The Effects of Thiazolidinediones (TZDs) on Aromatase Enzyme Kinetics in Human Granulosa Cells

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

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

5. Introduction (continued) • 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. • 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. • 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: • Human granulosa cells were obtained during in-vitro fertilization. • 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 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. • Measurements: • Estrone or estradiol concentrations in the medium were examined by radioimmunoassay or ELISA. • Protein concentrations were measured by modified Lowry method. • Analysis: • Pairwise t test was used to assess the statistical significance of mean values for the Km and Vmax in the presence or absence of TZDs.

7. Effects of TZD and Aromatase Inhibitor on Estrone Production ~ Androstenedione as a substrate ~ 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.

8. Effects of TZD and Aromatase Inhibitor on Estradiol Production ~ Testosterone as a substrate ~ 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.

9. aromatase aromatase b-actin b-actin Effects of TZD on Aromatase mRNA Insulin (ng/ml) 0 10 102 103 0 10 102 103 0 10 102 103 Rosiglitazone (25 mM) + + + + Pioglitazone (25 mM) + + + + Androstenedione Testosterone 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 b-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.

10. Aromatase enzyme expression Insulin (ng/ml) 0 10 102 103 0 10 102 103 0 10 102 103 Rosiglitazone (25 mM) + + + + Pioglitazone (25 mM) + + + + Androstenedione Testosterone Effects of TZD on Aromatase enzyme protein expression 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.

11. Effects of TZD on Substrate binding to Aromatase enzyme • Binding of125I-androstenedione to aromatase was decreased by up to 20% by rosiglitazone or pioglitazone. • Binding of 125I-testosterone to aromatase was decreased by 38% by rosiglitazone and 32% by pioglitazone. • There was no significant difference between the effects of both TZDs.

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

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

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

15. Effects of TZDs on circulating androgen & estrogen concentrations in-vivo • Androgen binding to aromatase  Insulin Testosterone  Estrogen In-vivo effects of TZDs to decrease Estrogen production based on TZD effects in vitro ?  Aromatase activity 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.

16. Discussion • 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 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%. • 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.

17. Conclusions • 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 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 from • Gerald J. and Dorothy Friedman Foundation • Empire Clinical Research Investigator Program of New York State Department of Health • Thanks Scandinavia Foundation • Chinese American Medical Association & Chinese American Independent Practice Association.