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The Journal of Clinical Endocrinology & Metabolism 90(9):5356 –5360
Copyright © 2005 by The Endocrine Society
doi: 10.1210/jc.2005-0399
Upstream Transcription Factor-1 Gene Polymorphism Is
Associated with Increased Adipocyte Lipolysis
Johan Hoffstedt, Mikael Rydén, Hans Wahrenberg, Vanessa van Harmelen, and Peter Arner
Department of Medicine, Karolinska Institute, SE-141 86 Stockholm, Sweden
Objective: Variations in lipid metabolism between individuals could
be due to genetic factors. A transmission of a haplotype of the upstream transcription factor-1 (USF-1) gene containing the minor alleles at the usf1s1 and usfs2 loci is described. We investigated whether
these polymorphisms are associated with adipocyte lipolysis.
Methods and Results: A total of 196 healthy obese women were
investigated for in vitro lipolysis regulation in sc fat cells, which was
set in relation to the usf1s1 C3 T and usf1s2 G3 A polymorphisms in
the usf1 gene. The two polymorphisms were in complete linkage
disequilibrium. The usf1s1/2 T/A allele was associated with increases
in the maximum lipolytic action of noradrenaline (P ⫽ 0.005), dobut-
A
FUNDAMENTAL ASPECT of adipocyte function is to
store energy in the form of triglycerides during surplus nourishment and to release this energy as free fatty acids
and glycerol during starvation. The latter process, termed
lipolysis, has in previous studies been found to be associated
with a large interindividual variation, above all with respect
to catecholamine-induced effects in fat cells (1). In the search
for underlying mechanisms, genetic variance may be important. Accordingly, genetic variance affecting adipose tissue
lipolysis has been found in several genes regulating the catecholamine-signaling pathway, as reviewed previously (2).
For instance, polymorphisms in the ␤2- and ␤3-adrenoceptor
(AR) genes associate with altered receptor sensitivity to agonist stimulation, and a dinucleotide repeat in the hormonesensitive lipase (HSL) gene markedly reduces the ability of
catecholamines to stimulate lipolysis.
In a recent study, Pajukanta et al. (3) showed an association
between familial combined hyperlipidemia (FCHL) and upstream transcription factor 1 (USF-1) in family studies. Transmission of a rare haplotype at the usf1s1 and usf1s2 loci was
reduced, suggesting a protective role of USF-1 in FCHL.
USF-1 has been shown to regulate several genes involved in
lipid metabolism, including apolipoprotein CIII (apo CIII)
(4), apo A5 (5), acetyl-coenzyme A carboxylase-␣ (6), and
fatty acid synthase (7). The usf1 gene may be of particular
importance for catecholamine-induced lipolysis in fat cells,
First Published Online June 28, 2005
Abbreviations: apo, Apolipoprotein; AR, adrenoceptor; ATGL, adipose triglyceride lipase; Ct, threshold cycle; DASH, dynamic allelespecific hybridization; FCHL, familial combined hyperlipidemia; HSL,
hormone-sensitive lipase; PRKAR1A, protein kinase A type 1␣ regulatory subunit; SNP, single nucleotide polymorphism; USF-1, upstream
transcription factor-1.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
amine (P ⫽ 0.008), terbutaline (P ⫽ 0.008), CGP12177 (P ⫽ 0.015),
and forskolin (P ⫽ 0.006). In contrast, no significant genotype effect
on lipolytic sensitivity (i.e. half-maximum effective concentration) for
any of the drugs was demonstrated. Analysis of adipose tissue mRNA
expression in 78 women from genes regulating lipolysis at the postadrenoceptor level showed an increased level of protein kinase A
subunit R1␣ in the T/A genotype (P ⫽ 0.02).
Conclusions: Polymorphism in the usf1 gene is associated with increased lipolytic effect of catecholamines in fat cells, which is localized
at the postadrenoceptor level, possibly, at least, involving protein
kinase A. (J Clin Endocrinol Metab 90: 5356 –5360, 2005)
because it is involved in the transcriptional regulation of
HSL (8).
We tested the hypothesis that usf1 gene polymorphism is
involved in lipolysis regulation by comparing lipolytic activities in vitro of sc fat cells with usf1s1 and usfs2 genotypes
of USF-1. For this purpose, we used a large and unique
material of 196 obese, otherwise healthy, female subjects.
This is the first genetic examination of this cohort.
Subjects and Methods
Subjects
A total of 196 obese women who were otherwise healthy and free of
medication were included. They were consequently recruited to study
the influence of genetic variance on adipocyte lipolysis regulation. Body
mass index ranged from 30 –52 kg/m2, and age ranged from 19 – 65 yr.
All were living in the Stockholm area and were at least second generation
Scandinavian. None was completely sedentary or involved in athletic
performances. All ate a standard Swedish diet. None had undergone a
slimming effort or experienced a change (⬎1 kg) in body weight during
the last 6 months before the study according to self report. They came
to the laboratory at approximately 0730 h after an overnight fast. A
venous blood sample was obtained for DNA and analysis of plasma
levels of fatty acids, glycerol, glucose, insulin, triglycerides, cholesterol,
high-density lipoprotein cholesterol, apo A-1, and apo B by the hospital’s
accredited chemistry laboratory. Thereafter, an adipose sample (1–2 g)
was obtained by needle biopsy from the abdominal sc area under local
anesthesia as previously described (9). The study was explained in detail
to each subject, and his or her informed consent was obtained. The study
was approved by the hospital’s committee on ethics.
Fat cell studies
In 78 women, there was enough adipose tissue for mRNA experiments (see below). This tissue was frozen in liquid nitrogen and kept
frozen at ⫺70 C. The remaining adipose tissue was collagenase treated,
and isolated fat cells were collected and subjected to lipolysis experiments exactly as previously described (10). In brief, fat cells were incubated in buffer (pH 7.4) containing albumin and glucose at 37 C in the
absence (basal) or presence of increasing concentrations of noradrenaline (natural hormone, nonselective ␣2- and ␤-AR agonist), dobutamine
5356
Hoffstedt et al. • USF-1 and Lipolysis
(a selective ␤1-AR agonist), terbutaline (a selective ␤2-AR agonist),
CGP12177 (a selective ␤3-AR agonist), and forskolin (an adenylyl cyclase
activator, increases cAMP). After 2-h incubation, the medium was removed for determination of glycerol, which is an indicator of lipolysis.
Glycerol release was related to the number of fat cells incubated. The
maximum lipolytic effects of the various drugs were determined as the
rate of glycerol release at maximum effective concentration of drug
minus basal glycerol release. Lipolytic sensitivity for noradrenaline,
dobutamine, terbutaline, and CGP12177 was assessed by estimating the
EC50 from the concentration-response curves. The EC50 was logarithmically transformed and expressed as the pD2 value. The accuracy of the
lipolysis method has been validated in detail previously (1). ARs in fat
cells act according to the so-called spare receptor hypothesis. Thus, a
maximum effect is obtained when only a fraction of receptors is occupied. Therefore, changes in pD2 reflect variations in AR agonist action
at or near the target receptors, whereas changes in maximum action
mirror events at more distal (postreceptor) levels.
Genotyping
DNA was extracted from frozen (⫺20 C) venous blood. Genotyping
of the two single nucleotide polymorphisms (SNPs) usf1s1 (C/T) and
usf1s2 (G/A) were performed using dynamic allele-specific hybridization (DASH) (11). Information on the two SNPs may be found at the
dbSNP website (www.ncbi.nlm.nih.gov/SNP) under their respective
identification numbers, rs3737787 (usf1s1) and rs2073658 (usf1s2). All
PCRs were run in 20-␮l volumes including 1.5 mm MgCl2 and 5 ng
genomic DNA as previously described (11). The annealing temperature
for all reactions was 60 C. For the usf1s1 polymorphism, the sequences
for the primers used were 5⬘-CGGCCTGCAGTGGTATGAAACA-3⬘
(sense) and 5⬘-GGGTGGGCAAGGCTGTCAGTGC-3⬘ (antisense), and
that for the probe was 5⬘-CAGTGCACGTCCACATT-3⬘. The primers
and probe used for the usf1s2 polymorphism were 5⬘-GAGACACCACACCTAGCTACCAT-3⬘ (sense), 5⬘-ACAAGATTTAGCAGGTATTAGGAC-3⬘ (antisense), and 5⬘-TAGGACCATTTATGGTA-3⬘ (probe). A biotin moiety was added at the 5⬘ end of the forward primer sequences.
The position of the polymorphic site is underlined in the probe sequence.
The genotyping of the usf1s1 polymorphism failed in 12 subjects.
mRNA analysis
Adipose tissue that was available for mRNA analyses (n ⫽ 78) was
used as follows. Total RNA was extracted from 300 mg adipose tissue
using the RNeasy minikit (QIAGEN, Hilden, Germany), and the RNA
concentration and purity were assessed spectrophotometrically. One
microgram of total RNA from each sample was reverse transcribed to
cDNA using the Omniscript RT kit (QIAGEN) and random hexamer
primers (Invitrogen Life Technologies, Tastrup, Denmark). The Agilent
2100 bioanalyzer (Agilent Technologies, Kista, Sweden) was used to
confirm the integrity of the RNA. In a final volume of 25 ␮l, 5 ng cDNA
was mixed with 2⫻ SYBR Green PCR MasterMix (Bio-Rad Laboratories,
Hercules, CA) and primers (Invitrogen Life Technologies). The primer
pairs were selected to yield a single amplicon based on dissociation
curves and analysis by agarose gel electrophoresis. The primers used
were 5⬘-CTCAGTGTGCTCTCCAAGTG-3⬘ (sense) and 5⬘-CACCCAGGCGGAAGTCTC-3⬘ (antisense) for HSL, 5⬘-GCAGGCACTCGTACAGACTC-3⬘ (sense) and 5⬘-CCGCATCTTCCTCCGTGTAG-3⬘ (antisense)
for protein kinase A type 1␣ regulatory subunit (PRKAR1A), 5⬘-TGTGATGGTGTTGGAAGATGTG-3⬘ (sense) and 5⬘-GAGAGGTAGCAGTGATTGTAGC-3⬘ (antisense) for protein kinase A type II␤ regulatory
subunit (PRKAR2B), 5⬘-GCGGATCGGAAGGTTCAG-3⬘ (sense) and 5⬘GCCCTGCTGGTCAATGAG-3⬘ (antisense) for protein kinase catalytic
subunit ␣ (PRKACA), 5⬘-GTGTCAGACGGCGAGAATG-3⬘ (sense) and
5⬘-TGGAGGGAGGGAGGGATG-3⬘ (antisense) for adipose triglyceride
lipase (ATGL) and 5⬘-TGACTCAACACGGGAAACC-3⬘ (sense) and 5⬘TCGCTCCACCAACTAAGAAC-3⬘ (antisense) for 18S. Quantitative real-time PCR was performed in an iCycler IQ (Bio-Rad Laboratories). The
mRNA levels were determined by a comparative threshold cycle (Ct)
method (see user bulletin 2, ABI PRISM 7700, Applied Biosystems,
Foster City, CA). The subject with the highest Ct value was used as a
reference; all other Ct values for the target gene and reference gene,
respectively, were subtracted from this Ct value. The Ct values were then
normalized to rRNA for 18S.
J Clin Endocrinol Metab, September 2005, 90(9):5356 –5360
5357
Statistical methods
Values are the mean ⫾ sd. Student’s unpaired t test or analysis of
covariance, using age as covariate, was used for statistical evaluation. A
value of P ⱕ 0.05 was considered statistically significant.
Power calculation
We made a calculation to estimate the smallest mean difference in the
lipolysis rate (maximal noradrenaline minus basal glycerol release/107
cells) between genotypes that can be detected in the present study
material of 196 subjects to have a power of 80% to yield a statistically
significant result. The calculation was performed assuming allele frequencies of 0.7 (A) and 0.3 (B) for two alleles of a specific SNP, which
would correspond to homozygous genotype AA (n ⫽ 96) and the homo/
heterozygous genotype BB/AB (n ⫽ 100). The criterion for significance
was set at 0.05. The test was two-tailed, which means that an effect in
either direction will be interpreted. On the average, a study of this design
would enable us to report a mean lipolysis rate difference of 20% corresponding to means of 10.0 (AA genotype) vs. 12.0 ␮mol glycerol/107
cells (BB/AB genotype) and a common within-group sd of 5.0 based on
sd estimates of 50% of the corresponding mean value. The calculation
was made using SPSS Sample Power program (www.spss.com/se).
Results
The allele frequency for the usf1s1 polymorphism was C
0.69 and T 0.31, and that for the usf1s2 polymorphism was G
0.69 and A 0.31. The two genotypes were in Hardy-Weinberg
equilibrium and were found to be in complete linkage disequilibrium (r2 ⫽ 1.0) (12). Consequently, in the following
analyses, only data for one of the polymorphisms, usf1s2, are
presented.
In Table 1, the effects of the usf1s2 polymorphism on clinical parameters are shown. Subjects either homozygous or
heterozygous for the A allele were compared with subjects
homozygous for the G allele. No effect of the usf1s2 A allele
on any of the examined clinical parameters was found.
With respect to lipolysis measurements, no effect of the
usf1s2 A-allele on basal (nonstimulated) lipolysis, 11.6 ⫾ 6.9
(A allele) vs. 12.1 ⫾ 7.2 (G allele) ␮mol glycerol/107 cells (P ⫽
0.59) was found. However, an effect was demonstrated on
the maximum lipolytic action of all the investigated drugs
(Table 2A). The relationship was particularly evident for
noradrenaline. As shown in Fig. 1, cells from the A allele
subjects had a 23% higher maximum lipolytic effect of norTABLE 1. Clinical data for the usf1s2 polymorphism
Measure
Age (yr)
BMI (kg/m2)
Waist (cm)
Pl-glucose (mmol/liter)
Pl-insulin (mU/liter)
Pl-TG (mmol/liter)
Pl-cholesterol (mmol/liter)
Pl-HDL cholesterol (mmol/liter)
Pl-Apo-A1 (g/liter)
Pl-Apo-B (g/liter)
Pl-glycerol (␮mol/liter)
Pl-fatty acids (mmol/liter)
Allele
A
G
40 ⫾ 10
39 ⫾ 5
115 ⫾ 14
5.6 ⫾ 1.7
14 ⫾ 7
1.5 ⫾ 0.9
5.1 ⫾ 1.2
1.2 ⫾ 0.3
1.3 ⫾ 0.3
1.1 ⫾ 0.3
112 ⫾ 50
0.7 ⫾ 0.2
37 ⫾ 10
38 ⫾ 5
114 ⫾ 11
5.4 ⫾ 0.9
14 ⫾ 7
1.5 ⫾ 1.0
5.1 ⫾ 1.0
1.2 ⫾ 0.3
1.3 ⫾ 0.2
1.0 ⫾ 0.3
108 ⫾ 45
0.7 ⫾ 0.2
Values are mean ⫾ SD and are compared using the Student’s unpaired t test. Pl, Fasting plasma; BMI, body mass index; TG, triglyceride; HDL, high-density lipoprotein. No significant differences between groups were observed (by Student’s unpaired t test). A-allele,
usf1s2 AA/AG-subjects; G-allele, usf1s2 GG-subjects.
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J Clin Endocrinol Metab, September 2005, 90(9):5356 –5360
Hoffstedt et al. • USF-1 and Lipolysis
TABLE 2. Lipolysis data for the usf1s2 polymorphism
Agent
Allele
A
G
P
A. Maximum rates of lipolysis stimulation (glycerol release/
107 cells)
Noradrenaline
14.6 ⫾ 7.2
11.9 ⫾ 5.9
0.005
Dobutamine
21.8 ⫾ 9.6
18.4 ⫾ 8.0
0.008
Terbutaline
23.1 ⫾ 9.8
19.6 ⫾ 8.3
0.008
CGP12177
9.5 ⫾ 6.5
7.4 ⫾ 4.5
0.015
Forskolin
23.6 ⫾ 10.7
19.5 ⫾ 8.8
0.006
B. Lipolytic sensitivity (pD2)
Noradrenaline
8.0 ⫾ 1.3
8.1 ⫾ 1.4
NS
Dobutamine
7.9 ⫾ 1.1
7.8 ⫾ 1.1
NS
Terbutaline
7.8 ⫾ 1.1
7.8 ⫾ 1.3
NS
CGP12177
9.0 ⫾ 1.6
9.0 ⫾ 1.6
NS
Values are mean ⫾ SD and were compared by Student’s unpaired
t test. A-allele, usf1s2 AA/AG-subjects; G-allele, usf1s2 GG-subjects.
adrenaline than those with the G allele had. The A allele effect
was also significant for ␤1- (dobutamine), ␤2- (terbutaline),
and ␤3- (CGP12177) AR-mediated lipolytic response. Furthermore, the maximum lipolytic rate for the postreceptor
active drug forskolin was also higher in subjects carrying the
usf1s2 A allele. In contrast, no effect of the usf1s2 A allele on
lipolytic adrenergic sensitivity (i.e. pD2) for noradrenaline,
dobutamine, terbutaline, or CGP12177 was demonstrated
(Table 2B).
To correlate these findings with possible alterations in
gene expression, we assessed the mRNA expression of a set
of genes regulating lipolysis at the postreceptor level. In 78
(NAA ⫽ 8, NAG ⫽ 33, and NGG ⫽ 37) women, adipose tissue
was still available for mRNA analysis. As shown in Fig. 2, a
significant effect of the usf1s2 SNP on mRNA levels of
PRKAR1 was found (P ⫽ 0.021, by analysis of covariance
adjusted for age, because a significant interaction among
usf1s2 genotypes, PRKAR1A, and age was found in this
FIG. 1. Relationship between usf1s2 genotypes and noradrenaline-induced lipolysis. Concentration-response
curves from three representative subjects carrying the
AA, AG, and GG genotypes are shown.
FIG. 2. Relationship between usf1s2 genotypes and adipose tissue
PRKAR1 mRNA levels. Values are the mean ⫾ SD.
subcohort). The adipose tissue PRKAR1A mRNA level from
AA subjects was 17% higher than from AG/GG carriers. In
contrast, there was no difference in mRNA levels of HSL (P ⫽
0.93), ATGL (P ⫽ 0.55), PRKAR2B (P ⫽ 0.28), or PRKACA
(P ⫽ 0.22) between usf1s2 genotypes (mRNA values not
shown).
Discussion
Polymorphisms in genes regulating lipolysis may be important factors in the chain of events leading to altered ad-
Hoffstedt et al. • USF-1 and Lipolysis
ipose tissue and lipid metabolism. Genetic variance could at
least in part explain the large interindividual variation in the
action of catecholamines on lipolysis in apparently healthy
subjects (1). A striking finding of the present study is the
close relationship between the usf1s1/s2 polymorphisms and
catecholamine-stimulated lipolysis in human fat cells. Carriers of the usf1s1/2 T/A allele have an approximately 25%
higher rate of catecholamine-stimulated lipolysis in vitro than
homozygous carriers of the G allele. The allele effect was also
independent of age and body mass index. To the best of our
knowledge the present study sample is the by far the largest
one available for genetic studies of lipolysis. According to the
power calculation, we can detect rather small variations in
lipolysis for relatively common polymorphisms such as the
ones in USF1 in a sample of about 200 subjects as our study
group. In addition, we do not need to make statistical adjustments for multiple comparisons with other genes, because USF1 is the first gene examined in this cohort. At
present, we do not know how strong the relationship between the usf1s1/2 polymorphisms and in vivo lipolysis is. We
are not aware of any large study sample that can be used for
genetic analysis of lipolysis in vivo. However, a recent direct
comparison of catecholamine-induced lipolysis in vitro (isolated fat cells) and in vivo (microdialysis) revealed a relatively
close correlation between these two lipolysis measures in sc
adipose tissue (13), which is the adipose depot examined in
the present study.
The mechanism for increased lipolysis in T/A carriers is
not known. However, the finding of increased maximum
␤1,2,3-AR-induced as well as increased maximum forskolinstimulated lipolysis (reflecting activation of adenylyl cyclase,
increasing cAMP) of T/A allele carriers suggests an effect of
the usf1s2 gene variation on postreceptor-related events and
not on agonist sensitivity (reflects receptor-related events).
We therefore measured the mRNA levels of several genes
implicated in regulating postreceptor-mediated adipocyte
lipolysis. A small (17%), but significant, effect of the usf1s2
genotype on mRNA levels of protein kinase A regulatory
subunit type 1␣ (PRKAR1A) was found, which indicates a
transcriptional role for USF-1 in PRKAR1A gene expression.
Interestingly, data from gene knockout studies in mice (14)
as well as data from human subjects (15) have demonstrated
that even small increases in PRKAR1〈 expression result in
a more pronounced activation of protein kinase and, thus, of
lipolysis. The molecular mechanisms underlying this effect
are probably due to the higher affinity of PRKAR1〈 for
cAMP compared with PRKAR2〉. The stoichiometry between PRKAR1〈 and PRKAR2〉 is therefore of importance
in fine-tuning lipolysis, suggesting that the slightly elevated
PRKAR1〈 expression demonstrated in this study could account for the differences in lipolytic activity.
Although USF-1 has been shown to induce transcription of
the human HSL promoter in 3T3-F442A cells (8), we failed to
demonstrate an effect of the usf1s2 gene variation on human
adipose tissue mRNA levels of the two lipases studied, i.e.
HSL and the recently discovered (16) ATGL. However, previous studies in humans have shown that catecholamineinduced lipolysis is directly proportional to the protein content and enzyme activity of HSL in sc fat cells (17). Thus, it
may be hypothesized that USF-1 has an indirect effect on
J Clin Endocrinol Metab, September 2005, 90(9):5356 –5360
5359
adipocyte lipase activity by inducing a step more proximal
in the lipolytic cascade, i.e. the cAMP-dependent, protein
kinase A-mediated phosphorylation of HSL/ATGL. Indirect
support for such an effect of the USF-1 polymorphism lies in
published results for FCHL. This condition is linked to the
investigated USF-1 polymorphism (3), and catecholamineinduced adipocyte lipolysis is decreased in FCHL due to
impaired HSL function (10).
It is of interest to compare the lipolytic effect of USF1
polymorphism with that of genetic variance in HSL (18).
Intronic variation in HSL was associated with a 50% decrease
in maximum lipolytic activation, a greater effect than that
presently observed for USF1, which is more than a 20%
increase. This might be due to the fact that events at the final
rate-limiting step of the lipolytic cascade, HSL, have more
pronounced effects on lipolysis than events at earlier steps,
such as protein kinase A.
Our data showed that the usf1s1/s2 SNPs do not associate
with variations in the clinical phenotypes, such as body mass
index, the plasma lipid profile, or circulating levels of free
fatty acids or glycerol. This is in contrast with two previous
studies showing association of USF1 SNPs with triglyceride
levels in families with FCHL (3) and with plasma lipid levels
and peak glucose during an oral glucose tolerance test in
male offspring of patients with premature myocardial infarction (16). The reason for this is probably that the present
study included healthy subjects only. Although obese, they
all had plasma levels of both lipids and glucose within the
normal range and showed no sign of cardiovascular or metabolic disease. It is quite possible that the adipocyte lipolysisrelated effects of genetic variance in USF1 lead to clinical
consequences when a pathophysiological state is present,
such as diabetes or FCHL.
The usf1s1 and usf1s2 SNPs both are localized within the
intron of the usf1 gene, which indicates that they may not be
functional themselves. Furthermore, these two SNPs were
found to be in tight linkage disequilibrium with the two SNPs
recently studied by Putt et al. (19), 475C⬎T and 1748C⬎T,
indicating that all these polymorphisms may be markers of
a hitherto unrecognized functional domain. However, by in
silico analysis, Pajukanta et al. (3) identified a putative internal promoter in intron 7 of the usf1 gene, in the vicinity of the
usf1s2 SNP, suggesting that genetic variation at the usf1s2
locus is of functional significance. The usf1s2 locus may thus
influence this promoter to initiate translation from the internal AUGs in exon 8 of the usf1 gene, thereby repressing the
normal function of the protein or vice versa, as previously
discussed (20).
The success rate for genotyping for usf1s1 was 94%, which
is not uncommon for crude design using the DASH technique (11). However, the success rate for usf1s2 was 100%.
Because the two SNPs were in complete linkage disequilibrium, we saw no reason to attempt successful genotyping of
the 12 failures using costly and elaborate improvements of
DASH (11).
In summary, a relatively common polymorphism in the
usf1 gene is associated with an increased ability of catecholamines to stimulate lipolysis in fat cells, which is most
likely explained by an increased postreceptor function, pos-
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J Clin Endocrinol Metab, September 2005, 90(9):5356 –5360
sibly at the level of protein kinase A, involving regulatory
subunit type 1␣.
Acknowledgments
We are grateful for the excellent technical assistance of Britt-Marie
Leijonhufvud, Katarina Sjöberg, Kerstin Wåhlén, Elisabeth Dungner,
and Eva Sjölin.
Received February 24, 2005. Accepted June 20, 2005.
Address all correspondence and requests for reprints to: Dr. Johan
Hoffstedt, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden. E-mail: [email protected].
This work was supported by grants from the Swedish Research Council, the Swedish Heart and Lung Foundation, the Swedish Diabetes
Association, the Novo Nordic Foundation, the Bergvall Foundation, the
Thuring Foundation, the Wiberg Foundation, and the Swedish Medical
Society.
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