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International Journal of Obesity (2000) 24, 1438±1444 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo Interactions between sex steroid and leptin in women. Studies in vivo and in vitro

K Kristensen1*, SB Pedersen1 and B Richelsen1

1Department of Endocrinology and , Aarhus University Hospital, Aarhus Amtssygehus, DK-8000, Denmark

OBJECTIVE: To investigate the associations between sex hormones and leptin. In addition, to investigate the direct effect of sex hormones by incubations of human subcutaneous adipose tissue explants, in vitro. DESIGN: Cross-sectional study and an experimental in vitro study. SUBJECTS: 36 women (age, 23 ± 65 y; body mass index, BMI, 19 ± 65 kg=m2) participated in the cross-sectional study. Subcutaneous abdominal biopsies were taken from nine women (age, 28 ± 46 y; BMI, 25.5 ± 36.0 kg=m2) for the in vitro study. MEASUREMENTS: Fat distribution parameters (by dual-energy X-ray absorptiometry and anthropometry), sex hormones, leptin and insulin. RESULTS: Leptin correlated signi®cantly with most estimates of adipose tissue mass (r ˆ 0.5 ± 0.9, P < 0.05). However, when the study group was divided in three equal groups (non-obese, obese, and very-obese) it revealed that the correlation predominantly was found in non-obese. In simple correlation analysis leptin was signi®cantly associated with estimates of adipose tissue, insulin and several sex hormones. However, in multiple regression analysis only insulin (partial correlation coef®cient ˆ 0.55, P < 0.004) and percentage fat mass (partial correlation coef®cient ˆ 0.72, P < 0.001) were signi®cantly and independently correlated with leptin without any independent effect of sex hormones. These ®ndings were in agreement with the in vitro studies where neither nor ( or DHT) affected subcutaneous adipose tissue leptin production. (10 nM) stimulated adipose tissue leptin production 3-fold (P < 0.001). CONCLUSION: In regression analysis, where both insulin and measurements of fat mass were taken into account, androgens or did not independently contribute to the variation in leptin levels. Estrogens and androgens had no direct effects on adipose tissue leptin production in vitro. Thus, the sexual dimorphism evident in serum leptin is not likely to be due to a direct in¯uence of sex hormones on leptin production in females. International Journal of Obesity (2000) 24, 1438±1444

Keywords: leptin; anthropometric measurements; DEXA; sex steroid hormones; insulin

Introduction is controlled for body mass index (BMI),10 percentage of body fat,11 total fat mass,12 different fat depots13 or A substantial variability in serum leptin concentra- skin-fold thickness. In addition, the sex difference is tions remains among subjects with equivalent amount evident at the ob mRNA level.14 In a study by of adipose tissue1,2 and obviously factors other than Wabitsch et al,15 the fat mass corrected leptin con- adipose tissue mass are important in the regulation of centrations were at the highest in boys during early serum leptin. In accordance, investigations have and declined thereafter, whereas the leptin shown that prolonged fasting decreases serum leptin concentrations in girls increased during the whole of and leptin expression,3 whereas only excessive food puberty. In trans-sexual subjects, leptin concentrations intake increases serum leptin.4 Furthermore, hor- were reversed according to sex steroid-induced mones such as dexamethasone5,6 and insulin lead to change in phenotype, suggesting an in¯uence of sex an increase in leptin concentrations,7,8 and adrenergic hormones on plasma leptin.16 This had led to the agonists to a decrease in serum leptin.9 suggestion that sex hormones are important in regu- The majority of investigations indicate a gender lating serum leptin. Furthermore, some investigations difference in leptin concentrations. The increased have shown that the ¯uctuation in estrogen during the leptin concentration in females has been con®rmed menstrual cycle has an in¯uence on leptin concentra- in several large studies and persists even when leptin tions.17 However, other studies have not been able to con®rm any signi®cant difference during the men- strual cycle,18,19 in pre-or postmenopausal states20 or *Correspondence: K Kristensen, Departments of Endocrinology in postmenopausal women treated with and Metabolism, Aarhus University Hospital, Aarhus replacement therapy.20 ± 22 Amtssygehus, DK-8000 Aarhus C, Denmark. The investigations regarding the effects of testos- E-mail: [email protected] Received 23 September 1999; revised 8 May 2000; accepted terone have been more convincing. In hypogonadal 12 June 2000 men, leptin concentrations adjusted for BMI, were Sex hormones and leptin in humans K Kristensen et al 1439 elevated when compared with those for healthy men, with underwater weighting and a reproducibility and normalized on testosterone substitution.23 How- of 98.7 ± 99.7%. Each scan can later be manipulated ever BMI is not a good estimate of adipose tissue to give body composition in speci®cally chosen mass and especially not when hormones areas of interest. The following measurements were are administered because this is followed by used to evaluate the effects of adipose tissue distribu- changes in both lean mass and adipose tissue tion on leptin measurements: total fat mass (FMDEXA), 24 mass. However, association studies have con®rmed percentage fat tissue ˆ FMDEXA=body weightÂ100% a negative correlation between leptin and different (FM%), lean body mass (LBMDEXA), fat tissue 13 androgens. localized on the trunk (TFDEXA), percentage of In the present study we have investigated the fat localized on the trunk ˆ TFDEXA=FMDEXA association between leptin and sex hormones in 100% (TF%), subcutaneous fat tissue localized 36 women. In addition, the direct effects of testo- on arms and legs (PERIFATDEXA) and percentage sterone, DHT and 17 b- were investigated of subcutaneous fat localized on arms and legs in female subcutaneous adipose tissue fragments (PERIFATDEXA%). in vitro. Finally, the correlation between leptin and estimates of body composition, evaluated by anthro- pometric measurements and by DEXA scans, was Incubation of adipose tissue investigated. Nine women (25.5 < BMI < 36.0 kg=m2) had a sub- cutaneous adipose tissue biopsy taken at the level of the umbilicus. The tissue was removed using sterile technique, placed in Medium 199 without red Materials and methods and all subsequent procedures were carried out under a laminar air¯ow hood. The tissue was minced into Subjects fragments of less the 10 mg each. All samples were Thirty-six healthy adult women with an age span from washed free of blood clots and free and placed in 23 to 65 y with a wide range of BMI (19.3 ± organ culture as previously described.22,25 In brief, 61.9 kg=m2) were selected to the study. Thirty-four 500 mg adipose tissue fragments ¯oated freely in were premenopausal and two were postmenopausal. 16 ml serum-free Medium 199 without phenol red in Twelve were considered as non-obese (BMI < 30), 12 50 ml plastic tubes. The cultures were placed in a as obese (30 < BMI < 40) and the last 12 as severely humidi®ed incubator and maintained at 37Cinan obese (BMI > 40). All the subjects were healthy apart atmosphere of 5% CO2 in O2. The medium was from being obese and none were diabetic. Body supplemented with 25 mM Hepes, 5% bovine albumin weight was stable for at least 2 months prior to the and 1 nM insulin (Novo Nordisk, Demark). The adi- study as indicated by history. The subjects were pose rissue inplants were incubated in duplicates, and included in the study after informed consent in accor- the medium collected at least every 24 h the medium dance with Helsinki Declaration II and the local was changed and the cumulative leptin secretion Ethics Committee approved the study. calculated. Because of the pronounced response to dexamethasone, all incubations with adipose tissue were made with dexamethasone as a control to assure Anthropometric measurements. the responsiveness of the cells. The culture medium Height and body weight were measured and body was kept at 720C until leptin was measured. All mass index (BMI ˆ weight in kg divided by the height reagents were obtained from Sigma Chemical Co., St in meters squared) was calculated. Waist circumfer- Louis, MO, USA. ence was measured in the supine position midway between the lower rib and the iliac crest. The hip circumference was measured at the widest part of the Blood parameters hip region and the waist ± hip ratio (WHR) was Leptin was measured as a single measurement calculated from the measurements. The sagittal dia- after fasting for 8 h in serum samples with a RIA meter (SD) was measured in maximal expiration with method from Linco Research Ltd., St Charles, a calliper halfway between the lower rib and the iliac MO. The range of the standard curve in this crest. assay was 0.5 ± 100 ng=ml. Intraassay coef®cient of variation was 3.7%. Full status was measured at the Statens Serum Institute, Copen- Dual energy X-ray absorbtiometry (DEXA) scans hagen, Denmark. The following hormones were Body composition was evaluated using a QDR- measured: sexual hormone binding globulin 1000 densiometre (Hologic, Waltham, MA, USA). (SHBG), 17b-estradiol, free estradiol, , Absorbance was measured in the patient alone and free testosterone, testosterone, together with a soft tissue and a bone standard, (DHT), androstendione and respectively. The DEXA scanner has an accuracy sulfate (DHAS). Insulin was measured with an of 3.3% in determining lean body mass compared RIA method.

International Journal of Obesity Sex hormones and leptin in humans K Kristensen et al 1440 Table 1 Subjects characteristics

All (n ˆ 36) Non-obese (n ˆ 12) Obese (n ˆ 12) Very obese (n ˆ 12) BMI (kg=m2) 33.1Æ 1.4 23.1Æ 0.9 36.0Æ 0.8 47.1Æ 1.9

Age (y) 39.2Æ 1.6 38.8Æ 2.3 39.5Æ 3.4 39.3Æ 2.7 Leptin (ng=ml) 32.4Æ 3.1 11.9Æ 2.5c 35.1Æ 2.9c 50.1Æ 3.3c a b Leptin=FMDEXA 0.71Æ 0.05 0.42Æ 0.06 0.82Æ 0.06 0.96Æ 0.07 c c c FMDEXA (kg) 37.8Æ 3.4 17.6Æ 2.6 44.0Æ 2.6 59.0Æ 4.5 FM% 38.1Æ 2.1 25.6Æ 2.0b 42.9Æ 1.9b 50.0Æ 2.0b WHR 0.88Æ 0.02 0.77Æ 0.01 0.89Æ 0.02a 0.94Æ 0.03b Waist (cm) 103.9Æ 4.2 76.8Æ 2.7c 107.3Æ 3.4c 127.5Æ 5.3c Sagittal (cm) 23.4Æ 1.9 17.5Æ 0.7 25.3Æ 2.8 32.8Æ 2.5b SHBG (nmol=l) 45.9Æ 4.0 71.5Æ 5.4 35.3Æ 3.0a 33.1Æ 5.5b Testosterone (nmol=l) 1.52Æ 0.11 1.52Æ 0.21 1.66Æ 0.16 1.37Æ 0.18 Free testosterone (nmol=l) 0.031Æ 0.003 0.021Æ 0.003 0.038Æ 0.005 0.034Æ 0.007 Estradiol (pmol=l) 351.6Æ 48.8 522.7Æ 85.6 326.7Æ 77.9 205.4Æ 65.5 Free estradiol (pmol=l) 9.8Æ 1.3 13.6Æ 2.2 9.6Æ 2.6 6.1Æ 1.7b DHT (nmol=l) 0.58Æ 0.04 0.76Æ 0.08 0.55Æ 0.06 0.26Æ 0.05b Glucose (mmol=l) 5.7Æ 0.1 5.4Æ 0.01 5.7Æ 0.2 5.9Æ 0.2 Insulin (mU=l) 29.3Æ 6.0 11.5Æ 1.2 22.9Æ 2.7 55.1Æ 15.7b

Non-obese (BMI < 30 kg=m2); obese (30 < BMI < 40 kg=m2), and very obese (BMI > 40 kg=m2). WHR ˆ waist-to-hip ratio; FM ˆ fat mass; FM% ˆ FM=body weight; SHBG ˆ sex hormone binding globulin, TG ˆ ; HDL ˆ high-density lipoprotein; meanÆ s.e.m. aObese signi®cantly different from non-obese; bvery obese signi®cantly different from non-obese; call three groups are signi®cantly different, P < 0.05, ANOVA with post hoc correction (Bonferroni).

Table 2 Pearson's correlation coef®cient between leptin and measurements of adiposity

All (n ˆ 36)

BMI 0.83** FM% 0.88** FMDEXA 0.81** TRUNKFATDEXA 0.81** PERIFATDEXA 0.80** WHR 0.52* Hip 0.82* Waist 0.76* Sagittal diameter 0.78*

n ˆ 36, *P < 0.05, **P < 0.01.

Statistical analysis Data are presented as meansÆ s.e.m. All analyses were performed using the Statistical Package for the Social Science (SPSS=PC ‡ , SPSS, Chicago, IL). The distributions of leptin, insulin and the various sex hormones were normalized by log transformations. Differences in mean values between obesity groups were assessed by ANOVA, followed by post hoc correction (Bonferroni). Differences between the two groups in the in vitro assay were assessed by students unpaired t-test. Simple and multiple regression analysis were used to identify the independent effects of variables associated with variations in leptin concentrations. P values below 0.05 were taken as statistically signi®cant.

Results

Associations between serum leptin and body composition Data on subjects participating in the study are given in Table 1. As seen, when the level of obesity increased, Figure 1 The correlation between leptin and dihydrotestoster- a number of well-known metabolic characteristics one (DHT), insulin and FM% in 36 women.

International Journal of Obesity Sex hormones and leptin in humans K Kristensen et al 1441 associated with adiposity were displayed, such as was signi®cantly correlated with leptin. If the leptin: increased concentrations of plasma leptin and insulin. FM-DEXA ratio was calculated as an index of the The mean serum leptin (s-leptin) concentrations in the plasma leptin per unit of adipose tissue, the leptin obese (30 < BMI < 40 kg=m2) was 35.1Æ 2.9 ng=ml secretion almost doubled from non-obese to obese and in the very-obese (BMI > 40 kg=m2) 50.1Æ (P < 0.05), whereas the increase from obese to very 3.3 ng=ml, corresponding to about three and four obese was only 17% (NS) (Table 1). times higher concentrations compared to the non- obese (BMI < 25 kg=m2; 11.9Æ 2.5 ng=ml), respec- tively. The range in the very obese group was varying Correlations between leptin, insulin and sex hormones from 37.2 to 72.4 ng=ml and in the non-obese group In Table 3 the correlations between leptin, insulin and from 1.9 to 34.6 ng=ml. sex hormones are shown. In the correlation analysis In Table 2 the correlations between s-leptin and there was a signi®cant negative correlation between different estimates of obesity are shown. s-Leptin was leptin and SHBG, total estradiol and free estradiol and highly correlated to estimates of obesity in the total among the androgens a signi®cantly negative correla- group and especially with the FM% determined by tion was found between leptin and DHT (r ˆ 70.57, DEXA scans (r ˆ 0.88). The correlations between P < 0.001; Figure 1) whereas no signi®cant correla- leptin and the different fat localizations determined tions were found to total and free testosterone or by DEXA scan were in the same range (Table 2). androstendione. A negative correlation was found According to the anthropometry the highest correla- between leptin and DHAS (70.31, P < 0.05). tion was obtained to total fatness determined by BMI Since the fat mass and the level of insulin may be (r ˆ 0.83) and the weakest to abdominal fatness interplayers in the associations between leptin and sex determined by WHR (r ˆ 0.52). However, the correla- hormones we also made the correlation analysis after tions to waist (r ˆ 0.76) and sagittal diameter controlling for these two variables. When the correla- (r ˆ 0.78) were high (Table 2). The association tions between leptin and sex hormones were con- between FM% and serum leptin is plotted in Figure 1. trolled for insulin, only minor changes were seen in Dividing our subjects according to BMI into three the correlation coef®cients but when leptin was con- groups revealed that the correlation between s-leptin trolled for fat mass using FM% no correlations were concentrations and measurements of adipose tissue found (Table 3). In multiple regression analysis using were only signi®cantly correlated in the non-obese various sex hormones, DHT, SHBG, insulin and individuals (results not shown). If all obese subjects FM%, it was found that only insulin and FM% (BMI > 30 kg=m2) were investigated in one group, remained independently and signi®cantly correlated leptin correlated with BMI (r ˆ 0.48, P ˆ 0.02) and to leptin without any signi®cant in¯uence of the sex FM% (r ˆ 0.56, P ˆ 0.01), whereas no other estimate hormones (Table 4).

Table 3 Correlations between leptin with levels of various sex hormones

123 4 Ð (insulin) (FM%) (insulin ‡ FM%)

SHBG 7 0.59** 7 0.66** 7 0.12 0.03 T-estradiol 7 0.52** 7 0.41* 0.07 0.07 F-estradiol 7 0.47** 7 0.37* 0.12 0.06 Estrone 7 0.23 7 0.15 0.01 7 0.13 T-testosterone 7 0.17 7 0.33 7 0.21 7 0.20 F-testosterone 0.21 7 0.03 7 0.13 7 0.19 DHT 7 0.57** 7 0.63** 7 0.32 7 0.19 Androstendione 7 0.19 7 0.33 7 0.09 7 0.24 DHAS 7 0.31* 7 0.41* 7 0.10 7 0.16

Partial correlation coef®cients (r-values): uncontrolled (1); controlled for insulin (2); and controlled for fat mass (FM%) (3); controlled for both insulin and FM% (4); n ˆ 36. *P < 0.05; **P < 0.01.

Table 4 In multiple regression only insulin and FM% were signi®cantly correlated with leptin

Partial correlation Variable P-value coef®cient Figure 2 Effects of sex hormones on leptin production in vitro. FM% 0.001 0.72 Subcutaneous adipose tissue (500 mg) was incubated for 24 h DHT 0.358 7 0.19 with testosterone, dihydroestosterone (DHT) and 17 b-estradiol Insulin 0.004 0.55 in the presence or absence of dexamethasone (10 nM). Dexa- SHBG 0.870 0.03 stimulated leptin production 3-fold (P < 0.001), whereas the sex hormones were without effect on leptin secre- R ˆ 0.93, F ˆ 37.96, P ˆ 0.001. tion (meanÆ s.e.m., n ˆ 9).

International Journal of Obesity Sex hormones and leptin in humans K Kristensen et al 1442 In vitro incubation of subcutaneous adipose tissue secretion after testosterone addition in females or Incubations with testosterone (50, 500 and 1000 nM), males, which is in agreement with our study using 17b-estradiol (50 and 500 nM) and DHT (50 and subcutaneous adipose tissue. However, several other 500 nM) were made for 24 h, but no effects on androgens (DHT, , and leptin secretion were found. To assure that adipose DHAS) inhibited leptin secretion in female omental tissue explants were viable all incubations were made adipose tissue samples by 27 ± 48% whereas no effects in parallel with a 10 nM dexamethasone incubation, were found in male samples in vitro. 27 This is hard to which caused an almost 3-fold (P < 0.001) increase in interpret as long as no sex differences in androgen leptin secretion after 24 h. To further investigate the receptor density28 or function have been reported in in¯uence of sex hormones 17 b-estradiol (500 nM), omental fat. Furthermore, the reported gender differ- testosterone (500 nM) and DHT (500 nM) were added ence between men and women cannot be explained by together with dexamethasone (10 nM) to the culture androgens, as they were reported not to affect male medium but no changes were seen in leptin secretion adipose tissue leptin secretion. The present ®nding compared to dexamethasone without sex hormone that sex hormones may not be directly involved in the addition (Figure 2). In a pilot study (n ˆ 3) incuba- sexual dimorphism in serum leptin is in agreement with tions for 72 h were made to investigate whether longer most recent studies in both rats30 and humans.31 ± 33 incubation time was necessary with both testosterone The signi®cance of leptin regulation in omental and 17 b-estradiol but without any in¯uence on the fat can also be discussed. Omental fat is a relatively results. small fat depot in comparison with subcutaneous fat. Investigations in women with different degrees of intra-abdominal fat depots have not revealed any differences in circulating leptin levels.29 This may Discussion re¯ect the fact that omental adipocytes secrete less leptin compared to subcutaneous adipocytes14 and There are well-known gender differences in the serum therefore have a rather small in¯uence on total concentrations of leptin and several studies have amount of circulating leptin. indicated that these differences are due to the in¯u- The in vivo studies, which indicate that sex hor- ence of sex hormones on leptin production.15 ± 17 In the mones may affect leptin production, seem to often present study we have investigated the relationship have problems in relation to controlling for differ- between leptin and sex hormones in a group of women ences in fat mass. Very often BMI is used instead of with a range of BMI from 19 to 48 kg=m2 as well as in more direct measurements of the fat mass.20,23,34 subcutaneous adipose tissue in vitro. Moreover, it has been well described that insulin In the in vivo investigations we found by simple stimulates human adipose tissue leptin production,8 correlation analysis signi®cant correlations between and, in addition, that insulin may inhibit the produc- several of the sex hormones and leptin. Previously, it tion of SHBG in the .35,36 Insulin level increases has been shown that insulin is an important regulator with obesity and the level of SHBG decreases.37 As of leptin secretion and leptin mRNA expression. most of the androgens are tightly bound to SHBG, Therefore the correlation between leptin and sex investigations with measurement of total testosterone hormones were controlled for both insulin and FM may be confounded by the lower SHBG level asso- and consequently the correlations disappeared (Table ciated with obesity. Thus, a negative correlation 4). The in vitro studies with incubations of subcuta- between leptin and a total measurement of androgens neous adipose tissue con®rmed that neither estrogen is likely to arise because of decreased SHBG levels. nor androgens (DHT or testosterone) had any direct The present ®nding of reduced DHT in obesity might effects on adipose tissue leptin production. actually re¯ect the reduction in SHBG, as DHT is A few other studies have investigated whether tightly bound to SHBG and is measured as total androgens have direct effects on adipose tissue amount of DHT in plasma. leptin secretion in humans. In subcutaneous preadipo- The present study reveals that s-leptin is closely cytes leptin secretion and leptin mRNA have been associated with different estimates of fat mass as seen reported to be moderately inhibited by testosterone in most other studies. We found the strongest correla- and DHT in vitro.15 However, these studies were tions in the lean individuals whereas no correlations made in preadipocytes from the female breast and were seen in obese or very obese subjects. These the doses used for the experiments were supra-phy- ®ndings might indicate that the regulation of s-leptin siological (100 ± 1000 ng=ml testosterone). In addi- differs in lean and obese individuals or may be due to tion, leptin secretion and leptin mRNA expression in the greater variability in s-leptin in obese subjects. In maximally differentiated preadipocytes are very low obese women we found a 2-fold greater leptin level and consist of only approximately 1% of that in per fat mass compared to non-obese (P < 0.05), which mature adipocytes26 making the relevance of this might be explained by an increase in leptin=fat cell regulation questionable. Recently, a study in adipose and=or an increase in fat cell numbers. However, no tissue explants from human omental fat cells has been differences were found in leptin=kg fat mass between performed. Omental fat cells did not change in leptin obese and the very obese subjects. These results might

International Journal of Obesity Sex hormones and leptin in humans K Kristensen et al 1443 indicate that in some very obese subjects an insuf®- 6 Miell JP, Englaro P, Blum WF. Dexamethasone induces an cient leptin level is present compared to the amount of acute and sustained rise in circulating leptin levels in normal human subjects. Horm Metab Res 1996; 28: 704 ± 707. adipose tissue, which may contribute to the develop- 7 Vidal H, Auboeuf D, De Vos P, Staels B, Riou JP, Auwerx J, ment of obesity. Ioffe et al 38 have tested the possibi- Laville M. The expression of ob gene is not acutely regulated lity that a relative low leptin production leads to by insulin and fasting in human abdominal subcutaneous obesity by mating carrying a weakly adipose tissue. J Clin Invest 1996; 98: 251 ± 255. expressed adipocyte-speci®c human transgene to 8 Dagogo-Jack S, Fanelli C, Paramore D, Brothers J, Landt M. Plasma leptin and insulin relationships in obese and nonobese ob=ob mice. 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