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
First couple of slides, I will briefly explain the background of our projects.

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)
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.

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
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

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
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.

6. Pregnenolone 17-hydroxypregnelone DHEA
Cholesterol
StAR/SCC
17 Alpha
17-20 lyase
Pregnenolone 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

7. 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.

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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.

14. 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

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
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