vation were excluded. Other exclusion criteria were known contrain- dications to HRT, recent use (1 yr) of oestrogen-containing compounds or tibolone, use of ...
0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society
Vol. 86, No. 5 Printed in U.S.A.
Angiotensin-I Converting Enzyme Genotype-Dependent Benefit from Hormone Replacement Therapy in Isometric Muscle Strength and Bone Mineral Density* DAVID WOODS, GLADYS ONAMBELE, ROGER WOLEDGE, DAWN SKELTON, STUART BRUCE, STEVE E. HUMPHRIES, AND HUGH MONTGOMERY Department of Cardiovascular Genetics (D.W., S.E.H., H.M.), Rayne Institute, University College London, London WC1E 6JJ, United Kingdom; Institute of Human Performance (G.O., R.W., S.B.), University College London, Royal National Orthapaedic Hospital, Stanmore HA7 4LP, United Kingdom; and Department of Cellular and Integrate Biology (D.S.), Division of Biomedical Sciences, Imperial College School of Medicine at St. Mary’s, London W2 1PG, United Kingdom ABSTRACT Low bone mineral density (BMD) and muscle weakness are major risk factors for postmenopausal osteoporotic fracture. Hormone replacement therapy (HRT) reverses the menopausal decline in maximum voluntary force of the adductor pollicis and reduces serum angiotensin-I converting enzyme (ACE) levels. The insertion (I) allele of the ACE gene polymorphism is associated with lower ACE activity and improved muscle efficiency in response to physical training. Therefore, we examined whether the presence of the I allele in postmenopausal women would affect the muscle response to HRT. Those taking HRT showed a significant gain in normalized muscle maxi-
L
OW MUSCLE STRENGTH and bone mineral density (BMD) and high body sway are independent and powerful synergistic predictors of fracture incidence in the elderly (1). A woman over 60 with high body sway and BMD in the lowest quartile has a 14-fold increased risk of fracture (8.4% per year) compared with a woman in the highest BMD quartile with low body sway. If we add to this equation low muscle strength there is a 13.1% risk of fracture per year (1). Although osteoporosis is a multifactorial condition, twin studies have shown that genetic factors may account for up to 80% of the variance in BMD (2). To date, research has focused on loci that may determine bone mass such as the vitamin D and oestrogen receptor genes as well as the collagen type 1 ␣1 locus. A polymorphism of the human angiotensin-I converting enzyme (ACE) gene has been identified in which the presence [insertion (I) allele] of a 287-bp fragment rather than the absence [deletion (D) allele] is associated with low ACE activity in both serum (3) and tissues (4). Studies from our laboratory have shown that the I allele is associated with improved muscle efficiency (5), a greater anabolic response (6), and an 11-fold greater increase in Received March 1, 2000. Revision received August 21, 2000. Rerevision received January 11, 2001. Accepted February 7, 2001. Address all correspondence and requests for reprints to: Major David Woods, M.D., Department of Diabetes and Endocrinology, Freeman Hospital, Newcastle Upon Tyne NE7 709, United Kingdom. * D.W. is supported by the Royal Army Medical Corps. S.E.H. and H.M. are supported by grants from the British Heart Foundation (RG95007 and SP97003). The original data were obtained in a study supported by Wyeth.
mum voluntary force slope, the rate of which was strongly influenced by ACE genotype (16.0 ⫾ 1.53%, 14.3 ⫾ 2.67%, and 7.76 ⫾ 4.13%, mean ⫾ SEM for II, ID, and DD genotype, respectively; P ⫽ 0.017 for gene effect, P ⫽ 0.004 for I allele effect). There was also a significant ACE gene effect in the response of BMD to HRT in Ward’s triangle (P ⫽ 0.03) and a significant I allele effect in the spine (P ⫽ 0.03), but not in the neck of femur or total hip. These data suggests that low ACE activity associated with the I allele confers an improved muscle and BMD response in postmenopausal women treated with HRT. (J Clin Endocrinol Metab 86: 2200 –2204, 2001)
duration of repetitive elbow flexion in response to physical training (7). A sudden decline in specific muscle strength of the adductor pollicis (AP) muscle [the ratio of its maximum voluntary force (MVF) to its cross-sectional area (CSA)] occurs with menopause but can be prevented (8) and partially regained (9) by hormone replacement therapy (HRT). HRT (9) and training (10), over a similar time course, are also both capable of increasing muscle strength without an increase in muscle size. Although HRT increases both muscle strength and BMD, the exact physiological mechanism is unknown. HRT reduces serum ACE activity (11), and we hypothesized that the I allele would be associated with greater strength gain due to HRT. Furthermore, because grip strength is an independent predictor of BMD (12) and improving strength has a positive effect on BMD (13) we also speculated that any genotype effect on strength gain may confer an additional advantage in the response of BMD to HRT. Materials and Methods The women used for this study were those from a previously described prospective, randomized, open-label, parallel-group trial (9) in which they were assigned to either a control group or HRT (Prempak C, 0.625 mg oestrogen per day and 0.15 mg norgestrel for 12 consecutive days in each cycle for 13 cycles of 4 weeks). Subjects who had pain or stiffness in the thumb, neuromuscular or generalized cardiovascular disease, or regular medication likely to effect muscle function or motivation were excluded. Other exclusion criteria were known contraindications to HRT, recent use (⬍1 yr) of oestrogen-containing compounds or tibolone, use of oestrogen implants within 3 yr, history of glucocor-
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AN ACE GENOTYPE-DEPENDENT BENEFIT FROM HRT Statistical analysis
ticoid use, coagulation disorders, malabsorption, alcohol or drug abuse, or any medication that would interfere with oestrogen metabolism. Blood testing was performed to screen for subclinical metabolic or endocrine abnormalities and subjects underwent a general and specific gynecological examination. Compliance throughout the study was assessed by serial blood samples. Of the 102 (52 controls and 50 HRT) women entering the study at baseline, 87 completed 39 weeks or more, and of these, 83 could be traced (45 control and 38 HRT). The higher drop-out rate among the HRT group was due to some subjects suffering adverse effects of treatment such as heavy bleeding and weight gain due to water retention, which are to be expected (14). MVF of the AP muscle was determined on eight occasions throughout the study period by measuring the force of adduction with the thumb in the plane of the palm of the hand and a transducer held between the proximal phalanx of the thumb and the metacarpal of the index finger. Multiple contractions of AP were recorded with the average of the best five contractions taken as the value for MVF (mean coefficient of variation for the repeated measurements was 5.1%). The slope of the linear regression of normalized MVF (i.e. values expressed relative to the mean for that subject, the simplest way to study proportional change in force) against time was calculated and used in the analysis. The CSA of the muscle was taken as the average of three measurements using an anthropometric technique (15) validated against computed tomography and magnetic resonance imaging with a coefficient of variation of 4.4%. BMD of the spine (L1-L4) and the hip (total, neck of femur, and Ward’s triangle) was measured using dual energy x-ray absorptiometry (QDR4500A; Hologic, Inc.) at initial screening, weeks 26 and 52. The in vivo coefficient of variation in the department performing the scans is 1% for the spine, 2% for the neck of femur and total hip, and 5% for Ward’s triangle. Absolute values for change in BMD over the study period for each individual at each site were used in statistical analysis, with the percentage change over baseline presented in the text and figures. Regular exercise levels for each participant were assessed by questionnaire recording number of minutes per week spent on different daily physical activities such as walking, swimming, and keeping fit. All 83 subjects who were traced provided a 5-mL sterile 0.9% saline mouthwash sample from which DNA was extracted. ACE genotype was determined using three-primer PCR amplification by two independent staff members from whom data on participants was concealed. The study was approved by the University of London Ethics Committee, and written informed consent was obtained from each participant.
TABLE 1. Baseline characteristics (mean ⫾ population randomized to HRT or control
SEM)
All results are presented as mean and se (sem). Allele frequencies were determined by gene counting. A 2 test was used to compare the observed numbers for each genotype with those expected for a population in Hardy-Weinberg equilibrium. Data were analyzed using a two-way analysis of covariance (ANCOVA) for the effects of treatment and genotype (or presence of I allele) on the rate of change per year of MVF for each subject. Various covariates were used to examine for confounding factors, including age, exercise, initial specific force, MVF at baseline, CSA at baseline, and change in CSA. Only age and initial specific force were ultimately used as covariates because they had significant effects and the latter includes force as a function of muscle size (although baseline cross-sectional area did have an effect, the change in cross-sectional area, exercise, and baseline MVF did not). Changes in BMD were also analyzed by two-way ANCOVA with treatment status and genotype (or presence of I allele) as factors with baseline bone density and age as covariates. P values of less than 0.05 were considered statistically significant.
Results
ACE genotype distributions were similar among the HRT and control groups and the null hypothesis that they were in Hardy-Weinberg equilibrium could not be rejected (22, 41, and 20 subjects of II, ID, and DD genotype, respectively). Baseline characteristics (Table 1) did not differ between those receiving HRT and the control group, and were independent of ACE genotype. The overall characteristics of the genotyped group did not differ significantly from that of the whole group (data not shown). Change in muscle force and BMD measurements are shown in Tables 2 and 3, respectively. As previously reported (9) the rate of change of MVF was significantly influenced by HRT (P ⬍ 0.001), but a significant ACE gene effect was also demonstrated (P ⫽ 0.02). Those taking HRT showed a significant gain in MVF slope, the rate of which was strongly influenced by ACE genotype (16.0 ⫾ 1.53%, 14.3 ⫾ 2.67%, and 7.76 ⫾ 4.13% for II, ID, and DD genotype, respectively). There was a significant difference comparing the II to DD and the ID to DD subjects (P ⫽ 0.005 and P ⫽ 0.01 respectively) but not between the II and ID subjects (P ⫽ 0.95). The presence of the I allele was decisive with the II and ID genotypes combined being significantly different to the DD subjects (P ⫽ 0.004, with age, initial specific force, and HRT all having significant effects; P ⫽ 0.036, P ⫽ 0.02, and P ⬍ 0.001) (Fig. 1). Those in the control group suffered a loss in muscle strength at a rate that was independent of genotype (3.18 ⫾ 2.33%, 1.92 ⫾ 1.88%, and 5.4 ⫾ 1.4%, mean ⫾ sem, for II, ID, and DD genotype, respectively). The increase in force was not due to an increase in the CSA of the AP, as the mean CSA remained within 2%
of the study
Treatment group
Control
HRT-treated
Group size Age (yr) Age at onset of menopause (yr) Body mass index (kg/m2) Oestrone level (pmol/L) Oestradiol level (pmol/L) Exercise (min/week)
n ⫽ 45 60.5 ⫾ 0.52 50.7 ⫾ 0.43 24.1 ⫾ 0.43 185.1 ⫾ 8.01 42.6 ⫾ 1.25 401 ⫾ 59
n ⫽ 38 60.7 ⫾ 0.51 50.3 ⫾ 0.56 24.5 ⫾ 0.57 183.7 ⫾ 11.34 42.0 ⫾ 1.28 343 ⫾ 53
TABLE 2. Baseline and changes in muscle parameters (mean ⫾
SEM)
by treatment status and ACE genotype
HRT
CSAa MVFb ISFc Change in CSAd Force changee a
Control
II
ID
DD
II
ID
DD
263.3 ⫾ 13.9 58.3 ⫾ 1.95 0.227 ⫾ 0.017 ⫺6.23 ⫾ 4.78 16 ⫾ 1.53
241.6 ⫾ 8.4 60.2 ⫾ 1.99 0.256 ⫾ 0.013 ⫺4.06 ⫾ 3.3 14.3 ⫾ 2.67
288.6 ⫾ 13.2 64 ⫾ 3.4 0.225 ⫾ 0.017 1.04 ⫾ 3.9 7.768 ⫾ 4.13
227.6 ⫾ 12 58.7 ⫾ 2.1 0.259 ⫾ 0.016 ⫺1.49 ⫾ 2.8 ⫺3.18 ⫾ 2.33
237 ⫾ 13.7 56.7 ⫾ 2.2 0.25 ⫾ 0.015 ⫺3.05 ⫾ 2.8 ⫺1.92 ⫾ 1.88
240.9 ⫾ 17.5 57.5 ⫾ 1.9 0.256 ⫾ 0.015 ⫺7.2 ⫾ 6.8 ⫺5.4 ⫾ 1.4
CSA of AP. MVF of AP in Newtons. ISF, Initial specific force; the ratio of the MVF of AP to its CSA (N/mm2). d Change in CSA over the study period. e Percentage change in the slope of normalized MVF over the study period. b c
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TABLE 3. Baseline and change in BMD (g/cm2) over 1 yr for spine, total hip, neck of femur, and Ward’s triangle by treatment status and ACE genotype HRT
Spine baseline Spine change Total hip baseline Hip change Neck of femur baseline Neck of femur change Ward’s baseline Ward’s change
Control
II
ID
DD
II
ID
DD
0.91 ⫾ 0.055 0.05 ⫾ 0.011 0.814 ⫾ 0.039 0.02 ⫾ 0.009 0.7 ⫾ 0.046 0.006 ⫾ 0.007 0.52 ⫾ 0.057 0.02 ⫾ 0.006
0.9 ⫾ 0.033 0.05 ⫾ 0.006 0.85 ⫾ 0.028 0.03 ⫾ 0.009 0.74 ⫾ 0.022 ⫺0.003 ⫾ 0.007 0.54 ⫾ 0.027 0.04 ⫾ 0.008
0.83 ⫾ 0.04 0.02 ⫾ 0.01 0.84 ⫾ 0.03 0.02 ⫾ 0.009 0.71 ⫾ 0.03 0.002 ⫾ 0.01 0.52 ⫾ 0.04 0.03 ⫾ 0.006
0.83 ⫾ 0.03 0.006 ⫾ 0.011 0.86 ⫾ 0.028 ⫺0.008 ⫾ 0.009 0.72 ⫾ 0.029 ⫺0.01 ⫾ 0.005 0.54 ⫾ 0.034 ⫺0.002 ⫾ 0.01
0.87 ⫾ 0.03 ⫺0.006 ⫾ 0.007 0.86 ⫾ 0.024 0.01 ⫾ 0.005 0.74 ⫾ 0.02 ⫺0.008 ⫾ 0.006 0.54 ⫾ 0.021 0.02 ⫾ 0.009
0.94 ⫾ 0.03 0 ⫾ 0.007 0.82 ⫾ 0.02 0.007 ⫾ 0.007 0.7 ⫾ 0.02 ⫺0.003 ⫾ 0.006 0.49 ⫾ 0.02 0.01 ⫾ 0.008
FIG. 2. BMD change per year as percentage of baseline by presence of I allele and treatment status for total hip, Ward’s triangle, neck of femur, and spine. Unadjusted data are presented but statistical significance is after adjustment for initial BMD and age. There was a significant effect of the I allele in the spine (P ⫽ 0.03) and evidence of interaction with HRT (P ⫽ 0.012).
FIG. 1. Rate of force change per year (mean ⫾ SEM) by presence of the I allele and treatment status. Unadjusted data are presented but statistical significance was estimated after adjustment for the baseline initial specific force and age. Two-way ANOVA revealed a significant I allele effect (P ⫽ 0.004).
of the initial value throughout the study period in both groups and there was no difference in change in CSA either between genotypes or treatment groups. In the analysis of BMD changes, two-way ANCOVA for the effect of HRT and the presence of the I allele revealed a significant HRT effect in the spine (P ⬍ 0.001), total hip (P ⫽ 0.002), and Ward’s triangle (P ⫽ 0.004), but not in the neck of femur (P ⫽ 0.21). The I allele had a significant effect in the BMD response of the spine (P ⫽ 0.03) and an interaction with HRT (P ⫽ 0.01) that wasn’t present in the total hip (P ⫽ 0.56),
neck of femur (P ⫽ 0.71), or Ward’s triangle (P ⫽ 0.51) (Fig. 2). Interaction between the ACE gene and HRT was also significant in the spine (P ⫽ 0.03), and a significant effect was seen in Ward’s triangle (P ⫽ 0.03), but not in the total hip (P ⫽ 0.28) or neck of femur (P ⫽ 0.75). There seemed to be a heterozygote advantage in the treated group for Ward’s triangle where BMD improved by 3.27 ⫾ 1.15%, 8.13 ⫾ 1.48%, and 5.6 ⫾ 1.16% (mean ⫾ sem, increase per year compared with baseline for II, ID, and DD genotypes, respectively). The difference between ID and II subjects was significant (P ⫽ 0.02) but not between the ID and DD (P ⫽ 0.19) or II and DD (P ⫽ 0.26). BMD of the spine increased by 5.06 ⫾ 1.21%, 5.64 ⫾ 0.66%, and 2.16 ⫾ 1.2% for the II, ID, and DD genotypes, respectively, in the treated groups. The BMD increase in ID vs. DD subjects was significant (P ⫽ 0.04), that of II vs. DD approached significance (P ⫽ 0.06), and no difference was seen between II and ID (P ⫽ 0.56). Discussion
This is the first report suggesting that the benefit to isometric strength from HRT is ACE genotype dependent. The II and ID subjects each had a significantly greater benefit from HRT than did the DD subjects. Analysis by presence of the I allele was also significant. Both analyses are required because although serum ACE levels correspond with the ACE genotype such that DD is greater than ID, which is
AN ACE GENOTYPE-DEPENDENT BENEFIT FROM HRT
greater than II (3), tissue ACE activity is lower in the presence of the I allele with no difference between the II and ID genotypes (4). This is also the first demonstration of a significant ACE genotype and I allele effect in the BMD response to HRT in Ward’s triangle and the spine, respectively. The suggestion of a heterozygote advantage evident in Ward’s triangle may be due to the small sample size and needs to be confirmed in larger studies. Important interactions between oestrogen and the reninangiotensin system exist, but the mechanism of the ACE gene polymorphism effect on muscle strength and BMD in response to HRT is unknown. The effect of lower ACE levels associated with the I allele could be due to direct or indirect actions through one of several physiologically relevant systems. HRT stimulates the synthesis of angiotensinogen (16), a potentially disadvantageous effect as renin cleaves angiotensinogen to generate angiotensin I, from which the vasoconstrictor angiotensin II is derived by the action of ACE. This rise in substrate may be countered by the reduction in renin and serum ACE activity that also occurs with HRT (11), and may reduce inactivation of the vasodilator bradykinin (16). The overall effect in the rat, where oestrogen treatment reduces tissue ACE messenger RNA, is a reduction in angiotensin II (17). The I allele is similarly associated with low ACE activity (3, 4), an increase in the half-life of bradykinin and reduced production of angiotensin II (18), effects that may be synergistic with those of HRT. Oestrogen-deficient rats increase vascular expression of the angiotensin II AT1 receptor, increasing the efficacy of angiotensin II on vasoconstriction, an effect reversed by oestrogen replacement (19). HRT (20) and the II genotype (21) are both associated with enhanced endothelium-dependent vasodilatation via stimulation of local nitric oxide and prostacyclin production, vascular effects that may represent a potential common, and perhaps synergistic, pathway to explain the observed genotype-dependent effects. The effect on muscle strength cannot be explained by an increase in muscle bulk as there was no significant increase in CSA, but it is possible that alterations in muscle substrate use and efficiency are important. Local skeletal muscle reninangiotensin-systems may modify substrate use and skeletal muscle cells contain a complete kallikrein-kinin system (22). Physiological doses of bradykinin, the half-life of which is prolonged in the II genotype, produce an increase in muscle blood flow and glucose extraction rate (23) and a stimulation of protein synthesis (24). It has also been speculated (9) that oestrogen may alter the sensitivity of the myosin cross-bridges to various metabolites. Oestrogen may regulate the expression of important genes in the rat myocardium, particularly myosin heavy chain (25), and oestrogen replacement in ovariectomized rats prevents the decline in the V1 myosin isoform that would otherwise occur (26). The exact mechanisms of the effect of HRT on BMD have not yet been fully elucidated. Resistance training can increase muscle strength and BMD over a year in postmenopausal women (13) in whom grip strength is an independent predictor of BMD (12). Whether the absolute increase in muscle
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strength of AP seen in our work (8.68 ⫾ 1.26 N and ⫺1.78 N, mean ⫾ sem, for HRT and control subjects, respectively) can be extrapolated to the changes in BMD are a matter of speculation. The apparent benefit conferred by the ACE genotype may be independent of ACE levels and the RAS and instead be due to linkage disequilibrium of the ACE I/D polymorphism with another gene or genes that may be responsible. The fact that the effects of the ACE genotype were confined to the spine and Ward’s triangle may be due to the greater benefit from HRT reported in the trabecular rich vertebral bodies of the spine (27) and Ward’s triangle compared with the neck of femur (28). Indeed our cohort did not demonstrate any significant benefit from HRT in the neck of femur, making it unlikely that the ACE genotype could be shown to confer any advantage. However, had the trial continued for longer, and perhaps with greater numbers, we may have been able to demonstrate a beneficial effect from HRT in these areas, as others have done, and perhaps reveal a coexistent effect of the ACE genotype. Reported increases in BMD with HRT compared with baseline and controls varies from 1.8% after 2.5 yr (29) to 5.5% after 10 yr (30). Therefore, the potential further 2.5–3% that the ACE genotype may account for in the spine and Ward’s triangle is likely to be biologically important. With the additional improvement in strength the predicted reduction in falls and osteoporotic fracture is likely to be clinically significant. Future prospective studies are needed to confirm these findings and clarify whether the effect is apparent in other muscle groups. If these effects are due to reduced ACE activity this may prompt further research and, one day, perhaps optimal combination therapy with HRT and ACE inhibition in fracture prevention. References 1. Nguyen T, Sambrook P, Kelly P, et al. 1993 Prediction of osteoporotic fractures by postural instability and bone density. Br Med J. 307:1111–1115. 2. Giguere Y, Rousseau F. 2000 The genetics of osteoporosis: “complexities and difficulties.” Clin Genet. 57:161–169. 3. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. 1990 An insertion/deletion polymorphism in the angiotensin-1-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 86:1343–1346. 4. Danser AHJ, Schalekamp MADH, Bax WA, et al. 1995 Angiotensin converting enzyme in the human heart: effect of the deletion/insertion polymorphism. Circulation. 92:1387–1388. 5. Williams AG, Rayson MP, Jubb M, et al. 2000 The ACE gene and muscle performance. Nature. 403:614. 6. Montgomery H, Clarkson P, Barnard M, et al. 1999 Angiotensin-convertingenzyme gene insertion/deletion polymorphism and response to physical training. Lancet. 353:541–545. 7. Montgomery HE, Marshall RM, Hemingway H, et al. 1998 Human gene for physical performance. Nature. 393:221–222. 8. Phillips SK, Rook KM, Siddle NC, Bruce SA, Woledge RC. 1993 Muscle weakness in women occurs at an earlier stage than in men, but strength is preserved by hormone replacement therapy. Clin Sci. 84:95–98. 9. Skelton DA, Phillips SK, Bruce SA, Naylor CH, Woledge RC. 1999 Hormone replacement therapy increases isometric muscle strength of adductor pollicis in postmenopausal women. Clin Sci. 96:357–364. 10. Jones DA, Rutherford OM, and Parker DF. 1989 Physiological changes in skeletal muscle as a result of strength training. Q J Exp Physiol. 74:233–256. 11. Proudler AJ, Hasib Ahmed AI, Crook D, Fogelman I, Rymer JM, Stevenson JC. 1995 Hormone replacement therapy and serum angiotensin-convertingenzyme activity in postmenopausal women. Lancet. 346:89 –90. 12. Kroger H, Tuppurainen M, Honkanen R, Alhava E, Saarikoski S. 1994 Bone mineral density and risk factors for osteoporosis: a population-based study of 1600 perimenopausal women. Calcif Tissue Int. 55:1–7. 13. Rhodes EC, Martin AD, Taunton JE, Donnelly M, Warren J, and Elliot J. 2000 Effects of one year of resistance training on the relation between muscular strength and bone density in elderly women. Br J Sports Med. 34:18 –22.
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