Download The Trp64Arg β3 -adrenergic receptor amino acid

Clinical Science (2002) 103, 397–402 (Printed in Great Britain)
The Trp64Arg β3-adrenergic receptor amino
acid variant confers increased sensitivity
to the pressor effects of noradrenaline
in Sardinian subjects
Maria G. MELIS, Giannina SECCHI, Patrizia BRIZZI, Cristiana SEVERINO,
Mario MAIOLI and Giancarlo TONOLO
Servizio Diabetologia, Istituto di Clinica Medica, Dipartimento Struttura Clinica Medica e Patologia Medica, Viale S Pietro 8,
07100 Sassari, Italy
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The β3-adrenergic receptor ( β3AR) plays a critical role in lipid metabolism, and thus alterations
in its function may be involved in the metabolic syndrome. Indeed, we have found previously that
the Trp64Arg amino acid variant of the β3AR is associated with hypertension and higher serum
triacylglycerol levels in the Sardinian population. The aim of the present study was to evaluate
the effect of the Trp64Arg β3AR variant on the regulation of triacylglycerol levels and blood
pressure during the exogenous infusion of noradrenaline. We studied groups of non-diabetic
normotensive subjects : eight with the wild-type Trp64Trp β3AR and eight with the Trp64Arg
variant. The subjects each received, on two different days (randomized, double-blind fashion), a
4 h infusion of either noradrenaline (0.147 nmol : min−1 : kg−1) or Emagel (subjects had fasted for
at least 12 h). The only available subject with a homozygous mutant Arg64Arg β3AR was also
studied. Blood pressure was measured every 10 min using a sphygmomanometer, and blood
samples were taken every 30 min from the contralateral vein for biochemical determinations.
After a 4 h noradrenaline infusion, significant increases in diastolic blood pressure (from 83p2
to 91p3 mmHg ; P 0.01) and serum triacylglycerol levels (from 1.69p0.4 to 1.79p0.6 mmol/l ;
P 0.05) were observed compared with basal values in subjects with the Trp64Arg β3AR variant,
whereas subjects with the Trp64Trp β3AR did not show any significant change over the infusion
period. Glycaemia had increased significantly only at the end of the first 1 h of infusion in subjects
with the Trp64Arg variant (from 5.0p0.1 to 5.8p0.3 mmol/l ; P 0.05), with no significantly
different behaviour compared with those subjects with the Trp64Trp β3AR during the remaining
infusion period. The effects of noradrenaline infusion were more marked in the subject with the
Arg64Arg variant. In conclusion, our data indicate that the Trp64Arg amino acid variant of
the β3AR confers increased sensitivity to the pressure effect of noradrenaline. Moreover, this
variant also influences blood triacylglycerol levels and, to a degree, glucose metabolism.
INTRODUCTION
A new class of β-adrenergic receptor ( βAR), the β AR,
$
has been described that differs in its molecular structure
and pharmacological properties from the β AR and the
"
β AR [1,2]. The β AR, which is expressed mainly in
#
$
visceral adipose tissue [3,4], has important local effects on
lipid metabolism by mediating lipolysis and thermogenesis [5]. The β AR is also expressed in other tissues,
$
and selective stimulation of the β AR results in sustained
$
Key words : metabolic syndrome, noradrenaline, triacylglycerol, Type II diabetes.
Abbreviations : βAR, β-adrenergic receptor ; HDL, high-density lipoprotein ; NEFA, non-esterified fatty acids.
Correspondence : Dr Giancarlo Tonolo (e-mail giantono2001!yahoo.it).
# 2002 The Biochemical Society and the Medical Research Society
397
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M. G. Melis and others
Table 1 Clinical and biochemical characteristics of the normotensive non-diabetic subjects,
grouped according to the presence or absence of the Arg64 allele
LDL, low-density lipoprotein ; HOMA-IR, insulin resistance as measured by homoeostasis model assessment ocalculated using the
formula [fasting glucose (mmol/l)ifasting insulin (µ-units/ml)/22.5]q. No significant differences were present between
subjects with the Trp64Arg and Trp64Trp β3ARs for any of the measured parameters (for triacylglycerol, P l 0.059).
Parameter
Trp64Trp
Trp64Arg
Arg64Arg
n
Sex (female/male)
Age (years)
Body mass index (kg/m2)
Waist/hip ratio
LDL-cholesterol (mmol/l)
Triacylglcerols (mmol/l)
NEFA ( µmol/l)
Fasting glycaemia (mmol/l)
HOMA-IR
Systolic/diastolic blood pressure (mmHg)
8
4/4
38p3
27p3
0.89p0.02
3.5p1.3
1.42p0.4
0.75p0.5
5.1p0.1
1.53p1.15
120p5/80p2
8
4/4
36p4
28p4
0.90p0.02
3.2p1.4
1.62p0.5
1.2p0.6
5.4p0.2
1.40p1.10
125p6/85p3
1
1/0
54
30
1.0
3.4
1.89
1.4
5.6
2.8
125/85
peripheral vasodilatation [6], predominantly in the skin
and fat, and a negative inotropic effect in the human heart
in vitro has also been described [7]. The Trp64Arg
polymorphism in the β AR has been reported to be
$
associated with clinical features of the insulin resistance
syndrome in Finns and Pima Indians with Type II
diabetes [8–10], and with essential hypertension and
elevated circulating triacylglycerol levels in the Sardinian
population [11]. The association with Type II diabetes
has been replicated in some, but not in all, studies [12,13].
Recently the Trp64Arg amino acid variant of the β AR
$
has been shown to modulate the effects of β-blockers on
levels of triacylglycerols and high-density lipoprotein
(HDL) cholesterol in a group of Indo–Mauritian subjects
with premature coronary heart disease [14], suggesting a
key role for the β AR in the regulation of lipid metab$
olism by endogenous catecholamines.
The principal aim of the present study was to evaluate
the effects of increasing the plasma noradrenaline level by
2-fold, by means of exogenous noradrenaline infusion
(0.147 nmol:min−" : kg−"), on the regulation of blood
pressure and serum triacylglycerol levels in normotensive
non-diabetic subjects.
METHODS
Subjects
Detection of the β AR Trp64Arg polymorphism was
$
performed as described previously [11]. From our large
database [15] we randomly selected eight normotensive,
normolipaemic non-diabetic subjects carrying the β AR
$
Trp64Arg polymorphism. Eight untreated non-diabetic
subjects with the Trp64Trp β AR, matched for age, blood
$
pressure and serum lipids, were studied as controls.
# 2002 The Biochemical Society and the Medical Research Society
Among more than 1800 subjects screened so far by our
group for the β AR Trp64Arg polymorphism, we found
$
only one homozygous mutant (Arg64Arg), who was also
studied. This subject was a female aged 54 years at the
time of study, who from the age of 44 years had had a
diagnosis of obesity (body mass index l 43 kg\m#), Type
II diabetes, hypertension and high circulating triacylglycerol levels. This subject was treated pharmacologically for all these conditions until 1997. At that time
a biliopancreatic diversion with preservation of the
stomach and pylorus was carried out for obesity, after
which she did not need any more pharmacological treatment and her body mass index decreased to 30 kg\m#.
All subjects had previously given approval to participate in the genetic study, and thereafter gave informed
consent to the study protocol, which was approved by
the local Ethics Committee.
Infusion study
Subjects had fasted for at least 12 h before the study, and
thus were in the post-absorptive state. After a 60 min
run-in period (Emagel ; 10 ml\h), all subjects received, on
two different days in a randomized double-blind fashion,
a 4 h infusion of either noradrenaline at 0.147 nmol :
min−" : kg−" (dissolved in Emagel ; 10 ml\h) or Emagel
(vehicle ; 10 ml\h), followed by an additional 60 min recovery period (Emagel ; 10 ml\h). This dose of noradrenaline was chosen on the basis of pilot experiments
as the highest dose that increased the circulating noradrenaline levels by 2-fold over basal, but was still within
the physiological range and did not cause significant
increases in blood pressure or metabolic changes in
normotensive subjects, as also described previously by
others [16].
Blood pressure was measured every 10 min using a
sphygmomanometer, and blood was taken every 30 min
β3-Adrenergic receptor : regulation of lipolysis and haemodynamics
from a vein in the contralateral arm into EDTA tubes for
measurement of plasma glucose, triacylglycerols, total
and HDL cholesterol and non-esterified fatty acids
(NEFA). Glucose, triacylglycerols and total and HDL
cholesterol were measured as described previously [17].
NEFA were measured using a commercial colorimetric
method (Boehringer Mannheim). Low-density lipoprotein cholesterol was calculated using the Friedewald
formula, since all subjects had a serum triacylglycerol
level lower than 300 mg\dl [18]. The study was begun at
08.30–09.00 hours after a 12 h fast. The researchers in
charge of noradrenaline\vehicle infusions were not aware
of the polymorphism status of the subjects. Emagel
(Hoechst Marion Russel) is a solution containing PoligelineTM, 145 mmol\l Na+, 5.1 mmol\l K+, 6.25 mmol\l
Ca#+ and 145 mmol\l Cl−, and is used as a synthetic
plasma substitute.
Statistical analysis
Values are meanspS.D. Differences between groups,
when significant after a multiple statistical comparison
(P 0n05 by ANOVA after a Bonferroni correction),
were tested using the unpaired Student’s t test. The paired
Student’s t test was used to compare differences from
basal within each group. Statistical calculations were
performed using the SIGMA STAT 1.0 statistical package.
RESULTS
The basal clinical characteristics of the subjects before
noradrenaline\vehicle infusion are presented in Table 1
(means of values on the two days). At the end of the 4 h
noradrenaline infusion, plasma noradrenaline levels were
3.15p1.55, 2.98p1.49 and 2.96 nmol\l in subjects with
the Trp64Arg, Trp64Trp and Arg64Arg β AR respect$
ively, while at the end of the 4 h vehicle infusion, plasma
noradrenaline levels were 1.52p0.91, 1.39p1.02 and
1.42 nmol\l respectively in the three groups.
After the 4 h noradrenaline infusion, significant increases in diastolic blood pressure (from 83p2 to
91p3 mmHg ; P 0.01) and triacylglycerol levels (from
1.69p0.4 to 1.79p0.6 mmol\l ; P 0.05) were observed
compared with basal values in subjects with the Trp64Arg
β AR, while Trp64Trp β AR subjects did not show any
$
$
significant changes during the infusion period. Glycaemia
was increased significantly only at the end of the first 1 h
of infusion in subjects with the Trp64Arg β AR (from
$
5.0p0.1 to 5.8p0.3 mmol\l ; P 0.05), with no significantly different behaviour compared with Trp64Trp
β AR subjects during the remaining period. The effects of
$
noradrenaline infusion were more marked in the subject
with the Arg64Arg β AR. These changes remained
$
significant when expressed as absolute changes from
Figure 1 Absolute changes in diastolic blood pressure,
serum triacylglycerols and plasma NEFA in the three groups
of subjects during infusion of noradrenaline (0.147 nmol :
min−1 : kg−1)
The groups comprised eight subjects with the wild-type Trp64Trp β3AR (open bars),
eight subjects with the Trp64Arg β3AR (closed bars) and one subject with the
homozygous mutant Arg64Arg β3AR (hatched bars). Values are meanspS.D.
Comparison between Trp 64Arg and Trp64Trp groups : *P 0.05, **P 0.01.
basal, as shown in Figure 1. The effects observed in
subjects with the Trp64Arg β AR were even more
$
marked in the subject with the homozygous Arg64Arg
β AR, in whom glycaemia also increased persistently
$
throughout the 4 h (Table 2). No significant changes
occurred during noradrenaline infusion for any of the
other parameters measured. Although systolic blood
pressure increased in subjects with the Trp64Arg β AR
$
during noradrenaline infusion, this barely reached statistical significance (P l 0.0549 ; Table 2). During vehicle
(Emagel) infusion, no significant changes from time zero
were observed in any of the metabolic or haemodynamic
# 2002 The Biochemical Society and the Medical Research Society
399
400
M. G. Melis and others
Table 2 Basal values and absolute changes from time 0 in the three groups of subjects during noradrenaline infusion
(0.147 nmol : min−1 : kg−1) for 4 h
SBP, systolic blood pressure (means of three measurements for each subject). Significance of differences : *P
subjects.
0.05 compared with basal and with Trp64Trp β3AR
Absolute changes from basal
Basal (time 0)
Noradrenaline 1 h
Noradrenaline 4 h
Recovery
Parameter
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
SBP (mmHg)
Heart rate (beats/min)
Glycaemia (mmol/l)
124p6
72p8
5.2p0.2
125p4
76p9
5.0p0.1
122
80
5.5
6p2
k6p1
0p0.1
3p2
k4p1
0.8p0.2*
0
3
1
5p1
k4p2
0p0.11
3p2
k3p1
0p0.1
15
6
1
0p1
k6p2
k0p0.11
1p1
k3p2
k1p0.11
9
8
0
Table 3
for 4 h
Basal values and absolute changes from time 0 in the three groups of subjects during vehicle (Emagel) infusion
SBP, systolic blood pressure ; DBP, diastolic blood pressure (means of three measurements for each subject). No significant differences were evident either compared
with basal for any group, or between the Trp64Trp and the Trp64Arg groups at any time.
Absolute changes from basal
Noradrenaline 1 h
Basal (time 0)
Noradrenaline 4 h
Recovery
Parameter
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
Trp64Trp
Trp64Arg
Arg64Arg
SBP (mmHg)
DBP (mmHg)
Heart rate (beats/min)
Glycaemia (mmol/l)
Triacylglycerols (mmol/l)
NEFA ( µmol/l)
123p7
80p3
75p6
5.3p0.1
1.51p0.4
1.3p0.7
126p5
84p2
79p7
5.1p0.1
1.71p0.5
1.6p0.6
124
85
84
5.7
1.81
1.9
0p0
0p1
1p1
0p0.11
0p1
0p1
1p2
0p2
1p0
0p0.2
0p0
0p0
1
0
2
0
2
0
2p1
1p1
k1p1
k1p0.2
k1p1
k1p1
2p1
0p1
k1p1
k1p0.1
1p1
1p1
2
0
1
0
1
1
1p2
0p1
k2p1
k1p0.1
k1p1
k1p1
k1p2
k1p1
k1p1
k1p0.1
1p1
1p1
2
1
k2
0
1
1
parameters considered in the eight subjects with the
Trp64Trp β AR, the eight subjects with the Trp64Arg
$
β AR or the one subject with the Arg64Arg β AR (Table
$
$
3).
DISCUSSION
The sympathetic nervous system plays an important role
in the regulation of energy expenditure. Although the
inhibition of resting sympathetic activity by α-adrenoceptor or βAR blocking agents affects the resting metabolic rate to only a small extent [19], the increase in
sympathetic activity above basal is associated with an
increase in energy expenditure, i.e. with thermogenesis.
We previously investigated the role of the Trp64Arg
amino acid variant of the β AR in the metabolic syndrome
$
in Sardinia, taking advantage of the population enrolled
by ‘ The Study Group for the Genetics of Diabetes in
Sardinia (SGGDS) ’ [15,20,21]. In the period 1994–2001, a
total of 2451 subjects were enrolled with different
phenotypes for parameters related to blood pressure,
diabetes and lipids ; the inclusion\exclusion criteria have
been detailed previously [14]. We previously reported, in
this population, a significant association of the Trp64Arg
β AR with essential hypertension [11], but we were not
$
able to find an association with Type II diabetes or
# 2002 The Biochemical Society and the Medical Research Society
obesity (G. Tonolo, unpublished work ; presented at the
37th EADSD Meeting, Glasgow, 9–13 September 2001).
In the present study we randomly selected eight nondiabetic normotensive subjects with the Trp64Arg β AR
$
variant from our large database, along with eight subjects
with the wild-type Trp64Trp β AR matched for sex, age,
$
blood pressure and circulating lipids ; additionally, we
studied the single subject with a homozygous Arg64Arg
β AR identified in our screening programme. By means
$
of a low-dose noradrenaline infusion protocol in these
three groups of subjects, we wanted to evaluate, in vivo,
the putative role of the β AR in the control of blood
$
pressure and lipolysis [5], mediated by catecholamines.
Although the β AR has binding capacity for synthetic
$
ligands, clearly differentiating it from both the β AR and
"
the β AR, noradrenaline is the natural ligand, being a full
#
β AR agonist in human omental adipocytes [22,23].
$
The β AR is the predominant receptor involved in adi#
pose tissue lipolysis, and also stimulates fat tissue blood
flow [24], but the β AR, whose mRNA has been isolated
$
in both white and brown adipose tissue, may have a
role in the control of lipolysis during excessive adrenergic
stimulation, when both the β AR and the β AR have been
"
#
desensitized [25]. In these situations the β AR might act
$
as an ‘ emergency ’ βAR, essentially under conditions of
strong and sustained sympathetic nervous system activation [26]. Our data suggest that the Trp64Arg amino
β3-Adrenergic receptor : regulation of lipolysis and haemodynamics
acid variant of the β AR confers increased sensitivity to
$
the pressure effect of noradrenaline, since a significant
increase in diastolic blood pressure occurred only in
Trp64Arg β AR carriers and in the Arg64Arg β AR
$
$
homozygote, but not in subjects with the wild-type
Trp64Trp β AR.
$
Moreover, in carriers of the Trp64Arg β AR and the
$
subject with the homozygous Arg64Arg β AR, nora$
drenaline significantly increased serum NEFA and triacylglycerols, whereas these were actually decreased in
carriers of the wild-type Trp64Trp β AR. These data
$
support the concept that the Trp64Arg amino acid variant
of the β AR has a role in the development of some
$
features of the metabolic syndrome, such as high blood
pressure and high serum triacylglycerols. Apparently
fewer β ARs must be occupied to stimulate lipolysis
$
compared with those needed to stimulate uncoupling
protein 1 [27]. Thus it is possible to speculate that, in the
presence of the Trp64Arg β AR variant, an imbalance is
$
created between lipolysis (which causes NEFA production) and thermogenesis via uncoupling protein 1
(which causes NEFA to be metabolized). The increase in
circulating NEFA makes more substrate available, which
in turn is transformed in the liver into triacylglycerol ; we
believe that this occurred in the present study during
noradrenaline infusion in subjects with the Trp64Arg and
Arg64Arg β AR variants.
$
The observed increase in NEFA also suggests an
alternative hypothesis to explain the rise in blood
pressure observed in subjects with the Trp64Arg β AR
$
during noradrenaline infusion. It is known that, in vivo,
NEFA may affect endothelial function [28]. This effect is
due, at least in part, to an inhibition of endothelial NO
synthase activity and thus of endothelium-dependent
vasodilatation [29]. Therefore the increase in NEFA will
be responsible not only for the increase in triacylglycerol,
but also for the observed elevation in blood pressure. We
can exclude a direct effect of the increase in triacylglycerols since, in vivo, using plethysmography in the
isolated human arm, NEFA, but not triacylglycerols,
have been shown to impair endothelium-dependent
vasodilatation [30,31].
Our new data, as well as confirming a link between the
Trp64Arg amino acid variant of the β AR and essential
$
hypertension and hypertriglyceridaemia, as described
previously [11], are in keeping with the observations of
Manraj et al. [14] on the modulatory effects of the
Trp64Arg β AR gene on triacylglycerol levels during β$
blocker treatment in Indo–Mauritian subjects. These
observations indicate that this polymorphism may influence the response to environmental factors (both drug
therapy with β-blockers, and stress mediated by circulating catecholamines) of the lipolytic process in both of
these populations (Indo–Mauritians and Sardinians),
which have very different genetic backgrounds. Our data
are also in keeping with the recent observation of
Carlsson et al. [32] in a familial study of an increase in
circulating NEFA in subjects with the Trp64Arg β AR
$
variant as compared with those with the wild-type
Trp64Trp β AR. We did not measure NEFA in all 1800
$
screened subjects, but in the present study basal NEFA
levels were somewhat higher in subjects with the
Trp64Arg and Arg64Arg β AR variants, and the increase
$
during noradrenaline infusion was significantly greater in
these subjects as compared with Trp64Trp β AR subjects.
$
The sib-pairs method used in the study of Carlsson et al.
[32], although robust, has a lower power than association
studies with unrelated case–controls for complex diseases
such as essential hypertension and Type II diabetes [33].
From this point of view, the high genetic homogeneity of
the Sardinian population [34,35] will lower the bias of a
case–control study such as the present one. The metabolic
syndrome is a multigenetic and multifactorial disease,
and thus it is not surprising that interactions with
different genetic backgrounds and environmental factors
may result in different outcomes of studies on the β AR.
$
For example, in our previous study [11] we found the
Tgrp64Arg β AR variant was associated with some
$
components of the metabolic syndrome (hypertension
and triacylglycerols), but not with diabetes or obesity.
A limitation of the present study is the relatively small
number of subjects studied. Despite this, our data suggest
an altered phenotypic function due to the described
β AR polymorphism, and are in keeping with other
$
data suggesting an association of this polymorphism
with blood pressure and plasma triacylglycerol levels
[11,14,32].
In conclusion, although a single gene cannot, by
definition, explain the aetiology of complex diseases such
as diabetes, hypertension and the metabolic syndrome,
screening for common polymorphisms in candidate genes
is a useful tool for obtaining improved phenotype
definition in complex diseases. In the present study we
have found that the Trp64Arg amino acid variant of the
β AR confers increased sensitivity to the pressure effect
$
of noradrenaline, and influences noradrenaline-mediated
lipolysis.
ACKNOWLEDGMENTS
We acknowledge the excellent technical work of Mrs
Francesca Fenu and Mrs Maristella Spissu.
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Received 4 March 2002/27 May 2002; accepted 8 July 2002
# 2002 The Biochemical Society and the Medical Research Society