1. SUMMARY AND CONCLUSIONS
MATERIALS AND METHODS
INTRODUCTION
RESULTS
ABSTRACT
Background: Reproductive aging is characterized by
fluctuations in estrogen level followed by a marked decline
after menopause. Loss of estrogen is known to dysregulate
cellular metabolism which may contribute to development of
several neurological disorders.
Purpose: To investigate the role of estrogen on the activities of
metabolic enzymes in Frontal cortex (FC), Striatum (STR),
Medial Basal Hypothalamus (MBH) and Hippocampus (HC) of
middle- aged ovariectomized female Sprague-Dawley rats.
Methods: Ovariectomized female Sprague-Dawley rats were
implanted with 17β-estradiol (E2) 30- day release pellets (0.6
and 300 µg). At the end of the treatment period, Frontal Cortex
(FC), Striatum (STR), Medial Basal Hypothalamus (MBH) and
Hippocampus (HC) were isolated and examined for the
activities of Hexokinase, Pyruvate kinase, Citrate synthase,
Cytochrome C Oxidase and Na+/K+-ATPase.
Results: In FC, high dose of E2 increased the activities of
glycolytic enzymes and Na+/K+-ATPase while low dose
enhanced the activity of citrate synthase (CS) and cytochrome c
oxidase (COX). In STR, an increase in pyruvate kinase (PK)
activity was observed when treated with low dose, while both
doses of estrogen increased the activities of Na+/K+-ATPase
and CS but downregulated COX activity. Both doses of
estrogen treatment upregulated hexokinase activity and low
dose alone increased PK, Na+/K+-ATPase and COX activities in
MBH while high dose of estrogen treatment alone enhanced
PK and CS activities in HC.
Conclusion: E2 treatment in Sprague-Dawley rats was found to
have beneficial effects on cellular metabolism by upregulating
the activities of metabolic enzymes in different brain areas
whereby it could promote neuronal survival mechanisms.
Keywords: Ovariectomy, Estrogen, Brain, Metabolic enzymes.
0
0.02
0.04
0.06
0.08
Hexokinaseactivity/
min/mgprotein
FC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.02
0.04
0.06
0.08
Hexokinaseactivity/
min/mgprotein
STR Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
Differential modulation of activities of the metabolic enzymes by 17β-estradiol
in the brain areas of ovariectomized female Sprague-Dawley rats
Ramanathan Chockalingam, Murali Mahadevan Hariharan, Vijayarengamani Prasanna, Uday P. Pratap,
Lalgi Hima and Srinivasan ThyagaRajan
Integrative medicine laboratory, department of biotechnology, school of bioengineering, srm university, tamil nadu, india.
0
0.02
0.04
0.06
0.08
Hexokinaseactivity/
min/mgprotein
MBH Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.02
0.04
0.06
0.08
Hexokinaseactivity/
min/mgprotein
HC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
#
Fig.1: Effect of E2 on Hexokinase activity in FC, STR, MBH, HC.
Hexokinase activity in brain areas Pyruvate kinase activity in brain areas Citrate synthase activity in brain areas Cytochrome C Oxidase activity in brain areas Na+/K+-ATPase activity in brain areas
0
0.2
0.4
0.6
Pyruvatekinaseactivity/
min/mgprotein
FC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.2
0.4
0.6
Pyruvatekinaseactivity/
min/mgprotein
MBH Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
Fig.3: Effect of E2 on Citrate Synthase activity in FC, STR, MBH, HC. Fig.4: Effect of E2 on Cytochrome C Oxidase activity in FC, STR, MBH, HC.
0
0.2
0.4
0.6
0.8
CytochromeCOxidaseActivity/
min/mgprotein
FC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
CytochromeCOxidaseActivity/
min/mgprotein
HC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
0
4
8
12
16
Na+/K+Activity/min/mgprotein
STR Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
4
8
12
16
Na+/K+Activity/min/mgprotein
FC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
Fig.5: Effect of E2 on Na+/k+-ATPase activity in FC, STR, MBH, HC.Fig.2: Effect of E2 on Pyruvate kinase activity in FC, STR, MBH, HC.
0
4
8
12
16
Na+/K+Activity/min/mgprotein
MBH Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E 300 µg
§
Reproductive aging in women is associated with the fluctuations and a concomitant decrease in
the estrogen level in the post-menopausal period. The marked decline in estrogen affects the brain functions by
deteriorating cognition, memory, and behavior. These alterations contribute to the development of age-associated and
neurodegenerative disorders such as Alzheimer’s disease. At cellular level, with aging, the activities of the key metabolic
enzymes involved in energy homeostasis are altered. In central nervous system, dysregulation in metabolism disturb the
cellular homeostasis, rendering the cells susceptible to damage which ultimately results in neurodegeneration. We have
shown the neuroprotective role of estrogen treatment by augmenting the tyrosine hydroxylase expression and intracellular
signaling markers of cell survival pathways in different brain areas. In addition, estrogens have crucial role in regulating
the activities of key rate limiting enzymes such as hexokinase, pyruvate kinase, citrate synthase, cytochrome c oxidase and
Na+/K+-ATPase, thereby influencing the neuronal survival mechanisms.
Therefore, examining the effects of ovariectomy and estrogen treatment on the activities of the
key enzymes involved in energy metabolism pathways in brain areas may reveal its role in regulating neuronal survival
mechanisms.
The decline in the cellular metabolism in ovariectomized rats was found to be reversed by E2 treatment. These findings suggest that estrogen promotes cellular metabolism by upregulating the
activities of enzymes of glycolysis and oxidative phosphorylation. These beneficial effects of estrogen may provide potential treatment strategies for age-associated and neurodegenerative
diseases predominantly in females.
§
- Significantly (p<0.05) different with respect to young control,
# - Significantly (p<0.05) different with respect to sham-operated group,
§ - Significantly (p<0.05) different with respect to placebo-treated group.
§ - Significantly (p<0.05) different with respect to placebo-treated group. § - Significantly (p<0.05) different with respect to placebo-treated group. - Significantly (p<0.05) different with respect to young control,
§ - Significantly (p<0.05) different with respect to placebo-treated group.
§ - Significantly (p<0.05) different with respect to placebo-treated group.
10
0
2
4
6
8
10
CytochromeCOxidaseActivity/
min/mgprotein
MBH
Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
0
2
4
6
8
10
CytochromeCOxidaseActivity/
min/mgprotein
STR Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
0
4
8
12
16
Na+/K+Activity/min/mgprotein
HC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
0
0.2
0.4
0.6
Pyruvatekinaseactivity/
min/mgprotein
STR Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
0
0.2
0.4
0.6
Pyruvatekinaseactivity/
min/mgprotein
HC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.004
0.008
0.012
CitrateSynthaseactivity/
min/mgprotein
FC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.004
0.008
0.012
CitrateSynthaseactivity/
min/mgprotein
STR Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.004
0.008
0.012
CitrateSynthaseactivity/
min/mgprotein
MBH Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
0
0.004
0.008
0.012
CitrateSynthaseactivity/
min/mgprotein
HC Young
MA Sham
MA OVX + Placebo
MA OVX + E2 0.6 µg
MA OVX + E2 300 µg
§
SD Rats
Ovariectomy
&
Estrogen
Treatment
Brain
Isolation
Enzyme
Assays
Sprague-Dawley Rats
8 Months old
(n=8)
Female
E2 30-day release pellets
0.6 µg and 300 µg
Brain Isolation
Frontal Cortex
Striatum
Medial Basal Hypothalamus
Hippocampus
Hexokinase
Pyruvate kinase
Citrate Synthase
Cytochrome C Oxidase
Na+/K+-ATPase