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International Journal of Obesity (2006) 30, 226232

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ORIGINAL ARTICLE
A polymorphism in the adiponectin gene influences
adiponectin expression levels in visceral fat in obese
subjects
J Fredriksson, E Carlsson, M Orho-Melander, L Groop and M Ridderstrale
Department of Clinical Sciences/Diabetes and Endocrinology, Malmo University Hospital, Lund University, Wallenberg
Laboratory, Malmo, Sweden
Objective: Reduced serum adiponectin levels have been found in obesity and type 2 diabetes and variations in the adiponectin
gene (APM1) have been associated with type 2 diabetes and features of the metabolic syndrome in different populations.
Study Design: Here, we investigated the expression of APM1 in adipose tissue and studied the relationship between variation
in APM1 expression, the APM1 G276T polymorphism, the common PPARG Pro12Ala polymorphism and clinical features of 36
morbidly obese (body mass index (BMI) 41.574.9 kg/m2) nondiabetic subjects.
Results: APM1 mRNA expression in visceral fat was correlated with serum adiponectin levels (r 0.54, P 0.012). In visceral, but
not in subcutaneous, adipose tissue APM1 mRNA level was 38% higher among carriers of the APM1 G276T T allele (G/T and T/T)
than among carriers of the G/G genotype (0.9170.06 for G/T and T/T carriers vs 0.6670.09 for G/G carriers, P 0.013).
Carriers of the T allele also had significantly higher body fat percent compared to G/G carriers (6576 vs 56710%, P 0.011).
Conclusion: Our results indicate that genetic variation in APM1 influences the expression of the gene in visceral adipose tissue
and suggest a potential role for such variation in regulation of body fat accumulation in obese subjects.
International Journal of Obesity (2006) 30, 226232. doi:10.1038/sj.ijo.0803138; published online 11 October 2005
Keywords: adiponectin; APM1; adipose tissue; gene expression

Introduction
The adipose tissue is a metabolically active tissue that
produces and secretes a variety of proteins, such as tumor
necrosis factor alpha, resistin, plasminogen activator inhibitor type 1, angiotensinogen, leptin and adiponectin, all
of which have been ascribed a role in the pathogenesis of
insulin resistance and obesity.15 Plasma adiponectin levels
are reduced in obesity, type 2 diabetes and cardiovascular
disease and low adiponectin concentration is an independent risk factor for progression to type 2 diabetes.58 In
analogy, high adiponectin plasma levels are associated with
enhanced insulin sensitivity in nondiabetic subjects.9 Variations in the adiponectin gene (APM1) have been associated
with type 2 diabetes in many populations.1016 The adiponectin gene has thus emerged as a susceptibility gene for

Correspondence: J Fredriksson, Department of Clinical Sciences/Diabetes and


Endocrinology, Lund University, Wallenberg Laboratory, Floor 3, UMAS
entrance 46, S-205 02 Malmo, Sweden.
E-mail: jenny.fredriksson@med.lu.se
Received 23 March 2005; revised 30 August 2005; accepted 12 September
2005; published online 11 October 2005

type 2 diabetes, as well as for cardiovascular disease and


obesity. The intronic G276T variant, located in intron 2, is
one of the most extensively studied variants within the
adiponectin gene and has the most consistent published
results. It has been associated with type 2 diabetes, insulin
resistance, coronary artery disease and adiponectin level in
several populations.10,14,1720 The majority of these studies
report the APM1 G276T G-allele as risk allele, with two
exceptions.14,19
Earlier studies have shown that APM1 expression is
reduced in visceral compared to subcutaneous adipose tissue
in normal-weight subjects.21 However, adiponectin secretion
was higher in cultured human omental adipocytes than
in subcutaneous adipocytes.22 Adiponectin serum levels
have a heritability of about 30%,18 indicating that genetic
variation within or outside the adiponectin gene could
affect the expression of the gene. Several studies have
reported an effect on serum adiponectin levels for different
single nucleotide polymorphisms (SNPs) in the APM1
gene,10,12,1719,23 but to our knowledge only one study has
focused on the effect of a genetic marker on the APM1
mRNA expression in omental adipose tissue, reporting
increased mRNA levels for the APM1 T94G G-allele.24

Adiponectin gene expression


J Fredriksson et al

227
Visceral adipose tissue is more metabolically active than
subcutanoeus adipose tissue and is of greater importance for
the development of obesity-related complications.25 Therefore it is important to investigate both these adipose depots
to detect differences in expression patterns and effects of
genetic variants.
The adiponectin gene is also regulated by the peroxisome
proliferator-activated receptor gamma (PPARg) gene (PPARG).
PPARg agonists increase adiponectin mRNA expression and
secretion in cultured 3T3-L1 adipocytes, in db/db mice and
the adiponectin concentrations in humans.26 The aim of
this study was to examine tissue-specific adiponectin mRNA
expression in morbidly obese nondiabetic patients and
whether this expression is influenced by the APM1 G276T
or the PPARG Pro12Ala polymorphisms.

using allelic discrimination on the ABI PRISM 7900 Sequence


Detection System (Applied Biosystems, Foster City, CA, USA)
according to the manufacturers instructions in a volume of
5 ml. Primers (APM1 G276T; Forward: 50 -TTCATCACAGACC
TCCTACACTGA-30 , Reverse: 50 -TCCCTGTGTCTAGGCCTTA
GTTAAT-30 PPARG Pro12Ala; Forward: 50 GTTATGGGTGAA
ACTCTGGGAGATT-30 Reverse: 50 -GCAGACAGTGTATCAGT
GAAGGAAT-30 ) and probes (APM1 G276T: Probe G: 50 -(VIC)ACTATATGAAGGCATTCAT-(nonfluorescent quencher)-30 ,
Probe T: 50 -(FAM)-AAACTATATGAAGTCATTCAT-(nonfluorescent quencher)-30 PPARG Pro12Ala; Probe C: 50 -(VIC)CTCCTATTGACCCAGAAAG-(nonfluorescent quencher)-30 ,
Probe G: 50 -(FAM)-CTATTGACGCAGAAAG-(nonfluorescent
quencher)-30 ) were ordered as Assays-by-Design (Applied
Biosystems).

Methods

Laboratory tests
Assessment of phenotypic characteristics has been described
previously.27 Briefly, blood was drawn from an antecubical
vein after a 12-h overnight fast and plasma glucose was
determined using an automated glucose oxidase method
(Glucose Analyzer 2; Beckman Instruments, Fullerton, CA,
USA). Serum insulin was determined using a radioimmuno
assay (RIA) (Pharmacia, Uppsala, Sweden). Serum adiponectin
and leptin levels were measured with human adiponectin
and leptin RIA kits (Linco Research Inc, St Charles, MO,
USA). BMI was calculated as weight (kg) divided by height
(m) squared. Triglyceride levels were measured on a Cobas
Mira analyzer (Hoffman-La Roche, Basel, Switzerland). Total
body fat percentage was calculated using bioimpedance
analysis.

Study subjects
The study material has been described previously.27 Visceral
and subcutaneous adipose tissue was obtained peroperatively
from 36 obese subjects, 32 females and 4 males, (body mass
index (BMI) 41.574.9 kg/m2, age 37.2710.1 years) undergoing bariatric surgery at Landskrona hospital, Sweden. All
subjects were Swedish, Caucasian and nondiabetic. Biopsies
were frozen in liquid nitrogen and stored in 801 until used
for analysis. The homeostasis model assessment (HOMA) was
used for assessment of insulin resistance (insulin resistance
index (fasting glucose (mmol/l)  fasting insulin (U/ml))/
22.5).28 DNA could not be obtained from five of the patients.
Clinical characteristics for the subjects are shown in Table 1.
The study was approved by local ethics committee and all
subjects gave informed consent.

Genotyping
The SNP located at position 276 (G4T) in intron 2 of APM1
and the PPARG Pro12Ala polymorphism were genotyped
Table 1

Clinical characteristics of the study subjects

Clinical variable [N]

All subjects

N (m/f)
Age (years)[36]
BMI (kg/m2)[35]
Waist (cm)
Males[4]
Females[26]
Fat %[31]
HOMA index[23]
Fasting glucose (mmol/l)[32]
Fasting insulin (mU/l)[24]
Leptin (mg/l)
Males[3]
Females[25]
Triglycerides (mmol/l)[27]
Adiponectin (mg/ml)[21]

4/32
37.2710.1
41.574.9
139.578.2
115.2711.1
60.079.1
4.074.2
4.970.6
19.5716.8
19.373.9
35.8710.7
1.971.4
14.476.9

Gene expression
Extraction of total RNA from the fat biopsies was performed
with the RNeasy Mini kit (Qiagen, Germany) and cDNA
synthesized using Superscript II RNase H- Reverse Transcriptase (Life Technologies, MD, USA) and random hexamer
primers (Life Technologies, MD, USA). APM1 mRNA levels
were quantified using real-time PCR on the ABI PRISM
7900 Sequence Detection System (Applied Biosystems) with
forward primer: 50 -AAAGGAGATCCAGGTCTTATTGGT-30 ,
reverse primer: 50 -CCTTCAGCCCCGGGTACT-30 , probe:
50 -(FAM)-AAGGGAGACATCGGTGAAACC-(TAMRA)-30 and
Cyclophilin A as endogenous control. All samples were run
in duplicate and data were calculated using the standard
curve method and expressed as a ratio to the Cyclophilin A
reference (arbitrary units).

Statistical analysis
Descriptive data are presented as means7s.d. and expression
data are presented as means7s.e.m. Differences were
considered significant when P was less than 0.05. Statistical
calculations were performed using the Number Cruncher
International Journal of Obesity

Adiponectin gene expression


J Fredriksson et al

Results
We investigated APM1 expression in subcutaneous and
visceral adipose tissue and tested whether APM1 expression
was influenced by variation in the gene in morbidly obese
subjects. Clinical characteristics of the study subjects are
found in Table 1. APM1 mRNA expression did not significantly differ between subcutaneous and visceral adipose
tissue (mean values 0.8970.08 vs 0.8370.08, P 0.92), and
no correlation was observed between the mRNA expression
in the two different adipose tissues. The mRNA expression in
visceral (r 0.54, P 0.012) but not in subcutaneous adipose
tissue (r 0.09, P 0.68) was correlated with serum adiponectin levels (Figure 1). The correlation in visceral adipose
tissue remained after excluding the male subjects (r 0.48,
P 0.036), and also after excluding outliers (r 0.48,
P 0.044). Moreover, visceral APM1 mRNA expression was
positively correlated with fasting plasma glucose concentrations (r 0.41, P 0.021) (in females only; r 0.39,
P 0.038). Subcutaneous APM1 expression correlated negatively with BMI (r 0.35, P 0.042), waist circumference
(r 0.34, P 0.042) and triglyceride level (r 0.55,
P 0.0028) (Table 2). When excluding male subjects,
subcutaneous APM1 expression correlated negatively with
triglyceride level (r 0.43, P 0.037). Adiponectin serum
levels did not significantly correlate with any of the
examined clinical variables, although a borderline inverse
relationship was seen with BMI (r 0.39, P 0.084).
Allele frequencies for the APM1 G276T variant were 0.73
(G) and 0.27 (T). All genotype frequencies were in Hardy
Weinberg equilibrium. Carriers of the APM1 G276T T allele
had significantly higher body fat percent (6576 vs 56710,
P 0.011) compared to homozygous carriers of the G-allele
(Table 3). This difference remained also after excluding male
subjects (6676 vs 57710, P 0.021 in female subjects only).
In an analysis including all three genotype groups, there was
a significant difference in visceral but not in subcutaneous
International Journal of Obesity

3
2.5
2
1.5
1
0.5
0

b
Subcutaneous APM1 mRNA

Statistical Software 2000 (NCSS, Kaysville, UT, USA). The


significance of difference in mean values was tested by
nonparametric statistics using P-values derived by Kruskal
Wallis or MannWhitney, and the significance of relationship between variables was tested using Spearmans correlation coefficient. Wilcoxon signed-rank test was used to
compare differences between paired variables. The w2 test was
used to detect deviations from the HardyWeinberg equilibrium. To obtain as much power as possible, the four male
subjects were included in analysis; however, significant
differences were reanalyzed in female subjects only. Owing
to few subjects carrying the APM1 G276 T T/T and the PPARG
Pro12Ala Ala/Ala genotypes, the APM1 G276 T T/T genotype
was combined with the G/T genotype and the PPARG Pro12
Ala/Ala genotype was combined with the Pro/Ala genotype
for most analyses.

Visceral APM1 mRNA

228

10

10

20
30
Adiponectin (ug/ml)

40

3
2.5
2
1.5
1
0.5
0
20

30

40

Adiponectin (ug/ml)
Figure 1 Correlation between APM1 mRNA expression and serum adiponectin. There was a correlation between adiponectin serum concentration and
APM1 expression in visceral adipose tissue (a), but not in subcutaneous
adipose tissue (b). (r 0.54, P 0.012 and r 0.09, P 0.68, respectively.
Spearman correlation coefficients).

Table 2 Correlations between visceral APM1 expression, subcutaneous APM1


expression and serum adiponectin levels, and clinical variables

BMI
Waist
HOMA
Fasting glucose
Triglycerides
Leptin
Fat %

Visceral APM1
mRNA

Subcutaneous
APM1 mRNA

Serum
adiponectin

r
0.12
0.11
0.07
0.41*
0.14
0.30
0.10

r
0.35*
0.38*
0.21
0.09
0.55**
0.12
0.07

r
0.39
0.13
0.28
0.18
0.38
0.32
0.33

Spearmans coefficients; *Po0.05, **Po0.01.

APM1 mRNA expression, with higher expression in carriers


of the T allele (P 0.014) than in G/G genotype carriers
(Figure 2) (in female subjects only: P 0.024). Visceral fat
APM1 mRNA levels were 38% higher among carriers of the
APM1 G276T T allele compared to G/G genotype carriers
(0.9170.06 vs 0.6670.09 for carriers of genotypes T/T
and G/T vs G/G; P 0.0065). This difference also persisted

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229
Table 3

Analysis of clinical variables according to APM1 G276T genotype


APM1 G276T G/G APM1 G276T G/T+T/T

Adipnectin mRNA expression

N
Age (years)
BMI (kg/m2)
Waist (cm)
Fat %
HOMA index
Fasting glucose (mmol/l)
Fasting insulin (mU/l)
Leptin (mg/l)
Triglycerides (mmol/l)
Adiponectin (mg/ml)

17
39.879.8
41.075.7
120.2712.8
55.579.8
4.775.3
4.770.5
23.0720.7
30.3711.3
2.371.7
13.878.1

14
36.9710.9
41.574.3
115.1714.4
65.376.3
2.771.0
5.170.6
12.875.2
37.9710.6
1.670.6
15.175.1

0.55
0.69
0.34
0.011
0.30
0.15
0.20
0.060
0.37
0.27

1.4
1.2
1
0.8
0.6
0.4
0.2
0
G276T G/G (N=17) G276T G/T (N=11) G276T T/T (N=3)

Figure 2 Adiponectin mRNA expression according to G276T genotype.


There was a difference in adiponectin gene expression when comparing the
three different APM1 G276T genotype groups in visceral adipose tissue (open
bars), P 0.014. No difference was seen in subcutaneous adipose tissue (filled
bars), P 0.90. In a two group comparison, P 0.0065 for APM1 G/G vs G/T
and T/T in visceral adipose tissue. The level of APM1 expression is normalized
to the mRNA level of endogenous cyclophilin A, and the ratios (APM1/
cyclophilin A) are presented in the figure. (MannWhitney, KruskalWallis
nonparametric test. Data presented as mean7s.e.m.).

Table 4

PPARG Pro12Ala genotype and expression of the adiponectin gene

N
Adiponectin (mg/ml) (N 16/5)
APM1 mRNA visceral
APM1 mRNA subcutaneous

N
Adiponectin (mg/ml) (N 8/10)
APM1 mRNA visceral
APM1 mRNA subcutaneous

PPARG ProPro

PPARG X/Ala

22
14.377.6
0.8770.50
0.9670.56

9
14.874.7
0.7370.34
0.7970.26

0.65
0.49
0.54

PPARG Pro/Pro
PPARG -/Ala
APM1 G276T G/ APM1 G276T-/T
G
13
18
13.979.1
14.674.7
0.31
0.7070.38
0.8270.27
0.13
0.9770.66
0.8570.37
0.78

when only female subjects were considered (0.9070.07 vs


0.6870.09, P 0.013).
We were also interested in whether the PPARG Pro12Ala
polymorphism would have an effect on the mRNA

expression of APM1. Allele frequencies of the PPARG


Pro12Ala variant were 0.81 (Pro) and 0.19 (Ala), and
genotype frequencies were in HardyWeinberg equilibrium.
We did not observe any difference in APM1 mRNA expression between the different PPARG genotype carriers (Table 4).
Neither was there any effect of combination of risk
genotypes in both genes on mRNA expression (Table 4).

Discussion
The key finding of the present study was that adiponectin
mRNA concentrations in visceral fat correlated with serum
adiponectin concentrations and that visceral APM1 mRNA
expression levels are influenced by an intronic polymorphism in the gene. The absence of a difference between
visceral and subcutaneous adiponectin mRNA levels in obese
subjects is in line with a previous study which showed
significantly lower APM1 mRNA levels in visceral compared
to subcutaneous adipose tissue in lean subjects, but only a
nonsignificant trend in obese subjects.21 In humans, subcutaneous adiponectin mRNA levels are reduced in obesity,29
independently of the degree of insulin resistance.30 In obese
type 2 diabetic subjects, both visceral and subcutaneous
APM1 mRNA levels have been reported to be reduced.31
Taken together, results suggest that there is no difference
in APM1 mRNA expression between subcutaneous and
visceral adipose tissue in obese subjects, possibly due to the
reduction of subcutaneous and perhaps also visceral APM1
mRNA levels, that accompany the obese state.
Obese carriers of the T allele of APM1 G276T had higher
adiponectin mRNA levels in visceral fat than carriers of the
G/G genotype. This finding is in line with previous studies
showing that the T allele of APM1 G276T has a positive
influence on serum adiponectin levels.10,17,18 Altered expression of genes due to cis-acting inherited allelic variations
has been reported for a large number of genes.32 It was
recently shown that the first intron of the adiponectin
gene is required for full transcriptional activation. This is
mediated by interaction between the CCAAT/enhancer
binding protein alpha and an intronic enhancer.33 It is
possible that similar enhancer or silencer regions could be
found also in intron 2, although this has not yet been
shown.
Subcutaneous and visceral adipose tissues differ at several
levels. For example, the expression levels of various adipokines34 as well as the expression of several receptors35 and
transcription factors35,36 differ between the two adipose
depots. Visceral fat is also more metabolically active than
subcutaneous fat and adipokines produced by visceral
adipose tissue have direct access to the liver. With these
differences in mind it is possible that a variant in a gene
could have effects in one but not in the other type of adipose
tissue. Interestingly, despite a higher percent body fat,
carriers of the APM1 G276T T allele had higher levels of
International Journal of Obesity

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J Fredriksson et al

230
visceral adiponectin gene expression. This may indicate that
T-allele carriers are protected against the negative metabolic
effects of obesity. From the data presented here, we are not
able to conclude whether this is an associating or secondary
effect. If this genotype is associated with elevated adiponectin expression it should also be associated with enhanced
insulin sensitivity, which has been shown to predict weight
gain.37 Although this is an appealing hypothesis we feel that
it is too soon to make this conclusion, not least in view of the
limited number of participants in this study, which was not
designed to answer this question primarily. In light of our
present data and previously published data on the T45G
polymorphism,17,24 we were also interested in combinatorial
effects between these polymorphisms and adiponectin
expression. However, the frequency of the T45G allele was
too low (0.08 for the G allele) in this material to perform
such analyses.
In a transgenic mouse model lacking adipose tissue,
adiponectin treatment normalized insulin sensitivity38 and
mice lacking adiponectin show impaired clearance of free
fatty acid and develop diet-induced insulin resistance.39 It
has been suggested that adiponectin enhances insulin
sensitivity by increasing fatty acid oxidation in muscle
which in turn will result in reduced intramyocellular
triglyceride content and improved glucose uptake.39 We
found a negative correlation between subcutaneous APM1
mRNA expression and fasting plasma triglyceride levels
which may be an indicator of insulin resistance, but we
were unable to detect significant correlations between
HOMA and serum adiponectin or subcutaneous or visceral
mRNA levels. This could be due to the use of HOMA as a
surrogate measure of insulin sensitivity. To our knowledge, it
has not been validated for this purpose in morbidly obese
subjects. Correlations between HOMA and serum adiponectin and subcutaneous mRNA levels have been reported
previously.30,40,41
Motoshima et al.22 showed that adiponectin secretion was
higher from cultured human omental than from subcutaneous adipocytes. In the same study, they also showed that
omental adiponectin secretion was increased after insulin
treatment while subcutaneous secretion was unaffected. To
that end, we found here that visceral APM1 expression
correlated with serum adiponectin. In accordance with
some,42,43 but not all 29,41,44 previous studies, we were not
able to show a correlation between serum adiponectin levels
and adiponectin mRNA levels in subcutaneous adipose
tissue. The reason for this is not apparent but could reflect
a greater role of visceral adipose tissue for adiponectin
secretion. Although only visceral APM1 mRNA expression
correlated with serum adiponectin levels, we did find
negative correlations between subcutaneous APM1 mRNA
expression and BMI and waist circumference. Subcutaneous
APM1 mRNA expression has been found to be unaffected by
diet in humans,45 which is in line with experiments in rats
showing increased levels of APM1 mRNA expression with
weight reduction in visceral but not in subcutaneous adipose
International Journal of Obesity

tissue.46 It is tempting to speculate that the two adipose


depots play different roles in adiponectin production and/or
secretion and the maintenance of insulin sensitivity.
There is convincing evidence that both adiponectin
secretion and adiponectin mRNA expression are regulated
by PPARg ligands26 and PPARg/RXR heterodimers transactivate the human adiponectin promoter in vitro where it binds
to PPARg-responsive element.47 The Ala-allele of the PPARG
Pro12Ala polymorphism has been associated with a reduced
risk for type 2 diabetes in several populations.48 The
Pro12Ala polymorphism has also been suggested to affect
the adiponectin plasma levels, Ala-carriers having lower
APM1 expression in a Japanese population.49 In line with
results on adiponectin expression from a German population,50 as well as on expression of other PPARg target genes in
obese Finnish subjects,51 we were not able to detect an effect
of the PPARG Pro12Ala polymorphism on the expression of
the adiponectin gene, neither in visceral nor in subcutaneous adipose tissue, suggesting that the effect of PPARg on
adiponectin gene expression is not influenced by this
polymorphism.
In conclusion, our results indicate that genetic variation in
the adiponectin gene influences the expression of the gene
in visceral adipose tissue and suggest a potential role for such
variation in regulation of body fat accumulation in obese
subjects.

Acknowledgements
This work was supported by Grants from the Foundation for
Strategic Research through the National Network for Cardiovascular Research, the Swedish Foundation for the Study
of Diabetes, the Albert Pahlssons Foundation, the Swedish
University Hospital
Medical Research Council, Malmo
m Foundation, the
Foundation, the Ernhold Lundstro
Anna-Lisa and Sven-Eric Lundgren Foundation, the Crafoord
Foundation, the Lundberg Foundation, the Magnus Bergvall
Foundation, the Fredrik and Ingrid Thurings Foundation, the
Novo Nordisk Foundation and the European Association for
the Study of Diabetes (Sankyo Pharma).

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