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Bioenergetic fuel shift of reproductive aging: implication for late-onset Alzheimer's disease
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Bioenergetic fuel shift of reproductive aging: implication for late-onset Alzheimer's disease
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Content
BIOENERGETIC FUEL SHIFT OF REPRODUCTIVE AGING:
IMPLICATION FOR LATE-ONSET ALZHEIMER’S DISEASE
by
FAN DING
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2013
Copyright 2013 FAN DING
i
Dedications
I dedicate this dissertation to my beloved parents, Lianzhi Ding and Liyan Fan, who
valued education and encourage me to reach my personal goals. Although we are
separated by the Pacific Ocean, their love is always with me.
I dedicate my dissertation to my husband, Yang Li, who always stands by me and
supports me through all difficult times.
ii
Acknowledgement
First of all, I want to thank my mentor, Professor Roberta Diaz Brinton. I still
remember the first time we met, when she discussed the biomedical imaging with great
passion. This discussion built the foundation for my doctoral research and I became a
member in Brinton lab. Robbie’s inspiring thoughts, passion and dedication to science
will always be treasured in my life. During my PhD years, Robbie not only provided me
with an incredible research environment and great learning opportunities, but also offered
me unwavering support and trust in my research.
I also want to thank my committee members, Dr. Enrique Cadenas, Dr. Ronald
Alkana and Dr. Helena Chui. Dr. Cadenas is a world-renowned scientist in the field of
aging process and age-related degenerative disease. He is truly a gentleman that is always
willing to offer me help at any time. Dr. Ronald Alkana is always nice and encouraging.
Dr. Helena Chui is the top neurologist and she provided me with valuable suggestions
from the clinical research field.
I would like to thank my colleague Dr. Jia Yao for his tremendous help in my
doctoral research. I would also thank my colleagues and friends in USC for their support,
especially Dr. Shuhua Chen, Dr. Liqin Zhao, Dr. Ronald Irwin, Jennifer Mao, Karren
Wong, Jamaica Rettberg, Lauren Klosinski, Christine Solinsky, Eric Hernandes, Jimmy
To, Claudia Lopez, Kathleen Cho, Dr. Hui Shen and Dr. Yujiao Sun.
iii
Table of Contents
Dedications i!
Acknowledgement ii!
Table of Contents iii!
List of Figures v!
Abbreviations vii!
Abstract viii!
Chapter I. Overview of female brain aging, ovarian hormone loss, brain metabolism and
Alzheimer’s disease 1!
Introduction 1!
Significance 9!
Specific Aims 10!
Chapter I References 11!
Chapter II. Early decline in glucose metabolism, transport and glycolytic capacity are
accompanied by a shift to alternative fuel transporters in the aging normal and
Alzheimer’s female mouse brain 19!
Abstract 19!
Introduction 21!
Materials and Methods 24!
Results 28!
Discussion 42!
Acknowledgements 53!
Chapter II References 54!
Chapter III. Ovariectomy induces a shift in fuel availability and metabolism in the
hippocampus of the female transgenic model of familial Alzheimer’s 60!
Abstract 60!
Introduction 61!
Materials and Methods 64!
Results 69!
Discussion 83!
Chapter III References 90!
Chapter IV. Ovariectomy induced a decline in brain glucose uptake associated with
peripheral glucose intolerance 97!
Abstract 97!
iv
Introduction 98!
Materials and Methods 100!
Results 106!
Discussion 115!
Chapter IV References 119!
Chapter V. Conclusions 125!
Chapter V References 129!
Complete References 131!
v
List of Figures
Figure I-1. Schematic of brain glucose utilization and bioenergetic fuel shift 6
Figure I- 2. Estrogen mediated neuroprotective action 8
Figure I- 3. Overview of specific aims 10
Figure II - 1. Decreased brain glucose uptake and glucose transporter expressions at
early stage of female aging 31
Figure II- 2. Decreased hexokinase activity at early stage in female aging 34
Figure II- 3. Increase in the phosphorylation of PDH at early stage of female aging 35
Figure II- 4. Reproductive transition paralleled a significant decrease in LDH5 and
LDH1 expressions in nonTg hippocampus 38
Figure II-5. Plasma β-hydroxybutyrate level and its neuronal transporter increase
with reproductive transition 41
Figure II- 6. Timeline of bioenergetic aging in female mammalian brain 52
Figure III-1. Experimental paradigm 65
Figure III-2 Ovariectomy (OVX) induced increase in body weight and tail skin
temperature and prevention by 17β-estradiol 70
Figure III-3. Ovariectomy (OVX) induced decline in brain glucose uptake and BBB
GLUT1
55Kda
expression: prevention by 17β-estradiol 73
Figure III-4. Ovariectomy (OVX) induced decrease in hexokinase expression and
activity: prevention by 17β-estradiol 75
Figure III-5. Ovariectomy (OVX) induced a shift in LDH1 and LDH5 ratio and
prevention by 17β-estradiol 77
Figure III-6. Ovariectomy (OVX) induced activation of ketogenic pathway and
prevention by 17β-estradiol 79
Figure III-7. Ovariectomy (OVX) induced increase in β-amyloid level: prevention by
17β-estradiol 80
Figure III-8. E2 treatment significantly regulated Bioenergetic gene expression 82
vi
Figure III-9. Impact of ovariectomy on bioenergetic system of 3xTgAD female brain 83
Figure IV-1. Experimental paradigm 101
Figure IV-2. Ovariectomy (OVX) induced increase in body weight and tail skin
temperature and prevention by 17β-estradiol 107
Figure IV- 3. Ovariectomy (OVX) induced glucose intolerance and prevention by
17β-estradiol 108
Figure IV- 4. Ovariectomy (OVX) induced a decline in brain glucose uptake:
prevention by 17β-estradiol 109
Figure IV- 5. Ovariectomy (OVX) induced a decline in neuronal GLUT4 and glial
GLUT1
45Kda
expression, prevention by 17β-estradiol 110
Figure IV- 6. No change in the LDH5 and LDH1 expression and the ratio of
LDH5/LDH1 112
Figure IV-7. Ovariectomy (OVX) induced an increase in serum β-hydroxybutyrate
concentration: prevention by 17β-estradiol 113
Figure IV- 8. E2 treatment significantly regulated Bioenergetic gene expression 114
Figure V-1. Timeline of bioenergetic aging in female mammalian brain 126
Figure V-2. Summary of changes in the expression level of key proteins involved in
glucose transport, glycolysis, alternative fuel transport and utilization in different
conditions. 128!
vii
Abbreviations
2-[
18
F]fluoro-2-deoxy-D-glucose FDG
17β-estradiol E2
Adenosine 5’-triphosphate ATP
Alzheimer’s disease AD
Amyloid precursor protein APP
Apolipoprotein E-ε4 APOE-ε4
Blood-brain-barrier BBB
Cache County Study CCS
Cerebral glucose metabolic rate CMRglu
Cytochrome c oxidase COX
Electron transport chain ETC
Familial AD FAD
Glucose transporter GLUT
Hydroxyacyl-Coenzyme A Dehydrogenase/
3-ketoacyl-Coenzyme A Thiolase/
Enoyl-Coenzyme A Hydratase (trifunctional protein), alpha subunit HADHA
Kilo/Milli/Micro gram kg/mg/µg
Kinmen Women-Health Investigation KIWI
Lactate dehydrogenase LDH
Mild cognitive impairment MCI
Milli/Micro molar mM/µM
Monocarboxylate transporter MCT
Non-transgenic wild-type nonTg
Ovariectomy/ovariectomized OVX
Posterior cingulate cortex PCC
Positron emission tomography PET
Presenilin 1 PSEN1
Presenilin 2 PSEN2
Pyruvate dehydrogenase PDH
Seattle Midlife Women’s Health Study SMWHS
Sporadic AD SAD
Study of Women’s Health Across the Nation SWAN
Subcutaneously SC
Succinyl-CoA:3-ketoacid Co Atransferase SCOT
Tail skin temperature TST
Taqman low-density arrays TLDA
Tricarboxylic acid cycle TCA
Triple-transgenic Alzheimer’s 3xTgAD
Women's Health Initiative Memory Study WHIMS
viii
Abstract
Previously, our lab have investigated the role of mitochondria in female brain
aging and Alzheimer’s disease. We demonstrated that mitochondrial bioenergetic deficit
precedes Alzheimer’s pathology. Further, reproductive senescence or ovariectomy
induced ovarian hormone loss exacerbates mitochondrial deficit, which can be prevented
by 17β-estradiol (E2) treatment. The purpose of the current project is 1) to characterize
the changes in brain fuel availability during female aging and reproductive transition; 2)
to determine the temporal relationship between changes in substrate availability and
mitochondrial deficits; 3) to investigate the mechanisms underlying such changes in brain
substrate fuel systems. We designed the following specific aims to address these
questions. Specific aim I (Chapter 2) seeks to characterize the female brain aging-
associated alteration in fuel availability in both female non-transgenic wild-type (nonTg)
and triple-transgenic Alzheimer’s (3xTgAD) brains. Specific aim II (Chapter 3) serves to
investigate the impact of ovarian hormone loss and E2 prevention on fuel availability and
bioenergetic fuel shift in 3xTgAD mouse model. Specific aim III (Chapter 4) serves to
determine the impact of ovarian hormone loss and E2 prevention on fuel availability and
the compensatory mechanism from a systems-biology perspective.
Data from this project demonstrated that: 1) the female brain aging associated
decline in glucose availability precedes the development of mitochondrial deficits and is
accompanied by the activation of the bioenergetic fuel shift. 2) Ovarian hormone loss
induces a decline in brain glucose availability and a shift towards utilization of alternative
fuel in 3xTgAD female brain, which can be partially prevented by E2 treatment. 3)
ix
Ovarian hormone loss induces dysregulated glucose homeostasis and decreased glucose
availability in female nonTg brain, which can be prevented by E2 treatment.
Mechanistically, female brain aging-associated decline in brain glucose uptake
was induced by a decline in neuronal glucose transporter (GLUT3) expression,
hexokinase activity and rise in phosphorylated pyruvate dehydrogenase. In nonTg brain,
the decline in glucose metabolism induced a rise in plasma β-hydroxybutyrate and
expression of monocarboxylate transporters in astrocytes and neurons. This activation of
ketogenic pathway occurred at an earlier age in 3xTgAD brain, which showed an
exacerbated bioenergetic deficit in alternative fuel supply/transport during reproductive
transition. Further, in 3xTgAD brain, ovariectomy-induced decline in brain glucose
uptake was attributed to the decline in blood-brain barrier glucose transporter expression,
hexokinase expression and activity. The decline in glucose metabolism activated the
expression of proteins required for lactate generation (lactate dehydrogenase 5) and
utilization (lactate dehydrogenase 1) as well as ketone body transport and utilization. In
nonTg brain, the ovariectomy-induced decline in brain glucose uptake is associated with
peripheral glucose intolerance.
Data from these studies expand our understanding of brain bioenergetic deficits in
female brain aging as well as the protective role of estrogen treatment. By elucidating the
trajectory of bioenergetic decline during female brain aging, the current study provides
preclinical evidence for understanding a “window of opportunity” in estrogen therapy. By
understanding the change in fuel availability, which is associated with ovarian hormone
x
dysregulation induced by female aging or ovariectomy, our research also provides new
information in early diagnosis and prevention of AD in menopausal women.
1
Chapter I. Overview of female brain aging, ovarian hormone loss, brain
metabolism and Alzheimer’s disease
Introduction
Previously we demonstrated that female brain aging resulted in a significant
decline in aerobic glycolysis and mitochondrial oxidative phosphorylation, which could
be attributed to decreased activities of bioenergetic enzymes, such as PDH and Complex
IV (cytochrome c oxidase). The decline in mitochondrial bioenergetics was exacerbated
in the female triple transgenic Alzheimer’s (3xTgAD) mice and preceded the
development of β-amyloid deposition in hippocampus. The decline in Complex IV
activity was accompanied by a significant increase in enzymes required for ketone body
utilization (SCOT) and long-chain fatty acid metabolism (HADHA), indicative of
compensatory fuel utilization. Further, the glucose metabolism associated bioenergetic
changes observed in the natural female brain aging was recapitulated in ovariectomy-
induced menopause. The current study builds on and expands our knowledge of the
impact of female brain aging and ovarian hormones on the brain bioenergetics. In the
current study, we sought to determine 1) the impact of female brain aging on fuel
availability and bioenergetic fuel shift in normal female non-transgenic brain and female
triple transgenic Alzheimer’s (3xTgAD) brain; 2) the impact of ovarian hormone
deprivation on fuel availability and bioenergetic fuel shift in female 3xTgAD brain; and 3)
the impact of ovarian hormone deprivation on brain fuel availability and the
compensatory mechanism in nonTg female brain.
2
Alzheimer’s disease – genetics and risk factors
Alzheimer’s disease (AD) is the most common type of dementia, symptomized by
loss of memory, apathy, depression, impaired judgment, behavioral and personality
change (Alzheimer’s Association, 2012). Pathologically, AD is characterized by the
presence of senile plaques, neurofibrillary tangles (NFTs), synaptic damage and cell
death in the brain (Blennow et al., 2006; Nakamura et al., 2012; Reddy et al., 2012).
Based on the genetics of the disease, AD can be categorized in to familial AD (FAD) and
sporadic AD (SAD). FAD, which only composes less than one percent of the AD
population, is mainly caused by mutations in three genes, including amyloid precursor
protein (APP), presenilin 1 (PSEN1) and/or presenilin 2 (PSEN2) (Alzheimer’s
Association, 2012; Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1995;
Tabaton and Tamagno, 2007; Tanzi et al., 1992). SAD, which represents the majority of
the AD population, is associated with multiple risk factors including aging, gender,
family history, apolipoprotein E-ε4 (APOE-ε4), mild cognitive impairment (MCI) and
cardiovascular disease risk factors. Advanced age is the greatest risk factor, as the
prevalence of dementia increases exponentially in elderly population (>65 years) and
continues to increase in population of more advanced age (> 95 years) (Alzheimer’s
Association, 2012; Plassman et al., 2007; von Strauss et al., 1999). Epidemiology study
also revealed that gender is important risk factor as the overall prevalence of AD is much
higher in women (Bachman et al., 1992; 1994; Launer et al., 1999; Plassman et al., 2007).
3
Brain metabolism, mitochondrial function and oxidative stress in Alzheimer’s disease
Brain glucose hypometabolism is an early and consistent event in Alzheimer’s
disease (Friedland et al., 1983; Minoshima et al., 1997; Mosconi, 2005; Mosconi et al.,
2007; Mosconi et al., 2009b; Mosconi et al., 2006; Reiman, 2007; Reiman et al., 1996).
Data emerging from clinical positron emission tomography (PET) with 2-[
18
F]fluoro-2-
deoxy-D-glucose (FDG-PET) analyses demonstrated a progressive reduction in cerebral
glucose metabolic rate (CMRglu), particularly in posterior cingulate (PCC) and parietal-
temporal cortex, in patients with Alzheimer’s disease (AD) (Friedland et al., 1983;
Minoshima et al., 1997). Further clinical studies indicated that brain glucose
hypometabolism temporally preceded and prospectively predicted the development of
AD. For example, in persons with increased risk of AD, such as APOE-ε4 carrier or
maternal family history descendants, the decline in CMRglu in PCC and parietal-
temporal cortex occurred before the onset of AD. (Mosconi et al., 2009a; Reiman et al.,
1996; Reiman et al., 2004). Clinical imaging also revealed a spatial correlation between
increased aerobic glycolysis and β-amyloid deposition in the “default mode network”
brain areas, suggesting that deficits in energy supply may underlie the vulnerability to the
AD pathogenic process in such areas (Vaishnavi et al., 2010; Vlassenko et al., 2010).
Although the molecular mechanism underlying the energy deficits in AD brain is still
under investigation, current data suggested that the energy deficits is induced by a
multifaceted process including 1) decreased glucose transport, 2) compromised glycolytic
capacity and 3) impaired mitochondrial function and oxidative stress (Brinton, 2008; Harr
et al., 1995; Horwood and Davies, 1994; Mattson, 2004; Piert et al., 1996; Vannucci et
al., 1998).
4
The pivotal role of mitochondria dysfunction in Alzheimer’s disease has been
well established in human beings and preclinical animal models (Brinton, 2008; Mattson,
2004; Swerdlow, 2009). Impaired mitochondrial function is not only associated with
diminished energy metabolism, but also closely related to disrupted redox homeostasis.
Mitochondria provide most of the cellular energy for proper functioning of brain cells
through oxidative phosphorylation (OXPHOS). During the process of OXPHOS, electron
leakage can occur and generate superoxide anion (O
2
!−
), which is further converted to
hydrogen peroxide (H
2
O
2
) (Melov, 2000). H
2
O
2
impairs OXPHOS efficiency and induces
increased oxidative stress, which is characteristic of AD brain (Atamna and Frey, 2007;
Dumont et al., 2010; Gibson and Shi, 2010; Lin and Beal, 2006)
While there is abundant evidence that mitochondrial dysfunction is crucial in AD,
little attention is given to the role of glucose transporter and glycolytic capacity. A few
studies reported decreased expressions of brain glucose transporters, including blood-
brain-barrier glucose transporter 1 (GLUT1) and neuronal glucose transporter 3
(GLUT3), as well as decreased activity of glycolytic enzymes, such as
phosphofructokinase, in the brain autopsy samples from AD patients (Bigl et al., 1999;
Bowen et al., 1979; Harr et al., 1995; Horwood and Davies, 1994).
Utilization of alternative fuels in the brain
The decline in brain glucose metabolism could activate alternative metabolic
pathways. Under metabolically challenging conditions, the brain can utilize
monocarboxylates such as lactate and ketone bodies as alternative fuel sources (Belanger
et al., 2011; Pellerin, 2003; Pellerin et al., 1998; Yao et al., 2010). Lactate is a well
5
described bioenergetic fuel supplied by astrocytes to neurons and is particularly
important under conditions of high synaptic activity, such as occurs during learning and
memory (Suzuki et al., 2011). Lactate can also serve as an auxiliary fuel to neurons
during glucose insufficiency by metabolism of glycogen stores to generate glucose and
subsequently lactate (Aubert et al., 2005). Besides serving as a compensatory fuel for
neurons, lactate is suggested to be utilized by oligodendrocytes for lipid/myelin synthesis
(Rinholm et al., 2011) (Figure I-1). Lactate generation and utilization depend on two
isoforms of lactate dehydrogenase (LDH): lactate dehydrogenase 5 (LDH5) that converts
pyruvate to lactate in glial cells and lactate dehydrogenase 1 (LDH1) that converts lactate
to pyruvate in neurons (Laughton et al., 2007; Venkov et al., 1976) (Figure I-1). The shift
in LDH5/LDH1 ratio can be indicative of elevated lactate level, which is consistent with
an aging phenotype in mouse and human brain (Ross et al., 2010; Yesavage et al., 1982).
Under conditions of prolonged glucose deprivation, the brain shifts to utilization
of ketone bodies, such as acetone, acetoacetic acid and β-hydroxybutyric acid, as the
alternative fuel (Costantini et al., 2008; Guzman and Blazquez, 2004; Morris, 2005).
Ketone bodies are produced by the liver or in neighboring astrocytes and can be
converted to acetyl-CoA by 3-oxoacid-CoA transferase (SCOT) to enter into citric acid
cycle (Figure I-1).
Clinical studies demonstrated that in people with AD, compromised brain glucose
metabolism is accompanied by parallel activation of alternative metabolic pathways, as
evidenced by a utilization ratio of 2:1 glucose to alternative substrate in persons with
incipient AD compared to a ratio of 29:1 in healthy elderly controls (Hoyer, 1991).
6
Figure I-1. Schematic of brain glucose utilization and bioenergetic fuel shift (adapted from
Rinholm et al., 2011, Pellerin, 2003 and Yao et al., 2011b). Glucose is transported through blood-
brain-barrier glucose transporter 1 (GLUT1
55Kda
). Glucose is transported into neurons by glucose
transporter 3 (GLUT3), phosphorylated by hexokinase and further metabolized into pyruvate.
Pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase to enter into TCA cycle to generate
ATP. Glucose is transported into astrocyte by glucose transporter 1 (GLUT1
45Kda
) and phosphorylated
by hexokinase to generate pyruvate, which can be converted to lactate by lactate dehydrogenase 5
(LDH5). Glucose is transported into oligodendrocyte to generate ATP or serve as carbon skeleton in
lipid/myelin synthesis. The alternative fuels, lactate or ketone bodies, are transported through blood-
brain-barrier monocarboxylate transporter 1(MCT1). Lactate generated by astrocyte is transported by
MCT1 or MCT4. Lactate and ketone bodies are transported by MCT2 into neuron, where lactate is
converted to pyruvate by lactate dehydrogenase 1 (LDH1) and ketone bodies is converted to acetyl-
CoA by 3-oxoacid-CoA transferase (SCOT) to generate ATP. In oligodendrocyte, lactate can be
transported by MCT1 and is suggested to serve important role in energy production and lipid/myelin
synthesis.
Ovarian hormone regulation of brain bioenergetic and therapeutic potential of
estrogen-induced neuroprotection
7
Bcientific analyses revealed that estrogen imposes a regulation of brain glucose
metabolism by promoting brain glucose uptake, glycolysis, glycolytic-coupled
tricarboxylic acid cycle (TCA) function, mitochondrial respiration and ATP generation
(Brinton, 2008; Nilsen et al., 2007; Yao et al., 2011a). From a translational perspective,
these basic science findings are supported by clinical analyses of glucose metabolism in
menopausal women. Postmenopausal women on estrogen therapy were reported to have
increased cerebral blood flow and cerebral metabolism relative to non-users while non-
users exhibited a significant decline in glucose metabolic rate, particularly in the
posterior cingulate and prefrontal cortex, which closely resembled the hypometabolic
profile of AD brains (Rasgon et al., 2005). Collectively, both preclinical analyses in
animal models and clinical observations provide compelling evidence in support of
decline in bioenergetic function in brain as an early indicator of neurodegenerative risk.
Multiple clinical studies demonstrated that menopause, both natural and
ovariectomy-induced, was associated with increased AD risk (Fuh et al., 2006; Greendale
et al., 2009; Rocca et al., 2007; Rocca et al., 2010; Sullivan Mitchell and Fugate Woods,
2001). Clinical studies of naturally menopausal women, such as Seattle Midlife Women’s
Health Study (SMWHS), Women’s Health Across the Nation (SWAN) and Kinmen
Women-Health Investigation (KIWI), reported cognitive decline in perimenopausal
women (Fuh et al., 2006; Greendale et al., 2009; Sullivan Mitchell and Fugate Woods,
2001). Besides natural menopause, surgically induced menopause is also associated with
increased risk of AD (Rocca et al., 2007; Rocca et al., 2010). In the Mayo Clinic Cohort
Study of Oophorectomy and Aging, women underwent either unilateral or bilateral
oophorectomy showed an increased risk of dementia (Rocca et al., 2007). Together, these
8
results indicated a close association between increased risk of AD and ovarian hormone
dysregulation or deprivation during natural menopause and surgically induced
menopause.
Estrogen is a well-known neuroprotective agent that protect against neuronal
injuries and damages. Preclinical studies revealed that estrogen-mediated neuroprotective
effect through multiple mechanisms including 1) regulation of mitochondrial
bioenergetics, 2) protection against oxidative stress, 3) protection against apoptosis
(Figure I-2), 4) increase neurogenesis and 5) regulation of neurotransmitter systems
(Barha and Galea, 2010; Brinton, 2008; Lee and McEwen, 2001; Luine, 1985; McEwen,
2002; Nilsen and Diaz Brinton, 2003; Nilsen et al., 2007; Pike, 1999; Yao et al., 2010;
Yao et al., 2011a; Yao et al., 2009).
Figure I- 2. Estrogen mediated neuroprotective action (Brinton, 2008). Estrogen mechanisms of
action converge upon the mitochondria. Estrogen (17 -estradiol; E2) binding to a membrane-
associated estrogen receptor (ER) undergoes a protein protein interaction with the regulatory subunit
9
of PI3K, p85, to activate the divergent but coordinated activation of the Akt and MAPk signaling
cascades. These E2-induced signaling pathways in hippocampal and cortical neurons converge upon
the mitochondria to enhance glucose uptake and metabolism, aerobic glycolysis and pyruvate
dehydrogenase to couple aerobic glycolysis to acetyl-CoA production and tricarboxylic acid cycle
(TCA) -coupled oxidative phosphorylation and ATP generation. In parallel, E2 increases antioxidant
defense andI-2 antiapoptotic mechanisms. Estrogen receptors at the membrane, in mitochondria and
within the nucleus are well positioned to regulate coordinated mitochondrial and nuclear gene
expression required for optimal bioenergetics. Enhancing and sustaining glycolysis, aerobic
metabolism and mitochondrial function would be predicted to prevent the shift to alternative fuel
sources and the hypometabolism characteristic of Alzheimer's disease
Clinical studies revealed that the effect of estrogen neuroprotection in women is
controversial, possibly due to the timing of hormone therapy. Results from the Mayo
Clinic Cohort Study of Oophorectomy and Aging indicated that hormone therapy reduced
risk of dementia in women underwent oophorectomy before menopause (Rocca et al.,
2007). Cache County Study (CCS) demonstrated that hormone therapy reduced AD risk
in postmenopausal women who had undergone the therapy within 5 years of menopause
(Shao et al., 2012). However, results from both CCS and Women's Health Initiative
Memory Study (WHIMS) showed that hormone therapy had no neuroprotective effect
and even increased AD risk in postmenopausal women who initiated the hormone therapy
after 5 years of menopause (Espeland et al., 2004; Shao et al., 2012).
Significance
Previously, our lab has elucidated the role of mitochondria in female brain aging
and Alzheimer’s disease. Data from the current study provide valuable insights in 1) the
trajectory of bioenergetic decline associated with female brain aging and ovarian
hormone loss; 2) the alternative fuel utilization in response to decreased brain
10
bioenergetics; and 3) the role of 17β-estradiol in preventing the decline in brain
bioenergetics associated with ovarian hormone loss.
By elucidating the trajectory of bioenergetic decline during female brain aging,
the current study provides preclinical evidence for improved understanding of the timing
issues of estrogen therapy, which is proposed to be associated with the controversial
effect of estrogen-induced neuroprotection in menopausal women. Further, findings from
the current study also provide new information for early diagnosis and prevention of AD
in menopausal women.
Specific Aims
Figure I- 3. Overview of specific aims. Specific aim I in Chapter II characterize the female brain
aging associated alteration in fuel availability in both female nonTg and 3xTgAD brains. Specific aim
II in Chapter III investigates the impact of ovarian hormone loss and prevention of E2 treatment on
fuel availability and bioenergetic fuel shift in 3xTgAD female brain. Specific aim III in Chapter IV
determines the impact of ovarian hormone loss and prevention of E2 treatment on fuel availability and
the compensatory mechanism in nonTg female brain.
In the current study, we sought to determine the impact of female brain aging and
ovarian hormone loss on brain bioenergetics in our preclinical animal models. Specific
aim I sought to characterize the female brain aging associated alteration in fuel
availability in both female nonTg and 3xTgAD brains. In order to investigate whether
11
these changes are induced by ovarian hormone dysregulation and can be prevented by
estrogen treatment, we further utilized ovariectomized mouse models. In specific aim II,
we investigated the impact of ovarian hormone loss and prevention of 17β-estradiol
treatment on fuel availability and bioenergetic fuel shift in female 3xTgAD brain. In
specific aim III, we determined the impact of ovarian hormone loss and prevention of
17β-estradiol treatment on fuel availability and the compensatory mechanism in nonTg
female brain (Figure I-3).
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19
Chapter II. Early decline in glucose metabolism, transport and
glycolytic capacity are accompanied by a shift to alternative fuel
transporters in the aging normal and Alzheimer’s female mouse brain
Fan Ding
1
, Jia Yao
1
, Jamaica R. Rettberg
2
, Shuhua Chen
1
, Roberta Diaz Brinton
1, 2*
1 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy,
University of Southern California, Los Angeles, California, United States of America,
2 Department of Neurology, Keck School of Medicine, University of Southern
California, Los Angeles, California, United States of America
* Email: rbrinton@usc.edu
Abstract
We previously demonstrated that during reproductive senescence, female non-
transgenic wild-type (nonTg) mice developed brain mitochondrial bioenergetic deficits,
which were exacerbated in triple-transgenic Alzheimer’s (3xTgAD) mouse. Further, the
decline in mitochondrial bioenergetics was accompanied by a shift towards a ketogenic
phenotype. Herein, we determined the impact of aging on systems of fuel supply,
transport and metabolic enzyme expression/activity. In nonTg-female brain, a significant
age-related decline in brain glucose uptake, assessed by microFDG-PET, occurred at 6-9
months and was sustained at decreased level thereafter. Mechanistic analyses indicated a
concomitant decline in neuronal glucose transporter (GLUT3) expression, hexokinase
20
activity and rise in phosphorylated / inactivated pyruvate dehydrogenase. Decline in
enzymes required for generation and utilization of lactate paralleled the decline in
glucose metabolism. Deficit in brain glucose metabolism was accompanied by a rise in
plasma ketone β-hydroxybutyrate and expression of monocarboxylate transporters
(MCTs) in astrocytes (MCT4) and neurons (MCT2). The adaptive transport of externally
derived alternative fuels was temporary, evidenced by a decreasing trend of blood-brain-
barrier (BBB)/glial MCT1 expression after 9-month. The 3xTgAD female demonstrated a
similar decline in brain glucose uptake, transport and glycolytic capacity but a different
profile in the adaptive response. Neither glial nor neuronal lactate dehydrogenase
expression changed with age. Activation of ketogenic pathway occurred at an earlier age
in the 3xTgAD female, evidenced by high plasma β-hydroxybutyrate level at 3-month,
which decreased at 6-9 months but rebounded at 12-15 months. 3xTgAD brain showed an
exacerbated bioenergetic deficit in alternative fuel supply/transport, indicated by
significantly decreased expression of BBB/glial MCT1 and astrocytic MCT4.
Collectively, these results indicate that during female brain aging, nonTg and 3xTgAD
brain differed in the adaptive response, although both genotypes showed compromised
glucose transport and glycolytic capacity preceding mitochondrial dysfunction, which
were coincident with compensatory activation of the ketogenic pathway.
21
Introduction
Previously we demonstrated that loss of ovarian hormones as well as the female
brain aging coincident with reproductive senescence were associated with a significant
decline in aerobic glycolysis and mitochondrial oxidative phosphorylation as well as
decreased activities of key bioenergetic enzymes, pyruvate dehydrogenase (PDH) and
Complex IV cytochrome c oxidase (COX) (Nilsen et al., 2007; Yao et al., 2010; Yao et
al., 2011a; Yao et al., 2009). The decline in mitochondrial bioenergetics was exacerbated
in the female triple transgenic Alzheimer’s (3xTgAD) mice and preceded the
development of β-amyloid deposition in hippocampus (Yao et al., 2009). During the
transition to reproductive senescence, COX activity decreased by 40% in both the nonTg
and 3xTgAD brains and is predictive of a decline in ATP generation (Yao et al., 2010).
The decline in PDH activity was accompanied by a significant increase in enzymes
required for ketone body utilization (SCOT) and long-chain fatty acid metabolism
(HADHA), which are indicative of compensatory fuel utilization (Yao et al., 2010).
These bioenergetic changes observed during age of reproductive senescence were
recapitulated in the ovariectomized rodent model of human menopause (Yao et al.,
2011a). Consistent with basic science findings, data emerging from clinical positron
emission tomography with 2-[
18
F]fluoro-2-deoxy-D-glucose (FDG-PET) analyses
demonstrate a significant decline in cerebral glucose metabolic rate (CMRglu) in the
posterior cingulate (PCC) in postmenopausal women (Rasgon et al., 2005).
Glucose hypometabolism and a shift to alternative substrates have been identified
as a metabolic phenotype characteristic of the Alzheimer s brain (Hoyer, 1991; Mosconi,
2005; Mosconi et al., 2009b; Mosconi et al., 2006; Reiman et al., 1996; Reiman et al.,
22
2004). FDG-PET imaging analyses have revealed a significant decline in CMRglu,
particularly in posterior cingulate (PCC) and parietal-temporal cortex, in persons with
Alzheimer s disease as well as those at increased risk for AD (Mosconi, 2005; Mosconi et
al., 2009b; Mosconi et al., 2006; Reiman et al., 1996; Reiman et al., 2004). Multiple
FDG-PET imaging studies in neurologically normal persons who are at risk for AD,
persons with an ApoE4 genotype (Reiman et al., 1996; Reiman et al., 2004) and persons
with maternal history of AD (Mosconi et al., 2007), present with significant
hypometabolism in brain prior to development of pathology. Further, in persons with AD,
brain glucose hypometabolism is accompanied by the activation of alternative metabolic
pathways, as evidenced by a utilization ratio of 2:1 glucose to alternative substrate in
persons with incipient AD compared to a ratio of 29:1 in healthy elderly controls (Hoyer,
1991).
As described above, our previous findings indicated a significant decline in the
bioenergetic system of the female brain that coincident with its transition through
reproductive senescence. Human reproductive senescence is characterized by three
stages, perimenopause, menopause and postmenopause (Brinton, 2010; Diaz Brinton,
2012). The reproductive transition in rodents occurs within a well-defined time frame and
shares multiple features and endocrine changes found in human perimenopause such as a
decline in follicles, irregular cycling and irregular fertility (Diaz Brinton, 2012; Finch et
al., 1984; Van Kempen et al., 2011). Cycle irregularity is a well-characterized indicator
for the onset of reproductive transition in both rodents and humans (Diaz Brinton, 2012;
Finch et al., 1984; Van Kempen et al., 2011). Previous reports identified that cycling
23
irregularity occurs in female mice between 7 and 12 months of age as evidenced by
change in both cycle frequency and length (Nelson et al., 1982).
Based on our earlier findings that the female aging brain developed deficits in
aerobic glycolysis and mitochondrial respiration during reproductive senescence that
were followed by increased expression of enzymes required for long-chain fatty acid
(HADHA) and ketone body (SCOT) metabolism (Yao et al., 2010; Yao et al., 2009), we
investigated the expression of substrate transporters during the reproductive senescence
transition. Specifically, we determined whether age-related decline in the bioenergetic
system of the brain was associated with changes in glucose uptake, substrate (glucose,
lactate/ketone body) transporter expression and/or enzyme systems required for
glycolysis. Further, we sought to determine whether there was a shift to the utilization of
alternative fuels, the adaptive mechanism in response to the decline in brain
bioenergetics. Outcomes of the current study indicate an early and significant impact of
female brain aging on the substrate transporter systems of the brain, which appear to be
initiating events that lead to dysfunctional bioenergetic system in brain.
24
Materials and Methods
Animal Treatments and Ethics
All rodent experiments were performed following National Institutes of Health
guidelines on use of laboratory animals and an approved protocol (protocol number:
10217) by the University of Southern California Institutional Animal Care and Use
Committee. The presented study has been approved by the University of Southern
California Institutional Animal Care and Use Committee (Ethics Committee).
Transgenic mice
Colonies of the 3xTgAD mice strain (129S; Gift from Dr. Frank Laferla,
University of California, Irvine) (Oddo et al., 2003) were bred and maintained at the
University of Southern California (Los Angeles, CA) following National Institutes of
Health guidelines on use of laboratory animals and an approved protocol by the
University of Southern California Institutional Animal Care and Use Committee. Mice
were housed on 12 hours light/dark cycles and provided ad libitum access to food and
water. The characterization of amyloid and tau pathologies, as well as synaptic
dysfunction in this line of mice has been described previously (Oddo et al., 2003) and
confirmed in our laboratory. Mice were genotyped routinely to confirm the purity of the
colony. To ensure the stability of AD-like phenotype in the 3xTgAD mouse colony, we
performed routine immunohistochemical assays every 3 to 4 generations. Only offspring
from breeders that exhibit stable AD pathology were randomized into the study. Only
intact female mice at the age of 3, 6, 9, 12 and 15 months were used for the experiment.
microFDG-PET and microCT imaging
25
Mice were maintained under anesthesia during microPET and microCT scans
with 2-2.5% isoflurane in oxygen. Scans were performed in an imaging chamber
equipped with a nose cone for anesthesia delivery and heating pad for body temperature
control. MicroPET imaging was performed with a microPET R4 rodent model scanner
(Concorde Microsystems Inc, Knoxville, TN) and micro CT imaging was performed on
MicroCAT II tomography (Siemens Preclinical Solutions, Knoxville, TN). Mice were
injected intravenously via the tail vein with radiotracer [
18
F] Fluoro-2-deoxy-2-D-
glucose (FDG, 200 Ci, 100uL). Radioactive dose was determined prior to injection by
radioisotope dose calibrator (Capintec, CRC-712M). At 40min post-injection of FDG,
each mouse was positioned in the MicroPET scanner in the center of the 10.8cm
transaxial and 8cm axial field of view (FOV). Brain microPET data were collected for
10min followed by a 10 min microCT scan for the purpose of co-registration.
Co-registration of microPET and microCT data was performed using the AMIDE
software package (http://amide.sourceforge.net/). After co-registration of the PET and CT
images, ROI (region of interest) was defined and used to measure the radioactivity
concentration in brains. Decay correction was used to adjust the actual radioactivity
dosage injected (Actual radioactivity dosage at time of injection = Initial radioactivity ×
!
!!"!×
!
!!"
e
!!"!×
!
!!"
, T = T minutes between injected time point and initial time point).
Brain tissue preparation and Western blot analysis
Upon completion of FDG-MicroPET imaging, mice were sacrificed and the
brains rapidly dissected on ice. Hippocampus was processed for protein extraction using
Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) with
phosphatase and protease inhibitors (Sigma, St. Louis, MO, USA), and protein
26
concentrations were determined with the Bio-Rad Bradford assay. Equal amounts of
protein (20mg/well) were loaded in each well of a 12.5% SDS PAGE criterion gel (Bio-
Rad, Hercules, CA) and electrophoresed with Tris/glycine running buffer. Proteins were
transferred to 0.45mm pore size polyvinylidene difluoride (PVDF) membranes and
immuneblotted with GLUT1 (glucose transporter 1) antibody (1:1500, Abcam,
Cambridge, MA, USA), GLUT3 (glucose transporter 3) antibody (1:1000, Abcam,
Cambridge, MA, USA), Hexokinase II antibody (1:1000, Millipore, Billerica, MD,
USA), MCT1 (Monocarboxylate transporter 1) antibody (1:1000, Millipore, Billerica,
MD, USA), MCT2 (Monocarboxylate transporter 2) antibody (1:1000, Millipore,
Billerica, MD, USA). HRP-conjugated anti-rabbit antibody and HRP-conjugated anti-
mouse antibody (Vector Laboratories, Burlingame, CA, USA) were used as secondary
antibodies. Immunoreactive bands were visualized with Pierce SuperSignal
Chemiluminescent Substrates (Thermo Scientific, Waltham, MA, USA) and captured by
Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).
All band intensities were quantified using the Un-Scan-it (version 6.0, Silk Scientific,
Orem, UT, USA) software.
Hexokinase activity assay
Hexokinase activity assay was measured by monitoring the conversion of NAD+
(nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine
dinucleotide) by following the change in absorption at 340 nm. The assay medium
contained: 0.1mg/mL of the hippocampal tissue protein, 0.05M Tris*HCl, PH8.0,
13.3mM MgCl2, 0.112M glucose, 0.227mM NAD+, 0.5mM Adenosine 5 Triphosphate,
and 1IU/mL glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides) in a final
27
volume of 150mL. The OD at = 340 nm was measured every 1 min for 30mins at a
temperature of 30 C. The increase in OD reflects the increase in NADPH concentration,
and the total hexokinase activity was calculated from the slope of the resulting curve.
Immunohistochemistry
For immunohistochemistry studies, fixed hemispheres were coronally sectioned at
30 mm, and then processed for immunohistochemistry using a standard protocol. Briefly,
every 12
th
section was blocked (1 h at RT, PBS with 5% goat serum and 0.3% trinton x-
100), immunolabeled using antibody directed against Ab for (PhosphoPDH, Covance,
1:1000 dilution 4°C overnight), followed by washing and secondary antibody Fluorescein
goat anti-mouse (1:500, Chemicon, Ramona, CA, 1 h at RT) and/or Cy3 conjugated goat
anti-rabbit (1:1000, Chemicon, Ramona, CA, 1 h at RT). Sections were mounted with
anti-fade mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Antigen
unmasking treatment, consisting of 5 min rinse in 99% formic acid was performed to
enhance Ab immunoreactivity (IR). Fluorescent images were taken using a fluorescent
microscope with the slide book software (Intelligent Imaging Innovations Inc, Santa
Monica, CA).
Plasma β-hydroxybutyric acid measurement
The concentration of plasma β-hydroxybutyric acid was determined by β-
hydroxybutyrate liquicolor assay kit (Stanbio laboratory, TX, USA) following
manufacturer s instructions.
Statistics
28
Statistically significant differences between groups were determined by an
ANOVA followed by a Newman Keuls post-hoc analysis. Statistical significance of the
correlation was determined by Pearson’s Correlation Analysis. Data are expressed as
mean ± SEM.
Results
Decline in brain glucose uptake and transporter expression at early stage of female
aging
– NonTg brain
To investigate the impact of female aging on brain glucose metabolism,
microFDG-PET/CT imaging was conducted to determine brain glucose uptake in female
nonTg and 3xTgAD mice at 3, 6, 9, 12, 15 months of age. In nonTg mice, there was an
age-related decline in brain glucose uptake, as indicated by semi-quantitative standard
uptake value (SUV), which reached statistical significance between 6 and 9 months of
age (Figure II- 1A; F (4, 19) = 13.16, p < 0.0001, n = 4 5/group). No evidence for
recovery of glucose uptake was evident as the deficit in glucose uptake was sustained at
12 and 15 months of age.
The age-related decline in brain glucose uptake could be attributed to multiple
levels of metabolic dysregulation, including reduced glucose transport, compromised
glycolysis and deficient mitochondrial capacity (Brinton, 2008; Nilsen and Diaz Brinton,
29
2003; Nilsen et al., 2007; Yao et al., 2011a). To assess the impact of female aging on the
expression of brain glucose transporters (GLUTs) in nonTg brain, we determined
hippocampal protein levels of blood brain barrier (BBB) GLUT1
55Kda
(GLUT1
55Kda
),
glial GLUT1
45Kda
(GLUT1
45Kda
) and neuronal GLUT 3 (GLUT 3) (Vannucci et al., 1998).
In nonTg hippocampus, expression of the BBB GLUT1
55Kda
showed a significant
increase after 3 months of age. The rise in GLUT1
55Kda
expression was sustained across 6,
9 and 12 months of age, followed by a significant decline at 15 months of age (Figure II-
1B; F (4, 20) = 5.956, p < 0.005, n = 5/group). The glial GLUT1
45Kda
showed a non-
significant trend towards increasing from 3 to 12 months of age (Figure II- 1C). GLUT3
expression demonstrated an age-related decline, which reached statistical significance
between 6 and 9 months of age (Figure II- 1D; F (4, 19) = 2.994, p < 0.05, n = 4
5/group). In addition, expression of GLUT3 was significantly positively correlated with
the decline in brain glucose uptake (Figure II-1D; Pearson s r = 0.89, p < 0.05, n = 4 –
5/group).
– 3xTgAD brain
In 3xTgAD mice, there was an age-related decline in brain glucose uptake, as
determined by microFDG-PET and indicated by semi-quantitative standard uptake value
(SUV), which reached statistical significance between 6 and 9 months of age (Figure II-
1E; F (4, 18) = 7.084, p < 0.005, n = 4 5/group). As with the nonTg brain, the deficit in
brain glucose uptake was sustained at 12 and 15 months indicating no recovery at these
ages.
30
To assess the impact of female aging on the expression of brain glucose
transporters (GLUTs) in 3xTgAD brain, we determined the hippocampal protein levels of
GLUT1
55Kda
(blood brain barrier (BBB) GLUT1
55Kda
), GLUT1
45Kda
(glial GLUT1
45Kda
)
and GLUT 3 (neuronal GLUT 3) (Vannucci et al., 1998).
In 3xTgAD hippocampus, there was an age-related decline in GLUT1
55Kda
, which
reached significance at 9 months of age (Figure II-1F; F (4, 19) = 19.08, p < 0.0001, n =
4 5/group). The decline in GLUT1
55Kda
at 9 months was sustained at 12 months.
However, unlike the nonTg mice, 3xTgAD mice GLUT1
55Kda
expression was not
maintained and underwent further significant decline at 15 months of age. The decline in
GLUT1
55KDa
showed a strong positive correlation with the brain glucose uptake (Figure
II-1F; Pearson’s r = 0.83, p = 0.08, n = 4 – 5/group). Hippocampal glial GLUT
45Kda
expression underwent a significant rise in expression with age in 3xTgAD mice that
reached asymptote at 12 months of age and was maintained at 15 months (Figure II-1G; F
(4, 19) = 29.14, p < 0.0001, n = 4 5/group). The rise in GLUT
45Kda
was negatively
correlated with the brain glucose uptake (Figure II- 1G; Pearson’s r = − 0.95, p < 0.05, n
= 4 – 5/group). In parallel, neuronal GLUT3 expression underwent an age-related
decline, which was significant at 9 months of age (Figure II- 1H; F (4, 19) = 16.08, p <
0.0001, n = 4 5/group). Further, GLUT3 expression was significantly positively
correlated with brain glucose uptake (Figure II- 1H; Pearson’s r = 0.96, p < 0.001, n = 4 –
5/group).
31
Figure II - 1. Decreased brain glucose uptake and glucose transporter expressions at early stage
of female aging. A. Representative FDG-microPET images showed an age-related decline in brain
glucose uptake in nonTg brain, which was maximal between 6 and 9 months of age. (Yellow indicates
32
higher values and red indicates lower value). B. Quantitative analysis demonstrated an age-related
decrease in brain glucose uptake in nonTg brain, which was significant between 6 and 9 months of age
(F (4, 19) = 13.16, p < 0.0001, n = 4 − 5). C. GLUT1
55Kda
expression showed a significant increase
after 3 months of age. This rise sustained across 6, 9 and 12 months of age and then decreased
significantly at 15 months of age (F (4, 20) = 5.956, p < 0.005, n = 5). D. In nonTg hippocampus,
there was a trend towards increased expression of GLUT1
45Kda
from 3 to 12 months of age. However,
it did not reach significance. E. GLUT3 expression in nonTg hippocampus demonstrated an age-
related decline, which reached statistical significance between 6 and 9 months of age (F (4, 19) =
2.994, p < 0.05, n = 4 − 5). F. Representative FDG-microPET images showed an age-related decline
in brain glucose uptake in 3xTgAD brain, which was maximal between 6 and 9 months of age.
(Yellow indicates higher values and red indicates lower value). G. Quantitative analysis demonstrated
an age-related decrease in brain glucose uptake in 3xTgAD mice, which was significant between 6
and 9 months of age (F (4, 18) = 7.084, p < 0.005, n = 4 − 5). H. 3xTgAD hippocampus demonstrated
an age-related decrease in the expression of GLUT1
55Kda
, which was significant after 6 months of age
(F (4, 19) = 19.08, p < 0.0001, n = 4 – 5). I. In 3xTgAD hippocampus, GLUT1
45Kda
increased
significantly during aging (F (4, 19) = 29.14, p < 0.0001, n = 4 – 5). J. The age-related decline in
GLUT3 expression reached significance after 6 months of age (F (4, 19) = 16.08, p < 0.0001, n = 4 –
5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, bars represent mean value ± SEM.
Decline in glycolytic capacity at early stage of female aging
Hexokinase irreversibly phosphorylates glucose to glucose-6-phosphate, which is
the first and rate-limiting step in glycolysis. The FDG uptake signal in FDG-PET imaging
positively correlates with hexokinase activity, as cellular FDG accumulation is mediated
by hexokinase that phosphorylates FDG to FDG-6-phosphate, which does not undergo
further metabolism (Pauwels et al., 2000). To investigate whether the decline in brain
glucose uptake is associated with compromised glucose phosphorylation, we analyzed the
activity of hexokinase and expression of hexokinase type II, the specific isozyme
sensitive to hormonal regulation (Wilson, 1995). The key enzyme linking aerobic
glycolysis to oxidative phosphorylation is pyruvate dehydrogenase (PDH).
Phosphorylation of the E1 subunit of PDH inactivates E1 that results in activation of the
entire PDH complex. PDHα has 3 phosphorylation sites, phosphorylation of site 1
(Ser293) reduces overall PDC activity by > 97% (Korotchkina et al., 2006). To
33
investigate the impact of female aging on PDH inactivation, we analyzed the
phosphorylation level of Ser293 on PDH protein.
– NonTg brain
In nonTg mice, hippocampal hexokinase II protein expression decreased
significantly at 15 months of age (Figure II- 2A; F (4, 20) = 12.79, p < 0.0001, n =
5/group), whereas hippocampal hexokinase activity was significantly decreased earlier at
9 months of age (Figure II- 2B; F (4, 20) = 9.775, p < 0.0005, n = 5/group). Further, the
decline in hexokinase activity was sustained at the decreased level across 9, 12 and 15
months of age. The decline in hexokinase activity was positively correlated with brain
glucose uptake (Figure II- 2B; Pearson s r = 0.77, p = 0.13, n = 5/group).
In parallel to the decline in hexokinase activity, immunofluorescent phosphoPDH
(Ser293) increased dramatically in the hippocampal CA3 region (Figure II-3A). Increased
phosphoPDH was detectable at 6 and 9 months of age and increased at 12 and 15 months
of age. Expression of phosphoPDH (Ser293) protein relative to total PDH expression
exhibited an age-related increase, which reached significance at 9 and 12 months of age
(Figure II- 3B; F (4, 18) = 4.002, p < 0. 05, n = 4 5/group). Further, the phosphorylated
state of PDH declined at 15 months. PDH phosphorylation was significantly negatively
correlated with brain glucose uptake in nonTg mice (Figure II- 3A; Pearson s r = - 0.93, p
< 0.05, n = 4 – 5/group).
– 3xTgAD brain
In 3xTgAD hippocampus, hexokinase II protein expression did not change with
age (Figure II- 2C). However, hexokinase activity significantly declined with age, which
34
was significant at 9 months of age (Figure II- 2D; F (4, 19) = 4.561, p < 0.001, n = 4
5/group) and was sustained at decreased level at later ages of 12 and 15 months.
Moreover, the decline in hexokinase activity was significantly positively correlated with
brain glucose uptake (Figure II- 2D; Pearson s r = 0.91, p < 0.05, n = 4 – 5/group).
In 3xTgAD hippocampus, there was an increased level of immunofluorescent
phosphoPDH (Ser293) signal in hippocampal CA3 region (Figure II-3C).
PhosphoPDH/PDH protein expression ratio exhibited an age-related increase, which was
significant at 9 months of age (Figure II- 3D; F (4, 20) = 7.956, p < 0. 001, n = 5/group)
and sustained at increased level at later ages of 12 and 15 months. Moreover, the
increased phosphoPDH/PDH protein expression increase was significantly negatively
correlated with brain glucose uptake (Pearson s r = - 0.94, p < 0.05, n = 4 – 5/group).
Figure II- 2. Decreased hexokinase activity at early stage in female aging. A. In nonTg
hippocampus, expression of hexokinase 2 decreased significantly at 15 months of age (F (4, 20) =
12.79, p < 0.0001, n = 5). B. Hexokinase activity in nonTg hippocampus decreased significantly after
6 months of age (F (4, 20) = 9.775, p < 0.0005, n = 5). C. There was no change in the expression of
hexokinase 2 in 3xTgAD hippocampus. D. Hexokinase activity in 3xTgAD hippocampus decreased
35
significantly after 6 months of age (F (4, 19) = 4.561, p < 0.001, n = 4 – 5). * p < 0.05, ** p < 0.01,
*** p < 0.001, **** p < 0.0001, bars represent mean value ± SEM.
Figure II- 3. Increase in the phosphorylation of PDH at early stage of female aging. A.
Immunofluorescent labeling of phosphoPDH (Ser293) in the nonTg hippocampal CA3. B. NonTg
hippocampus demonstrated an age-related increase in phosphoPDH/PDH ratio, which was significant
after 6 months of age (F (4, 18) = 4.002, p < 0. 05, n = 4 – 5). C. Immunofluorescent labeling of
phosphoPDH (Ser293) in the 3xTgAD hippocampal CA3. D. 3xTgAD hippocampus demonstrated an
age-related increase in phosphoPDH/PDH ratio, which was significant after 6 months of age (F (4, 20)
= 7.956, p < 0. 001, n = 5). * p < 0.05, ** p < 0.01, bars represent mean value ± SEM.
Decline in lactate generation and utilization at early stage of female aging
A decline in brain glucose utilization and associated metabolic pathways should
induce mechanisms to compensate for the decline in glucose. Lactate utilization would be
36
the first such adaptive response to support brain energy demand (Pellerin, 2003; Pierre
and Pellerin, 2005; Yao et al., 2010). To investigate whether the decline in brain glucose
uptake is associated with a shift in utilization of lactate, we first determined the
expression of lactate dehydrogenase (LDH) protein in nonTg and 3xTgAD female mice
of 3, 6, 9, 12 and 15 months of age. Glial and neuronal cells have different LDH
isoforms. LDH5 is the major isoform expressed in glial cells and converts pyruvate to
lactate to generate lactate whereas LDH1 is the major isoform in neurons and functions to
convert lactate to pyruvate thereby providing an indicator of lactate utilization. The ratio
of LDH5/LDH1 ratio provides an indicator of whether the two systems are functioning in
a coordinated manner or whether there is a dysregulation between the system of
generation and utilization of lactate predictive of lactate accumulation.
– NonTg brain
In nonTg hippocampus, an age-related decrease in glial LDH 5 protein expression
was apparent at 9 months of age and was sustained across 12 and 15 months of age
(Figure II- 4A; F (4, 20) = 15.89, p < 0.0001, n = 5). In parallel, neuronal LDH1
exhibited a comparable pattern of age-related decline beginning at 9 months, which was
sustained at the decreased level at later ages of 12 and 15 months (Figure II- 4B; F (4, 20)
= 12.72, p < 0.0001, n = 5). Moreover, LDH5/LDH1 ratio decreased significantly at 12
months of age (Figure II- 4C; F (4, 20) = 6.006, p < 0.01, n = 5). LDH5 and LDH1
expression was significantly positively correlated with glycolytic enzymes, especially
with hexokinase activity (Figure II- 4A (LDH5); Pearson s r = 0.97, p < 0.005, n =
5/group; Figure II- 4A (LDH1); Pearson s r = 0.92, p < 0.05, n = 5/group). The parallel
37
decline in both glucose and lactate pathways suggested a coordinated regulation of
glycolytic capacity and lactate generation/utilization. The decline of LDH5 and LDH1
expression in nonTg mice indicates that lactate is unlikely to be the alternative fuel to
compensate brain glucose hypometabolism during female aging process in nonTg brain.
– 3xTgAD brain
In 3xTgAD hippocampus, there was no change at any age in LDH5 and LDH1
expression (Figure II- 4D and Figure II- 4E). However, the ratio of LDH5/LDH1
decreased significantly at 15 months of age (Figure II- 4F; F (4, 20) = 3.365, p < 0.05, n
= 5). These data suggest that the lactate system is not coordinated with the glucose
system.
38
Figure II- 4. Reproductive transition paralleled a significant decrease in LDH5 and LDH1
expressions in nonTg hippocampus. A. NonTg hippocampus demonstrated a an age-related decrease
in LDH5 expression from 6 to 15 months of age, which was significant between 6 and 9, 9 and 12
months of age (F (4, 20) = 15.89, p < 0.0001, n = 5). B. LDH1 expression decreased with age in
nonTg hippocampus from 6 to 15 months of age, which was significant between 6 and 9, 9 and 15
months of age (F (4, 20) = 12.72, p < 0.0001, n = 5). C. LDH5/LDH1 ratio decreased significantly at
12 months of age (F (4, 20) = 6.006, p < 0.01, n = 5). D. There was no change in 3xTgAD LDH5
expression. E. There was no change in 3xTgAD LDH1 expression. F. LDH5/LDH1 ratio decreased
significantly at 15 months of age (F (4, 20) = 3.365, p < 0.05, n = 5). * p < 0.05, ** p < 0.01, *** p <
0.001, **** p < 0.0001, bars represent mean value ± SEM.
Activation of ketogenic pathway at early stage of female aging
In the nonTg brain, lactate is unlikely to be an alternative fuel to compensate for
the decline in glucose metabolism, we therefore investigated whether ketone bodies were
39
generated and utilized during female brain aging. Our previous analyses demonstrated
that the key enzyme in ketone body metabolism, 3-oxoacid-CoA transferase (SCOT),
increased with reproductive senescence (Yao et al., 2010), suggesting that ketone bodies
are a potential alternative fuel. To determine whether a shift in utilization of ketone
bodies paralleled the decline in brain glucose uptake, we first determined ketone body
level in plasma followed by analysis of monocarboxylate transporters (MCTs) for
transporting lactate/ketone bodies.
– NonTg brain
In pooled nonTg plasma samples (n = 5), β-hydroxybutyrate level increased with
age and reached maximum at 12 months of age. Plasma β-hydroxybutyrate level was
negatively correlated with brain glucose uptake level (Figure II- 5A; Pearson’s r = - 0.85,
p = 0.06).
MCTs transport monocarboxylates such as lactate, pyruvate and ketone bodies
across the cell membrane. MCT1 is specifically expressed in glial cells and at the blood
brain barrier (BBB) whereas MCT4 is expressed in astrocytes. MCT2 is mainly
expressed in neurons and has been found in cell bodies and in postsynaptic densities
(Aubert et al., 2005; Pierre and Pellerin, 2005). To determine the ability of the brain to
transport alternative substrates, we assessed protein expression level for BBB/glial
MCT1, glial MCT4 and neuronal MCT2.
In nonTg hippocampus, BBB/glial MCT1 expression exhibited no significant
change with age (Figure II- 5B). In contrast, astocytic MCT4 expression significantly
increased with age and was maximal at 15 months of age (Figure II- 5C; F (4, 18) =
40
12.51, p < 0.0001, n = 4 – 5/group). There was a significant trend towards increase in
neuronal MCT2 expression with age (Figure II- 5D; F (4, 19) = 3.555, p < 0.05, n = 4 –
5/group).
– 3xTgAD brain
In pooled 3xTgAD plasma samples (n = 5), β-hydroxybutyrate concentration at 3
months of age was the highest among all 3xTgAD age groups, indicating an early
ketogenic phenotype in 3xTgAD female mouse, which is consistent with our earlier
findings (Yao et al., 2010). The β-hydroxybutyrate concentration declined after 3 months
of age and increased again 12 and 15 months of age (Figure II- 5E).
Expression of BBB/glial MCT1 and astrocytic MCT4 in 3xTgAD hippocampus
showed an opposite profile to that of neuronal MCT2. BBB/glial MCT1 expression
decreased significantly at 12 and 15 months of age whereas significant decline in
astrocytic MCT4 occurred later at 15 months of age (Figure II- 5F (MCT1); F (4, 18) =
10.45, p < 0.0005, n = 4 – 5/group; Figure II- 5G (MCT4); F (4, 19) = 5.079, p < 0.01, n
= 4 – 5/group). In contrast, neuronal MCT2 increased significantly after 9 months of age
and remained significantly elevated at 12 and 15 months of age (Figure II- 5H; F (4, 19)
= 7.29, p < 0.005, n = 4 − 5/group). The opposing pattern of MCT1/MCT4 and MCT2
expression suggest a disconnection between BBB/glial transport of alternative fuel supply
and the alternative fuel requirements from neurons.
41
Figure II-5. Plasma β-hydroxybutyrate level and its neuronal transporter increase with
reproductive transition. A. Plasma β-hydroxybutyrate level increase with age in nonTg mice (plasma
samples were pooled from 5 different animals, n = 1-2). B. MCT1 expression showed a trend towards
increasing from 3 to 9 months of age and decreased afterwards in nonTg hippocampus. C. The age-
related decrease in MCT4 expression was significant between 3 and 6, 6 and 15 months of age (F (4,
18) = 12.51, p < 0.0001, n = 4 − 5). D. MCT2 increased significant from 3 to 12 months with age
(linear regression: slope = 0.07, R
2
= 0.3273, p < 0.05). E. In 3xTgAD mice, plasma β-
hydroxybutyrate level was highest at 3 months of age. The β-hydroxybutyrate level decreased after 3
months and increased again after 9 months of age (plasma samples were pooled from 5 different
animals, n = 1-2). F. In 3xTgAD hippocampus, MCT1 expression showed an age-related decline,
42
which was significant at 12 and 15 months of age (F (4, 18) = 10.45, p < 0.0005, n = 4 − 5). G. The
age-related decrease in MCT4 expression was significant at 15 months of age (F (4, 19) = 5.079, p <
0.01, n = 4 − 5). H. The age-related increase in MCT2 expression was significant between 6 and 9
months of age (F (4, 19) = 7.29, p < 0.005, n = 4 − 5). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
0.0001, bars represent mean value ± SEM.
Discussion
In this study, we sought to determine whether 1) the system of brain fuel supply
and transport changed with aging in the female brain and 2) whether the changes in this
vital system were antecedent to or subsequent to the decline in mitochondrial function
that we had previously observed (Yao et al., 2010; Yao et al., 2009). Results of analyses
reported herein indicated that 1) decline in brain glucose uptake occurred early in the
process of female brain aging; 2) decline in brain glucose uptake is paralleled by decline
in glucose transporter expression in neurons and rise in glial associated glucose
transporters; 3) decline in brain glucose uptake and neuronal glucose transporter
expression are paralleled by decline in key metabolic enzymes required for glucose
metabolism; 4) decline in glucose metabolism is paralleled by increases in alternative
substrate supply (ketone bodies) and associated transporters (MCT2 and MCT4); and
lastly 5) these changes in brain glucose supply and the system required for glucose
transport and metabolism precede development of mitochondrial dysfunction. These
changes in the systems required for substrate supply for production of ATP to support
energetic demand temporally mapped onto the earliest phase of reproductive senescence
(6-9 months). The decline in brain glucose uptake and neuronal glucose transport
preceded the shift to alternative, less efficient fuels, which was coincident with the age of
reproductive senescence (9-12 months).
43
Results of analyses of the aging female nonTg brain demonstrated a significant
decline in brain glucose uptake at an early stage in female brain aging. The decline in
brain glucose uptake, indicated by microFDG-PET imaging, could be attributed to
multiple levels of metabolic dysregulation, including reduced glucose transport,
compromised glycolysis, deficient mitochondrial capacity and/or impaired ATP
generation. In the nonTg brain, blood-brain-barrier (BBB) GLUT1
55Kda
increased
significantly relative to 3 month old female hippocampus then declined at 15months. In
parallel to the significant rise in BBB GLUT1
55Kda
, there was a trend toward increased
glial GLUT1
45Kda
expression. Coincident with the rise in BBB GLUT1
55Kda
and glial
GLUT
45Kda
transporters, neuronal GLUT3 decreased over the same time frame and then
rebounded at 15 months. GLUT3 expression coupled to metabolic demand or cerebral
glucose utilization (Fattoretti et al., 2001; Ferreira et al., 2011; Vannucci and Simpson,
2003). In the aging female brain, the pattern of glucose uptake and glucose transporter
expression is consistent with the decrease in microFDG-PET signal and thus largely
driven by the decline in neuronal glucose uptake via GLUT3. Further, the rise in BBB
GLUT1
55KDa
and glial GLUT1
45Kda
expression, likely a compensatory strategy to provide
glucose to the brain, does not obviate the decline in microFDG-PET signal.
GLUT1
45Kda
is mainly expressed in oligodendrocytes and astrocytes with
localization including the astrocytic endfeet adjacent to the BBB endothelial cell
(Simpson et al., 2007; Yu and Ding, 1998). GLUT1
45Kda
could increase glucose uptake
into astrocytes, where glucose would be converted to lactate to serve as a compensatory
fuel for neurons or into oligodendrocytes to address energetic demands of lipid / myelin
synthesis (Rinholm et al., 2011; Sanchez-Abarca et al., 2001). However, the decline in
44
both LDH5 to generate lactate in astrocytes and LDH1 in neurons to utilize lactate makes
this compensatory option unlikely. Further, the rise in phosphorylated / inactivated PDH
compromises the utilization of lactate by neurons as lactate is converted to pyruvate,
which requires active PDH for conversion to acetyl-CoA for entry into the TCA cycle.
These findings suggest that the increase in GLUT1
45Kda
is more likely associated with
transport of glucose for lipid / myelin synthesis (Rowe et al., 2007).
Cerebral glucose utilization is also controlled by hexokinase, which is the first
rate-limiting step in glycolysis and is critical in mediating the FDG-PET signal (Pauwels
et al., 2000). We found that a decline in hippocampal hexokinase activity paralleled the
decline in brain glucose uptake and GLUT3 expression. Compromised glucose transport
and hexokinase activity could be further exacerbated by decreased activity of PDH,
which converts pyruvate to acetyl-CoA and is the key enzyme linking glycolysis and
TCA cycle. Coincident with the decline in glucose uptake, GLUT3 expression and
hexokinase activity, we observed an age dependent increase in phosphorylated PDH, a
mechanism of PDH inactivation. Taken together, these data are indicative of a system-
wide deficit, from transport to cerebral glucose metabolism in the hippocampus at an
early stage of female brain aging that precedes both reproductive senescence and the
associated decline in mitochondrial function.
The 3xTgAD brain also exhibited an age-related decline in microFDG-PET that
occurred between 6 and 9 months of age. In parallel to the decline in brain glucose
uptake, expression of the blood-brain-barrier (BBB) GLUT1
55Kda
and neuronal GLUT3
also decreased. The decline in BBB GLUT1
55Kda
is consistent with earlier reports of
reduced BBB glucose transporter in postmortem AD cerebral cortex (Kalaria and Harik,
45
1989). The inability of the 3xTgAD brain to mount a compensatory rise in GLUT1
55Kda
expression may be related to accumulation of vascular β-amyloid, which has been
reported to impair BBB GLUT1
55Kda
expression (Merlini et al., 2011). Further, the
decline in both glucose uptake and hippocampal GLUT1
55Kda
expression in the female
3xTgAD brain is consistent with our previous findings that ovariectomy induced a
significant decline in both these indicators of brain glucose metabolism (Ding et al.,
2013). Further, the β-amyloid mechanism of reduced GLUT1
55Kda
expression is
consistent with the age-related and ovariectomy-associated increase in β-amyloid
deposition in the 3xTgAD brain (Yao et al., 2011a; Yao et al., 2009). In stark contrast to
the decline in GLUT1
55Kda
, there was a dramatic rise in glial GLUT1
45Kda
expression. The
significant rise in GLUT1
45Kda
was unique to the 3xTgAD hippocampus although a
similar trend was observed in the nonTg hippocampus.
Under normal conditions, glucose transport across the BBB is not considered to
be rate limiting (Leybaert, 2005b; Lund-Andersen, 1979). However, under pathological
conditions BBB glucose transport can be a rate-limiting step in brain metabolism, leaving
neurons and glial cells vulnerable to glucose deprivation (Qutub and Hunt, 2005). This
appears to be the case in the 3xTgAD female brain. The pattern of a concomitant decline
in BBB and neuronal glucose transporters in parallel to a dramatic rise in the glial
GLUT1
45Kda
glucose transporter, present in oligodendrocytes and the endfeet of
astrocytes, suggests either: 1) a compensatory upregulation of astrocytic endfeet glucose
transporters to compensate for the decline in glucose transport through the BBB or 2)
preferential transport of glucose into oligodendrocytes to meet the energetic demand of
46
white matter generation and maintenance (Harris and Attwell, 2012; Rinholm et al.,
2011).
In addition to significant changes in glucose transporter expression, the 3xTgAD
brain also exhibited a significant decrease in hippocampal hexokinase activity at an early
stage in female aging. This age-associated decline is consistent with our previous finding
that ovarian hormone loss in 3xTgAD mice induced a significant decline in hexokinase
activity, which was partially prevented by 17β-estradiol treatment (Ding et al., 2013).
Compared to nonTg mice, 3xTgAD mice exhibited a lower hippocampal hexokinase
activity at each age. Of note, β-amyloid in the 3xTgAD brain is detectable in the
mitochondria at 9 months of age and thereafter! (Yao et al., 2009), and is temporally
coincident with a significant decline in hexokinase activity 3xTgAD hippocampus!(Ding
et al., 2013). The decline in hexokinase activity and the appearance of β-amyloid in
mitochondria are consistent with previous in vitro assays in cultured neurons
demonstrating that β-amyloid triggers the release of neuronal hexokinase 1 from
mitochondria and inactivation of hexokinase 1 (Saraiva et al., 2010). The decline in BBB
and neuronal glucose transporters coupled with decreased hexokinase activity was
accompanied by increased phosphorylation of PDH, consistent with decreased PDH
activity. We found that in 3xTgAD hippocampus, there was an increase in the
phosphorylation of the PDH complex at the early stage of female aging, which is
consistent with previous findings of reproductive senescence-associated and
ovariectomy-induced decline in PDH activity (Yao et al., 2010; Yao et al., 2011a).
Collectively, these data indicate a system wide decline in glucose metabolism
evidenced in glucose uptake into the brain, glucose transporters and glucose metabolism.
47
Further, the data show that the glucose transporter system in hippocampus is dynamic in
its compensatory response potential with the nonTg and 3xTgAD brains generating
different adaptive responses. The nonTg brain favors a strategy to increase glucose
transport across the blood brain barrier while lowering glucose availability to
energetically demanding neurons in favor of a modest increase of glucose transport into
glial cells, either oligodendrocyte or astrocytic endfeet. In contrast, the 3xTgAD brain
favors a glial compensatory response to increase glucose transport either into
oligodendrocyte or astrocytic endfeet.
In response to decreased glucose uptake, the brain can utilize lactate or ketone
bodies as alternative fuels. Lactate is known to be a metabolic substrate for brain and is
particularly important under conditions of high synaptic activity, which occurs during
learning and memory (Suzuki et al., 2011). Further, during glucose insufficiency, lactate
can serve as a secondary fuel through glycogen metabolism (Aubert et al., 2005). In
nonTg mice, expression of LDH5 and LDH1 decreased during aging, suggesting
decreased generation and utilization of lactate in nonTg hippocampus during female
aging. The decrease in LDH5/LDH1 ratio also suggested that there was less lactate
accumulation in nonTg hippocampus during female aging. In nonTg brain, the pattern of
decreasing LDH5 and LDH1 expression is similar to the decreasing pattern of the system
required for glucose transport and metabolism, indicative of coordinated regulation
between the glucose and lactate systems which is consistent with glucose as the
metabolic precursor to lactate in aerobic glycolysis for conversion of pyruvate to lactate
via LDH5. The parallel decline in glucose uptake and LDH isoforms 5 and 1, predict that
astrocyte-derived lactate would not serve as an alternative fuel source to compensate for
48
the decline in glucose availability. However, transport of lactate from the periphery
remains a possibility, as BBB/glial MCT1 did not decline until 15 months of age and the
astrocytic MCT4 significantly increased with age. However, the rise in phosphorylated /
inactivated PDH compromises the utilization of lactate by neurons as lactate is converted
to pyruvate, which requires active PDH for conversion to acetyl-CoA for entry into the
TCA cycle.
In 3xTgAD mice, expression of LDH5 and LDH1 did not change during the aging
process, suggesting a lack of response in lactate compensatory system and loss of
dynamic adaptation during 3xTgAD female brain aging. The findings in lactate
generation/utilization system in 3xTgAD hippocampus were in contrast to our earlier
findings that in the ovariectomized female 3xTgAD mouse hippocampus, expression of
LDH isoforms 5 and 1 along with the LDH5/LDH1 ratio were increased (Ding et al.,
2013). These, data indicate an essential difference between the gradual loss of ovarian
hormone homeostasis during 3xTgAD female brain aging and an acute loss of ovarian
hormones due to surgical ovariectomy. The sustained expression of LDH5/LDH1
throughout the aging process suggests that in the 3xTgAD hippocampus, the tight
coupling between glucose and lactate metabolic systems that characterizes the nonTg
hippocampus is not expressed in the 3xTgAD hippocampus. Further, the data suggest that
transport of lactate from the periphery into the astrocytes and oligodendrocytes would be
compromised by the dramatic decline in MCT1 expression at 12 and 15 months and the
concomitant decline in MCT4 during the same period. The dramatic rise in neuronal
MCT2, when BBB and glial MCTs are decreasing, suggest a disconnection between
BBB/glial transport of alternative fuel supply and the alternative fuel requirements from
49
neurons. Although lactate could be provided to the brain from the periphery, the rise in
phosphorylated / inactivated PDH comprises the utilization of lactate in neurons as lactate
requires conversion to pyruvate which in turn requires active PDH for conversion to
acetyl-Co-A for entry into the TCA cycle. Together, the data suggest that lactate could
serve as an alternative fuel in some cells but that in hippocampus utilization of lactate
will be limited in magnitude.
In addition to lactate, ketone bodies can also serve as an alternative fuel for brain
during early development and starvation (Hawkins et al., 1971). In the current study, we
found that in the nonTg mouse, plasma concentration of the ketone body, β-
hydroxybutyrate, increased in parallel with the decline in glucose uptake. There was no
change in the BBB MCT1 and only a modest rise in neuronal MCT2 expression until 12
months of age. In contrast, astrocytic MCT4 expression increased significantly at 15
months. Although neuronal MCT2 and astrocytic MCT4 are actively involved in lactate
transport (Morris and Felmlee, 2008; Pierre and Pellerin, 2005), increased MCT2/MCT4
expression in nonTg aging hippocampus also has the potential to increase ketone body
transport. The rise in astrocytic MCT4 is consistent with our previous findings that
reproductive senescence is associated with activation of the ketogenic pathway indicated
by an increase in the rate limiting enzyme for ketone body utilization, 3-oxoacid-CoA
transferase (SCOT) (Yao et al., 2010). The rise in astrocytic MCT4, together with the
earlier findings, are indicative of a compensatory response in the aging female brain to
increase systems required for generation, metabolism and transport ketone bodies in
response to the decline in glucose uptake (Yao et al., 2010).
50
In 3xTgAD mice, plasma β-hydroxybutyrate concentration was maximal at an
early age (3 months of age), decreased at 6 and 9 months of age, and rose again at 12 and
15 months of age. The elevation of peripheral ketone bodies at 3 months of age is
consistent with our previous findings that hippocampal SCOT expression which is
required for catabolism of ketone bodies to generate acetyl-CoA (Yao et al., 2010). In
3xTgAD brain, the early activation of ketogenic pathway was also evident as an increase
in gene expression for enzymes involved in ketone body metabolism at 6 months of age
(Chou et al., 2011). Consistent with the maximal level of plasma β-hydroxybutyrate,
expression of MCT1 and MCT4 were highest at 3 months of age, sustained until 9
months of age and decreased dramatically thereafter. The decline in MCT1 and MCT4 at
12 and 15 months would limit the transport of ketone bodies into astrocytes and
oligodendrocytes. However, the rise in neuronal MCT2 would preferentially transport
either lactate and or ketone bodies into neurons. We and others have documented that
neurons can utilize ketone bodies as an alternative fuel (Edmond et al., 1987; Yao et al.,
2010).
We previously demonstrated a significant age-related decline in aerobic
glycolysis and mitochondrial respiration in female nonTg-wildtype brain between 9 and
12 months of age (Yao et al., 2010; Yao et al., 2009). Findings from the current study
demonstrate that the decline in brain glucose uptake, neuronal glucose transporter
expression and glycolytic capacity in the aging female brain temporally preceded the
decline in mitochondrial respiration. Further, the decline in brain glucose bioenergetics
was paralleled by a shift toward ketone body utilization, a metabolic phenotype that
51
precedes memory impairment, which is characteristic of early Alzheimer’s disease
(Figure II- 6A) (Kadish et al., 2009; Yao et al., 2010; Yao et al., 2009).
The 3xTgAD brain exhibited early activation of the ketogenic pathway and a lack
of adaptive response during female aging, indicated by a decline in alternative fuel
transport system (Figure II- 6B). Compared to 3xTgAD brain, the nonTg brain has a
more robust compensatory and dynamic adaptive system in terms of alternative fuel
supply and transport. However, because mitochondria are metabolizing glucose, lactate
and ketone bodies to produce ATP, both nonTg-wildtype and 3xTgAD brain reach a
stable state of diminished bioenergetic capacity after collapse of mitochondrial function.
52
Figure II- 6. Timeline of bioenergetic aging in female mammalian brain. A. In normal nonTg
brain, the decline in brain glucose transport and glycolytic capacity occurred between 6 and 9 months
of age, which temporally preceded mitochondrial dysfunction. The decline in brain bioenergetic was
paralleled with the increase in peripheral ketone body concentration. The expression of BBB and glial
ketone body transporter decreased after 9 months of age whereas the astrocytic ketone body
transporter increased at 15 months of age. B. In 3xTgAD brain, the decline in brain glucose transport
and glycolytic capacity also occurred between 6 and 9 months of age, which temporally preceded
mitochondrial dysfunction. The activation of the ketogenic pathway occurred at both early age and
early stage of female aging. The expression of BBB/glial and astrocytic ketone transporters was
maximal at early age (3 months) but decreased at early stage of female aging (9 to 15 months).
Ovarian hormones are known to enhance or maintain brain glucose metabolism
by increasing the expression of glucose transporters, and key rate limiting processes
required for aerobic glycolysis and oxidative phosphorylation (Brinton, 2008, 2009). In
primate cerebral cortex 17β-estradiol increased the mRNA expression of GLUT1 and
53
GLUT3 (Cheng et al., 2001). In addition to glucose transporters, estradiol and
progesterone significantly increased hexokinase activity in aged female rat brain
(Moorthy et al., 2004). Further, ovarian hormone deprivation induced a significant
decline in PDH activity, which was prevented by 17β-estradiol treatment (Yao et al.,
2011a).
Together, these findings provide a sequence of age-related events that are first
evident as glucose hypometabolism and a shift to alternative fuels followed by
mitochondrial dysfunction. Glucose hypometabolism appears to be the first and defining
step in female brain aging, followed by mitochondrial dysfunction, leads to a bioenergetic
phenotype consistent with the bioenergetic phenotype of persons at risk for AD (Del Sole
et al., 2008; Mosconi, 2005; Mosconi et al., 2008a; Reiman et al., 2004). From a
translational perspective, these findings suggest development of strategies that 1) prevent
transition to bioenergetic deficits and 2) target fuel and enzyme system of a brain that
was transitioned to alternative fuels.
Acknowledgements
Research reported herein was supported by National Institute on Aging
2R01AG032236 (to RDB), National Institute on Aging 5P01AG026572 (Project 1 to
RDB) and Eileen L. Norris Foundation (to RDB). The funders had no role in study design,
data collection or analysis, decision to publish, or preparation of the manuscript. We
gratefully acknowledge gift of triple-transgenic Alzheimer’s disease mouse model from
Frank M. LaFerla.
54
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Chapter III. Ovariectomy induces a shift in fuel availability and
metabolism in the hippocampus of the female transgenic model of
familial Alzheimer’s
Fan Ding, Jia Yao, Liqin Zhao, Zisu Mao, Shuhua Chen, Roberta Diaz Brinton
Published in PLoS One. 2013;8(3):e59825. Epub 2013 Mar 26.
Abstract
Previously, we demonstrated that reproductive senescence in female triple
transgenic Alzheimer’s (3xTgAD) mice was paralleled by a shift towards a ketogenic
profile with a concomitant decline in mitochondrial activity in brain, suggesting a
potential association between ovarian hormone loss and alteration in the bioenergetic
profile of the brain. In the present study, we investigated the impact of ovariectomy and
17β-estradiol replacement on brain energy substrate availability and metabolism in a
mouse model of familial Alzheimer’s (3xTgAD). Results of these analyses indicated that
ovarian hormones deprivation by ovariectomy (OVX) induced a significant decrease in
brain glucose uptake indicated by decline in 2-[
18
F]fluoro-2-deoxy-D-glucose uptake
measured by microPET-imaging. Mechanistically, OVX induced a significant decline in
blood-brain-barrier specific glucose transporter expression, hexokinase expression and
activity. The decline in glucose availability was accompanied by a significant rise in glial
LDH5 expression and LDH5/LDH1 ratio indicative of lactate generation and utilization.
In parallel, a significant rise in ketone body concentration in serum occurred which was
61
coupled to an increase in neuronal MCT2 expression and 3-oxoacid-CoA transferase
(SCOT) required for conversion of ketone bodies to acetyl-CoA. In addition, OVX-
induced decline in glucose metabolism was paralleled by a significant increase in Aβ
oligomer levels. 17β-estradiol preserved brain glucose-driven metabolic capacity and
partially prevented the OVX-induced shift in bioenergetic substrate as evidenced by
glucose uptake, glucose transporter expression and gene expression associated with
aerobic glycolysis. 17β-estradiol also partially prevented the OVX-induced increase in
Aβ oligomer levels. Collectively, these data indicate that ovarian hormone loss in a
preclinical model of Alzheimer’s was paralleled by a shift towards the metabolic pathway
required for metabolism of alternative fuels in brain with a concomitant decline in brain
glucose transport and metabolism. These findings also indicate that estrogen plays a
critical role in sustaining brain bioenergetic capacity through preservation of glucose
metabolism.
Introduction
Previously we demonstrated that a decline in mitochondrial bioenergetics
precedes the development of AD pathology in the female triple transgenic Alzheimer’s
(3xTgAD) mouse model (Yao et al., 2009). In both normal and 3xTgAD mice,
reproductive senescence, both natural and ovariectomy-induced, resulted in a significant
decline in aerobic glycolysis, PDH, and Complex IV cytochrome c oxidase activity, and
mitochondrial respiration. Following transition through reproductive senescence,
enzymes required for long-chain fatty acid (HADHA) and ketone body (SCOT)
62
metabolism were significantly increased whereas cytochrome c oxidase (Complex IV)
collapsed by 40% in both the nonTg and 3xTgAD brain which was predictive of a
concomitant decline in ATP generation. These bioenergetic changes, observed during
natural reproductive senescence, were recapitulated in an ovariectomy model of
menopause (Yao et al., 2011a).
Consistent with findings from basic science discoveries, data emerging from
clinical positron emission tomography with 2-[
18
F]fluoro-2-deoxy-D-glucose (FDG-PET)
analyses indicate a progressive reduction in cerebral glucose metabolic rate (CMRglu),
particularly in posterior cingulate (PCC) and parietal-temporal cortex, in persons with
Alzheimer’s disease as well as those at increased risk for AD (Mosconi, 2005; Mosconi
et al., 2009b; Mosconi et al., 2006; Reiman et al., 1996; Reiman et al., 2004). Clinical
imaging has also indicated a spatial correlation between increased aerobic glycolysis and
β-amyloid deposition in the “default mode network” brain areas, suggesting that deficits
in energy supply may underlie the vulnerability to the AD pathogenic process in such
areas (Vaishnavi et al., 2010; Vlassenko et al., 2010). Further in persons with AD,
compromised brain glucose metabolism is accompanied by parallel activation of
alternative metabolic pathways, as evidenced by a utilization ratio of 2:1 glucose to
alternative substrate in persons with incipient AD compared to a ratio of 29:1 in healthy
elderly controls (Hoyer, 1991).
Earlier studies indicated that 17β-estradiol (E2) promoted glucose uptake (Bishop
and Simpkins, 1995), glycolysis (Kostanyan and Nazaryan, 1992), glycolytic-coupled
tricarboxylic acid cycle (TCA) function, mitochondrial respiration and ATP generation
(Brinton, 2008; Nilsen and Diaz Brinton, 2003; Nilsen et al., 2007; Yao et al., 2011a).
63
Results of these discovery analyses demonstrate that 17β-estradiol sustains the ability of
the brain to transport and utilize glucose as its primary fuel source. From a translational
perspective, these basic science findings are supported by clinical analyses of glucose
metabolism in menopausal women. Postmenopausal women on estrogen therapy were
reported to have increased cerebral blood flow and cerebral metabolism relative to non-
users (Eberling et al., 2000; Maki and Resnick, 2001; Rasgon et al., 2005; Yao et al.,
2011a). Further, non-users exhibited a significant decline in glucose metabolic rate,
particularly in the posterior cingulate and prefrontal cortex, which closely resembled the
hypometabolic profile of AD brains (Eberling et al., 2000; Rasgon et al., 2005).
Collectively, both preclinical analyses in animal models (Yao et al., 2010; Yao et al.,
2011a; Yao et al., 2009) and clinical observations (Mosconi et al., 2006; Rasgon et al.,
2005; Reiman et al., 1996; Reiman et al., 2004) provide compelling evidence in support
of decline in bioenergetic function in brain as an early indicator of neurodegenerative
risk.
In the present study, we sought to investigate the impact of loss of ovarian
hormones and the efficacy of 17β-estradiol to sustain aerobic glycolysis as a primary
bioenergetic system of the brain. Specifically, we determined whether these bioenergetic
deficits were in response to a decline in substrate availability as evidenced by capacity for
glucose uptake, substrate (glucose, lactate/ketone body) transporter expression and/or
enzyme systems required for glucose metabolism. The goal of these analyses was to
determine the initiating events that lead to a dysfunctional bioenergetic system in brain.
As a translational preclinical model relevant to reproductive aging in the human, we
utilized temperature and body weight dysregulation in the ovariectomized 3xTgAD
64
mouse model as the trigger to initiate analyses of brain metabolism. Results presented
herein demonstrate that in 3xTgAD mice, ovarian hormone loss caused by OVX induces
a decline in brain glucose uptake, which could be partially attributed to decreased glucose
transporter expression and compromised hexokinase activity. The OVX induced decline
in cerebral glucose metabolism was accompanied by the activation of alternative
metabolic pathways. Further, E2 treatment initiated at the time of OVX prevented the
bioenergetic fuel shift by sustaining key elements in glucose metabolism in brain.
Materials and Methods
Animal Treatments and Ethics
All rodent experiments were performed following National Institutes of Health
guidelines on use of laboratory animals and an approved protocol (protocol number:
10217) by the University of Southern California Institutional Animal Care and Use
Committee. The presented study has been approved by the University of Southern
California Institutional Animal Care and Use Committee (Ethics Committee).
Transgenic mice
Colonies of the 3xTgAD mice strain (129S; Gift from Dr. Frank Laferla,
University of California, Irvine) (Oddo et al., 2003) were bred and maintained at the
University of Southern California (Los Angeles, CA) following National Institutes of
Health guidelines on use of laboratory animals and an approved protocol by the
University of Southern California Institutional Animal Care and Use Committee. Mice
were housed on 12 hours light/dark cycles and provided ad libitum access to food and
65
water. The characterization of amyloid and tau pathologies, as well as synaptic
dysfunction in this line of mice has been described previously (Oddo et al., 2003) and
confirmed in our laboratory. Mice were genotyped routinely to confirm the purity of the
colony. To ensure the stability of AD-like phenotype in the 3xTgAD mouse colony, we
performed routine immunohistochemical assays every 3 to 4 generations. Only offspring
from breeders that exhibit stable AD pathology were randomized into the study.
Experimental design
To investigate the change in substrate availability following removal of ovaries
and treatment with 17β-estradiol(E2), 6-month-old 3xTgAD mice were randomly
assigned into the following three treatment groups (n =7 per group): sham
ovariectomized (Sham-OVX), ovariectomized (OVX), and OVX plus 17β-estradiol
(OVX+E2). Mice were bilaterally OVX and injected with corn oil or coin oil containing
E2 (50µg/kg, Subcutaneously) daily for 5 weeks (Figure III-1).
Figure III-1. Experimental paradigm. 6-month-old 3xTgAD mice were randomly assigned into the
following three treatment groups (n = 7 per group): sham ovariectomized (Sham-OVX),
ovariectomized (OVX), and OVX plus 17β-estradiol (OVX+E2). Mice were bilaterally OVX and
injected with corn oil or corn oil with E2 (50µg/kg, Subcutaneously) daily for 5 weeks. Weight and
tail skin temperature were monitored twice per week during the sleep cycle before FDG-microPET
66
imaging. After FDG-microPET imaging, mice were sacrificed for in vitro analyses of substrate
transport and utilization.
Body weight and tail skin temperature (TST) measurement
Body weight and tail skin temperature (TST) were monitored twice per week
during the sleep cycle. To allow mice recover from the OVX surgery, measurements
started one week after the surgery. TST measurements were conducted using an infrared
thermometer (BIO-152-IRB, Bioseb, Chaville, France) designed for small rodents. TST
was measured twice per week until an increase of 1 degree or higher of temperature was
detected and sustained for 1 week. Body weight was also determined twice per week
subsequent to skin tail temperature measurement. Following one week of sustained
temperature dysregulation, mice underwent FDG-PET imaging.
FDG-microPET and microCT imaging
Mice were maintained under anesthesia during microPET and microCT scans
with 2-2.5% isoflurane in oxygen. Scans were performed in an imaging chamber
equipped with a nose cone for anesthesia delivery and heating pad for body temperature
control. MicroPET imaging was performed with a microPET R4 rodent model scanner
(Concorde Microsystems Inc, Knoxville, TN) and micro CT imaging was performed on
MicroCAT II tomography (Siemens Preclinical Solutions, Knoxville, TN). Mice were
injected intravenously via the tail vein with radiotracer [
18
F] Fluoro-2-deoxy-2-D-
glucose (FDG, 200µCi, 100uL). Radioactive dose was determined prior to injection by
radioisotope dose calibrator (Capintec, CRC-712M). At 40min post-injection of FDG,
each mouse was positioned in the microPET scanner in the center of the 10.8cm
67
transaxial and 8cm axial field of view (FOV). Brain microPET data were collected for
10min followed by a 10 min microCT scan for the purpose of co-registration.
Co-registration of microPET and microCT data was performed using the AMIDE
software package (http://amide.sourceforge.net/). After co-registration of the PET and CT
images, ROI (region of interest) was defined and used to measure the radioactivity
concentration in brains. Decay correction was used to adjust the actual radioactivity
dosage injected (Actual radioactivity dosage at time of injection = Initial radioactivity ×
, T = T minutes between injected time point and initial time point). Data were
analyzed using Student’s t-test with differences between groups with a p value <0.05
considered statistically significant.
Brain tissue preparation and Western blot analysis
Upon completion of FDG-microPET imaging, 3xTgAD mice (n = 7 per group)
were sacrificed and the brains rapidly dissected on ice. Hippocampus was processed for
protein extraction using Tissue Protein Extraction Reagent (Thermo Scientific, Rockford,
IL, USA) with phosphatase and protease inhibitors (Sigma, St. Louis, MO, USA), and
protein concentrations were determined with the Bio-Rad Bradford assay. Equal amounts
of protein (20 µg/well) were loaded in each well of a 12.5% SDS PAGE criterion gel
(Bio-Rad, Hercules, CA) and electrophoresed with Tris/glycine running buffer. Proteins
were transferred to 0.45µm pore size polyvinylidene difluoride (PVDF) membranes and
immuneblotted with GLUT1 (glucose transporter 1) antibody (1:1500, Abcam,
Cambridge, MA, USA), GLUT3 (glucose transporter 3) antibody (1:1000, Abcam,
Cambridge, MA, USA), Hexokinase II antibody (1:1000, Millipore, Billerica, MD,
USA), MCT2 (Monocarboxylate transporter 2) antibody (1:1000, Millipore, Billerica,
e
−ln2×
T
110
68
MD, USA), SCOT (3-oxoacid-CoA transferase) antibody (1:100, Santa Cruz
Biotechnology, Santa Cruz, CA, USA), LDHB (lactate dehydrogenase B) antibody
(1:1000, Abcam, Cambridge, MA, USA), and LDH V (lactate dehydrogenase V)
antibody (1:1000, Abcam, Cambridge, MA, USA). HRP-conjugated anti-rabbit antibody
and HRP-conjugated anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA)
were used as secondary antibodies. Immunoreactive bands were visualized with Pierce
SuperSignal Chemiluminescent Substrates (Thermo Scientific, Waltham, MA, USA) and
captured by Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules,
CA, USA). All band intensities were quantified using the Un-Scan-it (version 6.0, Silk
Scientific, Orem, UT, USA) software.
Hexokinase activity assay
Hexokinase activity assay was measured by monitoring the conversion of NAD+
(nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine
dinucleotide) by following the change in absorption at 340 nm. The assay medium
contained: 0.1 µg/µL of the hippocampal tissue protein, 0.05M Tris*HCl, PH8.0, 13.3
mM MgCl2, 0.112M glucose, 0.227 mM NAD+, 0.5mM Adenosine 5’Triphosphate, and
1IU/mL glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides) in a final
volume of 150µL. The OD at λ = 340 nm was measured every 1 min for 30mins at a
temperature of 30°C. The increase in OD reflects the increase in NADPH concentration,
and the total hexokinase activity was calculated from the slope of the resulting curve.
Serum β-hydroxybutyrate measurement
69
The concentration of serum β-hydroxybutyrate was determined by β-
hydroxybutyrate liquicolor assay kit (Stanbio laboratory, TX, USA) following
manufacturer’s instructions.
Real-time RT-PCR gene expression profiling
Total RNA was isolated from mouse hippocampal tissues using the RNeasy kit
(Qiagen, Valencia, CA). The quality and quantity of RNA samples were determined
using the Experion RNA analysis kit (Bio-Rad, Hercules, CA). RNA samples were
reverse-transcribed to cDNA using the high capacity cDNA reverse transcription kit
(Applied Biosystems, Foster City, CA) and stored at -80°C for gene expression analysis.
Taqman low-density arrays (TLDA) were custom manufactured at Applied Biosystems.
Real-time RT-PCR experiment and data analysis were conducted following the methods
as described in our previous report (Zhao et al., 2012).
Results
Ovariectomy induced increase in body weight and tail skin temperature (TST):
prevention by 17β-estradiol
It is well documented that ovarian hormone loss is associated with increased body
weight and temperature dysregulation in menopausal women (Deecher and Dorries,
2007). In this study, we used OVX mice as a translational model of surgically
oophorectomized premenopausal women. In this model, body weight and tail skin
temperature served as surrogate markers of metabolic and temperature dysregulation
(Williams et al., 2010)
70
OVX rapidly induced a significant increase in body weight (Figure III-2A; 2.94 ±
0.39 (OVX group) vs. 0.27 ± 0.54 (Sham-OVX group), F (2, 13) = 12.61, p < 0.001, n =
5 − 6) one week after surgery and this increase was sustained throughout the duration of
the experiment. E2 treatment initiated at the time of OVX prevented OVX-induced body
weight increase. OVX also induced a significant rise in tail skin temperature 4 weeks
after the OVX surgery, which reached asymptote by week 5. Asymptotic temperature
triggered in vivo brain glucose imaging followed by in vitro analyses (Figure III-1). Tail
skin temperature of OVX animals increased 1 °C ± 0.03 °C (Figure III-2B; 23.24 ± 0.08
(OVX group) vs. 22.28 ± 0.07 (Sham-OVX group), F (2,13) = 42.21, p < 0.001, n = 5 − 6)
by week 5 relative to Sham-OVX animals. OVX-induced rise in tail skin temperature was
prevented by E2 treatment initiated at the time of OVX.
Figure III-2 Ovariectomy (OVX) induced increase in body weight and tail skin temperature and
prevention by 17β-estradiol. A. OVX induced a significant increase in body weight (0.27 ± 0.54 vs.
2.94 ± 0.39, F (2, 13) = 12.61, p < 0.001, n = 5 − 6) one week after surgery, which was sustained
through the duration of the experiment. E2 treatment (50µg/kg, SC) initiated at time of OVX
prevented the OVX-induced body weight gain. B. OVX induced a significant rise (22.28 ± 0.07 vs.
23.24 ± 0.08, F (2,13) = 42.21, p < 0.001, n = 5 − 6) in tail skin temperature 4 weeks after surgery, and
E2 treatment (50µg/kg, SC) initiated at time of OVX prevented the OVX-induced increase in tail skin
temperature.
71
Ovariectomy induced decrease in brain glucose uptake: partial prevention by 17β-
estradiol
Ovarian hormone loss is associated with cerebral metabolic decline (Maki and
Resnick, 2001; Rasgon et al., 2005). In the current study, we sought to determine whether
loss of ovarian hormones and associated decline in brain metabolism were accompanied
by a change in brain glucose uptake. To address this question, FDG-microPET imaging
was conducted to determine the impact of ovarian hormone loss and E2 treatment
(50µg/kg, daily SC) on brain glucose uptake in 6-month 3xTgAD female mice. We
demonstrated that OVX induced a decline in brain glucose uptake, which can be partially
prevented by E2 treatment (Figure III-3). Quantitative analysis reveal that compared to
the Sham-OVX group, 5 weeks OVX induced a significant 25% decline in brain glucose
uptake (Figure III-3B; 74.1 ± 1.9% (OVX group) vs. 100 ± 5.4% (Sham-OVX group), p
< 0.05, n = 7). Decline in brain glucose uptake was coincident with thermal dysregulation
in the OVX condition (Figure III-2B). E2 treatment (50µg/kg, daily SC) at the time of
OVX partially prevented the OVX-induced decline in brain glucose uptake (Figure III-3B;
84.2 ± 2.9% (E2 group) vs. 74.1 ± 1.9% (OVX group), p < 0.05, n = 4 − 7), consistent
with the prevention of thermal dysregulation and weight gain (Figure III-2).
Ovariectomy induced decrease in brain glucose transporter expression: prevention by
17β-estradiol
The OVX-induced decrease in brain glucose uptake could be attributed to
multiple levels of metabolic dysregulation, including reduced glucose transport,
compromised glycolysis, deficient mitochondrial capacity and impaired ATP generation
72
(Brinton, 2008; Nilsen and Diaz Brinton, 2003; Nilsen et al., 2007; Yao et al., 2011a). In
these experiments, we determined the impact of ovarian hormone deprivation and E2 on
the expression of glucose transporters in brain and neural cells via specific glucose
transporters (GLUT) expressed in blood brain barrier (BBB) endothelial cells (GLUT1,
55KDa), glial cells (GLUT1, 45KDa) and neurons (GLUT3) (Vannucci et al., 1998).
To investigate whether the OVX-induced decline in brain glucose uptake is
correlated with compromised glucose transport, expression of BBB GLUT1
55Kda,
glial
GLUT1
45Kda
and neuronal GLUT3 was assessed. BBB GLUT1
55Kda
expression was
significantly decreased 38% by OVX (Figure III-3C; 62.16 ± 13.59% (OVX group) vs.
100 ± 8.37% (Sham-OVX group), p < 0.05, n = 5 – 6) in 3xTgAD female mice.
Treatment with E2 completely prevented the OVX-induced decline in BBB GLUT1
55Kda
expression (Fig 3C; 96.42 ± 5.56% (E2 group) vs. 62.16 ± 13.59% (OVX group), p <
0.05, n = 5 − 6). However, the expression level of GLUT1
55Kda
did not correlate with
complete reversal of brain glucose uptake relative to Sham-OVX (Figure III-3B). This
finding indicated that other key enzymes involved in brain glucose metabolism are
required for complete reversal of the OVX-induced decline in glucose uptake.
OVX also induced a 20% decline (Figure III-3D; 80.63 ± 10.68% (OVX group)
vs. 100 ± 10.28% (Sham-OVX), p > 0.1, n = 5 − 6) in neuronal GLUT3 expression,
which was partially prevented by E2 (Figure III-3D; 88.11 ± 8.05% (E2 group) vs. 80.63
± 10.68% (OVX group), p > 0.1, n = 5 − 6). Neither OVX nor E2 had a significant effect
on the glial transporter GLUT1
45Kda
protein expression (Figure III-3E). Collectively,
these data indicate that ovarian hormone loss compromised glucose transport through the
blood brain barrier (BBB) and neurons and spared glial glucose transporter expression.
73
E2 prevented the OVX-induced decline of BBB GLUT1
55Kda
expression, with a partial
preventative effect on neuronal GLUT3 expression. Further, the glial glucose transporter
is not regulated by ovarian hormones as evidenced by no change in response to OVX and
E2.
Figure III-3. Ovariectomy (OVX) induced decline in brain glucose uptake and BBB GLUT1
55Kda
expression: prevention by 17β-estradiol. A. Representative FDG-microPET images showed a
decline in brain glucose uptake in OVX condition, which was partially prevented by E2 treatment
(50µg/kg, SC). (Red and yellow indicate higher values; green and blue indicate lower values). B.
Quantitative analysis of brain glucose uptake demonstrated a significant decrease in OVX condition
(**, p < 0.01, bars represent mean value ± SEM, n = 5 − 7), which was partially prevented by E2
treatment (50µg/kg, SC) (*, p < 0.05; bars represent mean value ± SEM, n = 5 − 6). C. OVX induced a
significant decrease in protein expression of blood brain barrier glucose transporter 1 (GLUT1
55Kda
),
which was prevented by E2 treatment (50µg/kg, SC). (*, p < 0.05, bars represent mean value ± SEM,
n = 5 − 6). D. OVX induced a 20% decrease in protein expression of neuronal glucose transporter
(GLUT3), which is partially prevented by E2 treatment (50µg/kg, SC). E. No change in expressions of
glial glucose transporter (GLUT1
45Kda
) between Sham-OVX, OVX and OVX+E2 groups.
Ovariectomy induced decrease in hexokinase expression and activity: prevention by
17β-estradiol
74
In neurons and glial cells, glucose is irreversibly phosphorylated to glucose-6-
phosphate by hexokinase, which is the first and rate-limiting step in glycolysis. The
glucose uptake signal in FDG-PET imaging is also dependent upon hexokinase activity,
as cellular FDG accumulation is mediated by hexokinase that phosphorylates FDG to
FDG-6-phosphate, which does not undergo further metabolism (Pauwels et al., 2000). To
investigate whether OVX-induced decline in glucose uptake is associated with
compromised glucose phosphorylation, we first analyzed the expression of hexokinase
type II, which is the isozyme sensitive to hormonal regulation (Wilson, 1995). In
3xTgAD female brain, OVX induced a significant 30% decline in hexokinase II protein
expression (Figure III- 4A; 68.49 ± 8.01% (OVX group) vs. 100 ± 8.03% (Sham-OVX
group), p < 0.05, n = 5 − 6), which was partially but not significantly prevented by E2
(Figure III- 4A; 86.17 ± 10.61% (E2 group) vs. 68.49 ± 8.01% (OVX group), p = 0.11, n
= 5 − 6). The OVX-induced decline in hexokinase expression was paralleled by a
significant decline in hexokinase activity, although this 15% decline was not as great as
the decline in protein expression (Figure III- 4B; 84.80 ± 5.26% (OVX group) vs. 100 ±
3.4% (Sham-OVX group), p < 0.05, n = 5 − 6). As with protein expression, E2 partially
but not significantly prevented the OVX-induced decline in hexokinase activity (Figure
III- 4B; 96.07 ± 7.43% (E2 group) vs. 84.80 ± 5.26% (OVX group), p = 0.14, n = 5 − 6).
The magnitude of the OVX-induced decline in hexokinase protein expression and
activity was positively correlated with the OVX-induced decline in brain glucose uptake
(Figure III-3B). These data are consistent with the key role of hexokinase in the FDG-
PET signal (Sharma et al., 2002). Further, the impact of E2 on preventing decline in HEX
75
protein expression and activity is consistent its partial prevention of the OVX-induced
decline in FDG-PET signal.
Figure III-4. Ovariectomy (OVX) induced decrease in hexokinase expression and activity:
prevention by 17β-estradiol. A. In female 3xTgAD brains, OVX induced a significant decrease in
protein expression of hexokinase 2 (*, P < 0.05, bars represent mean value ± SEM, n = 5 − 6), which
was partially prevented by E2 treatment. B. OVX induced a significant decrease in hexokinase activity
(*, p < 0.05, bars represent mean value ± SEM, n = 5 − 6), which was partially prevented by E2
treatment.
Ovariectomy induced a shift in LDH1 and LDH5 ratio: prevention by 17β-estradiol
Decreased glucose supply and utilization could activate alternative metabolic
pathways to compensate for decline in brain glucose metabolism. As we and others have
shown, neurons can utilize lactate or ketone bodies as alternative fuels (Belanger et al.,
2011; Pellerin, 2003; Pellerin et al., 1998; Yao et al., 2010). In response to energetic
demand, brain can utilize lactate to sustain synaptic transmission, whereas under
prolonged glucose deprivation, the brain will utilize ketone bodies generated from the
liver to support the energetic demand (Yao et al., 2011b). To investigate whether OVX
induced a shift in utilization of alternative fuels, we first determined lactate
76
dehydrogenase protein expression level in the Sham-OVX, OVX and OVX+E2 mice.
Multiple isoforms of lactate dehydrogenase are expressed in glial and neuronal cell types,
which enable these cells to generate and utilize lactate. The LDH5 isoform, which is
composed of four A subunits, converts pyruvate to lactate in glial cells. LDH1 isoform,
which is formed by four B subunits, converts lactate to pyruvate for ATP generation in
neurons (Laughton et al., 2007; Venkov et al., 1976). In the OVX condition, LDH5 glial
cell expression was dramatically increased (Figure III- 5A; 178 ± 33.34% (OVX group)
vs. 100 ± 14.51% (Sham-OVX group), p< 0.05, n = 5 − 6). In neurons, LDH1 expression
exhibited a similar increase in expression, although not significant (Figure III- 5B; 125.22
± 16.32 % (OVX group) vs. 100 ± 8.69 % (Sham-OVX group), p = 0.09, n = 5 − 6).
An increased LDH5/LDH1 ratio can be indicative of elevated lactate in the mouse
and human brain, and is consistent with an aging phenotype (Ross et al., 2010; Yesavage
et al., 1982). Based on the elevated expression of LDH5 to LDH1, we tested whether
there was a significant increase in LDH5 to LDH1 ratio. The ratio of LDH5/LDH1
expression was significantly increased in the OVX condition relative to Sham-OVX
(Figure III- 5C; 139.70 ± 9.37 % (OVX group) vs.100 ± 10.72 % (Sham-OVX group), p
< 0.05, n = 5 − 6). Animals treated with E2 at the time of OVX did not exhibit an
increase in the LDH5/LDH1 ratio and were indistinguishable from Sham-OVX (Figure
III- 5C; 76.36 ± 10.90 % (E2 group) vs.100 ± 10.72 % (Sham-OVX group), p = 0.16, n =
5 − 6). Under OVX condition, the increased expression of the enzyme LDH5 required for
conversion of pyruvate to lactate by glial cells is predicative of greater lactate generation
relative to consumption in neurons, suggesting the potential of lactate accumulation in the
hippocampus. Prevention of the rise in LDH5 expression and prevention of the increase
77
in LDH5/LDH1 ratio by E2 is predicative of balanced lactate generation to utilization in
hippocampus.
Figure III-5. Ovariectomy (OVX) induced a shift in LDH1 and LDH5 ratio and prevention by
17β-estradiol. A. OVX induced a significant increase (*, p < 0.05, bars represent mean value ± SEM,
n = 5 − 6) in the expression of glial lactate dehydrogenase 5 (LDH5) expression. B. OVX induced a
25% increase in neuronal lactate dehydrogenase 1 (LDH1). C. The lactate production indicator,
LDH5/LDH1 ratio increased significantly (*, p < 0.05, bars represent mean value ± SEM, n = 5 − 6).
All OVX-induced changes were prevented by E2 treatment.
Ovariectomy activated the ketogenic pathway: prevention by 17β-estradiol
Under prolonged glucose deprivation, the brain can utilize ketone bodies as
energy substrates. To determine whether OVX induced a shift in utilization to ketone
bodies, we determined ketone body level and the expression of the ketone body
transporter MCT2 and the metabolic enzyme Succinyl-CoA: 3-ketoacid CoA transferase
78
(SCOT) required to convert ketone bodies to acetyl-CoA. To assess liver generation of
ketone bodies as an alternative fuel for utilization by brain, we determined the level of β-
hydroxybutyrate in the plasma (Bentourkia et al., 2009). OVX induced a significant 150%
rise in the serum level of β-hydroxybutyrate (Fig 6A; 0.2886 ± 0.0897 (OVX group) vs.
0.1142 ± 0.0132 (Sham-OVX group), p < 0.05, n = 5 − 6), the major circulating ketone
body generated by the liver. In parallel with the increased ketone levels, protein
expression of neuronal monocarboxylate transporter (MCT2), which transports ketone
bodies or lactate, was increased by 50% in the OVX condition (Fig 6B; 153.34 ± 7.63%
(OVX group) vs. 100 ± 4.41% (Sham-OVX group), p < 0.05, n = 5 − 6). SCOT, the key
enzyme that catabolizes ketone bodies to acetyl-CoA for subsequent ATP generation, was
also increased by OVX (Fig 6C; 130.40 ± 11.60% (OVX group) vs. 100 ± 10.60%
(Sham-OVX group), p = 0.09, n = 5 − 6). Treatment with E2 initiated at the time of OVX
prevented OVX-induced increase in β-hydroxybutyrate level (Fig 6A; 0.1304 ± 0.0232
(E2 group) vs. 0.2886 ± 0.0897 (OVX group), p = 0.06, n = 5 − 6) and expression of
MCT2 (Fig 6B; 126.42 ± 9.48% (E2 group) vs. 153.34 ± 7.63% (OVX group), p = 0.06,
n = 5). E2 treatment also significantly prevented the protein expression of SCOT (Fig 6C;
95.84 ± 5.54% (E2 group) vs. 125.14 ± 14.25% (OVX group), p < 0.05, n = 5 − 6).
Collectively, the data indicate that OVX induces a significant rise in the generation of
ketone bodies, the enzyme required to convert ketone bodies to acetyl-CoA and transport
of ketone bodies into neurons. E2 treatment at the time of OVX prevented the rise in the
ketogenic system.
79
Figure III-6. Ovariectomy (OVX) induced activation of ketogenic pathway and prevention by
17β-estradiol. A. OVX induced a significant increase (*, p < 0.05, bars represent mean value ± SEM,
n = 5 − 6) in serum β-hydroxybutyrate level, which was prevented by E2 treatment. B. OVX induced a
significant increase (*, p < 0.05, bars represent mean value ± SEM, n = 5 − 6) in the expression of
neuronal monocarboxylate transporter 2 (MCT2), which was prevented by E2 treatment. C. OVX
induced a significant increase (*, p < 0.05, bars represent mean value ± SEM, n = 5 − 6) in the
expressions of 3-oxoacid-CoA transferase (SCOT), which was prevented by E2 treatment.
Ovariectomy induced increase in β-amyloid levels in brain: prevention by 17β-estradiol
Previous analyses demonstrated that female 3xTgAD mouse model showed an
age-related increase in β-amyloid deposition in brain (Yao et al., 2009). To investigate
whether an increase in β-amyloid level is associated with ovarian hormone loss, we
assessed the β-amyloid oligomers in the 3xTgAD cortex samples. We characterized three
major Aβ related bands: full length APP band (~100 Kda), Aβ dodecamers (56Kda) and
Aβ hexamers (27Kda) (Figure III- 7A). There was no significant difference in APP
80
expression across all three groups (Figure III-7B). Compared to the Sham OVX, OVX
induced a significant increase in the protein level of Aβ oligomers (Figure III-7C and
Figure III-7D; 56Kda Aβ: 241 ± 32.3% (OVX group) vs. 100 ± 13.3% (Sham-OVX
group), p < 0.01; 27Kda Aβ: 197 ± 8.7% (OVX group) vs. 100 ± 8.7% (Sham-OVX
group), p < 0.01; n = 4 - 6). E2 treatment partially prevented the OVX-induced increase
in Aβ oligomer levels (Figure III-7C and Figure III-7D; 56Kda Aβ: 180 ± 33.7% (E2
group) vs. 241 ± 32.3% (OVX group), p = 0.11; 27Kda Aβ: 120 ± 32.3% (E2 group) vs.
100 ± 8.7% (OVX group), p < 0.05; n = 4 - 5). Data of analyses suggest that changes in
ovarian hormone status, such as OVX and E2 treatment, induced alteration in the
amyloidogenic pathway and/or Aβ clearance pathway, rather than directly regulating
APP availability.
Figure III-7. Ovariectomy (OVX) induced increase in β-amyloid level: prevention by 17β-
estradiol. A. Three Aβ related bands were characterized: full length APP band (~100 Kda), Aβ
dodecamers (56Kda) and Aβ hexamers (27Kda). B. There is no significant change of APP expressions
between Sham-OVX, OVX and OVX+E2 groups. C. OVX induced a significant increase (**, p <
81
0.01, bars represent mean value ± SEM, n = 4 – 6) in the expression of 56Kda Aβ. D. OVX induced a
significant increase (**, p < 0.01, bars represent mean value ± SEM, n = 4 – 6) in the expression of
27Kda Aβ, which was partially prevented by E2 treatment (*, p < 0.05; bars represent mean value ±
SEM, n = 4 − 5).
17β-estradiol upregulated bioenergetic gene expression: a broader impact on brain
energy production
To investigate at a systems level the impact of ovarian hormone loss and E2
treatment on the bioenergetic profile in 3xTgAD mouse brains, we designed a custom
bioenergetic low-density gene array and conducted real time RT-PCR analyses to assess
the expression of key genes involved in brain bioenergetics (Figure III-8A). Gene
expression data presented in the volcano plot displays fold change vs. p-value (Figure III-
8B and Figure III-8C), with significantly changed genes listed respectively (Figure III-8D
and Figure III-8E). Results presented in the volcano plot demonstrated that when
compared to Sham-OVX, OVX had a relatively small impact with 4 genes (Sdhc, Idh1,
Cpt1c, Star) that exhibited significant changes (Figure III-8B and Figure III-8D). E2
treatment induced a much broader response with a total of 10 genes significantly changed
when compared to the OVX group, including 1 gene involved in glycolysis (Pdhb), 4
genes involved in TCA and electron transport chain (ETC) (Sdha, Sdhb, Uqcrc2, Cyb5b),
3 genes involved in fatty acid metabolism (Acaa2, Acat1, Hadha), and 2 genes involved
in cellular redox and insulin signaling (Prdx3 and Irs4 respectively) (Figure III-8C and
Figure III-8E).
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Figure III-8. E2 treatment significantly regulated Bioenergetic gene expression. A. Gene list of
custom bioenergetic genearray. B. Gene expression data presented in the valcano plot which displays
fold changes vs p-values between Sham-OVX and OVX demonstrated that OVX had little impact on
the expression of the bioenergetic genes. C. Gene expression data presented in the valcano plot which
displays fold change vs p-values between OVX+E2 and OVX demonstrated that E2 treatment exerted
a broad impact on the expression of genes involved in bioenergetic pathways. Each dot representes
one individual gene; Red: up-regulated gene expression compared to OVX; Green: down-regulated
gene expression compared to OVX; Dots above the bule line represent genes that are significantly
regulated. D. Compared to OVX group, genes that were significantly changed in Sham-OVX group
were categorized into 3 different functional groups: TCA/ETC/Oxphos (tricarboxylic acid
cycle/electron transport chain/oxidative phosphorylation), fatty acid metabolism, and cholesterol
trafficking. Data is presented as fold change relative to the OVX group with the corresponding p value
listed for each individual gene. E. Compared to OVX group, genes that were significantly changed in
OVX+E2 group were categorized into 5 different functional groups: glycolysis, TCA/ETC/Oxphos,
fatty acid metabolism, redox and IGF signaling. Data is presented as fold change relative to the OVX
group with the corresponding p value listed for each individual gene.
83
Discussion
In this study, we demonstrated that loss of ovarian hormones induced a decline in
glucose-driven brain bioenergetics, which was associated with dysregulated body weight
and temperature control. We further identified a compensatory shift from glucose-driven
brain bioenergetics towards an alternative substrate pathway (lactate and ketone bodies)
induced by ovarian hormone loss. Although E2 treatment completely prevented the
dysregulation of body weight and temperature control, the efficacy of E2 to prevent
decline in on bioenergetic capacity of the brain was partial (Figure III- 9).
Figure III-9. Impact of ovariectomy on bioenergetic system of 3xTgAD female brain.
Ovariectomy (OVX) induced a decline in brain glucose metabolism as shown by the functional
microPET imaging and biochemical analyses. This decline is paralleled by an activation of the
lactate/ketone compensatory pathway. E2 treatment partially prevented the OVX-induced decline in
brain glucose metabolism and partially prevented the activation of ketogenic pathway.
Classic indicators of ovarian hormone loss occurred in the ovariectomized
3xTgAD mice, evidenced by an increase in body weight and tail skin temperature within
5 weeks, a time frame comparable to 2.5 years of human life (Klepper, 2004; Lazic,
2011). These results are consistent with clinical observations of the metabolic and
84
thermal dysregulation in surgically oophorectomized premenopausal women (Gallicchio
et al., 2006; Haney and Wild, 2007).
It is well known that the decline in brain glucose metabolism is associated with
decreased cognitive function in mild cognitive impairment and Alzheimer’s disease
(Chen et al., 2010; Mosconi, 2005; Mosconi et al., 2007; Mosconi et al., 2009b; Mosconi
et al., 2006; Reiman et al., 1996; Reiman et al., 2004; Silverman et al., 2008) and, at least,
partially contributes to the neurodegenerative process (Yao et al., 2011b). ApoE ε4
carriers, who are cognitively normal but at increased risk for AD, exhibited deficits in
cerebral metabolic rate for glucose (CMRglu) in posterior cingulate (PCC) and parietal
temporal cortex several decades prior to onset of dementia (Reiman et al., 2004). Further,
cognitively normal people with a maternal family history AD, who have a higher risk for
AD (Edland et al., 1996), exhibited glucose hypometabolism in brain regions identified in
prodromal AD (Mosconi et al., 2007; Mosconi et al., 2009a). Collectively, these data
indicate that hypometabolism in brain is an early and persistent indicator of both risk for
and severity of Alzheimer’s disease (Mosconi, 2005; Mosconi et al., 2007; Mosconi et
al., 2009b; Mosconi et al., 2006; Reiman et al., 1996; Reiman et al., 2004).
Results of our analyses to determine the impact of OVX on expression of glucose
transporters in blood brain barrier, neurons and glial cells indicated a significant
reduction in blood brain barrier specific glucose transporters. The 55Kda form of GLUT1
mediates glucose transport across the blood brain barrier (BBB) and is a rate-limiting step
for brain glucose utilization under metabolic demand (Barros et al., 2005; Leybaert,
2005a). Findings from the current study in a mouse model of familial AD are consistent
with a decline in BBB GLUT1
55Kda
expression in late stage AD and compromised BBB
85
glucose transport (Kalaria and Harik, 1989). In patients with GLUT1 deficiency
syndrome, GLUT1 haplo-insufficiency leads to a global decrease in cortical glucose
uptake and multiple neurological deficits (De Vivo et al., 1991; Pascual et al., 2002). In
addition to GLUT1, GLUT3 is also associated with the pathogenesis of Alzheimer’s
(Simpson et al., 1994). In the current report, 5-week OVX induced a significant (38%)
decrease in the BBB GLUT1
55Kda
protein, whereas E2 treatment initiated at the time of
OVX prevented the OVX-induced decline. In contrast to the BBB GLUT1
55Kda
, the
neuronal glucose transporter GLUT3 and the glial glucose transporter GLUT1
45Kda
were
not significantly affected by 5-week OVX or E2 treatment. The decline in BBB
GLUT1
55Kda
in the face of sustained glial and neuronal GLUT expression is indicative of
compromised BBB glucose transport as an early response to ovarian hormone loss. These
results indicate a key role of BBB GLUT1
55Kda
in mediating the decline in the FDG-PET
detected brain glucose uptake induced by ovarian hormone loss.
In addition to glucose transporters, cerebral glucose utilization is under tight
control by hexokinase, which serves as the first rate-limiting step in glycolysis. In the
current study, ovarian hormone deprivation induced a significant decline in hexokinase
protein expression as well as activity, which was prevented by E2 treatment in 3xTgAD
female mice. Hexokinase interaction with the mitochondrial VDAC also plays an
important role in preventing mitochondria-mediated apoptosis and promoting cell
survival in neurons and other cell types (Azoulay-Zohar et al., 2004; Gimenez-Cassina et
al., 2009; Vyssokikh and Brdiczka, 2003). AD patients exhibit declined hexokinase
activity in the brain, cerebral microvessel, leukocytes and fibroblasts (Antuono et al.,
1995; Marcus and Freedman, 1997; Sorbi et al., 1990). Previous studies have
86
demonstrated that ovarian hormone loss led to compromised-brain hexokinase activity,
whereas short-term estrogen treatment was efficacious to increase hexokinase activity in
rat brains (Kostanyan and Nazaryan, 1992). Collectively, these data demonstrate that the
reduction in BBB GLUT1
55Kda
expression and hexokinase activity are key drivers for the
decline in brain glucose uptake.
Lactate is a well described bioenergetic fuel supplied by glia to neurons and is
particularly important under conditions of high synaptic activity, such as occurs during
learning and memory (Suzuki et al., 2011). Further, lactate can serve as an auxiliary fuel
during glucose insufficiency by metabolism of glycogen stores to generate glucose and
subsequently lactate (Aubert et al., 2005). In the current study, we found that OVX
induced an 80% increase in LDH5, suggestive of increased production of lactate in glial
cells for use as a compensatory substrate in neurons. However, LDH1 expression,
presumably neuronal, was modestly although not significantly increased following OVX.
These findings suggest that glial cells are likely to produce lactate in excess to its
utilization by neurons. Increased LDH5 expression relative to LDH1 is consistent with an
accelerated aging phenotype (Ross et al., 2010). This postulate is supported by data
derived from a premature aging model of mitochondrial mutation with high levels of
mtDNA point mutations and linear deletions. In this model, increased LDH5/LDH1 ratio
was associated with high levels of lactate in brain, which paralleled that observed in
aging (Ross et al., 2010). In the current study, the increased LDH5/LDH1 ratio in the
OVX condition suggests that ovarian hormone loss leads to lactate accumulation and an
accelerated-aging phenotype in the 3xTgAD female brain.
87
Under conditions of prolonged glucose deprivation, the brain shifts to utilization
of ketone bodies as an alternative fuel. Previous findings from our group demonstrated
that the bioenergetic fuel shift occurred in the 3xTgAD brain from the outset and
represents an AD metabolic phenotype (Yao et al., 2010). In the current report, we
observed an increase in serum ketone body concentration, which was accompanied by a
simultaneous increase in hippocampal expression of SCOT, the rate-limiting enzyme for
conversion of ketone bodies to acetyl-CoA for entry into the TCA cycle. In addition, the
neuronal MCT2 transporter for ketone bodies and lactate was also increased. These
changes indicate a coordinated upregulation of peripheral ketone supply, cellular
transport mechanism and conversion of ketone bodies to compensate for decline in
glucose metabolism in brain. E2 treatment prevented this shift to the ketogenic pathway
in brain, likely via preventing decline of the aerobic glycolytic pathway. Collectively,
these data demonstrated that ovarian hormone loss induced an accelerated AD phenotype,
which was partially prevented by E2 treatment.
β-amyloid deposition is the pathological marker of AD and is proposed to be
responsible for cognitive deficits in AD (Klein et al., 2004; Klyubin et al., 2005; Lesne et
al., 2006). Consistent with the previous findings in hippocampus, the current study
demonstrated that OVX activates amyloidogenic pathway in cortex, indicating that
ovarian hormone loss and E2 treatment induced a brain-wide systems response in the
amyloidogenic pathway as well as the Aβ clearance pathway (Yao et al., 2011a). In
addition, increased β-amyloid levels paralleled the decline in hexokinase activity.
Previous studies reported that β-amyloid triggers the release of neuronal hexokinase-1
from mitochondria and inactivates hexokinase-1 (Lesne et al., 2006). Taken together,
88
these results provide a plausible mechanism underlying clinical observations that decline
in brain glucose metabolism is associated with an increase in β-amyloid deposition
(Belanger et al., 2011; Pavlides et al., 2010).
Gene expression analyses demonstrated that genes involved in the TCA cycle
were increased in the OVX group (Figure 8D), suggesting a potential compensatory
activation of acetyl-CoA utilization in response to decreased glucose transport and
compromised glycolysis induced by OVX. E2 treatment significantly increased the
expression of genes in Complex II (Sdha and Sdhb) and Complex III (Uqcrc2) (Figure
8E), suggesting an up-regulation in ETC function. Together with the observation that E2
treatment prevented decline in glucose transporters and hexokinase activity in cytosolic
compartment, these data suggest a system-level regulation of brain glucose metabolism
and bioenergetic function by E2 in multiple function domains spanning from glucose
transport to glycolysis and to ETC.
In the current study, E2 treatment in the 3xTgAD brain partially prevented OVX-
induced decline in whole brain glucose uptake and a shift to alternative substrates in the
hippocampus, whereas E2 treatment completely prevented dysregulation in temperature
and body weight. The E2 prevention of dysregulation of temperature and body weight
indicates that the E2 dose and regimen was appropriate to regulate hypothalamic
functions. The dissociation between hypothalamic and hippocampal response to E2 could
be explained by: 1) a different E2 dose requirement; 2) the 3xTgAD female brain has a
diminished response to E2; 3) the necessity for another ovarian hormone, progesterone,
as was shown for prevention of OVX-induced rise in β-amyloid (Rosario et al., 2012).
89
The purpose of the current study was to determine whether bioenergetic deficits in
mitochondrial function previously observed (Yao et al., 2011a) were accompanied by
changes in glucose availability to sustain aerobic glycolysis and whether there was
activation of compensatory responses for the transport and metabolism of alternative
fuels. Collectively, the data demonstrate that loss of ovarian hormones led to a significant
decline of glucose uptake, glucose transporter expression and in the enzymes required for
glucose metabolism. Coincident with the decline in glucose uptake, transport and
metabolism induced by OVX, there was a significant rise in the transporter for alternative
fuels and the enzymes required for their metabolism. These results together with our
previous findings demonstrate that loss of ovarian hormones is associated with a system
wide decline in glucose transport and utilization in brain and a compensatory rise in the
systems required to utilize alternative, less efficient fuels and induced an accelerated AD
phenotype.
90
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Chapter IV. Ovariectomy induced a decline in brain glucose uptake
associated with peripheral glucose intolerance
Abstract
Previous studies demonstrated that female brain aging was associated with a
significant decline in brain glucose uptake, glucose transport, glycolytic capacity and
mitochondrial function in both female non-transgenic (nonTg) and triple-transgenic
(3xTgAD) mouse models. In the present study, we investigated the impact of
ovariectomy and 17β-estradiol replacement on brain energy substrate availability and
metabolism from a systemic perspective. Results of these analyses indicated that ovarian
hormone deprivation by ovariectomy (OVX) induced potential insulin resistance, as
evidenced by an increase in fasting serum insulin level and glucose intolerance,
suggesting dysregulation of glucose homeostasis. Further, OVX induced a significant
decline in brain glucose uptake measured as decline in 2-[
18
F]fluoro-2-deoxy-D-glucose
uptake measured by microPET-imaging. Mechanistically, OVX induced a decrease in
neuronal glucose transporter 4 (GLUT4) and glial GLUT1
45Kda
expression. The decline in
glucose availability was accompanied by an increase in serum ketone β-hydroxybutyrate
level, indicating the activation of peripheral alternative fuel supply for the OVX brain.
17β-estradiol prevented the OVX-induced dysregulation of glucose homeostasis and the
OVX-induced decline in brain glucose availability. Compared to the OVX group, 17β-
estradiol treatment increased glucose uptake, glucose transporter expression and
expression of genes associated with aerobic glycolysis. Collectively, these data indicate
that ovarian hormone loss could be associated with dysregulation of glucose homeostasis
98
and a concomitant increase in alternative fuel supply. These findings also suggest a
critical role of estrogen in sustaining glucose homeostasis and brain substrate availability.
Introduction
Previous study demonstrated that female brain aging is associated with a decline
in brain glucose metabolism, including decreased brain glucose uptake and transport,
compromised glycolytic capacity and decreased mitochondrial function (Yao et al., 2010;
Yao et al., 2009). Parallel to the decline in brain glucose metabolism, there was an
activation of ketogenic pathway, reflected in the increase in plasma ketone body level,
ketone body transporter expression and enzymes expression in ketone body metabolism
(Ding et al., 2013; Yao et al., 2010; Yao et al., 2009). Brain glucose hypometabolism is
associated with increased risk of Alzheimer’s disease (AD). Clinical studies in persons
with increased risk of AD, such as APOE-ε4 carrier or maternal family history
descendants, revealed that brain glucose hypometabolism temporally precedes the onset
of AD (Mosconi, 2005; Mosconi et al., 2007; Reiman et al., 1996; Reiman et al., 2004).
Further in persons with AD, compromised brain glucose metabolism is accompanied by
the activation of alternative metabolic pathways, as evidenced by a utilization ratio of 2:1
glucose to alternative substrate in persons with incipient AD compared to a ratio of 29:1
in healthy elderly controls (Hoyer, 1991). These findings suggested that the decline in
brain glucose metabolism and shift to utilization of alternative fuels during female brain
aging are associated with increased risk of AD.
Basic scientific analyses revealed that estrogen imposes a regulation of brain
glucose metabolism by promoting brain glucose uptake, glycolysis, glycolytic-coupled
99
tricarboxylic acid cycle (TCA) function, mitochondrial respiration and ATP generation
(Brinton, 2008, 2009; Nilsen et al., 2007; Yao et al., 2011a). We previously demonstrated
that ovarian hormone loss induced a significant decline in mitochondrial function in
nonTg mouse, which can be prevented by 17β-estradiol (E2) treatment (Yao et al.,
2011a). From a translational perspective, these basic scientific findings are supported by
clinical analyses of glucose metabolism in menopausal women (Rasgon et al., 2005).
Postmenopausal women on estrogen therapy were reported to have increased cerebral
blood flow and cerebral metabolism relative to non-users, while non-users exhibited a
significant decline in glucose metabolism, particularly in the PCC and prefrontal cortex,
which closely resembled the hypometabolic profile of AD brains (Rasgon et al., 2005).
Both preclinical and clinical studies suggest an important role of ovarian hormones in
preventing female aging associated brain glucose hypometabolism and AD-like
bioenergetic phenotype.
Multiple clinical studies demonstrated that diabetes and insulin resistance are
associated with increased risk of Alzheimer’s disease (Biessels et al., 2006; Matsuzaki et
al., 2010; Ott et al., 1999). Findings from both preclinical study and clinical study
indicate that ovarian hormone loss increase the risk of developing impaired glucose
tolerance and diabetes whereas estrogen treatment increases insulin sensitivity and
promotes glucose metabolism, suggesting an important role of estrogen in regulating
glucose homeostasis (Margolis et al., 2004; Mauvais-Jarvis et al., 2013; Pirimoglu et al.,
2011; Saengsirisuwan et al., 2009). These findings suggest that loss of ovarian hormones-
associated dysregulation in glucose homeostasis could contribute to the decline in
cognition and increased risk of AD. Consistently, epidemiological studies revealed an
100
increased risk of dementia in menopausal women with metabolic syndromes, particularly
diabetes and insulin resistance, in (Gregg et al., 2000; Komulainen et al., 2007;
Logroscino et al., 2004).
In the current study, we sought to investigate the impact of ovarian hormone loss
on brain glucose metabolism from a systemic perspective, including glucose tolerance,
brain glucose availability and glycolytic capacity. We also investigated the efficacy of
17β-estradiol in preventing compromised glucose tolerance, brain glucose availability
and further promoting mitochondrial function. The goal of these analyses was to
determine the initiating events that lead to a dysfunctional bioenergetic system in the
brain. As a translational preclinical model relevant to reproductive aging in the human,
we utilized temperature and body weight dysregulation in ovariectomized nonTg mouse
model as the trigger to initiate analyses of brain metabolism. Results presented herein
demonstrate that ovarian hormone loss induced a decline in glucose tolerance and brain
glucose uptake, which could be attributed to decreased neuronal GLUT4 and glial
GLUT1
45Kda
expression. The OVX-induced decline in cerebral glucose metabolism was
accompanied by an increase in serum β-hydroxybutyrate concentration. Further, E2
treatment initiated at the time of OVX prevented the OVX-induced changes.
Materials and Methods
Animal Treatments and Ethics
All rodent experiments were performed following National Institutes of Health
guidelines on use of laboratory animals and an approved protocol (protocol number:
10217) by the University of Southern California Institutional Animal Care and Use
101
Committee. The presented study has been approved by the University of Southern
California Institutional Animal Care and Use Committee (Ethics Committee).
Experimental design
To investigate the change in substrate availability following removal of ovaries
and treatment with 17β-estradiol(E2), 6-month-old 3xTgAD mice were randomly
assigned into the following three treatment groups (n = 7 per group): sham
ovariectomized (Sham-OVX), ovariectomized (OVX), and OVX plus 17β-estradiol
(OVX+E2). Mice were bilaterally OVX and injected with corn oil or coin oil containing
E2 (50µg/kg, Subcutaneously) daily for 5 weeks (Figure IV-1).
Figure IV-1. Experimental paradigm. 6-month-old nonTg mice were randomly assigned into the
following three treatment groups (n = 7 per group): sham ovariectomized (Sham-OVX),
ovariectomized (OVX), and OVX plus 17β-estradiol (OVX+E2). Mice were bilaterally OVX and
injected with corn oil or corn oil with E2 (50µg/kg, Subcutaneously) daily for 5 weeks. Weight and
tail skin temperature were monitored twice per week during the sleep cycle before FDG-microPET
imaging. After FDG-microPET imaging, mice were sacrificed for in vitro analyses of substrate
transport and utilization.
Body weight and tail skin temperature (TST) measurement
Body weight and tail skin temperature (TST) were monitored twice per week
during the sleep cycle. To allow the mice to recover from the OVX surgery,
102
measurements were started one week after the surgery. TST measurements were
conducted using an infrared thermometer (BIO-152-IRB, Bioseb, Chaville, France)
designed for small rodents. TST was measured twice per week until an increase of 1
degree or higher of temperature was detected and sustained for 1 week. Body weight was
also determined twice per week subsequent to skin tail temperature measurement.
Following one week of sustained temperature dysregulation, mice underwent FDG-PET
imaging.
FDG-microPET and microCT imaging
Mice were maintained under anesthesia during microPET and microCT scans
with 2-2.5% isoflurane in oxygen. Scans were performed in an imaging chamber
equipped with a nose cone for anesthesia delivery and heating pad for body temperature
control. MicroPET imaging was performed with a microPET R4 rodent model scanner
(Concorde Microsystems Inc, Knoxville, TN) and micro CT imaging was performed on
MicroCAT II tomography (Siemens Preclinical Solutions, Knoxville, TN). Mice were
injected intravenously via the tail vein with radiotracer [
18
F] Fluoro-2-deoxy-2-D-
glucose (FDG, 200µCi, 100uL). Radioactive dose was determined prior to injection by
radioisotope dose calibrator (Capintec, CRC-712M). At 40min post-injection of FDG,
each mouse was positioned in the microPET scanner in the center of the 10.8cm
transaxial and 8cm axial field of view (FOV). Brain microPET data were collected for
10min followed by a 10 min microCT scan for the purpose of co-registration.
Co-registration of microPET and microCT data was performed using the AMIDE
software package (http://amide.sourceforge.net/). After co-registration of the PET and CT
images, ROI (region of interest) was defined and used to measure the radioactivity
103
concentration in brains. Decay correction was used to adjust the actual radioactivity
dosage injected (Actual radioactivity dosage at time of injection = Initial radioactivity ×
, T = T minutes between injected time point and initial time point). Data were
analyzed using Student’s t-test with differences between groups with a p value <0.05
considered statistically significant.
Brain tissue preparation and Western blot analysis
Upon completion of FDG-microPET imaging, 3xTgAD mice (n = 7 per group)
were sacrificed and the brains rapidly dissected on ice. Hippocampus was processed for
protein extraction using Tissue Protein Extraction Reagent (Thermo Scientific, Rockford,
IL, USA) with phosphatase and protease inhibitors (Sigma, St. Louis, MO, USA), and
protein concentrations were determined with the Bio-Rad Bradford assay. Equal amounts
of protein (20 µg/well) were loaded in each well of a 12.5% SDS PAGE criterion gel
(Bio-Rad, Hercules, CA) and electrophoresed with Tris/glycine running buffer. Proteins
were transferred to 0.45µm pore size polyvinylidene difluoride (PVDF) membranes and
immuneblotted with GLUT1 (glucose transporter 1) antibody (1:1500, Abcam,
Cambridge, MA, USA), GLUT3 (glucose transporter 3) antibody (1:1000, Abcam,
Cambridge, MA, USA), Hexokinase II antibody (1:1000, Millipore, Billerica, MD,
USA), MCT2 (Monocarboxylate transporter 2) antibody (1:1000, Millipore, Billerica,
MD, USA), SCOT (3-oxoacid-CoA transferase) antibody (1:100, Santa Cruz
Biotechnology, Santa Cruz, CA, USA), LDHB (lactate dehydrogenase B) antibody
(1:1000, Abcam, Cambridge, MA, USA), and LDH V (lactate dehydrogenase V)
antibody (1:1000, Abcam, Cambridge, MA, USA). HRP-conjugated anti-rabbit antibody
and HRP-conjugated anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA)
e
−ln2×
T
110
104
were used as secondary antibodies. Immunoreactive bands were visualized with Pierce
SuperSignal Chemiluminescent Substrates (Thermo Scientific, Waltham, MA, USA) and
captured by Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules,
CA, USA). All band intensities were quantified using the Un-Scan-it (version 6.0, Silk
Scientific, Orem, UT, USA) software.
Hexokinase activity assay
Hexokinase activity assay was measured by monitoring the conversion of NAD+
(nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine
dinucleotide) by following the change in absorption at 340 nm. The assay medium
contained: 0.1 µg/µL of the hippocampal tissue protein, 0.05M Tris*HCl, PH8.0, 13.3
mM MgCl2, 0.112M glucose, 0.227 mM NAD+, 0.5mM Adenosine 5’Triphosphate, and
1IU/mL glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides) in a final
volume of 150µL. The OD at λ = 340 nm was measured every 1 min for 30mins at a
temperature of 30°C. The increase in OD reflects the increase in NADPH concentration,
and the total hexokinase activity was calculated from the slope of the resulting curve.
Serum β-hydroxybutyrate measurement
The concentration of serum β-hydroxybutyrate was determined by β-
hydroxybutyrate liquicolor assay kit (Stanbio laboratory, TX, USA) following
manufacturer’s instructions.
Real-time RT-PCR gene expression profiling
Total RNA was isolated from mouse hippocampal tissues using the RNeasy kit
(Qiagen, Valencia, CA). The quality and quantity of RNA samples were determined
using the Experion RNA analysis kit (Bio-Rad, Hercules, CA). RNA samples were
105
reverse-transcribed to cDNA using the high capacity cDNA reverse transcription kit
(Applied Biosystems, Foster City, CA) and stored at -80°C for gene expression analysis.
Taqman low-density arrays (TLDA) were custom manufactured at Applied Biosystems.
Real-time RT-PCR experiment and data analysis were conducted following the methods
as described in our previous report (Zhao et al., 2012).
Glucose tolerance test
After overnight fasting, mice were injected with D-glucose (2g/kg,
Intraperitoneally). To measure glucose concentration, tail veins were punctured and a
small amount of blood was released and applied onto a glucometer (Freestyle Freedom
Lite Meter, Abbott Diabetes Care, Alameda, CA). Blood glucose levels were measured at
0, 5, 15, 30 and 60 minutes after injection of glucose.
Respiratory measurement
Mitochondrial oxygen consumption was measured polarographically using a
Clarke-type electrode. 100 µg of isolated mitochondria were placed in the respiration
chamber at 37 °C in respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES,
2 mM MgCl
2
, 1 mM EGTA) to yield a final concentration of 200 µg/ml After a 1-min
baseline recording, mitochondria were energized by the addition of glutamate (5 mM)
and malate (5 mM) as substrates. State 3 respiration was stimulated by the addition of
ADP (410 µM). State 4
0
respiration was induced by the addition of three pulses of the
adenine nucleotide translocator (ANT) inhibitor atractyloside (50 µM) to deplete ADP.
The rate of oxygen consumption was calculated based on the slope of the response of
isolated mitochondria to the successive administration of substrates. The respiratory
control ratio (RCR) was defined by dividing the rate of oxygen consumption/min for state
106
3 (presence of ADP) by the rate of oxygen consumption/min for state 4
0
respiration
(Absence of ADP by addition of atractyloside).
Results
Ovariectomy-induced increase in body weight and tail skin temperature (TST):
prevention by 17β-estradiol
It is well documented that ovarian hormone loss is associated with increased body
weight and temperature dysregulation in menopausal women. In this study, we used OVX
mice as a translational model of surgically oophorectomized premenopausal women. In
this model, body weight and tail skin temperature served as surrogate markers of
metabolic and temperature dysregulation.
OVX rapidly induced a significant increase in body weight (Figure IV-2A; 2.80 ±
0.25 (OVX group) vs. 1.64 ± 0.40 (Sham-OVX group), F (2, 13) = 6.839, p < 0.01, n = 5
− 6) one week after surgery and this increase was sustained throughout the duration of the
experiment. E2 treatment initiated at the time of OVX prevented OVX-induced body
weight increase. OVX also induced a significant rise in tail skin temperature 4 weeks
after the OVX surgery, which reached asymptote by week 5. Asymptotic temperature
triggered in vivo brain glucose imaging followed by in vitro analyses. Tail skin
temperature of OVX animals increased 1 °C ± 0.03 °C (Figure IV- 2B; 23.74 ± 0.09
(OVX group) vs. 22.42 ± 0.06 (Sham-OVX group), F (2,13) = 55.29, p < 0.0001, n = 5 −
6) by week 5 relative to Sham-OVX animals. OVX-induced rise in tail skin temperature
was prevented by E2 treatment initiated at the time of OVX.
107
Figure IV-2. Ovariectomy (OVX) induced increase in body weight and tail skin temperature and
prevention by 17β-estradiol. A. OVX induced a significant increase in body weight (2.80 ± 0.25
(OVX group) vs. 1.64 ± 0.40 (Sham-OVX group), F (2, 13) = 6.839, p < 0.01, n = 5 − 6) one week
after surgery, which was sustained through the duration of the experiment. E2 treatment (50µg/kg, SC)
initiated at time of OVX prevented the OVX-induced body weight gain. B. OVX induced a significant
rise (23.74 ± 0.09 (OVX group) vs. 22.42 ± 0.06 (Sham-OVX group), F (2,13) = 55.29, p < 0.0001, n
= 5 − 6) in tail skin temperature 4 weeks after surgery, and E2 treatment (50µg/kg, SC) initiated at
time of OVX prevented the OVX-induced increase in tail skin temperature.
Ovariectomy-induced glucose intolerance and increase in fasting insulin level:
prevention by 17β-estradiol
Both preclinical and clinical studies showed that ovarian hormone loss is
associated with impaired glucose tolerance (Kumagai et al., 1993; Pirimoglu et al., 2011;
Saengsirisuwan et al., 2009). In the current study, we sought to investigate the impact of
ovariectomy and E2 treatment (50µg/kg, daily SC) on glucose tolerance in 6-month
female mice. Compared to the Sham-OVX group, OVX group showed a significant
higher peak glucose concentration at 15 min (Figure IV- 3A; 198 ± 9.8 (OVX group) vs.
131.3 ± 7.3 (Sham-OVX group), p < 0.01, n = 4 - 5). E2 treatment (50µg/kg, daily SC) at
the time of OVX prevented the OVX-induced glucose intolerance (Figure IV- 3A; 124 ±
16.4 (E2 group) vs. 198 ± 9.8 (OVX group), p < 0.01, n = 4 - 5).
To investigate whether the OVX-induced glucose tolerance is associated with
altered insulin level, we measured the fasting serum insulin concentration. Compared to
108
the Sham-OVX group, OVX group showed higher fasting serum insulin concentration
(Figure IV- 3B; 0.49 ± 0.03 (OVX group) vs. 0.38 ± 0.05 (Sham-OVX group), p = 0.07,
n = 4 - 5). E2 treatment (50µg/kg, daily SC) at the time of OVX significantly prevented
the OVX-induced increase in fasting serum insulin concentration (Figure IV- 3B; 0.29 ±
0.07 (E2 group) vs. 0.49 ± 0.03 (OVX group), p < 0.05, n = 4 - 5).
Figure IV- 3. Ovariectomy (OVX) induced glucose intolerance and prevention by 17β-estradiol.
A. OVX induced a significant higher peak glucose concentration at 15 min as well as significant
higher glucose concentrations at 30 and 60 minitutes. E2 prevented the OVX induced changes. B.
OVX induced an increase in fasting seum insuin concentration, which can be prevented by E2
treatment (50µg/kg, SC) initiated at the time of OVX. *, p < 0.05, **, p < 0.01, bars represent mean
value ± SEM, n = 4 – 5.
Ovariectomy-induced decrease in brain glucose uptake: prevention by 17β-estradiol
Ovarian hormone loss is associated with cerebral metabolic decline. In the current
study, we sought to determine whether loss of ovarian hormones and associated decline
in brain metabolism were accompanied by a change in brain glucose uptake. To address
this question, FDG-microPET imaging was conducted to determine the impact of ovarian
hormone loss and E2 treatment (50µg/kg, daily SC) on brain glucose uptake in 6-month
female mice. Compared to the Sham-OVX group, 5 weeks OVX induced a significant 16%
decline in brain glucose uptake (Figure IV- 4A; 83.8 ± 4.5% (OVX group) vs. 100 ± 5.4%
109
(Sham-OVX group), p < 0.05, n = 7). Decline in brain glucose uptake was coincident
with thermal dysregulation in the OVX condition (Figure IV- 4B). E2 treatment (50µg/kg,
daily SC) at the time of OVX prevented the OVX-induced decline in brain glucose
uptake (Figure IV- 4A; 97.8 ± 2.4% (E2 group) vs. 83.8 ± 4.5% (OVX group), p < 0.05, n
= 4 − 7), consistent with the prevention of thermal dysregulation and weight gain (Figure
IV- 2).
Figure IV- 4. Ovariectomy (OVX) induced a decline in brain glucose uptake: prevention by 17β-
estradiol. A. Representative FDG-microPET images showed a decline in brain glucose uptake in
OVX condition, which was prevented by E2 treatment (50µg/kg, SC). (Yellow indicates higher
values; Red indicates lower values). B. Quantitative analysis of brain glucose uptake demonstrated a
significant decrease in OVX condition, which was prevented by E2 treatment (50µg/kg, SC) *, p <
0.05; bars represent mean value ± SEM, n = 5 – 7.
Ovariectomy-induced decline in brain glucose transporter expression: prevention by
17β-estradiol
The OVX-induced decrease in brain glucose uptake could be attributed to multiple levels
of metabolic dysregulation, including reduced glucose transport, compromised glycolysis,
deficient mitochondrial capacity and impaired ATP generation (Brinton, 2008; Nilsen
110
and Diaz Brinton, 2003; Nilsen et al., 2007; Yao et al., 2011a). In these experiments, we
determined the impact of ovarian hormone deprivation and E2 treatment on the
expression of neuronal insulin-sensitive glucose transporter 4 (GLUT4) and glial glucose
transporter 1 (GLUT1
45Kda
)!(Vannucci et al., 1998).
To investigate whether the OVX-induced decline in brain glucose uptake is
correlated with compromised glucose transport, expression of neuronal GLUT4 and
glial
GLUT1
45Kda
was assessed. Neuronal GLUT4 expression was decreased 13% by OVX
(Figure IV-5A; 87.43 ± 5.23% (OVX group) vs. 100 ± 3.40% (Sham-OVX group), p =
0.08, n = 5 – 6) in female nonTg mice. Treatment with E2 prevented the OVX-induced
decline in neuronal GLUT4 expression (Figure IV- 5A; 100.2 ± 3.14% (E2 group) vs.
87.43 ± 5.23% (OVX group), p = 0.08, n = 5 − 6). Moreover, OVX induced a significant
decline in glial GLUT1
45Kda
expression (Figure IV- 5B; 86.74 ± 3.73% (OVX group) vs.
100 ± 4.33% (Sham-OVX group), p < 0.05, n = 5 – 6), which was prevented by E2
treatment.
Figure IV- 5. Ovariectomy (OVX) induced a decline in neuronal GLUT4 and glial GLUT1
45Kda
expression, prevention by 17β-estradiol. A. OVX induced a decline in neuronal GLUT4 expression,
which was prevented by E2 treatment (50µg/kg, SC). B. OVX induced a significant decline in glial
GLUT1
45Kda
expression, which was prevented by E2 treatment (50µg/kg, SC). *, p < 0.05; bars
represent mean value ± SEM, n = 5 – 6.
111
No Change in lactation generation and utilization is associated with ovarian hormone
loss
A decline in brain glucose uptake and associated metabolic pathways should
induce mechanisms to compensate for the decline in glucose. Lactate utilization would be
the first such adaptive response to support brain energy demand (Pellerin, 2003; Pierre
and Pellerin, 2005; Yao et al., 2010). To investigate whether the decline in brain glucose
uptake is associated with a shift in utilization of lactate, we first determined the
expression of lactate dehydrogenase (LDH) protein. Glial and neuronal cells have
different LDH isoforms. LDH5 is the major isoform expressed in glial cells and converts
pyruvate to lactate to generate lactate whereas LDH1 is the major isoform in neurons and
functions to convert lactate to pyruvate thereby providing an indicator of lactate
utilization (Pellerin, 2003). The ratio of LDH5/LDH1 ratio provides an indicator of
whether the two systems are functioning in a coordinated manner or whether there is a
dysregulation between the system of generation and utilization of lactate predictive of
lactate accumulation. In the current study, we found that there was no change in LDH5
(Figure IV- 6A) and LDH1 expression (Figure IV- 6B) as well as LDH5/LDH1 ratio
(Figure IV- 6C). These data suggest that lactate is not likely to be the compensatory fuel
for the OVX brain.
112
Figure IV- 6. No change in the LDH5 and LDH1 expression and the ratio of LDH5/LDH1. A.
There was no change in LDH5 expression. B. There was no change in LDH1 expression. C.
LDH5/LDH1 ration did not change.
Ovariectomy-induced increase in serum ketone body level: prevention by 17β-estradiol
Because lactate is unlikely to be an alternative fuel to compensate for the decline
in glucose metabolism, we investigated whether ketone bodies were generated and
utilized during female brain aging. To determine whether a shift in utilization of ketone
bodies paralleled the decline in brain glucose uptake, we determined serum β-
hydroxybutyrate level. OVX induced an increase in serum level of β-hydroxybutyrate
(Figure IV- 7; 0.5637 ± 0.0512 (OVX group) vs. 0.4009 ± 0.0652 (Sham-OVX group), p
= 0.09, n = 5), the major circulating ketone body generated by the liver. Treatment with
E2 initiated at the time of OVX prevented OVX-induced increase in β-hydroxybutyrate
113
level (Figure IV- 7; 0.4070 ± 0.0424 (E2 group) vs. 0.5637 ± 0.0512 (OVX group), p =
0.08, n = 4 − 5).
Figure IV-7. Ovariectomy (OVX) induced an increase in serum β-hydroxybutyrate
concentration: prevention by 17β-estradiol. OVX induced an increase in serum β-hydroxybutyrate
concentration, which was prevented by E2 treatment initiated at the time of OVX.
17β-estradiol upregulated bioenergetic gene expression: a broader impact on brain
energy production
To investigate at a systems level the impact of ovarian hormone loss and E2
treatment on the bioenergetic profile in female nonTg brain, we designed a custom
bioenergetic low-density gene array and conducted real time RT-PCR analyses to assess
the expression of key genes involved in brain bioenergetics (Figure IV- 8A). Gene
expression data presented in the volcano plot displays fold change vs. p-value, with
significantly changed genes listed respectively.
Gene expression data presented in the volcano plot displays fold change vs. p-
value (Figure IV- 8B and Figure IV- 8C), with significantly changed genes listed
respectively (Figure IV- 8D and Figure IV- 8E). Results presented in the volcano plot
demonstrated that when compared to Sham-OVX, OVX had a small impact with only 1
gene (Prdx5) that exhibited significant change (Figure IV- 8B and Figure IV- 8D). E2
114
treatment induced a much broader response with a total of 10 genes significantly changed
when compared to the OVX group, including 1 gene involved in glycolysis (HK1), 5
genes involved in TCA and electron transport chain (ETC) (Uqcrc1, Atp5a1, Atp5b,
Cyb5b, Ogdh), 1 gene involved in insulin signaling (Irs1), 1 gene involved in
mitochondrial biogenesis (Sirt1) and 2 genes involved in cholesterol trafficking (Abca1
and Npc1 respectively) (Figure IV- 8C and Figure IV- 8E).
Figure IV- 8. E2 treatment significantly regulated Bioenergetic gene expression. A. Gene list of
custom bioenergetic genearray. B. Gene expression data presented in the valcano plot which displays
fold changes vs p-values between Sham-OVX and OVX demonstrated that OVX had little impact on
the expression of the bioenergetic genes. C. Gene expression data presented in the valcano plot which
displays fold change vs p-values between OVX+E2 and OVX demonstrated that E2 treatment exerted
a broad impact on the expression of genes involved in bioenergetic pathways. Each dot representes
one individual gene; Red: up-regulated gene expression compared to OVX; Green: down-regulated
gene expression compared to OVX; Dots above the bule line represent genes that are significantly
regulated. D. Compared to OVX group, there was only one gene involved in Redox that was
significantly changed. E. Compared to OVX group, genes that were significantly changed in OVX+E2
group were categorized into 5 different functional groups: glycolysis, TCA/ETC/Oxphos, insulin
signaling, mitochondrial biogenesis and cholesterol trafficking.
115
Discussion
In this study, we demonstrated that loss of ovarian hormones induced
dysregulation of glucose homeostasis, accompanied by dysregulated body weight and
temperature control. We further identified a decline in brain glucose availability and an
increase in serum ketone β-hydroxybutyrate induced by ovarian hormone loss. E2
treatment initiated at the time of OVX completely prevented the dysregulation in body
weight, temperature control and glucose homeostasis as well as the decline in brain
glucose availability.
Classic indicators of ovarian hormone loss occurred in the ovariectomized mice,
evidenced by an increase in body weight and tail skin temperature within 5 weeks, a time
frame comparable to 2.5 years of human life (Klepper, 2004; Lazic, 2011). These results
are consistent with clinical observations of metabolic and thermal dysregulation in
surgically oophorectomized premenopausal women (Gallicchio et al., 2006; Haney and
Wild, 2007). In addition to dysregulated body weight and temperature control, OVX also
induced dysregulation in glucose homeostasis, evidenced by glucose intolerance and
increased fasting serum insulin. The result is consistent with preclinical and clinical
studies showing that ovarian hormone loss increases the risk of developing impaired
glucose tolerance and diabetes whereas estrogen treatment increases insulin sensitivity
and enhances glucose metabolism (Margolis et al., 2004; Mauvais-Jarvis et al., 2013;
Pirimoglu et al., 2011; Saengsirisuwan et al., 2009). These findings suggest an important
role of estrogen in the systemic regulation of glucose homeostasis and metabolism.
In addition to the systemic regulation of glucose metabolism, estrogen regulates
brain glucose metabolism by promoting brain glucose uptake, glycolysis, glycolytic-
116
coupled tricarboxylic acid cycle (TCA) function, mitochondrial respiration and ATP
generation (Brinton, 2008, 2009; Nilsen et al., 2007; Yao et al., 2011a). Consistent with
these basic scientific findings, in our preclinical animal model, we found that OVX
induced a significant decline in brain glucose uptake, which can be prevented by E2
treatment. Clinical study also revealed that postmenopausal women on estrogen therapy
showed higher cerebral glucose metabolism relative to non-users while non-users
exhibited a significant decline in glucose metabolic rate (Rasgon et al., 2005). It is well
known that the decline in brain glucose metabolism is associated with decreased
cognitive function in mild cognitive impairment and Alzheimer’s disease (Chen et al.,
2010; Mosconi, 2005; Mosconi et al., 2007; Mosconi et al., 2009b; Mosconi et al., 2006;
Reiman et al., 1996; Reiman et al., 2004; Silverman et al., 2008) and, at least, partially
contributes to the neurodegenerative process (Yao et al., 2011b). These data suggest that
ovarian hormone loss associated brain glucose hypometabolism could contribute to the
increased risk of Alzheimer’s disease.
Results of our analyses to determine the impact of OVX on expression of glucose
transporters indicated a decline in neuronal GLUT4 and glial GLUT1
45Kda
expression.
Neuronal GLUT4 responds to insulin stimulation by translocating to plasma membrane
and thus increasing glucose uptake (Simpson and Cushman, 1986). In the current study,
we found that ovarian hormone loss induced a reduction in neuronal GLUT4 expression,
suggesting compromised potential to mediate glucose uptake during insulin stimulation.
In addition to neuronal GLUT4, OVX also induced a decline in the expression of
GLUT1
45Kda
, which is also reported to be partially insulin-sensitive (Clarke et al., 1984;
Schulingkamp et al., 2000; Schwartz et al., 1992). GLUT1
45Kda
is mainly expressed in
117
oligodendrocytes and astrocytes with localization including the astrocytic endfeet
adjacent to the BBB endothelial cell (Simpson et al., 2007; Yu and Ding, 1998). The
OVX-induced decline in GLUT1
45Kda
could decrease glucose uptake into astrocytes,
where glucose would be converted to lactate to serve as a compensatory fuel for neurons
or into oligodendrocytes to address energetic demands of lipid / myelin synthesis
(Rinholm et al., 2011; Sanchez-Abarca et al., 2001). However, the unchanged LDH5 to
generate lactate in astrocytes and LDH1 in neurons to utilize lactate makes this
compensatory option unlikely. The evidence suggest that decreased GLUT1
45Kda
is more
likely to induce a decline in lipid / myelin synthesis in oligodendrocytes.
Reduced glucose tolerance and increased starvation insulin level indicate a
potential insulin resistance, which is manifested by decreased cellular glucose uptake.
Insulin/ insulin-like growth factor-1 (IGF-1) signaling plays an important role in
regulating brain glucose metabolism (Duarte et al., 2012; Duarte et al., 2008; Schwartz et
al., 1992). Insulin resistance can lead to decreased PI3K/Akt signaling in brain (de la
Monte, 2012). PI3K/Akt signaling pathway is well known to be associated with
insulin/IGF1 regulation of glucose transport/metabolism. PI3K/Akt pathway activates the
translocation of GLUT4 from intracellular to cell membrane (Czech and Corvera, 1999;
Ueki et al., 1998). In addition, Akt-mediated pathway increases the expression of GLUT1
as well as its translocation to cell membrane (Edinger and Thompson, 2002; Rathmell et
al., 2003). These findings suggest the molecular mechanism underlying the glucose
hypometabolism observed in our female OVX mice. The OVX-induced decline in brain
glucose uptake could be attributed to decreased PI3K/Akt signaling pathway induced by
potential insulin resistance.
118
In response to decreased glucose uptake, the brain can utilize lactate or ketone
bodies as alternative fuels. Lactate is known to be a metabolic substrate for brain and is
particularly important under conditions of high synaptic activity, which occurs during
learning and memory (Suzuki et al., 2011). Further, during glucose insufficiency, lactate
can serve as a secondary fuel through glycogen metabolism (Aubert et al., 2005). In the
current study, there was no change in LDH5 or LDH1 expression, suggesting that lactate
is not likely to be the alternative fuel to compensate for the decline in brain glucose
uptake. In addition to lactate, ketone bodies can also serve as an alternative fuel for brain
during early development and starvation (Hawkins et al., 1971). In the current study, we
found that OVX induced an increase in serum ketone β-hydroxybutyrate level, indicating
an activation of peripheral compensatory fuel supply.
Gene expression analyses demonstrated that one gene (Prdx5) involved in the
redox was increased in the OVX group, which could be the adaptive response to
increased oxidative stress in the OVX group (Yao et al., 2011a). E2 treatment
significantly increased the expression of genes in glycolysis (HK1), mitochondrial
biogenesis (Sirt1) and TCA/ETC (Uqcrc1, Atp5a1, Atp5b, Cyb5b, Ogdh) (Figure IV- 8E),
suggesting an up-regulation in glycolysis and mitochondrial function. Together with the
observation that E2 treatment prevented decline in glucose transporters, these data
suggest a system-level regulation of brain glucose metabolism and bioenergetic function
by E2 in multiple function domains spanning from glucose transport to glycolysis and to
mitochondrial function.
Taken together, data in the current study demonstrate that loss of ovarian
hormones induced a significant decline of brain glucose uptake paralleled by potential
119
insulin resistance in the peripheral. Coincident with the decline in glucose uptake, there
was an increase in serum ketone body concentration. These results together with our
previous findings demonstrate that loss of ovarian hormones was associated with a
decline in glucose metabolism, which was possibly due to peripheral insulin resistance,
and a compensatory rise in the supply of alternative, less efficient fuels.
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Chapter V. Conclusions
The above chapters summarize my research on the impact of female brain aging
and estrogen regulation on brain fuel availability. The project is based on our existing
knowledge of brain aging in females and estrogen action on mitochondrial function and
brain bioenergetics (Brinton, 2008, 2009; Nilsen et al., 2007; Yao et al., 2010; Yao et al.,
2011a; Yao et al., 2009). The current project not only expands our understanding of the
trajectory of bioenergetic decline during female brain aging, but also characterizes the
shift in brain fuel availability associated with ovarian hormone dysregulation. As we and
others identified the association between brain bioenergetics and AD pathogenesis
(Mosconi, 2005; Mosconi et al., 2008b; Reiman et al., 1996; Reiman et al., 2004; Yao et
al., 2009), findings from the current project provide new information in the early
diagnosis and prevention of AD in menopausal women from a bioenergetic perspective.
In specific aim I, we demonstrated that brain aging in females is associated with a
decline in brain glucose uptake, glucose transport and aerobic glycolytic capacity, which
occurred early in the process of brain aging in both nonTg and 3xTgAD brains (Figure V-
1 and Figure V-2). The decrease in glucose availability and glycolytic capacity is also
accompanied by a shift towards utilization of alternative fuel (Figure V-1). These
findings indicated the impact of ovarian hormone dysregulation on brain fuel availability
during female aging. Thus, we continued to investigate the impact of ovarian hormone
loss and estrogen regulation on brain fuel availability in specific aim II. By using the
ovariectomized 3xTgAD mouse model, we demonstrated that ovarian hormone loss
induced a significant decline in brain glucose availability, which is accompanied by an
activation of compensatory fuel, including lactate and ketone bodies (Figure V-2).
126
Further, the OVX-induced bioenergetic fuel shift was paralleled by a significant increase
in Aβ oligomer levels. 17β-estradiol (E2) treatment partially prevented OVX-induced
bioenergetic fuel shift and reduced Aβ oligomer levels. Findings from specific aim I
suggest a “window of opportunity” at or around the early stage of brain aging in females.
In addition, data also suggest stepwise therapeutic strategies that target bioenergetic
deficits at different stages for prevention against a series of bioenergetic deficits.
Findings from specific aim II further establish the association between ovarian hormone
loss, bioenergetic fuel shift, and the development of AD pathology.
Figure V-1. Timeline of bioenergetic aging in female mammalian brain. A. In normal nonTg brain,
the decline in brain glucose transport and glycolytic capacity occurred between 6 and 9 months of age,
which temporally preceded mitochondrial dysfunction. The decline in brain bioenergetic was
paralleled with the increase in peripheral ketone body concentration. The expression of BBB and glial
ketone body transporter decreased after 9 months of age whereas the astrocytic ketone body
transporter increased at 15 months of age. B. In 3xTgAD brain, the decline in brain glucose transport
and glycolytic capacity also occurred between 6 and 9 months of age, which temporally preceded
mitochondrial dysfunction. The activation of the ketogenic pathway occurred at both early age and
early stage of female aging. The expression of BBB/glial and astrocytic ketone transporters was
maximal at early age (3 months) but decreased at early stage of female aging (9 to 15 months).
127
In specific aim III, using ovariectomized nonTg mouse model, we investigate the
impact of ovarian hormone loss on fuel availability from a systemic perspective. We
demonstrated that ovarian hormone loss induced glucose homeostatic dysregulation and
decreased brain glucose uptake, which is accompanied by an increase in serum ketone β-
hydroxybutyrate level (Figure V-2). Findings from specific aim III demonstrate that
ovarian hormone loss is associated not only with a decline in brain glucose availability,
but also with a systemic dysregulation of glucose homeostasis.
Taken together, from a translational perspective, our study suggests stepwise
therapeutic strategies that target bioenergetic deficits at different stages for prevention
against a series of bioenergetic deficits. Because compromised brain glucose uptake and
glycolytic capacity occur in the first place, the therapeutic strategy for preventing brain
glucose hypometabolism, such as hormone therapy, should be at or around the early stage
of female brain aging. Compromised brain glucose uptake is followed by mitochondrial
dysfunction, suggesting that mitochondria function could be the second-stage target.
Currently several mitochondrial enhancers such as R-α-lipoic acid and resveratrol are
under clinical trials (Packer and Cadenas, 2011; Wollen, 2010). While preventing glucose
hypometabolism or enhancing brain glucose bioenergetics could be the target at early
stage of female brain aging, our results suggested a potential therapeutic option for the
late stage. At late stage of female brain aging, there was a shift to utilize alternative fuels
such as ketone bodies in the brain. Therefore, increasing the alternative fuel (ketone
128
body) supply and transport could provide a fuel source that the brain is able to utilize at
this stage and prevents further exacerbation of brain bioenergetic deficits.
Figure V-2. Summary of changes in the expression level of key proteins involved in glucose
transport, glycolysis, alternative fuel transport and utilization in different conditions.
129
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Yao, J., Hamilton, R.T., Cadenas, E., and Brinton, R.D. (2010). Decline in mitochondrial
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131
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Abstract (if available)
Abstract
Previously, our lab have investigated the role of mitochondria in female brain aging and Alzheimer's disease. We demonstrated that mitochondrial bioenergetic deficit precedes Alzheimer's pathology. Further, reproductive senescence or ovariectomy induced ovarian hormone loss exacerbates mitochondrial deficit, which can be prevented by 17β-estradiol (E2) treatment. The purpose of the current project is 1) to characterize the changes in brain fuel availability during female aging and reproductive transition
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Ding, Fan
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Core Title
Bioenergetic fuel shift of reproductive aging: implication for late-onset Alzheimer's disease
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School of Pharmacy
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Doctor of Philosophy
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Molecular Pharmacology and Toxicology
Publication Date
07/16/2013
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03/26/2013
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Alzheimer's disease,brain metabolism,Estrogen,female aging,glucose transporter,OAI-PMH Harvest,ovarian hormone
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Brinton, Roberta D. (
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), Alkana, Ronald L. (
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), Cadenas, Enrique (
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), Chui, Helena (
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dingfan84@gmail.com,fding@usc.edu
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Tags
Alzheimer's disease
brain metabolism
female aging
glucose transporter
ovarian hormone