1. The Effects of Thiazolidinediones (TZDs) on Aromatase Enzyme Kinetics in Human Granulosa Cells Takako Araki, Michael Goldman, Miroslava Varadinova, Grishma Parikh, Prajesh Joshi, Antoine Chaanine, Amit Seth, Yun Feng, Shumei Kato, Zev Rosenwaks, Leonid Poretsky, Donna Seto-Young Department of Medicine Division of Endocrinology & Metabolism Beth Israel Medical Center Albert Einstein College of Medicine New York, NY

2. Introduction Aromatase is a cytochrome P450 super- family enzyme that converts androgens to estrogens.Aromatase is a cytochrome P450 super- family enzyme that converts androgens to estrogens. It is the major source of estrogen production in womenIt is the major source of estrogen production in women  Aromatase is an important factor in sexual development.  Abnormalities in aromatase may play a major role in reproductive dysfunction, including in the development of the polycystic ovary syndrome (PCOS).  Aromatase is an important factor in the development and progression of breast cancer.  Decline in estrogen production by aromatase is thought to be the cause of postmenopausal osteoporosis.

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

4. TZDs are a group of medications used as insulin sensitizers in treatment of diabetes TZDs, acting as PPAR-  agonists, enhance insulin sensitivity of tissues and improve glucose tolerance in patients with insulin resistant states Thiazolidinediones (TZDs)

5. Introduction (continued) TZDs reduce androgen levels in women and may improve ovulatory rates. TZDs reduce androgen levels in women and may improve ovulatory rates. These effects have been attributed to systemic insulin-sensitizing effects of TZDs and consequent reduction in hyperinsulinemia.These effects have been attributed to systemic insulin-sensitizing effects of TZDs and consequent reduction in hyperinsulinemia. However, TZDs also directly affect androgen and estrogen production in human ovarian cell culture.However, TZDs also directly affect androgen and estrogen production in human ovarian cell culture. We recently reported that the inhibitory effects of TZDs on estrogen production is due to inhibition of activity of the aromatase enzyme.We recently reported that the inhibitory effects of TZDs on estrogen production is due to inhibition of activity of the aromatase enzyme. In the present study, we investigated the effect of TZDs on substrate affinity to aromatase and enzyme kinetic properties in human granulosa cells.In the present study, we investigated the effect of TZDs on substrate affinity to aromatase and enzyme kinetic properties in human granulosa cells.

6. Materials & Methods Granulosa cell culture:Granulosa cell culture: Human granulosa cells were obtained during in-vitro fertilization.Human granulosa cells were obtained during in-vitro fertilization. Purified 2x on Percoll gradient.Purified 2x on Percoll gradient. Incubated for 48 h in M199 tissue culture medium, supplemented with 10% FBS, 10 μg/ml gentamicin, and 250 ng/ml amphotericin B;Incubated for 48 h in M199 tissue culture medium, supplemented with 10% FBS, 10 μg/ml gentamicin, and 250 ng/ml amphotericin B; Incubated for 24 h in the same medium, supplemented with 2% FBS;Incubated for 24 h in the same medium, supplemented with 2% FBS; Incubated in culture medium supplemented with various concentrations of androstenedione (A) or testosterone (T) (0.025, 0.05, 0.1, 0.13, 0.25, 0.5 or 1 µM) with either pioglitazone or rosiglitazone (25 or 50 µM) for 150 min.Incubated in culture medium supplemented with various concentrations of androstenedione (A) or testosterone (T) (0.025, 0.05, 0.1, 0.13, 0.25, 0.5 or 1 µM) with either pioglitazone or rosiglitazone (25 or 50 µM) for 150 min. Measurements:Measurements: Estrone or estradiol concentrations in the medium were examined by radioimmunoassay or ELISA.Estrone or estradiol concentrations in the medium were examined by radioimmunoassay or ELISA. Protein concentrations were measured by modified Lowry method.Protein concentrations were measured by modified Lowry method. Analysis:Analysis: Pairwise t test was used to assess the statistical significance of mean values for the K m and V max in the presence or absence of TZDs.Pairwise t test was used to assess the statistical significance of mean values for the K m and V max in the presence or absence of TZDs.

7. In the absence of AI, insulin stimulated estrone production by 114% while rosiglitazone and pioglitazone inhibited the estrone production by 22%. AI inhibited estrone production by 55% and in the presence of AI, the inhibitory effects of TZDs were reduced significantly. Effects of TZD and Aromatase Inhibitor on Estrone Production ~ Androstenedione as a substrate ~

8. In the absence of AI, insulin stimulated estradiol production by up to 124%, while rosiglitazone and pioglitazone inhibited the estradiol production by up to 20% in the absence of insulin and 34% in the presence of insulin. AI inhibited estradiol production by up to 60% and in the presence of AI, the inhibitory effects of TZDs was reduced significantly. Effects of TZD and Aromatase Inhibitor on Estradiol Production ~ Testosterone as a substrate ~

9. Testosterone A When androstenedione or testosterone was used as substrate, rosiglitazone or pioglitazone had no significant effect on aromatase mRNA compared to control either in the absence or in the presence of insulin. Similar results were observed when house protein  -actin primers were used to demonstrate the relative abundance of mRNA under each set of conditions. B A 148 bp DNA fragment, observed when Bam HI restriction enzyme was added, proved that RT-PCR generated fragments which were in the position corresponding to that of aromatase gene. C No genomic DNA contamination was observed in the total RNA preparation. Testosterone  -actin aromatase Insulin (ng/ml) 0 10 10 2 10 3 0 10 10 2 10 3 0 10 10 2 10 3 Rosiglitazone (25  M) + + + + Pioglitazone (25  M) + + + + Androstenedione  -actin aromatase Effects of TZD on Aromatase mRNA

10. Insulin (ng/ml) 0 10 10 2 10 3 0 10 10 2 10 3 0 10 10 2 10 3 Rosiglitazone (25  M) + + + + Pioglitazone (25  M) + + + + Testosterone Aromatase enzyme expression Androstenedione When androstenedione or testosterone was used as substrate, rosiglitazone or pioglitazone had no significant effect on aromatase enzyme protein expression compared to control, either in the absence or in the presence of insulin. Effects of TZD on Aromatase enzyme protein expression

11. A.Binding of 125 I-androstenedione to aromatase was decreased by up to 20% by rosiglitazone or pioglitazone. B.Binding of 125 I-testosterone to aromatase was decreased by 38% by rosiglitazone and 32% by pioglitazone. There was no significant difference between the effects of both TZDs. Effects of TZD on Substrate binding to Aromatase enzyme

12. 1/V 1/S 0 Competitive Inhibitor No Inhibitor 1/Km 1/V max 1/S 1/V 0 Non-competitive Inhibitor No Inhibitor 1/Km 1/V max 1/V 1/S 0 Uncompetitive Inhibitor No Inhibitor 1/Km 1/V max

13. When androstenedione was used as substrate, V max was reduced by 19% and by 31% and the K m was reduced by 14% and by 20% in the presence of rosiglitazone or pioglitazone, respectively. Effects of TZD on K m and V max of Aromatase ~ In the Presence of Androstenedione ~

14. When testosterone was used as substrate, both rosiglitazone and pioglitazone inhibited V max by 41% and K m by 36%. Effects of TZD on K m and V max of Aromatase ~ In the Presence of Testosterone ~

15. Effects of TZDs on circulating androgen & estrogen concentrations in-vivo  Aromatase activity  Estrogen ? TZDs reduce testosterone (estrogen substrate) levels by three disparate mechanisms:  Indirectly, by decreasing circulating insulin level through their insulin-sensitizing action.  Indirectly, by interfering with androgen binding to aromatase.  Directly,by reducing aromatase activity through uncompetitive inhibition.  Testosterone  Androgen binding to aromatase  Insulin In-vivo effects of TZDs to decrease Estrogen production based on TZD effects in vitro

16. Discussion We demonstrated that TZDs inhibit estrogen production in human granulosa cells via inhibition of aromatase activity.We demonstrated that TZDs inhibit estrogen production in human granulosa cells via inhibition of aromatase activity. TZDs had no effect on either aromatase mRNA or protein expression, meaning that inhibitory effects of TZDs on aromatase do not involve transcription or translation of the aromatase gene.TZDs had no effect on either aromatase mRNA or protein expression, meaning that inhibitory effects of TZDs on aromatase do not involve transcription or translation of the aromatase gene. TZDs acted as uncompetitive inhibitors of aromatase and therefore interfered with substrate (androgen) binding to aromatase and altering enzymatic kinetic properties by reducing both the Vmax and Km.TZDs acted as uncompetitive inhibitors of aromatase and therefore interfered with substrate (androgen) binding to aromatase and altering enzymatic kinetic properties by reducing both the Vmax and Km. Type 1 aromatase inhibitors in clinical use for breast cancer are competitive inhibitors. Thus, by combining the TZDs and type 1 aromatase inhibitors, further inhibition of aromatase by TZDs can be expected. Indeed, while type 1 aromatase inhibitors reduced the estrogen production by up to 65%, TZDs inhibited the remaining aromatase activity by an additional 20%.Type 1 aromatase inhibitors in clinical use for breast cancer are competitive inhibitors. Thus, by combining the TZDs and type 1 aromatase inhibitors, further inhibition of aromatase by TZDs can be expected. Indeed, while type 1 aromatase inhibitors reduced the estrogen production by up to 65%, TZDs inhibited the remaining aromatase activity by an additional 20%. Therapeutic applications of this effect may allow for the use of existing TZDs in a broad class of other diseases, including PCOS and breast cancer.Therapeutic applications of this effect may allow for the use of existing TZDs in a broad class of other diseases, including PCOS and breast cancer. Inhibition of aromatase activity by TZDs may, at least in part, explain their in-vivo effects on bone density on the postmenopausal women.Inhibition of aromatase activity by TZDs may, at least in part, explain their in-vivo effects on bone density on the postmenopausal women.

17. Conclusions TZDs inhibit estrone and estradiol production in human granulosa cells.TZDs inhibit estrone and estradiol production in human granulosa cells. TZDs inhibit production of estrogens by interfering with androstenedione and testosterone binding to aromatase.TZDs inhibit production of estrogens by interfering with androstenedione and testosterone binding to aromatase. TZDs act as un-competitive inhibitors, which is demonstrated by the reduction of the Vmax and Km of aromatase enzyme.TZDs act as un-competitive inhibitors, which is demonstrated by the reduction of the Vmax and Km of aromatase enzyme.

18. Acknowledgements We are thankful for the support fromWe are thankful for the support from Gerald J. and Dorothy Friedman FoundationGerald J. and Dorothy Friedman Foundation Empire Clinical Research Investigator Program of New York State Department of HealthEmpire Clinical Research Investigator Program of New York State Department of Health Thanks Scandinavia FoundationThanks Scandinavia Foundation Chinese American Medical Association & Chinese American Independent Practice Association.Chinese American Medical Association & Chinese American Independent Practice Association.