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

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

Background- Estradiol Estradiol (E 2 ) and progesterone (P 4 ) 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

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

Background- Glucose Oxidation When glucose is oxidized, energy is derived through the process of glycolysis and the Krebs cycle 6O 2 + C 6 H 12 O 6 → 6CO 2 + 6H 2 O + ATP(energy)

Previous observations Estradiol (E 2 ) stimulated an increase in the metabolism of glucose to CO 2 in rat uteri (Stormshak et al) Increase in Glucose-6-Phosphate Dehydrogenase due to estrogen stimulation (Barker et al)

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

Hypothesis tested Does estradiol (E 2 ) stimulate nuclear-induced signaling of glucose oxidation in the ovine endometrium?

Days 1-2 injection of E2 (25 µg), sc Days 3-7 injection of P4 (10 mg), sc Days 8-9 injection of E2 (25 µg), sc Days 8-9 injection of corn oil (vehicle), sc Day 10 collection of endometrial tissue 10 day treatment schedule of ovariectomized ewes

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 Removal of endometrial tissue during surgery Endometrium

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 ControlE2 treated H2OH2O H2OH2O H2OH2O H2OH2O H2OH2O

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

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

Methods- nuclear estrogen receptors Tissue was homogenized and the nuclear pellet was isolated To determine specific binding of [ 3 H]-estradiol the nuclear pellet was treated with either 2 x M [ 3 H]-estradiol (for total binding) 2 x M [ 3 H]-estradiol and 2 x 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 [ 3 H]-estradiol

Results- Nuclear Estrogen Receptors *P< 0.05 E 2 bound (fmol)/DNA (µg)

Experiment #3-Glucose Oxidation Glucose oxidation was assayed to determined if E 2 would cause endometrial tissue to take in glucose and metabolize it through glycolysis and the Krebs cycle Ultimately produce CO 2 and energy

Methods-Glucose Oxidation 1. Tissue is placed into an Erlenmeyer flask containing MEM and U- 14 C D-Glucose (atmosphere of 95% O2-5% CO2) 2. Flasks were incubated for 1 hour at 37° C 3. Hyamine hydroxide was added to each cup containing filter paper 4. H2SO4 is added to the solution (stops reaction) 5. After 2 hours filter paper was placed in LSC fluid and amount of 14 CO2 was determined by scintillation counter

Results- Glucose Oxidation *P< CO 2 (cpm)/DNA (µg)

Conclusion Estrogen stimulates a nuclear-induced signal to cause the metabolism of glucose to CO 2 in the ovine endometrium E2E2 E2E2 Glucose CO 2 Nucleus Energy

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