We examined the association between the FTO rs9939609 polymorphism and serum leptin concentrations in adolescents. The FTO rs9939609 polymorphism was genotyped, and fasting serum leptin and insulin were measured in 655 European adolescents (365 females) aged 14.6±1.2 years. We measured weight, height, triceps and subscapular skinfolds and waist circumference, and body fat percentage was calculated. Sex, pubertal status, center, physical activity (accelerometry), total or central adiposity and serum insulin concentrations were entered as confounders in the analyses. The minor A allele of the FTO rs9939609 was significantly associated with higher serum leptin concentrations independently of potential confounders including adiposity (+3.9 ng ml−1 per risk allele (95% confidence interval: 2.0, 5.9); adjusted P<0.001). These findings could link the FTO gene with serum leptin and consequently with the control of energy balance. Leptin could be a possible intermediary contributing to the association between the FTO rs9939609 polymorphism and adiposity.
Adiposity development and its complications are determined by lifestyle, genetic mechanisms and their interactions. Among the obesity-related genes, polymorphisms in the fat mass and obesity associated (FTO) gene are strongly associated with body fat estimates in different populations.1
The function and signaling pathways of the FTO gene in energy homeostasis are unknown, but several studies conducted in mice focusing on the nature of this locus showed the ubiquitous expression of this protein in both fetal and adult tissues with highest expression in the hypothalamus.2, 3, 4 It was suggested as a possible mechanism that single-nucleotide polymorphisms in the FTO gene may exert functional effects in energy balance through altered expression level of FTO mRNA, particularly in the hypothalamus.5
Leptin is a circulating hormone synthesized and secreted into the circulation primarily by white adipocytes. Leptin exerts, among other important actions, regulatory control on food intake and energy expenditure through a variety of central and peripheral actions. In obesity, the existence of an endogenous leptin-resistance mechanism limiting its regulatory effect may explain the strong correlation between serum leptin concentrations and body fat mass.6 In addition to its role in the management of energy balance, there is evidence that leptin has a role in the neonatal development in key areas of the hypothalamus that are involved in the central regulation of energy balance.7
The aim of this study was to examine the association between the FTO rs9939609 polymorphism and serum leptin concentrations in adolescents participating in the Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study.
Materials and methods
The HELENA study was designed to examine the interactions between personal, environmental and lifestyle influences on the risk factors for future cardiovascular diseases.8 A detailed description of the HELENA study sampling and recruitment approaches, standardization and harmonization processes, data collection, analysis strategies and quality control activities was published elsewhere.9, 10 The protocol was approved by the Human Research Review Committee of the Universities of the centers involved. Written informed consent to participate was obtained from both parents and adolescent.
The present study comprises 655 adolescents with valid data on the FTO rs9939609 polymorphism, serum leptin concentration, body mass index (BMI) and physical activity (PA). The study sample did not differ in key characteristics (that is age or BMI) from the original HELENA sample (N=3546 all P-values >0.1).
Harmonization and standardization of anthropometric measurements used to assess body composition in the HELENA study were strictly controlled and were described elsewhere.11 Body weight and height, waist circumference and subscapular and tricipital skinfold thicknesses were measured in triplicate. BMI was calculated as body weight (kg) divided by the square of height (m). Body fat percentage was calculated using Slaughter's equations, which showed the best agreement with total body fat percentage measured by DXA in adolescent population.12
Identification of sexual maturation was assessed by a trained physician according to Tanner and Whitehouse.13 PA was objectively measured by accelerometry (Actigraph GT1M, Pensacola, FL, USA).14 Average PA expressed as total counts per minute was used in the analyses.
The FTO rs9939609 genotyping was done by an Illumina system, using the GoldenGate technology. The genotyping success rate was 100%. Genotyping was performed once for each sample. Serum fasting leptin concentrations were measured using the RayBio Human Leptin enzyme-linked immunosorbent assay. The sensitivity of leptin assay is typically <6 pg ml−1, with intra- and inter-assay coefficients of variation of <10 and <12%, respectively. Serum fasting insulin concentrations were measured using the Dako Diagnostics Ltd, Ely, UK, respectively, in duplicate.
We determined the increase in fasting serum leptin levels per A risk allele of the FTO rs9939609 polymorphism in the whole sample of adolescents using the additive model after controlling for confounders (that is sex, age, pubertal status, PA and center) and BMI, waist circumference or body fat percentage, and insulin. Trend tests were performed by adding genotype categories in the regression analysis as ordinal variables.
We compared the frequencies of both the FTO A risk allele and the AA genotype between non-overweight and overweight adolescents using the χ2 test. In order to better examine the possible association between the FTO rs9939609 polymorphism and serum leptin levels, regardless of adiposity content, we analyzed the study sample taking into account body weight categories. Likewise, we compared fasting serum leptin levels between A allele carriers and non-A allele carriers (that is TT genotype) of the FTO rs9939609 polymorphism in the whole sample, in non-obese adolescents and in non-overweight adolescents by analysis of covariance after adjusting for sex, age, pubertal status, PA, center (random variable) and BMI, waist circumference or body fat percentage.
Serum leptin level presented a skewed distribution and was therefore logarithmically transformed. Analyses were performed using the SPSS, v. 17.0 (SPSS Inc., Chicago, IL, USA), and the level of significance was set to 0.05.
Characteristics of the study sample are shown in Table 1. The frequency of the minor A allele was 0.40. Genotype frequencies did not differ significantly (χ2=0.51, P=0.474) from Hardy–Weinberg equilibrium expectations (TT=0.36, TA=0.47 and AA=0.17).
The minor A allele was significantly associated with serum leptin concentrations (+5.1 ng ml−1 per risk allele (95% confidence interval: 2.8, 7.5); P=0.002) independently of sex, age, pubertal status, PA and center. This association persisted after further controlling for BMI (P=0.015), waist circumference (P=0.010) or body fat percentage (P=0.013). Additional adjustment for insulin did not substantially alter the results (+3.9 ng ml−1 per risk allele (95% confidence interval: 2.0, 5.9); sex, age, pubertal status, PA, center, body fat percentage and insulin adjusted P<0.001).
Both the A allele frequency (70.4% in overweight vs 60.5% in non-overweight, P=0.004) and the frequency of the AA genotype (24.1% in overweight vs 14.1% in non-overweight, P<0.001) were higher in overweight than in non-overweight participants. We observed that adolescents carrying the A allele of the FTO rs9939609 polymorphism had significantly higher levels of serum leptin than those carrying the TT genotype, when the analysis was controlled for confounders and BMI in either the whole sample (17.4±1.1 vs 22.4±0.9 for TT and A allele carriers, respectively, P=0.009), in non-obese adolescents (15.8±1.1 vs 20.2±0.9 for TT and A allele carriers, respectively, P=0.007) or in non-overweight adolescents (14.1±1.1 vs 17.1±0.8 for TT and A allele carriers, respectively, P=0.036) (Figure 1a). Moreover, serum leptin levels were higher in adolescents carrying the A allele of the FTO rs9939609 polymorphism than in those carrying the TT genotype after controlling for confounders and waist circumference) in the whole sample (16.9±1.2 vs 22.3±0.9 for TT and A allele carriers, respectively, P=0.004). The outcome did not substantially differ when obese participants (n=30) were excluded from the analysis (15.6±1.1 vs 20.0±0.9 for TT and A allele carriers, respectively, P=0.003), and was slightly attenuated (P=0.021) after the exclusion of overweight individuals (n=81) (Figure 1b). Finally, the A allele of the FTO rs9939609 polymorphism was associated with higher leptin levels after adjusting for confounders and body fat percentage in both the whole sample (17.4±1.1 vs 22.4±0.9 for TT and A allele carriers, respectively, P=0.012) and in non-obese adolescents (17.4±1.2 vs 22.9±0.9 for TT and A allele carriers, respectively, P=0.014), whereas the difference was diminished and became non-significant (P=0.079) after further exclusion of overweight adolescents (14.1±1.1 vs 17.1±0.8 for TT and A allele carriers, respectively) (Figure 1c). The outcome did not substantially change after further adjusting for serum insulin levels (data not shown), and the results were similar in girls and boys (data not shown).
The present study shows a statistically significant association between the FTO rs9939609 polymorphism and serum leptin concentrations in the whole sample of adolescents independently of several confounders including either total or central adiposity. Indeed, the A risk allele of the FTO rs9939609 polymorphism was associated with an increase of ∼3.9 ng ml−1 of serum leptin regardless of sex, pubertal status, PA, insulin and body fat percentage.
To our knowledge, this is the first study reporting this association in adolescents and taking into account confounders affecting FTO gene effects, leptin or adiposity estimates. Thus, the association between the A risk allele of the FTO rs9939609 polymorphism and leptin was independent of total or central adiposity, of insulin which participates together with leptin in the control of food-associated reward mechanisms6 and of objectively measured PA, known to modulate the deleterious effect of the FTO rs9939609 polymorphism on adiposity in adolescents.15 Therefore, the association between the A allele risk and serum leptin concentrations, regardless of BMI and waist circumference, persisted after the exclusion of both obese and overweight adolescents from the analysis, and was only slightly diminished after adjustment for body fat percentage in the non-overweight participants.
Three previous studies have examined the association of the FTO rs9939609 polymorphism with serum leptin concentrations. Qi et al.16 showed a borderline association between the A allele and serum leptin levels, which became non-significant after adjusting for BMI. Andreasen et al.17 in a cohort of 17 508 Danes adults reported that the A allele significantly increased fasting serum leptin levels, but it was not adjusted for BMI and was considered as a result of increased adiposity. Zabena et al.18 did not find any significant difference in serum leptin level among genotypes of the FTO rs9939609 polymorphism in a small sample of 75 obese adults. Finally, Do et al.19 showed that two other single-nucleotide polymorphisms of the FTO gene (rs17817449 and rs1421085) were not associated with serum leptin after controlling for BMI in a cohort of 908 adult from the Quebec Family study. Nevertheless, differences in study design, such as age (all the study participants were adults), weight and health status, make comparisons between studies difficult.
Although this study includes several strengths, such as its relatively large sample size, we acknowledge some limitations. First, we used the study center as a surrogate estimate of ethnicity, which is not ideal owing to the possibility of residual confounding factors and misclassification. However, we stratified the analyses by center, and we checked for a possible center-by-genotype interaction. The results were homogeneous across centers, and we did not find any significant interactions. Second, we used proxies to estimate adiposity content, and so adjustments may not fully account for fat mass; we did not measure visceral adiposity, which might be an important confounder. Nevertheless, as the reference methods are costly for the studies that involve large numbers of subjects, we used the skinfold thicknesses, a valid alternative for the measurement of total adiposity.20 Finally, due to the strong correlation between leptin level and fat mass, it is difficult to establish the directionality and causality of the association between the FTO polymorphism and leptin. Therefore, we cannot exclude that other mechanism(s) affected by FTO polymorphism could influence independently both adiposity and serum leptin concentrations. Further studies with bigger sample sizes, in children and adults, and in other ethnicities are needed to confirm these findings.
Our results seem to link the FTO gene with serum leptin and consequently with the energy balance control, suggesting that the A allele variant could somehow be associated with leptin-resistance mechanisms. These findings could at least partially explain the reported associations of the FTO polymorphism with increased dietary consumption5 and with a hyperphagic phenotype.21, 22 Likewise, leptin could well be a possible intermediary contributing to the association between the FTO rs9939609 polymorphism and adiposity.
The HELENA study was carried out with the financial support of the European Community Sixth RTD Framework Programme (Contract FOODCT-2005-007034). This work was also partially supported by grants from the Spanish Ministry of Education (EX-2008-0641) and by the Swedish Council for Working Life and Social Research (FAS).
The HELENA study
Co-ordinator: Luis A Moreno.
Core Group members: Luis A Moreno, Fréderic Gottrand, Stefaan De Henauw, Marcela González-Gross, Chantal Gilbert.
Steering Committee: Anthony Kafatos (President), Luis A Moreno, Christian Libersa, Stefaan De Henauw, Jackie Sánchez, Fréderic Gottrand, Mathilde Kersting, Michael Sjöstrom, Dénes Molnár, Marcela González-Gross, Jean Dallongeville, Chantal Gilbert, Gunnar Hall, Lea Maes, Luca Scalfi.
Project Manager: Pilar Meléndez.
1. Universidad de Zaragoza (Spain): Luis A Moreno, Jesús Fleta, José A Casajús, Gerardo Rodríguez, Concepción Tomás, María I Mesana, Germán Vicente-Rodríguez, Adoración Villarroya, Carlos M Gil, Ignacio Ara, Juan Revenga, Carmen Lachen, Juan Fernández Alvira, Gloria Bueno, Aurora Lázaro, Olga Bueno, Juan F León, Jesús Ma Garagorri, Manuel Bueno, Juan Pablo Rey López, Iris Iglesia, Paula Velasco, Silvia Bel.
2. Consejo Superior de Investigaciones Científicas (Spain): Ascensión Marcos, Julia Wärnberg, Esther Nova, Sonia Gómez, Esperanza Ligia Díaz, Javier Romeo, Ana Veses, Mari Angeles Puertollano, Belén Zapatera, Tamara Pozo.
3. Université de Lille 2 (France): Laurent Beghin, Christian Libersa, Frédéric Gottrand, Catalina Iliescu, Juliana Von Berlepsch.
4. Research Institute of Child Nutrition Dortmund, Rheinische Friedrich-Wilhelms-Universität Bonn (Germany): Mathilde Kersting, Wolfgang Sichert-Hellert, Ellen Koeppen.
5. Pécsi Tudományegyetem (University of Pécs) (Hungary): Dénes Molnar, Eva Erhardt, Katalin Csernus, Katalin Török, Szilvia Bokor, Mrs Angster, Enikö Nagy, Orsolya Kovács, Judit Repásy.
6. University of Crete School of Medicine (Greece): Anthony Kafatos, Caroline Codrington, María Plada, Angeliki Papadaki, Katerina Sarri, Anna Viskadourou, Christos Hatzis, Michael Kiriakakis, George Tsibinos, Constantine Vardavas, Manolis Sbokos, Eva Protoyeraki, Maria Fasoulaki.
7. Institut für Ernährungs- und Lebensmittelwissenschaften – Ernährungphysiologie. Rheinische Friedrich Wilhelms Universität (Germany): Peter Stehle, Klaus Pietrzik, Marcela González-Gross, Christina Breidenassel, Andre Spinneker, Jasmin Al-Tahan, Miriam Segoviano, Anke Berchtold, Christine Bierschbach, Erika Blatzheim, Adelheid Schuch, Petra Pickert.
8. University of Granada (Spain): Manuel J Castillo, Ángel Gutiérrez, Francisco B Ortega, Jonatan R Ruiz, Enrique G Artero, Vanesa España-Romero, David Jiménez-Pavón, Palma Chillón.
9. Istituto Nazionalen di Ricerca per gli Alimenti e la Nutrizione (Italy): Davide Arcella, Giovina Catasta, Laura Censi, Donatella Ciarapica, Marika Ferrari, Cinzia Le Donne, Catherine Leclerq, Luciana Magrì, Giuseppe Maiani, Rafaela Piccinelli, Angela Polito, Raffaela Spada, Elisabetta Toti.
10. University of Napoli ‘Federico II’ Dept of Food Science (Italy): Luca Scalfi, Paola Vitaglione, Concetta Montagnese.
11. Ghent University (Belgium): Ilse De Bourdeaudhuij, Stefaan De Henauw, Tineke De Vriendt, Lea Maes, Christophe Matthys, Carine Vereecken, Mieke de Maeyer, Charlene Ottevaere
12. Medical University of Vienna (Austria): Kurt Widhalm, Katharina Phillipp, Sabine Dietrich, Birgit Kubelka Marion Boriss-Riedl.
13. Harokopio University (Greece): Yannis Manios, Eva Grammatikaki, Zoi Bouloubasi, Tina Louisa Cook, Sofia Eleutheriou, Orsalia Consta, George Moschonis, Ioanna Katsaroli, George Kraniou, Stalo Papoutsou, Despoina Keke, Ioanna Petraki, Elena Bellou, Sofia Tanagra, Kostalenia Kallianoti, Dionysia Argyropoulou, Katerina Kondaki, Stamatoula Tsikrika, Christos Karaiskos.
14. Institut Pasteur de Lille (France): Jean Dallongeville, Aline Meirhaeghe.
15. Karolinska Institutet (Sweden): Michael Sjöstrom, Patrick Bergman, María Hagströmer, Lena Hallström, Mårten Hallberg, Eric Poortvliet, Julia Wärnberg, Nico Rizzo, Linda Beckman, Anita Hurtig Wennlöf, Emma Patterson, Lydia Kwak, Lars Cernerud, Per Tillgren, Stefaan Sörensen.
16. Asociación de Investigación de la Industria Agroalimentaria (Spain): Jackie Sánchez-Molero, Elena Picó, Maite Navarro, Blanca Viadel, José Enrique Carreres, Gema Merino, Rosa Sanjuán, María Lorente, María José Sánchez, Sara Castelló.
17. Campden and Chorleywood Food Research Association (United Kingdom): Chantal Gilbert, Sarah Thomas, Elaine Allchurch, Peter Burguess.
18. SIK – Institutet foer Livsmedel och Bioteknik (Sweden): Gunnar Hall, Annika Astrom, Anna Sverkén, Agneta Broberg.
19. Meurice Recherche and Development asbl (Belgium): Annick Masson, Claire Lehoux, Pascal Brabant, Philippe Pate, Laurence Fontaine.
20. Campden and Chorleywood Food Development Institute (Hungary): Andras Sebok, Tunde Kuti, Adrienn Hegyi.
21. Productos Aditivos SA (Spain): Cristina Maldonado, Ana Llorente.
22. Cárnicas Serrano SL (Spain): Emilio García.
23. Cederroth International AB (Sweden): Holger von Fircks, Marianne Lilja Hallberg, Maria Messerer
24. Lantmännen Food R&D (Sweden): Mats Larsson, Helena Fredriksson, Viola Adamsson, Ingmar Börjesson.
25. European Food Information Council (Belgium): Laura Fernández, Laura Smillie, Josephine Wills.
26. Universidad Politécnica de Madrid (Spain): Marcela González-Gross, Agustín Meléndez, Pedro J Benito, Javier Calderón, David Jiménez-Pavón, Jara Valtueña, Paloma Navarro, Alejandro Urzanqui, Ulrike Albers, Raquel Pedrero, Juan José Gómez Lorente.
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The writing group takes sole responsibility for the content of this article. The content of this paper reflects only the authors’ views, and the European Community is not liable for any use that may be made of the information contained therein.
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