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Original Studies |
2B-Adrenergic Receptor That Is Associated with Reduced Basal Metabolic Rate in Obese Subjects
Department of Pharmacology and Clinical Pharmacology, University of Turku (P.H., M.K., U.P., M.K.K., M.S.), FIN-20520 Turku; the Eating Disorder Unit, University Hospital of Helsinki (A.R.), FIN-00250 Helsinki; and the Departments of Medicine (M.L.) and Clinical Nutrition (R.V., M.U.), University of Kuopio, FIN-70211 Kuopio, Finland
Address all correspondence and requests for reprints to: Dr. Markku Koulu, Department of Pharmacology and Clinical Pharmacology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: markku.koulu{at}utu.fi
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Abstract |
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2-adrenergic receptors mediate part of the actions
of the catecholamines noradrenaline and adrenaline on the regulation of
energy balance. As part of an ongoing study on the genetics of obesity,
the entire coding sequence of the 2B-adrenoceptor gene
was screened in 58 obese, nondiabetic Finns by PCR-single stranded
conformational analysis (PCR-SSCA). A polymorphism that leads to a
deletion of 3 glutamic acids from a glutamic acid repeat element
(Glu x 12, amino acids 297–309) present in the third
intracellular loop of the receptor protein was identified. This repeat
element has previously been shown to be important for agonist-dependent
receptor desensitization. Of 166 genotyped subjects, 47 (28%) had 2
normal (long) alleles (Glu12/Glu12), 90 (54%)
were heterozygous (Glu12/Glu9), and 29 (17%)
were homozygous for the short (Glu9/Glu9) form.
The basal metabolic rate, determined by indirect calorimetry and
adjusted for fat-free body mass, fat mass, sex, and age, was 94 Cal/day
(5.6%) lower (95% confidence interval for difference, 32, 156)
in subjects homozygous for the short allele than in subjects with two
long alleles (F = 4.84; P = 0.009, by ANOVA).
Thus, a genetic polymorphism of the 2B-adrenoceptor
subtype can partly explain the variation in basal metabolic rate in an
obese population and may therefore contribute to the pathogenesis of
obesity. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A low basal metabolic rate (BMR) has been shown to be a risk factor for obesity (6), but the genetic regulation of BMR is largely unknown. The activity of the autonomic nervous system, particularly that of the sympathetic nervous system, has been shown to contribute to the control of BMR (7, 8). A ß3-adrenoceptor gene polymorphism, a missense mutation in codon 64 (Trp64Arg), has been associated with lowered BMR (9, 10, 11, 12) and appears to constitute a susceptibility factor to explain obesity and noninsulin-dependent diabetes mellitus (13). Quite recently it was reported that Glu instead of Gln in both alleles at codon 27 of the ß2-adrenoceptor gene was associated with a 7-fold relative risk for obesity in female Swedish subjects (14).
There are three known 2-adrenoceptor subtypes,
2A, 2B, and 2C, which are
encoded by distinct genes located on different chromosomes and have
different tissue and cellular distributions, but have relatively
similar, although not identical, pharmacological properties. Currently
available synthetic drug molecules are incapable of activating or
antagonizing the receptors in a subtype-selective manner. The most
obvious functionally important differences between the
2-adrenoceptor subtypes are based on their different
cellular and tissue distributions (15, 16, 17, 18). The full significance of
these differences remains to be established, as the expression patterns
of the receptor genes are still incompletely known, especially in
humans.
Relatively little is known about the significance of
2-adrenoceptors in obesity.
2-Adrenoceptors mediate inhibition of sympathetic
activity and are also known to influence energy metabolism through
inhibition of insulin secretion and lipolysis (19). Previously, a
polymorphism in the noncoding region of the
2A-adrenoceptor gene has been reported to be associated
with salt-sensitive hypertension in an African-American population
(20). Activation of 2B-adrenoceptors present in vascular
smooth muscle cells causes vasoconstriction (21), but no studies are
available to connect genetic variants of the
2B-adrenoceptor to the development of hypertension,
obesity, or other specific pathologies.
We investigated the entire coding region of the
2B-adrenoceptor gene in a patient group used for genetic
studies on obesity and report a deletion of three glutamic acid
residues in the third intracellular loop of the
2B-adrenoceptor that is associated with reduced mean BMR
and reduced mean heart rate in our study population of 166 obese
Finnish subjects.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All subjects participating in this study were Finnish. The
Finnish population is genetically quite homogeneous, descending mainly
from a small number of founders of Baltic Finnish and German origin
(22). The study population used for association analysis consisted of
166 obese (BMI, >27) subjects (137 women and 29 men) participating in
a weight reduction program, recruited from primary health care in
Kuopio and Helsinki (23). DNA samples from 58 of them were randomly
selected to screen the entire coding region of the
2B-adrenoceptor gene for sequence variants. All subjects
had normal liver, kidney, and thyroid function, and none had excessive
alcohol intake. None of the subjects was taking drugs known to affect
BMR or glucose metabolism, and none had diabetes, as evaluated by
fasting serum glucose or an oral glucose tolerance test (n = 105).
The mean age of the study subjects was 43 ± 0.6 yr (range, 24–61
yr), and their mean BMI was 34.8 ± 0.3 kg/m2 (range,
28.6–43.8 kg/m2). All physical and biochemical
measurements were determined in the morning after a 12-h fast, using
standardized methods that have been described previously in detail. The
BMR was determined by indirect calorimetry (Dealtatrac, TM Datex,
Helsinki, Finland) and adjusted for fat-free mass, body fat mass, sex,
and age as described previously (24). In brief, gas exchange was
measured for 30 min, of which the first 10 min were discarded, and the
mean value of the last 20 min was used in calculations. The energy
production rate (calories per min) was calculated according to
Ferrannini as follows: BMR (Cal/min) = 3.91 x
VO2(mL) + 1.10 x VCO2 (mL) -
3.34 x N (mL/min) and expressed as kilocalories per day. For each
subject, the adjusted BMR was calculated as follows: (the group mean
BMR) + (measured BMR - the predicted BMR), where the group mean
BMR is the mean absolute metabolic rate calculated according to
Ferrannini (kilocalories per day), the measured BMR is the rate
(kilocalories per day) measured in each subject, and the predicted BMR
is the calculated rate (kilocalories per day obtained using the
individual lean body mass, age, and sex in the linear regression
equation generated from the initial examinations of 170 subjects
(24).
The genotype frequency of the deletion mutation of 3 glutamates was
compared with that in 54 healthy normoglycemic subjects with normal
body weight (BMI, 
27), who were used as population controls in a
study investigating the relationship between insulin resistance and
dyslipidemia (25).
The protocols followed the principles of the Helsinki Declaration and were approved by the ethics committees of the Universities of Kuopio and Helsinki. All subjects gave their informed consents.
PCR-SSCA analysis
The genomic DNA encoding the 2B-adrenergic
receptor was amplified in two parts specific for the intronless
2B-adrenoceptor gene sequence (26). The PCR primer
pairs for PCR amplification were as follows: pair 1,
5'-GGGGCGACGCTCTTGTCTA-3' and 5'-GGTC-TCCCCCTCCTCCTTC-3' (product
size, 878 bp); pair 2, 5'-GCAGCAACCGCAGAGGTC-3' and
5'-GGGCAAGAAGCAGGGTGAC-3' (product size, 814 bp). The primers were
delivered by KeboLab (Helsinki, Finland). PCR amplification was
conducted in a 5-µL volume containing 100 ng genomic DNA (isolated
from whole blood), 2.5 mmol/L of each primer, 1.0 mmol/L deoxy-NTPs, 30
nmol/L [33P]deoxy-CTP, and 0.25 U AmpliTaq DNA polymerase
(Perkin Elmer/Cetus, Norwalk, CT). PCR conditions were
optimized using the PCR Optimizer kit (Invitrogen, San
Diego, CA). Samples were amplified with a GeneAmp PCR System 9600
(Perkin Elmer/Cetus). PCR products were digested with
restriction enzymes for SSCA analysis. The product of primer pair 1 was
digested with DdeI and DraIII (Promega Corp., Madison, WI). The product of primer pair 2 was digested
with AluI and HincII (Promega Corp.). The digested samples were mixed with SSCA buffer
containing 95% formamide, 10 mmol/L NaOH, 0.05% xylene cyanol, and
0.05% bromophenol blue (total volume, 25 µL). Before loading, the
samples were denatured for 5 min at 95 C and kept 5 min on ice. Three
microliters of each sample were loaded on MDE high resolution gel (FMC
BioProducts, Rockland, ME). The gel electrophoresis was performed twice
at two different running conditions, 6% MDE gel at 4 C and 3% MDE gel
at room temperature, both at 4 watts constant power for 16 h. The
gels were dried, and autoradiography was performed by apposing to Kodak
BioMax MR film (Eastman Kodak Co., Rochester, NY) for
24 h at room temperature.
Sequencing and genotyping
DNA samples migrating at different rates in SSCA were sequenced with the Thermo Sequenase Cycle Sequencing Kit (Amersham Life Science, Cleveland, OH).
For genotyping the identified 3-glutamic acid deletion, the 220 DNA samples (166 obese and 54 normal weight subjects) were amplified with a third pair of primers (OliGold, Eurogentec, Belgium): 5'-AGGGTGTTTGTGGGGCATCTCC-3' and 5'-CAAGCTGAGGCCGGAGACACTG-3' (product size, 112 bp for the long allele, 103 bp for the short allele). The conditions for this PCR reaction were the same as those previously described. The amplified samples were mixed with 4 µL stop solution (Thermo Sequenase Cycle Sequencing kit), heated to 95 C for 2 min, and loaded hot onto sequencing gels (Long Ranger, FMC BioProducts). The gels were dried, and autoradiography was performed as previously described. The long (Glu12) and short (Glu9) alleles were identified based on their different electrophoretic migration rates.
Statistical analysis
The genotype frequency distributions were tested for Hardy-Weinberg equilibrium. Computations concerning the association analysis were performed using SPSS/WIN programs, version 6.0 (SPSS, Inc., Chicago, IL). The statistical significance of differences between the group means was assessed using one-way ANOVA, followed by Tukey’s test for multiple comparisons. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A deletion polymorphism comprising nine nucleotides leading to a
deletion of three glutamic acids from a glutamic acid repeat element
(Glu x 12, amino acids 297–309) present in the putative third
intracellular loop of the receptor protein was identified. A
representative sequencing result is shown in Fig. 1
. Figure 2
shows the location of this polymorphic site in the receptor
protein.
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Of the 166 obese subjects, 47 (28%) had 2 long alleles (Glu12/Glu12), and 90 (54%) were heterozygous (Glu12/Glu9) and 29 (17%) were homozygous for the short (Glu9/Glu9) form. Of the control population, 14 (28%) were homozygous for the long form, and 33 (61%) were heterozygous and 7 (13%) were homozygous for the short form. These genotype frequencies were not different from those predicted by the Hardy-Weinberg equilibrium hypothesis. Furthermore, allelic frequencies of the polymorphism were similar in lean and obese subjects.
Association analysis
The results of an ANOVA-based association analysis of
obesity-related phenotypic variables with the identified deletion
polymorphism are shown in Table 1. BMR
was 94 (95% confidence interval for difference, 32, 156) Cal/day
(5.6%) lower in subjects homozygous for the short allele than
in subjects with two long alleles (F = 4.84; P =
0.009, by ANOVA). The mean BMR of heterozygous subjects was
intermediate between those of the two homozygous groups. In addition,
the subjects with two short alleles had a slower average resting heart
rate than subjects with two long alleles (mean ± SEM,
60 ± 2 vs. 66 ± 1 beats/min; F = 3.0;
P = 0.05; 95% confidence interval for difference, 1.3,
11). Systolic blood pressure was also statistically significantly
different between the groups, but post-hoc comparisons
failed to identify significant differences between the two homozygous
groups. Demographic variables and other clinical and biochemical
variables related to obesity or energy metabolism were not
statistically significantly different between the groups. A subgroup
analysis was performed separately in women. Women with two short
alleles (n = 26) had significantly lower BMR (adjusted for fat
mass, fat-free mass, and age; 1577 ± 27 vs. 1645
± 12; F = 5.612; P = 0.019) and lower heart rate
(61 ± 2 vs. 66 ± 1; F = 5.142;
P = 0.025) than women with either one or two long
alleles (n = 111).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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2B-adrenoceptor
gene that is associated with reduced BMR in obese, nondiabetic Finns.
Although the physiological functions and precise distribution pattern
of the 2B-adrenoceptor in humans are still poorly known,
recent results from gene-modified mice point to an important role for
the 2B-adrenoceptor, particularly in the regulation of
vascular tone. Mice with a disrupted 2B-adrenoceptor
gene failed to show the typical blood pressure increase after iv
injection of subtype-nonselective 2-adrenoceptor
agonists; instead, the centrally mediated hypotensive action of these
agents was significantly accentuated (21). It should be noted in this
context that the mice with 2B-adrenoceptor gene
disruption are not obese, but their energy metabolism has not been
investigated (Kobilka, B. K., personal communication).
The 2B-adrenoceptor gene is located on human chromosome
2 (26). Previous studies have shown strong genetic linkage of markers
in the vicinity of the uncoupling protein 2 gene (located on 11q13) and
melanocortin receptor 5 gene (located on 18p11.2) with BMR in humans
(27, 28). Thus, several genes and chromosomal loci seem to affect the
regulation of BMR in man.
Altered regulation of vascular resistance may influence BMR either directly through redistribution of blood flow or through reflex modulation of autonomic nervous system activity. Information on regional and tissue-specific blood flow patterns or on sympathetic or parasympathetic neuronal activity is not available from our study subjects. The slower average heart rate observed in the subjects with two short alleles can equally support either alternative, but both mechanisms may be acting in parallel.
Redistribution of blood flow away from metabolically active tissues,
striated muscle in particular, caused by any mechanism leading to
regional alterations in vascular resistance would be expected to reduce
BMR. The lack of consistent associations of the identified
2B-adrenoceptor polymorphism with blood pressure is not
unexpected, as blood pressure is subject to complex physiological
regulation. The observed slower mean heart rate in subjects with two
short 2B-adrenoceptor alleles could be a consequence of
either increased parasympathetic (vagal) activity or reduced
sympatho-adrenal activity. Both effects could follow from increased
vascular resistance and are possible explanations for the reduced mean
BMR observed in the subjects with two short alleles.
A possible mechanistic explanation for the association of this variant
receptor form and increased vascular resistance or other mechanism
mediated via 2B-receptors is offered by a study
employing site-directed mutagenesis and transfected Chinese hamster
ovary (CHO) cells (29). In this study, mutant receptors with the
glutamic acid repeat element either deleted or replaced with an equal
number of charge-neutral glutamine residues were found to be resistant
to short term, agonist-dependent, phosphorylation-mediated
desensitization (29). Thus, prolonged agonist exposure, which normally
results in gradual waning of the evoked physiological response as the
receptors become desensitized, might lead to sustained responsivity in
subjects with a genetic variant of this sequence that is incapable of
being phosphorylated and desensitized in the normal manner.
The magnitude in the difference in adjusted BMR between the subject
groups with two long alleles and two short alleles was approximately
5.6%, or 94 Cal/day. Such a difference in BMR may be considered
clinically significant (6). Nevertheless, the identified receptor
polymorphism is clearly only a risk factor or a codeterminant of
clinical obesity, as a large proportion of the obese subjects in our
study population had two normal receptor alleles, and no significant
differences were observed between the genotypic groups in the
severity of obesity. This does not, however, preclude the
2B-adrenoceptor from being a possible therapeutic target
in the pharmacological prevention and treatment of obesity.
Before clinical studies on yet to be developed subtype-selective
pharmacological agents acting on 2B-adrenoceptors are
warranted in patient populations, the molecular and cellular mechanisms
responsible for the observed association between the identified
2B-adrenoceptor polymorphism and BMR need to be
investigated in suitable preclinical test models.
Received August 27, 1998.
Revised March 2, 1999.
Accepted March 15, 1999.
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-2 adrenergic receptor subtypes in rat
brain: an in situ hybridization study. J Comp Neurol. 328:575–594.[CrossRef][Medline]
-2 adrenergic receptor subtype gene expression in
rat brain. Brain Res Mol Brain Res. 21:133–149.[Medline]
2-adrenoceptor subtype function. Trends Pharmacol Sci. 18:211–219.[Medline]
2-adrenoceptor subtypes. Annu Rev Pharmacol Toxicol. 32:243–279.
2–Adrenergic receptor gene polymorphism and
hypertension in blacks. Am J Hypertens. 8:390–394.[CrossRef][Medline]
2-adrenergic receptor
subtypes b and c. Science. 273:803–805.[Abstract]
-2 adrenergic receptor family: cloning and
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