Plasma adiponectin levels in postmenopausal women with or without long-term hormone therapy

Maturitas 54 (2006) 65–71

Plasma adiponectin levels in postmenopausal women with or without long-term hormone therapy Jee-Aee Im a , Ji-Won Lee b , Hye-Ree Lee b , Duk-Chul Lee b,∗ a

Department of Laboratory Medicine, MizMedi Hospital, 701-4 Naebalsan-dong, Gangseo-Gu, Seoul, Republic of Korea b Departments of Family Medicine, Yonsei University, College of Medicine, Yongdong Severance Hospital, Kangnam, P.O. Box 1217, Seoul, Republic of Korea Received 6 May 2005; received in revised form 18 August 2005; accepted 29 August 2005

Abstract Objective:: Recent large, prospective, randomized studies show that hormone therapy (HT) does not confer a protective effect against cardiovascular disease (CVD) but may in fact increase cardiovascular events. Low plasma adiponectin levels are considered to be related to the development of atherosclerosis and CVD. The purpose of this study was to determine the effect of long-term hormone therapy on plasma adiponectin levels in postmenopausal women. Methods:: We recruited a total of 88 postmenopausal women aged 55–69 years old. Our sample consisted of 44 women who had undergone estrogen plus progestogen therapy (EPT) for more than 5 years and 44 age-matched women who had not received HT. We measured plasma adiponectin levels, the serum levels of their lipid profiles, high-sensitivity C-reactive protein (hs-CRP), fasting glucose levels, fasting insulin levels and estradiol levels. Their medical histories including their age at menopause, vitamin use, exercise, alcohol ingestion and cigarette smoking were also assessed by a questionnaire. Results:: The mean duration (mean ± S.D.) of HT was 8.4 ± 2.4 years. The mean serum estradiol level (mean ± S.D.) of the HT group was 47.9 ± 36.8 pg/L, significantly higher than that of the non-HT group (p < 0.01). Plasma adiponectin levels were significantly lower in the HT group than in the non-HT group (p < 0.05). Plasma adiponectin levels were inversely correlated to cholesterol, triglycerides and HOMA-IR (r = −0.33, p < 0.05; r = −0.40, p < 0.01 and r = −0.30, p < 0.05, respectively) in the non-HRT group, but such correlations were not seen in the HT group. In the multivariate analysis, hormone therapy and serum estradiol levels were the independent factors associated with plasma adiponectin levels after adjustments were made for potential confounders. Conclusion:: Plasma adiponectin levels were significantly lower in postmenopausal women with long-term HT than in those without HT, suggesting that long-term HT may modulate plasma adiponectin level in postmenopausal women. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Adiponectin; Estrogen; Menopause; Hormone therapy

∗

Corresponding author. Tel.: +82 2 2019 3483; fax: +82 2 3463 3287. E-mail address: [email protected] (D.-C. Lee).

0378-5122/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2005.08.008

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1. Introduction

2. Patients and methods

Recent results from a randomized trial in women without coronary heart disease (CHD) conducted by the Women’s Health Initiative (WHI) [1] show a 30% increased risk of CHD in women assigned to estrogen plus progestin therapy compared with women assigned a placebo. The increased CHD risk in the treatment group appeared soon after randomization and persisted through 6 years of follow-up. A Heart and Estrogen/progestin Replacement Study (HERS) shows a 50% increased risk of CHD among women assigned to estrogen plus progestin therapy during the first year after randomization [2,3]. Interestingly, women who received hormone therapy (HT) showed increased risk for coronary disease, despite favorable changes in serum lipoprotein levels. Therefore, HT appears to worsen coronary atherosclerosis through mechanisms that are not counteracted by the potentially beneficial effects on lipoproteins. Mediators of this negative effect on cardiovascular events remain poorly understood. However, administration of HT (0.625 mg of conjugated equine estrogen plus 2.5 mg of medroxyprogesterone acetate) in postmenopausal women was shown to decrease insulin sensitivity significantly in a recent, randomized, double-blind, placebo-controlled study [4]. Considering the established atherogenic effects of insulin resistance [5,6], it is postulated that insulin resistance and/or the associated pro- and anti-inflammatory cytokines, such as adipokines, may contribute to the findings of increased cardiovascular events seen in recent clinical trials studying the effects of HT [1–3]. Adiponectin is identified as the most abundant protein produced in adipose tissue [7] and is present in large quantities in the human bloodstream [8]. Adiponectin has a protective role against atherosclerosis via its anti-atherogenic and anti-inflammatory properties [9,10]. Additionally, hypoadiponectinemia is known to be associated with decreased insulin sensitivity [11] and increased risk for the development of cardiovascular disease [12]. Based on these observations, it is hypothesized that HT in postmenopausal women increases insulin resistance and/or atherogenesis through the mechanism of modulating adiponectin secretion. The aim of this study was to investigate the effect of long-term HT on the plasma adiponectin levels in postmenopausal women.

2.1. Patients Study subjects were recruited through an advertisement at the health promotion center in a women’s hospital in Seoul, South Korea. All subjects signed an informed consent form approved by the hospital’s Ethical Committee. Two groups of study subjects were created on the basis of hormone therapy history: the HT group and the age-matched non-HT group. The HT group was defined as postmenopausal women aged 55–69 years who had been taking estrogen (0.625 mg of conjugated equine estrogen or 2 mg of estradiol combined with progestogen) for more than 5 years. The non-HT group was defined as postmenopausal women aged 55–69 years who had not taken hormone therapy after menopause. Because the reduction of plasma adiponectin levels is known to be associated with chronic diseases, such as hypertension and diabetes mellitus, and the liver is known to be one of the main action sites of lipid and estrogen metabolism, we excluded subjects with high blood pressure (mean systolic blood pressure ≥150 mmHg or mean diastolic blood pressure ≥95 mmHg after two consecutive measurements), high fasting blood glucose levels (≥126 mg/dL), abnormal liver function tests (AST > 35 IU/L or ALT > 35 IU/L), and/or medical histories of surgical hysterectomy or current illnesses (hypertension, diabetes mellitus and liver disease). We used a questionnaire to assess the medical histories of the study subjects, and included questions about illness, current medications, age at menopause, duration of HT and vitamin use, as well as lifestyle choices, such as exercise regimen, alcohol ingestion and cigarette smoking. Vitamin use was defined as daily use of Vitamin C, Vitamin E or a multivitamin. Jogging, walking, swimming, cycling, dancing or participating in alternate forms of exercise more than three times a week for at least 30 min defined the exercise criteria. Alcohol ingestion was defined as the ingestion of alcohol more than one time each week. Smoking was defined as current cigarette smoking. Forty-four postmenopausal women who had been taking HT and 44 age-matched, postmenopausal women with no history of HT were eligible for participation in this study after assessing the inclusion and exclusion cri-

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teria. The mean duration of HT (±S.D.) was 8.4 ± 2.4 years. 2.2. Anthropometric evaluation Body weight was measured to the nearest 0.1 kg on an electronic scale. Subjects were weighed in light clothing and without shoes. Height was measured to the nearest 0.1 cm using a wall mounted stadiometer. Body mass index (BMI) was defined as weight/height2 in kg/m2 . For the evaluation of fat distribution, waist and hip circumferences were measured and the waist-to-hip ratio (WHR) was calculated. Percent body fat (PBF) was assessed by the bioelectrical impedance method (Inbody 3.0, Biospace Ltd., Seoul, Korea). 2.3. Biochemical assays Biochemical tests were performed on blood samples collected after fasting for more than 12 hours. Venous blood was drawn, and after centrifugation of the specimen, serum and plasma were frozen immediately in a refrigerator at −80 ◦ C. Serum levels of blood glucose, total cholesterol, HDL-cholesterol and triglyceride were assayed using an ADVIA 1650 Chemistry system (Bayer, Tarrytown, NY, USA). LDLcholesterol was calculated by Friedewald’s formula [13]. Fasting insulin and estradiol were assayed by electrochemi-luminescence immunoassay using Elecsys 2010 (Roche, Indianapolis, IN, USA). Highsensitivity C-reactive protein (hs-CRP) was measured by a latex-enhanced immunoturbidimetric assay using an ADVIA 1650 Chemistry system (Bayer, Tarrytown, NY, USA); the inter-assay and intra-assay reproducibilities were 2.70 ± 1.13 and 2.55 ± 1.0%, respectively. Insulin resistance was estimated by the homeostasis model assessment of insulin resistance (HOMAIR) index (fasting insulin [U/mL] × fasting glucose [mmol/L])/22.5). Plasma adiponectin levels were measured by EIA kit (Komed, Seoul, Korea); the inter-assay and intra-assay reproducibilities were 4.63 ± 0.82 and 2.72 ± 0.52%, respectively.

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and the Chi-square (χ2 ) test or Fisher’s Exact Test for the categorical variables. Because of the skewed distributions of plasma adiponectin levels, hs-CRP, triglyceride levels, fasting insulin levels and estradiol levels, logarithmically transformed values of these parameters were used in the t-test, correlation analysis and multiple linear regression analysis, but untransformed raw data for the values of the mean ± S.D. are presented in tables. Plasma adiponectin levels were compared between HT and non-HT groups using the t-test. Pearson’s correlation coefficients were calculated to evaluate the relationship between plasma adiponectin levels and the continuous variables in this study. A multiple linear regression analysis was performed to determine the association between plasma adiponectin levels and hormone therapy after adjusting potential confounders. Significance was defined at the 0.05 level of confidence. All data were analyzed using the statistical program SAS 8.01 (SAS Institute, Cary, NC, USA). 3. Results 3.1. Clinical characteristics The clinical characteristics of the study subjects are shown in Table 1. No statistical differences were shown to exist in mean age, age at menopause, blood pressure, serum levels of total cholesterol, triglycerides, fasting insulin levels, HOMA-IR and hs-CRP levels between the two subject groups. In addition, there were no differences in anthropometric measures, such as BMI, WHR and PBF, and health behaviors, such as smoking habits, alcohol ingestion, exercise and vitamin intake between two groups. Women using HT had significantly higher serum estradiol and HDL-cholesterol levels than women who were not using HT (p < 0.01 and p < 0.05, respectively). In contrast, serum fasting glucose and LDL-cholesterol level were significantly lower in the HT group than in the non-HT group (p < 0.01 and p < 0.05, respectively).

2.4. Statistical analysis

3.2. Plasma adiponectin levels in the HT group and the non-HT group

Data are expressed as means ± S.D. Clinical characteristics were compared between the HT and the non-HT groups using t-tests for continuous variables

Plasma adiponectin levels were significantly lower in the HT group than in the non-HT group (p < 0.05) as seen in Fig. 1.

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HOMA-IR, fasting glucose levels, total cholesterol, triglycerides, smoking history, alcohol ingestion, vitamin use and exercise as shown in Table 3. When serum estradiol was combined with hormone therapy in a multivariate analysis, hormone therapy was no longer shown to have a significant association with adiponectin levels, but serum estradiol was shown to have a significant association with plasma adiponectin levels.

4. Discussion Fig. 1. Plasma adiponectin concentration in postmenopausal women with hormone therapy with estrogen (conjugated equine estrogen 0.625 mg or estradiol 2 mg) plus progestogen for more than 5 years compared with postmenopausal women without hormone therapy after menopause. Adiponectin levels were significantly decreased in HT group.

3.3. Correlation between plasma adiponectin levels, lipids and other parameters Plasma adiponectin levels were inversely correlated with serum levels of total cholesterol and triglycerides (r = −0.33, p < 0.05 and r = −0.40, p < 0.01, respectively) and HOMA-IR (r = −0.30, p < 0.05) in the non-HT group but these relationships were not seen in the HT group. Fasting glucose levels, however, were shown to have a significant inverse correlation with plasma adiponectin levels in the HT group (r = −0.43, p < 0.01), but not in the non-HT group. Plasma adiponectin levels were shown to have an insignificant negative correlation with anthropometric measures, such as BMI, PBF and WHR in the nonHT group (r = −0.30, p = 0.05; r = −0.28, p = 0.06 and r = −0.24, p = 0.12, respectively) and with serum estradiol levels in the HT group (r = −0.26, p = 0.08) as seen in Table 2. 3.4. Multiple linear regression analysis to assess independent relationships between adiponectin and clinical variables Hormone therapy was found to be an independent factor associated with plasma adiponectin levels after adjustments were made for the potential confounders, such as subject age, blood pressure, BMI, PBF, WHR,

In the present study, we reported that plasma adiponectin levels in postmenopausal women who undergo HT were significantly lower than in women who had never taken HT. We have also stated that HT was an independent factor associated with plasma adiponectin levels after adjusting for potential confounders in a multivariate analysis. Sites et al. [4] recently reported that continuous, combined oral estrogen and progestin therapy in postmenopausal women reduced insulin sensitivity by 17% within 6 months and that these effects persisted with treatment for 2 years. Moreover, the reduction in insulin sensitivity occurred without affecting body composition and fat distribution, suggesting an alternative mechanism for insulin resistance other than increased regional, visceral fat. Given the established insulinsensitizing effect of adiponectin, insulin sensitivity could be attenuated by decreased plasma adiponectin levels. Our results support the research of Sites et al. [4] as well as suggest a mechanism for decreased insulin sensitivity. The mechanism of reduced plasma adiponectin levels in postmenopausal women with HT is poorly understood. Previous studies in postmenopausal women suggest that estradiol production and bioavailability have important implications for circulating adiponectin and whole-body insulin resistance [14–16]. Tanko et al. [14] reported that estradiol produced by adipocytes reduces the secretion of anti-inflammatory cytokine, e.g. adiponectin, and that this mechanism is directly involved in the inflammatory response and aggravation of atherosclerosis. Kalish et al. [15] reported that high plasma bioavailable estradiol levels predicted insulin resistance after adjusting for body composition in early postmenopausal women. Gavrila et al. [16] reported

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Table 1 Clinical characteristics of study subjects Characteristics

HT groupa (N = 44)

Non-HT groupb (N = 44)

p-Value

Age (years) Age at menopause (years) BMIc (kg/m2 ) PBFd (%) WHRe Systolic BPf (mmHg) Diastolic BPg (mmHg) Fasting glucose (mg/dL) Cholesterol (mg/dL) Triglycerides (mg/dL) HDL-cholesterolh (mg/dL) LDL-cholesteroli (mg/dL) Fasting insulin (uIU/mL) HOMA-IRj hs-CRPk (mg/dL) Estradiol (pgl/L) Smokingl N (%) Alcoholn N (%) Exerciseo N (%) Vitamin intakep N (%)

59.9 ± 3.3 49.8 ± 3.4 23.3 ± 2.4 30.5 ± 3.8 0.84 ± 0.04 126.1 ± 12.1 73.8 ± 10.5 83.7 ± 9.7 186.0 ± 38.6 117.0 ± 51.8 59.2 ± 11.3 103.5 ± 35.7 6.62 ± 5.9 1.36 ± 1.25 0.23 ± 0.71 47.9 ± 36.8 0 (0) 12 (27.2) 24 (54.5) 29 (65.9)

59.9 ± 4.2 49.8 ± 3.8 24.1 ± 2.6 31.1 ± 5.0 0.85 ± 0.07 127.0 ± 11.8 75.2 ± 8.6 95.1 ± 8.3 199.3 ± 24.2 133.7 ± 78.0 53.8 ± 8.9 118.7 ± 22.0 4.71 ± 3.1 1.11 ± 0.72 0.087 ± 0.18 7.9 ± 2.6 1 (2.27) 6 (13.6) 22 (50.0) 25 (47.7)

0.93 0.96 0.12 0.53 0.63 0.73 0.49 <0.01 0.06 0.24 <0.05 <0.05 0.06 0.26 0.06 <0.01 0.49m 0.11 0.09 0.09

Data are shown as means ± the standard deviation. p-Values are calculated by t-test and X2 -test. a Postmenopausal women with hormone therapy with estrogen (conjugated equine estrogen 0.625 mg or estradiol 2 mg) plus progestogen for more than 5 years. b Postmenopausal women without hormone therapy after menopause. c Body mass index. d Percent body fat. e Waist-to-hip ratio. f Systolic blood pressure. g Diastolic blood pressure. h High density of lipoprotein cholesterol. i Low density of lipoprotein cholesterol. j Homeostasis model assessment of insulin resistance. k High-sensitivity C-reactive protein. l Smoking habit was defined as active smoking at the present. m Cells have expected count less than 5, p-value is calculated by Fisher’s Exact Test. n Alcohol ingestion ≥1 time a week. o Doing regular exercise ≥3 times a week. p Use of more than one vitamin among Vitamin C, Vitamin E or multivitamin.

that estradiol levels are negatively and independently associated with adiponectin levels. In animal studies, the administration of nonovarian estrogen induced the reduction of adiponectin, while the administration of the estrogen receptor antagonist reversed this effect [17,18]. Taken together, the results of these previous studies and the results of our study suggest that high plasma estradiol levels in HT users might inhibit the adiponectin secretion of adipocytes. In this study, plasma adiponectin levels were inversely correlated to cholesterol, triglycerides, BMI,

CRP, fasting insulin levels and HOMA-IR in the nonHT group, which is consistent with previous studies [8,19–22]. These correlations, however, were not shown in the HT group. Moreover, HT users showed more favorable lipid profiles and lower fasting glucose levels in spite of lower plasma adiponectin levels compared to the non-HT group. The mechanism that would explain this discrepancy is not clear. However, it is well-known that estrogen lowers LDLcholesterol by upregulating apo B100 receptors and increases HDL-cholesterol by inhibiting hepatic lipase

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Table 2 Correlations between adiponectin and various parameters in the two groups Parameters

Age (years) Systolic BPc (mmHg) Diastolic BP (mmHg) BMId (kg/m2 ) PBFe (%) WHRf Fasting glucose (mg/dL) Cholesterol (mg/dL) Triglyceride (mg/dL) HDL-cholesterolg (mg/dL) Fasting insulin (␮IU/mL) HOMA-IRh hs-CRPi (mg/L) Estradiol (pg/L)

HT groupa

Non-HT groupb

Adiponectin

Adiponectin

r

p

r

p

0.22 0.10 0.16 −0.004 −0.04 0.04 −0.43 −0.02 −0.10 0.09 −0.07 −0.14 0.03 −0.26

0.15 0.54 0.29 0.98 0.81 0.80 <0.01 0.88 0.50 0.55 0.67 0.37 0.84 0.08

0.02 −0.13 −0.16 −0.30 −0.28 −0.24 −0.18 −0.33 −0.40 0.10 −0.28 −0.30 −0.29 −0.10

0.90 0.39 0.31 0.05 0.06 0.12 0.25 <0.05 <0.01 0.50 0.06 <0.05 0.06 0.51

Coefficients (r) and p-values are calculated by Pearson correlation model. a Postmenopausal women with hormone therapy with estrogen (conjugated equine estrogen 0.625 mg or estradiol 2 mg) plus progestogen for more than 5 years. b Postmenopausal women without hormone therapy after menopause. c Blood pressure. d Body mass index. e Percent body fat. f Waist-to-hip ratio. g High density lipoprotein cholesterol. h Homeostasis model assessment insulin resistance. i High sensitivity C-reactive protein.

Table 3 Multivariate analysis to assess relationships between adiponectin, hormone therapy and serum estradiol Variables Model 1a Hormone therapyb

Regression coefficient

S.E.

p

−1.85

0.55

<0.01

−0.42 −0.85

0.90 0.42

0.64 <0.05

2c

Model Hormone therapy Serum estradiol

a Regression coefficients were adjusted for age, systolic blood pressure, diastolic blood pressure, body mass index, percent body fat, waist-hip circumference ratio, homeostasis model assessment insulin resistance(HOMA-IR), fasting glucose, low density lipoprotein, and a current history of smoking, alcohol ingestion, vitamin use, and exercise (R2 ; 0.28, F-value 2.07, p < 0.05). b Hormone therapy = 1, non-hormone therapy = 0. c Added serum level of estradiol as a independent variable to the Model 1 (R2 ; 0.32, F-value 2.29, p < 0.01).

activity [23], this favorable effect of HT might counteract the negative effects of hypoadiponectinemia on lipid metabolisms. Adiponectin has been shown to have direct effects on the vasculature in addition to having an inverse relationship with the measures of CVD [19–22]. Adiponectin accumulates in injured vessel walls and dose-dependently inhibits TNF-␣-induced cell adhesion in human aortic endothelial cells, an early step of atherosclerosis [21,24,25]. Cross-sectional studies report that the relationship between hypoadiponectinemia and CVD remains intact after adjustment for cardiovascular risk factors, such as diabetes, dyslipidemia, hypertension, smoking and BMI [12,26]. Based on this knowledge, it could be postulated that the reduction of plasma adiponectin levels during HT might have implications for CHD risks, regardless of the cardiovascular risk factors. There are some limitations to this study. The retrospective design of this study did not permit us to

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confirm the effect of HT on changes in adiponectin. Further studies are needed to explore these relationships and their implications. Second, there may be a selection bias of enrolled subjects due to the relatively small sample size in this study. Third, we do not provide data specific to each HT regimen evaluated, especially progestogen therapy. Varied types and doses of progestogen might modulate the insulin resistance and plasma adiponectin levels in different ways. In conclusion, this study revealed that in postmenopausal women, long-term HT reduces plasma concentration of adiponectin, the adipose tissuederived antiatherogenic protein in postmenopausal women. References [1] Writing group for the Women’s Health Initiative investigators. Risk and benefits from the Women’s Health Initiative randomized controlled trial. JAMA 2002;288:321–33. [2] Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 1998;280:605–13. [3] Grady D, Herrington D, Bittner V, et al., HERS Research Group. Cardiovascular disease outcomes during 6.8 years of hormone therapy: Heart and Estrogen/progestin Replacement Study follow-up (HERS II). JAMA 2002;288:49–57. [4] Sites CK, L’Hommedieu GD, Toth MJ, Brochu M, Cooper BC, Fairhurst PA. The effect of hormone replacement therapy on body composition, body fat distribution, and insulin sensitivity in menopausal women: a randomized, double-blind, placebocontrolled trial. J Clin Endocrinol Metab 2005;90:2701–7. [5] Reaven G. Role of insulin resistance in human disease. Diabetes 1988;37:1595–607. [6] Stout R. Insulin and atheroma, 20-yr perspective. Diab Care 1990;13:631–54. [7] Maeda K, Okubo K, Shimomura I, Funahashi T, Matsuzawa Y, Matsubara K. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1 (adipose most abundant gene transcript 1). Biochem Biophys Res Commun 1996;221:286–9. [8] Arita Y, Kihara S, Ouchi N, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999;257:79–83. [9] Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocytederived plasma protein, inhibits endothelial NF-kappa B signaling through a cAMP-dependent pathway. Circulation 2000;102:1296–301. [10] Zoccali C, Mallamaci F, Tripepi G, et al. Adiponectin, metabolic risk factors, and cardiovascular events among patients with endstage renal disease. J Am Soc Nephrol 2002;13:134–41. [11] Weyer C, Funahashi T, Tanaka S, et al. Hypoadi-ponectinemia in obesity and type 2 diabetes: close association with insulin

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