Thiazolidinediones Inhibit Aromatase Activity in Human Granulosa Cells by Interfering with Androgen Binding to Aromatase Takako Araki M.D. Dimiter Avtanski PhD Grishma Parikh M.D. Michael Goldman M.D. Zev Rosenwaks M.D. Leonid Poretsky M.D. Donna Seto-Young PhD. Department of Medicine Division of Endocrinology & Metabolism Beth Israel Medical Center Albert Einstein College of Medicine New York, NY

Role of Insulin in Human Ovary Insulin plays a role in human ovarian function Among other effects, insulin regulates steroidogenesis, upregulates insulin-like growth factor 1 receptors, and inhibits insulin-like growth factor binding protein 1 production in human ovary First couple of slides, I will briefly explain the background of our projects.

Hyperinsulinemia stimulates ovarian growth and cyst formation, probably contributing to the pathogenesis of polycystic ovary syndrome (PCOS) Treatment of PCOS Weight loss: lifestyle modifications via diet and exercise Regulation of menstrual cycles: oral contraceptives Anti-androgens: spironolactone Insulin-sensitizing agents: metformin, thiazolidinediones (TZDs) Our main project in the lab originated from interest to the mistery of polycystic ovary syndrome which is still unknown pathogenesis. One key factor is insulin. It means insulin level affects steroidogenesis in human ovary. So in our previous studies, (click) hyperinsulinemia stimulates ovarian growth and cyst formation, probably contributing to the pathogenesis of polycystic ovary syndrome. As you know, treatment are weight loss, oral contraceptives, spironolactone, and insulin sensitizer, such as metformin or thiazolidinediones.

Thiazolidinediones (TZDs) TZDs are a group of medications used as insulin sensitizers in treatment of diabetes TZDs, acting as PPAR-g agonists, enhance insulin sensitivity of tissues and improve glucose tolerance in patients with insulin resistant states TZDs reduce hyperandrogenemia and restore ovulation in PCOS Thiazolidinediones is an insulin sensitizer and widely used for the treatmetn of diabetes. TZDS acts as agonist of PPAR gamma which is nuclear receptor superfamily (ex. steroid, retinoid, vitamin D, thyroid receptor etc) and controls glucometabolism, lipid metabolism, atherogenesis, inflammation, cell differentiation

Aromatase Cytochrome P-450 superfamily Product of the CYP19 gene Heme structure is responsible for binding androgenic steroid Highly expressed in placenta and granulosa cells Key enzyme that regulates estrogen synthesis in ovary Present in subcutaneous fat (adipose tissue), fetal liver, muscle, brain, bone, breast, and testis Aormatase is cytochrome P450 superfamily located CYP number 19 gene. Whole structure has not obtained yet, but there is a heme structure which is responsible for binding androgenic steroid. Aromatase is highly expressed in aplacenta and human granulosa cells, also present in adipose tissue, fetal liver, muscle, brain, bone, breast, and testis.

Pregnenolone 17-hydroxypregnelone DHEA Cholesterol StAR/SCC 17 Alpha 17-20 lyase Pregnenolone 17-hydroxypregnelone DHEA 3 Beta HSD 3 Beta HSD 3 Beta HSD 17 Alpha 17-20 lyase Progesterone 17-hydroxyprogesterone Androstenedione 17 Beta HSD Aromatase Testosterone Estrone This is the steroid genesis pathway. Aromatase is our main focuse of our projects, this works in 2 important steps, one is from androstenedione to estrone, the other is from testosterone to estradiol. We focused this aromatase enzyme in our project. ( In human ovarian cells TZDs activate progesterone production and inhibit testosterone and estradiol production) Aromatase 17 Beta HSD Estradiol 17b-hydroxysteroid dehydrogenase:17b-HSD

Aromatase Inhibitor Type 1 Inhibitors: Steroidal Inhibitors Type 2 Inhibitors: Non-steroidal Inhibitors There are several commercially available products to inhibit aromatase, which we call aromatase inhibitor. There are 2 types, one is steriodal inhibitor which are similar structure to the substrate, and non steroidal inhibitor which usually contains triazole structures.

TZDs inhibit estrone production Androstenedione Estrone This is the comparison enzyme product with or without aromatase inhibitor and TZDs. Substrate is androstenedione and measured estrone production by ELISA. Graph A is the comparison with control, rosiglitazone and pioglitazone with differenct concentration of insulin. TDZs supress Estrone productions. Graph B is the comparison with control and aromatase inhibitor. With adding aromatase inhibitor, estrone produc tion decreased. Graph C is the combination of TZDs and aromatase inhibitor. Compare to graph A, TZDs effects are abolished with adding aromatase inhibitor. TZDs inhibit estrone production TZDs effect was abolished when aromatase activity was inhibited

TZDs inhibit estradiol production Testosterone Estradiol Same methods using testosterone as a substrate. We can see the similar resutls to the previous slide. Here is control, with Rosi and Pioglitazone, middle is with aromatase inhibitor, right side is with TZDs and aromatase inhibitor, again aromatase inhibitor abolished TZDs effects. TZDs inhibit estradiol production TZDs effect was abolished when aromatase activity was inhibited

Aromatase mRNA Expression This is the aromatase m RNA expression under different concentration of insulin, with or without rosiglitazone and pioglitazone. Bands above is with androstenedione media, and bands below is the testosterone media. You can recognize that there is no significant change in thickness of the bands, which suggests that aromatase m RNA expression was not changed by TZDs. Aromatase mRNA expression was not changed by TZDs

Aromatase Enzyme Expression This is the result of enzyme expression in similar experimental settings. Again you can recognize that there is no significant change in thickness of the bands, which suggests that aromatase enzyme expression was not changed by TZDs. Aromatase enzyme expression was not changed by TZDs

TZD Effect on Substrate binding to Aromatase We measured binding activity with or without TZDs. With both rosiglitazone and pioglitazone decrease binding activities in each substrates, which suggests that TZDs inhibit aromatase activity by interfering with substrate bindings. TZDs inhibit aromatase activity by interfering with substrate binding

Competitive Inhibitor Non-competitive Inhibitor 1/S Competitive Inhibitor No Inhibitor 1/Km Vmax 1/S 1/V Non-competitive Inhibitor No Inhibitor 1/Km Vmax 1/V 1/S Uncompetitive Inhibitor No Inhibitor 1/Km Vmax In order to prove this enzyme inhibitory effects, we did enzyme kinetic studies. There are 3 different type of enzyme inhibitors, compatitive, non competitive and uncompetitive inhibitors. These are the lineweaver-burk plots, 1 over substrate, 1 over velocity, Km is the substrate concentration which requires to produce half of maximum velocity.

Kinetic Study - Estradiol - This is our results with testosterone media, measuring estradiol. this study is not complites yet, but you can guess the pattern of parallel lines, which suggests TZDs works as a uncompetitive inhibitors. (Km 6.4(control), Km 11.3(rosi 25), Km 7.3(rosi 50), Km 10.6(pio 25), Km 9.8(pio 50)) Km 6.4 Km 11.3 Km 7.3 Km 10.6 Km 9.8

Summary ~in Human Granulosa Cells~ TZDs inhibit estrogen production TZDs do not change aromatase mRNA or enzyme protein expression TZDs inhibit aromatase activity by interfering with androgen binding to aromatase, acting as uncompetitive inhibitors TZDs inhibit estrogen production. TZDs do not change aromatase mRNA or enzyme protein expression. TZDs inhibit aromatase activity by interfering with androgen binding to aromatase, acting as uncompetitive inhibitors.

TZDs as Aromatase Inhibitors ~ Future Directions and Possible Clinical Implications ~ Potential use in estrogen – dependent diseases: Breast cancer Endometriosis Our findings may explain in part the relationship between TZDs and osteoporosis

Thank you We are thankful for the support from Gerald J. and Dorothy R. Friedman Foundation