From the Clinical Pharmacology and Metabolic Research Unit (I.J.D., A.N.T., A.T., E.T.P.), Department of Medicine, College of Medicine, University of Vermont, Burlington, Vermont; and the Division of Endocrinology, Diabetes, and Nutrition (T.I.P., D.A., D.G., A.R.S.), Department of Medicine and Baltimore Veterans Affairs Geriatric Research and Education Clinical Center, Department of Medicine, University of Maryland, Baltimore, Maryland.
Address correspondence and reprint requests to Eric T. Poehlman, PhD, Given Bldg. C-247, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail: epoehlma{at}zoo.uvm.edu .
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ABSTRACT |
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INTRODUCTION |
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Our laboratory has participated in a series of prospective interventional studies that examined the association between the Trp64Arg ß3AR variant in the ß3-adrenoceptor gene, with several obesity-related phenotypes. We and others have shown reduced energy expenditure (3,4,5,6), insulin resistance (7), a reduced ability to mobilize visceral fat (8), a lower rate of glucose disposal (9), and greater visceral obesity (7) and BMI (6) in individuals harboring the Trp64Arg variant. On the other hand, others have found no meaningful effect of this variant on obesity-related phenotypes (10,11).
Because it is recognized that the genetic component of human obesity is complex and polygenic, it is reasonable to hypothesize that the examination of only one genetic variant provides insufficient information regarding genetic susceptibility to obesity. Moreover, we would suggest that discrepancies found in aforementioned studies regarding the effect of the Trp64Arg ß3AR variant on obesity-related phenotypes may be partially attributed to the presence of other genetic variants that may interact with the Trp64Arg ß3AR to influence obesity and obesity-related phenotypes. For instance, several studies have shown additive effects of the Trp64Arg ß3AR variant and a variant in the uncoupling protein-1 gene on obesity-related phenotypes (12,13,14,15).
The ß3-adrenoceptor is known to activate lipolysis and
energy expenditure. Because other adrenoreceptors are expressed in adipocytes
and play a regulatory role in energy balance and lipolysis, the presence of
other genetic variants affecting adrenergic function may be interactive with
the Trp64Arg ß3AR variant. Recently, a newly described variant, a
threeaminoacid deletion in the 2b-adrenoceptor
(Glu12/Glu9
2bAR), has been shown to be
associated with a lower basal metabolic rate, but not increased BMI
(16). The high frequency of
this variant
(16,17)
and the potential of the
2b-adrenoceptor variant to interact
with the Trp64Arg ß3AR variant make it a potential candidate to influence
obesity-related phenotypes. The deletion of three glutamic acid residues
occurs within a polyglutamic tract at codons 297-309, which is within the
acidic domain of the third intracellular loop of this seven-membrane-spanning
G-proteincoupled receptor. The functional consequence of this deletion
is currently unknown. The purpose of this study was to examine the independent
and interactive effect of the Glu12/Glu9
2bAR and
Trp64Arg ß3AR variants on body composition and energy balance in a large
cohort of unrelated Caucasian women (n = 909).
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RESEARCH DESIGN AND METHODS |
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Genotyping
Detection of the Trp64Arg ß3AR variant. The
ß3-adrenoceptor gene is located on chromosome 8. Polymerase
chain reaction (PCR) was performed from 20 ng genomic DNA with upstream
primer 5'-CGCCCAATACCGC CAACAC-3' and downstream primer
5'-CCACCAGGAGTCCCATCACC-3' in the presence of 10%
dimethylsulfoxide. The resulting 210-bp product was digested with the
restriction endonuclease BST N1. The digested products were subjected to
electrophoresis through a 4% agarose gel (Metaphor agarose; FMC Bioproducts,
Rockland, ME). The gel was stained with ethidium bromide and DNA was
visualized by ultraviolet (UV) transillumination. The expected sizes were 99,
62, 30, 12, and 7 bp for Trp64 homozygotes; 161, 30, 12, and 7 bp for Arg64
homozygotes; and 161, 99, 62, 30, 12, and 7 bp for heterozygotes.
Detection of the Glu12/Glu9 2bAR
variant. The
2b-adrenoceptor gene is located on
chromosome 2. PCR was performed from
20 ng genomic DNA with upstream
primer 5'-AGGGTGTTTGTGGGGCATCTCC-3' and downstream primer
5'-CAAGCTGAGGCCGGAGACACTG-3'. The PCR product was subjected to
electrophoresis through a 4% agarose gel (Metaphor agarose; FMC Bioproducts).
The gel was stained with ethidium bromide and DNA was visualized by UV
transillumination. The expected sizes were 112 bp for Glu12
homozygotes, 103 bp for Glu9 homozygotes, and 112 and 103 bp for
heterozygotes.
Resting metabolic rate. Resting metabolic rate (RMR) was measured by indirect calorimetry for 45 min as previously described (3,18), using the ventilated hood technique, after a 12-h overnight fast at the University of Vermont General Clinical Research Center. Respiratory gas analysis was performed using a Deltatrac metabolic cart (Sensormedics, Yorba Linda, CA). RMR (kcal/d) was calculated from the equation of Weir (19). The test-retest correlation coefficient within 1 week is 0.90 for RMR in our laboratory.
Body composition
Dual-energy X-ray absorptiometry. Fat mass and fat-free
mass were assessed in the supine position, using dual-energy X-ray
absorptiometry (DEXA) at the University of Vermont (model DPX-L; Lunar
Radiation, Madison, WI) as previously described
(20,21).
Computed tomography. Visceral adipose tissue and subcutaneous adipose tissue were measured by computed tomography (CT) as previously described (3) using a High-Speed Advantage CT scanner (General Electric Medical Systems, Milwaukee, WI). The subjects were examined in the supine position with both arms stretched above their head. The position of the scan was established at the L4-L5 level using a scout image of the body. Visceral adipose tissue area was quantified by delineating the intra-abdominal cavity at the internal-most aspect of the abdominal and oblique muscle walls surrounding the cavity and the posterior aspect of the vertebral body. Adipose tissue was highlighted and computed using an attenuation range of -190 to -30 Hounsfield units. The subcutaneous adipose tissue area was quantified by highlighting adipose tissue located between the skin and the external-most aspect of the abdominal muscle wall.
VO2max. VO2max was determined from an incremental exercise test to exhaustion on a treadmill, as previously described (22). Briefly, after an initial 3-min warmup, the speed was set so that the heart rate would not exceed 70% of the agepredicted maximal heart rate (220 - age [years]). Thereafter, the speed was held constant and the grade was increased by 2.5% every 2 min. The criteria for achieving VO2max were 1) a respiratory exchange ratio >1.0, or 2) a heart rate at or above the age-predicted workload. Test-retest conditions for nine individuals (on two occasions retested 1 week apart) yielded an intraclass correlation of 0.94 using Haggard's model (23) and a coefficient of variation of 3.8% in our laboratory.
Energy intake. Energy intake was measured as previously described (24). Briefly, total daily food intake was assessed using the 3-day food record (2 weekdays and 1 weekend day). Subjects were trained to measure, weigh, and record portion sizes. Food scales and measuring instruments were provided to all subjects. Nutritional assessment was conducted using the Nutritionist III software package (Version 4.0; N-Squared Computing, Salem, OR). Although the measurement of energy intake using the 3-day food record may provide an approximation of habitual energy intake, we have shown that this instrument underestimates true energy intake (20).
Leisure time physical activity. Leisure time physical activity (LTA) was measured by the Minnesota LTA questionnaire (25), a commonly used interviewer-administered questionnaire that assesses daily physical activity accumulated during leisure time and household physical activity over the past 12 months. Trained personnel administered the questionnaire during a 20-min interview. LTA was calculated based on the number of months spent completing the specific activity per year, the average number of times for the specific activity each month, the total time of each physical activity session, and the activity-specific intensity code. The test-retest correlation coefficient over a month has been shown to be 0.92 in older women and men (26).
Statistical analyses. All values are presented as means ± SE,
unless otherwise specified. We performed an analysis of covariance to examine
the independent effect of the Glu12/Glu9 2bAR
variant on dependent variables, using appropriate covariates. Because age,
aerobic fitness, and substrate oxidation influence body weight, BMI, and fat
mass, we used age, VO2max, and respiratory quotient as
covariates for body composition. Visceral fat was adjusted for age and fat
mass and resting energy expenditure was adjusted for age, fat mass, and
fat-free mass. We then performed a univariate analysis of variance using a
general linear model procedure to examine the interactive effect of the two
variants on dependent variables using the aforementioned covariates. All
analyses were performed using Statistical Package for Social Science software,
version 9.0.0 (Chicago).
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RESULTS |
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Table 1 shows the individual
effect of the Glu12/Glu9 2bAR variant on age,
height, body weight, and BMI in the entire cohort (n = 909). There
was no independent association of the variant with any of these variables.
Furthermore, in the subset of subjects (n = 214) for whom detailed
phenotypes were obtained, there was no association of the
Glu12/Glu9
2bAR variant with fat mass, fat-free
mass, visceral fat, RMR, respiratory quotient, physical fitness, or energy
intake (Table 2).
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Because a subgroup of obese subjects (n = 82) was selected based
on their Trp64Arg ß3AR genotype for the purpose of a weight-loss
intervention study
(3,8),
the independent effect of this variant on obesity-related phenotypes is
confounded by a selection bias and therefore not reported. However, the
increase in the number of subjects with the Trp64Arg ß3AR variant
enhanced our power to examine the interactive effect of the Trp64Arg ß3AR
and Glu12/Glu9 2bAR variants on obesity-related
phenotypes. For these analyses, because the number of homozygotes was small,
and previous studies suggest that heterozygosity for each of the variants is
associated with their respective phenotypes, heterozygotes and homozygotes for
each of the variants were combined. We found a significant interaction effect
between the two variants on fat mass (P = 0.009) and percent fat
(P = 0.016) (Table 3).
Although the Glu12/Glu9
2bAR variant did not
associate with fat mass or percent fat when examined individually (Tables
1 and
2), subjects with both the
Glu12/Glu9
2bAR and Trp64Arg ß3AR variants
had 9.3 kg and 4.8% greater fat mass and percent fat, respectively, than
subjects who carried only the Trp64Arg ß3AR variant. Age, height, body
weight, BMI, fat-free mass, visceral fat, energy expenditure, respiratory
quotient, physical fitness, and energy intake were not different between
groups. Collectively, our findings support an interaction effect of the two
variants on body fatness.
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DISCUSSION |
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Controversies exist among studies pertaining to the singular effect of the
Trp64Arg ß3AR polymorphism on obesity-related phenotypes
(3,6,7,8,9,10,11,28,29).
This controversy is likely due to the moderate effect of the variant and the
failure to use highly controlled experimental conditions to control for
important covariates (e.g., diet, fluctuations in body weight, sex, ethnicity,
and even obesity itself) that may obscure the effect of the variant on
obesity-related phenotypes. Another potential reason for discrepancies among
investigators is that the Trp64Arg ß3AR variant may interact with other
variants to influence body fatness. We specifically examined the interaction
between the Glu12/Glu9 2bAR and Trp64Arg
ß3AR variants on body composition in a relatively large cohort of
well-characterized women with a wide range of body habitus. The
Glu12/Glu9
2bAR variant was selected because of
its previously reported association with a lower RMR and its potential
association with obesity-related variables
(16).
Since both the ß3AR and 2bAR are expressed in adipocytes and
reciprocally regulate lipolysis, we hypothesized that the simultaneous
existence of the Trp64Arg ß3AR and Glu12/Glu9
2bAR variants would be associated with an unfavorable metabolic profile
when compared with individuals who carry only one of the variants. To address
this issue, we performed direct measurements of body fatness, energy
expenditure, and energy intake under inpatient conditions, using
state-of-the-art methods. We found an interactive effect of both variants on
fat mass in which carriers of both variants displayed significantly higher fat
mass. That is, we observed a difference of
9.3 kg fat mass between the
group harboring only the Trp64Arg ß3AR variant and the group harboring
both variants. These results suggest an interaction effect of the Trp64Arg
ß3AR and Glu12/Glu9
2bAR on body fatness and
suggest the occurrence of a chronic energy imbalance at some point in these
women's lives.
As with most population-based association studies, the present study has potential statistical biases. First, the issue of multiple comparisons in the interpretation of P values is widely debated (30). Allison and Beasley (30) have suggested the use of an approach for multiple comparisons that utilizes simulations to take into account correlations between the variables. With use of this approach, the corrected P values for associations between fat mass and percent fat, and the interactive effects of the gene variants were P = 0.09 (from an uncorrected P value of 0.009) and P = 0.15 (from an uncorrected P value of 0.016), respectively. However, the selection of the phenotypes that we examined was based on specific mechanism-based hypotheses. That is, we selected the major determinants of energy balance, and examined whether each phenotype was specifically influenced by the two variants. Of course, it should be pointed out the method used to account for the multiple comparisons in adjusting our P value, as well as all classical frequentist P values approaches, do not take into account the fact that the hypotheses tested were based on specific postulated mechanistic/functional relationship. Therefore, the likelihood of the genetic variants observed having effects on the phenotypes studied should be viewed in light of both the P values obtained and the a priori nature of the hypotheses. Second, population admixture can be another source of bias leading to false-positive associations. However, the present study included only Caucasian women of European ancestry, thus minimizing this possibility.
One potential mechanism by which the Trp64Arg ß3AR variant may
influence adiposity is an impairment of the
ß3-adrenoceptormediated lipolysis. Indeed, several
studies have shown that the Trp64Arg substitution results in decreased
signaling and impaired lipolysis
(15,31,32,33,34,35,36,37,38).
The Glu12/Glu9 2bAR variant has also been
suggested to influence the rate of lipolysis. Indeed, it has been suggested
that the variant observed in the
2b-adrenoceptor gene may
impair receptor desensitization in response to prolonged agonist exposure
(39). Because of this
receptor's inhibitory role in lipolysis, this variant may potentially cause an
increase in the
2b-adrenoceptormediated lipolysis
inhibition. Thus, it is possible that the presence of both variants favors a
pattern of fat storage that is greater than lipolysis and fat oxidation.
We examined several physiological mechanisms by which the Trp64Arg
ß3AR and Glu12/Glu9 2bAR variants may
influence energy balance by measuring energy intake, energy expenditure, and
fasting substrate oxidation. Our study, however, did not provide evidence of
an interactive effect of Trp64Arg ß3AR and
Glu12/Glu9
2bAR variants on these phenotypes.
Therefore, the results of the present study do not permit us to identify which
component of energy balance may have been altered to cause a greater fat mass
in women carrying both variants. It is possible that the experimental design
of our study may have limited our ability to detect differences among
genotypes in energy expenditure, energy intake, and substrate oxidation that
had the potential to impact on body fat accumulation. That is, by definition,
the subjects who were eligible for the present study were weight stable,
suggesting that individuals were in energy (energy intake = energy
expenditure) and macronutrient balance. We thus assume that the energy
imbalance that led to an accumulation of fat mass in individuals with both
variants had already taken place and was not detectable when these phenotypes
were actually measured. Experimental studies using a weight loss/regain design
would be needed to investigate the effect of the combination of Trp64Arg
ß3AR and Glu12/Glu9
2bAR variants on dynamic
changes in body weight and fat mass during a period of energy and
macronutrient imbalance.
In conclusion, although the physiological mechanisms remain to be
determined, we found an interaction effect of Glu12/Glu9
2bAR and Trp64Arg ß3AR variants on body fatness. These results
suggest that the combined effect of both variants on fat mass is significantly
greater than the separate effects of the polymorphisms. Further studies are
needed to confirm and extend these findings.
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ACKNOWLEDGMENTS |
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Gratitude is expressed to all volunteers who participated in the study.
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FOOTNOTES |
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Received for publication January 18, 2000 and accepted in revised form September 21, 2000
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REFERENCES |
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