Metformin alters insulin signaling and viability of human granulosa cells

Metformin alters insulin signaling and viability of human granulosa cells Barbara Sonntag, M.D., Martin Götte, Ph.D., Pia Wülfing, M.D., Andreas N. Schüring, M.D., Ludwig Kiesel, M.D., and Robert R. Greb, M.D. Department of Obstetrics and Gynecology, University Hospital of Münster, Münster, Germany

Objective: To study whether insulin signaling pathways in the ovary are altered by metformin. Design: In vitro human granulosa cell culture system. Setting: Academic research environment. Patient(s): Infertility patients undergoing oocyte retrieval for IVF/ICSI. Main Outcome Measure(s): Cell viability and phosphorylated protein kinase B (PKB/AKT) and p44/42 mitogenactivated protein kinase (MAPK) expression of human primary and HGL5 granulosa cells. Result(s): Basal cell viability of primary granulosa cells was significantly increased relative to control by metformin preincubation, without an additional stimulatory effect of insulin or IGF. Phosphorylated AKT expression in lysates of the human granulosa cell line HGL5 was significantly increased in contrast to decreased phosphorylated MAPK expression by metformin preincubation. Conclusion(s): Besides systemic effects, the ovulation inducing action of metformin may at least partially be due to direct effects on insulin signaling intermediates and follicular growth patterns in the ovary. (Fertil Steril威 2005; 84(Suppl 2):1173–9. ©2005 by American Society for Reproductive Medicine.) Key Words: AKT, granulosa, insulin signaling, MAPK, metformin, polycystic ovary syndrome

Metformin is an antidiabetic drug of the biguanides class, which is widely used as an insulin-sensitizing agent in the treatment of type 2 diabetes mellitus. Although metformin has been clinically used for over 40 years, its mechanism of action is not fully elucidated (1). Most studies show a decrease in endogenous glucose production by the liver (2, 3) followed by a reduction in hyperinsulinemia. Suggested alternative mechanisms include direct changes in peripheral tissue insulin sensitivity, e.g., by changes in insulin receptor tyrosine kinase activity (4). These alterations in insulin signaling may also be applicable to ovarian tissue, where insulin and insulin-like growth factors (IGF-1 and -2) act as “cogonadotropins” and intraovarian growth factors during steroidogenesis and follicular development. Folliculogenesis is impaired in polycystic ovary syndrome (PCOS), the most common cause of anovulation and infertility, affecting 5%–10% women of reproductive age (5). The use of metformin in the treatment of patients with PCOS has rapidly increased over the past years (6). Numerous effects have been described which conclusively, in a majority of patients, lead to a decrease in peripheral androgen levels, a restoration of menstrual cycles, and increased spontaneous and clomiphene-induced ovulation rates (7–9). DeReceived January 4, 2005; revised and accepted April 13, 2005. Supported by a research grant of the German Research Foundation (DFG SO 464/2). Presented at the 20th Annual Meeting of the European Society of Human Reproduction and Embryology, Berlin, Germany, June 27–30, 2004. Reprint requests: Barbara Sonntag, M.D., Department of Obstetrics and Gynecology, University Hospital of Münster, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany (FAX: ⫹49-251-8348267; E-mail: sonnta@ uni-muenster.de).

0015-0282/05/$30.00 doi:10.1016/j.fertnstert.2005.04.043

spite its increasingly common and effective use in the treatment of PCOS there are only two recent studies on the direct effect of metformin on the ovary, both focusing on the reduction of ovarian androgen production (10, 11). Paradoxically, steroidogenesis remains responsive to insulin in PCOS despite peripheral insulin resistance (12). Besides ovarian hyperandrogenemia, which is clearly a hallmark of PCOS, its pathogenic features include follicular growth arrest, and a number of recent publications suggest an effect of metformin on the amelioration of PCO symptoms that may be independent of androgen levels (13–15). We hypothesized that metformin can directly act on insulin and IGF-dependent signaling pathways and viability of granulosa cells, thereby potentially improving follicular growth and promoting ovulation. MATERIALS AND METHODS Human Subjects Granulosa luteal cells (GC) were obtained from women undergoing ovarian hyperstimulation and IVF/ICSI for infertility treatment. All patients underwent a routine diagnostic work-up including baseline hormonal values and had to fulfill the following selection criteria: healthy female partner in infertile couple, age at the time of treatment ⱖ18 but ⬍45 years, body mass index (BMI) between 18 and 29 kg/m2, and regular menstrual cycles. Diagnosis included male factor, tubal factor, or idiopathic infertility. Patients with known ovulatory disorders including anovulation and PCOS were excluded from the study.

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Ovarian stimulation was performed according to standard protocols, employing a GnRH analog for the prevention of premature luteinization and recombinant or highly purified FSH preparations. At 32–36 hours prior to oocyte retrieval, hCG was injected for ovulation induction. The project was approved by the Institutional Review Board of the University, and all patients had given written informed consent prior to the use of cells. Cell Culture Serum-free cell culture of human GC was performed as described previously (16) with modifications. Briefly, follicular fluid was collected from patients at the time of follicular aspiration for IVF/ICSI. Cells from pooled Graafian follicles from one or more patients were used for each experiment. After removal of the oocyte-cumulus complex, the follicular fluid was centrifuged and the pellet resuspended and layered onto a 50% Percoll gradient (Amersham Pharmacia Biosciences, Freiburg, Germany) to pellet the red blood cells. For additional purification, erythrocyte lysis with ammonium chloride (NH4Cl; Sigma, Taufkirchen, Germany) and immunomagnetic separation (Dynal Biotech, Hamburg, Germany) of CD15- and CD45-positive leucocytes was performed. After washing, the GC were resuspended in 1 mL tissue culture medium (McCoy’s 5a medium; Invitrogen, Karlsruhe, Germany) supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 mg/L streptomycin (PAA Laboratories, Cölbe, Germany), and 1 ␮mol/L 4-androstene-3,17-dione (A4; Sigma). Cell numbers were assessed in a Neubauer chamber after trypan blue staining. GC were cultured at a final concentration of 5 ⫻ 104 cells/mL in 96-well plates in 95% air/5% CO2 at 37°C. In addition to primary human GC with only limited availability, immortalized HGL5 cells of human luteinized granulosa cell origin (17), kindly donated by Dr. William Rainey, University of Texas Southwestern Medical Center, USA, were employed for molecular signaling studies. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with 10% FCS, penicillin/streptomycin, and 2% Ultroser G (Ciphergen Biosystems, Fremont, CA) in tissue culture flasks at 37°C in an atmosphere of 5% CO2. Cell Viability Primary GC were incubated for up to 48 hours with or without metformin (1 mmol/L), human insulin, human IGF-1, or recombinant IGF-2 at 100 ng/mL final concentration. Regulatory molecules (all from Sigma) were prepared as stock solutions, stored at ⫺70°C, and diluted to their final concentrations immediately prior to use. GC viability was determined by the colorimetric cell proliferation assay WST-1 (Roche Diagnostics, Penzberg, Germany). It is based on the conversion of WST-1 tetrazolium reagent by metabolically active cells into a colored formazan product. At the end of each experiment 20 ␮L of diluted reagent solution 1174

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was added into each well, and after 4 hours incubation the absorbance was recorded at 460 nm with a 96-well plate reader, according to the manufacturer’s instructions. Western Immunoblot Analysis HGL5 cells were cultured at a concentration of 5 ⫻ 105 cells/mL in 6-well plates in serum-free media overnight followed by a 24-hour preincubation period with or without metformin (10 mmol/L). Proteins were isolated after stimulation with insulin (100 ng/mL) for 0 – 60 minutes using whole-cell lysis buffer containing 10% glycerol, ethylenediaminetetraacetic acid (EDTA) 5 mmol/L, Triton-X 100 1%, NP-40 0.5%, and 1% freshly added protease inhibitor cocktail (1:100, Sigma) and sodium orthovanadate (100 mmol/L). The total protein concentration was determined by a BCA protein assay (Pierce, Rockford, IL). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of a reducing agent (5% ␤-mercaptoethanol). Samples were loaded onto a 12% Trispolyacrylamide gel and electrophoresed at 20 mA for 1 hour. Proteins were transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia Biotech, Piscataway, NJ) for 90 minutes at 16 V. Molecular weights were estimated by comparison with prestained standards. Immunoblotting was performed by treating nitrocellulose membranes with blocking buffer (5% skim milk in 0.1% TBS-Tween (TBST)) for 1 hour at room temperature, followed by incubation with the primary antibody overnight at 4°C. Rabbit antihuman polyclonal antibodies (1:1000 in 5% BSA in TBST) were used for the detection of phosphorylated AKT (phospo-Ser473) and p44/42 MAP kinase (MAPK, phospho-Thr202/Tyr204; Cell Signaling, Beverly, MA). After washing in TBST buffer three times, the membranes were incubated for 1 hour with horseradish peroxidase– conjugated antirabbit IgG diluted 1:2000 (Cell Signaling) in blocking buffer. The membranes were washed and treated with enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech) for 1 minute and exposed to Hyperfilm-ECL. Chemiluminescence signal intensity on developed and scanned films was analyzed using National Institutes of Health image software for PC (Scion, Frederick, MD). For the detection of total AKT and MAPK protein, membranes were stripped with 0.87% NaCl and 0.75% glycin at pH 2.5 three times, washed, and reincubated with the primary antibodies (Cell Signaling) followed by the detection procedure as described previously. Statistical Analysis Experiments to measure cell viability were done in triplicates for each treatment condition. Data are presented as the mean ⫾ standard error of at least three independently performed experiments. Because of considerable variability in basal values between individual experiments, the results were expressed relative to the control. Statistical analysis was performed using the software package Sigmastat 2.03

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(Jandel Scientific, Erkrath, Germany). Basal cell viability was analyzed by t-test. Two-way analysis of variance (ANOVA) was used for analysis of stimulated cell viability and protein expression data. Logarithmic transformation was performed prior to statistical analysis for nonnormally distributed data. A P value ⬍.05 was considered statistically significant. RESULTS Metformin Significantly Increases Basal GC Viability The percentage of viable primary GC estimated by trypan blue staining at the beginning of the culture period was 67.5% ⫾ 4.7%, remaining stable for at least 48 hours of culture period. At present nothing is known about the effect of metformin on GC viability. Human GC were cultured under serum-free conditions with metformin at a final concentration of 1 mmol/L, which had previously been determined as the maximun effective dose (data not shown), for 48 hours in the absence of any other ligands. Cell viability determined by the cell proliferation assay WST-1 was significantly increased relative to control (1.448 ⫾ 0.202; P⫽.041; Fig. 1). Lack of Additional Stimulatory Effect of Insulin or IGF on GC Viability Following Metformin Preincubation Insulin and IGF are well-known intraovarian growth factors. Addition of insulin (Ins) and IGF-1 and -2 at doses of 100 ng/mL for 24 hours to cultured human primary granulosa cells after a 24-hour preincubation period significantly increased cell viability relative to control (Ins: 1.461 ⫾ 0.097; IGF-1: 1.668 ⫾ 0.128; IGF-2: 1.513 ⫾ 0.083; P⬍.001; Fig. 1). To test whether the observed stimulatory effect of metformin on basal GC viability is further increased by insulin or IGF, preincubated cells (metformin 1 mmol/L, 24 hours preincubation and 24 hours stimulation period) were stimulated at doses of 100 ng/mL with the respective ligands. Metformin (Met) coincubation did not result in an additional stimulatory effect compared to nonpreincubated cells (⫺Met vs. ⫹Met, P⫽.018) with no significant differences between stimulated compared to basal GC viability (Met: 1.448 ⫾ 0.202; Ins: 1.672 ⫾ 0.167; IGF-1: 1.814 ⫾ 0.219; IGF-2: 1.458 ⫾ 0.134; P⬎.05; Fig. 1). Divergent Effects of Metformin on Phosphorylated AKT and p44/42 MAP Kinase Protein Expression in HGL5 Cells AKT and p44/42 MAP kinase (MAPK) are important signaling intermediates of growth factor signaling pathways. Insulin significantly increased phosphorylated AKT protein expression relative to basal levels at 5 and 15 minutes (P⫽.002; Fig. 2). However, no statistical significance could be assigned to the observed stimulatory effect of insulin on phosphorylated MAPK protein expression (P⫽.624; Fig. 3). Basal and insulin-stimulated levels of phosphorylated AKT were significantly increased by metformin preincubation (10 mmol/L, 24 hours; ⫺Met vs. ⫹Met, P⫽.028; Fig. 2). In Fertility and Sterility姞

FIGURE 1 Human basal granulosa cell viability is significantly increased by metformin with no additional stimulatory effect of insulin and IGF. Primary granulosa luteal cells (GC) from IVF patients were cultured for 48 hours in serum-free medium with or without metformin (1 mmol/L) in the absence of any other ligands, or with the addition of insulin (Ins), IGF-1, or IGF-2 after a 24-hour preincubation period. Cell viability was determined by a colorimetric proliferation assay (WST-1). Addition of metformin (Met) significantly increases basal granulosa cell viability (P⫽.041) and significantly changes the stimulatory effect of ligands on granulosa cell viability (⫺Met vs. ⫹Met, P⫽.018). Insulin, IGF-1, and IGF-2 significantly increase granulosa cell viability relative to control only when not coincubated with metformin (P⬍.001). The results are presented as the mean ⫾ SEM relative to control arbitrarily set as 1. Bars with no common superscripts are significantly different (P⬍.05).

Sonntag. Metformin and granulosa insulin signaling. Fertil Steril 2005.

contrast, basal as well as insulin-stimulated phosphorylated MAPK was detectable at significantly lower levels in metformin-pretreated cells (⫺Met vs. ⫹Met, P⫽.008; Fig. 3). The unphosphorylated AKT and MAPK protein expression was unchanged by either insulin stimulation or metformin preincubation and served as an internal control. DISCUSSION In our study we can demonstrate a direct effect of metformin on granulosa cell viability and phosphorylated AKT and MAPK protein expression in human granulosa cells. It is well known that metformin can modulate insulin receptor tyrosine kinase activity itself in different cell types (18 –20). The phosphorylation of insulin and other receptor tyrosine kinases not only activates a single subordinate signaling 1175

FIGURE 2 Metformin preincubation significantly increases basal and insulin-induced phosporylated AKT protein expression in HGL5 cells. Following preincubation with or without metformin for 24 hours, total cell lysates were prepared from the human granulosa cell line HGL5 after stimulation with insulin for up to 60 minutes. Differences between total and phosphorylated protein were detected by immunoblotting using phosphospecific antibodies. Metformin significantly increases basal and stimulated phosphorylated AKT protein expression (P⫽.028). A significant increase following insulin stimulation was present after 5 and 15 minutes only when cells were not coincubated with metformin (P⬍.05). (Upper panel) Typical immunoblotting of phosporylated AKT and total AKT protein. (Lower panel) Data (mean ⫾ SEM) for the ratios of protein levels relative to unstimulated control arbitrarily set as 1. Bars with no common superscripts are significantly different (P⬍.05).

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FIGURE 3 Metformin preincubation significantly decreases basal and insulin-induced phosphorylated p44/42 mitogenactivated protein kinase (MAPK) protein expression in HGL5 cells. Following preincubation with or without metformin for 24 hours, total cell lysates were prepared from the human granulosa cell line HGL5 after stimulation with insulin for up to 60 minutes. Differences between total and phosphorylated protein were detected by immunoblotting using phosphospecific antibodies. Metformin significantly decreases basal and insulin-induced phosphorylated MAPK protein expression (P⫽.008). Insulin stimulation did not significantly increase phosphorylated MAPK expression with or without metformin preincubation. (Upper panel) Typical immunoblotting of phosporylated MAPK and total MAPK protein. (Lower panel) Data (mean ⫾ SEM) for the ratios of protein levels relative to unstimulated control arbitrarily set as 1. Bars with no common superscripts are significantly different (P⬍.05).

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molecule but through a diligent recruitment of docking proteins modulates the divergent activation of several signaling pathways (21–22). Based on the observed increase in basal human granulosa cell viability and the lack of an additional stimulatory effect of insulin and IGF, we hypothesized and could demonstrate a modulatory effect of metformin on or downstream of the insulin receptor leading to preferred activation of the AKT compared with the MAPK pathway. The importance of AKT for granulosa cell survival has previously been demonstrated in studies on granulosa cells of different species (23–27). The role of MAPK in granulosa cells is less well defined but has been associated with hormone signaling and cell proliferation (28). The increased basal granulosa cell viability after metformin preincubation in our study may be interpreted as indicative of reduced susceptibility of the cells to undergo apoptosis, combined with a missing additive effect of potentially mitogenic ligands such as insulin or IGF. Thus, metformin as well as insulin or IGF appears to have similar effects on cell viability but via separate pathways. In contrast to the results in our study, metformin at comparable concentrations has been found to increase MAPK phosphorylation with no effect on AKT in adipocytes (29). However, tissue specificity of downstream signaling cascades is a typical feature of the insulin and IGF receptor system owing to the tissue-specific distribution of docking molecules such as insulin receptor substrates (30). Although a direct inhibitory effect of metformin on androgen production in human theca cells has already been described (10), the observed changes in insulin signaling pathways and granulosa cell viability point to a novel and additional direct involvement of metformin on ovarian follicular growth patterns. Folliculogenesis in the human ovary is characterised by the selection process of a healthy dominant follicle destined to ovulate, whereas the remaining follicle cohort undergoes atresia. In PCOS, the ovary contains an excessive number of small antral follicles and for so far unknown reasons the selection process does not occur leading to a follicular arrest (31). The underlying mechanisms of follicular growth and atresia are granulosa cell proliferation and apoptosis, which are under the regulatory control of FSH, modulated by local ovarian growth factors such as IGF or insulin (32). Interestingly, two recent studies have described a higher number of early growing follicles as an intrinsic defect of PCOS ovaries (33, 34). Because peripheral insulin resistance in PCOS has previously been linked to defective insulin receptor autophosphorylation (35, 36), this may also occur in ovarian granulosa cells (37, 38). Future studies could address the comparison of metformin effects on AKT and MAPK pathways in granulosa cells from normal and PCOS ovaries to elucidate the impact of these possible defects on the observed changes in signaling cascades. In conclusion, our data give rise to the concept that besides well known systemic effects, the ovulation-inducing action of metformin may at least partially be due to direct 1178

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effects on the ovary, particularly on basal and stimulated granulosa cell viability mediated via MAPK and AKT pathways. Although the in vitro setting may not allow drawing any definite conclusions for the in vivo situation with a multitude of follicles at different stages of development, our study provides a potential molecular mechanism for the clinical improvement of follicular growth patterns and thereby reestablishment of ovulatory cycles in PCOS patients using metformin. Acknowledgments: We thank William Rainey, M.D., University of Texas Southwestern Medical Center, Dallas, for providing the HGL5 cell line. The authors are very grateful to Monika Offers, Birgit Pers, and Tanja Terhörst for excellent technical assistance during the study.

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