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

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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)
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Increases hyperemia
Increases imbibition of water
Increases glucose oxidation and lipid synthesis
Increases RNA polymerase activity
Long term (6-48 hours after exposure)
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Continued stimulation of RNA polymerase activity
Increases DNA synthesis
Increases in cell hypertrophy and hyperplasia
BackgroundGlucose 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- continued
Glucose
*Glucose Transporter
GT-Glucose
hexokinase
G-6-P
Glycolysis
*Glucose-6-phosphate
dehydrogenase
Pentose phosphate
pathway
*Been shown to be
stimulated by estrogen
Hypothesis tested

Does estradiol (E2) stimulate nuclear-induced
signaling of glucose oxidation in the ovine
endometrium?
10 day treatment schedule of
ovariectomized ewes
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
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
Experiment # 1- DNA assay

Estradiol causes imbibition of water into cells
Control
E2 treated
H2O
H2O
H2O

H2O
H2O
Results for both the amount of estrogen receptors
and glucose metabolism are expressed as per µg
of DNA
Results- DNA Assay
DNA (µg)/ (mg) tissue
DNA
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
*P< 0.05
Control
1
Treated
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


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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
Nuclear Estrogen Receptors
6.00
E2 bound (fmol)/DNA (µg)
5.00
*P< 0.05
4.00
3.00
2.00
1.00
0.00
Control
1
Treated
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
700.00
14 CO (cpm)/DNA (µg)
2
600.00
*P< 0.05
500.00
400.00
300.00
200.00
100.00
0.00
Control
1
Treated (E2)
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
Acknowledgements
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
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Howard Hughes Medical Institute (HHMI)
Undergraduate Research Innovation Scholarship
and Creativity (URISC)
Dr. Fred Stormshak
Reed Reeve
Tari Tan
Jared Deitz
Cecily Bishop