The influence of hormone replacement therapy (HRT) on serum leptin concentration in postmenopausal women

Maturitas 37 (2000) 105 – 111 www.elsevier.com/locate/maturitas

The influence of hormone replacement therapy (HRT) on serum leptin concentration in postmenopausal women Peyman Hadji a,*, Kay Go¨rke a, Olaf Hars b, Thomas Bauer a, Gu¨nther Emons a, Klaus-Dieter Schulz a a

Department of Gynecology and Obstetrics, Philipps Uni6ersity Marburg, Pilgrimstein 3, 35037 Marburg, Germany b Department of Human Biology, Uni6ersity of Hamburg, Hamburg, Germany Received 14 June 2000; accepted 14 September 2000

Abstract Objecti6e: This study aimed to evaluate the influence of hormone replacement therapy (HRT), the estradiol concentration and body mass index (BMI, kg/m2) on the serum leptin concentration in postmenopausal women. Subjects and methods: 352 healthy postmenopausal women (mean age, 60.9 98.5 years) participated in this comparative study. 71 (30%) women (mean age 55.9 98.3 years) had taken HRT, while 281 (70%) women (mean age, 59.1910.6 years) had not. Baseline characteristics -age, weight, height, BMI ( ] 25 or B 25), follicle stimulating hormone, estradiol, and leptin values-were compared in the two groups. In a second analysis to evaluate the influence of HRT, estradiol concentrations, and BMI on leptin concentrations, these data were analysed in women allocated to one of four groups: (a) postmenopausal women not on HRT with a BMI B 25 (n= 130); (b) postmenopausal women not on HRT with a BMI ]25 (n=151); (c) postmenopausal women on HRT with a BMI B 25 (n = 48); and (d) postmenopausal women on HRT with a BMI ]25 (n =23). Leptin concentrations were subsequently analysed in relation to BMI and age and BMI and estradiol concentrations to determine any independent effect of these variables. Results: The women taking HRT had a significantly lower mean age, weight, BMI and follicle stimulating hormone concentration than those who were not taking HRT. Furthermore, they had a higher mean height and serum estradiol value, but a significantly lower serum leptin concentration. After controlling for BMI, neither the use of HRT nor the estradiol concentration was found to be related to the leptin value (group (a) versus (c) and group (b) versus (d)), but there were significant differences in leptin concentrations between HRT users with BMI ] 25 and BMI B 25 and between women not taking HRT with BMI ] 25 and BMI B 25 (groups (a) versus (b) and (c) versus (d)). Furthermore, women with a BMI ]25 had significantly higher leptin concentrations than women with a BMI B 25, irrespective of the HRT use. Conclusions: Leptin concentrations are significantly higher in obese postmenopausal women than in their non-obese counterparts. Serum leptin concentrations are not influenced by HRT use or estradiol concentrations. Further studies are needed to elucidate the role of HRT and estrogen on serum leptin concentrations. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

* Corresponding author. Tel.: + 49-61-72459938; fax: + 49-61-72459938. E-mail address: [email protected] (P. Hadji). 0378-5122/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 0 0 ) 0 0 1 6 6 - 3

106

P. Hadji et al. / Maturitas 37 (2000) 105–111

Keywords: Leptin; Body mass index; Estradiol; Hormone replacement therapy (HRT)

1. Introduction An increase in body weight after the menopause, has been observed in several clinical trials [1–3]. It is a common clinical observation that central obesity increases after menopause, indicating that the loss of estrogen may not only increase body fat and weight but also change the distribution of fat [4]. Long term use of HRT is reported to prevent the increase in body weight and skinfold thickness observed after the menopause [5]. How HRT affects the body weight and fat distribution after the menopause has not been fully elucidated. However, a link between HRT and leptin metabolism in postmenopausal women has been suggested. Leptin is the product of the OB gene and is expressed in adipose tissue [6 – 9]. It seems to regulate appetite and weight loss [10 – 12]. Serum leptin concentrations are closely related to body fat content [13], are regulated by the fasting plasma insulin concentration [15 – 17], and have a circadian rhythm [14]. Leptin concentrations have been reported to be two-fold to four-fold higher in women than in men [18 – 21]. Most data indicate that the gender differences in leptin concentrations are not explained by differences in obesity. The mechanism for the gender dichotomy in the leptin-fat mass relation and for the greater sensitivity of leptin to changes in energy balance in women is not explained [20,22]. Since there is a residual variability in the leptin level dependent on the body mass index (BMI), environmental factors, genetics, and gender, other factors besides obesity may also be influential. A link between sex steroids and leptin secretion/action in humans has been suggested [23]. Results in the few studies evaluating the relation between serum estradiol and leptin concentrations however were conflicting [18–20,23,25]. Different proportions of men and women were investigated, in some studies only postmenopausal women and in others only premenopausal women were evaluated, and in most studies sample sizes were small.

In this study, we aimed to evaluate the influence of HRT, serum estradiol, and BMI on serum leptin concentrations in a large sample of obese and normal weight postmenopausal women who were taking and not taking HRT. We hypothesised that if HRT has a regulatory role in leptin secretion, leptin concentrations should vary as a function of HRT status.

2. Methods

2.1. Subjects Study subjects were recruited from women attending for routine gynecological check up at the department of obstetrics and gynecology, University of Marburg. Before entry to the study, all women had answered a detailed questionnaire on important risk factors for treatments or diseases known to effect metabolism. Women were considered to be postmenopausal if they had had a hysterectomy and bilateral oophorectomy and/or had no menstrual periods in the preceding year and/or had a serum follicle stimulating hormone (FSH) of 30 \ IU/l and a serum estradiol level of B10 pg/ml. Only women who had been currently taking either 0.625 mg of conjugated estrogens and progestogen or 2 mg of 17 b-estradiol and progestogen were included and allocated to the HRT group. Exclusion criteria included known cardiovascular disease, diabetes, and any medication that could effect the metabolism. Women wore an examination gown and removed their shoes while their weight and height were being measured. BMI was calculated as the weight (kg) divided by height (m2). Women with a BMI ] 25 were classified as obese and those whose BMI was B25 were considered normal. Premenopausal women were excluded from the study.

2.2. Study design In the initial analysis, we compared baseline characteristics-age, weight, height, BMI, FSH,

P. Hadji et al. / Maturitas 37 (2000) 105–111

estradiol, and leptin-in women taking HRT with those who had never used HRT. In order to reduce any bias introduced by confounding variables such as weight, height, and BMI (kg/m2), we then analysed these data in women grouped according to their HRT status and BMI as follows: 1. postmenopausal women not on HRT with a body mass index B25 2. postmenopausal women not on HRT with a body mass index ]25 3. postmenopausal women using HRT with a body mass index B25 4. postmenopausal women using HRT with a body mass index ]25. Leptin concentrations were subsequently analysed in relation to BMI and age and BMI and HRT use as well as serum estradiol concentrations to determine any independent effect of these variables.

2.3. Assays All blood samples were drawn in the morning to ensure comparability. Serum leptin was measured by a commercial radioimmunoassay (Linco Research, Inc, St. Louis, MI.). The intra-assay coefficient of variation was 3.4 – 8.3 %, and the inter-assay coefficient of variation was 3.6 – 6.2 %. Serum estradiol was measured by a solid phase radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA). The lower limit of detection was 5 pg/ml, and the intra-assay and inter-assay coefficients of variation were 5.3 and 6.4 %, respectively. Serum FSH was measured by a highly sensitive IEMA assay. (Biochem Immunosystems Co, Freiburg, Germany). Intra-assay and inter-assay coefficients of variation were 6.1 and 6.5%, respectively. The detection limit was 0.5 IU/l.

107

2.5. Statistical analysis Data analyses were performed using SPSS for Windows 9.0. The skewness and kurtosis of all variables were checked and were not normally distributed. Non-parametric statistical tests were therefore performed. Differences in baseline characteristics and hormone concentrations in postmenopausal women taking and not taking HRT as well as in women with a BMI ] 25 or B 25 were calculated by Mann-Whitney U test.

3. Results

3.1. Subjects A total of 352 healthy postmenopausal women (mean age, 60.99 8.5 years) participated in the study. Seventy one (30%) women (mean age 55.99 8.3 years) had taken HRT, while 281 (70%) women (mean age, 59.19 10.6 years) had not.

3.2. Leptin concentrations in relation to BMI and HRT use Women with a BMI ] 25 had higher serum leptin concentrations across all age groups irrespective of HRT use (Fig. 1). Table 1 shows the baseline characteristics of women grouped according to HRT use. Postmenopausal women using HRT showed a significantly lower mean age, weight, BMI, and FSH concentration, a higher

2.4. Ethical appro6al The study received the approval of the ethical committee of the University of Marburg, and was carried out according to the requirements of the Declaration of Helsinki. Each patient gave informed written consent beforehand.

Fig. 1. Serum leptin concentrations in relation to BMI (kg/m2) and age in the 352 study women, irrespective of their HRT use.

P. Hadji et al. / Maturitas 37 (2000) 105–111

108

Table 1 Baseline characteristics in postmenopausal women taking and not taking HRTa Characteristics

Age (years) Weight (kg) Height (cm) BMI (units) FSH (IU/l) Estradiol (pg/ml) Leptin (ng/ml) a

Women not taking HRT (n = 281)

Women taking HRT (n = 71)

Mean

9S.D.

Mean

9S.D.

59.1** 69.7*** 164.1 25.9*** 54.3* 5.1*** 7.7**

10.6 10.9 6.5 3.8 20.9 0.7 11.5

55.9 64.8 164.6 24.0 47.6 38.6 16.7

8.3 9.6 5.7 3.5 19.6 32.6 16.6

*P50.05; **P50.01; ***P50.001

mean height and serum estradiol concentration (P B 0.001) but a significantly lower serum leptin concentration (PB 0.01) than postmenopausal women who were not taking HRT. When women were divided into four groups according to their HRT use and BMI (Table 2), postmenopausal HRT users with a BMI B 25 had a significantly lower mean age, weight, BMI and serum leptin concentration (P B0.05 and P B 0.001) than postmenopausal HRT users with a BMI ]25 (groups (c) versus (d)). There were no significant differences between these groups in serum estradiol and FSH concentrations or in mean height. Postmenopausal women with a BMI B 25 who had never taken HRT had a significantly lower weight, BMI, and serum leptin concentration (PB 0.001), as well as a higher mean height (P B 0.05) and a higher mean serum FSH concentration (PB0.001) than postmenopausal women with a BMI ]25 who were not taking HRT (groups (a) versus (b)). There were no significant differences between these groups in the mean age and mean serum estradiol concentration (Fig. 2). We analysed separately differences between postmenopausal HRT users and non-users with a BMI B 25 (groups (c) versus (a)). Differences between the groups were significant in relation to age and FSH and estradiol concentrations (P 5 0.05; P 50.001), but not for serum leptin concentrations (P=0.186). When we analysed differences between postmenopausal HRT users and non-users with a BMI ] 25 (groups (d) ver-

sus (b)), only estradiol concentrations were significantly different (P 5 0.001), while serum leptin concentration were not (P = 0.394).

3.3. Leptin in relation to estradiol The serum leptin concentrations of postmenopausal users and non-users of HRT in relation to the BMI is given in Fig. 3. Our results showed no independent effect of serum estradiol on the serum leptin concentration.

4. Discussion Our results show that obese postmenopausal women have significantly higher leptin concentrations than their counterparts of normal weight. They indicate further that neither HRT use nor serum estradiol concentrations affect the serum leptin concentration. This effect persisted even after adjustment for BMI. There is still debate over the role of leptin in humans. The serum leptin concentration is increased in obese women, and is closely related to the fat mass, BMI, and the amount of body fat [13,19]. The leptin concentration is regulated by the serum insulin concentration [25] and declines with weight loss [13,16,17]. A number of studies have reported higher leptin concentration in women than in men [18,23]. Variability in the degree and distribution of body fat can explain only part of the gender difference

P. Hadji et al. / Maturitas 37 (2000) 105–111

109

Table 2 Baseline characteristics of postmenopausal women in relation to BMI and use of HRT Characteristics

Women not taking HRT BMI B25 (n=130)

Age (years) Weight (kg) Height (cm) BMI (kg/m2) FSH (IU/l) Estradiol (pg/ml) Leptin (ng/ml)

Women taking HRT BMI ]25 (n = 151)

BMIB25 (n =48)

BMI]25 (n =23)

Mean

9S.D.

Mean

9S.D.

Mean

9S.D.

Mean

9S.D.

58.0 61.5*** 165.3* 22.5*** 60.3*** 5.2 12.7***

11.8 6.3 6.2 1.6 22.3 0.9 10.8

60.0 76.5 163.1 28.8 49.6 5.1 22.2

9.3 9.0 6.6 2.7 18.4 0.6 10.2

54.4§ 60.3§§§ 165.5 22.0§§§ 47.2 41.0 11.4§§§

8.2 5.6 5.3 1.8 18.1 31.9 9.8

59.4 74.2 162.7 28.0 48.8 34.1 23.2

7.8 9.3 6.2 2.7 23.1 35.0 12.5

* P50.05; ***P50.001 Differences between postmenopausal women without HRT and with BMI under or over 25. §P50.05; P50.0001 Differences between postmenopausal women with HRT and with BMI under or over 25.

§§§

in serum leptin concentrations. Women tend to have more body fat overall than men. Furthermore, they generally have more subcutaneous fat than fat around the viscera, while the opposite is true in men. Lo¨nnqvist et al. recently reported that subcutaneous fat produces more leptin mRNA than visceral fat-a fact that may help explain part of the gender differences in leptin concentrations [8]. Ostlund et al. and Haffner et al. reported higher leptin concentrations in women than in men, even after adjusting for obesity (assessed by subcutaneous skinfold thickness) [21,23]. In contrast, two reports by Consodine et al. and Maffei et al. showed no difference between leptin values in women and men after adjusting for obesity (assessed by bioimpedance) [13,26]. It has also been suggested that gender differences of serum leptin concentrations can be partly explained by differences in estrogen values. A study of Barash et al. suggested that the leptin concentration seems to modulate gonadal activity [27]. Ovarian and testicular weight increased after treatment with leptin, and histological examination showed more follicular development in ovarian tissue and raised cellular activity in seminiferous tubules in the testis [27]. More recently, Ahima et al. suggested that leptin acts as a signal triggering puberty [28]. A report by Shimizu et al. also indicated that estrogen raises leptin levels in humans [29]. The leptin concentration was about

33% higher during the luteal phase of the menstrual cycle than in the follicular phase, and was accompanied by an estrogen concentration that was two-fold higher [29]. Hardie et al., using a similar study design, suggested that leptin levels are more closely associated with serum progesterone concentrations. These authors also reported that during gestation serum leptin concentrations correlate closely with estrogen and not with progesterone [30]. The influence of HRT on the leptin concentration is still poorly understood. Hickey et al. recently analyzed data on postmenopausal women participating in a cardiovascular risk factor identification program. Despite a four-fold variation

Fig. 2. Serum leptin concentrations in postmenopausal users and non-users of HRT further subdivided in relation to their BMI (kg/m2) B 25 or ] 25 (n =352).

110

P. Hadji et al. / Maturitas 37 (2000) 105–111

trations and HRT. If, how, and why HRT influences leptin concentrations should be investigated further in longitudinal or interventional studies.

References

Fig. 3. Serum Leptin concentration in healthy postmenopausal women in relation to BMI in users (n= 71, estradiol \ 38pg/ ml) and non-users of HRT(n= 281, estradiol B 10 pg/ml).

in the estradiol concentration, the leptin concentration was similar in HRT users and non-users even after matching for age, aerobic capacity, and adiposity [20]. A previous study by Haffner et al. on a small but well matched number of Mexican American women selected from the San Antonio heart study also showed no relation between the leptin concentration and menopausal status or postmenopausal HRT use [23]. These findings, including our results, argue against the hypotheses that differences in leptin concentrations between men and women can be explained by differences in estrogen concentrations. In contrast, a recent study by Paolisso et al. on a much younger population showed strong correlation between leptin and estradiol/testosterone concentrations in men and estradiol concentrations in women after adjusting for age, amount of body fat, and waist to hip ratio [24]. These authors indicated that the relation between sex hormones and leptin concentration could also be mediated through the effect of sex hormones on the body fat content and concentration, since in vitro studies showed a direct relation between serum estradiol levels and BMI [24,31]. In conclusion, our study, in a large sample of healthy postmenopausal users and non-users of HRT, shows that neither HRT nor serum estradiol concentrations have an effect on leptin concentrations, even after adjustment for BMI. However, the role of leptin in humans is still poorly understood. Existing reports show inconclusive results on the interaction of leptin concen-

[1] Lindquist O, Bengsston C. Serum lipids, arterial blood pressure and body weight in relation to the menopause: results from a population study of women in Goeteborg, Sweden. Scan J Clin Labor Invest 1990;40:629 – 36. [2] Peiris A, Sothmann M, Hoffman R, Wilson C, Gustafson A, Hennes M, Kisselbach A. Adipositiy, fat distribution, and cardiovascular risk. Ann Intern Med 1989;110:867 – 72. [3] Pasquali R, Casimirri F, Labate AM, Tortelli O, Pascal G, Anconetani B, et al. Body weight, fat distribution and the menopausal status in women. The VMH collaborative group. Int J Obes Related Metab Disord 1994;18:614 – 21. [4] Bjo¨rkelund C, Lissner L, Andersson S, Lapidus L, Bengtsson C. Reproductive history in relation to relative weight and fat distribution. Int J Obes Related Metab Disord 1996;20:213 – 9. [5] Hassager C, Christiansen C. Estrogen/gestagen therapy changes soft tissue body composition in postmenopausal women. Metabolism 1989;38:662 – 5. [6] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedmann JM. Positional cloning of the mouse obese gene and ist human homologue. Nature 1994;372:425 – 32. [7] Masuzaki H, Ogawa Y, Isse N. Human obese gene expression: Adipocyte-specific expression and regional differences in the adipoe tissue. Diabetes 1995;44:855 – 8. [8] Lo¨nnqvist F, Arner P, Nordfors L, Schalling M. Overexpression of the obese gene in adipose tissue of human obese subjects. Nature Med 1995;1:950 – 3. [9] Hamilton BS, Paglia D, Kwan AYM, Deitel M. Increased obese mRNA expression in omental fat cells from massively obese humans. Nature Med 1995;1:953 – 6. [10] Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995;269:546 – 9. [11] Pellymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269:540 – 3. [12] Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995;269:543 – 6. [13] Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, et al. Serum immunoreactive-leptine concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292 – 5. [14] Sinha MK, Opentanova I, Ohannesian JP, et al. Evidence of free and bound leptin in human circulation: Studies in

P. Hadji et al. / Maturitas 37 (2000) 105–111

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

lean and obese subjects and during short-term fasting. J Clin Invest 1996;98:1277–82. Kolaczynski JW, Nyce MR, Considine RV, Boden G, Nolan JJ, Henry R, et al. Acute and chronic effect of insulin on leptin production in humans. Studies in vivo and in vitro. Diabetes 1996;45:699–701. Utriainen T, Malmstrom R, Makimattila S, Yki-Jarvinen H. Supraphysiological hyperinsulinemia increases plasma leptin concentration after 4 h in normal subjects. Diabetes 1996;45:1364 – 6. Wabitsch M, Jensen PB, Blum WF, Christoffersen CT, et al. Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes 1996;45:1435–8. Havel PJ, Kasim-Karakas S, Dubuc GR, Mueller W, Phinney SD. Gender differences in plasma leptin concentration (letter). Nature Med 1996;2:949–50. Rosenbaum M, Nicolson M, Hirsch J, Heymsfield SB, Gallagher D, Chu F, Leibel RL. Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab 1996;81:3424–7. Hickey MS, Gardiner SN, Thomson DP, Barakat HA. Gender differences in plasma leptin concentration are not influenced by menopause or hormone replacement therapy. Med Sci Res 1998;26:271–3. Ostlund RE, Jr., Yang JW, Klein S, Gingerich R. Relation between plasma leptin concentration and body fat, gender, diet, age, and metabolic covariates. J Clin Endocrinol Metab 1996;81:3909–13. Nicklas BJ, Toth MI, Goldberg AP, Poehlman ET. Racial differences in plasma leptin concentrations in obese postmenopausal women. J Clin Endocrinol Metab 1997;82:315 – 7. Haffner SM, Mykka¨nen L, Stern MP. Leptin concentra-

.

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

111

tions in women in the San Antonio heart study: effect of menopausal status and postmonopausal hormone replacement therapy. Am J Epidemiol 1997;146:581 – 5. Paolisso G, Rizzo MR, Mone CM, Tagliamonte MR, Gambardella A, et al. Plasma sex hormones are significantly associated with plasma leptin concentration in healthy subjects. Clin Endocrinol 1998;48:291 – 7. Segal KR, Landt M, Klein S. Relationship between insulin sensitivity and plasma leptin concentration in lean and obese men. Diabetes 1996;45:988 – 91. Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nature Med 1995;1:1155 – 61. Barash IA, Cheung CC, Weigle DS, Hongping R, Kabigting EB, et al. Leptin is a metabolic signal to the reproductive system. Endocrinology 1996;137:3144– 7. Ahima RS, Prabakaran D, Mantzoros C. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250 – 2. Shimizu H, Shimomura Y, Nakanishi Y, Futawatari T, Ohtani K, Sato N, Mori M. Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 1997;154:285 – 92. Hardie L, Trayhurn P, Abramovich D, Fowler P. Circulating leptin in women: a longitudinal study in the menstural cycle and during pregnancy. Clin Endocrinol 1997;47:101 – 6. Evans DJ, Hoffmann RG, Kalkhoff RK, Kissebah AH. Relationship of androgenic activity to body fat topography, fat cell morphology and metabolic aberration in premenopausal women. J Clin Endocrinol Metab 1983;57:304 – 10.