Download Fat Mass and Obesity- Associated Gene (FTO) Is Linked to Higher

Diabetes Volume 63, November 2014
3955
Christian Benedict,1 Tomas Axelsson,2 Stefan Söderberg,3 Anders Larsson,4 Erik Ingelsson,5,6 Lars Lind,7
and Helgi B. Schiöth1
Diabetes 2014;63:3955–3959 | DOI: 10.2337/db14-0470
The mechanisms through which common polymorphisms
in the fat mass and obesity-associated gene (FTO) drive
the development of obesity in humans are poorly understood. Using cross-sectional data from 985 older people
(50% females) who participated at age 70 years in the
Prospective Investigation of the Vasculature in Uppsala
Seniors (PIVUS), circulating levels of ghrelin and leptin
were measured after an overnight fast. In addition, subjects were genotyped for F TO rs17817449 (AA, n = 345
[35%]; AC/CA, n = 481 [48.8%]; CC, n = 159 [16.1%]).
Linear regression analyses controlling for sex, selfreported physical activity level, fasting plasma glucose,
and BMI were used. A positive relationship between the
number of F TO C risk alleles and plasma ghrelin levels
was found (P = 0.005; relative plasma ghrelin difference
between CC and AA carriers = ∼9%). In contrast, serum
levels of the satiety-enhancing hormone leptin were inversely linked to the number of F TO C risk alleles (P =
0.001; relative serum leptin difference between CC and
AA carriers = ∼11%). These associations were also
found when controlling for waist circumference. The
present findings suggest that F TO may facilitate weight
gain in humans by shifting the endocrine balance from
the satiety hormone leptin toward the hunger-promoting
hormone ghrelin.
1Department
6Wellcome
of Neuroscience, Uppsala University, Uppsala, Sweden
of Medical Sciences, Molecular Medicine and Science for Life
Laboratory, Uppsala University, Uppsala, Sweden
3Department of Public Health and Clinical Medicine and Heart Center, Umeå
University, Umeå, Sweden
4Department of Medical Sciences, Clinical Chemistry, Uppsala University,
Uppsala, Sweden
5Department of Medical Epidemiology and Science for Life Laboratory, Uppsala
University, Uppsala, Sweden
2Department
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
Trust Centre for Human Genetics, University of Oxford, Oxford, U.K.
of Medical Sciences, Cardiovascular Epidemiology, Uppsala University, Uppsala, Sweden
7Department
Corresponding author: Christian Benedict, [email protected].
Received 21 March 2014 and accepted 27 May 2014.
© 2014 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
GENETICS/GENOMES/PROTEOMICS/METABOLOMICS
Fat Mass and ObesityAssociated 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
3956
FTO and Ghrelin and Leptin
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 populationbased 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, selfreported physical activity, sex, and fasting plasma glucose
levels.
RESEARCH DESIGN AND METHODS
Design Overview, Participants, and Genotyping
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 x2 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.
Diabetes Volume 63, November 2014
Clinical and Biochemical Investigation
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 selfreported light (non2sweat-inducing) and hard (sweatinducing) 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.
Statistical Analysis
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 6 SE. Overall, two-tailed
P values , 0.05 were regarded as significant.
RESULTS
Descriptive Characteristics
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 x2 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 (b [SE] = 20.167 [0.071], P = 0.018,
adjusted for sex, BMI, and self-reported physical activity
level).
Plasma Ghrelin and Serum Leptin Concentrations
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: b [SE] =
20.83 [0.035], P = 0.017; ghrelin: b [SE] = 0.124 [0.049],
P = 0.011; adjusted for sex [when appropriate], waist
circumference, self-reported physical activity level, and
diabetes.diabetesjournals.org
Benedict and Associates
3957
Figure 1—Link between the obesity-risk FTO rs17817449 C allele and circulating levels of ghrelin and leptin in a Swedish population of men
and women at age 70 years. A: Genotype frequency of FTO rs17817449 in participants at age 70 years from PIVUS. B: Distribution of
standardized circulating levels of ghrelin and leptin (standardized by sex due to the large sex differences in circulating levels [17]) following
natural log-transformation. C: Significant association between the number of obesity-risk FTO rs17817449 C alleles and circulating levels of
ghrelin and leptin (raw values are shown). 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 (i.e., b) are unstandardized coefficients 6 SE.
Overall, two-tailed P values < 0.05 were regarded as significant.
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 = 20.07,
P = 0.830) were significantly correlated with BMI.
DISCUSSION
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 appetitestimulating hormones in the circulation may increase the
susceptibility for energy surplus in subjects carrying the
common FTO obesity-risk rs17817449 C allele.
Comparison With the Literature
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
3958
FTO and Ghrelin and Leptin
Diabetes Volume 63, November 2014
Table 1—Raw data of study participants at age 70 years, stratified by the number of obesity-risk FTO rs17817449 C alleles
FTO rs17817449 genotype
AA
AC/CA
CC
345
481
159
Women, n (% genotype group)
164 (48)
240 (50)
88 (55)
Self-reported physical activity level, n (% genotype group)
Very low
Low
Medium
High
Missing values
27 (8)
213 (62)
71 (20)
16 (5)
18 (5)
66 (14)
267 (56)
97 (20)
24 (5)
27 (5)
17 (11)
100 (63)
28 (17)
6 (4)
8 (5)
BMI (kg/m2)
27.3 6 4.4
27.0 6 4.5
26.8 6 3.9
Blood glucose levels (mmol/L)
5.6 6 2.1
5.2 6 1.2
5.2 6 1.6
n
Data are mean 6 SD, unless indicated otherwise.
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).
Potential Mechanisms for the Observed Associations
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 obesityassociated 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.
Limitations
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.
Conclusions
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 appetitestimulating 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
diabetes.diabetesjournals.org
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.
References
1. Fall T, Ingelsson E. Genome-wide association studies of obesity and metabolic syndrome. Mol Cell Endocrinol 2014;382:740–757
2. Ho AJ, Stein JL, Hua X, et al.; Alzheimer’s Disease Neuroimaging Initiative. A
commonly carried allele of the obesity-related F TO gene is associated with reduced
brain volume in the healthy elderly. Proc Natl Acad Sci USA 2010;107:8404–8409
3. Melka MG, Gillis J, Bernard M, et al. F TO, obesity and the adolescent brain.
Hum Mol Genet 2013;22:105021058
4. Hess ME, Hess S, Meyer KD, et al. The fat mass and obesity associated
gene (Fto) regulates activity of the dopaminergic midbrain circuitry. Nat Neurosci
2013;16:1042–1048
5. Karra E, O’Daly OG, Choudhury AI, et al. A link between FTO, ghrelin, and
impaired brain food-cue responsivity. J Clin Invest 2013;123:3539–3551
6. Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central
regulation of feeding. Nature 2001;409:194–198
Benedict and Associates
3959
7. Friedman JM, Halaas JL. Leptin and the regulation of body weight in
mammals. Nature 1998;395:763–770
8. Baicy K, London ED, Monterosso J, et al. Leptin replacement alters brain
response to food cues in genetically leptin-deficient adults. Proc Natl Acad Sci
USA 2007;104:18276–18279
9. Lind L, Fors N, Hall J, Marttala K, Stenborg A. A comparison of three different methods to evaluate endothelium-dependent vasodilation in the elderly:
the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study.
Arterioscler Thromb Vasc Biol 2005;25:2368–2375
10. Benedict C, Brooks SJ, Kullberg J, et al. Association between physical
activity and brain health in older adults. Neurobiol Aging 2013;34:83–90
11. Gonzalez M, Lind L, Söderberg S. Leptin and endothelial function in the
elderly: the Prospective Investigation of the Vasculature in Uppsala Seniors
(PIVUS) study. Atherosclerosis 2013;228:485–490
12. Dickson SL, Egecioglu E, Landgren S, Skibicka KP, Engel JA, Jerlhag E. The
role of the central ghrelin system in reward from food and chemical drugs. Mol
Cell Endocrinol 2011;340:80–87
13. Maffei M, Halaas J, Ravussin E, et al. Leptin levels in human and rodent:
measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med 1995;1:1155–1161
14. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;
372:425–432
15. Do R, Bailey SD, Desbiens K, et al. Genetic variants of FTO influence adiposity, insulin sensitivity, leptin levels, and resting metabolic rate in the Quebec
Family Study. Diabetes 2008;57:1147–1150
16. Wang P, Yang FJ, Du H, et al. Involvement of leptin receptor long isoform
(LepRb)-STAT3 signaling pathway in brain fat mass- and obesity-associated
(FTO) downregulation during energy restriction. Mol Med 2011;17:523–532
17. Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG. Central nervous
system control of food intake. Nature 2000;404:661–671
18. Smemo S, Tena JJ, Kim KH, et al. Obesity-associated variants within FTO
form long-range functional connections with IRX3. Nature 2014;507:371–375
19. Nogueiras R, Tschöp MH, Zigman JM. Central nervous system regulation
of energy metabolism: ghrelin versus leptin. Ann N Y Acad Sci 2008;1126:
14–19