Iranian Journal of Reproductive Medicine Vol. 7, No. 2, Spring, 2009, pp. 53-58
Relationships between free leptin and insulin resistance in women with polycystic ovary syndrome
Javad Mohiti-Ardekani1 Ph.D., Nasim Tarof1 M.Sc., Abbas Aflatonian2 M.D.
1 Department of Biochemistry and Molecular Biology, Shahid
Sadoughi University of Medical Sciences, Yazd, Iran.
Received: 1 December 2008; accepted: 11 April 2009
Code Number: rm09010
Background: Leptin is an
adipokine that circulates in a free form and bound to a soluble leptin receptor.
Patients with polycystic ovary syndrome have increased insulin resistance and
high incidence of obesity.
Key words: Free leptin, Polycystic Ovarian Syndrome (PCOS), Insulin resistance.
Leptin,the gene product of the ob gene, is thought to provide the centralnervous system with feed-back information about fat storageof the body (1). Thus, leptin is thought to be a part of the regulationof appetite, food intake and the lipid metabolism (2).
Obese humanspresent with hyperleptinemia as an indicator of leptin resistancewhichplays a major role in thepathogenesis of obesity (3).Polycystic ovary syndrome (PCOS) is among the most common endocrinedisorders, affecting more than 5% of women of reproductive age(4, 5). An association of PCOS with peripheral insulin resistance,compensatory hyperinsulinemia and alterations in ß-cellfunction as the cause of its predisposition to develop a metabolicsyndrome (type 2 diabetes mellitus, hypertension, lipid disorders,obesity) has been established (6). According to the majority of studies, most PCOS women are insulinresistant and overweight or obese (7-11).In mice, a genetic defect in the ob gene results in severe obesityand type 2 diabetes mellitus, as well as in infertility (6).
In this mouse model, leptin injections restore fertility. Leptintreatment in normal female mice accelerates puberty (12). Inhumans, granulosa cells have been shown to secrete leptin (13)and leptin seems to influence adrenal androgen formation (14).
However, the role of leptin in ovarian function or on the reproductivesystem remains unclear. It was shown that leptin levels did notinfluence follicular maturation and ovulation in PCOS patients(10) while Carmina et al demonstrateda negative correlation between leptin and dehydroepiandrosteronesulphate (DHEAS) levels in 21 lean PCOS women, suggesting thatandrogens may play a role in suppressing serum leptin (15).
Another study showed that leptin concentrations correlated inverselywith luteinizing hormone (LH) levels, independent of body weightand insulin resistance (16). In contrast again, Laughlin etal (17) proposed that leptin is neither involved in the hypersecretionof LH nor in the regulation of LH pulsatility. During lactationalamenorrhea Sir-Petermann et al (18) also showed that leptinsecretion is not related to LH secretion. Plasma leptin is in free and bound forms in circulation (19).
The free leptin hasbeen shown to correlate with total serum leptin levelsand BMI in women of reproductive age. Kado et al (20)hypothesized that obese women are able to conversely increasetheir leptin activity by minimizing the bound leptin fraction,thus influencing the biological activity of leptin. In linewith this, Sinha et al (21) reported that in lean subjects,leptin circulates predominantly in the bound form, whereas inobese patients, leptin circulates mostlyin the free form. Similar results were found for Japanese menand women, showing a direct correlation between free leptin leveland BMI and parameters of insulin resistance (22).
Weight loss, following gastric restrictive surgery,leads to a reduction in circulating leptin levels and to adecrease free leptin (23). In adolescent girls with anorexia nervosa,free leptin and Free Leptin Index (FLI) but not total leptin levels were closely relatedto parameters of sexual maturation and the onset of puberty(26). Therefore, to elucidate the free leptin role in PCOS, we purified the free leptin in PCOS patients and healthy control and analyzed the interrelations between serum total leptin and free forms with insulin and insulin resistance and the other metabolic factors.
Materials and methods
The study was carried out on 54 women (27 PCOS subjects and 27 voluntary BMI, and age matched healthy women with normal menstrual cycle as controls). Clinical diagnosis of PCOS was made from the 1990 National Institutes of Health (NIH) conference,when either oligomenorrhea(cycles lasting longer than 35 days) or amenorrhea (less thantwo menstrual cycles in the past 6 months) and either clinicalsigns of hyperandrogenism (hirsutism or obvious acne or alopeciaand/or an elevated total testosterone) were found, and other pituitary,adrenal or ovarian diseases could be excluded. PCOS as well as controlsubjects had not taken any medication known to affect carbohydratemetabolism or endocrine parameters for at least 3 months beforeentering the study. In controls, LH, FSH and insulin levels were estimated in the early follicular phase (second or third day of menstruation) from fasting blood samples.
In PCOS subjects and control women, clinical parameters wereassessed by physical examination, and BMI wascalculated as (weight/height2)(kg/m2).
Insulin resistancewas calculated by homeostasis assessment model (HOMA) using the formula HOMA-IR = Fasting insulin (μU/ml) × Fasting glucose (mg/dl)/22.5 (27). Low-IR values indicate high insulin sensitivity, whereas high-HOMA values indicate low insulin sensitivity (insulin resistance).
Leptin were measured using ELISA kits [DiagnosticSystems Laboratories (DSL), Webster, TX, USA]. The DSL ACTIVEHuman Leptin ELISA is an enzymatically amplified two-stepsandwich-type immunoassay.The theoretical sensitivity or minimum detection limit as calculatedby interpolation of the mean plus two standard deviations of12 replicants of the zero standard was 0.05 ng/ml. Intra-assay variation was <5% andinterassay variation was <8% for all measured parameters.Insulin concentrations were measured by sandwich ELISA (Webster, Texas 77598-4217 USA, DSL).
Free leptin form was purified by Gel filtration methods as bellow (20). This study was approved by the local ethical committee and each patient gave written informed consent.
Purification of free leptin form in serum
To obtain a standard curve, one vial containing 1 mg lyophilized leptin (Sigma) was dissolved in 0.5 ml HCL (1.5mM) and neutralized with 0.25 ml NaOH (7.5 mM) and applied with Marker Gel filtration Bludextran (Product Brand: Sigma Product Number: D4772) to Sephadex G-100 column chromatography (Amersham Biosource (1908-7101) (9/30 column).
The leptin was eluted with 0.25 mM phosphate buffer (Ph=7.4). Fractions eluting were collected and assayed by a Sensitive ELISA Kit (Catalogue Number: kap 2281: 96 determinations. Manufactured by: Biosource Europe S-A). It showed a single bound indicating free leptin fraction.
Serum sample (0.5ml) was fractionated by Sephadex G-100 gel filtration chromatogheraphy. Fractions eluting between void and bed volumes were assayed by the ELISA kit.
Data are presented as median plus range for non-parametric data.For better comparison, means ±S.D. is also shown. Correlations between variables were examined by Spearmans correlationcoefficient (rs) because analyzed data were not normally distributed.Differences between the groups were evaluated with the MannWhitneyU test. p values <0.05 were considered significant.
Patients with PCOS showed significantly higher concentrations of leptin (mean 21±9 ng/ml) vs. control (16±10 ng/ml, p=0.042), free leptin (mean 7.3±1.2) vs. control (mean 4.3±1.3 ng/ml, p=0.011) and LH (mean 76±18 IU/ml) vs. control (mean 2.87±1.93, p=0.001) in plasma (Table I).
We observed no marked difference in insulin level between patients with PCOS (mean 26±13µIU/ml) vs. control (mean 25±13) and FSH level between patients with PCOS (8.69±15 IU/ml) vs. control (mean 6.56±1.46) (Table I). Furthermore, we analyzed the relationships between leptin, free leptin with BMI, insulin, insulin resistance and LH in PCOS and control groups.
Significant correlations were observed between free leptin and BMI (rs=0.78, p=0.000),insulin resistance (IR) (r=0.53, p=0.004) and insulin (r=0.93, p=0.000) in PCOS patients. Significant correlations were also observed between free leptin and BMI (r=0.86, p=0.00), IR (r=0.57, p=0.003) and insulin (r=0.91, p=0.000) in control group (Table II).No significant correlation was observed between total and free leptin with LH.
Table I. Characteristic of control (n=27) and PCOS patients (n=27). Values are means ±SD (median and range).
Table II. Correlation of leptin and free leptin levels with metabolic parameters in PCOS and control women.
Leptin levels are increased in obesity and may play a role inthe development of insulin resistance(7). Our study showed a significant high total and free leptin in PCOS patients as it is compared with controls (Table I) (p<0.05). Total leptin levels correlated significantly withBMI in both PCOS women and controls. Leptin correlated with other metabolic parameters includinginsulin resistance and insulin level in both groups. Similar results were found in PCOS patientsfrom Australia (29), Brazil (16), Canada (10), Finland (30),Italy (15), Sweden (8), Turkey (11) and USA (17, 31). Significant correlation of total leptin with HOMA IR reflected a degree of insulin resistance in both controls and test groups (Table II). We can place our results along the large group of studies, which declare an important role of leptin in pathogenesis of insulin resistance (1, 2, 4, 6, 8, 11, 15, 20, 28, 29).
In addition, we found free leptin levels in PCOS patient and control group correlate with parameters of insulin, and insulin resistance (Table II).
Thus free leptin appears to be the appropriate form of leptin which it contributes to insulin resistance and leptin resistance. Potential mechanisms that may mediateleptin resistance include impairment of brain leptin transportor leptin receptor internalization, and receptor post-receptorsignaling defects. Furthermore, the active hormone may be reducedby binding proteins or soluble receptors (32). Soluble leptin receptor represents the main leptin-binding compound in plasma, thusregulating its free fraction in the circulation (33).
A considerable portion of circulating leptin is free form which is affected by the degree of adiposity and nutritional state (35-37). We found a positive correlation between free leptin with BMI in PCOS patients and controls.Yannakoulia and co-workers (24) demonstrated that Free leptin index iscorrelated with total energy intake and inversely correlatedwith energy intake from dietary fat. They speculated that themacronutrient composition of the diet influences serum concentrationsof free leptin. The authors hypothesizedthat the increase of free leptin levels representsa compensatory mechanism to overcome leptin resistance.
A recently published twin study of pubertal females studyingthe genetic and environmental influences on the variations ofleptin and free form levels showed that leptin is mostly influencedby body composition (25, 34). We have demonstrated the correlation of free leptin with insulin resistance in diabetes patients (38). In conclusion, a considerable part of plasma leptin is in free form in circulation.
Although the physiological function of bound and free leptin are not well understood, it has been hypothesized that leptin is more active in its free form because this form is present in cerebrospinal fluid (CSF) (34). We found a relationship between total leptin levels, andfree leptin with BMI, insulin resistance, and insulin levels in overweight PCOS patients and their matched control group.
However, further studiesare needed to investigate the effect of factors including food or body composition,and genetic on high level of free leptin inPCOS patients.
We thank Members of the Dept. of Biochemistry and Molecular Biology for helpful discussins. This research was supported by Shahid Sadoughi University of Medical Science and Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
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