Metformin: direct inhibition of rat ovarian theca-interstitial cell proliferation

Abstract

Objective: To determine whether metformin has direct effects on ovarian theca-interstitial (T-I) cell proliferation through activation of adenosine monophosphate-activated protein kinase (AMPK).

Design: In vitro experimental study.

Setting: Academic medical center laboratory.

Animal(s): Immature Sprague-Dawley female rats.

Intervention(s): Ovarian T-I cells were isolated, purified, and cultured in the absence (control) or presence of insulin (1 μg/mL) with or without metformin or other activators/inhibitors of AMPK (AICAR, compound C).

Main outcome measure(s): Proliferation assessed by determination of expression levels of proteins involved in cell cycle progression, cyclin D3, and cyclin-dependent kinase 4 (CDK4) with Western blot analysis, and determination of DNA synthesis with bromodeoxyuridine (BrdU) incorporation assay; activation of AMPK, Erk1/2, and S6K1 determined by Western blot analysis with the use of antibodies specific for the phosphorylated (activated) forms.

Result(s): Metformin inhibited insulin-induced ovarian T-I cell proliferation and the up-regulation of the cell cycle regulatory proteins, cyclin D3 and CDK4. Metformin independently activated AMPK in a dose-dependent manner. Treatment with metformin inhibited insulin-induced activation of Erk1/2 and S6K1. This effect was reversed with the addition of compound C, a known AMPK inhibitor.

Conclusion(s): Metformin directly inhibits proliferation of ovarian T-I cells via an AMPK-dependent mechanism. These findings further validate the potential benefits of metformin in the treatment of conditions associated with hyperinsulinemia and excessive growth of ovarian T-I cells (such as polycystic ovary syndrome).

Figure 1

Effect of metformin on insulin-induced cell proliferation and cell cycle regulatory protein expression. A, T-I cells were incubated in 0.1% BSA-containing McCoy’s media for 24 hours in 96 well plates at a concentration of 2 × 10⁴ cells/well. Cells were labeled with BrdU, and cell proliferation was assessed by BrdU incorporation as described in Materials and Methods. B–C, Cells were treated without or with metformin (3mM) for 18 h in the presence or absence of insulin (1µg/mL). Cell lysates were analyzed by Western blotting for cyclin D3, CDK4, and for β-tubulin to verify equal protein loading. The graphs in B–C represent densitometric scans of cyclin D3 and CDK4, respectively, normalized for β-tubulin as seen in the representative Western blots. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. Each mean is labeled with a superscript (a or b). b, p < 0.05 vs other treatment groups (a).

Figure 2

Dose-response study of effect of metformin on phosphorylation of AMPK. T-I cells (2 × 10⁶ cells/plate) were incubated in 0.1% BSA-containing McCoy’s media for 24 hours and then treated with 0, 0.3, 1, 3, 10 mM Metformin for 18 hours. Cell lysates were analyzed by Western blotting for AMPK phosphorylated at Thr172 and protein loading was normalized for total AMPK as seen in the representative Western blots. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. Each mean is labeled with a superscript (a, b or c). b, P < 0.05 vs a; c, P < 0.01 vs a.

Figure 3

Effect of metformin on insulin-induced phosphorylation of Erk1/2 and S6K1. T-I cells (2 × 10⁶ cells/plate) were pretreated without or with metformin (3mM) for 18 h or AICAR (1mM), a specific AMPK activator, for 1 h followed by insulin (1µg/mL) treatment for 10 min. Control groups were treated with vehicle (acid water, pH 2). Cell lysates were analyzed for phospho-specific Erk1/2 (Thr²⁰²/Tyr²⁰⁴) in panel A and S6K (Thr³⁸⁹) in panel B and protein loading was normalized for Erk1/2 by Western blotting. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. Each mean is labeled with a superscript (a or b). b, P < 0.05 vs other treatment groups (a).

Figure 4

Effect of metformin on insulin-induced phosphorylation of Erk1/2 and S6K1 in cells treated or not treated with compound C, a specific AMPK inhibitor. T-I cells (2 × 10⁶ cells/plate) were incubated without or with metformin (3mM) for 18 hours followed by compound C (20µM) or vehicle (dimethylsulfoxide) for 2 h and insulin (1µg/mL) or vehicle (acid water, pH 2) for 10 min. Cell lysates were analyzed for phospho-specific Erk1/2 (Thr202/Tyr204) in panel A and S6K (Thr389) in panel B and protein loading was normalized for Erk1/2 by Western blotting. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. Each mean is labeled with a superscript (a or b). b, P < 0.05 vs other treatment groups (a).