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

Human Granulosa CellsHuman Granulosa Cells

Takako Araki, Michael Goldman, Miroslava Varadinova, Grishma Parikh, Takako Araki, Michael Goldman, Miroslava Varadinova, Grishma Parikh, Prajesh Joshi, Antoine Chaanine, Amit Seth, Yun Feng, Shumei Kato,Prajesh Joshi, Antoine Chaanine, Amit Seth, Yun Feng, Shumei Kato,

Zev Rosenwaks, Leonid Poretsky, Donna Seto-YoungZev Rosenwaks, Leonid Poretsky, Donna Seto-Young

Department of MedicineDivision of Endocrinology & Metabolism

Beth Israel Medical CenterAlbert Einstein College of Medicine

New York, NY

IntroductionIntroduction• Aromatase is a cytochrome P450 super- family enzyme Aromatase is a cytochrome P450 super- family enzyme

that converts androgens to estrogens. 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. Aromatase is an important factor in sexual development. Abnormalities in aromatase may play a major role in Abnormalities in aromatase may play a major role in

reproductive dysfunction, including in treproductive dysfunction, including in the development of the he development of the polycystic ovary syndrome (PCOS). polycystic ovary syndrome (PCOS).

Aromatase is an important factor in the development and Aromatase is an important factor in the development and progression of breast cancer.progression of breast cancer.

Decline in estrogen production by aromatase is thought to be the Decline in estrogen production by aromatase is thought to be the cause of postmenopausal osteoporosis. cause of postmenopausal osteoporosis.

Cholesterol

Estradiol

Estrone

Pregnenolone 17-hydroxypregnelone DHEA

17-hydroxyprogesterone

Aromatase

Aromatase

3 HSD 3 HSD 3 HSD

17 Alpha

17

17-20 lyase

17-20 lyase

17 HSD

17 HSD

StAR/SCC

Progesterone

Testosterone

17-hydroxysteroid dehydrogenase:17-HSD

Androstenedione

• 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)

Introduction (continued)Introduction (continued)• TZDs reduce androgen levels in women and may improve ovulatory TZDs reduce androgen levels in women and may improve ovulatory

rates.rates.

• These effects have been attributed to systemic insulin-sensitizing These effects have been attributed to systemic insulin-sensitizing effects of TZDs and consequent reduction in hyperinsulinemia.effects of TZDs and consequent reduction in hyperinsulinemia.

• However, TZDs also directly affect androgen and estrogen However, TZDs also directly affect androgen and estrogen production in human ovarian cell culture.production in human ovarian cell culture.

• We recently reported that the inhibitory effects of TZDs on estrogen We recently reported that the inhibitory effects of TZDs on estrogen production is due to inhibition of activity of the aromatase enzyme.production is due to inhibition of activity of the aromatase enzyme.

• In the present study, we investigated the effect of TZDs on substrate In the present study, we investigated the effect of TZDs on substrate affinity to aromatase and enzyme kinetic properties in human affinity to aromatase and enzyme kinetic properties in human granulosa cells.granulosa cells.

Materials & MethodsMaterials & Methods• Granulosa cell culture:Granulosa cell culture:

• Human granulosa cells were obtained during Human granulosa cells were obtained during in-vitroin-vitro fertilization. fertilization. • Purified 2x on Percoll gradient.Purified 2x on Percoll gradient.• Incubated for 48 h in M199 tissue culture medium, supplemented with Incubated for 48 h in M199 tissue culture medium, supplemented with

10% FBS, 10 10% FBS, 10 μμg/ml gentamicin, and 250 ng/ml amphotericin B;g/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 Incubated in culture medium supplemented with various concentrations

of androstenedione (A) or testosterone (T) (0.025, 0.05, 0.1, 0.13, 0.25, 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 0.5 or 1 µM) with either pioglitazone or rosiglitazone (25 or 50 µM) for 150 min. 150 min.

• Measurements:Measurements:• Estrone or estradiol concentrations in the medium were examined by Estrone or estradiol concentrations in the medium were examined by

radioimmunoassay or ELISA. radioimmunoassay or ELISA. • Protein concentrations were measured by modified Lowry method.Protein concentrations were measured by modified Lowry method.

• Analysis:Analysis:• Pairwise Pairwise tt test was used to assess the statistical significance of mean test was used to assess the statistical significance of mean

values for the Kvalues for the Kmm and V and Vmaxmax in the presence or absence of TZDs. in the presence or absence of TZDs.

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 ~

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 ~

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 10 102 103 0 10 102 103 0 10 102 103

Rosiglitazone (25 M) + + + +Pioglitazone (25 M) + + + +

Androstenedione-actin

aromatase

Effects of TZD on Aromatase mRNA

Insulin (ng/ml) 0 10 102 103 0 10 102 103 0 10 102 103

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

A. Binding of125I-androstenedione to aromatase was decreased by up to 20% by rosiglitazone or pioglitazone.

B. 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.

Effects of TZD on Substrate binding to Aromatase enzyme

1/V

1/S0

Competitive Inhibitor

Competitive Inhibitor

No Inhibitor

1/Km

1/Vmax

1/S

1/V

0

Non-competitive Inhibitor

Non-competitive Inhibitor

No Inhibitor

1/Km

1/Vmax

1/V

1/S0

Uncompetitive Inhibitor

Uncompetitive Inhibitor

No Inhibitor

1/Km

1/Vmax

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.

control50 M rosiglitazoneregression line for controlregression line for 50 M rosiglitazone

Effects of TZD on Km and Vmax of Aromatase ~ In the Presence of Androstenedione ~

When testosterone was used as substrate, both rosiglitazone and pioglitazone inhibited Vmax by 41% and Km by 36%.

control50 M rosiglitazoneregression line for controlregression line for 50 M rosiglitazone

control50 M pioglitazoneregression line for controlregression line for 50 M pioglitazone

Effects of TZD on Km and Vmax of Aromatase ~ In the Presence of Testosterone ~

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

DiscussionDiscussion• We demonstrated that TZDs inhibit estrogen production in human We demonstrated that TZDs inhibit estrogen production in human

granulosa cells via inhibition of aromatase activity. granulosa cells via inhibition of aromatase activity. • TZDs had no effect on either aromatase mRNA or protein TZDs had no effect on either aromatase mRNA or protein

expression, meaning that inhibitory effects of TZDs on aromatase expression, meaning that inhibitory effects of TZDs on aromatase do not involve transcription or translation of the aromatase gene. do not involve transcription or translation of the aromatase gene.

• TZDs acted as uncompetitive inhibitors of aromatase and therefore TZDs acted as uncompetitive inhibitors of aromatase and therefore interfered with substrate (androgen) binding to aromatase and interfered with substrate (androgen) binding to aromatase and altering enzymatic kinetic properties by reducing both the Vmax and altering enzymatic kinetic properties by reducing both the Vmax and Km. Km.

• Type 1 aromatase inhibitors in clinical use for breast cancer are Type 1 aromatase inhibitors in clinical use for breast cancer are competitive inhibitors. Thus, by combining the TZDs and type 1 competitive inhibitors. Thus, by combining the TZDs and type 1 aromatase inhibitors, further inhibition of aromatase by TZDs can be aromatase inhibitors, further inhibition of aromatase by TZDs can be expected. Indeed, while type 1 aromatase inhibitors reduced the expected. Indeed, while type 1 aromatase inhibitors reduced the estrogen production by up to 65%, TZDs inhibited the remaining estrogen production by up to 65%, TZDs inhibited the remaining aromatase activity by an additional 20%.aromatase activity by an additional 20%.

• Therapeutic applications of this effect may allow for the use of Therapeutic applications of this effect may allow for the use of existing TZDs in a broad class of other diseases, including PCOS existing TZDs in a broad class of other diseases, including PCOS and breast cancer. and breast cancer.

• Inhibition of aromatase activity by TZDs may, at least in part, explain Inhibition of aromatase activity by TZDs may, at least in part, explain their in-vivo effects on bone density on the postmenopausal women. their in-vivo effects on bone density on the postmenopausal women.

ConclusionsConclusions• TZDs inhibit estrone and estradiol production in TZDs inhibit estrone and estradiol production in

human granulosa cells. human granulosa cells.

• TZDs inhibit production of estrogens by TZDs inhibit production of estrogens by interfering with androstenedione and interfering with androstenedione and testosterone binding to aromatase. testosterone binding to aromatase.

• TZDs act as un-competitive inhibitors, which is TZDs act as un-competitive inhibitors, which is demonstrated by the reduction of the Vmax and demonstrated by the reduction of the Vmax and Km of aromatase enzyme. Km of aromatase enzyme.

AcknowledgementsAcknowledgements• 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 Empire Clinical Research Investigator

Program of New York State Department of Program of New York State Department of HealthHealth

• Thanks Scandinavia FoundationThanks Scandinavia Foundation• Chinese American Medical Association & Chinese American Medical Association &

Chinese American Independent Practice Chinese American Independent Practice Association. Association.