J Appl Physiol 99: 344–348, 2005. First published February 10, 2005; doi:10.1152/japplphysiol.00037.2005.

HIGHLIGHTED TOPIC Role of in Reducing the Risk of Diabetes and Obesity PPARGC1A genotype (Gly482Ser) predicts exceptional endurance capacity in European men

Alejandro Lucia,1 Fe´lix Go´mez-Gallego,2 Ineˆs Barroso,3 Manuel Rabada´n,4 Fernando Bandre´s,1 Alejandro F. San Juan,1 Jose´ L. Chicharro,5 Ulf Ekelund,6 Soren Brage,6 Conrad P. Earnest,7 Nicholas J. Wareham,6 and Paul W. Franks8 1European University of Madrid, Madrid, Spain; 2Department of Toxicology, Complutense University, Madrid, Spain; 3The Wellcome Trust Sanger Institute, Metabolic Disease Group, Cambridge, United Kingdom; 4Department of Sports Medicine, Higher Sports Council, Madrid, Spain; 5School of Nursing, Complutense University, Madrid, Spain; 6Medical Research Center Epidemiology Unit, Cambridge, United Kingdom; 7The Cooper Institute Center for Human Performance and Nutrition Research, Dallas, Texas; and 8Phoenix Epidemiology and Clinical Research Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Phoenix, Arizona Submitted 11 January 2005; accepted in final form 6 February 2005

Lucia, Alejandro, Fe´lix Go´mez-Gallego, Ineˆs Barroso, Manuel 37) and early death (16–18, 38). V˙ O2 max is determined by Rabada´n, Fernando Bandre´s, Alejandro F. San Juan, Jose´L. modifiable factors that include physical activity level (1), Chicharro, Ulf Ekelund, Soren Brage, Conrad P. Earnest, Nich- cigarette smoking (31), and micro- and macrovascular disease olas J. Wareham, and Paul W. Franks. PPARGC1A genotype (14), and by unmodifiable factors such as genotype (6, 27) and (Gly482Ser) predicts exceptional endurance capacity in European the intrauterine environment (5). Several candidate have men. J Appl Physiol 99: 344–348, 2005. First published February 10, ˙ 2005; doi:10.1152/japplphysiol.00037.2005.—Animal and human been identified that appear to influence VO2 max (27), of which data indicate a role for the peroxisome proliferator-activated recep- one, PPARGC1A, is a particularly promising candidate, owing tor-␥ 1␣ (PPARGC1A) product in the development to the consistency of supportive data in rodents (3, 12, 19, 32, of maximal oxygen uptake (V˙ O2 max), a determinant of endurance 33) and humans (23, 25, 28, 30, 34). capacity, diabetes, and early death. We tested the hypothesis that the Several studies have reported association between a com- frequency of the minor Ser482 allele at the PPARGC1A locus is lower mon variant in the PPARGC1A gene (Gly482Ser) and Type 2 in World-class Spanish male endurance athletes (cases) [n ϭ 104; diabetes, in which the frequency of the Ser482 allele is higher mean (SD) age: 26.8 (3.8) yr] than in unfit United Kingdom (UK) in diabetic patients than normal glucose-tolerant controls (9, ϭ Caucasian male controls [n 100; mean (SD) age: 49.3 (8.1) yr]. In 15, 24). More recently, healthy older Danish carriers of the cases and controls, the Gly482Ser genotype met Hardy-Weinberg Ser482 allele have also been reported to have lower levels of expectations (P Ͼ 0.05 in both groups tested separately). Cases had Ϫ1 Ϫ1 PPARCG1A and PPARGC1B mRNA by comparison with significantly higher V˙ O2 max [73.4 (5.7) vs. 29.4 ml⅐kg ⅐min (3.8); P Ͻ 0.0001] and were leaner [body mass index: 20.6 (1.5) vs. Gly482 allele homozygotes (20). 27.6 kg/m2 (3.9); P Ͻ 0.0001] than controls. In unadjusted ␹2 The PPARGC1A gene is a coactivator of a subset of genes analyses, the frequency of the minor Ser482 allele was significantly that control oxidative phosphorylation, which have been lower in cases than in controls (29.1 vs. 40.0%; P ϭ 0.01). To assess termed the OXPHOS genes. Control of oxidative phosphory- the possibility that genetic stratification could confound these obser- lation by the OXPHOS genes is achieved through the regula- vations, we also compared Gly482Ser genotype frequencies in Span- tion of , glucose and lipid transporta- ish (n ϭ 164) and UK Caucasian men (n ϭ 381) who were unselected tion and oxidation (32, 34), and fiber-type for their level of fitness. In these analyses, Ser482 allele frequencies formation (19). V˙ O2 max is positively correlated with OXPHOS were very similar (36.9% in Spanish vs. 37.5% in UK Caucasians, mRNA levels in human skeletal muscle (23), and OXPHOS P ϭ 0.83), suggesting that confounding by genetic stratification is unlikely to explain the association between Gly482Ser genotype and is coordinately downregulated in diabetic endurance capacity. In summary, our data indicate a role for the skeletal muscle (23). Gly482Ser genotype in determining aerobic fitness. This finding has In animals (3, 12, 32, 33) and humans (25, 26, 28, 30, 34), relevance from the perspective of physical performance, but it may also exercise training increases PPARGC1A mRNA levels, and be informative for the targeted prevention of diseases associated with low transgenic overexpression of PPARGC1A mRNA corresponds fitness such as Type 2 diabetes. with an increased resistance by contracting muscle to fatigue single nucleotide polymorphism; maximal oxygen uptake; metabo- (19), indicating that PPARGC1A and exercise are part of a lism; genetics; diabetes coregulatory feedback loop. The use of extreme phenotype cohorts (i.e., groups of people who exhibit extreme levels of a given outcome trait) can help A LOW MAXIMAL OXIDATIVE CAPACITY, as defined by maximal maximize statistical power in studies that seek to test hypoth- oxygen uptake (V˙ O2 max), independently predicts diabetes (22, eses of genetic association. In the present study, we employed

Address for reprint requests and other correspondence: P. Franks, Phoenix Epidemiology & Clinical Research Branch, National Institute of Diabetes and The costs of publication of this article were defrayed in part by the payment Digestive and Kidney Diseases, 1550 E. Indian School Rd., Phoenix, AZ of page charges. The article must therefore be hereby marked “advertisement” 85014 (E-mail: [email protected]). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

344 http://www.jap.org PPARGC1A GENOTYPE PREDICTS ENDURANCE CAPACITY 345 this approach to test the hypothesis that the minor Ser482 allele Genetic Analyses at the PPARGC1A locus is less frequent in world-class endur- DNA extraction and genotyping was undertaken using previously ance athletes than in unfit controls. described methods (4). Genotyping was performed using an adapta- tion of the fluorescence polarization template-directed incorporation METHODS method described by Chen et al. (7). In short, primer extension preamplified DNA samples were polymerase chain reaction amplified Selection of the Participants and Metabolic Tests in 8-␮l reactions with primers flanking the variant site; unincorporated Cases. Written consent was obtained from each subject according 2-deoxynucleotide 5’-triphosphate and remaining unused primer were the guidelines of the local institutional ethics committee (Universidad degraded by exonuclease I and shrimp alkaline phosphatase at 37°C Europea de Madrid, Spain), which approved this study. The study for 45 min before the enzymes were heat inactivated at 95°C for 15 adhered to the principles of the Declaration of Helsinki. min. At the end of the reaction, the samples were held at 4°C. Single The case group comprised 50 unrelated male World-class Spanish base primer extension reactions were performed as previously de- riders from the four best professional cycling teams who ranked scribed (7), and allele detection was performed by measuring fluores- among the top 65–70 Spanish cyclists in terms of a 3-wk stage race cence polarization on an LJL Analyst fluorescent reader (Molecular performance in this country. We specifically chose professional cy- Devices, Sunnyvale, CA). The primer extension preamplified protocol clists for this study who met the following criteria: each cyclist 1) had was specifically developed and tested to ensure that allele bias was not previously been enrolled on a professional cycling team, 2) had at introduced during the amplification process. least 2 yr experience in the professional category of the International Statistical Analysis Cycling Union, and 3) had participated in a classic 3-wk stage race from 1999 to 2004. During this period, 11 cyclists won at least one All analyses were conducted using the SAS statistical software mass-start stage or time trial of the Tour de France, Giro d’Italia, or (SAS Institute, Cary, NC). The means and standard deviations of Vuelta a Espan˜a, and 7 and 11 were among the top 3 and top 10 anthropometric and fitness data were stratified by case and control finishers, respectively, in these classic cycling races. status, and two-tailed independent samples t-tests were performed to We recruited 54 male runners for this study who were the best detect differences between groups. Mantel-Haenszel ␹2 test and World- and Olympic-class Spanish middle- and long-distance track Fisher’s exact test with 2 df were used to test differences in Gly482Ser runners, as determined by finishing positions during international genotype frequencies between groups. The association between ge- competitions for the 1999–2004 period. Their training loads typically notype and V˙ O2 max (for the athlete, fit, and unfit groups combined) included more than 150 km/wk (150–200 km/wk) and approached was tested using linear regression models (PROC REG in SAS), 250 km/wk in marathoners during some periods of the year. Athletes where genotype was the predictor variable and V˙ O2 max was the within this group included individuals who had placed within the top continuous trait outcome. three at World, European, and Olympic Championships during the years 1999–2004. V˙ O2 max was measured during an incremental load RESULTS exercise stress test until exhaustion on a cycle-ergometer (cyclists) or treadmill (runners), as described in detail previously (21). Participant characteristics stratified by case and control sta- Spanish reference population. Written consent was obtained from tus are presented in Table 1. Cases were young [mean age: 26.8 each subject according to the guidelines of the local institutional (3.8) yr], normal weight, Spanish male athletes (n ϭ 104). ethics committee (Universidad Europea de Madrid, Spain), which Controls were unfit, middle-aged [mean age: 49.1 (8.3) yr] men approved this study. The study adhered to the principles of the selected from an existing population-based longitudinal study Declaration of Helsinki. A group of 164 healthy unrelated Spanish of UK Caucasians (n ϭ 100) who were free of electrocardio- men (aged 18–55 yr) served as the Spanish reference population for graphic abnormalities and other serious health ailments. As the ethnic comparison of Gly482Ser genotype frequencies. These demonstrated by the characteristics in Table 1, controls were individuals were anonymous blood donors in whom information was older, had higher body mass index [20.6 (1.5) vs. 27.6 kg/m2 only available for age, ethnicity, genotype, and general health status (3.9); P Ͻ 0.0001] and were less fit than cases [73.4 (5.7) vs. and were thus unselected for their level of fitness. ⅐ Ϫ1 ⅐ Ϫ1 Ͻ Ͻ United Kingdom reference population and controls. The United 29.4 ml kg min (3.8); P 0.0001] (P 0.0001). In cases Kingdom (UK) reference and control groups were selected from the and unfit controls, the Gly482Ser genotype met Hardy-Wein- Medical Research Council (MRC) Ely Study, a continuing popula- berg expectations (P Ͼ 0.05 in both groups tested separately). tion-based cohort study in Ely, Cambridgeshire, the design of which has been described previously (10, 11). In brief, volunteers attended the clinic at 8:30 AM having fasted since 10 PM the previous evening. Table 1. Participant characteristics stratified After an explanation of laboratory procedure, all participants provided written, informed consent. Height and weight were measured in light by case-control status clothing, and blood was drawn through a cannulated artery. Ethical Cases: Spanish Fit Controls: Unfit Controls: permission for the study was granted by the Cambridge Local Re- (n ϭ 104) UK (n ϭ 100) UK (n ϭ 100) search Ethics Committee, which approved this study. The study adhered to the principles of the Declaration of Helsinki. Trait Mean SD Mean SD Mean SD Anthropometric and genetic data were available in 381 men. This Age, yr 26.8 3.8 46.2 8.2 49.3 8.1* group was unselected for their level of cardiorespiratory fitness and Height, cm 176.5 6.1 176.2 6.6 175.3 6.1 constituted the sample for the comparison of allele frequencies be- Weight, kg 64.2 6.7 80.6 12.1 85.0 13.2* tween ethnic groups (i.e., the UK reference population). BMI, kg/m2 20.6 1.5 25.9 3.4 27.6 3.9* ˙ Of the 381 participants in the UK reference population, V˙ O2 max VO2max, ⅐ Ϫ1⅐ Ϫ1 was measured in a subset of 200 individuals. V˙ O was assessed ml kg min 73.4 5.7 41.2 6.2 29.4 3.8* 2 max ˙ during an incremental load exercise stress test on a bicycle ergometer, VO2max, l/min 4.70 0.6 3.32 0.7 2.49 0.5* as described previously in detail (10, 11). This sample was stratified V˙ O2max, maximal oxygen uptake; BMI, body mass index; UK, United above and below the group median for V˙ O2 max, to constitute the “fit” Kingdom. Student’s t-test for difference between cases and unfit controls: *P (n ϭ 100) and “unfit” (n ϭ 100) controls groups. Ͻ 0.0001.

J Appl Physiol • VOL 99 • JULY 2005 • www.jap.org 346 PPARGC1A GENOTYPE PREDICTS ENDURANCE CAPACITY Ser482 allele, in conjunction with other environmental and genetic factors, predicts aerobic capacity. This finding is inter- esting from the perspective of physical performance but may also have relevance in identifying individuals who, by virtue of their genetic predisposition to low cardiorespiratory fitness, may be at increased risk when sedentary of related disorders such as Type 2 diabetes. Thus, because aerobic capacity can be increased through exercise training, knowledge of the geno- types that predict those with low fitness presents an opportunity for targeted diseases prevention. PPARGC1A mRNA is expressed predominantly in tissues with high metabolic activity, the majority of which are rich in mitochondria. These include heart, skeletal muscle (during exercise), brown fat, kidney, liver, and brain, and other tissues of low metabolic activity such as white adipose (29, 35). Through coactivation of the OXPHOS genes, PPARGC1A transiently controls glucose transportation through SLC2A4 regulation, and lipid and glucose oxidation through the regu- lation of the PPARs ␣, ␦, and ␥ (2). PPARGC1A also chroni- Fig. 1. PPARGC1A Ser482 allele frequency in unfit UK men (Unfit), Spanish cally modulates muscle oxidative capacity. This is achieved men unselected for fitness (Unselected), fit UK men (Fit), and World-class Spanish athletes (Athletes). P values denote statistical significance (Mantel- primarily via the coactivation of the nuclear respiratory tran- Haenszel ␹2 test) for difference in allele frequencies between groups. scription factors (NRF-1 and NRF-2), cytochrome-c oxidase 4 (COX4), and the mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2), which in a coordinated manner powerfully ␹2 In Mantel-Haenszel analyses, the frequency of the minor induce mitochondrial biogenesis (36). Inactivation of this sig- Ser482 allele was significantly lower in cases than in unfit naling cascade is likely to result in attenuation of oxidative ϭ controls (29.1 vs. 40.0%; P 0.01) (Fig. 1). Owing to the capacity and disordered that manifests as an insu- small sample size, we also undertook Fisher’s exact test. In this lin resistance-like syndrome (8). ϭ analysis, the results remained statistically significant (P In human Type 2 diabetic skeletal muscle, OXPHOS genes 0.037). These models also remained statistically significant are coordinately downregulated (23), and in morbid obesity after the inclusion of “fit” controls, whose Ser482 allele fre- PPARGC1A mRNA is threefold lower in subcutaneous and ϭ quency was 33.8% (P 0.01 with 3 df). In post hoc regression omental by comparison with tissue of lean ˙ models, we combined VO2 max data from the athlete, fit, and controls (29). However, because data from these studies are ˙ unfit groups and tested whether VO2 max differed by genotype. cross sectional, one cannot determine whether downregulation ˙ In these models, mean (SD) level of VO2 max was highest in the of OXPHOS genes is a cause or consequence of diabetes and ϭ Gly482Gly genotype group [n 122; 3.7/l/min/(SD 1.1)], obesity. ϭ moderate in the Gly482Ser genotype group [n 154; 3.4 In humans, a single 2-min bout of exhaustive exercise l/min (SD 1.1)], and lowest in the Ser482Ser genotype group corresponds with increased PPARGC1A mRNA expression (7- ϭ ϭ [n 27; 1.7 l/min (SD 1.1)] (P 0.003). to 10-fold), which peaks at around 2 h (26). Furthermore, by To assess the possibility of genetic stratification between restricting blood flow to the exercising limb, PPARGC1A Spanish and UK Caucasians, which could confound the asso- mRNA overexpression is prolonged (to ϳ6 h) (25), suggesting ciation between genotype and endurance capacity, we also that metabolic perturbation influences PPARGC1A mRNA compared Gly482Ser genotype frequencies in two reference expression. Short et al. (30) recently reported 1.5-fold in- ϭ ϭ populations of Spanish (n 164) and UK Caucasian men (n creases in PPARGC1A mRNA levels in men and women after 381) who were unselected for their level of fitness. Allele a 16-wk exercise training program consisting of stationary frequencies in both groups met Hardy-Weinberg expectations cycling (3–4 sessions/wk at 70–80% maximum heart rate), Ͼ (P 0.05 in both groups tested separately). In Mantel-Haen- and Russell et al. (28) reported chronic increases in ␹2 szel analyses, Ser482 allele frequencies did not differ PPARGC1A mRNA expression (2.7-fold) after 6 wk of run- ϭ significantly (36.9 vs. 37.5%, P 0.83), suggesting that ning training. Moreover, these changes in expression are inde- confounding by genetic stratification is unlikely to explain the pendent of increased type 1 fiber quantity or preferential association between Gly482Ser genotype and endurance ca- fiber-type switching. However, others have been unable to pacity. detect changes in PPARGC1A expression after 9 days of cycle DISCUSSION ergometry (60 min/day) (34) or after a single bout of one- legged knee extension exercise (25). The fact that PPARGC1A The present study compared the allele frequencies at the expression levels increase after exercise is important, because Gly482Ser locus of the PPARGC1A gene in elite-level endur- the presence of functional genetic variation at this locus could, ance athletes and unfit controls. In athletes, the Gly482 allele therefore, result in differential levels of expression after exer- was significantly more common than in unfit controls. Given cise, which would depend on genotype. that V˙ O2 max is a major determinant of endurance performance, Several studies have reported an increased frequency of the and the unfit controls in the present study are defined by their Ser482 allele in Type 2 diabetic patients (9, 15, 24). Given the low level of V˙ O2 max, our data indicate that the presence of the metabolic characteristics of PPARGC1A and the manner in

J Appl Physiol • VOL 99 • JULY 2005 • www.jap.org PPARGC1A GENOTYPE PREDICTS ENDURANCE CAPACITY 347 which exercise training modulates its expression, it is plausible REFERENCES that variation at the Gly482Ser locus modifies the protective 1. A˚ strand PO, Rodahl K, Dahl HD, and Stromme SB. Textbook of Work effect of physical activity on risk of diabetes-related traits. For Physiology: Physiological Bases of Exercise (4th ed.). Champaign, IL: example, in a recent study of Danish adults (20), the Ser482 Human Kinetics, 2003. PPARGC1A 2. Attie AD and Kendziorski CM. PGC-1alpha at the crossroads of type 2 allele was inversely associated with mRNA level diabetes. Nat Genet 34: 244–245, 2003. and V˙ O2 max. Furthermore, we have previously described the 3. Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, Kelly interaction between physical activity and the Gly482Ser vari- DP, and Holloszy JO. 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