results from subgroup analyses. Although we found a significant interaction between PPARGC1A Gly482Ser and age in the men but not in the women, we did not observe any interaction between LTPA and the polymorphism. This is likely to be due to the lack of power to detect such an interaction in our subgroups. However, we feel that our findings are also firmly supported by other data. Replication of association studies is subject to several obstacles and confounding factors that may preclude replication. Two studies on the possible association between PPARGC1A Gly482Ser and type 2 diabetes and the metabolic syndrome reported negative results [11, 12]. The fact that the level of physical activity modifies the 482Ser-associated risk may explain these inconsistencies. Our investigation was performed as a population-based study in a random sample of all residents aged between 30 and 74 years in a community, giving the genetic analysis a broader generalisation compared with studies based on single families. The genotype distribution of the PPARGC1A Gly482Ser polymorphism found here was similar to that observed in other studies [5, 6, 10, 12]. Considering the high prevalence and possible dominant effect of 482Ser, our data may have implications for public health and prevention. A recent publication from the Study to Prevent Non-Insulin-Dependent Diabetes Mellitus (STOP-NIDDM) substantiates this view [13]. In this diabetes prevention trial, 482Ser-carriers in the placebo group had a 1.6-fold higher risk of developing type 2 diabetes, and it was only in these subjects that acarbose prevented diabetes. Although our findings are consistent with these studies, given that they are based on a subgroup, the risk of a chance finding should be considered. Further studies are warranted to confirm or disprove our results.

Although a sedentary leisure time showed a strong association with obesity in both men and women, it was only in males that 482Ser was associated with this risk. This is not an unexpected finding. There are several examples of genetic variation that exhibit such sexual dimorphism, not least the PPARGC1A Gly482Ser polymorphism [7]. It was recently shown that PPARGC1A mRNA levels are reduced in women compared with those in men [8]. It is possible that the association between PPARGC1A Gly482Ser and PPARGC1A mRNA expression, which is positively correlated with the degree of physical activity, is of greater importance in men. Since this is a cross-sectional study, such interpretation should be treated with caution until further studies, preferably prospective interventional studies, have been undertaken. Meanwhile, it is likely that the age-dependent association with obesity in the present study is explained by an age-dependent reduction in PPARGC1A gene expression and protein function [8]. Physical activity is known to facilitate weight loss and prevent obesity by increasing the fat oxidation rate and energy expenditure. People with a high level of LTPA often have a healthier lifestyle, including a more frequent intake of healthy food, and both a healthy diet and physical activity are known to improve insulin sensitivity. PPARGC1A drives the formation of oxidative type 1 myofibres and activates genes involved in mitochondrial oxidative metabolism [14]. A reduction in type 1 myofibres and an increase in glycolytic type 2 myofibres has been associated with type 2 diabetes [15]. Differences in PPARGC1A mRNA expression, mitochondrial metabolism and energy expenditure are likely explanations for the protective effect of 482Gly [1].

In conclusion, our findings highlight the importance of accounting for physical activity, particularly LTPA, when studying the epidemiology of obesity. We found that the PPARGC1A Gly482Ser polymorphism was associated with an increased risk of obesity in physically inactive elderly males. Our results therefore support the emphasis on physical activity in the prevention of obesity.