Preliminary findings on the influence of FTO rs9939609 and MC4R rs17782313 polymorphisms on resting energy expenditure, leptin and thyrotropin levels in obese non-morbid premenopausal women
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- Arrizabalaga, M., Larrarte, E., Margareto, J. et al. J Physiol Biochem (2014) 70: 255. doi:10.1007/s13105-013-0300-5
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Given that leptin, ghrelin and thyrotropin play a major role in the regulation of resting energy expenditure (REE) and that the FTO rs9939609 and the MC4R rs17782313 polymorphisms have been proposed to affect energy homeostasis, we hypothesized that both polymorphisms are associated with REE and that these relationships can be mediated by leptin, ghrelin and thyrotropin in obesity. Therefore, the present study aimed to examine the relationships between FTO rs9939609 and the MC4R rs17782313 with REE, leptin, ghrelin and thyrotropin levels in obese women. The study comprised 77 obese (body mass index 34.0 ± 2.8 kg/m2) women (age 36.7 ± 7 years). We measured body composition by dual-energy X-ray absorptiometry and REE by indirect calorimetry. We analysed fasting leptin, ghrelin and thyrotropin levels and the ratio of leptin to fat mass was calculated. Genotype distributions of the polymorphisms did not deviate from Hardy–Weinberg expectations (P values >0.2). Women carrying the A allele of the FTO rs9939609 had lower REE (1,580 ± 22 vs. 1,739 ± 35 kcal/day, P < 0.001) and higher leptin to fat mass ratio (1.33 ± 0.05 vs. 1.13 ± 0.08 ng/ml kg, P < 0.05) and thyrotropin levels (1.93 ± 0.10 vs. 1.53 ± 0.16 μU/ml, P < 0.05) regardless of age and body mass index. We found no significant influence of the MC4R rs17782313 on energy metabolism or biochemical variables. Our findings confirm that the A allele of the FTO rs9939609 is associated with lower REE and increased plasma leptin levels. We also found an association between the FTO rs9939609 and thyrotropin, suggesting the possible influence of FTO in the hypothalamic–pituitary–thyroid axis as a potential mechanism of the increased adiposity.
KeywordsFTO rs9939609MC4R rs17782313Resting energy expenditureLeptinThyrotropinObesity
Common variants in several genes encoding proteins participating in the hypothalamic control of energy balance have been associated with excess adiposity; among them, the most robust associations in populations of European descents have been observed for the fat mass and obesity associated (FTO) rs9939609 and the melanocortin-4 receptor (MC4R) rs17782313 polymorphisms [11, 43]. Likewise, it was estimated that each copy of the FTO rs9939609 polymorphism A allele corresponds to ∼1.5 kg heavier weight in adults  and with 0.42 kg/m2 higher body mass index (BMI) in adolescents . Similarly, each copy of the rs17782313 C allele in the MC4R gene was associated with an 8 % increase in the odds of being overweight and 12 % of being obese in adults, and with an increase of 1.4 times the risk of obesity in children [30, 31]. The function and signalling pathways in energy homeostasis for these genotype–adiposity associations are unknown; nevertheless, subjects with variants in these two single nucleotide polymorphisms (SNPs) may have an altered control of energy homeostasis, either by greater appetite and energy intake or reduced energy expenditure .
Leptin and ghrelin play key roles in the control on energy balance control . 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. One previous study in adolescents proposed a link between the FTO rs9939609 polymorphism and energy balance control through a leptin resistance mechanism . Likewise, in this study, the influence of the A allele risk on leptin levels was independent of adiposity content and insulin levels. On the other hand, thyroid hormones have been demonstrated to modulate the behaviour of many metabolic pathways relevant for the resting energy expenditure (REE) . Although thyroid function is usually normal in obese subjects, thyrotropin (TSH) levels and BMI are usually positively correlated . Furthermore, leptin concentrations influence TSH release. A previous report in children observed that the A allele of the FTO rs9939609 polymorphism was positively associated with serum TSH levels, suggesting an influence of the FTO on the hypothalamic–pituitary–thyroid axis . Evidence suggests that ghrelin is involved in the regulation of energy homeostasis by increasing energy intake, while there is little information about its influence on energy expenditure . Nevertheless, St-Pierre et al.  reported that higher ghrelin levels were associated with lower REE and studies in animal models suggest that ghrelin signalling is required for the control of energy expenditure . Given that leptin, ghrelin and TSH play a major role in the regulation of REE and that both FTO rs9939609 and the MC4R rs17782313 polymorphisms have been proposed to affect REE, we hypothesized that both polymorphisms are associated with REE and that these relationships could be mediated by leptin, ghrelin and/or TSH in obesity. Therefore, the present study aimed to examine the associations of the FTO rs9939609 and the MC4R rs17782313 polymorphisms with REE in a well-characterized sample of Spanish obese women. In addition, we explored the influence of these polymorphisms on leptin, ghrelin and TSH levels, in order to understand whether FTO rs9939609 and MC4R rs17782313 play a role in these key mechanisms of body weight and adiposity control.
Materials and methods
A total of 77 obese women (BMI 30.0–39.9 kg/m2) from Vitoria (North Spain), euthyroid (fasting TSH levels between 0.3 and 4.5 μU/ml), premenopausal (age 19–49 years) and sedentary (<20 min on <3 days/week) volunteered to participate in the study and underwent a comprehensive medical examination. Participants were Caucasian and showed stable weight (body weight changes <3 kg in the last 3 months). Exclusion criteria included history of cardiovascular disease, diabetes, cholesterol and triglyceride levels >300 mg/dl, blood pressure >140/90 mmHg and pregnancy. All of them were also free of medication (except oral contraceptives) for hypertension, hyperlipidemia, hyperuricemia or other illness. The study examinations were performed in the Unit of Clinic Assays of LEIA Foundation (Txagorritxu Hospital, Vitoria, Spain). All women received verbal and written information about the nature and purpose of the study, and all of them gave written consent to participate. The study was in accordance with the Helsinki II Declaration and was approved by the Ethical Committee of the Hospital of Txagorritxu (Vitoria, Spain).
Dual X-ray absorptiometry measurements (HOLOGIC, QDR 4500 W, with QDR software for windows version 12.4) were performed to estimate fat mass (FM) and lean tissue mass (LM). Thereafter, FM percentage was calculated.
Resting energy expenditure
Respiratory exchange measurements by indirect calorimetry were used to estimate REE following the recommended measurements conditions . The participants were asked not to perform any intense physical activity the day immediately before the measurement. For each examination day (at baseline and 12 weeks after), participants arrived by car or bus at the hospital at 8–9 a.m. in a fasting condition of at least 12 h. The measurements were taken in peaceful and relaxing environment and at a constant temperature (∼24 °C) and humidity (∼50 %). Women were in a supine position and awake. After 30 min of rest, respiratory exchange measurements were determined by means of an open-circuit computerized indirect calorimeter (Vmax, Sensormedics, Germany) using a transparent, ventilated canopy-hood system and after daily calibration with a reference gas mixture (95 % O2, 5 % CO2). The first and final 5 min of each set were routinely discarded and the mean value of the remaining 20 min was used for the calculations, once the steady-state conditions were obtained. The coefficient of variation (CV) was <10 %. If steady state could not be maintained that long, a 10-min segment with CV <5 % was accepted. This instrument has shown to be valid to assess REE and respiratory exchange ratio . Urine was collected in the postabsorptive state to determine nitrogen output. REE was calculated from O2 and CO2 volumes, as well as from urine excretion nitrogen values, by using the formula of Weir and expressed as kilocalories/day as reported elsewhere [23–25].
Blood samples were taken following a 12-h overnight fast. Serum leptin (in nanogram per millilitre) and ghrelin (in nanogram per millilitre) were measured by Enzyme-Linked Immunosorbent Assay kits (EZHL-80SK from LINCO Research, Missouri, USA and EK-031-30 from Phoenix Europe, GMBH, Karlsruhe, Germany, respectively) and TSH by immunoradiometric assay (Immunotech, Beckman Coulter). All samples were measured in duplicate and the mean was scored. The ratio of leptin to FM was scored . Fasting plasma glucose (in milligram per decilitre), total cholesterol (in milligram per decilitre), and triglyceride levels (in milligram per decilitre) were measured by enzymatic spectrophotometric technique with an autoanalyzer (COBAS FARA; Roche Diagnostics, Basel, Switzerland). Insulin (in microunit per millilitre) was measured by Enzyme-Linked Immunosorbent Assay kits (LINCO Research, Missouri, USA). Insulin sensitivity was assessed by the homeostasis assessment model (HOMA-IR).
Genomic DNA was isolated from the buffy coat of centrifuged whole blood using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. The FTO rs9939609 and the MC4R rs17782313 genotyping were carried out using Taqman probes and Applied Biosystems 7300 Sequence Detection System (Applied Biosystems, Foster City, CA). Genotyping success rate was 100 % and no discordant genotypes were observed in duplicate samples.
Analyses were performed using the Statistical Package for Social Sciences (SPSS, v. 20.0 for WINDOWS; SPSS, Chicago, IL) and a two-tailed probability test of 0.05 or less was considered statistically significant. Hardy–Weinberg Equilibrium was tested using Pearson’s Chi-squared test for each polymorphism. Variables with skewed distribution were logarithmically transformed to obtain a more symmetrical distribution. We grouped carriers of the less common alleles and compared with the wild-type genotypes (dominant models) . We analyzed mean differences of the study variables across FTO rs9939609 (TT vs. A-carriers) and MC4R rs17782313 (TT vs. C-carriers) polymorphisms using non-parametric tests (Mann–Whitney U test). Differences in REE, leptin and TSH were further analysed by analyses of covariance (ANCOVA) adjusting for age (model 1); age and BMI (model 2); and age, LM and FM (model 3) using ANCOVA. We used ANCOVA to test for the existence of an interaction effect between the two polymorphisms on body composition, REE and biochemical variables. When significant interaction effects were found, the combined influence of both polymorphisms was examined. For this purpose, women were categorized as non-risk allele carriers, carriers of the C allele risk of the MC4R rs17782313 polymorphism, carriers of the A allele of the FTO rs9939609 polymorphism and carriers of both risk alleles, i.e. women carrying the A allele of the FTO rs9939609 and the C allele of the MC4R rs17782313 polymorphisms.
Genotype frequencies were as follows: 22 (28.6 %), 43 (55.8 %) and 12 (15.6 %) for the TT, TA and AA genotypes of the FTO rs9939609 polymorphism, respectively, and 53 (68.8 %), 21 (27.3 %) and 3 (3.9 %) for the TT, TC and CC genotypes, respectively, of the MC4R rs17782313 polymorphism. Genotype distributions of the two polymorphisms did not deviate from Hardy–Weinberg expectations (P values >0.2).
Characteristics of participants across FTO rs9939609 and MC4R rs17782313 polymorphisms
TT (N = 22)
TA + AA (N = 55)
TT (N = 53)
TC + CC (N = 24)
Body mass index (kg/m2)
Fat mass (kg)
Fat mass (%)
Lean mass (kg)
Resting energy expenditure (kcal/day)
Leptin (ng/ml)/fat mass (kg)
Total cholesterol (mg/dl)
We found no significant influence of the MC4R rs17782313 polymorphism on body composition, energy metabolism or biochemical variables (Table 1). The outcome did not substantially change when the influence of the MC4R rs17782313 polymorphism on body composition, energy metabolism or biochemical variables was tested with additive (P values >0.1) or recessive (P values >0.3) models (data not shown).
Combined influence of the FTO rs9939609 and the MC4R rs17782313 polymorphisms
The present study shows that obese women carrying the A allele of the FTO rs9939609 polymorphism had lower REE and higher leptin and TSH levels than non-A allele carriers. In contrast, we found no influence of the MC4R rs17782313 polymorphism on energy metabolism, leptin, ghrelin or TSH levels in obese women.
Previous studies have explored the association of the FTO rs9939609 polymorphism on energy homeostasis examining its influence on energy intake and/or energy expenditure in either human or animal studies [5, 15, 18, 19, 29, 33, 41]. Overall, the majority of the studies performed in animal models suggest that the FTO gene influences the “output” side of the energy balance equation [15, 33, 41], while human studies showed inconsistent associations with energy intake [5, 18, 19, 29]. We observed that women carrying the A allele of the FTO rs9939609 polymorphism had lower REE than non-A allele carriers independently of body size. These results could at least partially explain the reported attenuation of the deleterious impact of the FTO rs9939609 polymorphism on adiposity by physical activity . Thus, A allele carriers might compensate the influence of the risk allele on lowering resting energy expenditure by increasing the energy expenditure derived from physical activity. As far as we are aware, there are very few previous studies that have investigated the association of the FTO rs9939609 polymorphism with REE in adults [3, 9]. Berentzen et al.  and Speakman et al.  showed lack of association of rs9939609 with energy expenditure in their reports. It is worth noting that there are remarkable differences in sample characteristics between our study and the above mentioned reports, such as sex, age range, BMI range or menopausal status which may explain discrepancies observed across studies. Likewise, in contrast to their hypothesis, Berentzen et al.  observed higher REE in men younger than 65 years with the AA genotype, though this association disappeared after adjustment for body size. Speakman et al.  examined the association of rs9939609 with body composition and REE in men and mixed of pre-menopausal and post-menopausal women adults with wide range of age (from 21 to 64 years) and BMI (from 16.4 to 49.3 kg/m2) and they found no association with REE. Of note is also that they did not find any significant association with adiposity, body weight or BMI. The results of the present study are in line with the findings of Do et al.  showing an association between the obesity risk alleles of FTO rs17817449 and FTO rs1421085 polymorphisms and lower REE in a large sample of obese adults; however, these relationships were found with different FTO SNPs and did not survive adjustment for body size.
Our findings suggest a link between the FTO gene with energy balance control through an effect on REE and that this influence could be mediated by leptin and TSH. Likewise, we observed that women carrying the A risk allele had higher levels of leptin regardless of BMI, and leptin to FM ratio in agreement with a previous report in adolescents . Our observations are also partially in agreement with previous studies reporting higher leptin levels in adults carrying risk alleles of the FTO rs9939609 or other polymorphisms [2, 9, 34], although these associations were abolished after adjustment for BMI. To our knowledge, this is the first study reporting higher TSH levels independently of BMI in adults carrying the A allele of the rs9939609. In line with our findings, Dwivedi et al.  observed significant associations of the FTO rs9939609 polymorphism with elevated levels of TSH in a large sample of adolescents. Previous studies showed that small increases in TSH levels are associated with body weight and adiposity gain and altered REE even in euthyroid individuals [1, 20, 36]. The biological function of FTO is largely unknown; however, our results could be partially explained because TSH is produced in the pituitary gland, where FTO is highly expressed . In addition, there are complex interactions between leptin and TSH . Likewise, leptin is also an important neuroendocrine regulator of the hypothalamic–pituitary–thyroid axis by regulation of TRH gene expression in the paraventricular nucleus, and TSH in turn will stimulate leptin secretion in adipose tissue . Our results showed that there was not any significant effect of the A allele of the FTO rs9939609 polymorphism on serum ghrelin levels, which is in agreement with one previous report in children .
Most of the evidence for a direct effect of MC4R on energy expenditure comes from rodent studies. Nevertheless, several studies in Hispanic and Pima Indian children [6, 21] suggested that MC4R may play a key role in the regulation of body weight, not only through energy intake but also through energy expenditure. In contrast, Kring et al.  did not find any significant relationship between two polymorphisms of the MC4R gene (one of them the rs17782313) with energy expenditure in adults, which concurs with our findings. One possible explanation for these discrepancies could be that the genetic effects of the MC4R gene are more pronounced during childhood . Our results did not show any significant influence of the MC4R rs17782313 polymorphism on REE. However, it might exert an additive effect together with the FTO rs9939609 polymorphism lowering REE. As we did not observe any significant combined influence of carrying both risk alleles on leptin, leptin to FM ratio, or TSH levels, this additive combined influence of both polymorphisms seems to be independent of leptin or TSH levels and explained by other unknown mechanism.
We acknowledge the limitations of our study. Because of our cross-sectional design, the associations between the FTO rs9939609 polymorphism and REE, leptin and TSH do not prove causality. Due to the relatively small sample size of our study, these findings should be taken as preliminary. Further studies with bigger sample sizes in other well-characterized populations, and in men, where the effect of these polymorphisms seems to be different  are warranted. To bear in mind is that the effects reported in this study would become statistically non-significant after correction for multiple testing. However, to conclude negatively from a purely statistical point of view would be too stringent. Definition of the modulating effects of candidate genes for obesity will allow the identification of individuals likely to be resistant to diet or physical activity interventions, due to for example to low REE.
In conclusion, our findings confirms that the A allele of the FTO rs9939609 is associated with lower REE and increased plasma leptin levels. We also found an association between the FTO rs9939609 and thyrotropin, suggesting the possible influence of FTO in the hypothalamic–pituitary–thyroid axis as a potential mechanism of the increased adiposity.
We thank the women for their participation in the study and Raquel Ares, Silvia Francisco, Izaskun Felipe and Emilio Sanz for their contribution to the participant’s recruitment and medical supervision of the study. This study was supported by the University of the Basque Country (UPV 05/80), Social Foundation of the Caja Vital-Kutxa and by the Department of Health of the Government of the Basque Country (2008/111062).
Compliance with ethics guidelines
The study was in accordance with the Helsinki II Declaration, as revised in 2000 and was approved by the Ethical Committee of the Hospital of Txagorritxu (Vitoria, Spain). All participants received verbal and written information about the nature and purpose of the study, and all of them gave written consent to participate.
Conflict of interest
Maria Arrizabalaga, Eider Larrarte, Javier Margareto, Sara Maldonado, Lourdes Barrenetxea and Idoia Labayen declare that they have no conflict of interest.