Measurements and assays:
Body weight was measured to the nearest 0.1Kg and height was measured without cap and shoes to the nearest 0.1cm. Waist and hip circumferences were determined to the nearest 0.1cm. The former was taken to the smallest standing horizontal circumference between the ribs and the iliac crest, while the latter was measured as the largest standing horizontal circumference of the buttocks. BMI calculation was weight(kilograms)/height(m)2, BMI>25 being considered as overweight or obese.
Serum leptin levels were determined by RIA with an internationally conventionally used leptin RIA Kit (Linco Research, St. Charles, MO U.S.A.). The immunopurified rabbit antibodies were raised against highly purified full-length recombinant human leptin. The intra-and inter- assay coefficients of variation were all less than 5%. Serum testosterone was determined by the Enzyme Immunoassay(magnetic solid phase) Kit supplied by the Biochem Immunosystems Inc. U.S.A.
Data Analysis
Percent body fat was calculated from BMI and age using gender-specific regression equations[8]:
%fat in male=1.2×BMI+0.23×age−16.2
%fat in female=1.2×BMI+0.23×age−5.4
Body fat distribution was estimated by the waist to hip measurements. WHR as waist/hip ratio is thought to be less than 0.9 in normal man and 0.85 in normal woman.
Data(reported as the mean±SD) analysis was performed with the assistance of SAS(the Statistical Analysis System). Statistical methods used in this study included t test, ANOVA, simple linear regression and multiply stepwise regression where indicated.
Results
The leptin levels of 105 lean and obese subjects comprised of 53 men and 52 women (BMI, 15.90-42.19Kg/m2; percent body fat, 9.24-55.27%) were found to be between 0.58-27.01ng/ml(shown in Tab1). 75% of the subjects were below 7.94ng/ml. And the mean level was 6.14±5.29ng/ml. If evaluated by percentile, 90% normal range of leptin level in Chinese were 0.66-5.63ng/ml in men and 2.67-13.66ng/ml in women.
A striking gender difference was observed(BMI<25: 2.15±1.46ng/ml in men and 7.86±3.60ng/ml in women; BMI>25: 4.87±3.47ng/ml vs 16.59±6.92ng/ml, P<0.001)(shown in Fig1). The mean leptin levels in women were 2-3 times higher than those in men. After adjusted for body fat, the difference remained highly significant(P<0.001).
Leptin was significantly related to BMI and percent body fat(r=0.69 and r=0.50 in men; r=0.63 and r=0.54 in women, all P<0.001) (shown in Fig2a and Fig2b). The increasing amplitude of serum leptin levels with increasing percent body fat was nearly twofold greater in women than in men (Leptin=0.41×%fat-1.85 vs Leptin=0.28×%fat-1.70), indicating that at any given body fat mass, women are likely to have a higher leptin level. There was a significant negative correlation between serum testosterone and leptin levels in men (Tab.2). However, this gender difference was attenuated, though still significant, when percent body fat was replaced by BMI (Leptin=0.75×BMI-9.31 in women vs Leptin=0.60×BMI-11.14 in men). Leptin was weakly related to age (r=-0.12,P=NS in men and r=0.17,P=NS in women), an extended research in 79 men aged from 17 to 80 yrs still did not reveal any significant correlation between age and leptin levels but the level of statistical significance increased after adjustment for body fat. The decreasing trend existed in both men and women. Besides, men had higher correlation of leptin with WHR than women (shown in Tab2) did.
In the multiple regression analysis containing independent contributions of percent body fat, gender, age, waist, WHR on leptin, gender was the first factor that associated with serum leptin. However, adding percent body fat in this model greatly increased the explained leptin variance from14.9% to 37.8%. And with age the model became the fittest one (shown in Tab3).
Discussion
It was until 1994 Zhang et al discovered the mouse ob gene and its human homologue by positional cloning that the research in the mechanisms of obesity had made a breakthrough[9]. The measurement of the level of the ob gene product in the peripheral blood is convenient and useful for the elucidation of its physiological effect and its relationship to the obesity. A specific and sensitive radioimmunoassay from Linco Research was chosen by most investigators in the literature. In our current study, the leptin levels were measured in Chinese by this worldwide accepted human leptin RIA method, which allows us to compare our data with those of western countries. As a matter of fact, we found the level of leptin in lean Chinese was similar to that in Caucasians, though the levels in overweight and obese patients were lower than those reported in Caucasians, probably due to the differences of the degree of obesity.
Adiposity, measured as BMI or percent body fat, was the dominant factor related to leptin level. With BMI, the correlation were highly significant and with percent body fat which was calculated from BMI according to gender and age, the correlation still remained significant. As an adipocyte-derived hormone, leptin helps burning body fat, reduces body weight in rodents, which once brought the cure hope to the obese. However, it did not turn out so optimistic. In fact, leptin levels, confirmed in this study, are higher in most obese humans, suggesting a state of leptin resistance. It was hypothesized that there may be a result of internal defects, such as leptin antibodies, leptin “antagonists” (e.g. neuropeptide Y, agouti protein, orexin etc.)[10] of the body, or increased production of leptin binding proteins which prevents free leptin from reaching the brain, these hypotheses need further proving. Nevertheless, it is reasonable to consider that the circulating leptin, being a 146-amino acid protein, is difficultly crossing the blood-brain barrier unless a specific transporter facilitates this step. Therefore, leptin resistance could be due to a defect in this putative transporter system. On the other hand, to some extent, the overlap of leptin levels in lean and obese subjects are evident. Although levels lower than 1.32ng/ml was only found in lean subjects, the leptin levels in 25%(7/28)of obese subjects(BMI>25) were lower than the mean level of normal-weight subjects, forming a group of leptin deficiency (relative), which lights the prospect of the Stage II leptin trial in humans[11].
Leptin levels are significantly different between men and women even after adjusting for body fat. There is a 3-4 fold elevation of serum leptin in women when comparing with men for both normal weight and obese subjects. Most studies demonstrated that there were independent relationship between leptin and total body fat content[12-14], it would probably exclude the possibility of the gender-based fat distribution and adipocyte differentiation. We accepted the hypothesis suggesting a role of the sex hormones. In fact, in vitro, estradiol stimulates leptin secretion and release from adipose tissue in women, but not in men[15]. Meanwhile, testosterone inhibits leptin secretion and release in mRNA and protein level either in vitro or in vivo[16]. Our current study indicates that leptin was also negatively correlated to the testosterone level.
Waist/hip ratio(WHR) is a parameter which roughly estimates the fat accumulation in abdomen. It has been shown in some prospective researches that WHR possesses many important relationships with several metabolic syndromes such as hyperinsulinemia, insulin resistance, glucose intolerance as well as dislipidemia. There is no consensus standard on WHR normal range available. The suggested value that normal men should be less then 0.9 and women less than 0.85 was adopted in our study. There was no independent relationship between leptin and fat distribution, as measured by the WHR, consistent with the previous reports in which the abdominal fat (mostly visceral) was estimated by the CT scanning[17]. However, after grouping by gender, serum leptin becomes correlated with WHR in men, whereas their relationship was less pronounced in women. This may be explained by the influence of androgen on fat deposition. On interpreting our data, we favor the hypothesis that leptin regulates overall body fat content, which can then be directed by prevailing androgen levels to an upper body distribution.
There is little relationship between serum leptin level and age. It was reported that circulating leptin was inversely related to age and was reduced 53% in subjects over age 60yr[18]. However, in our study, although a slight decreasing trend of leptin by age is observed, obviously no such reduction in the elderly.
In summary: For the first time we measured serum leptin level in Chinese by an internationally comparable RIA method. The results were similar to the people of the western countries. The mean levels were higher in overweight or obese subjects. BMI, body fat mass, gender and age were the factors which influence serum leptin level, especially important, were the first three factors. We found that 25% of the obese subjects were relatively deficient of leptin, they may become the good candidates for the exogenous leptin treatment in the future.
References
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- Halaas JL, Gagiwala KS, Maffei M et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 1995,269:543-546
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Tables
Tab.1 Clinical Characteristics
lean (BMI<25) Overweight and obese (BMI≥25)
male n= 33 female n= 44 male n= 20 female n= 8
age(Yr) 32.33±11.73 35.45±11.04 34.35±12.00 41.00±15.78∙
(17-65) (17-67) (15-63) (20-68)
BMI(Kg/m2) 21.70±2.67✧ 20.48±2.25* 27.57±2.34 30.11±5.24
(15.90-24.91) (15.43-24.61) (25.03-33.86) (25.27-42.19)
%fat 16.30±3.19✧ 23.13±3.34*° 23.44±2.85 37.39±7.76∙∙
(9.24-20.21) (15.67-29.25) (20.36-31.11) (30.23-55.27)
WHR 0.83±0.05✧ 0.78±0.05*° 0.89±0.05 0.88±0.05
(0.73-0.95) (0.67-0.91) (0.80-0.99) (0.79-0.94)
leptin 2.15±1.46✧✧7.86±3.60**° 4.87±3.47 16.59±6.92∙∙
(ng/ml) (0.58-5.69) (1.72-20.63) (1.42-17.59) (6.59-27.01)
T 379.4±125.9✧✧✧ 334.4±143.3
(ng/dl) (159.20-710.8) (187.6-764.9)
Notes:all data are shown as mean±SD (range),
compared with the obese males,✧ p<0.001;✧✧ p<0.01;✧✧✧ p>0.05
compared with the obese females,* p<0.001;** p<0.01
compared with the lean males,° p<0.001
compared with the obese males,∙ p<0.01;∙∙ p<0.001
Tab.2.Pearson and partial correlation analysis for the association between leptin and adiposity
Pearson’s correlation coefficient
male n= 53 female n=52 total n=105
body measurements
BMI 0.69c(0.72c) 0.63c(0.63c) 0.40c(0.66c)
%fat 0.50c(0.72c) 0.54c(0.63c) 0.65c(0.66c)
WAIST 0.59c 0.64c 0.29b
WHR 0.57c 0.41b 0.05a
age -0.12a(-0.61c) 0.17a(-0.40b) -0.06a(-0.47c)
sex hormone
testosterone -0.29b -0.29b
a.p=NS b.p<0.05 c.p<0.001
(partial correlation coefficients after adjustment are in the parentheses)
Tab.3.Multiple regression analysis for independent contribution of gender ,%fat and age on leptin
p r2
gender <0.001 0.149
gender <0.001
+%fat <0.001 0.378
gender <0.001
+%fat <0.001
+age <0.001 0.419
Figure legends
Fig1.Gender-based differences of leptin
Notes:compared with the females in the same group, ** p<0.001
compared with the lean males, # p<0.01
compared with the lean females, + p<0.01
Fig2(a) The relationship between leptin and BMI,dotted line indicating the board line between lean and obese(overweight) subjects
Fig2(b) The relationship between leptin and percent body fat
Fig3. The relationship between leptin and age in men
Fig.1
Fig.2(a)
Fig.2(b)
Fig.3