Takako Araki M.D. Dimiter Avtanski PhD

1. 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

2. 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

3. 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)

4. 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

5. 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

6. Pregnenolone 17-hydroxypregnelone DHEA Cholesterol StAR/SCC 17 Alpha 17-20 lyase 3 Beta HSD 3 Beta HSD 3 Beta HSD 17 Alpha 17-20 lyase Progesterone 17-hydroxyprogesterone Androstenedione 17 Beta HSD Aromatase Testosterone Estrone Aromatase 17 Beta HSD Estradiol 17b-hydroxysteroid dehydrogenase:17b-HSD

7. Aromatase Inhibitor Type 1 Inhibitors: Steroidal Inhibitors Type 2 Inhibitors: Non-steroidal Inhibitors

8. Androstenedione Estrone TZDs inhibit estrone production TZDs effect was abolished when aromatase activity was inhibited

9. Estradiol Testosterone TZDs inhibit estradiol production TZDs effect was abolished when aromatase activity was inhibited

10. Aromatase mRNA Expression Aromatase mRNA expression was not changed by TZDs

11. Aromatase Enzyme Expression Aromatase enzyme expression was not changed by TZDs

12. TZD Effect on Substrate binding to Aromatase TZDs inhibit aromatase activity by interfering with substrate binding

13. Competitive Inhibitor 1/V 1/V Non-competitive Inhibitor Vmax Vmax No Inhibitor No Inhibitor 0 1/S 1/Km 1/Km 0 1/S Competitive Inhibitor Non-competitive Inhibitor 1/V Uncompetitive Inhibitor Vmax No Inhibitor 0 1/S 1/Km Uncompetitive Inhibitor

14. Kinetic Study - Estradiol - Km 6.4 Km 11.3 Km 7.3 Km 10.6 Km 9.8

15. 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

16. 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

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