6
Mechanisms behind gender differences in circulating leptin levels L. HELLSTRO ¨ M 1 , H. WAHRENBERG 2 , K. HRUSKA 2 , S. REYNISDOTTIR 2 & P. ARNER 2 From the 1 Department of Medicine, Danderyd Hospital, Stockholm, and the 2 Karolinska Institute, Department of Medicine and Clinical Research Centre at Huddinge Hospital, Stockholm, Sweden Abstract. Hellstro ¨m L, Wahrenberg H, Hruska K, Wahrenberg H, Reynisdottir S, Arner P (Danderyd Hospital and Karolinska Institute, Stockholm, Swe- den). Mechanisms behind gender differences in circulating leptin levels. J Intern Med 2000; 247: 457–462. Objectives. To investigate gender differences in circulating leptin levels and adipose tissue production of leptin. Design setting and subjects. Thirty-two men and 63 women with a large interindividual variation in body mass index (BMI), but otherwise healthy, were investigated after an overnight fast. Body fat (bioimpedance), abdominal subcutaneous adipose tissue secretion of leptin in vitro and serum leptin were determined. Results. Although there was no gender difference in mean BMI or fat cell size, mean percentage body fat was 49 in women and 36 in men (P , 0.001). At each level of BMI, serum leptin levels were about two times higher in women than in men (P , 0.001). Adipose tissue secretion rate of leptin in men was two- thirds of that in women (P , 0.05). The gender differences in body fat content, serum leptin and leptin secretion were observed in obese (BMI . 27 kg m 22 ) as well as non-obese subjects. Serum leptin levels (P , 0.001) and leptin secretion rate (P , 0.01) correlated positively with body fat content in either sex. However, the gender differences in serum leptin (P , 0.001) and leptin secretion rate (P , 0.01) remained statistically significantly different even when the values were adjusted for body fat. Conclusion. The gender difference in circulating leptin concentrations can be due to at least two different mechanisms. A higher proportion of adipose tissue and increased production rate of leptin per unit mass of adipose tissue might explain why women have higher circulating leptin levels than men. Keywords: adipose tissue, body fat, fat cell, obesity, plasma, sex. Introduction Leptin, secreted from adipose tissue, appears to play a major role in regulating the body fat mass in animal models [1–4]. Genetic studies strongly suggest such a role of leptin in humans as well, since deletion mutations in leptin or in leptin receptors associate with morbid obesity [5, 6]. A particular feature with leptin in humans is the marked gender difference in the circulating hor- mone level (see Saad et al. [7] and Licinio et al. [8] for detailed discussion). Women have much higher leptin concentrations than men at all levels of body mass index (BMI). The mechanism behind this sex variation is not known, although an influence of sex hormones is considered [9, 10]. It is possible that gender variations in circulating leptin levels explain why most women have a higher body fat content than do men. We recently demonstrated that the leptin secre- tion rate from adipose tissue is a major denominator of the circulating level in women [11]. The rate of secretion was markedly increased in obese subjects, which corresponded to increased circulating leptin levels in the obese state. In the present study we have compared adipose tissue secretion rate of leptin Journal of Internal Medicine 2000; 247: 457–462 # 2000 Blackwell Science Ltd 457

Mechanisms behind gender differences in circulating leptin levels

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Mechanisms behind gender differences in circulating leptin

levels

L. HELLSTROÈ M1

, H. WAHRENBERG2

, K. HRUSKA2

, S. REYNISDOTTIR2

& P. ARNER2

From the1

Department of Medicine, Danderyd Hospital, Stockholm, and the2

Karolinska Institute, Department of Medicine and Clinical Research Centre at

Huddinge Hospital, Stockholm, Sweden

Abstract. HellstroÈm L, Wahrenberg H, Hruska K,Wahrenberg H, Reynisdottir S, Arner P (Danderyd

Hospital and Karolinska Institute, Stockholm, Swe-

den). Mechanisms behind gender differences incirculating leptin levels. J Intern Med 2000; 247:

457±462.

Objectives. To investigate gender differences in

circulating leptin levels and adipose tissue productionof leptin.

Design setting and subjects. Thirty-two men and 63

women with a large interindividual variation in bodymass index (BMI), but otherwise healthy, were

investigated after an overnight fast. Body fat

(bioimpedance), abdominal subcutaneous adiposetissue secretion of leptin in vitro and serum leptin were

determined.

Results. Although there was no gender difference inmean BMI or fat cell size, mean percentage body fat

was 49 in women and 36 in men (P , 0.001). At

each level of BMI, serum leptin levels were about two

times higher in women than in men (P , 0.001).Adipose tissue secretion rate of leptin in men was two-

thirds of that in women (P , 0.05). The gender

differences in body fat content, serum leptin and leptinsecretion were observed in obese (BMI . 27 kg m22)

as well as non-obese subjects. Serum leptin levels

(P , 0.001) and leptin secretion rate (P , 0.01)correlated positively with body fat content in either

sex. However, the gender differences in serum leptin

(P , 0.001) and leptin secretion rate (P , 0.01)remained statistically significantly different even

when the values were adjusted for body fat.

Conclusion. The gender difference in circulatingleptin concentrations can be due to at least two

different mechanisms. A higher proportion of adipose

tissue and increased production rate of leptin per unitmass of adipose tissue might explain why women have

higher circulating leptin levels than men.

Keywords: adipose tissue, body fat, fat cell, obesity,

plasma, sex.

Introduction

Leptin, secreted from adipose tissue, appears to play

a major role in regulating the body fat mass in

animal models [1±4]. Genetic studies stronglysuggest such a role of leptin in humans as well,

since deletion mutations in leptin or in leptin

receptors associate with morbid obesity [5, 6].A particular feature with leptin in humans is the

marked gender difference in the circulating hor-mone level (see Saad et al. [7] and Licinio et al. [8] for

detailed discussion). Women have much higher

leptin concentrations than men at all levels of body

mass index (BMI). The mechanism behind this sexvariation is not known, although an influence of sex

hormones is considered [9, 10]. It is possible that

gender variations in circulating leptin levels explainwhy most women have a higher body fat content

than do men.

We recently demonstrated that the leptin secre-tion rate from adipose tissue is a major denominator

of the circulating level in women [11]. The rate of

secretion was markedly increased in obese subjects,which corresponded to increased circulating leptin

levels in the obese state. In the present study we

have compared adipose tissue secretion rate of leptin

Journal of Internal Medicine 2000; 247: 457±462

# 2000 Blackwell Science Ltd 457

in vitro in men and women who were either obese or

non-obese in order to elucidate the mechanisms

behind gender variations in circulating leptin.

Materials and methods

Subjects

The study is an ongoing project with the aim ofstudying leptin secretion from human adipose tissue

[11]. All subjects (n = 96) with no overt disease

except uncomplicated obesity were included. Thisstudy group consisted of healthy volunteers with

varying BMI and obese, otherwise healthy subjects

who later underwent weight reduction treatment(conservative or surgical). None of the subjects was

on a diet or had undertaken a weight reduction

programme within 6 months of the investigation.None was completely sedentary and none was

involved in athletic performance. Thirty-two men

aged 23±59 years and 64 women aged 20±64 yearswere included. BMI ranged from 23 to 53 kg m22 in

men and from 21 to 53 kg m22 in women. Obesity

was defined as BMI . 27 kg m22.The subjects came to the laboratory at about

08.00 h after an overnight fast. Height and weight

and body fat were determined and BMI andpercentage body fat were calculated. The bioimpe-

dance method used to determine body fat content

has been described earlier [12]. In the supineposition, waist-to-hip ratio (WHR) was determined

and a blood sample was obtained for measurement

of serum leptin; thereafter, a specimen of subcuta-neous adipose tissue was removed from the middle

part of the abdominal region. During the first period

of the project, adipose tissue was removed by openbiopsy under local anaesthesia as described pre-

viously [13]. Recently, a needle biopsy procedure

has been described and evaluated [14]. Since thelatter method is more convenient for patients, it was

considered unethical to continue with open biopsies.

Therefore all biopsies in the later part of the studywere performed with the needle aspiration technique

(ISO-Caro).The study was approved by the hospital's

committee on ethics and it was explained in detail

to each participant and his or her consent obtained.

Adipose tissue experiments

Adipose tissue was cut into fragments (about 10±20 mg each). One part was used for isolation of fat

cells and determination of fat cell volume exactly as

described previously [15]. Another part of theadipose tissue fragments was incubated in vitro

exactly as described previously [11]. In brief,

approximately 300 mg of tissue was incubated in3 mL of an albumin-containing buffer for 2 h at

378C. The ingredients in the buffer were from the

same batches throughout the study. An aliquot ofthe medium was freeze-dried and stored at ±70 8C

for subsequent leptin analysis. The incubated

adipose tissue was collected, lipids were extractedand the measured leptin secretion was related to the

whole incubation volume and lipid weight of the

incubated adipose sample as described earlier [11].

Determination of leptin protein

Serum (stored at ±70 8C) was thawed and freeze-

dried incubation medium was redissolved in distilled

water. Leptin in serum or incubation medium wasdetermined with a radioactive immune assay kit

(Linco Research, St Charles, USA)

Statistics

All values are mean 6 SE (standard error of mean).

The data were analysed statistically using linear

regression, analysis of covariance and Student'sunpaired two-tailed t-test. The values for adipose

tissue leptin secretion rate were not normally

distributed, and were transformed to the logarithmicform before analysis.

Results

The relationship between serum leptin and BMI is

depicted in Fig. 1. A significant (P , 0.001) rela-tionship between the two parameters was found in

both women (r = 0.82) and men (r = 0.87). There

was, however, a gender effect on the relationship(F = 11.8, P , 0.001 by analysis of covariance). At

each level of BMI, higher serum leptin values wererecorded in women. A significant relationship

between adipose tissue secretion rate of leptin and

serum leptin levels was also found in both men

4 5 8 L . H E L L S T R OÈ M E T A L .

# 2000 Blackwell Science Ltd Journal of Internal Medicine 247: 457±462

(r = 0.57, P , 0.001) and women (r = 0.52,P , 0.0001).

The clinical data are shown in Tables 1±3.

Women had significantly higher mean values ofpercentage body fat and significantly lower WHR

than men. However, the means for fat cell volume,

BMI and age were comparable in the two groups.Women had 2±3 times higher leptin concentrations

than men. Similar results were recorded in lean and

obese subjects as regards the gender effects.Figure 2 depicts the influence of gender leptin

secretion. The adipose tissue secretion rate inwomen was increased by one-third as compared

with that of men (P , 0.05).

Clinical and adipose secretion data were alsoanalysed after the cohort was separated into lean

and obese subjects (Tables 1±3, Fig. 2). The gender

differences observed in the whole cohort were alsopresent in non-obese or obese subjects.

The influence of body fat content (kg) has also

been taken into account. Total body fat correlated

positively with serum leptin (r = 0.83, P , 0.001

for women, and r = 0.89, P , 0.001 for men) andleptin secretion rate (r = 0.50, P , 0.01 for wo-

men, and r = 0.80, P , 0.001 for men). We also

analysed the gender influence of total body fat onleptin using analysis of covariance. This analysis

showed that gender interacted with the relationships

between total body fat and serum leptin (F = 12.8,P , 0.001) and between total body fat and leptin

secretion (F = 6.9, P = 0.01). However, the gender

differences in serum leptin (P , 0.001) and leptinsecretion rate (P , 0.05) remained when the

influence of body fat content was taken into

account.

Discussion

To the best our knowledge, we have, in the present

study, for the first time demonstrated some mechan-isms which can explain why circulating leptin levels

are higher in women than in men. This was done by

comparing adipose tissue leptin secretion rates invitro in a large population of apparently healthy

men and women (except for obesity).

We could confirm that plasma leptin is related toBMI and adipose secretion rate of leptin. In

agreement with numerous pervious findings [7, 8],

we observed higher serum leptin levels in women. Ateach level of BMI, plasma leptin concentrations were

2±3 times higher in women than in men.At least two factors ± the production rate in

adipose tissue and the fat mass ± might explain the

gender differences in circulating level of leptin. Theincreased rate of leptin secretion in women was

observed in obese as well as lean subjects, and it was

L E P T I N I N M E N A N D W O M E N 4 5 9

120

020

BMI (kg m–2)

Lept

in (

ng m

L–1)

5030 40

100

80

60

40

20

60

Female

Male

Fig. 1 Bivariate plot of serum leptin versus body mass index (BMI)

in women (open symbols) and men (filled symbols).

Table 1 Clinical data on the whole study group

Measure

Females

(n = 63)

Males

(n = 32) P

Age (years) 40 � 1 39 � 2 0.84

BMI (kg m22) 36.7 � 1.2 35.1 � 1.6 0.45WHR 0.893 � 0.011 0.993 � 0.011 <0.0001

Percentage body fat 52 � 2 32 � 2 <0.001

Fat cell volume (pL) 764 � 32 776 � 39 0.81

Serum leptin (mg L21) 38 � 3 17 � 2 <0.001

Values are mean � SE. They were compared using Student's two-tailed unpaired t-test.

BMI, body mass index; WHR, waist-to-hip ratio; n = number of subjects.

# 2000 Blackwell Science Ltd Journal of Internal Medicine 247: 457±462

not due to a gender difference in adipose tissuecellularity, because mean fat cell size was similar in

men and women.

The relative amount of adipose tissue was muchhigher in women, regardless of BMI, than it was in

men. The data on body fat content indicate that one

important reason behind gender variation in plasmaleptin concentration is an adipose tissue mass effect.

This mass effect, in combination with an increased

rate of leptin secretion per unit mass in women,seems to be the major mechanism responsible for the

fact that women, at all levels of body fat, have

higher circulating leptin levels than do men.It should be noted that serum leptin levels vary

more directly with total body fat content than withBMI [16]. In the present study, total body fat was a

strong regressor for serum leptin and leptin secretion

rate. Furthermore, gender interacted significantlywith both relationships. However, the results of

covariance analysis showed that the gender differ-

ences in serum leptin and leptin secretion remainedafter the influence of total body fat content was

accounted for. Thus, gender variations in circulatingleptin and leptin secretion are not only due to the

fact that women in general have more body fat than

do men.The presently recorded secretion rates of leptin are

much higher than reported values for leptin secre-

tion from abdominal subcutaneous tissue in vivo[17]. Methodological reasons most probably explain

the reported differences in leptin secretion rates in

vitro versus in vivo. The in vivo investigatorsexpressed leptin production per total weight of

adipose tissue and we used lipid weight as denomi-

nator. In our hands leptin values differ approxi-mately two times using total weight as compared to

lipid weight as denominator. The in vivo investiga-tors used arterial plasma, which has lower leptin

concentrations than the levels in serum of blood

from the antecubital vein. Finally, some leptin mightbe retained by adipose tissue in vivo, thereby

underestimating the true production rate in vivo

[18]. Because of the observed strong correlationbetween secretion rate of leptin in vitro and the

4 6 0 L . H E L L S T R OÈ M E T A L .

Table 2 Clinical data on the obese subjects

Measure

Females

(n = 44)

Males

(n = 23) P

Age (years) 41 � 2 39 � 2 0.66

BMI (kg m22) 40.4 � 0.8 39.3 � 1.5 0.60WHR 0.927 � 0.010 1.016 � 0.011 <0.0001

Percentage body fat 64 � 2 40 � 2 <0.001

Fat cell volume (pL) 866 � 28 866 � 38 0.99

Serum leptin (mg L21) 52 � 3 22 � 11 <0.001

Values are mean � SE. They were compared using Student's two-tailed unpaired t-test.

Obesity was defined as BMI > 27 kg m22.BMI, body mass index; WHR, waist-to-hip ratio; n, number of subjects.

Table 3 Clinical data on the lean subjects

Measure

Females

(n = 19)

Males

(n = 9) P

Age (years) 38 � 2 40 � 3 0.71

BMI (kg m22) 23.5 � 0.4 24.3 � 0.4 0.27

WHR 0.813 � 0.016 0.929 � 0.017 0.0002Percentage body fat 33 � 1 20 � 1 <0.0001

Fat cell volume (pl) 508 � 39 556 � 37 0.42

Serum leptin (mg L21) 13 � 2 6 � 2 <0.01

Values are mean � SE. They were compared using Student's two-tailed unpaired t-test.

Obesity was defined as BMI > 27 kg m22.

BMI, body mass index; WHR, waist-to-hip ratio; n, number of subjects.

# 2000 Blackwell Science Ltd Journal of Internal Medicine 247: 457±462

serum concentration of leptin, we believe that oursecretion values are representative of true release of

the hormone from fat tissue.

Recently, we have shown that there are regionalvariations in leptin secretion; the rates are higher in

subcutaneous than in visceral adipose tissue [19]. Itis unclear at present if gender variations in leptin

secretion rate also exist in visceral adipose tissue.

The observation that women, in spite of a largerfat mass, have an increased production rate of leptin

compared with men suggests that women have a

less efficient leptin signalling system than do men. Ifwe accept that leptin is a major regulator of body fat

stores [18], it may be inferred that the centres in the

brain that control body fat stores have a higher set-point for leptin in women than in men. This could

explain a higher body fat content in women than in

men. Alternatively, other factors could be respon-sible for gender differences in body fat content, so

that the difference in circulating leptin concentra-

tions between the sexes is merely an adaptivephenomenon.

In summary, this study provides evidence that

gender differences in circulating leptin levels arecaused by at least two different mechanisms: a larger

adipose tissue mass and a higher production rate of

leptin per unit mass of adipose tissue in women than

in men.

Acknowledgements

The study was supported by grants from the Knutand Alice Wallenberg Foundation, the Swedish

Medical Society, the Swedish Foundation for Strate-

gic Research, the Karolinska Institute, the SwedishDiabetes Association, the Swedish Medical Research

Council, the Swedish Heart and Lung Foundation,

Novo Nordisk Foundation, Bergwall Foundation andSoÈderberg Foundation.

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L E P T I N I N M E N A N D W O M E N 4 6 1

425

50

Lept

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ecre

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(ng

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g–1

lipi

d)

ObeseAll Non-obese

350

275

200

125

P < 0.05P < 0.05

P < 0.05

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Received 13 September 1999; accepted 25 November 1999.

Correspondence: Peter Arner, professor, MD, Department ofMedicine, CME (M61), Huddinge Hospital, S-141 86 Huddinge,

Sweden (fax: + 46 8 58582407; e-mail: [email protected]

ki.se).

4 6 2 L . H E L L S T R OÈ M E T A L .

# 2000 Blackwell Science Ltd Journal of Internal Medicine 247: 457±462