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BOR - Papers in Press, published online ahead of print June 7, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.050831
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BIOLOGY OF REPRODUCTION 75, 342–351 (2006)
DOI: 10.1095/biolreprod.106.050831
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Metformin-Induced Stimulation of Adenosine 5' Monophosphate-Activated Protein Kinase (PRKA) Impairs Progesterone Secretion in Rat Granulosa Cells1

Lucie Tosca 3, Patricia Solnais 3, Pascal Ferré 4, Fabienne Foufelle 4, and Joëlle Dupont 2 3

Unité de Physiologie de la Reproduction et des Comportements,3 Institut National de la Recherche Agronomique, 37380 Nouzilly, France U671 INSERM,4 Centre Biomédical des Cordeliers, 75270 Paris, France

ABSTRACT

Metformin is an anti-diabetic drug commonly used to treat cycle disorders and anovulation in women with polycystic ovary syndrome. However, the effects and molecular mechanism of metformin in the ovary are not entirely understood. We investigated the effects of this drug on steroidogenesis and proliferation in rat granulosa cells. Metformin (10 mM) treatment for 48 h reduced progesterone and estradiol (E2) production in both basal conditions and under FSH stimulation. It also decreased the levels of the HSD3B, CYP11A1, STAR, and CYP19A1 proteins in response to FSH (10–8 M) and of HSD3B in the basal state only. Metformin treatment (10 mM, 24 h) also reduced cell proliferation and the levels of CCND2 and CCNE proteins without affecting cell viability, both in the basal state and in response to FSH. Furthermore, metformin treatment for 1 h simultaneously increased the Thr172 phosphorylation of PRKAA (adenosine 5' monophosphate-activated protein kinase alpha) and the Ser79 phosphorylation of ACACA (acetyl-Coenzyme A carboxylase alpha). The adenovirus-mediated production of dominant-negative PRKAA totally abolished the effects of metformin on progesterone secretion, HSD3B and STAR protein production, and MAPK3/1 phosphorylation. Conversely, total inhibition of PRKAA Thr172 phosphorylation with the dominant-negative PRKAA adenovirus did not restore the decrease in E2 production and cell proliferation induced by metformin. Our results therefore strongly suggest that metformin reduces progesterone production via a PRKAA-dependent mechanism, whereas PRKAA activation is not essential for the decrease in E2 production and cell growth induced by metformin in rat granulosa cells.

adenosine 5', monophosphate-activated protein kinase, follicle-stimulating hormone, granulosa cells, kinases, mechanisms of hormone action, ovary, progesterone, steroid hormones

INTRODUCTION

Metformin, a derivative of biguanide, is an insulin-sensitizing agent used to treat type 2 diabetes mellitus [1] and polycystic ovary syndrome (PCOS). This syndrome is the most common cause of anovulation and infertility, affecting 5% to 10% of women of reproductive age [2]. It is characterized by hyperandrogenism, chronic anovulation, and, occasionally, obesity [2]. In addition to its reproductive consequences, PCOS is a metabolic disorder associated with insulin resistance and hyperinsulinemia [3]. In women with PCOS, metformin treatment restores the cyclic nature of menstruation [4] and increases ovulation (by improving follicular growth), fertilization, and pregnancy rates [5]. These improvements have been attributed to decreases in the level of insulin, subsequently attenuating a hyperandrogenic status. However, the decrease in insulin concentration is not large enough to account for the decrease in androgen levels and improvements in several aspects of PCOS. Thus, although metformin has been used in clinical practice for several years, its mechanism of action remains unclear. Most of the suggested mechanisms include changes in peripheral tissues. For example, metformin is known to increase peripheral glucose utilization [6] and to decrease hepatic glucose output [7] through effects on the muscle and liver [8, 9]. However, this drug may also have direct effects on ovary cells. Indeed, metformin decreases androgen production in human-cultured theca cells [10]. However, to our knowledge, the molecular mechanism of action of metformin in ovary cells has not been investigated.

Adenosine 5' monophosphate-activated protein kinase (PRKA; formerly known as AMPK) is a key regulator of cellular energy homeostasis involved in the regulation of fatty acid and cholesterol synthesis [11]. It is a heterotrimeric enzyme, consisting of one catalytic subunit, {alpha} (PRKAA1 and PRKAA2; formerly known as AMPK isoforms {alpha}1 and {alpha}2), and two regulatory subunits, ß (PRKAB1 and PRKAB2; formerly known as AMPK isoforms ß1 and ß2) and {gamma} (PRKAG1, PRKAG2, and PRKAG3; formerly known as AMPK isoforms {gamma}1, {gamma}2, and {gamma}3) [12]. Phosphorylation of the threonine 172 residue in the catalytic subunit {alpha} is essential for activity and is regulated by the upstream PRKA kinase STK11 (LKB1) [13]. PRKA is activated by changes in the cellular AMP:ATP ratio and by metabolic stress due to exercise [14], hypoxia [15], cell nutrient deficiency [16], hormones such as adiponectin [17] and leptin [18], and drugs such as 5-aminoimidazole-4-carboxamide-1-ß-D-ribonucleoside (AICAR) [19] and rosiglitazone [20]. PRKA is a multisubstrate enzyme well characterized in many tissues, including the liver, muscle, lung, heart, kidney, and brain [21]. We recently detected PRKA in rat ovary, in the granulosa and theca cells [22]. We also showed that the activation of PRKA by AICAR in rat primary granulosa cells reduces progesterone secretion and the production of HSD3B, a key enzyme in steroidogenesis, through the MAPK3/1 signaling pathway [22]. Recent studies have shown that metformin activates PRKA in several cell types. For example, metformin-mediated PRKA activation decreases glucose production and increases fatty acid oxidation in rat primary hepatocytes [23] and increases glucose uptake in human skeletal muscle [24]. It also increases PRKAA1 and PRKAA2 activity in mouse skeletal muscle H-2Kb cells [20] and activates PRKA in rat liver [25].

We hypothesized that metformin could act directly on granulosa cells to decrease steroid production. We therefore investigated the effects and molecular mechanism of metformin on rat primary granulosa cell steroidogenesis and cell proliferation. We investigated whether the decreases in progesterone and estradiol (E2) production and cell proliferation observed in response to metformin treatment were PRKAA-dependent by means of an adenovirus-based system to produce a dominant-negative form of PRKAA in rat granulosa cells.

MATERIALS AND METHODS

Hormones and Reagents

The purified ovine FSH-20 (oFSH) (batch AFP-7028D, 4453 IU/mg, FSH activity = 175 times the activity of oFSH-S1) used in cultures was kindly provided by the National Hormone Pituitary Program (NIDDK, NIH, Bethesda, MD). McCoy A modified culture medium, penicillin, and streptomycin were purchased from Invitrogen (Cergy Pontoise, France). Thymidine methyl-H3 was purchased from Perkin Elmer Life and Technological Sciences (Boston, MA). Metformin and diethylstilbestrol were obtained from Sigma (Saint Quentin Fallavier, France).

Antibodies

Rabbit polyclonal antibodies against phospho-PRKAA Thr172, acetyl-Coenzyme A carboxylase alpha (ACACA), and phospho-MAPK3/1 (Thr202/Tyr204) were purchased from New England Biolabs Inc. (Beverly, MA). Rabbit polyclonal antibodies against MAP1 (C14), CCND2 (C17), and CCNE (C19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against PRKAA1 and phospho-ACACA Ser79 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal antibodies against CYP11A1, STAR, and HSD3B were generously provided by Dr. Dale Buchanan Hales (University of Illinois, Chicago, IL) and Dr. Van Luu-The (CHUL Research Center and Laval University, Quebec, Canada), respectively. Mouse monoclonal antibody against TUBA (alpha tubulin) was obtained from Calbiochem (Fontenay sous Bois, France). Mouse monoclonal antibody against CYP19A1 was purchased from Serotec (Varilhes, France). All antibodies were used at a dilution of 1:1000 in Western blotting.

Animals; Isolation and Culture of Rat Granulosa Cells

All procedures were approved by the Agricultural Agency and the Scientific Research Agency and were conducted in accordance with the guidelines for Care and Use of Agricultural Animals in Agricultural Research and Teaching.

Three-week-old immature female rats of the Wistar strain were purchased from Janvier Laboratories (Genest St. Isle, France). They were housed in controlled temperature and photoperiod (10D:14L and lights-on from 0600 to 2000 h) conditions. The animals had ad libitum access to food and water. They were injected subcutaneously with DES (diethylstilbestrol, 1 mg/day) every day for 3 days. On the fourth day of DES treatment, the animals were killed, and the ovaries were removed aseptically and transferred to culture medium. Granulosa cells were harvested by puncturing the follicles, expelling the cells. Cells were recovered by centrifugation, washed with fresh medium, and counted in a hemocytometer. The culture medium used was McCoy 5A supplemented with Hepes (20 mmol/L), penicillin (100 U/ml), streptomycin (100 mg/L), L-glutamine (3 mmol/L), 0.1% BSA, androstenedione (0.1 µmol/L), transferrin (5 mg/L), selenium (20 µg/L), and 10% fetal bovine serum (FBS). The cells were initially cultured for 48 h with no other treatment and then were incubated in fresh culture medium with or without test reagents for the times indicated. All cultures were performed under a water-saturated atmosphere containing 95% air:5% CO2, at 37°C.

Thymidine Incorporation into Granulosa Cells

Granulosa cells (2 x 105 viable cells/500 µl) were cultured in 24-well dishes in McCoy 5A medium supplemented with 10% FBS for 48 h. They were deprived of serum for 24 h, and 1 µCi/µl of [3H-thymidine (Amersham Life Science, Arlington Heights, IL) was then added in the presence or absence of metformin (10 mM) and/or FSH (10–8 M). Cultures were maintained at 37°C under an atmosphere consisting of 5% CO2 in air. After 24 h of culture, excess thymidine was removed by washing twice with PBS. The cells were fixed by incubation with 50% cold trichloroacetic acid for 15 min and lysed in 0.5 N NaOH. Radioactivity was determined in scintillation fluid (Packard Bioscience, now Perkin Elmer) in a ß-photomultiplier.

Adenoviruses and Infection of Rat Granulosa Cells

A dominant-negative PRKA adenovirus (Ad. DN) was constructed from PRKAA1, carrying the Asp-157 to Ala (D157A) mutation, as previously described [26]. Recombinant adenovirus was propagated in HEK293 cells, purified by cesium chloride density centrifugation, and stored as previously described [27]. Rat granulosa cells were infected with adenovirus (20 plaque forming units [pfu]/cell) in serum-starved McCoy 5A medium for 24 h. They were then cultured for a further 24 h in the presence or absence of FSH (10–8 M) and metformin (10 mM). Preliminary studies revealed that within 24 h of infection (20 pfu/cell) by a green fluorescent protein-expressing virus, the majority of granulosa cells (>90%) expressed green fluorescent protein [22].

Western Blot

Total protein was extracted from granulosa cells on ice in lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 0.5% Igepal) containing various protease inhibitors (2 mM PMSF, leupeptin [10 mg/ml], and aprotinin [10 mg/ml]) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate [Sigma, l'Isle d'Abeau Chesnes, France]). Lysates were centrifuged at 13 000 x g for 20 min at 4°C, and the protein concentration in the supernatants was determined by a colorimetric assay (BC Assay Kit, Uptima Interchim, Montluçon, France).

Cell extracts were subjected to electrophoresis in 10% (w:v) SDS-PAGE under reducing conditions. The proteins were then electrotransferred onto nitrocellulose membranes (Schleicher and Schuell, Ecquevilly, France) for 1 h 30 min at 80 V. Membranes were incubated for 1 h at room temperature with Tris-buffered saline (TBS; 2 mM Tris-HCl [pH 8.0] and 15 mM NaCl [pH 7.6]) containing 5% nonfat dry milk powder (NFDMP) and 0.1% Tween-20 to saturate nonspecific binding sites. Membranes were then incubated overnight at 4°C with the appropriate primary antibodies (final dilution, 1:1000) in TBS containing 0.1% Tween-20 and 5% NFDMP. After washing in TBS-0.1% Tween-20, the membranes were incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (final dilution, 1:10 000; Diagnostic Pasteur, Marnes-la-Coquette, France) in TBS-0.1% Tween-20. The membranes were washed again in TBS-0.1% Tween-20, and the signal was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Orsay, France). The films were analyzed, and the signals were quantified with MacBas V2.52 software (Fuji Photo Film).

Progesterone and E2 Radioimmunoassay

The concentrations of progesterone and E2 in the culture medium of granulosa cells were determined by radioimmunoassay (RIA), as previously described [22]. The limit of detection for progesterone was 12 pg/tube (60 pg/well), and the intra- and interassay coefficients of variation were less than 10% and 11%, respectively. The limit of detection for E2 was 1.5 pg/tube (7.5 pg/well), and the intra-and interassay coefficients of variation were less than 7% and 9%, respectively. Results were initially expressed as the amount of steroids (nanograms per milliliter) secreted per 100 µg of protein. They were then expressed as a percentage of the values obtained in the absence of treatment to eliminate the effects of culture.

Statistical Analysis

All experimental data are presented as means ± SEM. A one-way analysis of variance (ANOVA) was used to test differences. If the ANOVA showed significant effects, the means were compared by the Newman test, with P < 0.05 considered significant. In the various graphs, bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin).

RESULTS

Effects of the Metformin Treatment on Basal and FSH-Stimulated Progesterone and E2 Production in Rat Granulosa Cells

We investigated the effect of metformin treatment on progesterone and E2 production by incubating rat granulosa cells with various concentrations of metformin (0, 0.5, 1, 5, and 10 mM) for 48 h or with 10 mM (0, 3, 6, 12, 24, and 48 h). The secretion of progesterone (Fig. 1A) and E2 (Fig. 1B) was inhibited by metformin (48 h) in a dose-dependent manner (P < 0.001). Furthermore, as shown in Figure 1, C and D, metformin treatment (10 mM) acted rapidly, as the production of progesterone and E2 reached a minimum after only 3 h of stimulation (P < 0.05). We also investigated whether metformin could affect the production of progesterone and E2 in response to FSH. In the presence of FSH (10–8 M), metformin (10 mM, 48 h) decreased progesterone secretion by a factor of about 4 (P < 0.001) (Fig. 1E) and halved E2 secretion (P < 0.001) (Fig. 1F). Thus, metformin decreased both basal and FSH-stimulated progesterone and E2 production in rat granulosa cells.


Figure 1
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  		FIG. 1. Effect of metformin treatment on basal and FSH-stimulated progesterone and E2 secretion by rat granulosa cells. Granulosa cells from immature rats were cultured for 48 h in medium with serum and then in serum-free medium in the presence or absence of various doses of metformin for 48 h (A, B) or with 10 mM metformin for various times (C, D) or in the presence or absence of 10 mM metformin ± 10–8 M FSH (E, F), as described in Materials and Methods. The culture medium was then collected, and its progesterone (A, C, E) and E2 (B, D, F) content was then analyzed by RIA. Results are expressed as means ± SEM of three groups of granulosa cells. Data are expressed as a percentage of the result obtained with unstimulated cells. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)

We then investigated whether this inhibitory effect of metformin on the production of both progesterone and E2 resulted from the production of smaller amounts of the three key enzymes in steroidogenesis (HSD3B, CYP11A1, and CYP19A1) and/or of STAR, an important cholesterol carrier. Metformin treatment (10 mM, 48 h) decreased HSD3B (Fig. 2A) and CYP19A1 (Fig. 2D) production by a factor of about 3 (P < 0.05) in the presence of FSH (10–8 M) and halved the production of CYP11A1 (Fig. 2B) and STAR (Fig. 2C) with respect to FSH treatment alone. In the basal state (no FSH), metformin treatment (10 mM, 48 h) decreased HSD3B protein levels by a factor of about 2 (P < 0.05) (Fig. 2A). Thus, the decrease in FSH-induced progesterone secretion in response to metformin treatment appears to result from a decrease in the amounts of HSD3B, CYP11A1, STAR, and CYP19A1. The inhibition of basal and FSH-induced E2 secretion in response to metformin treatment may result from decreases in progesterone production, CYP19A1 protein levels, or both.


Figure 2
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  		FIG. 2. Effect of metformin treatment on the amounts of HSD3B, CYP11A1, STAR, and CYP19A1 proteins in rat granulosa cells. Protein extracts from rat granulosa cells cultured for 48 h in the presence or absence of 10 mM metformin ± 10–8 M FSH were subjected to SDS-PAGE, as described in Materials and Methods. The membranes were incubated with antibodies raised against the HSD3B (A), CYP11A1 (B), STAR (C), and CYP19A1 (D). Equal protein loading was checked by reprobing the membrane with an anti-TUBA antibody. Results are representative of at least three independent experiments. Blots were quantified, and the HSD3B, CYP11A1, STAR, and CYP19A1:TUBA ratios are shown. The results are expressed as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)

Effects of the Metformin Treatment on the Phosphorylation of AMPK{alpha} and Acetyl CoA Carboxylase in Rat Granulosa Cells

Metformin has been shown to activate AMPK in various types of cells, including hepatocytes and skeletal muscle cells [23]. We investigated the pattern of PRKAA phosphorylation after treatment with metformin (10 mM) for various lengths of time (0, 5, 10, 30, 60, and 120 min) with an anti-phospho-Thr172 antibody specific for the phosphorylated catalytic {alpha} subunit in rat granulosa cells. Metformin treatment increased PRKAA phosphorylation after 60 min of stimulation (Fig. 3A, P < 0.05). Metformin effects were observed with concentrations of 5 and 10 mM after 120 min of stimulation (Fig. 3B, P < 0.05). We also indirectly assessed PRKA activity by assessing the phosphorylation of the downstream target of this enzyme, ACACA. Metformin (10 mM) increased the phosphorylation of ACACA on the Ser79 residue in a time- and dose-dependent manner, paralleling the stimulation of Thr172 phosphorylation for PRKAA at 60 and 120 min (Fig. 3C) and at 5 and 10 mM (Fig. 3D). Thus, PRKA activation by metformin affected downstream targets in rat granulosa cells.


Figure 3
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  		FIG. 3. Effect of metformin treatment on PRKAA and ACACA phosphorylation in rat granulosa cells. Granulosa cell lysates were prepared from cells incubated with 10 mM metformin for various times (0, 5, 10, 30, 60, or 120 min) or with various doses of metformin (0, 0.1, 0.5, 1, 5, or 10 mM) for 120 min. Lysates (50 µg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with anti-PRKAA and anti-PRKAA1 (A, B) or anti-phospho-ACACA and anti-ACACA (C, D) antibodies. Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. The results are presented as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)

Effects of the Overexpression of a Dominant-Negative PRKAA1 on Progesterone and E2 Production and on MAPK3/1 Phosphorylation in Rat Granulosa Cells

We previously demonstrated that PRKAA activation by AICAR, a potent activator of the PRKA system, decreases basal and FSH-induced progesterone production by inhibiting MAPK3/1 signaling in rat granulosa cells [22]. We therefore investigated whether the metformin-induced decreases in progesterone and E2 production and in the levels of HSD3B, CYP11A1, STAR, and CYP19A1 and in MAPK3/1 phosphorylation were mediated by PRKA. We infected rat granulosa cells by incubation for 24 h with the dominant-negative (Ad. DN) PRKAA1, and cells were then incubated in the presence or absence of FSH (10–8 M) ± metformin (10 mM) for a further 24 h. After infection, granulosa cell proteins were Western blotted for analysis of the production of mutant and endogenous PRKAA1 subunits. The mutant dominant-negative PRKAA1 proteins were produced, and they significantly decreased the levels of basal and FSH-stimulated PRKAA Thr172 phosphorylation in response to metformin treatment (Fig. 4A). The infection of granulosa cells with a control (Ad. GFP) had no effect on PRKAA1 production or PRKAA Thr172 phosphorylation (Fig. 4A). The production of the mutant dominant-negative PRKAA1 in rat granulosa cells strongly reduced the metformin-induced decrease in progesterone production in the absence (Fig. 4B, left panel) or presence (Fig. 4B, right panel) of FSH, whereas it had no effect on E2 production (Fig. 4C). A quantitative analysis of Western blots indicated that the decreases in basal and FSH-stimulated MAPK3/1 phosphorylation induced by metformin were reversed by Ad. DN (Fig. 4D). The production of the mutant dominant-negative PRKAA1 in the cells also reduced the metformin-induced decrease in FSH-induced HSD3B (Fig. 5A, right panel) and STAR (Fig. 5B, right panel) levels but not those in CYP11A1 protein levels (Fig. 5C, right panel). In the basal state (no FSH), Ad. DN restored only HSD3B protein levels (Fig. 5A, left panel). The infection of cells with a control GFP virus had no effect on progesterone and E2 production (Fig. 4, B and C), on the levels of HSD3B, CYP11A1, and STAR proteins (Fig. 5, A, B, and C) or on MAPK3/1 phosphorylation (Fig. 4D).


Figure 4
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  		FIG. 4. Metformin-induced decreases in progesterone and E2 secretion in rat granulosa cells infected with adenovirus expressing PRKAA1 mutant constructs. A, D) The production and level of phosphorylation of PRKAA and MAPK3/1 in rat granulosa cells infected with adenovirus constructs expressing a mutant PRKAA1 construct (Ad. DN) or a GFP construct (Ad. GFP). Granulosa cells were infected with virus (20 pfu/cell) and were stimulated 24 h later in the presence or absence of 10 mM metformin ± 10–8 M FSH, as indicated in Materials and Methods. After 24 h, cell lysates were prepared and resolved by SDS-PAGE. The proteins were transferred to nitrocellulose membranes and probed with anti-phospho-PRKAA Thr172 (A) and anti-PRKAA1 (A) or anti-phospho-MAPK3/1 (D) and anti-MAP1 (D) antibodies. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. The immunoblots shown are representative of three independent experiments; bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin). B, C) Progesterone (B) and E2 (C) secretions in granulosa cells producing the mutant dominant-negative PRKAA1 or the GFP protein in the presence or absence of 10 mM metformin ± 10–8 M FSH (24 h). Granulosa cells were infected or not infected with Ad. DN or Ad. GFP virus (20 pfu/cell). GFP virus for 24 h and then were stimulated or not stimulated with 10 mM metformin ± 10–8 M FSH (24 h). The culture medium was then collected, and its progesterone and E2 content was analyzed by RIA. The data shown represent means ± SEM from three independent experiments for the concentrations of progesterone and E2. Data are expressed as a percentage of the result obtained with unstimulated cells. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)


Figure 5
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  		FIG. 5. Metformin-induced decreases in HSD3B, CYP11A1, and STAR protein levels in rat granulosa cells infected with adenovirus expressing PRKAA1 mutant constructs. Granulosa cells were infected or not infected with the Ad. DN or GFP virus for 24 h and then were left unstimulated or were stimulated with 10 mM metformin ± 10–8 M FSH for a further 24 h, as indicated in Materials and Methods. Cell lysates were then prepared and resolved by SDS-PAGE. The proteins were transferred to nitrocellulose membranes and probed with anti-HSD3B (A), anti-STAR (B), or anti-CYP11A1 (C) antibodies. Blots were quantified, and the ratios of HSD3B, CYP11A1, and STAR to TUBA levels are shown. The immunoblots shown are representative of three independent experiments, and the results are expressed as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)

Effects of the Metformin Treatment on Granulosa Cell Proliferation and Viability

We also investigated whether the metformin treatment affected the number of granulosa cells in culture by inducing mitosis or altering cell viability. [3H]-Thymidine incorporation into granulosa cells treated with various doses of metformin (0, 0.1, 0.5, 1, 5, and 10 mM) and with 10 mM metformin ± FSH (10–8 M) was determined after 24 h of culture. Metformin treatment significantly decreased [3H]-thymidine incorporation by about 10% and 20%, with concentrations of 5 and 10 mM, respectively (Fig. 6A). As expected, FSH treatment significantly increased [3H]-thymidine incorporation (P < 0.001) (Fig. 6B). However, metformin treatment decreased both basal and FSH-stimulated [3H]-thymidine incorporation (Fig. 6B) by about 20% and 40%, respectively (P < 0.001). These effects of metformin on cell proliferation were confirmed by evaluating CCND2 and CCNE protein levels by Western blotting (Fig. 6, C and D). Indeed, metformin treatment reduced basal CCND2 protein levels by 39% and FSH-stimulated CCND2 protein levels by 41% (P < 0.05, Fig. 6C). It also decreased basal CCNE protein levels by 43% and FSH-induced CCNE protein levels by 44% (P < 0.05, Fig. 6D). However, the production of the mutant dominant-negative PRKAA1 in rat granulosa cells did not restore the metformin-induced decrease in both basal and FSH-induced [3H]-thymidine incorporation, despite the total inhibition of metformin-induced PRKAA Thr172 phosphorylation (Fig. 6E). Trypan blue staining demonstrated that metformin treatment had no effect on cell viability in the presence or absence of FSH (data not shown).


Figure 6
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  		FIG. 6. Effect of metformin on rat granulosa cell proliferation. A, B) Thymidine incorporation was determined in rat granulosa cells cultured for 24 h with various doses of metformin (0, 0.1, 0.5, 1, 5, and 10 mM) or in the presence or absence of 10 mM metformin ± 10–8 M FSH, as described in Materials and Methods. Results are representative of at least three independent experiments. The results are expressed as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin). C, D) Protein extracts from rat granulosa cells cultured for 24 h in the presence or absence of 10 mM metformin ± 10–8 M FSH were subjected to SDS-PAGE, as described in Materials and Methods. The membranes were incubated with antibodies against CCND2 (C) or CCNE (D). Equal protein loading was checked by reprobing the membrane with an anti-TUBA antibody. Blots were quantified, and the CCND2 and CCNE:TUBA ratios are shown. A representative blot from three independent experiments is shown. The results are expressed as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin). E) PRKAA Thr172 phosphorylation (left panel) and thymidine incorporation (right panel) were determined in rat granulosa cells either uninfected or infected with the Ad. DN or Ad. GFP virus and cultured for 24 h in the presence or absence of 10 mM metformin ± 10–8 M FSH, as described in Materials and Methods. After 24 h, some cells were used to prepare lysates, which were resolved by SDS-PAGE. The resulting proteins were transferred to nitrocellulose membranes and probed with anti-PRKAA Thr172 and anti-PRKAA1 antibodies. The other cells were lysed for the assessment of thymidine incorporation. Results are representative of at least three independent experiments. The results are expressed as means ± SEM. Bars with different superscripts are significantly different (P < 0.05). The superscript a indicates values that are not significantly different from the control (without FSH and metformin)

DISCUSSION

We show in the present study that metformin inhibits progesterone secretion both in the basal state and in response to FSH, via a PRKAA-dependent mechanism, in rat granulosa cells (Fig. 7A). We also show that this drug decreases both E2 secretion and cell proliferation, without affecting cell viability. However, PRKA activation does not seem to be essential for these effects, because the overproduction of a dominant-negative PRKAA adenovirus does not restore the metformin-induced inhibition of E2 production and cell growth, despite the strong inhibition of PRKAA Thr172 phosphorylation (Fig. 7B).


Figure 7
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  		FIG. 7. Schematic representation of the PRKAA-dependent (A) and PRKAA-independent (B) effects of metformin in response to FSH on steroidogenesis and proliferation in rat granulosa cells. A) The activation of PRKA by metformin treatment (10 mM) led to a decrease in MAPK3/1 phosphorylation. As previously reported [22], a decrease in MAPK3/1 phosphorylation may lead to the inhibition of HSD3B (and possibly of STAR) protein production and progesterone secretion in rat granulosa cells. B) Metformin treatment (10 mM), independently of PRKAA activation, inhibited the production of the CYP11A1 and CYP19A1 proteins, together with E2 secretion in rat granulosa cells. Metformin also reduced rat granulosa cell proliferation by acting on the G1/S transition of the cell cycle and decreasing the levels of the CCND2 and CCNE proteins

The decrease in progesterone and E2 production observed in rat granulosa cells treated with metformin is consistent with in vivo and in vitro results for humans. Indeed, the treatment with metformin of women with PCOS leads to decreases in serum progesterone and E2 concentrations [27, 28]. However, the dose of metformin used in our study in rat granulosa cells (10 mM) is high, corresponding to about 100 times that used in patients. Metformin has also been shown to decrease basal and FSH-stimulated progesterone and E2 secretion in a dose-dependent manner in cultured human granulosa cells [29]. Thus, our results and those obtained for human granulosa cells suggest that metformin directly affects steroidogenesis in ovary cells. However, in rat granulosa cells, high doses of metformin (5–10 mM) are required to obtain a significant decrease in progesterone and E2 secretion. The precise sites of action of metformin on steroidogenic enzymes and the mechanisms involved are not fully understood. We found that metformin treatment (10 mM, 48 h) decreased both basal and FSH-stimulated HSD3B protein levels and FSH-stimulated STAR, CYP11A1, and CYP19A1 protein levels. Thus, metformin may decrease basal progesterone production by inhibiting HSD3B protein production and FSH-stimulated production by inhibiting HSD3B, STAR, and CYP11A1 protein production. To our knowledge, the present study is the first to show a direct effect of metformin on the production of steroidogenesis factors in granulosa cells. However, other insulin-sensitizing agents, including troglitazone and rosiglitazone, are known to reduce the activities of certain steroidogenic enzymes. For example, troglitazone treatment inhibits HSD3B activity in porcine granulosa cells but has no effect on the production of this protein. Some reports have indicated that different insulin-sensitizing agents have different effects. Indeed, troglitazone inhibits the HSD3B2 activity in microsomes from transformed yeast, whereas metformin does not [30]. The decrease in FSH-stimulated E2 production in response to metformin in the present study may result from a decrease in the amount of CYP19A1 protein. These results are consistent with those of La Marca et al. [31], who reported that metformin reduces CYP19A1 activity in response to FSH in human granulosa cells from PCOS patients. Thus, the effects of the metformin, decreasing serum androgen concentration in women with PCOS, may be secondary to the increase in insulin sensitivity or result from a direct effect on steroidogenesis.

In some cell types, the effects of metformin are known to be mediated by PRKA activation [23–25]. We recently characterized PRKA and studied its role in rat granulosa cells [22]. The various subunits of PRKA are produced (protein and mRNA) in the oocyte, corpus luteum, granulosa cells, and, to a lesser extent, theca cells. In granulosa cells, the AICAR treatment activates PRKA, decreasing progesterone secretion [22]. We show for the first time in this study that metformin treatment increases PRKAA Thr172 phosphorylation after 60 min of stimulation, consistent with an increase in ACACA phosphorylation at Ser79. ACACA is one of the main substrates of PRKA, and assessment of the phosphorylation of this molecule can be used as an indirect assay for PRKA activation [32]. Metformin significantly decreased the production of progesterone and E2 from 3 h of stimulation onward. We therefore suggest that the effects of metformin on steroid production are mediated by PRKAA in rat granulosa cells. We tested this hypothesis by overproducing a mutant dominant-negative PRKAA1, with an adenovirus vector, in rat granulosa cells, assessing progesterone and E2 production and analyzing the levels of certain proteins known to be involved in steroidogenesis. The metformin-induced decrease in progesterone secretion was reversed by the Ad. DN, whereas the metformin-induced decrease in E2 secretion was not. Thus, PRKAA is essential for the effects of metformin on progesterone production but not on E2 production. These results are consistent with our previous studies showing that AICAR-induced PRKA activation inhibits progesterone secretion by rat granulosa cells but not E2 secretion [22]. Thus, in these cells, the activation of PRKA by metformin or AICAR decreases progesterone production. PRKA is a key regulator of cellular energy homeostasis that inhibits both fatty acid and cholesterol synthesis in skeletal muscle and liver [11]. In women with PCOS, metformin decreases the high levels of total cholesterol [33], triglycerides [34], and low-density lipoprotein cholesterol [35] and the high serum concentrations of free fatty acid [33]. In women with PCOS, metformin decreases the production of both steroids [27, 28]. Thus, in women with PCOS, metformin may modify both glucose and lipid metabolism, together with steroidogenesis in the ovary, through a PRKAA-dependent mechanism.

The overproduction of the mutant dominant-negative PRKAA1 showed that PRKA was essential for the basal inhibition of HSD3B protein production and for the inhibition of FSH-stimulated HSD3B and STAR protein production mediated by metformin. In our study with Ad. DN, some effects in response to FSH or FSH and metformin are low although significant. One possible explanation for this is the shorter FSH stimulation (24 h) of rat granulosa cells in the presence or absence of metformin since we did not want to let the virus have too much time on the cells. Metformin treatment (24 h) also inhibited MAPK3/1 phosphorylation, consistent with PRKA activation, as this inhibition was restored by Ad. DN. The phosphorylation of MAPK p38 and Akt was unaffected by the treatment (data not shown). We previously showed that the MAPK3/1 signaling pathway positively regulates progesterone production [22]. Moreover, the results obtained in the present study are consistent with those of our previous study showing that PRKA activation by AICAR treatment reduces progesterone secretion and HSD3B through the MAPK3/1 signaling pathway in rat granulosa cells [22]. Furthermore, a recent study showed that metformin treatment for 24 h inhibits the basal phosphorylation of MAPK3/1 in the human granulosa cell HGL5 [36]. However, the authors of this study observed an increase in Akt phosphorylation in response to metformin treatment, whereas we found no effect of metformin on this signaling pathway (data not shown). One explanation for this discrepancy is that HGL5 granulosa cells are an immortalized human granulosa-lutein cell line, whereas the cells used in the present study were immature rat granulosa cells.

We found that metformin treatment inhibited cell growth in basal conditions and in response to FSH without affecting cell viability. These effects were associated with a decrease in the levels of CCND2 and CCNE, two positive regulators of the G1/S transition of the cell cycle in rat granulosa cells [37]. However, PRKAA does not seem to be essential for these effects, as the overproduction of a dominant-negative form of PRKAA1 did not restore the decrease in cell growth in response to metformin, despite the strong inhibition of PRKAA Thr172 phosphorylation. Thus, metformin appears to stimulate or inhibit at least one molecule other than PRKA in rat granulosa cells, leading to a decrease in cell numbers. The inhibitory effects of metformin on cell proliferation have been well documented in various cell types, including smooth muscle [38, 39] and pancreatic cells [40]. Like other anti-diabetic drugs, such as thiazolidinediones, metformin may inhibit cell proliferation by activating the peroxisome proliferator-activated receptor gamma (PPARG). We previously showed that rosiglitazone treatment inhibits sheep primary granulosa cell proliferation [41]. Furthermore, PPARG has been shown to downregulate CCND1 [42, 43], CCNE [44], or both [45, 46] in various cell types.

In conclusion, the activation of PRKA by metformin in rat granulosa cells decreases basal and FSH-induced progesterone secretion by decreasing HSD3B and STAR protein levels. However, PRKAA is not essential for the decrease in metformin-induced E2 secretion and cell proliferation. These findings significantly increase our understanding of the mechanism of action of metformin on ovary cells. However, further investigations with human granulosa cells are required to determine whether the effects of metformin treatment in women with PCOS involve the activation of PRKA.

ACKNOWLEDGMENTS

We thank M. Peloille for the sequencing and C. Cahier and J.C. Braguer for animal care. We thank J. Sappa from the Alex Edelman Company for editing the manuscript.

FOOTNOTES

1 Supported by the Région Centre to L.T. Back

2 Correspondence. FAX: 33 2 47 42 77 43; jdupont{at}tours.inra.fr Back

Received: 8 January 2006.

First decision: 18 February 2006.

Accepted: 13 May 2006.

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