Clinical Science (2005) 108, 323–329 (Printed in Great Britain)
Change in β 1-adrenergic receptor protein
concentration in adipose tissue correlates
with diet-induced weight loss
Mads RASMUSSEN∗ , Anita BELZA†, Thomas ALMDAL∗ , Søren TOUBRO†,
Palle BRATHOLM∗ , Arne ASTRUP† and Niels J. CHRISTENSEN∗
∗
Department of Endocrinology, Herlev Hospital, University of Copenhagen, 2730 Herlev, Denmark, and †Department of
Human Nutrition, The Royal Veterinary and Agricultural University, 2000 Frederiksberg, Denmark
A
B
S
T
R
A
C
T
The aim of the present study was to examine gene expression and protein concentrations of
β 1 - and β 2 -adrenergic receptors in subcutaneous adipose tissue in obese subjects in response to
weight loss. Eighteen obese subjects were studied during diet-induced weight loss. β-Adrenergic
receptor mRNA levels were quantified by reverse transcription-PCR–HPLC. β-Adrenergic receptor protein concentrations were measured by Western blotting using fluorescence laser
scanning for detection. Subjects lost 12.8 +
− 0.8 kg (mean +
− S.E.M.) during diet treatment. There
was a 34 % decrease in the β 1 -adrenergic receptor mRNA level (0.92 +
− 0.09 compared with
0.06
amol/µg
of
DNA;
P
<
0.002).
β
0.61 +
-Adrenergic
receptor
mRNA
did
not decrease signifi2
−
cantly. β 2 -Adrenergic receptor protein concentration decreased 37 % (25.5 +
− 7.1 compared with
-adrenergic
receptor
protein con16.0 +
5.6
arbitrary
units/ng
of
DNA;
P
=
0.008),
whereas
β
1
−
centration did not decrease significantly. The degree of weight loss was correlated with the
concentration of β 1 -adrenergic receptor protein (r = 0.65, P < 0.003) and changes in receptor
protein concentration (r = 0.50, P = 0.035) during the very-low-calorie diet. In conclusion, the
present study demonstrates a relationship between β 1 -adrenergic receptor protein concentration
in adipose tissue and the degree of weight loss. This relationship is not directly related to energy
expenditure and deserves further investigation.
INTRODUCTION
The role of catecholamines in obesity and weight loss is a
controversial subject. Catecholamines are powerful regulators of lipolysis and act through the α 2 -, β 1 - and β 2 adrenergic receptors [1,2]. A decreased sensitivity of fat
cells to adrenergic stimulation and subsequent lipolysis
has been demonstrated repeatedly in obese subjects (for a
review, see van Baak [3]). However, the molecular mechanism has not been fully elucidated yet. One in vitro
study [4], performed on isolated subcutaneous fat cells,
indicated that the lipolytic response induced by isoprenaline (a mixed β 1 - and β 2 -adrenergic agonist) was dimi-
nished after fasting. In contrast, two in vivo studies have
demonstrated an increased responsiveness to catecholamines during fasting [5,6]. Alterations in β-adrenergic
signalling can occur directly at the receptor level (through
altered gene expression or receptor protein concentration) or at the post-receptor level. In a previous study
performed in our laboratory [7], we found that the
increase in NEFA (non-esterified fatty acids) during 60 h
of fasting (an indirect indicator of lipolysis) was diminished in obese subjects compared with lean subjects. The
decreased lipolytic response was accompanied by a higher
protein concentration of β 2 -adrenergic receptors in obese
subjects compared with lean subjects, both before and
Key words: adipose tissue, catecholamine, reverse transcription–PCR–HPLC, weight loss, Western blot.
Abbreviations: BMI, body mass index; HOMA, homoeostasis model assessment; RT, reverse transcription.
Correspondence: Dr Mads Rasmussen (email [email protected]).
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M. Rasmussen and others
Table 1 Subject characteristics before and after a very-lowcalorie diet for 8 weeks
Values are means +
− S.E.M.
Body weight (kg)
BMI (kg/m2 )
Waist circumference (cm)
Body fat (%)
Fat-free mass (kg)
Heart rate (beats/min)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Resting energy expenditure
(kcal/24 h)
Fasting plasma glucose (mmol/l)
Fasting plasma insulin (pmol/l)
HOMA
Fasting triacylglycerols (mmol/l)
Before diet
After diet
P value
99.6 +
− 2.7
32.4 +
− 0.4
108.3 +
− 2.4
35.3 +
− 2.1
65.0 +
− 3.1
65.5 +
− 2.2
126.4 +
− 2.5
77.7 +
− 1.7
1873 +
− 66
86.8 +
− 2.5
28.2 +
− 0.5
98.7 +
− 2.1
30.0 +
− 2.4
61.0 +
− 2.9
55.6 +
− 2.0
121.3 +
− 3.4
74.8 +
− 2.0
1655 +
− 57
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.16
0.11
< 0.001
5.3 +
− 0.1
49.3 +
− 5.1
1.93 +
− 0.2
1.80 +
− 0.18
4.9 +
− 0.1
18.5 +
− 2.2
0.70 +
− 0.09
1.15 +
− 0.05
< 0.001
< 0.001
< 0.001
0.002
after fasting, but a comparable decrease in β 2 -adrenergic
receptor protein concentration was observed in response
to the fasting period. During long-term weight reduction,
pre-existing hypertrophy of fat cells is reversed to some
degree. An in vitro study [8] has shown an increased β 2 adrenergic lipolytic response following weight reduction,
and several in vivo studies [9,10] have shown an increased
lipolytic response to β-adrenergic stimulus following
weight reduction. The molecular mechanism underlying
increased lipolytic response following weight reduction
is unknown, but is likely to involve β-adrenergic receptor
level and/or function. The aim of the present study was
to test the hypothesis that the degree of weight loss that
can be obtained during a very-low-calorie diet is related
to the level of β-adrenergic receptor gene expression and
protein concentration.
METHODS
Subjects
Twenty volunteers (ten female) with a BMI (body mass
index) over 30 kg/m2 were initially invited to participate
in the study, but two dropped out (one female) leaving
18 subjects to complete the study. Participant age was
40.3 +
− 2.1 years (mean +
− S.E.M.), and the physical characteristics of the subjects are summarized in Table 1. All
subjects were in good health as assessed by medical history and physical examination and were weight stable for
at least 6 months prior to the study. None of the study
participants had diabetes or were taking any medication.
All subjects gave informed consent before participating,
and the study protocol was reviewed and approved by the
Ethics Committee of Copenhagen and Frederiksberg, and
was in compliance with the Declaration of Helsinki II.
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2005 The Biochemical Society
Experimental design
Subjects were studied before and after diet-induced
weight loss. The study was performed as a substudy of a
longer weight maintenance programme and, therefore,
the examination after weight loss was performed while the
subjects were still in negative energy balance. The participants achieved weight loss on an 8-week very-low-energy
formula diet [slimming powder giving 800 kcal/day
(where 1 kcal ≡ 4.184 kJ); Speasy® ; Dansk Droge, Ishøj,
Denmark]. A dietician individually supervised subjects
every other week. On each study day, the subjects met
after an overnight fast and underwent blood sampling
and dual X-ray absorptiometry (DPX-IQ; Lunar Corp,
Madison, WI, U.S.A.) for determination of fat percentage,
fat mass and lean body mass. Resting energy expenditure
was measured by indirect calorimetry with an openair circuit, computerized, ventilated hood system before
and after weight loss. A percutaneous abdominal subcutaneous fat sample was obtained under local anaesthesia
(1 % lidocaine without adrenaline) using a 16-gauge liver
biopsy cannula (Hepafix Luer Lock; Braun, Melsungen,
Germany). Approx. 500 mg of adipose tissue was obtained on each occasion. Samples were rinsed in sterile
saline and frozen in liquid nitrogen within 2 min after
removal and stored at − 80 ◦ C until assayed.
Analytical methods
Triacylglycerols (triglycerides) and glucose were determined by in-house colorimetric methods (Boehringer
Mannheim, Mannheim, Germany), and insulin was measured by a two-site sandwich ELISA method (AutoDELFIA Insulin Kit; Wallac, Turku, Finland).
Western blotting
Membrane protein was extracted from adipose tissue
biopsies (adapted from [11]). Approx. 30 mg of adipose
tissue were homogenized by hand and centrifuged at
10 000 g for 15 min at 4 ◦ C. The fatcake (of adipose tissue)
was discarded and the pellet was resuspended and washed.
The extract was centrifuged at 10 000 g for 15 min at 4 ◦ C
and the pellet was extracted. The extract was stored at
− 20 ◦ C until further analysis. Protein concentration in
the extract was determined using the BCA Protein Assay
Kit (Pierce, Rockford, MD, U.S.A.).
PAGE was performed as described by Laemmli [12].
A portion (5 µg) of adipose tissue membrane protein was
applied per lane. As a positive control for verification of
blotting, incubation, detection and quantification, 10 µg
of mononuclear cell membrane protein (corresponding to
1.7 × 106 mononuclear cells) was applied. Tank blotting
of separated proteins to nitrocellulose was performed
according to standard protocols, with transfer for 3 h at
70 V and 15 ◦ C.
The blots were incubated with 0.5 µg/ml rabbit anti(human β 1 -adrenergic receptor) IgG or 1 µg/ml rabbit
anti-(human β 2 -adrenergic receptor) IgG (Santa Cruz
β-Adrenergic receptors and weight loss
Biotechnology, Santa Cruz, CA, U.S.A.) and were reincubated with an alkaline phosphatase-labelled goat
anti-rabbit antibody. Detection was performed using
DDAO [9H-(1,3-dichloro-9,9-dimethylacridin-2-one7-yl)] phosphate (Pro-Q Western Blot Stain Kit; Molecular Probes, Leiden, The Netherlands) on a Fujifilm
FLA-3000.
Quantification of β-adrenergic receptor protein was
performed by determining the fluorescence intensity of
β-adrenergic-receptor-antibody-immunoreactive bands
as described previously [7]. The concentration of β-adrenergic receptor protein is expressed as the intensity of
the immunoreactive bands of the tissue samples in relation to the intensity of the immunoreactive bands of the
mononuclear cell membrane protein internal standard.
Western blots of β 1 -adrenergic receptor revealed only
one band, whereas blots of the β 2 -adrenergic receptor
revealed several immunoreactive bands. Quantification
of the β 2 -adrenergic receptor is based on the total band
area, as described previously [7]. β-Adrenergic receptor
protein concentration is expressed in relation to cell size
(per ng of DNA) or as the relative amount of the receptor
in the membrane (per mg of membrane protein). It is
indicated in the text where results expressed differently
gave discrepancies. The coefficient of variation based on
13 determinations in duplicate was 28 %.
Determination of receptor mRNA
The technique for determining specific mRNA by RT
(reverse transcription)–PCR–HPLC has been described
in detail previously [13,14]. Briefly, RNA was isolated
from approx. 30–40 mg of adipose tissue by the QIAamp
kit (Qiagen, Hilden, Germany). Total RNA concentration was measured with an Eppendorf Biophotometer,
and DNA concentration was measured by H33258 fluorescence (DyNAQuant 200 apparatus; Hoefer Pharmacia Biotech, Freiburg, Germany). Oligonucleotide primers were synthesized at DNA Technology (Aarhus,
Denmark). β 1 - and β 2 -Adrenergic receptor primer sequences have been published previously [7].
Internal standard DNAs for β 1 - and β 2 -adrenergic
receptor mRNA were constructed using the sets of primers reported previously [7] and the PCR-MIMICTM
construction kit (BD Biosciences Clontech, Palo Alto,
CA, U.S.A.). An internal standard RNA for the β 1 - and
β 2 -adrenergic receptor mRNA was constructed as described previously [13,14]. The resulting internal standard
RNA was quantified by UV detection (Gene-Quant II,
Pharmacia Biotech, Uppsala, Sweden). The RT–PCR products were indistinguishable from the internal standard
DNA.
For the PCR reactions, cDNA was combined with
each primer and amplified, as reported previously [7]. The
resulting PCR products were quantified by HPLC and
UV detection at 254 nm. The PCR product was quantified
relative to the internal standard using areas and corrected
for the different sizes of the two products. mRNA content
is expressed in relation to the total number of cells in the
biopsy, i.e. as amol/µg DNA, since the DNA concentration can be measured very precisely and it is well
established that the cell content of DNA is constant in
most tissues (in contrast with RNA).
Statistics
The statistical analysis was performed by the SigmaStat
program version 1.02 (SPSS, Chicago, IL, U.S.A.). Paired
Student’s t test was performed on data measured before
and after weight loss, whereas Student’s t test was used
to compare sex differences. Pearson correlation (r) or
the Spearman rank order test (Rs ) were used and leastsquares linear regression (R2 ), where applicable. A P value
< 0.05 was considered significant. Results are presented
as means +
− S.E.M.
RESULTS
Anthropometric measurements,
hormones and metabolites
Table 1 shows the characteristics of the participants before
and after weight loss induced by 8 weeks of a very-lowcalorie diet. As expected, subjects had a marked weight
loss (12.8 +
− 0.8 kg) during the diet. There was no statistical difference in the weight loss in men and women
(13.7 +
− 1.4 compared with 11.9 +
− 0.7 kg respectively).
The mean decrease in fat mass and fat-free mass was
9.3 +
− 0.6 and 4.0 +
− 0.5 kg respectively.
Values for insulin, glucose and triacylglycerols are also
shown in Table 1. Eight of the subjects had some degree of
insulin resistance prior to the weight loss [as evaluated by
HOMA (homoeostasis model assessment): fasting insulin
(pmol/l) × fasting glucose (mmol/l)/(22.5 × 6) exceeding
the 75th percentile of a normal population, i.e. 2.0].
After weight loss, none of the test subjects had a
HOMA level more than 2. There was a close correlation
between percentage weight change and the decrease in
plasma insulin (r = 0.65, P = 0.003) or HOMA (r = 0.61,
P = 0.008).
β-Adrenergic receptor mRNA
Gene expression of the β 1 -adrenergic receptor decreased
34 % in response to diet treatment (0.92 +
− 0.09 compared
with 0.61 +
− 0.06 amol/µg DNA; P = 0.002; Figure 1).
Before weight loss, fat-free mass (kg) and β 1 -adrenergic
receptor mRNA were correlated negatively (Rs = − 0.80,
P < 0.001). Corresponding to the higher fat-free mass,
men had significantly lower values of β 1 -adrenergic receptor mRNA than women before and after diet treatment (P < 0.001 and P = 0.04 respectively).
The β 2 -adrenergic receptor mRNA level did not decrease significantly in response to weight loss (0.36 +
−
0.03 compared with 0.31 +
− 0.02 amol/µg DNA; P = 0.18).
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Figure 1 Gene expression of the β 1 - and β 2 -adrenergic
receptors in subcutanous adipose tissue before and after
8 weeks of a very-low-calorie diet
Figure 3 Protein concentration of the β 1 - and β 2 -adrenergic receptors in adipose tissue before and after 8 weeks
of a very-low-calorie diet
∗
Values are means +
− S.E.M. P < 0.05 compared with before weight loss.
The β-adrenergic receptor protein concentration was determined by laser scanning the intensity of DDAO-immunoreactive band or bands in relation to the intensity of a mononuclear cell membrane standard. Values are means +
− S.E.M.
∗
P < 0.05 compared with before weight loss.
Figure 2 Relationship between BMI and gene expression of
the β 2 -adrenergic receptor after 8 weeks of a very-lowcalorie diet
R s = 0.70, P < 0.002.
After the diet treatment, β 2 -adrenergic receptor mRNA
levels and BMI were negatively correlated (Rs = − 0.70,
P < 0.002; Figure 2). The correlation after weight loss was
similar in both sexes, although the regression coefficient
was slightly different (r = − 0.79, P = 0.01 for men; and
r = − 0.74, P = 0.02 for women). There was no significant
relationship between β 2 -adrenergic receptor mRNA
levels and BMI before weight loss (P = 0.22).
The mean DNA concentration in adipose tissue was
increased significantly after 8 weeks of treatment from
37.6 +
− 1.2 to 50.3 +
− 2.6 ng of DNA/mg of adipose tissue
(P < 0.001), reflecting the decrease in fat cell size. We
found a strong correlation between increments in DNA/
mg of adipose tissue and the corresponding decrease in
the waist/hip ratio (Rs = 0.79, P < 0.005), but not with
other anthropometric measures such as weight loss or
change in BMI.
β-Adrenergic receptor protein
Whether expressed as relative protein concentration
(receptor protein relative to total membrane protein
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concentration) or as receptor protein concentration/cell
(estimated from the DNA content in the sample), the β 1 adrenergic receptor protein concentration did not change
significantly after weight loss [0.63 +
− 0.14 compared
with 0.64 +
0.13
arbitrary
units/mg
of
membrane
protein
−
+
(P = 0.92) and 10.5 +
3.3
to
7.1
1.5
arbitrary
units/ng
of
−
−
DNA (P = 0.2) respectively; Figure 3]. When expressed
in relation to the DNA content of the sample, the β 2 adrenergic receptor protein concentration decreased by
37 % [from 25.5 +
− 7.1 to 16.0 +
− 5.6 arbitrary units/ng
of DNA (P = 0.008); Figure 3], but when expressed as
relative protein concentration in the membrane there
was only a trend toward a decrease [from 1.01 (0.70–
1.74) to 0.85 (0.39–1.27) arbitrary units/mg of membrane
protein (P = 0.06); values are means (range)]. There was
no correlation between β 2 -adrenergic receptor protein
concentration and BMI as was found between β 2 -adrenergic receptor mRNA and BMI. We did not find a
direct relationship between β 2 -adrenergic receptor gene
expression and protein level; however, multiple regression
analysis indicated an inverse relationship between gene
expression and protein concentration of the β 1 -adrenergic receptor. This relationship involved several other factors (including insulin levels), indicating a more complex
regulation of receptor protein concentration levels than
directly via gene expression.
Weight loss and β-adrenergic
receptor protein
β 1 -Adrenergic receptor protein concentration (expressed/cell or relative to membrane protein) before diet
treatment did not predict weight loss (P = not significant).
After diet treatment, however, there was a close correlation between β 1 -adrenergic receptor protein concentration and weight loss (per ng of DNA: r = 0.65, P =
0.003; per mg of membrane protein: r = 0.55, P = 0.02).
Furthermore, we found a relationship between the change
β-Adrenergic receptors and weight loss
Figure 4 Relationship between relative protein concentration of the β 1 -adrenergic receptor (receptor protein in
arbitrary units/mg of membrane protein) and total weight
loss during 8 weeks of a very-low-calorie diet
r = 0.50, P = 0.035.
in relative β 1 -adrenergic receptor protein concentration
and weight loss (r = 0.50, P = 0.035; Figure 4), i.e. the
greatest weight loss was achieved in individuals who
maintained the highest levels of β 1 -adrenergic receptor
protein during diet treatment. There was no correlation between β 2 -adrenergic receptor protein concentration and weight loss.
DISCUSSION
The major findings of the present study are the close relationships between gene expression of the β 2 -adrenergic
receptor and BMI after weight loss and the correlation between β 1 -adrenergic receptor protein, whether
expressed in relation to cell size or relative to total membrane protein, and very-low-calorie diet-induced weight
loss.
We found that gene expression of β 1 -adrenergic receptors, but not β 2 -adrenergic receptors, decreased after
weight loss. The decrease in β 1 -adrenergic receptor
mRNA may be part of a regulatory response involved in
limiting the reduction of energy stores observed in relation to weight loss. We also found that gene expression of the β 2 -adrenergic receptor was closely and negatively related to the BMI after weight loss. This may
suggest that larger adipose tissue cells (biopsies with low
DNA concentration) had the greatest adaptation (lowest
mRNA levels) in response to diet intervention limiting
the effects of energy restriction. Deficient gene expression, or overcompensation during energy restriction, of a
receptor closely linked to lipolysis could be a pathogenic
factor leading to obesity or a contributing factor in the
difficulty for obese individuals to lose weight.
β 2 -Adrenergic receptor protein concentration decreased after weight loss. In contrast, β 1 -adrenergic receptor protein concentration remained unchanged after
weight loss. This may suggest a differential regulation of
β 1 - and β 2 -adrenergic receptor protein during very-lowcalorie diet treatment. We cannot exclude the possibility
that there was a small decrease in β 1 -adrenergic receptor
protein after diet-induced weight loss which was not
significant due to the relatively limited number of subjects examined. In the present study, we found close correlations between the magnitude of the weight loss and
both β 1 -adrenergic receptor protein concentration
and the change in β 1 -adrenergic receptor level. One
explanation for this correlation is that high levels of
β 1 -adrenergic receptors mediate lipolysis and, hence,
increased weight loss. An alternative is that increased
levels of β 1 -adrenergic receptors induce thermogenesis
and an increase in energy expenditure and, hence, weight
loss. If this is true, our results may suggest a deficiency
in β 1 -adrenergic receptor-mediated energy expenditure in
obesity. The most compelling evidence for a link between
deficits in energy expenditure and obesity comes from
monogenic animal models. When food intake of ob/ob,
db/db and melanocortin-4 receptor gene knockout mice
is restricted to that of wild-type mice, marked obesity
still develops, due to decreased energy expenditure and
not decreased lipolysis. The molecular mechanism behind
this phenomenon most probably involves reduced β-adrenergic signalling [15,16]. Unfortunately, our present
results cannot corroborate this hypothesis, as we did not
find a correlation between β 1 -adrenergic receptor protein
concentration and energy expenditure.
It is surprising that β 1 - and β 2 -adrenergic receptor proteins responded differently to weight loss. This may be
related to changes in insulin secretion or insulin sensitivity, i.e. insulin having a more pronounced inhibitory
effect on β 2 -adrenergic receptor-mediated energy response compared with the β 1 -adrenergic receptor through
differential phosphorylation or sequestration [17,18].
Furthermore, we cannot exclude the possibility that the
two adrenergic receptors to some extent are located on
different fat cells. These suggestions are, however, rather
speculative and deserve further investigation.
In contrast with the findings in our present study, a
previous study [8] using isolated fat cells from women
found unchanged amounts of β 1 - and β 2 -adrenergic
receptors after weight loss, as evaluated by radioligand
binding. That study used receptor density expressed in
relation to cell membrane area and cannot be directly
compared with our present study. Furthermore, it only
included women, and gender differences in adrenergic
receptor function have been described [19].
The present study was carried out as part of an ongoing
weight-loss trial, and therefore we could not examine
participants in steady state after weight loss, as they were
still in negative energy balance. It is therefore uncertain
whether the described changes in receptor gene expression and protein concentrations are due to acute energy
restriction, a fasting response, or to the long-term
weight loss and, hence, reduction in fat cell size. In our
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previous study [7], 12 or 60 h of fasting did not alter
β-adrenergic receptor gene expression in the same way,
and it is therefore unlikely that the mechanisms behind
β-adrenergic receptor protein reduction during shortterm energy restriction and the present weight loss study
are the same. The present changes are more likely to be
related to the reduction in cell size and/or membrane
remodelling.
The present study was limited by the fact that we only
obtained samples from the abdominal subcutaneous adipose tissue. Regional differences in β-adrenergic receptor concentrations and lipolytic responses have been observed [20,21]. Catecholamine-induced lipolysis rates in
subcutaneous and visceral fat cells are interrelated though
[22]. The present study has been conducted on whole
adipose tissue and not isolated adipocytes. Our goal was
to preserve levels of mRNA and membrane receptors
as intact as possible and we therefore chose to freeze tissue
samples as quickly after extraction as possible. Recent
evidence has shown that standard isolation of primary
adipocytes from adipose tissue alters the expression of
genes important for insulin action and triacylglycerol
synthesis [23].
We applied the DNA content of the tissue specimen
(DNA/mg of tissue) as an index of cell number and,
therefore, indirectly of the cell size. The tissue was frozen
immediately after it was collected and therefore we
could not apply more conventional parameters (direct
measurement of cell size from isolated adipocytes). It is
clear, however, that our index reflects the number of all
cells in the tissue specimen and not only the adipocytes.
The possible influence of β 3 -adrenergic receptors
on weight loss has not been included in our present
study. Investigators have found functional β 3 -adrenergic
receptors in human adipose tissue [24], and stimulation
with specific β 3 -adrenergic receptor agonists can stimulate lipolysis [25], but the relative importance for
lipolysis is probably very small under physiological conditions [26].
In conclusion, the main finding of the present study
is the observed relationship between the concentration
of the β 1 -adrenergic receptor protein and changes in the
receptor protein concentration during a very-low-calorie
diet and the degree of weight loss. This relationship is
not directly related to energy expenditure and deserves
further investigation.
ACKNOWLEDGMENTS
We thank Karen Andersen, Gurli Habekost, Ulla
Kjærulff Hansen, Tonni Løve Hansen and Debbie
Nadelmann for excellent technical assistance. This study
was supported by grants from Niels and Desirée Ydes
Foundation, Toyota Fonden, Denmark, Jacob Madsens
and Wife Olga Madsens Foundation and the NOVO
Nordisk Foundation.
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2005 The Biochemical Society
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Received 10 August 2004/4 November 2004; accepted 1 December 2004
Published as Immediate Publication 1 December 2004, DOI 10.1042/CS20040238
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