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 (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

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

6O2 + C6H12O6 → 6CO2 + 6H2O + ATP(energy)

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)

Previous Observations- continuedGlucose

*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 (E2) 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

Control E2 treated

H2O

H2O H2OH2O

H2O

Results- DNA Assay

DNA

0.000.050.100.150.200.250.300.350.400.45

1Control Treated

*P< 0.05

DN

A (

µg)

/ (m

g) ti

ssue

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 [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

Results- Nuclear Estrogen Receptors

*P< 0.05

Nuclear Estrogen Receptors

0.00

1.00

2.00

3.00

4.00

5.00

6.00

1

Control Treated

E2 bo

und

(fm

ol)/

DN

A (

µg)

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

Methods-Glucose Oxidation 1. Tissue is placed into an Erlenmeyer flask containing MEM

and U-14C 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 14CO2 was determined by scintillation counter

Results- Glucose Oxidation

Glucose Oxidation

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

1Control Treated (E2)

*P< 0.05

14 C

O2(

cpm

)/D

NA

(µ

g)

Conclusion

Estrogen stimulates a nuclear-induced signal to cause the metabolism of glucose to CO2 in the ovine endometrium

E2

E2

Glucose

CO2

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