Human Reproduction, Vol. 18, No. 4, 715-720,
April 2003
© 2003 European Society of Human Reproduction and Embryology
Relationships between the serum levels of soluble leptin receptor and free and bound leptin in non-pregnant women of reproductive age and women undergoing controlled ovarian hyperstimulation
Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, Kyoto 602–8566, Japan
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602–8566, Japan. e-mail: kitawaki{at}koto.kpu-m.ac.jp
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Abstract |
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BACKGROUND: The aim of this study was to investigate the relationships between the serum levels of soluble leptin receptor (SLEPR), and total, free and bound leptin, and the change in the serum SLEPR level during an IVF cycle. METHODS: Serum concentrations of leptin and SLEPR were measured in 50 Japanese women of reproductive age, and 20 patients participating in an IVF programme. The total leptin was fractionated into free and bound portions by gel filtration chromatography. RESULTS: The SLEPR level was negatively correlated with the body mass index (BMI) (r = –0.548, P < 0.0001), total leptin (r = –0.433, P < 0.0001), the percentage of free leptin (r = –0.732, P < 0.0001) and the absolute free leptin concentration (r = –0.506, P < 0.0001). The SLEPR level was positively correlated with the percentage of bound leptin (r = 0.730, P < 0.0001), whereas there was little variation in the absolute bound leptin concentration, regardless of the BMI or SLEPR concentration. During the IVF cycle, total and free leptin elevated during maximal ovarian stimulation, whereas there was no significant difference in the SLEPR concentration. CONCLUSIONS: The results demonstrate a skilful mechanism where a change in the serum SLEPR level regulates, in part, the biological activity of leptin in the circulation.
Key words: bound leptin/controlled ovarian hyperstimulation/free leptin/IVF/soluble leptin receptor
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Introduction |
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Leptin, the product of the obese (ob) gene, is a 16 kDa hormone principally secreted by the adipose tissue (Zhang et al., 1994
). This protein regulates the body fat mass by decreasing food intake through interaction with hypothalamic leptin receptors (LEPR) (Tartaglia et al., 1995). In humans, the circulating level of leptin is positively correlated with the amount of total body fat mass (Maffei et al., 1995; Considine et al., 1996). Leptin also modulates various reproductive functions which are mediated mainly through brain LEPR (Barash et al., 1996; Chehab et al., 1996; Ahima et al., 1997). In addition to the central action, leptin and LEPR are expressed in peripheral reproductive tissues (Moschos et al., 2002) including granulosa cells (Antczak et al., 1997; Cioffi et al., 1997; Karlsson et al., 1997), where leptin directly affects estrogen production (Spicer and Francisco, 1997; Zachow and Magoffin, 1997; Kitawaki et al., 2000). LEPR, but not leptin, is expressed in human endometrium and the amount of LEPR fluctuates during the menstrual cycle (Kitawaki et al., 2000), partly due to the action of progesterone (Koshiba et al., 2001).
In serum, leptin circulates as free and protein-bound forms (Sinha et al., 1996). A soluble LEPR (SLEPR) has been shown to account for the majority of the serum leptin binding activity (Lammert et al., 2001; Maamra et al., 2001). SLEPR consists entirely of the extracellular ligand-binding domain and lacks the transmembrane residues and intracellular domain responsible for signal transduction (Lee et al., 1996). SLEPR plays a role in the regulation of the biological activity of leptin.
Recent studies have shown a reverse relationship between the serum SLEPR level and the body mass index (BMI) (Chan et al., 2002; Lahlou et al., 2002; Shimizu et al., 2002; Van Dielen et al., 2002; Wu et al., 2002). However, the relationships between the serum levels of SLEPR, and total, free and bound leptin have not been fully clarified. In the present study, we investigated these serum levels in non-pregnant Japanese women of reproductive age. In addition, the serum level of total leptin has been shown to be elevated under conditions of ovarian hyperstimulation (Mannucci et al., 1998; Messinis et al., 1998; Strowitzki et al., 1998; Butzow et al., 1999; Stock et al., 1999; Lindheim et al., 2000; Unkila-Kallio et al., 2001). We investigated the change in the serum level of SLEPR during an IVF cycle.
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Materials and methods |
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Subjects
The study protocol was approved by the Kyoto Prefectural University of Medicine Institutional Review Board, and informed consent was obtained from each subject. A total of 50 Japanese women of reproductive age [36.3 ± 8.1 years (mean ± SD), range 24–48], who had neither a malignant tumour or pregnancy, was enrolled. None of the subjects had a history of diabetes mellitus, thyroid or another endocrine disease. Neither were they receiving any steroids or lipid-reducing drugs. Blood samples were collected during the early follicular phase if the patients had a spontaneous menstrual cycle. To investigate changes during the menstrual cycle, 20 women (32.6 ± 3.8 years, range 26–42; BMI 21.2 ± 3.3 kg/m2, range 13.9–26.7), participating in an IVF programme, who were otherwise healthy but complained of infertility, were enrolled. Each patient underwent a long standard protocol treatment starting with an intranasal spray of buserelin acetate (Sprecur®; Aventis Pharma, Tokyo, Japan, two shots of 150 µg/shot, three times daily) during the mid-luteal phase of the preceding cycle. After the onset of menstruation, ovarian stimulation was initiated by giving 225–300 IU daily of either hMG (Humegon®; Organon, Oss, The Netherlands) or purified urinary FSH (Fertinom P®; Serono, Geneva, Switzerland). Ovarian stimulation was monitored by follicular diameter and serum estradiol concentration. When at least three follicles were
16 mm in diameter, the administration of buserelin acetate was stopped and 10 000 IU hCG was injected. Oocyte retrieval was carried out by transvaginal aspiration 36 h after the hCG injection. Oocytes were fertilized in vitro and 1–3 embryos were transferred 3 days after oocyte retrieval. After the embryo transfer, an intravaginal tablet of progesterone was given (200 mg, twice daily for 11 days). Blood was drawn in the morning after an overnight fast. For the patients undergoing IVF, blood samples were collected at the following five points during the treatment: (i) the mid-luteal phase before the start of buserelin acetate (cycle day 21.6 ± 3.3); (ii) in the presence of ovarian suppression but before gonadotrophin administration; (iii) during maximal ovarian stimulation 1–2 days before the hCG injection; (iv) at the time of oocyte retrieval; and (v) 14 days after oocyte retrieval. Sera were separated by centrifugation (1700 g for 10 min) and kept frozen at –20°C until analysis. Serum concentration of estradiol was assayed using radioimmunoassay (Diagnostic Products Co, Los Angeles, CA, USA).
Assay of total, bound and free leptin in serum
Total leptin concentrations were initially measured by enzyme-linked immunosorbent assay (R & D Systems, Minneapolis, MN, USA). The detection limit was 7.8 pg/ml, and the intra- and inter-assay coefficients of variation were 3.0–3.3% at 64.5–621 pg/ml and 3.5–5.4% at 65.7–581 pg/ml respectively. Each concentration was determined by duplicate assays followed by averaging. Bound and free portions of leptin were subsequently separated by gel filtration chromatography as described by Sinha et al. (1996) with modifications. All experiments were performed at 4°C. A 2.0 ml aliquot of the serum sample was pre-incubated with [125I]-leptin (PerkinElmer Life Sciences Inc., Boston, MA, USA, 
36 000 cpm, 0.27 ng) for 24 h, loaded onto a Sephadex G-100 column (1.5x40 cm) and then eluted with 25 mmol/l phosphate-buffered saline, pH 7.4, containing 0.01% sodium azide. The radioactivity in 50 fractions (1.0 ml each) formed two peaks. The first peak eluted in the void volume and represented the bound fraction, and the second peak represented the free fraction. The recovery of leptin from the column was 93.4 ± 3.0%. The percentages of bound and free leptin to total leptin were calculated by dividing by the total [125I]-leptin eluted. The absolute concentrations of bound and free leptin were calculated by multiplying the percentage of bound and free leptin, respectively, by the total leptin concentration and dividing by 100.
Assay of the soluble form of LEPR (SLEPR) in serum
The SLEPR concentration in serum was measured by enzyme-linked immunosorbent assay (BioVendor Laboratory Medicine Inc., Brno, Czech Republic). The detection limit was 0.4 unit/ml, and the intra- and inter-assay coefficients of variation were 5.6–8.2% at 8.45–22.73 units/ml and 3.9–5.1% at 16.65–35.15 units/ml respectively. Since the assay data were not influenced by the addition of up to 100 units/ml leptin to the serum, standards and controls, the kit appeared to measure the total SLEPR, either bound or free. Each concentration was determined by duplicate assays followed by averaging.
Statistics
Correlations between variables were examined by Pearson’s correlation coefficients. Simple regression lines were made in which the maximal coefficient of determination (R2) was obtained. Variables during the IVF cycles were expressed as the mean ± SEM, and analysed using one-way ANOVA followed by multiple comparisons using Bonferroni/Dunn’s procedure. A P-value of <0.05 was considered significant.
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Results |
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The serum concentration of total leptin showed a positive correlation with the BMI (r = 0.711, P < 0.0001) (Figure 1A), whereas the serum concentration of SLEPR showed a negative correlation with the BMI (r = –0.548, P < 0.0001) in 50 women of reproductive age (Figure 1B). Consequently, the serum concentration of SLEPR was negatively correlated with the total serum leptin concentration (r = –0.433, P < 0.0001) (Figure 1C). However, the SLEPR level almost reached the minimal level of
16 units/ml at a total leptin level of 20 ng/ml, which corresponded to a BMI of less than 30 kg/m2, and remained almost constant in the presence of >20 ng/ml of total leptin.
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The total leptin was fractionated into free and bound portions by gel filtration chromatography after incubation of each serum sample with radiolabelled leptin. The percentage of free leptin was positively correlated with the BMI (r = 0.640, P < 0.0001), whereas the percentage of bound leptin was negatively correlated with the BMI (r = –0.638, P < 0.0001) (Figure 2A). Accordingly, the serum concentration of SLEPR was negatively correlated with the percentage of free leptin (r = –0.732, P < 0.0001), and positively correlated with the percentage of bound leptin (r = 0.730, P < 0.0001) (Figure 2B).
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When the percentages of the leptin proportions were calculated into absolute concentrations, the free leptin level was positively correlated with the BMI (r = 0.728, P < 0.0001) (Figure 2C), whereas the SLEPR level was negatively correlated with the absolute concentration of free leptin (r = –0.506, P < 0.0001) (Figure 2D). However, the SLEPR level almost reached the minimal level of
16 units/ml at a free leptin level of 16 ng/ml, which corresponded to 30 kg/m2 BMI, and remained almost constant in the presence of >16 ng/ml of free leptin (Figure 2D). Interestingly, although there was a positive correlation between the absolute concentrations of bound leptin and the BMI (r = 0.701, P < 0.0001) (Figure 2E), and a negative correlation between SLEPR and the absolute concentrations of bound leptin (r = –0.433, P < 0.0001) (Figure 2F), little variation was observed in the absolute concentrations of bound leptin compared with the free leptin levels, regardless of the BMI or SLEPR concentration. The mean concentration of bound leptin in all serum specimens was 2.5 ± 1.3 ng/ml.
The variations in the total leptin and SLEPR concentration in the serum during the IVF cycle are summarized in Figure 3. The total leptin levels at the time of maximal ovarian stimulation (group c) were significantly higher than the levels in the presence of ovarian suppression (group b) (P < 0.05) (Figure 3B) in accordance with the increase in serum estradiol levels (Figure 3A). In contrast, there was no significant difference in the serum level of SLEPR among the five different points measured (Figure 3D). There was no significant difference when the patients were divided into three groups according to the BMI. Accordingly, the absolute concentration of bound leptin was almost constant during the IVF cycle, while the absolute concentration of free leptin was higher at the time of maximal ovarian stimulation (group c) than in the presence of ovarian suppression (group b) (P < 0.05) (Figure 3C).
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Discussion |
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The present results indicate that the serum level of SLEPR is inversely correlated with both the total serum leptin level and the BMI in non-pregnant women of reproductive age. In lean women, the serum conditions of a lower total leptin level and a higher SLEPR level results in a higher percentage of bound leptin and a lower percentage of free leptin, and exhibits a suppressed activity of leptin. Conversely, in obese women, due to the higher total leptin level and lower SLEPR level, the percentage of bound leptin was low and the percentage of free leptin was high, leading to a higher activity of leptin. A similar relationship was recently reported by other groups (Chan et al., 2002
; Lahlou et al., 2002; Shimizu et al., 2002; Van Dielen et al., 2002; Wu et al., 2002). Notably, our results demonstrate that, as a consequence of the inverse relationship between the levels of leptin and SLEPR, the absolute level of bound leptin appeared to be almost constant, regardless of the leptin levels or BMI. In serum, leptin and SLEPR bind and dissociate in a simple ligand-receptor fashion (Quinton et al., 1999) and SLEPR is capable of binding to leptin at a 1:1 ratio (Devos et al., 1997). Therefore this is a reasonable regulatory mechanism by which lean women can suppress the biological activity of leptin and retain leptin in their serum as the bound form. Obese women can conversely increase the activity of leptin by minimizing the bound leptin level. This may suggest that SLEPR plays an important role in modulating the biological activity of leptin as a reservoir or blocker. Huang et al. (2001) observed an increase of circulating leptin without affecting the leptin expression after injecting rats with adenoviruses encoding SLEPR. They concluded that the elevation of leptin level resulted from delayed clearance in the presence of overexpressed SLEPR. However, it is still not known where SLEPR is produced or how SLEPR is regulated at the transcriptional level. It may be possible that free leptin regulates the SLEPR level.
Our results show that the approximate serum levels of free leptin can be calculated simply by subtracting the constant value of the bound leptin level (2.5 ± 1.3 ng/ml) from the total leptin level. This amplifies the difference in free leptin levels between lean and obese women. Furthermore, our results show that, while the total serum leptin level increases with the BMI, the serum SLEPR levels are minimal in women with a BMI of 30 kg/m2 or higher, indicating that only the increase of free leptin accounts for the increase of total leptin in obese women. This suggests that the extremely high levels of leptin in obese women are over the programmed range within which the serum leptin activity can be regulated by SLEPR.
In the present study, the gel chromatographic separation of the serum specimens after incubation with radiolabelled leptin resulted in two major peaks, the bound and free peaks. This suggests that SLEPR is the major leptin binding component in human serum. The proportions of free and bound leptin in human serum were analysed by other groups using gel filtration chromatography (Sinha et al., 1996; McConway et al., 2000), and were comparable with ours. Landt et al. (2000) and Wu et al. (2002) reported similar results using a different methodology to measure the bound form.
The present results show little variation in the serum level of SLEPR during the IVF cycle. In contrast, the serum level of total leptin is elevated during ovarian hyperstimulation, being consistent with previous reports by other groups (Butzow et al., 1997; Mannucci et al., 1998; Messinis et al., 1998; Strowitzki et al., 1998; Stock et al., 1999; Lindheim et al., 2000; Unkila-Kallio et al., 2001). Although the exact mechanism is still unknown, these authors have postulated that the rise in the estrogen level is involved in the elevation of the leptin level. Again, our data show that the elevation of free leptin accounts for increases in total leptin and that the level of bound leptin is almost constant. Lewandowski et al. (1999) measured the serum levels of SLEPR in pregnant women after 20 weeks gestation and showed no significant change, although the levels were 3-fold higher than our non-pregnant subjects. In contrast, the mRNA expression of the long form of LEPR in the endometrium was reduced during the secretory phase (Kitawaki et al., 2000) and inhibited by the addition of progesterone in vitro (Koshiba et al., 2001).
In conclusion, the serum level of SLEPR is inversely correlated with both the serum level of total leptin and the BMI in non-pregnant women of reproductive age. We have also demonstrated that the absolute level of bound leptin is almost constant, regardless of the leptin levels or the BMI. The serum SLEPR level does not change under the conditions of ovarian hyperstimulation during IVF treatment. These results demonstrate a skillful mechanism where a change in the serum SLEPR level regulates, in part, the biological activity of leptin in the circulation.
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Submitted on August 29, 2002; resubmitted on November 27, 2002; accepted on January 9, 2003.
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