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Keywords:

  • physical activity;
  • BMD;
  • trabecular;
  • men;
  • polymorphism;
  • estrogen

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

In this large population-based study in young men, we show that the COMT val158met polymorphism modulates the association between physical activity, aBMD (DXA), and trabecular vBMD (pQCT).

Introduction: Peak BMD is an important predictor of future risk of osteoporosis and is largely determined by genetic factors but also by environmental factors, among which physical activity (PA) is a strong contributor. Estrogens are believed to influence the mechanical strain signal generated by bones subjected to mechanical loading. Catechol-O-methyltransferase (COMT) is involved in the degradation of estrogens. A functional polymorphism in the COMT gene (val158met), results in a 60–75% difference in enzyme activity between the val (high activity = H) and met (low activity = L) variants. The aim of this study was to determine if the COMT val158met polymorphism modulates the association between PA and BMD in young men.

Materials and Methods: The Gothenburg Osteoporosis and Obesity Determinants (GOOD) study consists of 1068 men (age, 18.9 ± 0.6 yr). Areal BMD (aBMD) was measured by DXA, whereas cortical and trabecular volumetric BMD (vBMD) were measured by pQCT. Study subjects were genotyped and classified as COMTLL, COMTHL, or COMTHH. The amount (h/wk) of PA was determined through questionnaires.

Results: Using a linear regression model (including age, height, weight, smoking, and calcium intake as covariates), significant interactions between the COMT genotype and PA were seen for aBMD at all sites and for trabecular vBMD in both the radius and the tibia. The difference in adjusted aBMD and trabecular vBMD between high (≥4 h/wk) and low PA (<4 h/wk) was greater in COMTLL subjects than in subjects homozygous for the COMTHH (total body aBMD: COMTLL 4.2% versus COMTHH 1.5%, p = 0.02; lumbar spine aBMD: COMTLL 7.8% versus COMTHH 3.9%, p = 0.04; tibia trabecular vBMD: COMTLL 7.1% versus COMTHH 1.0%, p < 0.01). The COMT polymorphism was associated with aBMD, at all sites and with trabecular vBMD in the low-PA subjects, but not in their high-PA counterparts.

Conclusions: We show that the COMT val158met polymorphism modulates the association between PA, aBMD, and trabecular vBMD, suggesting that this polymorphism is of importance for BMD in subjects with a low level of PA.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

A plethora of evidence indicates that the highest attained bone mass in life (i.e., peak bone mass) has a strong impact on bone mass at old age and affects the risk of developing osteoporosis.(1,2) Low BMD has been shown to be a strong independent predictor of fractures, both in men and in women.(3,4) Twin and family studies have shown that as much as 60–80% of the variation in the age-specific BMD can be attributed to genetic factors.(5–7) Several environmental factors also contribute considerably to the level of attained peak bone mass. Among these factors, physical activity (PA) and calcium intake are believed to have a stimulatory role on bone mass accretion, whereas smoking has been suggested to reduce bone acquisition early in life.(8–10)

A large number of studies have investigated genetic polymorphisms in several candidate genes for osteoporosis and their association with bone mass,(11) but only a few reports are available on how genetic polymorphisms in these candidate genes interact with environmental factors. In a small (n = 140) controlled and randomized study of middle-aged Finnish men, the PvuII polymorphism of estrogen receptor α (ERα) was associated with a BMD increase in subjects enrolled in a 4-yr aerobic exercise program, whereas no association was seen between this polymorphism and BMD in the reference group.(12) Suuriniemi et al.(13) showed that the association between exercise and pubertal BMD in girls is modulated by ERα genotype. Increasing evidence implicates estrogen status as an important determinant of male bone mass.(14–18) Several reports indicate that bone cells respond to mechanical strain and estrogen through the same signaling pathways.(19–22)

We have previously shown that both PA and a polymorphism in the estrogen degrading enzyme catechol-O-methyltransferase (COMT) are associated with areal BMD (aBMD) and trabecular volumetric BMD (vBMD) in young men.(23,24) The investigated COMT polymorphism constitutes a functional G to A polymorphism, which results in a valine to methionine substitution at codon 158. This amino acid replacement leads to a 60–75% difference in enzyme activity between the Val (high activity) and Met (low activity) variants.(25) The alleles are therefore termed COMTL (L = low activity) and COMTH (H = high activity), respectively.

We hypothesized that the COMT polymorphism could alter estrogen status, in the bone microenvironment and/or in the circulation, and therefore modulate the association between PA and bone parameters. The aim of this study was to determine if the COMT polymorphism modulates the association between PA and BMD in young adult men in the Gothenburg Osteoporosis and Obesity Determinants (GOOD) study.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Subjects

The GOOD study was initiated with the aim to determine both environmental and genetic factors involved in the regulation of bone and fat mass. Study subjects were randomly identified using national population registers, contacted by telephone, and asked to participate in this study. A total of 1068 men (age, 18.9 ± 0.6 yr) from the greater Gothenburg area were included. To be included in the GOOD study, subjects had to be >18 and <20 yr of age and willing to participate in the study. There were no other exclusion criteria; 48.6% of the contacted study subject candidates agreed to participate and were included in this study. A standardized questionnaire was used to collect information about amount of present PA (h/wk), nutritional intake (dairy products), and smoking. Calcium intake was estimated from dairy product intake and semiquantitated into quintiles.

The GOOD study was approved by the ethics committee at Gothenburg University. Written and oral informed consent was obtained from all study participants. We have previously presented baseline data on aBMD and bone parameters measured with pQCT in this cohort.(26)

Anthropometrical measurements

Height and weight were measured using standardized equipment. The CV values were <1% for these measurements.

DXA

aBMD (g/cm2) of the whole body, total femur, trochanter, femoral neck (of the left leg), and the lumbar spine (L2—L4) were assessed using the Lunar Prodigy DXA (GE Lunar Corp., Madison, WI, USA). The CVs for the aBMD measurements ranged from 0.5% to 3%, depending on application.

pQCT

A pQCT device (XCT-2000; Stratec Medizintechnik, Pforzheim, Germany) was used to scan the distal leg (tibia) and the distal arm (radius) of the nondominant leg and arm, respectively. A 2-mm-thick single tomographic slice was scanned with a voxel size of 0.50 mm. The cortical vBMD (not including the bone marrow; mg/cm3) and cortical cross-sectional area (CSA, mm2) were measured using a scan through the diaphysis (at 25% of the bone length in the proximal direction of the distal end of the bone) of the radius and tibia. Trabecular vBMD (mg/cm3) was measured using a scan through the metaphysis (at 4% of the bone length in the proximal direction of the distal end of the bone) of these bones. Tibia length was measured from the medial malleolus to the medial condyle of the tibia, and length of the forearm was defined as the distance from the olecranon to the ulna styloid process. The CVs were <1% for all pQCT measurements.

DNA isolation and genotyping

Genomic DNA was isolated from EDTA stabilized whole blood using the DNA Purigene Kit (Gentra Systems). The val158met polymorphism (rs 4680) of the COMT gene was successfully genotyped in 1050 subjects, as previously described.(23) Using this assay, we failed to genotype 18 of the 1068 subjects. The genotypes were classified as COMTLL (AA:Met), COMTHL (AG:Met/Val), or COMTHH (GG:Val).

Serum analyses of sex hormone binding globulin and sex steroids

Total estradiol was measured using an ultrasensitive radioimmunoassay (RIA; Orion Diagnostics, Esboo, Finland; intra-assay CV 3%, interassay CV 6%). Total testosterone was measured using RIA (Orion Diagnostics; intra-assay CV 6%, interassay CV 6%). Sex hormone binding globulin (SHBG) was measured using IRMA (Orion Diagnostics; intra-assay CV 3%, interassay CV 7%).

Free testosterone and free estradiol were calculated according to the method described by Vermeulen et al.(27) and Van den Beld et al.,(28) taking the concentrations of total testosterone, total estradiol, and SHBG into account and assuming a fixed albumin concentration of 43 g/liter.

Statistical analysis

Values are given as mean ± SD, unless otherwise indicated. All calculations were performed with the SPSS Statistical Software (version 14.0; SPSS, Chicago, IL, USA). Hardy-Weinberg equilibrium for the COMT polymorphism was calculated using a χ2 test.

χ2 tests were used to determine whether the distribution of smokers differed between the COMT genotype groups.

Continuous variables were compared between individuals with different COMT genotypes using ANOVA, followed by Bonferronís correction for multiple comparisons.

The independent predictors of the various bone parameters were tested using multiple linear regression analysis, including the investigated genetic polymorphism, height, weight, age, calcium intake (quintiles), smoking (yes/no), and PA (h/wk).

The general linear model (GLM) was used to perform linear regression analysis and to calculate interactions between the COMT polymorphism and PA. The percentage of the variation of each bone parameter (r2) explained by the COMT gene polymorphisms was calculated, using linear regression models. To illustrate the magnitude of the discrepancies in bone parameters between the high- and low-PA groups in subjects homozygous for COMTHH versus the corresponding discrepancies in subjects homozygous for COMTLL, we performed two-way analysis of covariance (ANCOVA), including an interaction term between PA groups and homozygous genotypes, with the same covariates as above.

A p value <0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Anthropometrics, genotype distribution, and sex hormone levels

Anthropometrics, sex hormone levels, and bone variables of the whole cohort are presented in Table 1. Three hundred fourteen subjects (29.9%) were homozygous for the low activity COMT allele (COMTLL), 527 subjects (50.2%) were heterozygous (COMTHL), and 209 (19.9%) subjects were homozygous for the high activity COMT allele (COMTHH). The genotype distribution was in Hardy-Weinberg equilibrium (p = 0.88).

Table Table 1.. Anthropometrics, Sex Hormone Levels, and Bone Variables
  1. Values are given as mean ± SD, n = 1068.

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There were no differences in age (LL, 18.9 ± 0.6 [SD] yr; HL, 18.9 ± 0.5 yr; HH, 18.9 ± 0.6 yr; p = 0.64), height (LL, 181.8 ± 6.7 cm; HL, 181.8 ± 6.7 cm; HH, 181.5 ± 6.8 cm; p = 0.25), weight (LL, 75.0 ± 13.5 kg; HL, 73.2 ± 10.7 kg; HH, 74.0 ± 10.0 kg; p = 0.09), frequency of smokers (LL, 8.9%; HL, 8.2%; HH, 10.5%; p = 0.59), daily calcium intake (LL, 1124 ± 728 mg/d; HL, 1104 ± 669 mg/d; HH, 1031 ± 849 mg/d; p = 0.34), or weekly PA (LL, 4.1 ± 5.0 h/wk; HL, 4.5 ± 5.3 h/wk; HH, 4.2 ± 5.6 h/wk; p = 0.49) between the genotype groups. Furthermore, the COMT polymorphism was not associated with total or free levels of either estradiol (free estradiol [pM]: LL, 1.39 ± 0.65; HL, 1.38 ± 0.66; HH, 1.51 ± 0.74; p = 0.30; data not shown) or testosterone (free testosterone [nM]: LL, 0.46 ± 0.17; HL, 0.46 ± 0.17; HH, 0.48 ± 0.16; p = 0.10; data not shown).

PA and COMT polymorphism as independent predictors of aBMD and trabecular vBMD

The independent predictive role of PA and COMT genotype for aBMD (DXA), as well as cortical and trabecular bone parameters (pQCT), were studied using linear regression, including age, weight, height, smoking, calcium intake, present amount of PA, and COMT polymorphism (Table 2). Both amount of PA and the COMT val158met polymorphism were independent predictors of aBMD of the total femur, trochanter, and femoral neck. Amount of PA also independently predicted aBMD of the total body and lumbar spine, whereas the COMT val158met polymorphism did not predict aBMD of the lumbar spine but showed a strong tendency toward being associated with aBMD of the total body (p = 0.05; Table 2). pQCT analyses showed that amount of PA and COMT val158met polymorphism were both independent predictors of trabecular vBMD, but only amount of present PA predicted cortical bone size (Table 2). Amount of present PA was a negative independent predictor of cortical vBMD in the radius but not in the tibia. The COMT val158met polymorphism was not associated with cortical vBMD (Table 2).

Table Table 2.. Amount of Present PA and COMT Genotype as Independent Predictors of aBMD and Trabecular vBMD
  1. Linear regression, including amount of present PA (h/wk), COMT genotype (coded as HH > HL > LL), age, height, weight, calcium intake, and smoking. Standardized β and p values are presented. n = 1050.

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COMT genotype modulates the association between PA and BMD

Using a linear regression model (including age, height, weight, smoking, and calcium intake as covariates), significant interactions between COMT genotype and PA were seen for aBMD at all sites and for trabecular vBMD in both the radius and the tibia (Table 2), whereas no interactions were seen for parameters of cortical bone size or cortical vBMD (Table 2).

We have previously shown that the lowest amount of PA associated with bone parameters in this cohort was 4 h/wk.(24) Based on this knowledge, subjects were divided into a low (<4 h/wk, n = 554) and high (≥4 h/wk, n = 514) PA group. To further characterize the interaction between PA and the COMT polymorphism for bone parameters, the high- and low-PA groups were further divided according to COMT genotype into a total of six subgroups (Fig. 1). Anthropometrics and sex hormone levels for each respective group are presented in Table 3. Because there were discrepancies between the different PA COMT genotype groups in anthropometric characteristics (Table 3), aBMD and trabecular vBMD values were adjusted for smoking status, calcium intake, age, height, and weight. After adjustment for these covariates, aBMD of the lumbar spine and total body, as well as trabecular vBMD of both the radius and tibia, were not higher in the high PA than in the low-PA COMTHH subjects (Figs. 1A, 1B, 1E, and 1F). In contrast, aBMD at all sites, as well as trabecular vBMD of both the radius and tibia, were higher in the high-PA groups than in the low-PA groups of both COMTHL and COMTHH subjects (Figs. 1A—1F). The difference in aBMD and trabecular vBMD between high and low PA was in general greater in COMTLL subjects than in subjects homozygous for the COMTHH (total body aBMD: COMTLL 4.2% versus COMTHH 1.5%, p = 0.02; lumbar spine aBMD: COMTLL 7.8% versus COMTHH 3.9%, p = 0.04; total femur aBMD: COMTLL 9.2% versus COMTHH 5.3%, p = 0.07; radius trabecular vBMD: COMTLL 9.9% versus COMTHH 2.3%, p = 0.02; tibia trabecular vBMD: COMTLL 7.1% versus COMTHH 1.0%, p < 0.01).

Table Table 3.. Anthropometric Characteristics and Sex Hormone Levels According to PA Level and COMT Genotype
  1. Means ± SD are given. p values are presented for comparison of the groups by one-way ANOVA, followed by Bonferroni's correction for multiple comparisons. Differences in smoking percentage between the groups were tested using χ2 tests. n = 1050.

  2. Low PA, <4 h/wk; high PA, ≥4 h/wk; COMTHH, homozygous for the high activity variant; COMTHL, heterozygous; COMTLL, homozygous for the low activity COMT variant; SHBG, sex hormone binding globulin; NS, not significant.

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Figure Figure 1. The association between PA amount, aBMD (A—D), and trabecular vBMD (E and F) is dependent on COMT genotype. +PA, physical activity ≥ 4h/wk; −PA, physical activity < 4 h/wk; HH, COMTHH; HL, COMTHL; LL, COMTLL. BMD values are adjusted for age, height, weight, calcium intake, and smoking. ANOVA was used to calculate p values for trend. Mean ± SE are presented.

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The COMT polymorphism was a clear predictor (COMTHH > COMTHL > COMTLL) of aBMD of the total body, lumbar spine, total femur, and trochanter (Figs. 1A—1D), as well as for trabecular vBMD (Figs. 1E—1F) in the subjects with low but not in the subjects with a high amount of PA.

To determine the magnitude of the variation of the various bone parameters explained in the low-PA subjects (<4 h/wk), by the COMT polymorphism, we used a multiple linear regression model, including age, height, weight, smoking, calcium intake, amount of PA as covariates. Addition of COMT genotypes to this regression model increased the percentage (aBMD total femur: 1.8%; trabecular vBMD tibia: 2.2%; data not shown) of the variation in aBMD and trabecular vBMD explained (r2) by the model. In contrast, in the subjects with high PA (≥4 h/wk), the inclusion of COMT genotypes in the model only marginally increased the percentage of the variation in bone parameters explained (aBMD total femur: 0.2%, not significant; trabecular vBMD tibia: 0.1%, not significant; data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

In this study, we confirmed our previous findings(23) in that the investigated COMT val158met polymorphism was associated with aBMD at several sites, as well as with trabecular vBMD, in an enlarged cohort of young men (the GOOD cohort). To the best of our knowledge, no other report studying the COMT val158met polymorphism in relation to BMD in men is available. A recent study found that the COMT val158met polymorphism was associated with bone loss of the radius but not to baseline aBMD at any site in Japanese postmenopausal women.(29) However, another report did not find any association between COMT val158met polymorphism and aBMD or bone loss in postmenopausal women.(30) In these reports on postmenopausal women, the possible interaction between the COMT val158met polymorphism and PA was not studied.

We recently reported that amount of PA was strongly independently associated with aBMD and trabecular vBMD in the GOOD cohort.(24) Given that both amount of PA and the COMT polymorphism were associated with both aBMD and trabecular vBMD, in addition to the fact that both PA and the estrogen system affect bone cells through common pathways,(19–21,31) we hypothesized that the COMT val158met polymorphism, possessing the ability to alter estrogen degradation, could interact with the association between PA and these bone parameters. The results of this study showed that COMT genotype modulates the association between PA and bone parameters, including aBMD at several locations and trabecular vBMD. The general pattern was that the COMT genotype predicts bone parameters in subjects with low amounts of PA but not in subjects with high amounts of PA. Our findings showed that the COMTHH genotype is associated with higher aBMD and trabecular vBMD than the COMTLL genotype, whereas the latter genotype displays a stronger association, than the former, with PA. These findings indicate that the COMTLL subjects have more to gain by PA, in terms of increased BMD, than their COMTHH counterparts. An alternative explanation is that subjects with a high amount of PA have a maximal response to mechanical loading that can not be further modulated by the COMT genotype, whereas subjects with a low amount of PA and submaximal response to mechanical loading are clearly affected by the COMT polymorphism.

We observed several-fold higher percentage differences between the high- and low-PA groups in the COMTLL subjects than in the COMTHH subjects (e.g., total body aBMD 4.2% versus 1.5% and tibia trabecular vBMD 7.1% versus 1.0%), suggesting that the COMT genotype might modulate the BMD response to mechanical loading. Our data indicate that early identification of COMTLL subjects and PA intervention could aid in maximizing peak BMD in young adult men. However, our results are based on young men and can therefore not be generalized and interpreted in regard to possible risk prevention in the population primarily subjected to osteoporosis (i.e., elderly women and men). The studied population in this study is primarily of white origin. Hence, the results from this study may not be transferable to populations of other ethnicity.

A mechanism, termed the mechanostat, has been proposed to sense the mechanical strain signal (which is produced when bones are subjected to loading), and as a result, regulate bone formation and resorption, to adapt the skeleton to the forces applied to it. Estrogen has been proposed to modulate the mechanostat.(32,33) Using intact and ovariectomized rats on earth and on the orbiting space station, Westerlind et al.(31) showed that estrogen governs the rate of bone turnover, but the greatest impact on the balance between bone formation and resorption is exerted by mechanical loading. It was recently shown that mice with a gene deletion of the ERα are unable to respond to PA with a periosteal bone expansion as seen in wildtype mice,(34) supporting the notion that estrogen signaling is involved in the skeletal response to mechanical loading induced by PA.

The roles of gene—environment interactions in the sex hormone system, and in general, have been inadequately studied. However, two recent studies report that the association between exercise and BMD in girls and in middle-aged men was modulated by ERα genotype.(12,13)

We have previously found that the functional COMT val158met polymorphism was associated with serum estradiol levels in middle-aged men,(35) but we failed to detect such a relationship in this study. Lack of this association was also reported from two separate cohorts of postmenopausal women.(29,36) It is not yet known whether the COMT gene is expressed in bone cells. 2-hydroxylation and 16α-hydroxylation constitute the two major pathways for inactivation of circulating estrogen.(33) COMT inactivates 2- and 4-hydroxyestradiol, suggesting that a polymorphism that reduces the activity of COMT could result in a shift toward a higher proportion of 2-and 4-hydroxy metabolites, leading to a shift toward the 16α-pathway.(25) These changes could possibly occur in the local bone environment, without affecting the serum levels of these metabolites. 16-hydroxy-estradiol retains estrogenic activity, whereas the 2-hydroxy-metabolites are devoid of estrogenic activity and may also possess anti-estrogenic activities.(33,37) Because of the lack of association between the COMT val158met polymorphism and serum sex hormone levels in this study, the mechanisms by which the COMT val158met polymorphism modulates the association between PA and BMD remains unclear. One could, however, speculate that the stronger association between PA and BMD seen in the COMTLL than in the COMTHH subjects can be explained by higher levels of bioactive estrogens in the bone microenvironment, giving rise to a favorable mechanical strain signal in response to PA in the COMTLL subjects. The COMT enzyme induced degradation of 2-hydroxyestradiol results in the metabolite 2-methoxyestradiol. This metabolite has been shown to be protective against bone loss after ovariectomy in rats.(38) Hence, an alternative explanation of how the COMT val158met polymorphism affects bone metabolism can be that the COMTLL subjects, in this study, could have lower levels of 2-methoxyestradiol, leading to lower BMD than the COMTHH subjects.

It is also possible that the COMT polymorphism affects peak bone mass through mechanisms not involving sex hormones and their metabolites. COMT is also involved in the degradation of catecholamines, and results from animal studies indicate that the sympathetic nervous system has a catabolic effect on bone.(39)

In conclusion, in this large population-based study, we showed that the COMT val158met polymorphism modulates the association between PA, aBMD, and trabecular vBMD in young men, suggesting that gene—environment interactions are of importance and can exert a substantial impact on the attainment of peak BMD in men.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This study was supported by the Swedish Research Council, the Swedish Centre for Research in Sports Medicine (CIF), the Swedish Foundation for Strategic Research, European Commission Grant QLK4-CT-2002–02528, the Lundberg Foundation, the Torsten and Ragnar Söderberg's Foundation, Petrus and Augusta Hedlunds Foundation, the ALF/LUA grant from the Sahlgrenska University Hospital, and the Novo Nordisk Foundation.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References