Impact of Estrogen on Uterine Glucose Metabolism

1. Impact of Estrogen on Uterine Glucose Metabolism Caitlin Murphy Dr. Fred Stormshak Department of Animal Science

2. Relevance • Human Impact • Estrogens and xenoestrogens (endocrine disruptors) can promote uterine and breast cancer • Animal Production • Better understanding of the mechanism of action of estrogens in regulation of reproductive cycles of livestock

3. Background- Estradiol • Estradiol (E2) and progesterone (P4) are two of the major reproductive or “sex” hormones present in females. • The ovary is the major production site • Through appropriately timed production of both hormones during the estrous or menstrual cycle, ovulation is stimulated

4. Responses of Target Cells to Estrogens • Short term (1-6 hours after exposure) • Increases hyperemia • Increases imbibition of water • Increases glucose oxidation and lipid synthesis • Increases RNA polymerase activity • Long term (6-48 hours after exposure) • Continued stimulation of RNA polymerase activity • Increases DNA synthesis • Increases in cell hypertrophy and hyperplasia

5. Background-Glucose Oxidation • When glucose is oxidized, energy is derived through the process of glycolysis and the Krebs cycle 6O2 + C6H12O6→ 6CO2 + 6H2O + ATP(energy)

6. Previous observations • Estradiol (E2) stimulated an increase in the metabolism of glucose to CO2 in rat uteri (Stormshak et al) • Increase in Glucose-6-Phosphate Dehydrogenase due to estrogen stimulation (Barker et al)

7. Previous Observations- continued Glucose *Glucose Transporter GT-Glucose hexokinase Glycolysis G-6-P *Glucose-6-phosphate dehydrogenase *Been shown to be stimulated by estrogen Pentose phosphate pathway

8. Hypothesis tested • Does estradiol (E2) stimulate nuclear-induced signaling of glucose oxidation in the ovine endometrium?

9. 10 day treatment schedule of ovariectomized ewes

10. Removal of endometrial tissue during surgery • Tissue is removed from the endometrium of left and right uterine horns • Tissue was kept at 4° C for transport to the laboratory and until processed • Once tissue is collected 3 assays were done to determine- • glucose oxidation • presence of nuclear estrogen receptors • amount of DNA Endometrium

11. Experiment # 1- DNA assay • Estradiol causes imbibition of water into cells • Results for both the amount of estrogen receptors and glucose metabolism are expressed as per µg of DNA Control E2 treated H2O H2O H2O H2O H2O

12. Results- DNA Assay *P< 0.05 DNA (µg)/ (mg) tissue

13. Experiment #2- Nuclear Estrogen Receptors • A radioreceptor exchange assay was used to determine the effect of treatments on the concentration of nuclear estrogen receptors • Stimulated in treatment animals • Low stimulation for control animals

14. Methods- nuclear estrogen receptors • Tissue was homogenized and the nuclear pellet was isolated • To determine specific binding of [3H]-estradiol the nuclear pellet was treated with either • 2 x 10-8 M [3H]-estradiol (for total binding) • 2 x 10-8 M [3H]-estradiol and 2 x 10-6 M diethylstilbestrol (for non-specific binding) • SB= total - NSB • Nuclear pellet was then incubated at 37º C, cooled and placed in EtOH overnight • EtOH was then measured in scintillation counter to determine concentration of [3H]-estradiol

15. Results- Nuclear Estrogen Receptors *P< 0.05 E2 bound (fmol)/DNA (µg)

16. Experiment #3-Glucose Oxidation • Glucose oxidation was assayed to determined if E2 would cause endometrial tissue to take in glucose and metabolize it • through glycolysis and the Krebs cycle • Ultimately produce CO2 and energy

17. Methods-Glucose Oxidation

18. Results- Glucose Oxidation *P< 0.05 14 CO2(cpm)/DNA (µg)

19. Conclusion • Estrogen stimulates a nuclear-induced signal to cause the metabolism of glucose to CO2 in the ovine endometrium Glucose E2 E2 Energy Nucleus CO2

20. Acknowledgements • Howard Hughes Medical Institute (HHMI) • Undergraduate Research Innovation Scholarship and Creativity (URISC) • Dr. Fred Stormshak • Reed Reeve • Tari Tan • Jared Deitz • Cecily Bishop