Leptin inhibits gonadotrophin-stimulated granulosa cell progesterone production by antagonizing insulin action

John D. Brannian1, Yulian Zhao and Michelle McElroy

Department of Obstetrics and Gynecology, University of South Dakota School of Medicine, 1400 West 22nd Street, Sioux Falls, SD 57105–1570, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent evidence has demonstrated that expression of leptin and leptin receptors is expected in the human ovary, and that leptin alters ovarian steroidogenesis in animal models. This study was designed to determine whether leptin modulates basal, gonadotrophin-, and insulin-stimulated progesterone production by human luteinized granulosa cells (GC). GC were recovered from follicular aspirates obtained during transvaginal ultrasound-guided oocyte retrieval for in-vitro fertilization–embryo transfer, and cultured in defined medium with various combinations of chorionic gonadotrophin (HCG; 0 or 100 ng/ml), insulin (0–30 µg/ml), and leptin (0–100 ng/ml). Progesterone concentrations in media were determined at various time points (2 h to 6 days). Leptin time- and dose-dependently inhibited (P < 0.05) HCG-stimulated progesterone production by human luteinized GC, but did not alter basal steroidogenesis. Moreover, the inhibitory effect of leptin on gonadotrophin-stimulated progesterone production was only manifested in the presence of insulin. Leptin suppression of insulin-supported steroidogenesis was also time- and dose-dependent. We conclude that leptin inhibits gonadotrophin-stimulated GC progesterone production apparently by antagonizing insulin action. Leptin suppression of progesterone production by human luteinized GC is consistent with recent data from animal models, and supports the possible role of leptin as a regulator of human ovarian function.

Key words: granulosa cells/insulin/leptin/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leptin is a tumour necrosis factor {alpha} (TNF{alpha})-related cytokine secreted by adipocytes. Since its recent discovery, leptin has received a great deal of attention as a metabolic regulator and appears to have an intimate interactive relationship with insulin (Bray and York, 1997Go). Initial interest in a potential reproductive role for leptin focused on its effects on the hypothalamic–pituitary axis. Leptin modulates gonadotrophin and gonadotrophin-releasing hormone secretion (Barash et al., 1996Go; Chehab et al., 1996Go; Yu et al., 1997Go), and may be an important signal in the onset of puberty (Hassink et al., 1996Go; Yu et al., 1997Go).

Recent evidence has revealed a possible role for leptin as a direct regulator of ovarian function. Leptin inhibited insulin-induced steroidogenesis by bovine granulosa (Spicer and Francisco, 1997Go) and thecal (Spicer and Francisco, 1998Go) cells, and suppressed insulin-like growth factor (IGF)-1 action in mediating follicle stimulating hormone (FSH) stimulation of rat granulosa cell steroid production (Zachow and Magoffin, 1997Go). Leptin and its receptor are expressed in the human ovary (Cioffi et al., 1997Go; Karlsson et al., 1997Go), and a high dose of leptin suppressed LH-stimulated oestradiol production in human granulosa cells (Karlsson et al., 1997Go). The objective of the present study was to determine whether leptin, at physiological doses, alters gonadotrophin- and insulin-stimulated progesterone production by luteinized human granulosa cells (GC) in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The experimental protocol was approved by the University of South Dakota Institutional Review Board (IRB). Luteinized GC were recovered from follicular aspirates of women (n = 16) during transvaginal ultrasound-guided oocyte retrieval for in-vitro fertilization (IVF) and embryo transfer. All women were down-regulated with gonadotrophin-releasing hormone (GnRH) agonist for 10–14 days, stimulated for 7–10 days with purified or recombinant FSH, and given human chorionic gonadotrophin (HCG; 10 000 units) 34 h prior to oocyte retrieval. Women with a diagnosis of polycystic ovaries were excluded.

Follicular aspirates from each patient were pooled, washed in Ham's F-10 medium supplemented with 0.1% bovine serum albumin (BSA), and centrifuged over 40% Percoll (Sigma, St Louis, MO, USA) to remove red blood cells. GC were washed again with Ham's F-10/BSA, resuspended in bicarbonate-buffered Dulbecco's modified Eagle medium (DMEM)/Ham's F-12 (1:1 v/v), and plated onto 96-well plastic culture plates (CoStar, Cambridge, MA, USA) coated with bovine fibronectin (Sigma) at a density of 2x104 cells/0.2 ml. The culture medium (DMEM/F-12) was supplemented with penicillin/streptomycin (50 000 units/50 mg/l; Sigma), low density lipoprotein (25 µg/ml; Sigma), transferrin (5 mg/l; Sigma), sodium selenite (0.25 nmol/l; Sigma), and bovine insulin (2 µg/ml; Sigma). For experiment 2 (see below), human insulin (0–30 µg/ml) was substituted for the bovine insulin. Cells were cultured in a humidified atmosphere of 5% CO2:95° air at 37°C.

Experiment 1
As a preliminary experiment to determine whether a high dose of leptin altered progesterone production, GC (n = four patients) cultures were treated in duplicate wells with human recombinant leptin (0 or 100 ng/ml; Linco Research, St Louis, MO, USA) ± HCG (100 ng/ml; CR-127, NHPP-NIDDK, Baltimore, MD, USA) for 24 or 48 h. To further characterize the time- and dose-dependency of leptin action, GC (n = 6 patients) were cultured for up to 48 hours with leptin (0–100 ng/ml) ± HCG (100 ng/ml). Progesterone concentrations in media were measured at 2, 4, 24, and 48 h.

Experiment 2
GC (n = 6 patients) were treated in triplicate wells with leptin (0 or 50 ng/ml) and human insulin (0–30 µg/ml; Sigma) ± HCG (100 ng/ml). Progesterone concentrations in media were measured after 1, 2, 4, and 6 days of culture.

Progesterone was assayed using Diagnostic Products Corporation (DPC, Los Angeles, CA, USA) Coat-a-Count radioimmunoassay kits. Media samples were diluted (1:100 or 1:200) in zero standard and measured in duplicate by direct assay following the manufacturer's protocol. Parallelism was demonstrated across multiple dilutions. Low, medium, and high range controls were run at the end of each standard curve and again at the end of each assay. Intra- and inter-assay coefficients of variation were 4.9 ± 1.1 and 7.7% respectively.

Data were log-transformed to achieve homogeneity of variance prior to analysis of variance (ANOVA). For experiment 1, one-way ANOVA with repeated measures blocked by patient was performed on data sets from each time point (i.e. 2, 4, 24, 48 h). For experiment 2, two-way ANOVA was performed on data sets from each time point (i.e. day 1, 2, 4, 6) with treatments (±leptin, ±HCG) as factor A and insulin concentrations as factor B. These analyses were followed by one-way ANOVA with repeated measures blocked by patient across treatments (±leptin, ±HCG) for each concentration of insulin, and across insulin concentrations within each treatment. In all cases, mean comparisons were made using Scheffé's f-test. Significance was assumed at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Leptin at high physiological concentration (100 ng/ml) suppressed (P < 0.05) HCG-stimulated progesterone production by luteinized GC at 24 and 48 h of culture (Figure 1Go), but did not alter basal progesterone production. The time- and dose-dependency of leptin inhibition of gonadotrophin steroidogenesis is shown in Figure 2Go. Leptin did not alter granulosal progesterone secretion after 2 h, but 10 and 50 ng/ml doses suppressed progesterone production by 4 h (P < 0.05). Very low (0.1 ng/ml) to high (100 ng/ml) doses of leptin inhibited progesterone production after 24 and 48 h of culture. Although not statistically different from other doses, 50 ng/ml consistently resulted in maximum inhibition of steroidogenesis (up to 60% in some cell cultures).



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Figure 1. Progesterone production by human luteinized granulosa cells treated with HCG (0 or 100 ng/ml) and leptin (0 or 100 ng/ml) after 24 and 48 h of culture. Culture medium was supplemented with bovine insulin (2 µg/ml). Means ± SE of four separate experiments (i.e. four different patients), each run with duplicate wells for each treatment. Bars with different letters are statistically different (P < 0.05) by ANOVA with Scheffé's f-test.

 


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Figure 2. Progesterone production (expressed as percentage of control, i.e. without leptin) by human luteinized granulosa cells treated with HCG (100 ng/ml) and leptin (0–100 ng/ml) after 2, 4, 24 and 48 h of culture. Culture medium was supplemented with bovine insulin (2 µg/ml). Means of six separate experiments, each run with duplicate wells for each treatment. Common estimates of variance (÷MSE/n) for each time point were: 2 h = 1.4%, 4 h = 2.4%, 24 h = 3.4%, and 48 h = 2.1%. Asterisks denote means (of raw data) that differ (P < 0.05) from the controls.

 
The effect of leptin on insulin responsiveness in HCG-stimulated GC cultures is depicted in Figure 3Go. After 24 h (day 1), leptin (50 ng/ml) suppressed (P < 0.05) HCG-stimulated progesterone production in the presence of 3 µg/ml insulin, but not in the absence of insulin. In combination with other concentrations of insulin (1, 10, 30 µg/ml), leptin tended to reduce progesterone secretion. Leptin continued to suppress (P < 0.05) HCG-stimulated steroidogenesis in the presence of low insulin concentrations (1 or 3 µg/ml) through 6 days of culture, but had no effect in the absence of insulin. Leptin inhibited (P < 0.05) HCG-supported progesterone production in the presence of all concentrations of insulin (1–30 µg/ml) after 4 and 6 days of culture. In general, leptin did not alter non-gonadotrophin-stimulated progesterone production in the presence or absence of insulin. However, leptin appeared to enhance unstimulated steroidogenesis at a few time points (e.g. day 1, 0 µg/ml insulin; day 2, 1 µg/ml insulin; Figure 3Go).



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Figure 3. Progesterone production by human luteinized granulosa cells treated with HCG (0 or 100 ng/ml), human insulin (0–30 µg/ml), and leptin (0 or 50 ng/ml) after 1, 2, 4 and 6 days of culture. Means of six separate experiments, each run with triplicate wells for each treatment. Common estimates of variance (÷MSE/n) for treatments ranged from 4.5–28.4 ng progesterone. Different letters denote statistical differences (P < 0.05) within each insulin concentration at a given time point. NS = not significant.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results demonstrate that leptin inhibits steroidogenesis by luteinized human GC by interfering with the stimulatory action of gonadotrophins. Moreover, leptin inhibition of gonadotrophin-stimulated steroidogenesis was only manifested in the presence of insulin. Since gonadotrophin stimulation of ovarian steroidogenesis is mediated in part by insulin and insulin-like growth factors (Adashi, 1993Go), it appears that leptin antagonizes insulin and/or IGF action in this stimulatory pathway.

Inhibition of HCG-stimulated progesterone production by luteinized human GC is consistent with the results of previous studies on rat (Zachow and Magoffin, 1997Go), bovine (Spicer and Francisco, 1997Go), and human (Karlsson et al., 1997Go) GC demonstrating leptin suppression of gonadotrophin-stimulated oestradiol production. That physiological doses of leptin (10–50 ng/ml) suppressed progesterone production by as early as 4 h in culture indicates that leptin can act in an acute manner. Moreover, GC appear to be very sensitive to leptin action as indicated by suppression of steroidogenesis by very low doses of leptin (as little as 0.1 ng/ml).

Our initial experiment showing leptin inhibition of HCG-stimulated progesterone production was conducted with bovine insulin present in the culture medium. The second series of experiments was conducted to investigate the effect of leptin on the insulin responsiveness of human GC steroidogenesis. It was reported that leptin impaired the synergistic effect of insulin-like growth factor (IGF-1) in potentiating FSH stimulation of rat GC oestradiol synthesis, but did not alter the stimulatory effect of FSH alone (Zachow and Magoffin, 1997Go). Similarly, it was reported that leptin inhibited FSH-stimulated steroid production by bovine GC in the presence of insulin, but had minimal or no effect in the absence of insulin (Spicer and Francisco, 1997Go). Our results confirm these animal studies in that leptin only suppressed HCG-stimulated progesterone production in the presence of insulin.

The degree of leptin inhibition varied with different concentrations of insulin and duration of treatment. However, there was not a clear dose- and time-dependent relationship. For example, the most pronounced effect of leptin was consistently at a concentration of 3 µg/ml insulin relative to higher or lower insulin concentrations. Moreover, leptin in the absence of insulin or at very low concentrations tended to enhance non-gonadotrophin-stimulated progesterone production at certain time points. Therefore the cellular signalling interactions among gonadotrophins, insulin, and leptin may be quite complex. Furthermore, we do not know what effect, if any, intrinsic IGF may have had in our culture system.

Insulin and IGF are known to enhance stimulable cyclic(c) AMP formation and enhance cAMP action within GC (Adashi, 1993Go). The exact mechanism by which insulin/IGF signalling pathways communicate with the adenyl cyclase pathway is not completely understood. Insulin receptor substrate-1 (IRS-1) is a ubiquitous protein in insulin/IGF-responsive cells that can act as a link between the insulin receptor and other signalling pathways (White, 1997Go). Thus leptin-induced suppression of insulin action in GC might involve IRS-1. Indeed, recent evidence has demonstrated that both TNF{alpha} (Hotamisligil et al., 1996Go) and leptin (Bjorbaek et al., 1997Go) can modulate IRS-1 activity in adipocytes. The fact that leptin did not alter insulin-supported progesterone production in the absence of gonadotrophin strongly implicates impairment of cAMP formation as the ultimate consequence of leptin action.

Leptin has been proposed to play a role in the aetiology of polycystic ovary syndrome (PCOS) (Brzechffa et al., 1996Go; Micic et al., 1997Go), although this possibility remains quite controversial (Gennarelli et al., 1998Go). Considering the importance of insulin resistance and hyperinsulinaemia in the aetiology of PCO (Homburg et al., 1996Go), antagonistic interaction between leptin and insulin at the ovarian level has intriguing implications. Although leptin may not be an obligatory factor in PCO (Gennarelli et al., 1998Go), it remains to be elucidated how leptin–insulin interactions in the ovary are manifested clinically.

Although leptin is primarily secreted by adipocytes, leptin mRNA expression has been reported to be present in granulosa and cumulus cells of pre-ovulatory human follicles (Cioffi et al., 1997Go). Moreover, in immunofluorescence studies, leptin exhibited a localized distribution in cumulus cells associated with regions of intense leptin immunofluorescence in the oocyte (Antczak and Van Blerkom, 1997Go). These authors proposed that leptin from cumulus cells may be transferred into oocytes and play a role in embryonic development. Thus leptin may have multiple, complex roles in ovarian function.

In summary, leptin at physiological levels suppresses gonadotrophin-stimulated progesterone production by luteinized human GC apparently through antagonism of insulin (and/or IGF) action. The cellular mechanism of this antagonism remains to be elucidated. The results suggest that leptin may be a physiological regulator of follicular and/or luteal function in humans.


    Acknowledgments
 
The authors thank Pamela Long and Carla Rickert for technical assistance. This work was supported by National Science Foundation (US) grant OSR-9452894 and NICHD grant HD-35333.


    Notes
 
1 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on October 28, 1998; accepted on February 15, 1999.