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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 12 4663-4670
Copyright © 2000 by The Endocrine Society

Original Studies

Effect of Estrogen Replacement Therapy on Insulin Sensitivity of Glucose Metabolism and Preresistance and Resistance Vessel Function in Healthy Postmenopausal Women1

Satu Vehkavaara, Jukka Westerbacka, Tiina Hakala-Ala-Pietilä, Antti Virkamäki, Outi Hovatta and Hannele Yki-Järvinen

Department of Medicine (S.V., J.W., A.V., H.Y.-J.), University of Helsinki, 00029 HUCH, Helsinki, Finland; Minerva Foundation Institute for Medical Research (A.V.), 00250 Helsinki, Finland; Family Federation of Finland (T.H.-A.-P.), 00100 Helsinki, Finland; and Karolinska Institut, Department of Obstetrics and Gynaecology (O.H.), Huddinge University Hospital, S-14186 Huddinge, Sweden

Address correspondence and requests for reprints to: Hannele Yki-Järvinen, M.D., Department of Medicine, University of Helsinki, P.O. Box 340, 00029 HUCH, Helsinki, Finland. E-mail: ykijarvi{at}helsinki.fi


   Abstract
Top
 Abstract
Introduction
 Subjects and Study Design
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we hypothesized that estradiol, via its ability to vasodilate in an endothelium-dependent manner, might enhance vascular effects of insulin. Basal and insulin-stimulated peripheral blood flow and resistance, arterial stiffness, and glucose metabolism were determined in 27 healthy postmenopausal women before and after 12 weeks of treatment with either transdermal or oral estradiol or corresponding placebo preparations. Whole body insulin sensitivity was determined using the euglycemic insulin clamp technique (rate of continuous insulin infusion 1 mU/kg·min), forearm blood flow with a strain-gauge plethysmography, and arterial stiffness using pulse wave analysis. Estradiol therapy increased basal peripheral blood flow (1.5 ± 0.1 vs. 1.9 ± 0.1 mL/dL·min, 0 vs. 12 weeks; P < 0.01), decreased peripheral vascular resistance (65 ± 3 vs. 52 ± 3 mm Hg/mL/dL·min, respectively; P < 0.01), and diastolic blood pressure (78 ± 2 vs. 75 ± 2 mm Hg, respectively; P < 0.05) but had no effect on large artery stiffness. Infusion of insulin did not acutely alter peripheral blood flow but diminished large artery stiffness significantly both before and after the 12-week period of estradiol therapy. No measure of acute insulin action (glucose metabolism, blood flow, or large artery stiffness) was altered by estradiol or placebo treatment. These data demonstrate that insulin and estradiol have distinct hemodynamic effects. Physiological doses of estradiol increase peripheral blood flow but have no effects on large artery stiffness, whereas physiological concentrations of insulin acutely decrease stiffness without changing peripheral blood flow. Putative vasculoprotection by estradiol is, thus, not mediated via alterations in arterial stiffness or insulin sensitivity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Study Design
 Materials and Methods
 Results
 Discussion
 References
 
INSULIN RESISTANCE and its accompanying features form a cluster that is associated with an increased risk of cardiovascular morbidity and mortality (1, 2, 3). Recent studies have also demonstrated insulin to have direct vascular actions, which include the ability of insulin to acutely decrease vascular stiffness (4, 5), as measured from a decrease in central aortic pressure augmentation using pulse wave analysis, and an increase in peripheral blood flow (6). The latter effect can be blocked by inhibiting nitric oxide synthesis (6, 7). Defects in the vascular actions of insulin have been suggested to provide a novel mechanistic link between insulin resistance and macrovascular disease (8).

Hormone replacement therapy (HRT) seems to have favorable effects on some aspects of vascular function. Several studies have reported improvements in in vivo endothelial function, as measured from an increase in flow-mediated brachial artery diameter by ultrasound techniques (9, 10, 11, 12, 13). As those of insulin, the favorable vascular effects of estradiol may be mediated via increased synthesis of nitric oxide (14). Cross-sectional studies (15, 16, 17) have also suggested that large artery stiffness, measured using Doppler techniques (17) or pulse wave analysis (15, 16), is greater in women using HRT than in nonusers. Withdrawal of HRT has been suggested to increase arterial stiffness (18, 19), but no placebo-controlled studies have hitherto examined effects of estradiol or HRT on arterial stiffness. No studies have examined whether estradiol or HRT alters the sensitivity of blood flow or arterial stiffness to insulin.

Regarding effects of sex steroids on insulin action on glucose metabolism, data are complex. Whereas women seem to be more insulin sensitive than equally fit men (20, 21), effects of HRT on insulin sensitivity have more often been neutral or negative than positive. In three studies in which the euglycemic insulin clamp technique, the golden standard for measuring whole body insulin sensitivity (22), was used, insulin sensitivity remained either unchanged (23, 24, 25) or even decreased (25). The decrease was observed in a study where a high dose of oral estradiol (2 mg) combined with norethisterone acetate was used as HRT (25). Similarly, in most other studies that used techniques such as the oral or the iv glucose tolerance test, or the iv insulin tolerance test, insulin sensitivity either remained unchanged (23, 24, 25, 26, 27, 28, 29, 30) or decreased (25, 27, 31, 32). In these studies, estradiol was used either alone (24, 26, 28, 29, 30, 31) or in combination with progestin (23, 25, 27, 31, 32) and was administered either via the transdermal (23, 24, 27, 30) or the oral (24, 25, 26, 27, 28, 29) route. There are, however, also studies that reported improvements in indirect measures of insulin sensitivity, using both transdermal (30, 31, 33, 34) and oral (32, 33, 35) estrogen preparations.

In the present study, we hypothesized, given the consisted reports of effects of estradiol on endothelium-dependent vasodilatation, that it might enhance vascular effects of insulin. We, therefore, measured actions of insulin on peripheral blood flow and resistance, arterial stiffness, and glucose metabolism in 27 postmenopausal women before and after 12 weeks of treatment with either transdermal or oral estradiol or corresponding placebo preparations.


   Subjects and Study Design
Top
 Abstract
 Introduction
 Subjects and Study Design
Materials and Methods
 Results
 Discussion
 References
 
Study protocol

Screening visit I (internist). Postmenopausal women were recruited using a newspaper advertisement and were first screened for eligibility by an internist (S.V.). To be acceptable for the study, the subjects had to fulfill the following inclusion criteria: 1) natural amenorrhea for at least 12 months or age greater than 52 yr; 2) FSH greater than 30 U/I; 3) no gynecological or other contraindications for estrogen treatment; and 4) no evidence of acute or chronic disease based on history and physical examination and standard laboratory tests (blood counts, serum creatinine, electrolyte concentrations, liver function tests, and electrocardiogram). None of the women used any medications, including vitamins and antioxidants.

Screening visit II (gynecologist). A total of 36 women visited a gynecologist (T.H-A.-P.). A gynecological history was obtained, and a gynecological examination including transvaginal ultrasound scan was performed. The size of the uterus and ovaries and the thickness of the endometrium were measured, and possible tumors were registered. A Pap smear was taken in case it had not been examined or had been abnormal during the past 12 months or the past 5 yr in case of hysterectomy. An endometrial biopsy was taken, and breasts were examined. To be included in the study, the subjects had to fulfill the following criteria: 1) no suspicion of gynaecological malignancy; 2) no submucous myomas or other myomas greater than 4 cm; 3) no endometrial pathology; 4) endometrial thickness less than 6 mm; 5) normal ovaries (no simple cysts >1.5 cm, no other tumors); and 6) normal breasts.

Of the 36 women attending the first gynecological examination 4 had had a bleeding after the first screening visit. Two of these women had an endometrial thickness greater than 6 mm, and two women had a suspicion of an endometrial polyp. These women were excluded from the study. Another three subjects were excluded from the study because of myomas. Two of the subjects did not wish to continue the study after the first gynecological visit. Thus, the remaining number of subjects after this visit was 27. All subjects were amenorrheic. The subjects gave written informed consent to participate in the study. The experimental protocol was designed and performed according to the principles of Helsinki Declaration and was approved by the Ethics Committee of the Helsinki University Central Hospital.

Randomization and metabolic studies. Subjects considered eligible to participate in the study were randomly assigned into three groups by using minimization of differences (calculated for the variables listed below) between the treatment groups as the method of randomization (36). The following variables (relative weight of each variable is given in parentheses) were included: age (2x), body mass index (2x), total cholesterol (1.5x), total triglycerides (1x), and smoking status (1x). The first group used an oral estradiol tablet of 2 mg (Estrofem; Novo Nordisk, Bagsværd, Denmark) and a placebo patch; the second group transdermal estradiol (50 µg/day) (Menorest; Noven Pharmaceuticals Inc., Miami, FL) and a placebo tablet; and the third group a placebo patch and a placebo tablet for 12 weeks. Measurements of insulin sensitivity, blood flow, and arterial stiffness were performed at 0 and 12 weeks. Because results regarding insulin sensitivity, blood flow, or arterial stiffness were not dependent on the route of estradiol administration, results from the transdermal and oral estradiol groups were pooled, and the combined group is referred to as estradiol group.


   Materials and Methods
Top
 Abstract
 Introduction
 Subjects and Study Design
 Materials and Methods
Results
 Discussion
 References
 
Whole body insulin sensitivity and insulin action on forearm blood flow and arterial stiffness. Whole body insulin sensitivity of glucose metabolism (the M-value) was determined using the euglycemic insulin clamp technique (22). The study was performed after an overnight fast starting at 0730 h. Two 18-gauge catheters (Venflon; Viggo-Spectramed, Helsingborg, Sweden) were inserted as described previously (37). Insulin (rate of continuous infusion 1 mU/kg·min) and 20% glucose were infused in a catheter inserted in the left antecubital vein. The left hand was kept in a heated chamber (65 C), and arterialized venous blood was withdrawn from a heated dorsal hand vein. Before and during the 120-min insulin infusion, metabolic and hemodynamic measurements (pulse wave analysis, heart rate, blood flow, and vascular resistance) were performed at 30-min intervals, as detailed below. We did not perform a time control study because we have previously shown that there are no changes in these hemodynamic or metabolic parameters during a 6-h saline infusion (4).

Pulse wave analysis. The technique of pulse wave analysis was used to determine central aortic pressure and the augmentation index, a measure of large artery stiffness (38). All measurements were made from the radial artery by applanation tonometry using a Millar tonometer (SPC-301; Millar Instruments, Houston, TX), as described previously (4). Data were collected directly into a desktop computer and processed with SphygmoCor Blood Pressure Analysis System (BPAS-1; PWV Medical, Sydney, Australia), which allows continuous on-line recording of the radial artery pressure waveform. The radial waveform was assessed visually to ensure that artifacts from movement and respiration were minimized. Pulse wave analyses were made basally and every 30 min during insulin infusion. The mean of three measurements, each consisting of 15–20 sequentially recorded radial artery waveforms, was used to calculate augmentation and the augmentation index, as well as other parameters at a given time point. The integral system software was used to calculate an average radial artery waveform and to generate the corresponding ascending aortic pressure waveform using a previously validated transfer factor (39, 40). The aortic waveform was then subject to further analysis for calculation of aortic augmentation, the augmentation index, and central blood pressure. The augmentation index is calculated by dividing augmentation with pulse pressure (38, 41). As suggested by O’Rourke and Gallagher (38), the radial blood pressure was calibrated against the sphygmomanometrically determined brachial pressure, ignoring the small degree of amplification between the brachial and radial sites. All measurements were made by a single operator (S.V.). The measurement of the augmentation index with applanation tonometry has shown to be highly reproducible (42, 43). This transfer function is not influenced by gender (44, 45).

Forearm blood flow and peripheral vascular resistance. Forearm blood flow was measured every 30 min with venous occlusion plethysmography using a mercury in silastic rubber strain-gauge apparatus (Model EC-4; Hokanson, Bellevue, WA), a rapid cuff inflator (Rapid Cuff Inflator model E20; Hokanson), and computerized analysis of flow curves (MacLab/4e; AD Instruments, Castle Hill, Australia), as described previously (37). Peripheral vascular resistance was calculated by dividing mean arterial pressure in the brachial artery by forearm blood flow.

Other measurements. Fat free mass and the percentage of body fat were determined using bioelectrical impedance analysis (BioElectrical Impedance Analyzer System model BIA-101A; RJL Systems, Detroit, MI) (46). Serum free insulin was measured before and at 30-min intervals during the insulin infusion by double antibody RIA (Pharmacia Insulin RIA kit; Pharmacia, Uppsala, Sweden) after precipitation with polyethylene glycol (47). The plasma glucose concentration was measured in duplicate with the glucose oxidase method (48) using a Beckman Glucose Analyzer II (Beckman Coulter, Inc., Fullerton, CA). Glycosylated hemoglobin (HbA1c) was measured by high-performance liquid chromatography using a fully automated Glycosylated Hemoglobin Analyzer System (Bio-Rad Laboratories, Inc., Richmond, CA). Serum total cholesterol and triglycerides and high-density lipoprotein cholesterol concentrations were measured as described previously (49, 50). Low-density lipoprotein cholesterol concentration was calculated by the formula of Friedewald. Serum concentrations of FSH and sex hormone-binding globulin (SHBG) (AutoDELFIA SHBG; Wallac, Inc., Turku, Finland) were measured by fluoroimmunometric assays (51). Serum estradiol (Estradiol-2; Sorin Biomedica, Saluggia, Italy), estrone (Estrone-RIA; Bühlman, Schönenbuch, Switzerland), and serum testosterone (Spectria; Orion Diagnostica, Espoo, Finland) concentrations were measured by RIA (52). Because estradiol is bound to SHBG and oral, but not transdermal, estradiol increases SHBG concentrations (53), free estradiol concentrations were calculated based on serum estradiol, estrone, SHBG, and testosterone concentrations as described by Dunn et al. (54).

Final visit at the gynecologist. After the second insulin sensitivity study, the patients underwent a gynecological examination, including transvaginal ultrasound scan. This visit revealed pathology in three patients; one had an endometrial polyp, one a submucous myoma, and one a simple ovarian cyst. All these women were referred for further treatment. Immediately after stopping the study medication, the women who had been randomized to either of the hormone treatment groups and were not hysterectomized received 2 mg estradiol (Progynova; Schering AG, Berlin, Germany) and 15 mg norethisteroneacetate (Primolut N; Schering AG) daily for 12 days to induce bleeding.

Statistical analysis

Analysis of group, time, and group x time effects between study groups were made using ANOVA for repeated measures, followed by the Bonferroni test. Correlation analyses were performed using Spearman’s nonparametric correlation coefficient. The results are expressed as means ± SEM. P values less than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Study Design
 Materials and Methods
 Results
Discussion
 References
 
Baseline characteristics of the study groups

At baseline the estradiol and placebo groups were comparable with respect to biological and menopausal ages, body weight, blood pressure, and circulating concentrations of glucose, insulin, lipids, and FSH (Table 1Go). Body weight remained unchanged in the estradiol (68.5 ± 2.1 kg vs. 69.1 ± 2.2 kg, 0 vs. 12 weeks) and placebo (67.8 ± 2.5 kg vs. 68.3 ± 2.6 kg, respectively) groups. Also, waist to hip ratio remained unchanged in both groups (0.82 ± 0.01 vs. 0.82 ± 0.01 and 0.83 ± 0.02 vs. 0.83 ± 0.02, respectively).


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  		Table 1. Baseline characteristics of the groups

 
Serum estradiol, SHBG, and testosterone concentrations

Serum estradiol concentrations were below the limit of detection in the placebo group (<20 pmol/L) at 0 and 12 weeks. In the estradiol group, serum total estradiol concentrations increased from undetectable at baseline to 310 ± 40 pmol/L at 12 weeks (P < 0.001). Serum SHBG concentrations remained unchanged in the placebo group (49 ± 10 vs. 48 ± 9 pmol/L, 0 vs. 12 weeks) but increased in the estradiol group by 84% from 65 ± 6 to 112 ± 13 pmol/L at 12 weeks (P < 0.001). This increase was due to an increase in the oral estradiol (72 ± 11 vs. 168 ± 11 nmol/L, 0 vs. 12 weeks, P < 0.001) group, whereas the concentrations remained unchanged in the transdermal group (58 ± 4 vs. 65 ± 7 nmol/L, 0 vs. 12 weeks). Serum free estradiol concentrations increased from undetectable to 3.1 ± 0.4 pmol/L in the estradiol group. Because of the increase in serum SHBG concentrations, serum free estradiol concentrations were similar in the oral (3.2 ± 0.4 pmol/L) and transdermal (3.1 ± 0.5 pmol/L) groups at 12 weeks. Serum testosterone concentrations remained unchanged in the estradiol (1.07 ± 0.10 vs. 1.01 ± 0.09 nmol/L, 0 vs. 12 weeks) and placebo (1.07 ± 0.12 vs. 1.01 ± 0.09 nmol/L, 0 vs. 12 weeks) groups.

Basal hemodynamic parameters

Peripheral blood flow and vascular resistance (Fig. 1Go). Basal (measured before insulin infusion) forearm blood flow (1.5 ± 0.1 vs. 1.9 ± 0.1 mL/dL·min, 0 vs. 12 weeks, P < 0.01) increased significantly in the estradiol group but not in the placebo group (Fig. 1Go). In the estradiol group, brachial diastolic blood pressure also decreased slightly (78 ± 2 vs. 75 ± 2 mm Hg, 0 vs. 12 weeks, P < 0.05). Consequently, peripheral vascular resistance decreased significantly (65 ± 3 vs. 52 ± 3 mm Hg/(mL/dL·min), 0 vs. 12 weeks, P < 0.01) in the estradiol group but not in the placebo group (Fig. 1Go). Forearm blood flow increased similarly in the women using oral (1.4 ± 0.1 vs. 1.8 ± 0.2 mL/dL·min, 0 vs. 12 weeks, P < 0.05) and transdermal (1.6 ± 0.1 vs. 1.9 ± 0.1 mL/dL·min, 0 vs. 12 weeks, P < 0.05) estradiol. Also, peripheral vascular resistance decreased similarly in the women using oral (63 ± 5 vs. 50 ± 3 mm Hg/(mL/dL·min), 0 vs. 12 weeks, P < 0.05) and transdermal (68 ± 4 vs. 55 ± 6 mm Hg/(mL/dL·min), 0 vs. 12 weeks, P < 0.05) estradiol. Basal brachial systolic blood pressure did not decrease in the estradiol group [129 ± 4 vs. 125 ± 5 mmHg, 0 vs. 12 weeks; not significant (NS)]. The basal brachial pulse pressures did not change in the estradiol (50.5 ± 3.1 mm Hg vs. 52.6 ± 3.3 mm Hg, 0 vs. 12 weeks, NS) or placebo (49.4 ± 3.5 mm Hg vs. 50.3 ± 4.8 mm Hg, respectively) groups.



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  		Figure 1. Basal brachial diastolic blood pressure, basal forearm blood flow, whole body insulin sensitivity (M-value), and basal peripheral vascular resistance in the estradiol and placebo groups at 0 and 12 weeks. *, P < 0.05; **, P < 0.01. BW, Body weight.

 
Central hemodynamic parameters

Aortic diastolic blood pressure decreased significantly (79 ± 2 vs. 76 ± 2 mm Hg, 0 vs. 12 weeks, P < 0.05) in the estradiol group but not in the placebo group (81 ± 4 vs. 79 ± 3 mm Hg, 0 vs. 12 weeks, NS). Augmentation (14.6 ± 1.7 vs. 13.1 ± 1.1 mm Hg, NS, 0 vs. 12 weeks) and aortic systolic blood pressure (data not shown) remained unchanged in the estradiol group. The basal augmentation index (31.2 ± 1.7 vs. 29.9 ± 1.5%, 0 vs. 12 weeks, NS) also remained unchanged in the estradiol group. Basal aortic systolic and diastolic blood pressure, augmentation, and the augmentation index remained unchanged in the placebo group (data not shown).

Insulin action on glucose metabolism

Fasting plasma glucose concentrations were 5.5 ± 0.1 and 5.4 ± 0.1 mmol/L at 0 and 12 weeks (NS) in the estradiol group and 5.3 ± 0.1 and 5.6 ± 0.1 mmol/L, respectively (NS) in the placebo group. Glycosylated hemoglobin (HbA1C) concentrations averaged 5.8 ± 0.1 and 5.6 ± 0.1% in the estradiol group (0 and 12 weeks) and 5.7 ± 0.1 and 5.7 ± 0.1% in the placebo group, respectively (NS for changes between groups). Serum free insulin concentrations were 29 ± 3 and 25 ± 3 pmol/L (0 and 12 weeks, NS) in the estradiol group and 26 ± 6 and 28 ± 4 pmol/L in the placebo group, respectively (NS). During the insulin infusion, serum free insulin concentrations averaged at 0 weeks 439 ± 12 (30–120 min) and at 12 weeks 407 ± 17 pmol/L in the estradiol group (NS), and 447 ± 22 and 435 ± 26 pmol/L in the placebo group, respectively (NS). During hyperinsulinemia plasma glucose concentrations were maintained at 4.8 ± 0.3 mmol/L (30–120 min) at 0 weeks and 5.1 ± 0.1 mmol/L at 12 weeks in the estradiol group and at 5.1 ± 0.1 and 5.1 ± 0.1 mmol/L in the placebo group, respectively (NS). Whole body insulin sensitivity averaged 4.99 ± 0.35 vs. 4.61 ± 0.32 mg/kg BW·min in the estradiol group (0 vs. 12 weeks, NS) and 5.10 ± 0.59 vs. 4.80 ± 0.56 mg/kg BW·min in the placebo group, respectively, NS) (Fig. 1Go).

Insulin action on vascular function

Insulin action on central hemodynamic parameters (Fig. 2Go). Augmentation was significantly acutely decreased by insulin within 30 min in the estradiol group both at 0 and 12 weeks (Fig. 2Go). The decrease in augmentation by insulin was not altered by estradiol treatment (Fig. 2Go). The augmentation index was also significantly decreased by insulin within 30 min both at 0 and 12 weeks (Fig. 2Go). The ability of insulin to decrease the augmentation index was not altered by estradiol treatment in the estradiol group (Fig. 2Go). The response of the augmentation and augmentation index to insulin were identical in the placebo group at 0 and 12 weeks (data not shown).



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  		Figure 2. Augmentation and the augmentation index (augmentation/pulse pressure) before and during insulin infusion in the estradiol group at 0 and 12 weeks. Euglycemia was maintained with the use of the insulin clamp technique. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 for change in augmentation or augmentation index at a given time point vs. 0 min.

 
Acute hyperinsulinemia did not change heart rate (62 ± 2 vs. 64 ± 1 beats/min, basal vs. 30–120 at 0 weeks and 60 ± 1 vs. 61 ± 1 beats/min, basal vs. 30–120 min at 12 weeks) or ejection duration (338 ± 4 vs. 337 ± 3 ms, basal vs. 30–120 min at 0 weeks and 349 ± 4 vs. 340 ± 3 ms, basal vs. 30–120 min at 12 weeks) in the estradiol or placebo groups (data not shown). Estradiol therapy also did not change heart rate (64 ± 1 and 61 ± 1 beats/min, 0 and 12 weeks, NS) or ejection duration (337 ± 3 and 340 ± 3 ms, respectively, NS) during hyperinsulinemia. These parameters also remained unchanged in the placebo group (data not shown).

Insulin action on peripheral hemodynamic parameters

Estradiol therapy did not change insulin action on peripheral blood flow (1.5 ± 0.1 vs. 1.5 ± 0.1 mL/dL·min, basal vs. 30–120 min at 0 weeks and 1.9 ± 0.1 vs. 1.8 ± 0.1 mL/dL·min, basal vs. 30–120 min at 12 weeks) or peripheral vascular resistance [65 ± 6 vs. 65 ± 4 mm Hg/(mL/dL·min), basal vs. 30–120 min at 0 weeks and 52 ± 3 vs. 53 ± 4 mm Hg/(mL/dL·min), basal vs. 30–120 min at 12 weeks] in the estradiol or placebo groups (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Study Design
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we determined whether 12 weeks of estradiol therapy changes basal arterial stiffness, peripheral vascular resistance, or the effects of insulin on these measures of vascular function in healthy postmenopausal women. Basally, estradiol decreased peripheral vascular resistance by increasing peripheral blood flow, and diastolic blood pressure also decreased significantly. Estradiol had, however, no effect on either basal arterial stiffness or on the actions of insulin on glucose metabolism, peripheral blood flow, or arterial stiffness. Insulin did not change peripheral blood flow but did diminish arterial stiffness. These data demonstrate that physiological concentrations of estradiol and insulin have different vascular effects and that any putative cardiovascular benefit of estradiol is not mediated via changes in insulin sensitivity.

The increase in peripheral blood flow by estradiol in the present study is consistent with a recent cross-sectional report, which suggested postmenopausal women to have lower forearm blood flow and vasodilator reserve than premenopausal women (55). Acute administration of estradiol has also been shown to decrease peripheral vascular resistance via an increase in peripheral blood flow (56). However, the peak estradiol concentrations were ~10-fold higher than those observed during chronic therapy. Regarding previous intervention studies, HRT has repeatedly been shown to improve endothelium-dependent vasodilatation (9, 10, 11, 12, 13). Because 30–40% of basal forearm blood flow is endothelium- dependent (57, 58), one would expect HRT to increase not only blood flow responses to various endothelium-dependent stimuli but also to increase basal blood flow. In previous endothelial function studies, basal flow tended to increase in two studies (10, 11) and was not reported in four studies (12, 13, 59, 60). As an expected consequence of the increase in blood flow, peripheral vascular resistance and diastolic blood pressure decreased significantly. This finding is in keeping with other data according to which estradiol and its active metabolite estrone, have either a small depressor (61, 62, 63, 64) or no (10, 65) effect on blood pressure.

The present study is the first placebo-controlled study to examine effects of estradiol on arterial stiffness, as measured using the augmentation index (38). Changes in the augmentation index reflect changes in stiffness provided peripheral vascular resistance, heart rate, and ejection duration all remain unchanged (38, 66). Basally, before the insulin infusion, peripheral blood flow increased and vascular resistance decreased significantly by the 12 weeks of estradiol therapy. This could be predicted to slightly decrease both augmentation and the augmentation index independent of stiffness, although it is well established that wave reflection mainly occurs at reflection points along the arterial tree before resistance vessels (38). The small nonsignificant decrease in augmentation and the augmentation index support this view of the anatomical location of reflection sites. Regarding heart rate and ejection duration, both heart rate and ejection duration remained unchanged by estradiol and, thus, did not confound interpretation of the augmentation index.

In previous cross-sectional studies, postmenopausal estrogen replacement therapy has been associated with higher common carotid artery distensibility (67, 68, 69) and lower aorto-femoral and leg pulse-wave velocity (19), with no differences in brachial pulse-wave velocity (16) and common carotid artery distensibility (68). In another cross-sectional study, central arterial compliance was similar in nonsmoking women using HRT and in those not using HRT. However, among smoking women, users of HRT had higher compliance than nonusers (68). The present study does not exclude the possibility that estradiol has beneficial effects on arterial stiffness in postmenopausal women with elevated cardiovascular risk. Also, the effects of progestins were not examined in this study. Regarding intervention studies, Waddell et al. (18) measured systemic arterial compliance and pulse wave velocity in postmenopausal women on and 4 weeks off HRT. Systemic arterial compliance decreased after discontinuation of HRT. This decrease did not seem to be due to a change in large artery compliance because the aorto-femoral pulse wave velocity remained unchanged while pulse wave velocity in the femoral-dorsalis pedis region increased significantly in response to cessation of HRT. In view of the present data, the latter might have been due to an increase in peripheral vascular resistance.

During insulin infusion, peripheral blood flow and vascular resistance remained unchanged. This finding is consistent with previous data demonstrating insulin to be a slow and relatively weak vasodilatator compared with classic endothelium-dependent vasodilatators (70, 71). Even so, the blood flow response to the classic agents such as acetylcholine is markedly enhanced by low concentrations of insulin (72). Given that both insulin (7, 73) and estradiol (74, 75, 76, 77) vasodilate via an endothelium-dependent mechanism, it seemed reasonable to hypothesize that estradiol and insulin have additive or even synergistic effects on peripheral blood flow. An additive effect was observed because blood flow was higher during hyperinsulinemia after than before insulin therapy. This effect could, however, be entirely attributed to the estradiol induced increase in basal blood flow.

We have previously demonstrated temporal dissociation between the effect of insulin on wave reflection and its effect on peripheral blood flow (4), and also that these actions of insulin are blunted in insulin-resistant obese subjects (5). In both of these studies, which were performed in young healthy volunteers, insulin markedly reduced wave reflection within 1 h whereas peripheral blood flow did not change significantly until after 2 h at an insulin infusion rate, which was twice that used in the present study (4). In keeping with stiffening of arteries by aging (78), the effect of insulin on the augmentation index was much smaller in the postmenopausal women than in the young volunteers studied previously (4). The ability of insulin to acutely diminish stiffness remained, however, unchanged during estradiol therapy. Although, and as discussed above, acute administration of large doses of estradiol diminishes stiffness, the present data suggest that this does not happen with physiological doses of estradiol and also that such doses also do not potentiate the ability of insulin to diminish stiffness.

A recent analysis of the European Group for the Study of Insulin Resistance of the relationship between age and insulin sensitivity of glucose metabolism in 320 women found no difference in insulin sensitivity, measured using the euglycemic insulin clamp technique, between women with a mean age of 45 yr (n = 76) and those aged 65 yr (n = 88) (79). Although no information of use of HRT was available, these data suggest that no abrupt change in insulin sensitivity occurs at menopause. Most (80, 81), although not all (32), studies directly comparing insulin sensitivity between pre- and postmenopausal women are compatible with this conclusion. Effects of HRT on insulin sensitivity of glucose metabolism have previously been examined in three placebo-controlled studies using the clamp technique. The present negative findings of effects of 2 mg oral estradiol or 50 µg transdermal estradiol on insulin stimulated glucose uptake add to the list of other negative studies which used either 100 µg transdermal estradiol or 1.25 mg oral conjugated estrogen (24), 50 µg transdermal estradiol and oral norethisterone (23), or continuous combined oral estradiol/norethisterone acetate (25). A limitation of our study is the relatively small number of subjects studied, although when subdivided into subjects using transdermal and oral estradiol similar to the previous clamp-studies (23, 24). If we assume that the size of data groups is doubled (40 + 14 subjects) and that in subsequent clamps insulin sensitivity improves in each estradiol-treated subject by 25% and that in the placebo group M-values either increase or decrease by 5%, which is the reproducibility of the clamp technique (82), we would still be unable to detect a significant difference in the change in insulin sensitivity between the estradiol and placebo groups.

In conclusion, physiological doses of estradiol increase peripheral blood flow, decrease peripheral vascular resistance and diastolic blood pressure, but have no effect on arterial stiffness. Insulin at a dose of 1 mU/kg·min did not alter peripheral blood flow, but diminished large artery stiffness significantly. Estradiol does not change either insulin sensitivity of glucose metabolism or the vascular effects of insulin, indicating that cardioprotective effects of estradiol are not mediated via changes in insulin sensitivity or arterial stiffness.


   Acknowledgments
 
We gratefully acknowledge Ms. Kati Tuomola and Ms. Sari Haapanen for excellent technical assistance.


   Footnotes
 
1 Supported by grants from the Academy of Finland (to H.Y.-J. and S.V.), Novo-Nordisk (to H.Y.-J.), Sigrid Juselius Foundations (to H.Y.-J.), and Liv och Hälsa (to A.V.). Back

Received March 3, 2000.

Revised July 18, 2000.

Accepted August 25, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Study Design
 Materials and Methods
 Results
 Discussion
 References
 

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