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Departments of Obstetrics and Gynecology (C.K.S., G.D.L., B.C.C., P.A.F.) and Medicine (M.J.T., M.B.), University of Vermont, Burlington, Vermont 05405
Address all correspondence and requests for reprints to: Cynthia K. Sites, M.D., Department of Obstetrics and Gynecology, University of Vermont College of Medicine, Given Building C250, 89 Beaumont Avenue, Burlington, Vermont 05405. E-mail: cynthia.sites{at}uvm.edu.
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Abstract |
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Methods: Seventy-six postmenopausal women, age 51.6 ± 3.9 yr with body mass index of 24.9 ± 3.2 kg/m2, were randomized to conjugated estrogens (0.625 mg) plus medroxyprogesterone acetate (2.5 mg) or placebo daily. Women received a computed tomography scan at the L4–L5 vertebral disk space, a dual x-ray absorptiometry scan, and a euglycemic hyperinsulinemic clamp at baseline, 6 months, 1 yr, and 2 yr.
Results: Fifty-one women completed the trial and were analyzed (n = 26 on HRT and n = 25 on placebo). Intraabdominal fat, sc abdominal fat, total fat, percent fat, fat-free mass, and weight did not differ between treatment groups by time. Insulin sensitivity did not change in the placebo group, but decreased by 17% in the HRT group by 6 months and persisted at 2 yr (P < 0.01 for treatment by time effect). One year after the trial, insulin sensitivity increased by 25% in women who had taken HRT (P = 0.006 for treatment by time effect), to a level similar to those women in the placebo group.
Conclusions: Conjugated estrogens plus medroxyprogesterone acetate reduce insulin sensitivity in menopausal women without affecting body composition or body fat distribution. The reduction in insulin sensitivity is reversible after discontinuing HRT.
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Introduction |
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The population is aging, and the number of postmenopausal women will increase dramatically over the next decades. Consequently, strategies that improve the metabolic profile could also be beneficial in preventing diabetes mellitus and cardiovascular disease. We hypothesized that combined estrogen plus progestin replacement therapy would reduce the central accumulation of body fat and thus improve insulin sensitivity in postmenopausal women. Previous studies have focused on short-term treatment with hormone replacement therapy (HRT) and its effects on body composition (7, 8, 9) and indirect measures of insulin sensitivity (10, 11). To our knowledge, no long-term, randomized, placebo-controlled trial has examined the effects of a common HRT regimen available in the United States on the temporal changes in body fat distribution and insulin sensitivity in postmenopausal women. Furthermore, no trial has examined the discontinuation of HRT to determine whether these metabolic changes persist.
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Subjects and Methods |
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One hundred five women were recruited by newspaper advertisement and screened for eligibility by phone from the Burlington, Vermont, community (Fig. 1
). Twenty-nine women were excluded due to failure to meet the inclusion criteria or refusal to participate. Seventy-six perimenopausal and early postmenopausal women, aged 51.6 ± 3.9 yr (mean ± SD) and from 6 months to 5 yr since last menstrual period, were randomized by block design to oral conjugated estrogens 0.625 mg plus medroxyprogesterone acetate 2.5 mg (n = 40) or placebo (n = 36) (Prempro; donated by Wyeth Ayerst Pharmaceuticals, Philadelphia, PA). Of these subjects, 74 were Caucasian, 1 was Abenaki Indian, and 1 was Asian. Fifty-one women completed the 2-yr trial (n = 25 on HRT, n = 26 on placebo). At the conclusion of the trial, all women discontinued HRT or placebo and were asked to consider returning in 2 yr for an additional visit. Seventeen women agreed to do so (n = 9 in HRT group and n = 8 in placebo group).
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Interventions
Insulin-stimulated glucose disposal. We performed euglycemic hyperinsulinemic clamps according to the method of DeFronzo et al. (12), with modifications as previously described by our group (13, 14, 15). After 3 d of standardized meals (55% carbohydrate, 30% fat, and 15% protein), volunteers were tested after a 12-h overnight fast. An iv catheter was placed in an antecubital vein for the infusion of insulin, [6,6 D2]glucose, and 20% dextrose. A second catheter was placed in the volunteer’s contralateral hand and kept in a hot box (60 C) for sampling of arterialized blood samples. A primed (60:1) continuous (2 mg/kg·h) infusion of [6,6 D2]glucose was started at 0700 h. A constant infusion of insulin (40 mU/min per m2) was started at 0900 h to approximate postprandial insulin levels. A 20% dextrose solution enriched with [6,6 D2]glucose was also started at 0900 h. Plasma glucose levels were measured every 5 min during the insulin infusion to adjust the dextrose infusion and maintain fasting glucose levels.
During hyperinsulinemia, steady-state was achieved for plasma glucose concentrations and glucose enrichment. Thus, the rate of insulin-stimulated glucose disposal corresponded to the removal of glucose from two sources: the mean dextrose infusion used to maintain euglycemia during the last 30 min of the clamp (milligrams per minute), and the residual endogenous glucose production (Ra). Ra was calculated as:
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Body composition. Fat mass, percent fat, and fat-free mass were measured by dual-photon x-ray absorptiometry (DXA) using a Lunar DPX-L densitometer (Lunar Corp., Madison, WI) as reported previously (13, 15). Scans were analyzed using the Lunar version 1.3y DPX-L extended analysis program for body composition. The coefficient of variation for repeated measurements in our laboratory is 1% for fat mass.
Computed tomography (CT). Intraabdominal fat and sc abdominal fat were measured at the L4–L5 vertebral disk space at an attenuation range of –190 to –30 Hounsfield units by CT using a GE High Speed Advantage scanner (General Electric Medical Systems, Milwaukee, WI) (13, 15). Sagittal diameter was measured as the anterior-to-posterior distance in millimeters at this disk level. The within-subject variation for repeated analysis of fat measurements in our laboratory is less than 1%.
Biochemical analysis. Plasma glucose concentrations were measured by the glucose oxidase method with an automated glucose analyzer (YSI Instruments, Yellow Springs, OH). Serum insulin concentrations were determined with a double-antibody RIA (Diagnostic Products Corp., Los Angeles, CA). Serum FSH was measured with a chemiluminescent assay (Bayer Diagnostics, Tarrytown, NY). Inter- and intraassay coefficients of variation for insulin were 10 and 4%, respectively, and for FSH were 2.2 and 0.3%, respectively.
Food intake. Study volunteers recorded food intake for 3 d at each time point to determine whether HRT affected body composition by affecting self-reported food intake. Diaries were analyzed by computer in the General Clinical Research Center using the Food Intake Analysis System program. Total calories, carbohydrate, fat, and protein intake were assessed (17, 18).
Physical activity. Leisure time physical activity at each time point was measured using the Minnesota Leisure Time Physical Activity Questionnaire (19). This validated, standardized, 60-question survey quantifies various activities performed over the previous year based on intensity of the exercise and time performed, and provides reliable group means for assessing physical activity (20). Test-retest correlations of this questionnaire are 0.79–0.88 for total activity (21).
Statistical analysis
Baseline clinical variables were analyzed using Fisher’s Exact test for categorical variables, Student’s t test for continuous variables, and the Wilcoxon test for nonparametric variables (22). Fifty-one subjects who completed the study were examined by intention to treat, and were those considered for statistical analyses. According to pharmacy records, subjects in the HRT group took 86.9 ± 6.7% of their study medication, and subjects in the placebo group took 86.6 ± 6.9% of their study medication (means ± SD). For analyses of variables over the four time periods, we used a repeated-measures analysis with orthogonal decomposition and baseline values as covariate to determine whether effects differed over time, and whether the two treatment groups differed (23). Baseline fasting glucose levels, baseline serum insulin levels during the last 30 min of the clamp, and the serum insulin levels during the last 30 min of the clamp at the corresponding time point were included as additional covariates when analyzing insulin sensitivity. In all cases, treatment effects were considered statistically significant at P < 0.05.
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Results |
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. Placebo and HRT groups were not significantly different with regard to any baseline variable except for fasting glucose (79.4 mg/dl or 4.4 mmol/liter in the HRT group vs. 82.8 mg/dl or 4.6 mmol/liter in the placebo group, both within normal limits). Clamp and body composition variables at baseline are shown in Table 2. Groups were not significantly different in any of these variables at baseline (Table 2).
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. There were no differences between groups during the treatment period in intraabdominal fat (P = 0.15), sc abdominal fat (P = 0.20), total fat (P = 0.54), or in fat-free mass (P = 0.13) (Fig. 2). Furthermore, we found no differences between groups during the treatment period in percent fat (P = 0.62) or sagittal diameter (P = 0.46) (data not shown). Waist circumference did not differ between groups during the treatment period (82.8 ± 6.8 to 84.9 ± 7.9 cm in the placebo group, and 79.5 ± 8.1 to 80.6 ± 8.4 cm in the HRT group, P = 0.42). Similarly, there were no differences in weight between groups during the treatment period (66.9 ± 8.8 to 67.4 ± 9.5 kg in the placebo group, and from 64.6 ± 9.1 to 64.9 ± 8.9 kg in the HRT group, P = 0.79).
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. Insulin sensitivity declined by 17% in the HRT group over 2 yr, with no significant change in the placebo group (P < 0.01 for differences between groups; Fig. 3A). Insulin sensitivity per fat-free mass also decreased with HRT (Fig. 3B). The decrease in insulin sensitivity was present at 6 months and remained significant at 24 months. HRT reduced insulin sensitivity compared with placebo by 14% more at 6 months, 15% more at 12 months, and 17% more at 24 months (Fig. 3C). Fasting endogenous glucose production before hyperinsulinemia was induced during the clamp increased in both HRT and placebo groups over the 2 yr study (P = 0.02), but was not different between groups (P = 0.31, data not shown). The suppression of endogenous glucose production by hyperinsulinemia during the clamp was not different between groups over time (Fig. 3D).
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Self-reported food intake during the study was limited to 28 subjects who returned both baseline and 2-yr diaries (14 on HRT and 14 on placebo). During the study, women on HRT reported a 12% decrease in caloric intake (212 kcal), with no change reported in the placebo group (P = 0.42 for differences between groups). Specifically, self-reported carbohydrate intake was decreased by 16% (38.4 ± 61.4 g/d) in the HRT group at 2 yr compared with a 1% increase (2.7 ± 72.7 g/d) in the placebo group (P = 0.07 for differences between groups). Self-reported protein intake decreased by 7.6 ± 16.6 g/d in the HRT group compared with a 2.2 ± 21.7 g/d increase in the placebo group at 2 yr (P = 0.09 for difference between groups). No changes in caloric intake between groups reached statistical significance. Leisure time physical activity during the study did not differ between groups (n = 25 on HRT and n = 23 on placebo; decreases of 57 kcal/d in HRT and 91 kcal/d in placebo groups, P = 0.69).
At the conclusion of the 2-yr study, all women discontinued HRT or placebo, and were asked to consider returning in 1 yr for an additional euglycemic hyperinsulinemic clamp and DXA scan. Seventeen women agreed to do so (n = 9 in HRT group and n = 8 in placebo group). In these women, the decrease in insulin sensitivity with HRT (at 24 months) completely reversed at 1 yr after HRT discontinuation (at 36 months, P = 0.006 for treatment by time effect) (Fig. 4).
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Discussion |
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The effect of combined estrogen plus progestin replacement therapy to decrease insulin sensitivity in menopausal women might be explained by the use of a progestin. Specifically, no studies report that estrogen alone decreases insulin sensitivity (24, 25). However, studies in which estrogen plus progestin were employed either show a decrease (26) or no change (27, 28, 29, 30) in insulin sensitivity. It appears that insulin sensitivity is decreased when estrogen and progestin are taken together daily (26), but is unaffected if progestin is taken only for a few days every 3 months along with daily estrogen (29), or if insulin sensitivity is measured during the estrogen alone phase of a sequential estrogen plus progestin regimen (30). Thus, sequential regimens that use a progestin infrequently to induce a withdrawal bleed may be preferable to continuous estrogen plus progestin regimens to minimize changes in insulin sensitivity. Results of these prior studies should be interpreted with caution, however, since some were of short-term duration (<7 months) (24, 25, 26, 30), did not include a control group or use a clamp to measure insulin sensitivity (25), and included open-label (29) or single-blinded (30) study designs.
The route of administration of HRT may also contribute to the decrease in insulin sensitivity. When transdermal estrogen is employed with an oral progestin, insulin sensitivity appears to be unaffected (27, 28). Furthermore, transdermal estrogen alone has no effect on insulin sensitivity (24, 25). In contrast, a continuous regimen of oral estradiol plus oral norethisterone acetate decreased insulin sensitivity in a 3-month study (26). Prior studies of transdermal regimens are also limited by their short-term duration (24, 27, 28), lack of a control group (28), and use of methods other than the clamp to measure insulin sensitivity (25). Our study, which employed a clamp to measure insulin sensitivity in women receiving a continuous combined regimen of oral conjugated estrogens plus oral medroxyprogesterone acetate or placebo, found a reduction of 17% in insulin sensitivity with HRT over 2 yr. The effect of transdermal estradiol plus transdermal progestin on insulin sensitivity has not been reported, but should be studied as an alternative regimen which may minimize changes in insulin sensitivity.
The ability of hyperinsulinemia to suppress endogenous glucose production was not affected by HRT. This suggests that the deterioration in insulin sensitivity with oral conjugated estrogens plus medroxyprogesterone acetate occurs at sites of insulin-stimulated glucose uptake (i.e. skeletal muscle and fat) rather than at sites of glucose production (i.e. liver and kidney). Effects on insulin sensitivity with other medications such as thiazolidinediones also occurs at the peripheral level (muscle) (31). The mechanism for the decrease in peripheral insulin sensitivity with HRT is not clear, but could involve a reduction in glucose uptake by skeletal muscle with a reduction in muscle glycogen synthase (32).
The reversibility of the decrease in insulin sensitivity with HRT may be reassuring for postmenopausal women considering using HRT for a relatively short period of time. The American College of Obstetricians and Gynecologists and the U.S. Food and Drug Administration now recommend that HRT be used for the shortest time possible and at the lowest dose in symptomatic individuals (33, 34). Clinical practice has responded to these recent guidelines, with many patients discontinuing HRT or tapering to lower doses since the publication of the Women’s Health Initiative findings (35, 36). Despite these recent changes in clinical practice, little information is available with regard to insulin sensitivity after discontinuation of HRT. In a cross-sectional study, we reported no difference in insulin sensitivity between recent HRT users compared with never users after 2 months of HRT discontinuation (37). Thus, the decrease in insulin sensitivity with HRT occurs early (by 6 months), and appears to reverse readily in postmenopausal women upon discontinuation of medication.
The health implications of a decrease in insulin sensitivity with ongoing HRT in postmenopausal women are unknown. Our findings of a decrease in insulin-stimulated glucose disposal with HRT but no change in endogenous glucose production mimic the changes noted by Weyer et al. (38) in the transition from normal to impaired glucose tolerance. Of note, none of the women in our study became diabetic during the 2-yr trial based on fasting glucose levels at each time point (for HRT, means ± SD = 79.3 ± 4.5, 78.9 ± 4.2, 77.4 ± 4.4, 78.5 ± 4.7 mg/dl and for placebo, means ± SD = 83.0 ± 6.1, 81.7 ± 5.4, 79.9 ± 4.5, 81.2 ± 7.1 mg/dl; conversion factor to SI units = 0.05551). In older postmenopausal women, HRT has been reported to reduce the incidence of diabetes mellitus by 17% in women without known heart disease (39) and by 35% in women with known heart disease (40). Whether this reduction in the incidence of diabetes occurs through changes in insulin resistance is unclear, because only 8% of the women without heart disease had insulin resistance reported by homeostasis model of assessment-insulin resistance (39), and too few women with heart disease had fasting glucose, fasting insulin, and homeostasis model of assessment-insulin resistance measurements performed (40). Our study would suggest that if HRT reduces the incidence of diabetes mellitus as reported, it may do so by mechanisms other than by improving insulin sensitivity. Furthermore, because insulin resistance is a factor in the progression to cardiovascular disease (41), it is possible that increased insulin resistance with HRT contributes to the finding of increased cardiovascular events seen early in the Women’s Health Initiative Trial (35).
Increases in intraabdominal fat are associated with metabolic abnormalities and cardiovascular disease (42). During our 2-yr trial, both women on continuous combined HRT and placebo gained similar amounts of intraabdominal fat (6.5 cm2 vs. 11.9 cm2, respectively). In a recent open-label trial in Sweden, estradiol valerate, 2 mg/d, with 10 mg medroxyprogesterone acetate given for 10 d every 3 months reduced intraabdominal fat by 11% compared with no change with placebo after 1 yr (29). sc fat did not change significantly in their study. It is possible that HRT regimen differences explain the differences in intraabdominal fat findings between studies. Higher doses of estrogen with an infrequent progestin may reduce intraabdominal fat, but continuous exposure to progestin may dilute the benefit of estrogen. Perhaps of practical interest, neither weight nor waist circumference were reduced significantly by HRT in our study. This finding contrasts to those of Espeland et al. (43), and Kanaya et al. (40), who reported that HRT reduced waist circumference significantly by 1.0–1.2 cm and weight significantly by 1.0 kg compared with placebo in large clinical trials. Our study was designed to examine the effect of HRT on body fat distribution by CT measured outcome variables rather than by anthropometric outcome variables. Thus, our inability to detect significant changes in weight or weight circumference with HRT was likely limited by our smaller sample size.
Increased total and regional body fat are associated with increased insulin resistance in postmenopausal women (13), yet our study indicates that these outcome variables may not necessarily be linked. HRT reduced insulin sensitivity without affecting body composition or body fat distribution, contrary to our hypothesis. It has been hypothesized that free fatty acids released from visceral fat to the liver may increase lipid and lipoprotein production by the liver, thus contributing to the metabolic syndrome with its associated insulin resistance (44, 45). Visceral fat increases were no different between HRT and placebo, suggesting an alternative mechanism for the insulin resistance we observed, and furthermore suggesting that a peripheral mechanism involving skeletal muscle is involved. Indeed, other factors such as exercise capacity are more important than are total and regional adiposity to the determination of insulin sensitivity in nonobese women (16). Furthermore, in other studies of late premenopausal and early postmenopausal women similar in age, we reported that postmenopausal women have greater intraabdominal and total fat than premenopausal women, although insulin sensitivity is not different between groups (3, 14). These data suggest that insulin sensitivity and body composition may not necessarily be linked, and that other factors determine variability in insulin sensitivity.
Self-reported leisure time activity did not change with HRT, suggesting that the decrease in insulin sensitivity with HRT is not related to a decrease in physical activity with HRT. We found a trend toward a self-reported decrease in carbohydrate and protein intake with HRT, although these changes were not significant. These dietary findings suggest that changes in insulin sensitivity with HRT are not due to changes in caloric intake.
In summary, we report that oral combined conjugated estrogens plus medroxyprogesterone acetate reduce insulin sensitivity without significantly affecting body composition or body fat distribution in nonobese, early postmenopausal women. The mechanism of insulin resistance with HRT involves a decrease in peripheral insulin sensitivity rather than a change in endogenous glucose production. The effect is reversible after discontinuing HRT. The majority of volunteers in our study were Caucasian; it is unknown if HRT affects other racial and ethnic groups in a similar manner. Our study is limited in that it involved a relatively small sample size which may have precluded finding differences between groups. Further studies are necessary to understand the cellular and molecular mechanisms involved in this insulin resistance, and to understand the effect of HRT on insulin sensitivity in obese postmenopausal women.
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Acknowledgments |
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Footnotes |
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First Published Online February 1, 2005
Abbreviations: CT, Computed tomography; DXA, dual x-ray absorptiometry; HRT, hormone replacement therapy.
Received July 27, 2004.
Accepted January 26, 2005.
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) or HRT (
). Values are means ± SEM, with baseline values adjusted for repeated measures ANOVA with baseline as covariate. There were no significant differences between groups over time in intraabdominal fat (A, n = 23 in placebo group and n = 21 in HRT group), sc abdominal fat (B, n = 23 in placebo group and n = 21 in HRT group), total fat (C, n = 22 in placebo group and n = 25 in HRT group), or fat-free mass (D, n = 22 in placebo group and n = 25 in HRT group).
