Fat Mass and Obesity-Associated Gene (FTO) Is Linked to Higher Plasma Levels of the Hunger Hormone Ghrelin and Lower Serum Levels of the Satiety Hormone Leptin in Older Adults

Population-based studies have repeatedly shown that subjects carrying specific single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated gene (FTO) are more prone to gain weight and to develop obesity and associated comorbidities (1–4). Using functional MRI, a recent small clinical trial involving normal-weight young men revealed that the FTO obesity-risk rs9939609 A allele—located in the first intron of this gene—is associated with an enhanced brain response to hedonic food stimuli (5). Furthermore, a divergent neural responsiveness to circulating acyl ghrelin within brain regions that regulate appetite, reward processing, and incentive motivation was observed between AA and TT subjects (5). Finally, by using cell models, the authors demonstrated that FTO overexpression reduced ghrelin mRNA N6-methyladenosine methylation, concomitantly increasing ghrelin mRNA and peptide levels (5). Ghrelin, which is mainly produced by the stomach, causes hyperphagia while decreasing the energy expenditure (6). Therefore, a ghrelin-driven shift in the energy balance to positive values could be hypothesized to promote weight gain in those who carry the FTO obesity-risk rs9939609 A allele. However, this previous observation that FTO is linked to ghrelin metabolism is based on a small sample of young men. Further, only carriers homozygous for either rs9939609 A allele or rs9939609 T allele were included in the study (5). However, approximately 50% of the general population carries only one copy of the FTO obesity-risk rs9939609 A allele (5). Thus, the finding that FTO is linked to ghrelin metabolism requires further validation in studies involving larger samples, as well as using all three available genotypes (i.e., AA, AT/TA, and TT). Contrary to ghrelin, leptin that mainly stems from subcutaneous adipose tissue leads to lower food intake (7). In addition, it reduces brain activation in regions linked to hunger (e.g., insula) while enhancing activation in regions linked to inhibition and satiety (e.g., prefrontal cortex) (8).

Against this background, in the present population-based study involving 985 men and women at age 70, we examined the link between the FTO obesity-risk rs17817449 C allele and circulating levels of ghrelin and leptin measured in the morning after an overnight fast. FTO rs17817449 was chosen instead of FTO rs9939609 as it reached a higher genotyping success rate in the current study (n = 985 vs. n = 800). Importantly, correlational analysis in the subsample of 800 participants revealed singularity (i.e., perfect linkage disequilibrium) between the FTO rs9939609 SNP and FTO rs17817449 SNP (r² = 1). All analyses were controlled for BMI, self-reported physical activity, sex, and fasting plasma glucose levels.

Details of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) have been reported previously (9). Briefly, all subjects aged 70 years and living in the community of Uppsala, Sweden, were eligible. The subjects were chosen from the Total Population Register and were invited in a randomized order from the start of the study in April 2001 to the last included subject in June 2004. Of the 2,025 subjects invited, 1,016 subjects (507 men and 509 women) were investigated (50%). Of these individuals, 985 (97%) were successfully genotyped for the FTO rs17817449 SNP (chromosome 16) as a part of a custom Illumina iSelect genotyping array (average call rate of 99.9%). Testing for Hardy-Weinberg equilibrium (using a χ² test, 1 df) revealed that the SNP did not deviate from expected genotype proportion (Fig. 1A).

The study was approved by the Uppsala University ethics committee. All participants gave their written informed consent. The study was conducted according to Declaration of Helsinki principles.

At the age of 70 years, blood samples were collected in the morning after an overnight fast. No medication or smoking was allowed after midnight. Plasma ghrelin and serum leptin were analyzed with commercially available assays (Linco Research, St. Charles, MO). Fasting plasma glucose levels were measured by standard laboratory techniques. Height and weight was used to calculate BMI (kg/m²).

As previously reported (10), the number of self-reported light (non−sweat-inducing) and hard (sweat-inducing) exercise activities with a duration of at least 30 min per week was used to assign each participant’s physical activity level to one of the four physical activity categories: very low, low, medium, and high.

Data were analyzed using linear regression models. Ghrelin and leptin variables were naturally log-transformed and subsequently standardized to approach normal distribution (Fig. 1B). In the case of leptin, serum levels were standardized by sex due to the large sex differences in circulating levels (11). If not otherwise specified, linear regression analyses were adjusted for sex (on models on ghrelin only), fasting plasma glucose, BMI, and self-reported physical activity level. Reported regression coefficients are unstandardized coefficients ± SE. Overall, two-tailed P values < 0.05 were regarded as significant.

Descriptive characteristics for the cohort are shown in Table 1. No differences in sex ratio and self-reported physical activity level were found between the three FTO rs17817449 groups (P > 0.05 for all χ² comparisons). For 51 individuals, no physical activity data were available (5%), i.e., their data were not entered into the main regression analysis. No association was found between BMI and the FTO rs17817449 SNP (P > 0.05, adjusted for sex). In contrast, fasting plasma glucose concentrations significantly differed between the three FTO rs17817449 groups (β [SE] = −0.167 [0.071], P = 0.018, adjusted for sex, BMI, and self-reported physical activity level).

Linear regression analysis demonstrated a positive association between the number of FTO rs17817449 risk alleles (i.e., allele C) and plasma ghrelin concentrations measured after an overnight fast (P = 0.005; Fig. 1C, left panel). In contrast, serum leptin concentrations were lower the more FTO rs17817449 risk alleles an individual had (P = 0.001; Fig. 1C, right panel). Importantly, the use of waist circumference instead of BMI as a measure of body adiposity did not change the direction and significance of the observed associations (leptin: β [SE] = −0.83 [0.035], P = 0.017; ghrelin: β [SE] = 0.124 [0.049], P = 0.011; adjusted for sex [when appropriate], waist circumference, self-reported physical activity level, and fasting plasma glucose levels). Moreover, imputation of missing values for self-reported physical activity did not change the results (data not shown). Finally, a Pearson correlational analysis revealed that serum leptin (r² = 0.719, P < 0.001) but not plasma ghrelin (r² = −0.07, P = 0.830) were significantly correlated with BMI.

We demonstrate in elderly subjects that a common obesity-susceptibility variant (rs17817449) located in an intron region of the FTO gene is linked to higher plasma levels of the hunger-promoting hormone ghrelin after an overnight fast. Concomitantly, the FTO risk allele was associated with lower serum levels of the satiety-enhancing adipokine leptin. Importantly, the observed associations were independent of body adiposity, as indicated by the robustness of our results when adjusting for waist circumference instead of BMI. Ghrelin is primarily released by the stomach and is integrally involved in the hedonic value of food (12). In contrast, leptin is an adiposity signal that circulates in proportion with fat mass (13). Leptin administration can decrease food intake, increase energy expenditure, and cause weight loss, whereas deficiencies in leptin are associated with obesity (14). Against this background, the results of the current study indicate that a shift from satiety-enhancing toward appetite-stimulating hormones in the circulation may increase the susceptibility for energy surplus in subjects carrying the common FTO obesity-risk rs17817449 C allele.

Our findings of increased plasma ghrelin concentrations after an overnight fast complement results of a previous experiment (5) involving 10 homozygous carriers of the FTO obesity-risk rs9939609 A allele and 10 homozygous carriers of the FTO rs9939609 T allele (this SNP occurred in perfect linkage in a subsample of our cohort with rs17817449). While fasting ghrelin levels did not significantly differ between FTO genotypes in this small clinical trial, homozygous carriers of the FTO obesity-risk rs9939609 A allele exhibited attenuated postprandial suppression of both hunger and circulating ghrelin levels compared with TT carriers (2). In this study, no genotypic differences were observed for either fasting or postprandial serum leptin levels (5). The discrepancies in results between this study and ours might be explained by differences in subject characteristics, e.g., our sample consisted of elderly men and women at age 70 years, whereas the other study involved young adult men (i.e., age <30 years) (5).

In another study from the Quebec City metropolitan area, it has been demonstrated in 359 men and women that the risk allele of FTO was associated with higher plasma leptin, but this was abolished after adjusting for BMI (15). The divergence between our results and those of the Quebec Family Study (15) may be explained by differences in participant age (70 years vs. 41 years), inclusion of confounders in the analysis, and sample size that was entered into the final analysis (ours was ∼2.7-fold greater).

Although we cannot establish causality in our observational study, there are findings from previous studies that may explain by which mechanisms FTO enhances plasma ghrelin in humans. For instance, in cell models, FTO overexpression reduced ghrelin mRNA N6-methyladenosine methylation, concomitantly increasing ghrelin mRNA and peptide levels (2).

Using double-immunofluorescence staining, FTO also has been colocalized with the leptin receptor long isoform in arcuate nucleus of hypothalamus and the nucleus of the solitary tract (16), two brain regions that are very important for the central nervous system control of food intake and energy expenditure (17). Interestingly in this previous study, leptin administration also downregulated FTO in vitro arcuate nucleus of hypothalamus cultures and in vivo wild-type mice (16). This suggests that lower circulating leptin levels—as observed in subjects carrying the obesity-risk FTO allele in our population—may concur with an increased gene expression of FTO in brain structures involved in appetite control. However, evidence for a regulatory effect of FTO on the expression, secretion, or degradation of leptin remains to be uncovered. Against this background, using animal models will help to decipher the molecular mechanisms through which an FTO overexpression or functional long-range targets of obesity-associated variants within FTO (e.g., IRX3 [18]) may predispose humans to exhibit higher plasma levels of ghrelin and lower serum levels of leptin.

The finding that serum levels of leptin were linked to both BMI and FTO rs17817449 and no link between FTO rs17817449 and BMI was found might indicate that this SNP is more closely related to leptin than BMI. One explanation could be that FTO or its functional long-range targets (18) exerts a more direct effect on fat mass (which can be better reflected by leptin) rather than overall obesity.

In contrast to large population-based genome-wide association studies (1), no link was observed between FTO and BMI, indicating that the lacking association between FTO and BMI is just a consequence of our relatively small sample size. While FTO was linked to higher plasma ghrelin and lower serum leptin concentrations after an overnight fast—a condition that is conducive for increased hunger (19)—we cannot draw firm conclusions by which extent such endocrine alterations would lead to energy surplus, as we neither measured acute food intake or hunger at the time when blood was sampled. Another limitation is that we measured total, but not active, ghrelin in blood. Finally, generalization of our findings to other age-groups or ethnic groups may not be appropriate.

The present cross-sectional analysis provides a strong rationale for hypothesizing that FTO may facilitate weight gain by tipping the scale of circulating signals involved in central nervous system food intake control toward appetite-stimulating factors. However, unless independent cohorts can replicate that the FTO risk allele is linked to higher plasma ghrelin and lower serum leptin levels, caution is needed before generalizing our results to other age-groups or ethnicities. For instance, in a study involving young men (5), FTO was linked to an altered postprandial plasma ghrelin response, whereas fasting levels of this hormone—contrary to our findings—were not linked to this gene.

Funding. This work is funded by the Swedish Research Council (E.I., L.L., H.B.S.), Swedish Brain Research Foundation (C.B., H.B.S.), Novo Nordisk Foundation (C.B.), and Åke Wibergs Foundation (C.B.). The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. T.A., S.S., A.L., E.I., and L.L. researched data. All authors wrote the manuscript. C.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.