Metformin directly inhibits androgen production in human thecal cells George R Attia, M.D., William E Rainey, Ph.D., Bruce R Carr, M.D.  Fertility and Sterility  Volume 76, Issue 3, Pages 517-524 (September 2001) DOI: 10.1016/S0015-0282(01)01975-6

Figure 1 Effect of metformin with and without forskolin on steroid production in human ovarian thecal-like androgen-producing tumor cells. Cells were treated for 48 hours with medium alone, forskolin (10 μM), metformin (50 μM and 200 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM). A, androstenedione production. B, progesterone. C, 17OHP production. All are expressed as a percentage of steroid production in control (medium-only treatment). Each data point represents mean ± SE of replicate dishes (n = 3). Data are from a representative experiment that was run six times. Statistical analysis was performed using results from all six experiments (∗P<.05, ∗∗P<.01 compared with forskolin alone.) Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)

Figure 2 Effect of metformin with and without forskolin on T production in human ovarian thecal-like androgen-producing tumor cells. Cells were treated for 48 hours with medium alone, forskolin (10 μM), metformin (50 μM and 200 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM). All are expressed as a percentage of steroid production in control (medium-only treatment). Each data point represents mean ± SE of replicate dishes (n = 3). Data are from a representative experiment that was run six times. Statistical analysis was performed using results from all six experiments (∗∗P<.01 compared with forskolin alone.) Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)

Figure 3 Concentration-dependent effect of metformin on combined steroid production by forskolin-stimulated human ovarian thecal-like androgen-producing tumor cells. Combined steroid production includes androstenedione, 17OH progesterone, and progesterone. Cells were treated for 48 hours with low-serum medium (basal), metformin (50 μM and 200 μM), forskolin (10 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM). Each data point represents the mean ± SE of replicate dishes (n = 3). Data are from a representative experiment that was run three times. Statistical analysis was performed using results from all three experiments. (∗P<.05, ∗∗P<.001, compared with forskolin alone). Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)

Figure 4 Effect of metformin with and without forskolin treatment on steroidogenic acute regulatory, CYP17, and 3βHSD expression in human ovarian thecal-like androgen-producing tumor cells. Cells were treated for 48 hours with medium only, forskolin (10 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM). Western analysis was performed as described in Materials and Methods, with 20 μg of cellular protein per lane. Lanes corresponding to each experimental condition are listed above the figure. Molecular weight markers are indicated with a dash. Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)

Figure 5 Effect of metformin and forskolin on CYP17 mRNA expression in human ovarian thecal-like androgen-producing tumor cells. Top, Northern analysis for CYP17 mRNA. Cells were treated with medium only (basal), forskolin (10 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM) for 24 hours. RNA was then extracted, and the Northern blot analysis was performed as described in Materials and Methods. Bottom, a diagrammatic presentation of the mean of duplicate data in the top portion of this figure. The data are expressed as percentage of CYP17 mRNA expression compared with basal (control) cells (∗P<.05, ∗∗P<.01). Phosphoimager counter was used to measure the density of each band. Probing for β-actin was used to standardize results from each lane. Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)

Figure 6 Effect of metformin and forskolin on steroidogenic acute regulatory mRNA expression in human ovarian thecal-like androgen-producing tumor cells. Top, Northern blot analysis for steroidogenic acute regulatory (StAR) protein mRNA. Cells were treated with medium only (basal), forskolin (10 μM), or forskolin (10 μM) plus metformin (50 μM and 200 μM) for 24 hours. RNA was then extracted, and the Northern blot analysis was performed as described in Materials and Methods. Bottom, a diagrammatic presentation of the mean of duplicate data in the top portion of this figure. Data are expressed as percentage of StAR protein mRNA expression compared with basal (control) cells. Phosphoimager counter was used to measure the density of each band. Probing with β-actin was used to standardize results from each lane. Attia. Metformin inhibits ovarian androgen. Fertil Steril 2001. Fertility and Sterility 2001 76, 517-524DOI: (10.1016/S0015-0282(01)01975-6)