Estrogen replacement therapy has been consistently associated with a reduced incidence of coronary heart disease in postmenopausal women in epidemiological studies. 1–3 Changes in serum lipids can only explain 25–50% of the cardioprotective effects due to estrogens. 4,5 Estrogens can have direct effects on carbohydrate metabolism by increasing or augmenting pancreatic insulin response to glucose and increasing peripheral insulin sensitivity. 6,7 There are conflicting findings over the effects of conjugated equine estrogens on glucose tolerance. Earlier studies have reported a deterioration in glucose tolerance and an increase in insulin levels, 8,9 whereas other studies have shown no significant alterations in carbohydrate metabolism. 10,11 Epidemiological studies have reported reduced fasting glucose and insulin levels in postmenopausal women receiving hormonal replacement therapy. 7 Differences in the route of administration along with the use of different formulations and dosage as well as a failure to delineate the individual or combined effects of estrogen and progestogen all contribute to the conflict in the published data.
The effect of hormone replacement therapy on carbohydrate metabolism has often been investigated in older women in whom it is safe to use unopposed estrogens. The actions of combined hormone therapy in younger postmenopausal women, who require an added progestogen to prevent endometrial hyperplasia and carcinoma, has not been well studied. Medroxyprogesterone acetate (MPA) is the most commonly used progestogen in clinical trials. Treatment with MPA has been associated with a deterioration in glucose tolerance compared with conjugated equine estrogen alone. 12,13 Few studies have examined the influence of oral human 17β-estradiol plus norethisterone acetate on insulin sensitivity. Altered fat distribution as well as lean body mass may influence insulin resistance or occur as a consequence of changed insulin sensitivity and in turn has been correlated with the increase in cardiovascular disease risk. Changes in body composition occur during menopause. 14 Estrogen deficiency in the perimenopausal years increases fat tissue mass and decreases lean tissue mass. The increase in body fat is predominantly due to an increase in abdominal fat. These changes seem to be more related to menopause than to age. 15–17 Oral conjugated equine estrogen increases fat mass and reduces lipid oxidation, which may be metabolically significant. 18 Progestogens with their androgenic properties are also thought to influence body fat distribution, which may or may not negate some of the beneficial effects of estrogen therapy. No previous studies seem to have directly compared changes in the percentage of total body fat and the percentage of truncal fat during hormonal replacement therapy with changes in insulin sensitivity.
In the present study, we evaluated the effects of a combined sequential oral regimen of human 17β-estradiol and norethisterone acetate, compared with placebo, for 6 months in 30 postmenopausal women, on insulin sensitivity and body composition.
MATERIALS AND METHODS
Thirty postmenopausal women aged 45–55 years were recruited from women in the Dunedin population who responded to an advertisement in the newspaper. Inclusion criteria were (1) nonsmoker, (2) no history of hypertension, ischemic heart disease, renal disease, or diabetes (normal fasting blood sugar), and (3) receiving no medications and no previous exposure to hormone replacement therapy (HRT). Postmenopausal women were identified using standard criteria, including an absence of menstruation for at least 6 months and elevated levels of follicle-stimulating hormone (FSH) (>35 IU/L) and low levels of estradiol (<55 IU/L). All women had undergone a natural menopause. The mean duration of menopause was 35 months with a range from 9 to 96 months. All subjects received a full medical review before participation in the study. Their demographic data is presented in Table 1. The study was approved by the Southern Regional Health Authority Ethics Committee. Informed written consent was obtained before participation.
The study was a randomized single blind placebo-controlled trial of HRT for 6 months in postmenopausal women. Randomization was undertaken by the hospital pharmacy using a random number code, with the investigators analyzing the data blinded to the treatment until after data entry was complete. The postmenopausal women (n = 30) were randomized to receive either hormone replacement therapy or placebo. Hormone replacement therapy consisted of 2 mg of 17β-estradiol for days 1–12, 2 mg of 17β-estradiol and 1 mg of norethisterone for 10 days, and 1 mg of 17β-estradiol for 6 days (Trisequens, Novo Nordisk, Auckland, New Zealand). The placebo medications were matched to look like Trisequens and were provided to the hospital pharmacy by Novo Nordisk. Clinical measurements were made in all women at baseline and again at 6 months after the start of HRT or placebo. Measurements included height, weight, and body mass index, and body composition as measured by dual-energy x-ray absorptiometry scanning; fasting blood glucose and insulin; plasma estradiol, FSH, and luteinizing hormone (LH); and plasma lipids and lipoproteins.
All studies took place after an overnight fast, at the same time of the day and on day 6 of the HRT or placebo cycle (estrogen-only phase) in the second phase of the study (postmenopausal women). The women were asked to maintain their current lifestyle with no change in their dietary or exercise habits. This was reviewed at each study visit along with a review of their pill-dispensing packs. All 30 women completed the study.
Subjects were studied after a 12-h overnight fast. All subjects reported to the study area at 8:30 a.m. Height, weight, and blood pressure were recorded at the start of each study. Blood pressure was measured using a mercury sphygmomanometer after 5 minutes of bed rest and recorded as the mean of three measurements, at 2-minute intervals. Blood was taken for glucose, insulin, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and apolipoprotein B1 in a fasting state at the start of each clamp study. Additional bloods were taken for plasma estradiol, FSH, and LH levels. Plasma and serum were immediately separated by low-speed centrifugation at 4°C.
Insulin sensitivity was measured using a 2-h hyperinsulinemic euglycemic clamp with a modified manual technique of primed continuous insulin infusion (40 mU/m2/min). 19–21 Arterialized blood was taken for blood glucose measurements at 10-minute intervals. Arterialization of venous blood was achieved by the heated hand method. 22 Blood glucose was analyzed using a bench top glucose analyser (YSI 1500 Sidekick glucose analyser). The intra-and inter-test coefficients of variation for glucose estimation using the YSI 1500 Sidekick glucose analyser were 3.2% and 3.8%, respectively. During the clamp study, glucose infusion rates were adjusted to maintain fasting glucose levels using a negative feedback algorithm calculated on a microcomputer. 23
At any level of insulin infusion, where euglycemia is maintained, the rate of infusion must be equivalent to the rate of glucose disposal. The rate of glucose infusion thus reflects the rate of insulin-mediated glucose uptake (M value − mg glucose/kg/min) for each subject. M values for the subjects were calculated from the mean 10-minute values of glucose over the final 60 minutes of the study. We had previously demonstrated that the insulin levels reach a steady state over the second 60 minutes of the study. 23 The insulin sensitivity index [ISI, M/mU/(lx100)] was determined by dividing the average M value by the mean plasma insulin concentration over the final 60 minutes. By correcting for differences in steady-state plasma insulin concentrations, this provides a better index for comparison of possible changes in tissue sensitivity to insulin. 24 Basal fasting insulin levels were measured before the clamp, and insulin levels were measured during the study at 60, 80, 100, and 120 minutes (radioimmunoabsorbant assay; Diagnostic Products Inc.).
Plasma estradiol, FSH, and LH were measured by immunofluorescence assays (National Hormone Assay Service). Plasma cholesterol was determined using an enzymatic kit (Boehringer Mannheim) with calibrators and controls from the Australian Quality Assurance Programme.
Body composition analyses
Each subject underwent a rectilinear body scan using the fast or medium scanning modes on a Lunar DPX-L scanner (Lunar Corporation) within 24 h of the clamp study. Scans were analysed with Software Version 1.3. Body composition included bone density, lean tissue mass, and body fat. Body fat was expressed in terms of weight (g) and as a percentage of total body composition plus distribution patterns of body fat. The total body lean mass expressed in terms of weight (g) and as a percentage of total body composition, as well as skeletal muscle mass as represented by the muscle mass of the lower limbs, was also measured. The in vivo scanning precision in our laboratory matches that reported by others. The coefficients of variation for dual-energy x-ray absorptiometry fat estimations in our unit are 2.64% for fat mass and 2.52% for percentage body fat. 25
The data were analysed using the Statistical Package for Social Sciences (SPSS) Release 4.1. The data were analyzed by repeated measures analysis of variance for each variable and when a significant change was detected, a paired t test was used to estimate the treatment effect in the HRT group. The data is presented as the mean ± SD. A p value less than 0.05 (two-sided) was considered statistically significant.
All 30 postmenopausal women completed the study. The two groups did not differ in chronological age or menopausal age. Body mass index was also similar in the two groups (Table 1). Mean values for each variable at each time point in the study are presented in Table 2. There were no significant differences in metabolic measures at baseline. All women demonstrated a degree of decreased insulin sensitivity that was not modified by 6 months of hormone replacement therapy. The predicted beneficial effects on the lipid profile were demonstrated by a significant lowering of cholesterol levels (6.25 ± 0.92 mmol/L vs. 5.75 ± 1.08 mmol/L, p < 0.003) (Table 2). Body composition remained unchanged over the 6-month period. In particular, there was no alteration in total body fat or the distribution of body fat. Specifically, the percentage central abdominal fat was not altered by hormonal replacement therapy in our study (Table 2). Using repeated measures analysis of variance for each variable measured, over each time point and comparing treatment with placebo, there was no significant correlation between percentage of total body fat or percentage of abdominal fat with insulin sensitivity, fasting glucose, or insulin concentrations. By contrast, analysis demonstrated a significant result with the changes in cholesterol as would be expected from previous studies.
A post-study analysis of data using the standard deviation for change in ISI of 2.1 M mU-1(lx100)-1 (Table 2) and power of 80% indicated that a 30% change in ISI could be detected at p = 0.05.
This randomized single blind placebo-controlled study demonstrated that 6 months of combined sequential oral hormonal replacement therapy using human 17β-estradiol and norethisterone acetate did not modify insulin sensitivity as measured by the euglycemic hyperinsulinemic clamp. A significant reduction in plasma cholesterol levels with HRT was evident, as has been previously published, 12,13 confirming that this form of HRT was eliciting the expected metabolic changes. Baseline values for insulin sensitivity in all the postmenopausal women (n = 30) were significantly lower than values for a group of 49 premenopausal women (aged 46 ± 3.6 years) studied at the same time [4.38 ± 2.37 M mU-1 (lx100)-1 vs. 5.88 ± 1.91 M mU-1 (lx100)-1;p = 0.003] as part of a larger cross-sectional study we have previously reported. 26 This study demonstrates that the menopause is associated with the development of insulin resistance, 11–13,27,28 which is not improved by 6 months of combined sequential estrogen-progestogen therapy. Apparently the increased insulin resistance in postmenopausal women is not directly due to estrogen deficiency. Equally as important is the observation that combined estradiol-progestogen therapy does not adversely affect carbohydrate metabolism.
The results reported here support previously published studies 29–31 using 17β-estradiol preparations over a 3-to 6-month period. Although our studies were carefully carried out in the estrogen-only phase of the cycle, to reduce the potential influence of progestogen on insulin sensitivity; a similar neutral response on insulin sensitivity and other parameters of carbohydrate metabolism using the combined 17β-estradiol norethisterone acetate preparation were reported in the study by Kimmerle and colleagues 31 and by Luotola and colleagues. 30 In both these studies insulin sensitivity was studied, in a similar number of women, during the combined estrogen-progestogen phase of therapy and insulin sensitivity was not significantly altered, when compared with placebo over 3 months 31 and 6 months. 30 These studies along with the results of this present study suggest that norethisterone in combination with 17β-estradiol has little impact on carbohydrate metabolism.
The study by Kimmerle 31 with 18 women in each group was powered to detect a 15% change in ISI but did not detect any significant change in ISI after 3 months of therapy. The changes observed in ISI 31 were of the same magnitude as reported in this study with 15 women in each group. Although the small numbers in this study may have limited the ability to detect a significant effect of HRT on insulin sensitivity, any potential change in the ISI would almost certainly be of a minor amount and the possible clinical relevance limited.
Alterations in carbohydrate metabolism have been linked, via a number of studies, to body composition. The development of a more android distribution of body fat is associated with the development of an increased cardiovascular risk profile. 32,33 However, the published literature is variable on the effects of HRT on body composition. This is in part due to the variations in methodology for assessing body composition as well as variations in the route of administration of HRT. For example, previous studies of oral administration of conjugated equine estrogens suggest an increase in fat mass occurs in association with a reduction in lipid oxidation, which is not evident after transdermal administration of 17β-estradiol. 18 In another study, where 15 women treated with sequential estradiol valerate and cyproterone acetate for 12 months were compared with a control group (n = 12), there was no significant modification of total body fat or percentage of total fat mass in the HRT group, but a significant increase in total body fat and the percentage of total fat mass was evident in the placebo group. 34 In our study, there was no alteration in the distribution of or in the amount of total body fat and the percentage of truncal adiposity in either group of women after 6 months of oral 17β-estradiol therapy plus cyclical progestogen or placebo. This may be due to the differences in menopausal status at the time of study. In the above study reported by Gambacciani and colleagues, 34 the women were very early into their menopause, whereas our study group had a slightly longer duration of menopause. It is possible that the increased deposition of fat associated with menopause had occurred to a greater extent in our study group, and hence any impact of HRT on percentage body fat or distribution is less likely to be apparent. Tremollieres and colleagues 15 have demonstrated an early increase in android fat distribution within the first years after menopause before a significant increase in total body fat. It is also possible that the 6-month period of observation was too short to observe any significant change in body composition and longer follow-up may be required to detect any difference.
From this study, it is not possible to state whether the lack of change in body fat is related to the type of estrogen or the effect of the progestogen. Norethisterone is mildly androgenic and therefore would be expected to enhance the development of a more android fat distribution, although weight loss with topical androgens alone has been reported. 35 Similarly, there was no correlation between insulin sensitivity and percentage of abdominal fat or total body fat in either the treated or placebo group at the start or after 6 months of treatment.
In conclusion, this study demonstrates that in a group of women who are less than 8 years postmenopausal and require combined sequential hormonal replacement therapy, sequential oral therapy with 17β-estradiol and norethisterone acetate is an acceptable form of therapy. Sequential oral therapy with 17β-estradiol and norethisterone acetate had the expected beneficial effects on lipids and lipoproteins; did not seem to promote excess weight gain or a more android distribution of body fat; and did not adversely affect insulin sensitivity, all of which can contribute to the increased cardiovascular risk profile that is evident in postmenopausal women.
This study was funded by the National Heart Foundation of New Zealand; the Laurenson Trust, Otago Medical Research Foundation (Dunedin New Zealand); and Novo Nordisk New Zealand. This study was an investigator-initiated study and was analyzed independently by the investigators.
1. Bush T, Barrett-Connor E, Cowan L. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987; 75:1102–9.
2. Stampfer M, Willett W, Colditz G, et al. A prospective study of postmenopausal estrogen therapy and coronary heart disease. N Engl J Med 1985; 313:1044–9.
3. Wolf P, Madans J, Finucane F, et al. Reduction of cardiovascular disease-related mortality among postmenopausal women who use hormones: evidence from a national cohort. Am J Obstet Gynecol 1991; 164:489–94.
4. Sullivan JM. Hormone replacement therapy and cardiovascular disease: the human model. Br J Obstet Gynaecol 1996; 103(Suppl 13):59–67.
5. Bush TL. The epidemiology of cardiovascular disease in postmenopausal women. Ann N Y Acad Sci USA 1990; 592:263–71.
6. Costrini N, Kalkhoff R. Relative effects of pregnancy, estradiol and progesterone on plasma insulin and pancreatic islet insulin secretion. J Clin Invest 1971; 50:992–9.
7. Barrett-Connor E, Lasko M. Ischemic heart disease risk in postmenopausal women: effects of estrogen use on glucose and insulin levels. Arteriosclerosis 1990; 10:531–4.
8. Buchler D, Warren JC. Effects of estrogen on glucose tolerance. Am J Obstet Gynecol 1966; 95:479–83.
9. Spellacy WN, Buhi WC, Birk SA. The effect of estrogens on carbohydrate metabolism: glucose, insulin, and growth hormone studies on one hundred and seventy-one women ingesting Premarin, mestranol, and ethinyl estradiol for six months. Am J Obstet Gynecol 1972; 114:378–92.
10. De Cleyn K, Buytaert P, Coppens M. Carbohydrate metabolism during hormonal substitution therapy. Maturitas 1989; 11:235–42.
11. Cagnacci A, Soldani R, Carriero PL, et al. Effects of low doses of transdermal 17β estradiol on carbohydrate metabolism in postmenopausal women. J Clin Endocrinol Metab 1992; 74:1396–400.
12. Lobo RA, Pickar J, Wild R, Walsh B, Hirvonen E. Metabolic impact of adding medroxyprogesterone acetate to conjugated estrogen therapy in postmenopausal women: The Menopause Study Group. Obstet Gynecol 1994; 84:987–95.
13. Writing group for the PEPI trial. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women: the postmenopausal estrogen/progestin interventions (PEPI) trial. JAMA
14. Ley CJ, Lees B, Stevenson JC. Sex-and menopause-associated changes in body-fat distribution. Am J Clin Nutr 1992; 55:950–4.
15. Tremollieres FA, Pouilles JM, Ribot CA. Relative influence of age and menopause on total and regional body composition changes in postmenopausal women. Am J Obstet Gynecol 1996; 175:1594–600.
16. Aloia JF, Vaswani A, Russo L, Sheehan M, Flaster E. The influence of menopause and hormonal replacement therapy on body cell mass and body fat mass. Am J Obstet Gynecol 1995; 172:896–900.
17. Svendsen OL, Hassager C, Christtiansen C. Age and menopause associated variation in body composition and fat distribution in healthy women as measured by dual energy x-ray absorptiometry. Metabolism 1995; 44:369–73.
18. O'Sullivan AJ, Crampton LJ, Freund J, Ho KKY. The route of estrogen replacement therapy confers divergent effects on substrate oxidation and body composition in postmenopausal women. J Clin Invest 1998; 102:1035–40.
19. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237:E214–23.
20. Poncher M, Heine RJ, Pernet A, et al. A comparison of the artificial pancreas (glucose controlled insulin infusion system) and a manual technique for assessing insulin sensitivity during euglycaemic clamping. Diabetologia 1984; 26:420–5.
21. Black PR, Brooks DC, Bessey PQ, Wolfe RR, Wilmore DW. Mechanisms of insulin resistance following trauma. Ann Surg 1982; 196:420–35.
22. Abumrad N, Rabin D, Diamond M, Lacy W. Use of heated superficial hand vein as an alternate site for the measurement of amino acid concentrations and for the study of glucose and alanine kinetics. Metabolism 1981; 30:936–40.
23. Walker RJ, Lewis-Barned NJ, Edwards EA, Robertson, MC. The effects of increasing doses of enalapril on insulin sensitivity in normotensive non-insulin dependent diabetic subjects. Aust N Z J Med 1995; 25:698–702.
24. Sherwin RS, Kramer KJ, Tobin JD, et al. A model of the kinetics of insulin in man. J Clin Invest 1974; 53:1481–92.
25. Goulding A, Gold E, Cannan R, Taylor RW, Williams S, Lewis-Barned NJ. DEXA supports the use of BMI as a measure of fatness in young girls. Int J Obesity 1996; 20:1014–21.
26. Lewis-Barned NJ, Sutherland WHF, Walker RJ, et al. Plasma cholesterol esterification and transfer, the menopause and hormone replacement therapy in women. J Clin Endocrinol Metab 1999; 84:3534–8.
27. Lindheim SR, Vijod MA, Presser SC, Stanczyk FZ, Ditkoff EC, Lobo RA. A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of adding progestin. Fertil Steril 1993; 60:664–7.
28. Godsland IF, Gangar K, Walton C, et al. Insulin resistance, secretion and elimination in postmenopausal women receiving oral or transdermal hormone replacement therapy. Metabolism 1993; 42:846–53.
29. O'Sullivan AJ, Ho KKY. A comparison of the effects of oral and transdermal estrogen replacement on insulin sensitivity in postmenopausal women. J Clin Endocrinol Metab 1995; 80:1783–8.
30. Luotola H, Pyorala T, Loikkanen M. Effects of natural oestrogen/progestogen substitution therapy on carbohydrate and lipid metabolism in postmenopausal women. Maturitas 1986; 8:245–53.
31. Kimmerle R, Heinemann L, Heise T, et al. Influence of continuous combined estradiol-norethisterone acetate preparations on insulin sensitivity in postmenopausal non-diabetic women. Menopause 1999; 6:36–42.
32. Lapidus L, Bengtsson C, Larsson B, et al. Distribution of adipose tissue and risk of cardiovascular disease and death: a 12 year follow up of participants in the population study of women in Gothenburg, Sweden. Br Med J 1984; 289:1257–61.
33. Witteman JCM, Grobbe DE, Kok FJ, et al. Increased risk of atherosclerosis in women after menopause. Br Med J 1989; 298:642–8.
34. Gambacciani M, Ciaponi M, Cappagli B, Piaggeso L, De Simone L, Orlandi R, Genazzani AR. Body weight, body fat distribution, and hormonal replacement therapy in early postmenopausal women. J Clin Endocrinol Metab 1997; 82:414–7.
35. Gruber DM, Sator MO, Kirchengast S, Joura EA, Huber JC. Effect of percutaneous androgen replacement therapy on body composition and body weight in postmenopausal women. Maturitas 1998; 29:253–9.