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