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Age dependent modulation of synaptic plasticity and insulin mimetic effect of lipoic acid on a 3xTg-AD mouse model of Alzheimer's disease: implications as a therapeutic/nutraceutical agent
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Age dependent modulation of synaptic plasticity and insulin mimetic effect of lipoic acid on a 3xTg-AD mouse model of Alzheimer's disease: implications as a therapeutic/nutraceutical agent
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1
AGE DEPENDENT MODULATION OF SYNAPTIC PLASTICITY AND INSULIN MIMETIC
EFFECT OF LIPOIC ACID ON A 3XTG-AD MOUSE MODEL OF ALZHEIMER’S DISEASE:
IMPLICATIONS AS A THERAPEUTIC/NUTRACEUTICAL AGENT
BY
HARSH SANCHETI
A Thesis 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)
May 2014
Copyright 2014 Harsh Sancheti
2
TABLE OF CONTENTS
Dedication 4
Acknowledgements 5
Abstract
6
CHAPTER 1: Overview of Alzheimer’s disease, its link to impaired energy
metabolism, and the role of lipoic acid
Introduction
Gaps in our understanding of Alzheimer’s disease
Substrate Supply to the brain
The major factors that need to be addressed in Alzheimer’s disease
Lipoic Acid
Hypothesis
Specific Aims
8
8
10
11
15
17
19
20
CHAPTER 2: Glucose uptake, PI3K/Akt cell signaling, and synaptic plasticity
in the 3xTg-AD mouse model and the effect of lipoic acid
Abstract
Introduction
Materials and Methods
Results
Discussion
Table
Figures
24
25
26
29
35
41
48
49
CHAPTER 3: Glucose hypometabolism in the old 3xTg-AD mouse model and
the effect of lipoic acid
Abstract
Introduction
Materials and Methods
Results
Discussion
Table
Figures
66
67
68
70
74
78
85
86
3
CHAPTER 4: Glucose hypermetabolism in the young 3xTg-AD mouse model
and the effect of lipoic acid
Abstract
Introduction
Materials and Methods
Results
Discussion
Tables
Figures
95
96
97
99
104
109
115
119
CHAPTER 5
Conclusions
128
References 131
4
DEDICATION
This thesis is dedicated to my grandfather Rajmal Sancheti (R.I.P), my parents Dinesh Sancheti
and Rajni Sancheti, my wife Mona Verma, my sister Prachi Parekh, and beloved young brother
Yash Sancheti.
‘Miles to go before I sleep and miles to go before I sleep’ - Robert Frost.
5
ACKNOWLEDGEMENTS
My sincere and heartfelt gratitude towards my mentor Sir Enrique Cadenas, M.D, Ph.D., for
his guidance, generosity and more importantly the trust and belief he bestowed upon me while I
completed my doctoral dissertation. His insightful scientific knowledge, expansive
understanding of the biological systems, well equipped laboratory, along with his noteworthy
macro-management skills has ensured my dissertation work to be a quest with excitement rather
than a task with tensions. My sincere thanks to all the collaborators, Keiko Kanamori, PhD,
Brian Ross, MD, PhD, Graeme Mason, PhD, Garnik Akopian, PhD, John Walsh, PhD, Eric
Hernandez, Roberta Diaz Brinton, PhD, Wei Zhang, PhD, and Ai-Ling Lin, PhD. These
collaborations between the USC School of Pharmacy, Huntington Medical Research Institute,
Yale School of Medicine, USC School of Gerontology, and the Research Imaging Institute
University of Texas Health Science Center at San Antonio made it possible to complete this
complex project. The assistance of Ishan Patil and Robert Martins, two graduate students who
worked with me was very helpful. I would like to thank David Carlson (Geronova, Inc) for
providing all the lipoic acid used in this study. My appreciation for Jerome Garcia, PhD, Li-Peng
Yap, PhD, Fei Yin, PhD, Derick Han, PhD, and all laboratory members for the intellectual
discussions, being very supportive, willing to teach and helping me understand the basics of
several laboratory techniques. I would like to thank my oral qualifying and doctoral thesis
committee members Wei Chiang Shen, PhD, Curtis Okamoto, PhD, Helena Chui, MD, and Brian
Ross, MD, PhD for their insightful suggestions and discussion.
Most importantly, my sincere thanks to Mona Verma for keeping me optimistic and cheerful
during my doctoral research work.
6
ABSTRACT
Alzheimer’s disease is a type of dementia that causes problems with memory, thinking and
behavior. Currently, there are no approved drugs to treat Alzheimer’s disease; however, certain
drugs that alter the course of the disease and improve quality of life have been approved by the
FDA (e.g. donepizil, rivastigmine, and galantamine). Symptoms usually develop slowly and
worsen over time, becoming severe enough to interfere with daily tasks and progressively disrupt
learning and memory-dependent activities. Synaptic plasticity is widely considered to be strongly
associated with learning and memory, two components that are extensively deregulated in
Alzheimer’s disease. On the other hand, human brain has the highest consumption of glucose
with respect to its size and longitudinal studies have shown that decreased brain glucose uptake
far precedes the pathology associated with Alzheimer’s disease. Additionally, several studies
have also shown the existence of brain insulin resistance in clinical and pre-clinical studies of
Alzheimer’s diseases. Importantly, brain insulin resistance can contribute to disturbances in brain
glucose uptake, brain glucose metabolism (that generates energy and neurotransmitters), and
ultimately lead to an impaired synaptic plasticity.
Thus, insulin resistance, disturbances in glucose uptake and metabolism, and impaired
synaptic plasticity are the major issues to be addressed for preventing the progression of
Alzheimer’s disease. Lipoic acid, a disulfide, has been shown to increase glucose uptake in
several tissues and improve age-related decline in synaptic plasticity; these effects of lipoic acid
are based on its participation in thiol/disulfide exchange reactions (e.g., activation of the insulin
receptor substrate). Owing to its participation in thiol/disulfide exchange reactions, lipoic acid is
expected to induce an insulin mimetic effect and overcome some of the major complications
associated with Alzheimer’s disease. The hypothesis to be tested here is that the insulin-like
7
effect of lipoic acid restores PI3K/Akt signaling and stimulates mitochondrial bioenergetics, thus
overcoming the energy deficit and impaired synaptic plasticity, inherent in AD.
The major experimental model used to examine the hypothesis is a triple transgenic mouse
model of Alzheimer's disease (3xTg-AD) that shows progression of pathology as a function of
age; two age groups: 7 months (young) and 13 months (old) were used in this study. Overall, it
was found that the 3xTg-AD mice showed disturbances in brain glucose uptake, insulin signaling,
glucose metabolism, and synaptic plasticity. The 3xTg-AD mice fed 0.23% w/v lipoic acid in
drinking water for 4 weeks showed restoration of brain glucose uptake, activation of the insulin
receptor substrate and of the PI3K/Akt signaling pathway. Additionally, there was restoration of
brain glucose metabolism and synaptic plasticity. It must also be noted that lipoic acid was more
effective in stimulating an insulin-like effect and reversing the impaired synaptic plasticity in the
old mice, wherein the impairment of insulin signaling and synaptic plasticity was more
pronounced than those in young mice.
Overall, these studies show a state of brain insulin resistance and impaired synaptic plasticity
in a very advanced pre-clinical mouse model of Alzheimer’s disease i.e., 3xTg-AD; importantly,
lipoic acid reverses those states. Thus, these studies present functional and mechanistic insights
that solidify the need for a large multi-center double-blinded clinical study of testing the efficacy
of lipoic acid as a therapeutic/nutraceutical agent in Alzheimer’s disease.
8
CHAPTER 1: Overview of Alzheimer’s disease, its link to impaired energy metabolism,
and the role of lipoic acid
INTRODUCTION
GAPS IN OUR UNDERSTANDING OF ALZHEIMER’S DISEASE
SUBSTRATE SUPPLY TO THE BRAIN
THE MAJOR FACTORS THAT NEED TO BE ADDRESSED IN ALZHEIMER’S DISEASE
LIPOIC ACID
HYPOTHESIS
SPECIFIC AIMS
INTRODUCTION
The significance of this study stems from the fact that, among the top ten causes of death in
United States, Alzheimer’s disease is the only cause of death that cannot be prevented, cured, or
even slowed down, and the number of deaths has risen by 68%
from 2000-2008 as shown in Fig. 1 (obtained from Alzheimer’s
Association (www.alz.org)). Worldwide it is estimated to affect
25-30 million people [1]. It is alarming that this number is
expected to triple by 2040.
Alzheimer’s disease is characterized by loss of brain
function and causes problems with memory, thinking and
behavior; ultimately leading to destruction of brain cells. Its risk
increases exponentially after 65 years. It has been categorized into two major forms: Familial
9
Alzheimer’s Disease (FAD) and Sporadic Alzheimer’s Disease (SAD). FAD has onset before 65
years of age mainly due to mutations in three genes, i.e., Amyloid Precursor Protein (APP),
preselin 1 (PSEN), and preselin 2 (PSEN2). Although SAD usually has a later onset, its etiology
is not fully understood [2] [3]. Alzheimer’s disease is characterized by progressive cortex and
hippocampus atrophy. In the cortex, marked atrophy is seen in the frontal, parietal, and temporal
lobes; whereas, the hippocampal atrophy can also extend into the amygdala [4,5]. The
pathologies seen in Alzheimer’s disease are centered on abnormalities in two proteins i.e.,
Amyloid β (Aβ) and tau [6].
Typically, misprocessing of amyloid precursor protein leads to formation of insoluble fibrils
of Aβ. Amyloid precursor protein can be processed by three different proteases i.e., α, β, and γ
sectretase. Cleavage by α-secretase typically leads to a non-amyloidogenic pathway. Herein
amyloid precursor protein is cleaved by α-secretase at a position 83 amino acids from the
carboxy terminus and thus results in a fragment that is further cleaved by γ-secretase to result in
non-amyloidgenic peptide [7]. However, cleavage by the β secretase within the extracellular
domain located at 99 amino acids from the carboxy terminus results in further cleavage by γ-
secretase results in Aβ. Most of the Aβ is 40 residues in length (Aβ40), however a small fraction
of ~10% is 42 residues in length (Aβ42). Aβ42 is more hydrophobic and prone to fibril
formation. These insoluble fibrils accumulate in the extracellular space to form amyloid plaques
(a classical pathology associated with Alzheimer’s disease) [8].
The other classical pathology in Alzheimer’s disease is the formation of neurofibrillary
tangles due to hyperphosphorylated tau protein. Tau is a microtubule-associated protein that
stabilizes neuronal microtubules. Microtubules are required for a number of cellular processes
such as cellular polarity and intracellular transport [9]. However, in Alzheimer’s disease there is
10
abnormal hyperphosphorylation of tau protein [10,11]. Hyperphosphorylated tau protein in
clumps forms neurofibrillary tangles which are mainly intracellular and interfere with numerous
cellular functions [12]. Another hallmark of Alzheimer’s disease is the enlargement of ventricles,
especially the temporal horns [13] [14]. The progressive shrinkage of cortex and hippocampus
affects thinking and formation of new memories. Eventually, there is loss of connections
between neurons and leads to cell death, all accumulating into massive brain atrophy.
GAPS IN OUR UNDERSTANDING OF ALZHEIMER’S DISEASE
The major problem with Alzheimer’s disease, besides the unavailability of a drug to treat it, is
an unknown causative factor or triggering factor for the majority of cases (SAD type) around
which all events in Alzheimer’s disease can be centered around. Various hypotheses have been
proposed: A) Amyloid hypothesis: It is a largely believed theory and it revolves around Aβ
plaques accumulation being central to Alzheimer’s disease [15]. B) Cholinergic hypothesis: It
points to the loss of neurotransmitter acetylcholine, thus resulting in synaptic deficits [16]. C)
Oxidative stress hypothesis: It centers on the accumulation of cellular oxidants contributing to
the pathology seen in Alzheimer’s disease [17]. D) Mitochondrial cascade hypothesis: This
hypothesis points to the precedence of mitochondrial dysfunction prior to Alzheimer’s disease
histopathology and symptoms [18].
The inadequacy in our current understanding of Alzheimer’s disease can be assessed from the
fact that none of the drugs tried so far have been successful in the treatment of Alzheimer’s
disease [19]. To further complicate matters, the current methods for diagnosis of Alzheimer’s
disease are inadequate in terms that they are either invasive, insensitive, or measure parameters
that are detected after Alzheimer’s disease has progressed into a late stage [20]. Some of the
11
current methods for detection of Alzheimer’s disease include CSF measurements of Aβ, total tau,
and p-tau, MRI to quantify the volumetric changes in brain, and PET imaging with Pittsburg
compound, which binds to certain forms of Aβ [21].
SUBSTRATE SUPPLY TO THE BRAIN
Brain is a high energy demanding organ as it needs to support the energy consuming
neurotransmission. To meet its high energy needs, the brain is primarily supported by glucose as
its primary source of fuel. Glucose enters brain though different isoforms of glucose transporters.
GLUT1 (55K) is present on capillaries in brain and allows glucose transport across blood brain
barrier. Glucose can enter neurons through GLUT1 (45K), GLUT3, and GLUT4 whereas it can
enter astroglia mainly through GLUT1 (45K). Besides, glucose can enter microglia through
GLUT1 (45K) and GLUT5 [22,23]. Majority (~90%) of the glucose that enters brain is fully
oxidized to CO
2
and water after sequential processing by glycolysis, TCA cycle, and oxidative
phosphorylation that ultimately results in generation of energy in form of ATP [24]. Brain also
has a ratio of ~1 in terms of its O
2
/CO
2
utilization, demonstrating that glucose is exclusively used
for oxidative metabolism [22]. A small fraction of brain glucose is metabolized through hexose
monophosphate shunt or synthesized into glycogen. Generally, the brain stores only small
amounts of glycogen and this storage is not a useful reservoir when glucose supply is depleted
[24]. During decrease in supply of glucose to the brain, majority of the glucose is supplied by
liver. This ensures a steady state supply of ATP, which is required to maintain intra/extracellular
ion homeostasis, synthesis and degradation of proteins, and most importantly, the maintenance of
synaptic transmission [25]. However, during periods of prolonged fasting or pathological
12
conditions, brain can use ketone bodies such as acetoacetate and 3-hydroxybutryate as energy
substrates. Typically their levels in the blood is low, however, during periods of prolonged
fasting, their levels increase by increasing fatty acid degradation [26]. The scheme for utilization
and metabolism of glucose and ketone bodies has been shown in Fig. 2
13
MITOCHONDRIAL ENERGY REDOX AXIS
Mitochondrial energy-redox axis [27] ensures regulation of energy and redox components
housed by the mitochondria and affect several cellular processes. It regulates the energy
components by maintaining steady levels of ATP in addition to its regulation of NAD
+
/NADH
and thus influences the activation of co-factor PGC1α via sirtuins. The levels of NAD
+
/NADH
also regulate the activation of the AMPK pathway [28]. AMPK (system) is a key player in
regulating energy balance at cellular and whole body levels. Once activated (by
phosphorylation), it switches on the catabolic pathways that generate ATP and switches off the
ATP-consuming anabolic process such as synthesis of lipids, carbohydrates and proteins
[29,30,31].
On the other hand, mitochondria regulates redox by generating secondary messengers such as
H
2
O
2
(largely involved in redox-sensitive signaling pathways). The mitochondrial redox system
includes a thiol-based antioxidant system that mainly is accounted by glutathione and
thioredoxin based systems. These support activities of glutathione peroxidases, peroxiredoxins,
and methionine sulfoxide reductase [32]. The generation of H
2
O
2
reports the mitochondrial
energy charge to cytosol [33] and is implicated in the regulation of the cell's redox status, thus
transducing redox signals into a wide variety of responses, such as proliferation, differentiation,
14
and cellular death pathways [34]. This interplay and regulation of energy and redox is critical to
cellular functions and survival and has been summarized in Fig. 3 taken from[32].
15
THE MAJOR FACTORS THAT NEED TO BE ADDRESSED IN ALZHEIMER’S DISEASE
Amidst our seemingly inadequate knowledge about Alzheimer’s disease, there are multiple
factors which have been shown to be affected in several studies of Alzheimer’s disease. These
factors provide a strong rationale for designing therapeutic approaches that would address these
factors. The major factors that have been seen to be affected in multiple clinical studies are:
(1) Decreased brain glucose uptake
(2) Brain insulin resistance
(3) Impaired synaptic plasticity
In addition to these factors, it is important to recognize mild cognitive impairment (MCI), a state
that may precede Alzheimer’s disease in a large number of patients. MCI is a high risk pre-
dementia state wherein, cognitive decline faster than expected for a particular age is seen.
Considering that a large number of patients with Alzheimer’s disease have preceding MCI, there
must be one or multiple causative factors that could be common to both conditions. It has been
observed by multiple independent clinical studies that decreased brain glucose uptake is a
common feature in patients with Alzheimer’s disease or MCI. Longitudinal studies in normal
individuals, who were started to be followed before onset of clinically diagnosed MCI or
Alzheimer’s disease until much later stages when some of them progressed to either MCI or
Alzheimer’s disease, clearly shows a reduction in brain glucose uptake much before any clinical
diagnosis of MCI or Alzheimer’s disease was possible. These studies were performed in two
parts, on one hand clinical scores for tests such as mini-mental state examination (MMSE) and
global deterioration scale (GDS) were determined and, on the other hand, PET imaging of brain
was performed for glucose uptake. Importantly, glucose uptake was progressively decreased in
patients who progressed to MCI and finally onto Alzheimer’s disease [35]. Interestingly, patients
16
who progressed from being clinical normal MCI Alzheimer’s disease had a distinctly low
brain glucose uptake even when they were diagnosed as clinically normal. This shows that the
current clinical diagnosis is less sensitive in detecting early changes in the brain. A summary of
the patient that progressed from
being cognitively normal (but low brain glucose uptake) MCI DAT (Alzheimer’s type
dementia) and finally detected with Alzheimer’s disease in postmortem tissue has been shown in
Fig. 4 (taken and slight modified from [35]). It must be noted that the current diagnosis of
probable Alzheimer’s can only be confirmed in post-mortem tissue. Besides, clinical studies
have shown that individuals with MCI and low brain glucose uptake have a 15-fold greater
chance of progressing to Alzheimer’s disease [36,37,38,39,40]. The prominent decrease of
glucose uptake in several types of dementias, i.e., MCI, DAT, frontotemporal dementia, and
dementia with Lewy bodies has been demonstrated in multicenter clinical studies of 548 patients
[41]. Reduction in brain glucose uptake (associated with Alzheimer’s disease) opens up a strong
possibility of impaired brain insulin signaling. Insulin and insulin receptors present in brain are
very important and have been suggested to play a role in metabolism, cell survival/death, and
17
synaptic plasticity [42]. Binding of insulin to insulin receptor regulates glucose uptake by
translocating the intracellular glucose transporters to the plasma membrane. Generally, insulin
promotes survival by direct inactivation of pro-apoptotic machinery. Majority of the functions of
insulin the brain involve the ‘PI3K’ route as shown in the Fig. 5 taken from [43].
LIPOIC ACID
Lipoic acid was first isolated and identified chemically by Lester Reed and colleagues in 1951
[44]. It is a naturally occurring disulfide compound as a co-factor in mitochondrial α-
ketoglutarate dehydrogenase reactions. However, the externally administered lipoic acid
(typically orally) is not expected to covalently bind to the mitochondrial α-ketoglutarate
18
dehydrogenases. Rather, it is a potent modulator of redox status and it regulates cell-signaling
and transcriptional factors by thiol/disulfide exchange reactions [45,46]. Cellular transportation
of lipoic acid probably involves several systems such as the medium chain fatty acid transporter
[47], H
+
-lined monocarboxylate transporter for intestinal uptake,[48] and a sodium dependent
vitamin transport system [49,50]. Even though lipoic acid occurs in R- and S-enantiomeric
structures, only the R-form is essential in biological systems and shown to be absorbed better in
human body [51]. Thus, for the purposes of this research, only R-α-lipoic acid will be used.
Externally administered lipoic acid has been shown to have several beneficial effects like
weight loss, metal chelation, activation of catabolic pathways, and increasing mitochondrial
biogenesis in multiple tissues. Studies in muscle and heart tissue have found that the insulin-like
19
effect of lipoic acid results in increased glucose uptake and utilization [52,53]. Lipoic acid was
able to partially recover Akt activation that decreased as a function of age in hepatocytes.
Moreover, it also inhibited phosphatase activities of PTEN and PP2A in 3T3-LI adipocytes [54].
Age-related decrease of LTP and memory deficits was reversed in rats fed with lipoic acid, thus
implying its potential to improve synaptic plasticity and improve cognition [55]. Further, it has
also been found to activate the catabolic AMPK pathway and increase energy availability [56].
The several known actions of lipoic acid have been summarized in Fig. 6. Several clinical trials
in Germany have found beneficial effects of lipoic acid in treating diabetic neuropathies
[57,58,59]. Importantly, a very small clinical study following patients over four years, looking at
the efficacy of lipoic acid in Alzheimer’s disease treatment found beneficial effects in terms of
stabilization of cognitive functions [60].
HYPOTHESIS
The hypothesis was formulated keeping cognizance of several keys points like:
The major factors associated with Alzheimer’s disease that need to be addressed i.e.,
disrupted glucose uptake, insulin resistance, and impaired synaptic plasticity.
The previously demonstrated abilities of lipoic acid to show an insulin mimetic effect in
muscle and heart tissues.
The ability of lipoic acid to stimulate/restore synaptic plasticity as shown in models of
aging.
Hypothesis: The hypothesis to be tested here was that the insulin-like effect of lipoic acid
restores PI3K/Akt signaling and stimulates mitochondrial bioenergetics, thus overcoming the
energy deficit and impaired synaptic plasticity inherent in AD. The hypothesis has been
schematically shown in Fig. 7
20
SPECIFIC AIMS
Four specific aims were formulated to test the hypothesis that lipoic acid –an insulin-mimetic
disulfide– increases brain glucose uptake and improves cognition in a triple transgenic mouse
model of AD; these effects of lipoic acid are based on its participation in thiol/disulfide exchange
reactions (e.g., activation of the insulin receptor substrate) that are expected to overcome some
complications associated with insulin resistance and prevent the hypometabolic state that
precedes and is associated with the neurodegeneration inherent in AD. The experimental design
consists of non-transgenic (nonTg) and 3xTg-AD mice fed 0.23% w/v lipoic acid in drinking
water for 4 weeks. Experiments were carried out in 6 (young) and 12 (old) month-old nonTg and
21
3xTg-AD mice to assess the optimum time point for preventive intervention. The mice are fed
with lipoic acid for a month (~4 weeks) and thus they are ~7-month-old (young) and ~13-month-
old when the experiments were carried out. The 3xTg-AD mouse model closely mimics the
pathology type (β-amyloid plaques and hyperphosphorylated tau resulting in neurofibrillary
tangles) and synaptic impairments in an age-dependent manner as seen in humans and represents
an advanced pre-clinical tool to study AD. It is also widely used to assess therapeutic efficacy of
potential drugs. Notably, the 3xTg-AD mice show some extra-cellular Aβ deposits in the
hippocampus by 6 months. However, tau alterations and human tau immunoreactivity are only
present post 12 months of age with extensive hyperphosphorylation of tau between 15-18
months. Thus, the period before 15 months of age might be important for preventive therapy in
3xTg-AD mice as it is not characterized by full-blown AD associated pathology. The hypothesis
of this study will be addressed via four specific aims as outlined below
SPECIFIC AIM 1. BRAIN GLUCOSE UPTAKE. The effects of lipoic acid on brain glucose
uptake and glucose transporters were assessed in this specific aim. The primary objective of this
specific aim was to characterize and quantify the amount of glycolytic energy deficit in the
3xTg-AD compared with the nonTg mice and evaluate the efficacy of lipoic acid to restore this
energy deficit. Experiments were carried out in mice of 7 and 13 month-old, 3xTg-AD and
nonTg ± lipoic acid. The brain glucose uptake was determined by functional 2-deoxy-2-
[
18
F]fluoro-D-glucose-microPET whereas glucose transporter levels were assessed in brain and
plasma membrane homogenate by western blotting. This specific aim has been addressed in the
chapter 2 below.
22
SPECIFIC AIM 2. CELL SIGNALING. Investigate the signaling pathways involved in
restoring the energy deficit by lipoic acid – The broad goal of this specific aim was to elucidate
the mechanisms that lead to the brain energy deficits and the cellular processes that could be
involved in the ability of lipoic acid to restore it. Experiments were carried out in 7 and 13-
month-old, 3xTg-AD and nonTg mice ± lipoic acid. They involved quantitative comparing of the
various activated and basal proteins involved in the IRS/PI3k/Akt signaling (carried out by
western blotting techniques). Further, it was shown that the effect of lipoic acid on mitochondrial
bioenergetics was upstream of PI3K, using an extracellular flux analyzer. This specific aim has
been addressed in the chapter 2 below.
SPECIFIC AIM 3. BRAIN GLUCOSE METABOLISM. Investigate the glycolytic
metabolism in the 3xTg-AD mice and the effect of lipoic acid. The broad aim of this specific aim
was to elucidate the fate of glucose once it is up taken by neurons and astrocytes in the 3xTg-AD
mice brain and the effect of lipoic acid. Additionally, neuronal and astrocytic metabolism was
studied by co-infusion of glucose (neuron preferential substrate) + acetate (astrocytes specific
substrate). Experiments were carried out in mice of 7 and 13-month-old, 3xTg-AD and nonTg ±
lipoic acid. The experimental procedure involved infusion of
13
C labeled glucose and acetate
followed by acid extraction of the metabolites,
13
C NMR, and HPLC. The levels of different
metabolites from the TCA cycle and the major neurotransmitters like glutamate could be
quantified in the different groups of mice using the above experimental approaches. This specific
aim has been addressed in Chapter 3 (old mice) and Chapter 4 (young mice) of this thesis.
23
SPECIFIC AIM 4. SYNAPTIC PLASTICITY. Efficacy of lipoic acid on synaptic plasticity –
The specific aim 4 evaluated the functional outcome of lipoic acid by measuring parameters
involved in learning and memory i.e., Input/Output (I/O) and Long Term Potentiation (LTP) was
measured by electrophysiology. The experimental approach again involves mice of 7 and 13-
month-old, 3xTg-AD and nonTg ± lipoic acid. I/O is a measure of the strength between neuronal
connections whereas LTP is a measure of the biochemical basis for the formation of new
memories. This specific aim has been addressed in the Chapter 2 of this thesis.
This study gives insights into Alzheimer’s disease by studying a very advanced pre-clinical
mouse model of Alzheimer’s disease (3xTg-AD mice). It also tests the efficacy of lipoic acid as
a therapeutic or a nutraceutical agent in the treatment of Alzheimer’s disease. Specifically, the
ability of lipoic acid to thwart insulin hyporesponsivity in the brain has been tested. Additionally,
the mechanisms and cell signaling pathways of lipoic acid action in the brain have been
explored. Although, there was previous evidence indicating the insulin-mimetic effects of lipoic
acid in heart, muscle and liver [54,61,62], there was no evidence showing these effects in the
brain. Finally, the functional outcome of lipoic acid treatment in terms of long term potentiation
has also been assessed.
24
CHAPTER 2: Glucose uptake, PI3K/Akt cell signaling, and synaptic plasticity in the 3xTg-
AD mouse model and the effect of lipoic acid
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
TABLE
FIGURES
25
ABSTRACT
Alzheimer’s disease is a progressive neurodegenerative disease that entails impairments of
memory, thinking and behavior and culminates into brain atrophy. Impaired glucose uptake
(accumulating into energy deficits) and synaptic plasticity have been shown to be affected in the
early stages of Alzheimer’s disease. This study examines the ability of lipoic acid to increase
brain glucose uptake and lead to improvements in synaptic plasticity on a triple transgenic mouse
model of Alzheimer's disease (3xTg-AD) that shows progression of pathology as a function of
age; two age groups: 7 months (young) and 13 months (old) were used in this study. 3xTg-AD
mice fed 0.23% w/v lipoic acid in drinking water for 4 weeks showed an insulin mimetic effect
that consisted of increased brain glucose uptake, activation of the insulin receptor substrate and of
the PI3K/Akt signaling pathway. Lipoic acid supplementation led to important changes in
synaptic function as shown by increased input/output (I/O) and long term potentiation (LTP)
(measured by electrophysiology). Lipoic acid was more effective in stimulating an insulin-like
effect and reversing the impaired synaptic plasticity in the old mice, wherein the impairment of
insulin signaling and synaptic plasticity was more pronounced than those in young mice.
26
INTRODUCTION
Alzheimer's disease is a neurodegenerative disorder characterized by brain accumulation of
senile amyloid-β plaques and hyperphosphorylated tau (neurofibrillary tangles) in the medial
temporal lobe and cortical areas of the brain [63]. Alzheimer's disease is the most prevalent
neurodegenerative disease among the aging population [63] and a leading cause of dementia with
progressive memory deficits, cognitive impairments, and personality changes. Support for an
early mitochondrial dysfunction that precedes the histopathological hallmarks described above in
Alzheimer's disease continues to increase [18,64,65,66]. Perturbations of mitochondrial function
in terms of altered morphology, compromised electron transfer complexes, and tricarboxylic acid
cycle deficiencies have been long identified in post-mortem tissues of Alzheimer's patients
[67,68].
Multiple levels of analyses indicate a dysfunction of glucose metabolism and mitochondrial
bioenergetics as antecedents to the development of Alzheimer's pathology [69,70,71,72]. A
decline in brain glucose uptake (and metabolism) can appear decades prior to the onset of
histopathological changes inherent in Alzheimer's disease: several independent clinical studies
showed that decreased brain glucose uptake is a common condition in patients with Alzheimer’s
disease and mild cognitive impairment (MCI) [73,74]. A prominent decrease of glucose uptake
in several types of dementias, i.e., MCI, dementia Alzheimer's type (DAT), frontotemporal
dementia, and dementia with Lewy bodies has been demonstrated in multicenter clinical studies
of 548 patients [75].
Human brain has the highest consumption of glucose with respect to its size (60% of body’s
resting state glucose) and the energy generated from glucose metabolism is essential to support
synaptic transmission [76]; as a corollary, synaptic transmission is susceptible to the bioenergetic
27
deficits associated with the progress of Alzheimer's disease [77,78]. The insulin-stimulated brain
glucose uptake knits and links brain insulin to synaptic transmission. Insulin has been shown to
influence synaptic transmission by modulating the cell membrane expression of NMDA (N-
methyl-D-aspartic acid) receptors and, thereby affect long-term potentiation (LTP) [79]. Hence,
insulin resistance (as it occurs in metabolic syndrome) can modulate cognition and deteriorate
other brain functions [43]. The disruption of brain glucose uptake and metabolism following
insulin resistance is linked to Alzheimer’s disease and ‘treatment of brain insulin resistance’ is
being widely considered as a therapeutic approach in Alzheimer’s disease [80]. Alzheimer’s
disease is widely associated with synaptic failure, resulting in the loss of declarative and
nondeclarative memory and is associated with massive brain atrophy over a period of time [81].
Long-term potentiation (LTP) is considered as a major cellular mechanism underlying learning
and memory [82]. The classical pathology associated with Alzheimer’s disease i.e., β-amyloid
oligomers, have been shown to impair synaptic plasticity by inhibiting LTP and enhancing long
term depression (LTD) [83]. Thus, impaired insulin signaling and subsequent decrease in brain
glucose uptake (leading to disturbances in bioenergetics) and compromised synaptic plasticity
(leading to disturbances in synaptic transmission) are major deficiencies associated with
Alzheimer’s disease.
Lipoic acid (1,2-dithiolane-3-pentanoic acid) was reported to increase glucose uptake in L6
muscle cells and 3T3-L1 adipocytes [84,85], induce the redistribution of GLUT4 to the plasma
membrane in 3T3-L1 adipocytes [85], and increase insulin sensitivity in diabetic patients [86].
R-α-lipoic acid -naturally occurring form of the cyclic disulfide- is involved in the regulation of
cellular energy (as catalytic cofactor of mitochondrial α-ketoacid dehydrogenases), of
transcriptional processes (such as activation of Nrf2 and phase II enzymes), and of kinases and
28
phosphatases involved in signal transduction pathways [87]. These numerous effects of R-α-
lipoic acid can be mechanistically viewed in terms of thiol/disulfide exchange reactions that
modulate the redox and energy status of the environment; hence, lipoic acid-driven
thiol/disulfide exchange reactions appear critical for the modulation of proteins involved in cell
signaling and transcriptional pathways [88]. Exogenous lipoic acid equilibrates among different
intracellular and extracellular compartments but cannot substitute for covalently bound lipoic
acid (as the cofactor of mitochondrial complexes such as pyruvate dehydrogenase and α-
ketoglutarate dehydrogenase).
This study is aimed at establishing the effects of lipoic acid, on glucose uptake, insulin
signaling through the PI3K/Akt pathway, and synaptic plasticity on a triple transgenic mouse
model of Alzheimer's disease (3xTg-AD). This transgenic model harbors PS1(M146V),
APP(Swe), and tau(P301L) transgenes, shows progressive development of both plaques and
tangles with increasing age in a region specific manner: ~7 month-old 3xTg-AD mice show
diffuse amyloid plaques in different regions but tangle pathology is established at ~12 months
[89]. The experimental model consisted of 3xTg-AD mice of these two ages with or without 4
weeks of lipoic acid supplementation in the drinking water. It is hypothesized that an insulin-
like effect of lipoic acid could overcome the decreased brain glucose uptake, restore the
PI3K/Akt signaling to stimulate the cellular bioenergetics, and reverse the impaired synaptic
plasticity in the 3xTg-AD model of Alzheimer’s disease.
29
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: 11211) 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).
Mice colonies and lipoic acid feeding – Colonies of 3xTg-AD and nonTg mouse strain
(C57BL6/129S; gift from Dr. Frank Laferla, University of California, Irvine) were bred and
maintained at the University of Southern California. Mice were housed on 12-h light/dark cycles
and provided ad libitum access to food and water. 7- and 13 month-old mice were used for
experiments. 3xTg-AD and nonTg mice were either fed with water containing 0.23% R sodium
lipoic acid (gift from Geronova Research, Inc.) or normal water for 4 weeks. Thus, at the time of
sacrifice, the mice were ~7 (young mice) or ~13 months (old mice). The terms “young mice” and
“old mice” are only for representing the data with simplicity. In regards to the nonTg mice, 13
months may not be technically considered as an old age, however, the 3xTg-AD mice show
substantial pathology at this age and thus we used age matched nonTg mice at 13 months
terming them as old mice. NonTg and 3xTg-AD mice were used to assess the effects of lipoic
acid on glucose uptake, the PI3K pathway of insulin signaling, and synaptic plasticity.
Brain glucose uptake – We employed positron emission tomography utilizing radiotracer fluoro-
2-deoxy-2-[
18
F]-fluoro-D-glucose (FDG-PET) in a clinical setting to measure the brain glucose
uptake. Utilizing microPET scanning after 40 min post-injection of [
18
F]-FDG-PET as a tracer,
30
the brain glucose uptake was determined by standard uptake value (SUV). SUV represents the
standardized uptake value taking into consideration the ratio of the actual radioactivity
concentration found in brain at a specific time point and the concentration of radioactivity,
assuming an even distribution of the injected radioactivity across the whole body. Briefly, mice
were fasted overnight and then sedated using 2% isoflurane by inhalation and were administered
the radiotracer 2-deoxy-2-(
18
F) fluoro-D-glucose intravenously. Mice were placed on the scanner
bed with a warming bed to maintain body temperature and underwent scanning using a Siemens
MicroPET R4 PET scanner with a 19 cm (transaxial) by 7.6 cm (axial) field of view and an
absolute sensitivity of 4% with a spatial resolution of ~1.3 mm at the center of view for a
duration no longer than 90 minutes. Blood for glucose baseline measurements was collected
before the administration of the tracer and measured to ensure that abnormalities in glucose
metabolism during [
18
F] FDG-PET imaging are not due to huge differences in starting blood
glucose levels but the intrinsic activity of the brain. Additionally, the animals underwent CT
scanning with intravenous contrast material. This provided (~1 mm) information of brain
structure. Structural imaging using CT scanning allowed the analysis of functional ([
18
F] FDG-
PET) and anatomical data. PET data were all reconstructed using the 2D-OSEM algorithm
supplied by microPET manager (Siemens Medical Solutions USA, Inc., Knoxville, TN) into
128×128×63 images with 0.084 mm × 0.084 mm × 1.21 mm resolution. CT scans were acquired
in two bed positions using the following settings: 80 kVp, 500 uA, 100 ms/180 steps covering
360 degrees and reconstructed into 768×768×923 images with 0.105 mm isotropic resolution.
PET and CT images were co-registered using rigid transformations as both scans were performed
using warmed multi-modality imaging chambers. Region of interest were drawn to calculate
SUV.
31
Blood glucose levels – Briefly, mice were fasted overnight and then sedated using 2% isoflurane
by inhalation. The mouse tail was warmed a bit using a lamp or a heating pad. The tail vein was
located and a small puncture with a 25mm gauge needle was made. The drops of blood that
oozed out were tested for the blood glucose levels using a glucose meter and strips (Abbott, Inc.)
as per manufacturers supplied instructions. Blood concentrations were considered basal if they
were below 45mg/dl after fasting overnight. Glucose standards were used regularly to ensure the
accuracy of the glucose meter.
Brain homogenate preparation – Mice at ~7 and ~13months, after 4 weeks of lipoic acid feeding
in drinking water, and age matched control animals fed normal water were used for the
experiments. Age matched groups (3xTg-AD and nonTg ± lipoic acid) were fasted overnight to
ensure basal glucose levels. Mice were sacrificed by decapitation and the brains were quickly
excised on ice. Whole brain was further washed with isolation buffer, and cut into small pieces
and finally homogenized using a loose Teflon homogenizer. The buffer consisted of 250 mM
sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 1.0% (w/v) BSA, and 25 μl/100 ml
protease inhibitor mixture (Sigma Aldrich, MO, USA; Catalogue #8340) at pH 7.4.
Crude plasma membrane preparation – Brain homogenate was prepared as explained above.
Further, the homogenate was spun at 1000 x g for 15 min to remove pelleted nuclear fraction.
The nuclear fraction was then discarded and the remaining supernatant was spun at 9000 x g for
30minutes to yield crude cytosol supernatant and a pellet of crude membrane. The pellet was re-
suspended in HEPES-Lysis buffer (50 mM HEPES pH 7.4, 2 mM EDTA, protease/phosphatase
32
inhibitors) and spun at 30,000 x g for 30min to yield membrane fraction as a pellet. The pellet
was further re-solubilized in 2% CHAPS and stored at -80°C until used.
Western blotting – Brain homogenates or membrane preparations were quantified by using BCA
protein assay kit (Thermo Scientific, IL). The samples were diluted using 2% CHAPS to equalize
the protein concentrations. 25% (of the total sample volume) lane marker non-reducing sample
buffer (Thermo Scientific, Inc) was added prior to denaturing the proteins by boiling the samples
at 95°C for 10 min. Equal amounts of brain homogenate or membrane proteins (20 μg/well) were
loaded in each well of a 10% SDS-PAGE gel, electrophoresed with a Tris/glycine running
buffer, and transferred to a 0.45 μm pore size polyvinylidene difluoride (PVDF) membrane and
immunobloted with the appropriate primary antibody. Primary antibody was incubated
overnight, followed by washing and probing with the appropriate HRP-conjugated anti-rabbit
secondary antibody or HRP-conjugated anti-mouse secondary antibody (Vector Laboratories,
Burlingame, CA). The immunoreactive bands were visualized by Pierce SuperSignal
Chemiluminescent Substrates or SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific, IL) and captured by Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules,
CA). All band intensities were quantified using Un-Scan-it software (Silk Scientific, UT).
Metabolic Flux Analysis: XF-Extraflux Analyzer – Primary cortical neurons from day 14 (E14)
embryos of non-Tg mice were cultured on Seahorse XF-24 plates at a density of 75,000
cells/well. Neurons were grown in Neurobasal Medium +B27 supplement for 7 days before
experiment. 18 hours before assay, lipoic acid (20 μM) and/or LY294002 (50 μM) were added to
medium. On the day of metabolic flux analysis, cells were changed to unbuffered DMEM
33
(DMEM base medium supplemented with 25 mM glucose, 2 mM sodium pyruvate, 31 mM
NaCl, 2 mM GlutaMax, pH 7.4) and incubated at 37°C in a non-CO
2
incubator for 1 h. All
medium and injection reagents were adjusted to pH 7.4 on the day of assay. Baseline
measurements of oxygen consumption rate (OCR, measured by oxygen concentration change)
and extracellular acidification rate (ECAR, measured by pH change) were taken before
sequential injection of treatments/inhibitors: oligomycin (ATP synthase inhibitor, 4 μM), FCCP
(mitochondrial respiration uncoupler, 1 μM), and rotenone (Complex I inhibitor, 1 μM). After
the assays, plates were saved and protein readings were measured for each well to confirm equal
cell number/well.
Long-Term Potentiation and I/O Curves – Preparation of hippocampal slices: Each animal was
decapitated after deep isoflurane anesthesia and the brain was rapidly removed and immersed in
sucrose-modified artificial cerebrospinal fluid (ACSF) containing (in mM): 105 sucrose; 62
NaCl, 3 KCl, 4 MgCl
2
, 1.25 NaH
2
PO
4
, 26 NaHCO
3
, 10 glucose. After 3-5 min of cooling, the
brain was cut to contain the hippocampus and coronal 350 µm thick hippocampal slices with
surrounding cortical tissue using a vibratome (Series 1000, St Louis, MO). Sections were then
transferred to an incubation chamber, where they remained submerged in oxygenated artificial
cerebrospinal fluid (aCSF), which consisted of (in mM), 124 NaCl, 3 KCl, 1.25 NaH
2
PO
4
, 1.3
MgSO
4
, 26 NaCO
3
, 2.4 CaCl
2
and 10 glucose at room temperature until used for recording.
Electrophysiological recordings: After at least 1 h of equilibrium, one slice was transferred to an
interface recording chamber and perfused with aCSF at a rate of 1.5–2 ml/min, with the surface
of slices exposed to warm, humidified 95% O
2
–5% CO
2
. Field EPSPs (fEPSPs) were recorded
from stratum radiatum of CA1 using a glass pipette filled with 2M NaCl (yielding a resistance of
34
2–3 MΩ) in response to orthodromic stimulation (twisted nichrome wires, 50 µm) of the
Schaffer collateral-commissural projections in CA1 stratum radiatum. Pulses of 0.1 ms duration
were delivered to the stimulating electrode every 20 sec. The responses were amplified with
Axoclamp 2A DC amplifier (Axon Instruments, Foster City, CA), filtered at 6 kHz and digitized
at 20 kHz. Data acquisition was controlled by Clampex 9.0 software (Axon Instruments, Foster
City, CA). Input/output (I/O) curves were generated using stimulus intensities from 100-350 µA
in increments of 50 µA. Baseline fEPSP were evoked at 30-50% of maximal fEPSP in 20 sec
intervals. LTP was induced at baseline intensity using Theta Burst Stimulation (TBS) consisting
of ten trains of five 100Hz stimulation repeated at 5 Hz. Recording continued for at least 30 min
following TBS. fEPSP slope magnitude was calculated as the difference between two cursors,
separated by 1 ms, and placed on the middle portion of the ascending phase of the fEPSP. Three
consecutive responses separated with 20 sec intervals were averaged and presented as a single
point to reduce deviations. LTP were expressed as a percentage of the average slope from the
baseline recordings. Comparison of theta burst-induced plasticity was performed between groups
using repeated measures ANOVA (across all post-theta burst time points). This analysis was
followed by a post-hoc t-test performed between groups for the average change in fEPSP
amplitude recorded during the final 5 minutes of recording (35-40 min post theta burst
stimulation).
Data analysis – Student's two-tailed t-test was used for statistical analysis of paired data. The
level of statistical significance and the values of n are indicated in the respective figures.
ANOVA- Groups were initially compared for differences in tetanus-induced plasticity by
35
performing a repeated measures analysis of variance (ANOVA) across the entire post-tetanus
sampling period.
RESULTS
[
18
F]-FDG-PET imaging
(dynamic microPET scanning) revealed a slight decline in glucose uptake of the young (7-month
old) 3xTg-AD compared to age matched nonTg mice (Fig. 1A) (SUV
3xTg-AD
= 2.2 versus
SUV
non-Tg
= 2.6; p ≤ 0.05). A prominent difference in glucose uptake was found in the older (12
month-old mice) 3xTg-AD compared to nonTg mice (Fig. 1B) (SUV
3xTg-AD
= 1.8 versus
SUV
non-Tg
= 2.9; p ≤ 0.01). This suggests that deregulation of glucose uptake occurs at a younger
age in the 3xTg-AD mice and that it increases with age. Lipoic acid feeding led to increased
brain glucose uptake in young and old 3xTg-AD mice as compared to age-matched 3xTg-AD
mice not fed on lipoic acid (Fig. 1A and 1B) (SUV
3xTg-AD + lipoic acid
= 2.7 versus SUV
3xTg-AD
= 2.2
SUV = 2.2; not significant) (SUV
3xTg-AD + lipoic acid
= 3.1 versus SUV
3xTg-AD
= 1.8; p ≤ 0.01). This
increase in SUV in lipoic acid-fed mice correlated to a net ~20 and ~60 % increase of net
glucose uptake in lipoic acid fed young and old 3xTg-AD mice, respectively (Fig. 1), thus
suggesting that lipoic acid can rescue the decreased whole brain glucose uptake.
Effects of lipoic acid on the total and plasma membrane-associated GLUT3 and GLUT4 –
The levels of GLUT3 and GLUT4 are critical for glucose transport and its subsequent
metabolism to generate energy in neurons. Total GLUT3 levels were not affected in the young
3xTg-AD mice (Fig. 2B), whereas old 3xTg-AD mice had significantly lower (~20% decrease)
36
total GLUT3 levels compared to the age matched nonTg mice (Fig. 2E). Total GLUT4 was
significantly lower by ~20% and ~35% in the young and old 3xTg-AD mice, respectively, as
compared to the age-matched nonTg mice (Fig. 2C and 2F). Lipoic acid feeding had no
significant effect on the total GLUT3 levels of the young nonTg and 3xTg-AD mice (Fig. 2B)
but it led to a slight increase (~10%) of the total GLUT4 in younger 3xTg-AD mice (Fig. 2C).
Lipoic acid feeding led to an increase in the total GLUT3 (~20%) (Fig. 2E) and GLUT4 (10%)
(Fig. 2F) in the old 3xTg-AD mice.
The total amount of GLUT3 and GLUT4 does not directly indicate the active glucose
transporters, for they need to be translocated to the cell surface to facilitate glucose transport in
the cell. The amount of plasma membrane-associated GLUT3 was decreased in the young and old
3xTg-AD mice by ~30% as compared to the age-matched nonTg mice (Fig. 3B and 3E). The
decrease of GLUT4 translocation was particularly drastic, both in the young 3xTg-AD mice
(~40% decrease) (Fig. 3C) and the old 3xTg-AD mice (~50% decrease) (Fig. 3F) as compared to
the age- matched nonTg mice. Lipoic acid feeding had no significant effect on the membrane-
associated levels of GLUT3 in the young nonTg and 3xTg-AD mice (Fig. 3B); however, it led to
a ~100% increase in the older nonTg and 3xTg-AD mice (Fig. 3E). GLUT4 was increased by
lipoic acid feeding in the younger 3xTg-AD mice (~40%) (Fig. 3C) but not in the age matched
nonTg mice. Among the older mice, lipoic acid feeding led to an increase of GLUT4 in nonTg
(~15%) and 3xTg-AD (~115%) mice (Fig. 3F).
Insulin receptor
substrate (IRS) is the immediate downstream substrate of the insulin receptor after activation of
the latter. One of the major effects of IRS activation is the downstream activation of the Akt
37
pathway through phosphatidylinositol 3-kinase (PI3K) [90]. Young 3xTg-AD mice showed ~45%
decrease in the phosphorylation of IRS on the Tyr
608
residue compared to age matched nonTg
mice (Fig. 4B), whereas old 3xTg-AD mice showed a prominent (~75%) decrease of pIRS Tyr
608
phosphorylation (Fig. 4G). Lipoic acid feeding did not lead to a statistically significant difference
of pIRS Tyr
608
phosphorylation among the young nonTg; however, it elicited a ~35% increase of
pIRS Tyr
608
phosphorylation among the young 3xTg-AD mice (Fig. 4B). Lipoic acid feeding was
found to increase this phosphorylation substantially by ~2.5 fold in the old 3xTg-AD mice (Fig.
4G). A slight, not statistically significant decrease of pIRS Tyr
608
phosphorylation was found in
the young and old nonTg mice supplemented with lipoic acid (Fig. 4B and 4G).
Tyrosine phosphorylation of IRS-1 leads to its activation, whereas, phosphorylation on the
Ser
307
residue leads to its inactivation. The serine phosphorylation, and thus the IRS-1
inactivation, is associated with c-Jun NH
2
-terminal Kinase (JNK) activation [91,92]. Young and
old 3xTg-AD mice showed an increase in Ser
307
phosphorylation compared to age-matched
controls. The younger 3xTg-AD mice showed an increase of ~40% (Fig. 4C), whereas the older
showed an increase of 150% (Fig. 4H). Lipoic acid feeding was clearly effective in reducing the
Ser
307
phosphorylation mediated IRS-1 inactivation as it decreased it by ~20% (Fig. 4C) and
~65% (Fig. 4H) in the young and old nonTg mice respectively. In the young and old 3xTg-AD
mice, lipoic acid feeding led to a decrease in Ser
307
phosphorylation by ~25% and ~70%
respectively (Fig. 4C and 4H). Because active JNK (bisphosphorylated) is mainly responsible for
the serine phosphorylation of IRS-1, the status of JNK1 phosphorylation in these mice was
assessed and found its activation to be in agreement with the IRS-1 serine phosphorylation data.
The levels of pJNK1 phosphorylated at Thr
183
-Tyr
185
(associated with its activation) in the 3xTg-
AD mice were increased by ~ 2 fold and ~ 2.8 fold in the young and old mice, respectively, as
38
compared to the age-matched nonTg mice. Lipoic acid supplementation decreased the levels of
pJNK in both young and old 3xTg-AD mice (Fig. 4E and 4J).
Akt activation, through
phosphorylation on Thr
308
and Ser
473
, leads to the translocation of GLUT3 and GLUT4 to the
plasma membrane, thus facilitating glucose transport [93,94]. Western blotting for activated Akt
(phosphorylated on Ser
473
) showed that there was no substantial difference among the young
nonTg and 3xTg-AD mice, and among those treated with lipoic acid (Fig. 5A). However, there
was ~60% decrease of Akt phosphorylated at Ser
473
in the older 3xTg-AD mice compared with
the age-matched nonTg (Fig. 5C). Moreover, feeding lipoic acid increased activated Akt by
~10% and ~40% in the old nonTg and 3xTg-AD mice, respectively (Fig. 5C).
An important downstream target of activated Akt is GSK-3β, phosphorylated (and inactivated)
at Ser
9
(46). GSK-3β is widely implicated in several cellular pathways and, in the context of
Alzheimer’s disease, associated with the hyperphosphorylation of the microtubule-associated
protein tau [95]. The extent of GSK-3β phosphorylated at Ser
9
decreases in the 3xTg-AD mice
compared to the nonTg mice. Young 3xTg-AD mice show ~20% less phosphorylation (Fig. 5B),
whereas older 3xTg-AD mice show ~50% lesser phosphorylation (Fig. 5D) when compared to the
nonTg mice of the same age. Lipoic acid feeding to the young and old 3xTg-AD mice lead to
increased phosphorylation of GSK3β (thus, increasing the extent of inactivation): a slight increase
in the young 3xTg-AD mice (Fig. 5B) and a substantial increase in the older mice (~35%) (Fig.
5D). These results are consistent with those observed in terms of Akt phosphorylation at Ser
473
.
39
Effect of PI3K/Akt signaling on neuronal energy metabolism – The oxygen consumption rate
(OCR) and extracellular acidification rate (ECAR) of primary cortical neurons of nonTg mice was
assessed with the extracellular flux analyzer. This approach permits gaining mechanistic insights
as to the site of lipoic acid action. OCR represents measurements of mitochondrial respiration
(i.e., metabolism of glucose to pyruvate and pyruvate metabolism in mitochondria), whilst ECAR
measures extracellular acidification and is indicative of glycolysis (i.e., lactate formation). Lipoic
acid pre-treated neurons showed a substantially increased basal OCR by ~2 fold and stimulated
ATP turnover, maximal respiratory capacity, and reserve respiratory capacity (Fig. 6A and Table
1) as compared to un-treated neurons. Inhibition of PI3K by LY294002 resulted in a slight
decrease in basal OCR but the inhibitor abolished the lipoic acid-mediated effect (Fig. 6A), thus
suggesting that lipoic acid action was upstream of PI3K. The increase in basal ECAR levels was
also observed upon treatment with lipoic acid; these effects were abolished by LY294002 (Fig.
6B).
The data shown above indicated that lipoic acid
enhanced glucose uptake (Fig. 1) and the translocation to the membrane of GLUT3 and GLUT4
as well as stimulated several components of the PI3K/Akt signaling pathways and increased
mitochondrial reserve capacity. However, whether or not lipoic acid treatment enhances
function, i.e., synaptic plasticity remains to be determined. Synaptic failure in Alzheimer’s
disease has been found to be associated with deficits in numerous neurotransmitters and
neurochemicals; these deficits ultimately impair synaptic plasticity and brain function [96,97].
Electrophysiology was employed in this experimental model to measure the I/O responses and
gauge the strength of synaptic connections. Young 3xTg-AD mice had smaller synaptic
40
responses compared to age-matched nonTg mice (Fig. 7A,B). Lipoic acid feeding was found to
have adverse effects in the younger nonTg mice leading to a decrease in the I/O responses (Fig.
7A), whereas in the young 3xTg-AD mice, it led to a substantial increase in I/O slopes (Fig. 7B).
In the 13 month-old mice, the I/O responses were considerably decreased in the 3xTg-AD mice
(Fig. 7D) as compared to the age-matched nonTg mice (Fig. 7C). Lipoic acid feeding had no
effect on the older nonTg mice but elicited a substantial increase in I/O responses of the 3xTg-
AD mice (Fig. 7D).
In the young 3xTg-AD mice, the minimum and maximum output was significantly decreased
compared to the age-matched nonTg mice by ~70% (Fig. 8A) and 50% (Fig. 8B), respectively.
Lipoic acid feeding elicited an increase of the minimum and maximum output in the young
3xTg-AD mice by ~800% and ~30% (Fig. 8A,B). Conversely, lipoic acid feeding elicited a
decrease in minimum and maximum output in the young nonTg mice (Fig. 8A,B). The young
3xTg-AD mice also required considerably greater stimulation intensity to reach a minimum of 1
mV response in comparison with the nonTg mice of the same age (Fig. 8C). A similar trend for
the minimum and maximum output and stimulation required to reach 1 mV was found for old
3xTg-AD mice (Fig. 8D, E, and F). Interestingly, old 3xTg-AD mice failed to reach 1 mV output
even at the maximal stimulation intensity, i.e., 350 µA.
Lipoic acid increases long-term potentiation (LTP) in the old 3xTg-AD mice – Long-term
potentiation in the CA1 region is widely believed to be a form of plasticity responsible for
learning and memory. It involves activation of NMDA receptors for its induction and increased
insertion of AMPA (α-amino-3-hydroxy-5-methyl-1,4-isoxazolepropionate) receptors for its
expression [82]. The young 3xTg-AD mice expressed reduced LTP (Fig. 9B) compared to the
41
nonTg mice (Fig. 9A). Lipoic acid feeding had no effect on LTP expression in either young
nonTg or 3xTg-AD mice (Fig. 9C), which meant that their comparison showed near identical
results after lipoic acid feeding.
The old 3xTg-AD mice expressed a substantially reduced LTP (Fig. 9E) compared to nonTg
mice (Fig. 9D). At variance with the results observed in young mice, lipoic acid feeding exerted a
profound increase in LTP in the old 3xTg-AD mice (Fig. 9E,F). These data show that dietary
lipoic acid was not effective in altering the deficits in hippocampal LTP seen in young 3xTg-AD
mice, while it was extremely potent in restoring LTP to control levels in 3xTg-AD mice. The old
3xTg-AD mice showed even more reduced LTP than that seen in young 3xTg-AD mice (p < 0.04;
F = 6.0 repeated measures ANOVA) (old 3xTg AD n = 6, old 3xTg-AD + lipoic acid; n = 7).
However, the improvement in LTP did not bring it to the levels observed in either young nonTg
or nonTg-lipoic acid (p < 0.05; F = 5.17 repeated measures ANOVA) (young 3xTg AD-lipoic
acid n = 7, old 3xTg-AD-lipoic acid; n = 6).
DISCUSSION
This is a comprehensive study aimed at establishing the effects of dietary lipoic acid on brain
function in a triple transgenic mouse model of Alzheimer's disease and addresses effects on
substrate supply assessed by brain glucose uptake ([
18
F]-FDG-PET imaging) and glucose
transporters translocation to the plasma membrane, the modulation of glucose metabolism by the
PI3K/Akt pathway of insulin signaling, mitochondrial oxidative metabolism capacity, and
synaptic plasticity. These effects of lipoic acid are summarized in Fig. 10.
42
Longitudinal studies carried out in human subjects before the onset of clinically diagnosed
MCI or Alzheimer's disease showed a pronounced decrease of brain glucose uptake in
individuals who progressed to MCI or Alzheimer's disease (compared to normal aging
individuals) before any clinical diagnosis was possible [74]. This study added an important
dimension to earlier studies showing the association of decreased brain glucose uptake in
Alzheimer’s disease by suggesting that the accentuated decrease of brain glucose uptake was an
early event in the transition from normal aging to MCI and/or to Alzheimer’s disease. Further
studies established associations between brain hypometabolism and cognition residing in the
parietal and temporal lobar regions in the early stages of the disease and of the frontal regions in
the late stages of the disease [98].
Data in the current study showed ~10% decrease in brain glucose uptake in the young 3xTg-
AD mice and ~35-40% decrease in the old 3xTg-AD mice, thus suggesting an age-dependent
decrease in brain glucose uptake in these mice, thus establishing similarities between the
progress of sporadic Alzheimer's disease and the transgenic 3xTg-AD mouse model used in this
study. This further validates the 3xTg-AD mouse model as a tool to study Alzheimer’s disease
and the use of PET-CT imaging to follow the progress of MCI and Alzheimer’s disease. Young
and old nonTg did not show statistically significant differences in brain glucose uptake. Lipoic
acid feeding was able to restore glucose uptake at both ages in the 3xTg-AD mice, however,
elicited a more pronounced increase of net glucose uptake in the old mice (Fig. 10; component
A), possibly due to the significant drop in glucose uptake at that age.
Glucose transport in neurons is facilitated by the different glucose transporters especially,
GLUT3 and GLUT4, which are exclusively present in neurons; moreover, GLUT4 is primarily
insulin sensitive and is activated after insulin stimulation [99,100,101]. GLUT3 and GLUT4
43
facilitate glucose transport after they are translocated to the cell surface by appropriate
stimulation that involves Akt activation. The decrease of active glucose transporters on the
plasma membrane could be the reason for the decrease in glucose uptake as a function of age in
the 3xTg-AD mice. Translocation to the plasma membrane of the insulin-sensitive GLUT4 was
decreased in young and old 3xTg-AD mice (with greater net drop in the older mice) compared to
the age matched nonTg mice; this suggests a decrease in insulin signaling-mediated translocation
of GLUT4. GLUT3 showed a slight decrease in the plasma membrane translocation in both
young and old 3xTg-AD mice. Total GLUT4 was also decreased in the young and old mice,
suggesting a decreased pool of glucose transporters available for translocation to the plasma
membrane. Only minor differences were seen in the total GLUT3 at young and old age, thus
implying that the capacity for translocation of GLUT3 was not substantially decreased in the
3xTg-AD mice. Lipoic acid feeding lead to increase of both GLUT3 and GLUT4 membrane
translocation in the old nonTg and 3xTg-AD mice, demonstrating that lipoic acid facilitates a
greater capacity for glucose transport in neurons. Feeding of lipoic acid did not significantly
increase the total GLUT3 and GLUT4 in old mice, thus the effect of lipoic acid in increasing
total glucose uptake (PET-CT data in Fig. 1) might be mainly due to its ability to increase
translocation of glucose transporters on the plasma membrane. Overall, brain glucose uptake was
decreased in the 3xTg-AD mice and lipoic acid was able to restore the glucose uptake by
increasing the translocation of GLUT3 and GLUT4 to the plasma membrane (Fig. 10;
components B and C). Lipoic acid has previously been shown to increase glucose uptake in L6
muscle cells and 3T3-L1 adipocytes [84,85], induce the redistribution of GLUT4 to the plasma
membrane in 3T3-L1 adipocytes [85], and increase insulin sensitivity in diabetic patients [86].
44
Insulin signaling is responsible for glucose transport in the brain [102] and control of energy
homeostasis. Disruption of insulin signaling is expected to affect glucose transport and the
subsequent energy generation from glucose oxidation. Samples of autopsied brains from patients
with Alzheimer’s disease have shown a deficiency in insulin signaling [103], thus further
confirming the link between disrupted insulin signaling and the well-established decrease of
brain glucose uptake associated with Alzheimer’s disease. Autophosphorylation of specific
tyrosine residues by tyrosine kinase activity of the insulin receptor is considered essential for its
activity [104]. IRS phosphorylation at Tyr
608
was found to ensue after insulin binding to the
receptor and is required for the full activation of PI3K [105] that results in phosphorylation of
Akt and the ensuing effects on translocation of GLUT4 to the plasma membrane [106] and
phosphorylation (inactivation) of GSK-3β. Impaired IRS-1 activation is associated with retarded
embroyonic and postnatal growth [107]. In Alzheimer’s disease, wherein loss of neurons is
apparent, this cell survival protein is increasingly deregulated or less activated [80].
Data in this study showed that IRS and Akt activation was decreased in the old 3xTg-AD
mice with the concomitant activation of JNK; these data might explain the decrease in both brain
glucose uptake and GLUT4 translocation to the plasma membrane in old 3xTg-AD mice. An
overall reduction of IRS activity in the young 3xTg-AD mice was suggested by the decreased
phosphorylation of IRS at Tyr
608
(activation) and increased phosphorylation of IRS at Ser
307
(inactivation); however, this could not be explained in lieu of the Akt data i.e., IRS activation
was decreased by ~40% in the young 3xTg-AD mice whereas the effect on Akt activation was
rather minimal. Conversely, the old 3xTg-AD mice showed a substantial decrease of both -
active IRS and Akt. Interestingly, there was ~10 fold difference of active/inactive IRS i.e., pIRS-
Tyr
608
/pIRS-S
307
between the young and old 3xTg-AD mice (Fig. 4D and 4I). This provokes a
45
speculation about the occurrence of a threshold for the levels of active/inactive IRS that is
required for Akt activation. The increase in brain glucose uptake exerted by lipoic acid feeding
poses the question as to whether or not the cyclic disulfide might stimulate insulin signaling:
lipoic acid was able to substantially stimulate insulin signaling in the old 3xTg-AD mice, i.e.,
increase of pIRS-Tyr
608
/pIRS-S
307
, pAkt-Ser
307
, and pGSK3β-Ser
9
(Fig. 10; components D-F).
The latter effects are probably a consequence of lipoic acid-mediated thiol/disulfide exchange on
IRS followed by an increased phosphorylation at Tyr
608
. This notion is strengthened by the
enhancing effect of lipoic acid on OCR in primary cortical neurons and its inhibition by
LY294002, suggests that this increase in mitochondrial efficiency is PI3K dependent and the site
of action probably upstream of PI3K. This effect of lipoic acid may be accounted for by
oxidation of critical cysteine residues in the insulin receptor and IRS [46,85]; this thiol-disulfide
exchange mechanism increases the activation of the IRS and it might be surmised that the site(s)
of action of lipoic acid might be at the insulin receptor and IRS.
Synaptic strength, measured by I/O, showed significantly lower response in the young 3xTg-
AD mice as compared to the young nonTg mice. The older 3xTg-AD mice showed a lower I/O
response at all the stimulus intensities and the maximum response is far lower than the older
nonTg mice, showing that the strength of synaptic connections is severely affected in the 3xTg-
AD mice.
LTP observed in the hippocampal CA1 pyramidal cells has been widely investigated and
considered critical for learning and memory. Release of the neurotransmitter glutamate from the
presynaptic terminal followed by its binding to the NMDA and AMPA receptors results in
depolarization and subsequently expelling of Mg
2+
[108], thus allowing for Ca
2+
and Na
+
to enter
the postsynaptic neuron. Ca
2+
is further believed to trigger several protein kinases and signal
46
transduction cascades involving Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) and
protein kinase C (PKC) that ultimately translate the signals to nucleus and initiate the formation
of new synapses [82,109]. In addition to kinase activity and signal transduction, protein synthesis
follows for maintenance of LTP. Learning and memory are primarily affected in Alzheimer’s
disease and thus it was of primary interest to assess LTP in these 3xTg-AD mice and examine the
effect that lipoic acid feeding might induce on this machinery of learning and memory. Several
Alzheimer’s disease transgenic mouse models have impaired synaptic plasticity, demonstrated by
measuring LTP and LTD (using electrophysiology) [89,110,111,112,113]. We examined the
effect of the 3xTg-AD genome as well as lipoic acid feeding on the expression of hippocampal
LTP induced with TBS. The LTP for the young and older 3xTg-AD mice was found to be lower
than the age matched nonTg mice, pointing towards the synaptic deficits. Interestingly, lipoic acid
did increase the I/O but had no effect on the LTP of the young 3xTg-AD mice. However, lipoic
acid lead to substantial increase in both I/O and LTP of the older 3xTg-AD mice (Fig. 10;
component G). It may be surmised that dietary lipoic acid that was more effective in reversing the
effects that occur later in life in the 3xTg-AD mice, in which a profound deficit in insulin
signaling was also observed. Hence, the synaptic plasticity of the older 3xTg-AD mice was
substantially decreased and was restored by lipoic acid. Although the mechanistic reasons for the
loss/gain of synaptic plasticity in the 3xTg-AD mice remain to be determined, it can be speculated
that the activation of insulin signaling via IRS/PI3K/Akt pathway may have increased NMDA
receptor conductance and/or AMPA receptor cycling. Previous studies have shown the effect of
PI3K/Akt pathway in stimulating LTP [43] and lipoic acid has been shown to reverse the age-
related decrease of LTP and memory deficits in aged rats [53,114], to reduce the hippocampal
47
memory deficits in the Tg2576 model of Alzheimer’s disease [115], and to stabilize cognitive
functions in patients afflicted with moderate Alzheimer’s disease [116].
CONCLUSIONS
Lipoic acid successfully reverted the age-associated decrease of glucose uptake and
stimulated the PI3K/Akt pathway of insulin signaling in the 3xTg-AD mouse model.
Importantly, the synaptic deficits associated with the old 3xTg-AD mice were also reversed by
lipoic acid feeding. To our knowledge, this is the first study that establishes an age-dependent
correlation between the decrease in glucose uptake in vivo assessed by PET-CT imaging along
with decrease in insulin cell signaling and concomitant decrease of synaptic plasticity in the
3xTg-AD mouse model of Alzheimer’s disease. Albeit, the study does not provide causal
relationship between decrease in glucose metabolism and impaired synaptic plasticity, but it
hints at a plausible hypothesis that decrease in glucose uptake and metabolism ultimately affects
the high energy-demanding synaptic transmission, leading to impaired synaptic plasticity.
Previous behavioral studies have shown the positive effects of lipoic acid on memory in different
mouse models of aging [117,118,119] and Alzheimer’s disease [115,120]. However, future
comprehensive studies on behavioral changes effected by lipoic acid in the 3xTg-AD model are
warranted.
48
TABLE 1
Oxygen consumption rates (OCR) by hippocampal neurons from nonTg mice
Control +lipoic acid +LY294002 +lipoic acid
+LY294002
Basal respiration 64 ± 3 110 ± 8 50 ± 2 57 ± 4
ATP turnover 35 ± 3 68 ± 8 26 ± 2 30 ± 4
H
+
leak-induced respiration 29 ± 1 42 ± 2 24 ± 1 27 ± 2
Maximal respiratory capacity 77 ± 4 157 ± 12 57 ± 2 68 ± 8
Non-mitochondrial respiration 13 ± 1 19 ± 1 9 ± 1 11 ± 3
Reserve capacity 13 ± 5 47 ± 15 7 ± 3 10 ± 9
Data expressed in pmoles O
2
/min
49
FIGURES
Fig. 1. Age-dependent decrease of whole brain glucose uptake and the restorative effect of lipoic
acid
50
Standard uptake value (SUV) was calculated after [
18
F]-FDG injection followed by PET
and CT scanning as described in the Materials and Methods section. (A) Young mice, n = 34, n ≥
6/group. (B) Old mice, n = 27, n ≥ 6/group. Upper panel: Representative combined images from
PET-CT scanning of nonTg and 3xTg-AD mice ± lipoic acid; lower panel: Average SUV values
with the error bar indicating ± SEM. *P ≤ 0.05, **P ≤ 0.01
51
Fig. 2. Brain GLUT3 and GLUT4 levels
The levels of total GLUT3 and GLUT4 in whole brain from nonTg and 3xTg-AD mice
+/– lipoic acid (young and old) were determined by western-blot analyses. Left panels (A, B, and
C) correspond to data from young mice; right panels (D, E, and F) to data from old mice.
Representative western blot images of GLUT3, GLUT4, and β-actin (loading control) are shown.
Bar graphs show the average GLUT3 or GLUT4 values after normalization with the loading
control and the error bars indicating ± SEM. Total n = 48, n ≥ 5/group. *P ≤ 0.05, **P ≤ 0.01.
52
Fig. 3. Membrane-associated GLUT3 and GLUT4 levels in brain
The levels of GLUT3 and GLUT4 in whole brain crude membranes from nonTg and
3xTg-AD mice +/– lipoic acid (young and old) were determined by western-blot analyses. Left
panels (A, B, and C) correspond to data from young mice, whereas right panels (D, E, and F)
correspond to data from old mice. Representative western blot images of GLUT3, GLUT4, and
Na,K-ATPase (loading control) in whole brain crude membrane are shown. Bar graphs show the
average membrane-associated GLUT3 and GLUT4 values after normalization with the loading
control and the error bars indicating ± SEM. Total n = 32, n = 4/group. *P ≤ 0.05, **P ≤ 0.01.
53
Fig. 4. IRS activation status in the 3xTg-AD mice and the effect of lipoic acid
54
The levels of pIRS-Tyr
608
(activated) and pIRS-Ser
307
(inactivated) in whole brain from
young and old nonTg and 3xTg-AD mice +/– lipoic acid were determined by western-blot
analyses. Left panels (A, B, C, D, and E) correspond to data from young mice; right panels (F, G,
H, I, and J) correspond to data from old mice. Bar graphs show the average pIRS Tyr
608
, pIRS
Ser
307
, and pJNK Thr
183
-Tyr
185
values after normalization with the loading control (IRS and
JNK) and the error bars indicating ± SEM Total n = 48, n ≥ 5/group. *P ≤ 0.05, **P ≤ 0.01.
55
Fig. 5. Effect of lipoic acid on age- G K 3β
Western blot analyses of the levels of pAkt Ser
473
and pGSK3β Ser
9
in whole brain from
nonTg and 3xTg-AD mice +/– lipoic acid. Left panels (A and B) correspond to data from young
mice and right panels (C and D) to data from old mice. Bar graphs show the average pAkt Ser
473
(normalized to loading control, Akt) and pGSK3β Ser
9
(normalized to loading control, GSK3β)
with error bars indicating ± SEM. Total n = 48, n ≥ 5/group. *P ≤ 0.05, **P ≤ 0.01.
56
Fig. 6. PI3K dependent effect of lipoic acid on cellular bioenergetics
Primary cortical neurons from nonTg mice were isolated and cultured for 7 days. 18
hours before the assay, lipoic acid (20 μM) and/or LY294002 (50 μM) were added to medium.
(A) OCR and (B) ECAR were determined using Seahorse XF-24 Metabolic Flux Analyzer.
Vertical dashed lines indicate time of addition of mitochondrial inhibitors: oligomycin (4 μM),
FCCP (1 μM), and rotenone (1 μM). (open circles) control; (closed circles) plus lipoic acid;
57
(open squares) plus LY294002; (closed squares) plus lipoic acid and LY294002. OCR and
ECAR readings were normalized to total protein concentration in each well.
58
Fig. 7. Age dependent changes in I/O of the 3xTg-AD mice and the effect of lipoic acid
I/O relationships after applying increasing stimulation to the stratum radiatum of the CA1
region in the hippocampus for nonTg and 3xTg-AD mice +/– lipoic acid and recording the
output (electrophysiology techniques as described in the Materials and Methods section). Left
59
panels (A and B) correspond to data from young mice and right panels (C and D) to data from
old mice. (open circles) Control; (closed circles) Plus lipoic acid. fEPSP slope (mV/ms) plotted
against the corresponding stimulation intensity for (A) young non-Tg mice (p < 0.001; F = 27.1
repeated measures ANOVA; young nonTg n = 4, young nonTg + lipoic acid n = 7); (B) young
3xTg-AD mice (p < 0.002; F = 20.4 repeated measures ANOVA; young 3xTg-AD n = 8, young
3xTg-AD + lipoic acid n = 6). (C) old nonTg mice (p < 0.004; F = 13.9 repeated measures
ANOVA; old nonTg n = 6, old nonTg + lipoic acid n = 6). (D) old 3xTg-AD mice (p < 0.00003;
F = 46.4 repeated measures ANOVA; old 3xTg-AD n = 6, old 3xTg-AD + lipoic acid n = 7). The
inserts in each panel are the corresponding representative I/O raw data as obtained during the
electrophysiology recordings: (open circles) control and (closed circles) plus lipoic acid. Total n
= 51 slices, n ≥ 5 slices/group and at least 3-4 animals/group.
60
Fig. 8. Minimum EPSP, maximum EPSP, and stimulation intensity required to reach 1mV
61
Bar graphs of the levels of minimum EPSP, maximum EPSP, and stimulation intensity
required to reach 1 mV as obtained during the I/O recordings in the stratum radiatum of the
hippocampal CA1 region for nonTg and 3xTg-AD mice +/– lipoic acid. Left panels (A, B, and
C) correspond to data from young mice and right panels (D, E, and F) to data from old mice. Bar
graphs showing the minimum EPSP or the fEPSP slope values at 100 μA and the error bars
indicating ± SEM for (A) young mice and (D) old mice. Bar graphs showing the maximum EPSP
or the fEPSP slope values at 350 μA and the error bars indicating ± SEM for (B) young mice and
(E) old mice. Bar graphs showing the stimulation intensity required to reach at least 1mV output
and the error bars indicating ± SEM for (C) young mice (p < 0.01; F = 8.9 repeated measures
ANOVA) (young nonTg n = 7, young 3xTg-AD n = 7) and (F) old mice (p < 0.003; F = 14.6
repeated measures ANOVA) (old nonTg n = 6, old 3xTg-AD n = 7). Total n = 51 slices, n ≥ 5
slices/group and at least 3-4 animals/group. *P ≤ 0.05, **P ≤ 0.01
62
Fig. 9. Age dependent changes in the LTP of the 3xTg-AD mice and the lipoic acid effect
LTP was induced at baseline intensity using theta burst stimulation (TBS) consisting of
ten trains of five 100 Hz stimulation repeated at 5 Hz. Slope of EPSPs was measured and results
normalized to the average value measured during the 10 min baseline period. Recording
continued for at least 30 min following TBS and the last 5 min was used to calculate the LTP.
Panels A, B, and C correspond to data from young mice, whereas, panels D, E, and F correspond
to data from old mice (gray circles/bars – control (nonTg or 3xTg-AD), black circles/bars – fed
lipoic acid (nonTg or 3xTg-AD + lipoic acid). A graph showing the first 10 min of baseline
followed by the percentage of the baseline response elicited after TBS for 30 min for (A) young
nonTg mice, (B) young 3xTg-AD mice, (D) old nonTg mice, (E) old 3xTg-AD mice. Bar graphs
showing the measured LTP using %EPSP for the last 5 min of the response to TBS stimulation
63
for (C) young mice and (F) old mice. Total n = 51 slices, n ≥ 5 slices/group and at least 3-4
animals/group. *P ≤ 0.05, **P ≤ 0.01
64
Fig. 10. Sites of action and effects of lipoic acid on brain glucose metabolism
65
The scheme shows the PI3K/Akt pathway of insulin signaling and the effects of lipoic
acid on the different components investigated in this study: (A) glucose uptake, (B) total GLUT3
and GLUT4 expression, (C) translocation of GLUT3 and GLUT4 to the plasma membrane from
intracellular vesicles, (D) changes in IRS-Tyr
608
/IRS-Ser
307
ratio, (E) activation of Akt, (F)
phosphorylation of GSK3β at Ser
9
, and (G) synaptic plasticity
66
CHAPTER 3: Glucose hypometabolism in the aged 3xTg-AD mouse model and the effect of
lipoic acid
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
TABLE
FIGURES
67
ABSTRACT
Alzheimer’s disease is an age-related neurodegenerative disease characterized by
deterioration of cognition and loss of memory. Several clinical studies have shown Alzheimer’s
disease to be associated with disturbances in glucose metabolism and the subsequent TCA cycle-
related metabolites like glutamate, glutamine, and N-acetylaspartate. These metabolites have
been viewed as biomarkers by (a) assisting early diagnosis of Alzheimer’s disease and (b)
evaluating the efficacy of a treatment regimen. In this study, 13 month-old triple transgenic mice
(a mouse model of Alzheimer's disease (3xTg-AD)) were given intravenous infusion of [1-
13
C]glucose followed by an ex vivo
13
C NMR to determine the concentrations of
13
C labeled
isotopomers of glutamate, glutamine, aspartate, GABA, myoinositol, and N-acetylaspartate.
Total (
12
C +
13
C) glutamate, glutamine, and aspartate were quantified by HPLC to calculate
enrichment. Furthermore, we examined the effects of lipoic acid in modulating these metabolites,
based on its previously established insulin mimetic effects. Total
13
C labeling and percent
enrichment decreased by ~50% in the 3xTg-AD mice. This hypometabolism was partially or
completely restored by lipoic acid feeding. The ability of lipoic acid to restore glucose
metabolism and subsequent TCA cycle-related metabolites further substantiates its role in
overcoming the hypometabolic state inherent in early stages of Alzheimer's disease.
68
INTRODUCTION
Human brain consumes ~60% of body’s resting-state glucose and the energy generated from
glucose metabolism is essential for maintaining synaptic transmission.[121] Hence, disturbance
of brain glucose uptake is expected to create a hypometabolic state that impinges on synaptic
plasticity, because glucose availability tightly regulates glutamate neurotransmission within the
TCA cycle and glutamine-glutamate cycles of neurons and glia. Reduction of brain glucose
metabolism in patients with Alzheimer’s disease has been shown by several clinical studies.[73]
Definite diagnosis of Alzheimer’s disease is conventionally only possible by detection of β-
amyloid plaques and neurofibrillary tangles in post-mortem tissues; a clinical diagnosis is
possible by testing mental status output in terms of different scales like the Mini-Mental State
Examination (MMSE) or the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-
cog).[122] However, the sensitivity of these scales is rather low, especially, during the early-
stages of Alzheimer’s disease.[123] Currently, early diagnosis and sensitive treatment
monitoring are the important challenges that hamper effective management of Alzheimer’s
disease. Thus, biomarkers that would assist in accomplishing those two goals would be
invaluable towards effectively managing Alzheimer’s disease. It is established that biochemical
changes accompanying energy metabolism (associated with N-acetylaspartate (NAA),
myoinositol (MI), glutamate (Glu), glutamine (Gln), and aspartate (Asp)) precede the structural
abnormalities in Alzheimer’s disease. These developments have renewed interest in the
metabolic aspect of Alzheimer’s disease. In that light, magnetic resonance spectroscopy (MRS)
(
1
H and
13
C) has diagnostic value and aids early diagnosis of Alzheimer’s disease. Using these
tools, Glu, Gln, Asp, GABA, MI, NAA, and other metabolites (or neurochemicals) can be
detected. These metabolites can serve as biomarkers because they are found to be dysregulated in
69
Alzheimer’s disease.[124] Beside diagnosis, assessment of therapeutic agents for Alzheimer’s
disease remains to be a challenge. Thus, there is a need for biomarkers of Alzheimer’s disease
that respond to the biochemical effects of the drug being tested. In this respect, MRS studies
measuring metabolites have been shown as viable measurements to dynamically monitor
therapeutic treatment.[125]
Lipoic acid (1,2-dithiolane-3-pentanoic acid), a disulfide compound, has been shown to have
an insulin mimetic effect that results in increased glucose uptake and activation of the PI3K/Akt
pathway, thus stimulating mitochondrial bioenergetics.[45,126,127] A small clinical study
following patients over four years, looking at the efficacy of lipoic acid in Alzheimer’s disease
treatment found beneficial effects in terms of stabilization of cognitive functions.[60] Moreover,
feeding lipoic acid increased glucose uptake and improved synaptic plasticity in the 3xTg-AD
mouse model of Alzheimer's disease[128], reduced hippocampal memory deficits in the Tg2576
model of Alzheimer’s disease,[129] improved long-term memory of aged NMRI mice,[130]
improved cognition in aged SAMP8 mice,[131] and improved memory in aged rats.[55] These
multifaceted effects of lipoic acid can be mechanistically ascribed to its participation in
thiol/disulfide exchange reactions that modulate the redox and energy status of the cellular
environment.[132]
This study examines the ability of lipoic acid to modulate brain glycolytic and mitochondrial
metabolic pathways in a triple transgenic mouse model of Alzheimer’s disease (3xTg-AD). This
mouse model closely mimics the pathology type (β-amyloid plaques and hyperphosphorylated
tau resulting in neurofibrillary tangles) and synaptic deficits in an age dependent manner as seen
in humans[133] and represents an advanced pre-clinical tool to study Alzheimer’s disease and
assess therapeutic efficacy of a candidate drug. However, the characterization of the metabolic
70
components of glucose metabolism and the downstream TCA cycle-related metabolites has not
been explored in this model. Experiments in this study were performed on whole brain of 13
month-old non-transgenic (nonTg) and 3xTg-AD mice; the study was aimed at (a) evaluating
glucose metabolism by quantification of the subsequent TCA cycle-related metabolites, such as
Glu, Gln, NAA, GABA, and Asp, and (b) assessing the ability of lipoic acid to modulate the
levels of these metabolites.
MATERIALS AND METHODS
Materials – [1-
13
C]Glucose (99%) was purchased from Sigma-Aldrich (MO, USA) and
Deuterium Oxide (99.9%) from Cambridge Isotope Laboratories, Inc (MA, USA). All other
chemicals were the purest grade available from Sigma-Aldrich (MO, USA)
Mice colonies and lipoic acid feeding – Colonies of 3xTg-AD and nonTg mouse strain
(C57BL6/129S; Gift from Dr. Frank Laferla, University of California, Irvine) 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. The triple
transgenic mouse model of Alzheimer's disease (3xTg-AD) was first developed by Oddo et al.
19
12 month-old 3xTg- -amyloid deposits in cortex and human tau
immunoreactivity.
19
Mice were housed on 12-h light/dark cycles and provided ad libitum access
to food and water. 12-month-old male mice were used for experiments. 3xTg-AD and nonTg
mice were either fed with water containing 0.23% R-sodium lipoic acid (gift from Geronova
71
Research, Inc.) or normal water for 4 weeks. Thus, at the time of glucose infusion, mice were
~13 months old.
Intravenous Glucose Infusion – The mouse to be infused was first restrained using a rotating
tail vein injection restrainer (Braintree Scientific, Inc, MA, USA). No anesthesia was used during
the entire procedure to assess metabolism in non-anaesthetized and awake mice; thus avoiding
anesthesia-related interferences on brain metabolism. After restraining the mice, we tested the
basal blood glucose levels as described below. The puncture made for testing the basal blood
glucose levels was also used for inserting a vein catheter in the mouse tail (Braintree Scientific,
Inc, MA, USA). The catheter was inserted following the manufacturer’s instructions and was
ensured to be in the tail vein by pushing some saline through the catheter. Any bulge at the
bottom of the tail, resistance, or back flow of saline was considered as an improper insertion of
catheter and performed again at a more proximal point in the same vein or the next vein was
used. The glucose infusion protocol,[134] previously shown in rat to achieve steady-state blood
glucose concentration and brain glucose
13
C enrichment rapidly was slightly modified by scaling
down glucose concentrations according to weight of the mouse. In short, infusion consisting of a
bolus, followed by exponentially decreasing amount of glucose for 8 min, and finally, infusion at
a constant rate was performed for different durations (0, 8, 30, 60, and 120 min) to determine the
time point for steady-state-like concentration. Sixty min was determined to be the approximate
time point to reach the steady-state for most metabolites in the non-anaesthetized mice. Thus,
further constant infusions were carried out for 60 min. Mice were kept in a quiet and warm
environment to avoid too much stress during the glucose infusion. The constant infusion was
72
carried out using a pump from Bio-Rad Laboratories Inc, CA. The labeling pattern for [1-
13
C]glucose has been well described earlier.[135]
Tissue Collection and Extraction – The infusion was stopped after 60 min and catheter
removed, followed by testing for final blood glucose levels as described below. The mouse was
then immediately taken to a cold room followed by decapitation and quick removal of whole
brain and dropping it in liquid nitrogen. The total time taken from end of infusion to start of
decapitation and the total time taken from decapitation to dropping the whole brain in liquid
nitrogen was ensured to be less than 1 min each for all mice (to minimize the post-mortem
metabolic changes). Ischemia is expected to rapidly change the lactate concentrations due to
decapitation and thus, relative concentrations were used to make any inferences about lactate
metabolism. Following freezing of the brain, it was weighed and perchloric acid extraction was
carried out as described previously.[136] Briefly, frozen brain was powdered using a mortar and
pestle (while keeping the brain cold by constantly adding liquid nitrogen). Powdered brain was
000g for 20 min using a
microcentrifuge, followed by neutralization of the supernatant with potassium hydroxide.
Following centrifugation at 22,000g for 20 min (to remove precipitates of potassium
perchlorate), the final brain extract supernatant was stored in -80 °C freezer until used for NMR
or HPLC analysis. Weighing was carried out at each step to calculate the neutralization factor for
each mouse brain extraction.
Blood glucose levels – Briefly, mice were fasted overnight (10-12 h); its tail was warmed
slightly using a lamp or a heating pad. The tail vein was located and a small puncture was made
73
with a 25G needle. The drops of blood that oozed out were tested for the blood glucose levels
using a glucose meter and strips (Abbott, Inc.) as per manufacturers supplied instructions. Blood
concentrations were considered basal if they were below 45mg/dl after fasting overnight. After
glucose infusion, the levels typically rose to ~150-200 mg/dl indicating successful tail vein
glucose infusion. Glucose standards were used regularly to ensure the accuracy of the glucose
meter. The
13
C enrichments of blood glucose were measured by quantifying [1-
13
C]glucose in
perchloric acid extract of the blood by NMR. The
13
C enrichments did not differ much between
the different groups and were ~65% in all groups.
NMR – μL
of D
2
chemical shift reference and internal standard) and 4-5 crystals of
sodium azide (preservative). All samples were analyzed using Varian VNMRS 600Mhz
instrument at 150.86 MHz for
13
C.
13
C spectra were acquired with proton-decoupling and nuclear
Overhauser enhancement with the following parameters: pulse angle of 45°, acquisition time of 1
sec and a relaxation delay of 5 sec, 251ppm spectral width with 32,768 spectral points. A total of
7312 scans were acquired at 25 °C. Peak identification was carried out using chemical shift
values from previous literature[106,137]
after adjusting the chemical shift reference peak of 1, 4
dioxane to 67.4ppm. Relevant peaks in the spectra were identified and integrated using
MestReNova software (Mestrelab Research, CA). The peak area of the internal standard 1,4-
dioxane was used to normalize peak areas. The quantification of each peak was carried out by
acquiring natural-abundance
13
C spectra of Glu, Gln, Asp, NAA, and GABA in a single solution
at different concentrations and constructing a standard curve of peak area vs.
13
C concentration
for each isotopomer of these metabolites.
74
HPLC – Total (
12
C +
13
C) Glu, Gln, and Asp concentrations in the brain extract were
measured, after precolumn derivatization with o-phthaldehyde (OPA) and 2-mercaptoethanol,
and separation on a reverse-phase column, by fluorometric detection as described
previously.[138] To achieve baseline separation of Asp, Glu and Gln from adjacent peaks while
minimizing the total elution time, the following chromatographic program was used: elution with
25% methanol and 75% aqueous sodium phosphate buffer (50 mM, pH 5.29) for 10 min
followed by increase in the percentage of methanol to 49% in 15 min and to 100% in 8 min. The
metabolites were quantified by comparison of the peak areas with those of standards. The
percentage
13
C enrichment of Glu C4 for example, was calculated from the concentration of [4-
13
C]Glu (after correction for natural-abundance
13
C) and the total Glu concentration in each
mouse.
Data analysis – Student's two-tailed t-test was used for statistical analysis of paired data. The
level of statistical significance and the values of n are indicated in the respective figures. *p ≤ 0.05,
**p ≤ 0.01.
RESULTS
13
C-labelling of brain metabolites and the time-course – Fig. 1 shows a representative
13
C
NMR spectrum of the nonTg brain extract after 1 h of [1-
13
C]glucose infusion. Well-resolved
peaks of
13
C labeled isotopomers of lactate, Glu, Gln, Asp, GABA, NAA, MI, and glucose (C1
α and β) were observed. To ensure near-steady state
13
C enrichment of cerebral metabolite pools,
the time-course of
13
C enrichment was examined: the concentrations of the major
13
C-enriched
75
brain metabolites were measured in animals sacrificed at the end-point brain after 8, 30, 60, and
120 min of [1-
13
C]glucose infusion. As shown in Fig. 2A and 2B, the concentrations of [4-
13
C]Glu and [4-
13
C]Gln in awake nonTg mice reached ~maximum values in 60 min and showed
no further increase. [3-
13
C] and [2-
13
C] Glu and Gln increased more slowly; and, little change
was observed after 60 min. Hence, 1 h infusion was deemed to be appropriate and used in
subsequent experiments to examine possible differences in the concentrations of the
13
C-
metabolites between nonTg and 3xTg-AD mice as well as the effect of lipoic acid treatment.
Awake animals were used to avoid the effect of anesthesia on cerebral glucose utilization.[139]
A similar trend of time dependent labeling was observed for the 3xTg-AD mice (Fig. 2C and 2D)
but the levels were much lower as compared to those in the nonTg mice.
Comparison of
13
C-metabolite concentrations among different mouse groups – The
concentrations of the
13
C-labelled metabolites after 1 h of
13
C-glucose infusion are shown in
Table 1, as the mean ± SEM for each mouse group. Within each group, the concentration is
highest for [4-
13
C]Glu followed by [3-
13
C]Glu and [2-
13
C]Glu. For Gln, the concentration is
highest for [4-
13
C]Gln followed by [3-
13
C]Gln and [2-
13
C]Gln. For Asp, the highest
concentration is observed for [3-
13
C]Asp followed by [2-
13
C]Asp. The results are in good
agreement with the known
13
C labeling pattern of brain metabolites from [1-
13
C]glucose infusion
in rat brain, indicating that these techniques are readily transferable to mice also.[140]
As shown in Table 1,
13
C isotopomers of Glu showed a marked decrease in the 3xTg-AD
mice compared with nonTg mice (values in parenthesis represents the % decrease); [4-
13
C]Glu
(~50%), [3-
13
C]Glu (~49%), [2-
13
C]Glu (~55%), [1-
13
C]Glu (~58%).
13
C isotopomers of Gln
also showed a trend of prominent decrease in the 3xTg-AD mice compared with nonTg mice: [4-
76
13
C]Gln (~49%), [3-
13
C]Gln (~63%), [2-
13
C]Gln (~50%), [1-
13
C]Gln (~71%). Overall, more than
50% decrease of
13
C Glu and Gln labeling was observed in the 3xTg-AD mice compared with
the nonTg mice. Similar trends were also observed in the labeling for the different isotopomers
of Asp (except [3-
13
C]Asp), NAA and GABA (except [1-
13
C]GABA) (Table 1).
Importantly, a pronounced recovery of the cerebral metabolites enriched from 1-
13
C glucose
was observed in the brain extract of 3xTg-AD mice fed with lipoic acid; all Glu isotopomer
levels were increased by lipoic acid feeding (values in parenthesis represents the % increase of
3xTg-AD mice fed lipoic acid in comparison to the 3xTg-AD mice not fed lipoic acid): [4-
13
C]Glu (~77%), [3-
13
C]Glu (~70%), [2-
13
C]Glu (~84%), [1-
13
C]Glu (~79%).
13
C labeling of Gln
isotopomers in the 3xTg-AD mice was also stimulated by lipoic acid feeding: [4-
13
C]Gln
(~89%), [3-
13
C]Gln (~137%), [2-
13
C]Gln (~94%), [1-
13
C]Gln (~250%). Moreover, it brought
levels of the different Gln isotopomers close to levels of nonTg mice.
Mice fed lipoic acid showed similar trends in regards to [2-
13
C]Asp, [1-
13
C]Asp, [3-
13
C]NAA
and all isotopomers of GABA. [3-
13
C]Lactate levels also decreased by about 50% in the 3xTg-
AD mice and was almost completely restored in the 3xTg-AD mice fed lipoic acid as shown in
supplemental Fig. 1S. At variance with the 3xTg-AD mice, lipoic acid administration to the
nonTg mice showed a minimal and statistically no significant effect (Table 1). Moreover, the
levels of different MI isotopomers (natural-abundance
13
C) did not show significant differences
among the different groups. It is difficult make conclusive remarks about myoinositol levels by
mainly looking at the natural enrichment levels due to very small concentrations.
Fractional
13
C (%) enrichments of Glutamate, Glutamine, and Aspartate - comparison
among different mouse groups – The total [
12
C +
13
C] concentrations of Glu, Gln and Asp in the
77
endpoint brain, measured by HPLC, are shown in Fig. 3 as the mean ± SEM for each group. The
concentrations are in good agreement with published values for rodent brain. There was no
statistically significant difference in total [
12
C +
13
C] Glu, Gln, and Asp concentrations among
different groups of mice (except the effect of lipoic acid in increasing Asp of the 3xTg-AD
mice).
From the concentration of each
13
C-metabolite (Table 1) and the total metabolite
concentration (Fig. 3), the %
13
C enrichments at each carbon of Glu, Gln, and Asp were
calculated for each mouse group, then the mean ± SEM taken for each group. The results are
shown in Figs. 4-6. As shown in Fig. 4, overall, %
13
C Glu enrichments of the different
isotopomers were significantly decreased in the 3xTg-AD mice as compared to the nonTg
(values in parenthesis represents % decrease): [4-
13
C]Glu enrichment (~48%) (Fig. 4A), [3-
13
C]Glu enrichment (~51%) (Fig. 4B), [2-
13
C]Glu enrichment (~56%) (Fig. 4C), and [1-
13
C]Glu
enrichment (~56%) (Fig. 4D). In comparison, the 3xTg-AD mice fed lipoic acid showed an
increase of ~50% enrichment for the four detectable Glu isotopomers over the untreated 3xTg-
AD mice (Fig. 4).
An even more pronounced decrease in the 3xTg-AD mice compared to the nonTg mice was
found when assessing %
13
C Gln enrichments of the different isotopomers (values in parenthesis
represents the % decrease); [4-
13
C]Gln enrichment (~61%) (Fig. 5A), [3-
13
C]Gln enrichment
(~65%) (Fig. 5B), [2-
13
C]Gln enrichment (~56%) (Fig. 5C), and [1-
13
C]Gln enrichment (~74%)
(Fig. 5D). Lipoic acid feeding increased the enrichment by ~90% for the different Gln
isotopomers of 3xTg-AD mice as shown in Fig. 5 (with the corresponding p value).
In terms of %
13
C Asp enrichments, a similar decrease in the 3xTg-AD compared to the
nonTg mice was found (values in parenthesis represents the % decrease; except [3-
13
C]Asp
78
wherein it represents an increase); [4-
13
C]Asp enrichment (~33%) (Fig. 6A), [3-
13
C]Asp
enrichment (~34%) (Fig. 6B), [2-
13
C]Asp enrichment (~50%) (Fig. 6C), and [1-
13
C]Asp
enrichment (~56%) (Fig. 6D). Lipoic acid feeding did not lead to an overall increase of the
different Asp isotopomers as shown in Fig. 6 (with the corresponding p value). However, it lead
to an increase of % [2-
13
C]Asp by ~40% (p = 0.16) and % [1-
13
C]Asp by ~100% (p ≤ 0.05).
Lipoic acid feeding to the nonTg mice did not elicit a statistically significant effect in either
isotopomers of Glu, Gln, or Asp (except % [1-
13
C]Asp enrichment) (6.4% in nonTg versus
13.2% in nonTg + lipoic acid mouse group, p ≤ 0.05) (Fig 6D).
DISCUSSION
Nuclear magnetic resonance (NMR) allows for quantification of the different glucose
metabolites and in essence allows monitoring of glucose metabolism and the downstream TCA
cycle-related metabolites. The current study using an ex vivo NMR approach has an advantage
over the in vivo approach in terms of facilitating the determination of metabolite levels in non-
anesthetized mice and avoids interferences due to anesthesia. Moreover, the ex vivo approach
permits quantification of the well-resolved
13
C metabolite peaks by high-resolution NMR. On the
other hand, the drawbacks of using our current ex vivo approach are possible post-mortem
changes in metabolite levels and inability to dynamically monitor the metabolite levels to
calculate the metabolic rates as described earlier.[116]
In this study, the metabolic status following [1-
13
C]glucose infusion was assessed in nonTg
and 3xTg-AD mice with and without lipoic acid feeding. Of special interest were Glu and NAA,
for Glu is the major excitatory neurotransmitter, whereas NAA is the most abundant amino-
molecule in the brain. Glu flux is believed to represent up to 80% of glucose metabolism, and to
79
directly define glutamate neurotransmitter rate in the intact brain.[141] The role of Glu in
memory and cognition has been well documented earlier[142] and NAA, while unlikely to be a
neurotransmitter per se, has been shown to be a neuronal and axonal marker.[143]
13
C enrichment in awake vs. anesthetized rodent brain – The %
13
C enrichments of Glu, Gln,
and Asp isotopomers after 1 h of [1-
13
C]glucose infusion indicate the fraction of
13
C labeling
from precursor
13
C glucose to the named
13
C-isotopomer. It indicates the fraction of
13
C labeling
with respect to the total metabolite levels and would be helpful in identifying the metabolic
differences among 3xTg-AD, nonTg mice, and the effect of lipoic acid feeding.
The lag between enrichment of [4-
13
C]Glu and [4-
13
C]Gln follows from the predominance of
glutamine synthesis in glia and confirms earlier studies. The delays in enrichment of [3-
13
C]Glu,
[3-
13
C]Gln, [2-
13
C]Glu, and [2-
13
C]Gln compared with [4-
13
C]Glu and [4-
13
C]Gln reflects the
enrichment of the isotopomers in the first and subsequent ‘turns’ of the TCA cycle.
Accordingly, results match the expected isotopomer, cell location and ‘turn’ through TCA cycles
in the rodent brain, previously described for rats and humans.
The
13
C enrichments of 20.7% at [4-
13
C]Glu and 16.3% at [4-
13
C]Gln observed in our awake
nonTg mice after 1 h of [1-
13
C]glucose infusion (Figs. 4,5) are in reasonable agreement with the
13
C enrichments of ~19% ([4-
13
C]Glu) and ~11 % ([4-
13
C]Gln) reported in anesthetized rat brain
in vivo after 1 h of [1-
13
C]glucose infusion using identical infusion protocol.[140] In their study,
the maximum
13
C enrichments attained after 3 h of infusion were 20-24% for [4-
13
C]Glu and 16-
20% for [4-
13
C]Gln. It is possible that near maximum
13
C enrichments were attained in our
control mice after 1 h (Fig. 2) because glucose uptake and metabolism through the TCA cycle
are faster in awake, than in anesthetized rodent brain. These results strongly suggests that 1 h
infusion is a reasonable time point at which to compare the percentage
13
C enrichments of [4-
80
13
C]Glu and [4-
13
C]Gln among different groups of awake mice with and without lipoic acid
treatment.
[1-
13
C]Glucose metabolism – The concentrations of the
13
C Glu, Gln, Asp, and NAA
isotopomers in Table 1 show the extent of the
13
C label transferred from the intravenously
infused [1-
13
C]glucose to the downstream TCA cycle-related metabolites (Glu, Gln, Asp, and
NAA). The prominent decrease of
13
C Glu, Gln, Asp, NAA, and GABA isotopomers levels in
the 3xTg-AD mice compared to the nonTg mice shows that significantly less
13
C glucose label
was transferred to the Glu, Gln, Asp, NAA, and GABA isotopomers. Interestingly, there was
~50% decrease of [3-
13
C]lactate in the 3xTg-AD compared to nonTg mice; this could be
explained by reduced brain glucose uptake in the 3xTg-AD mice, as shown previously.[128]
Importantly, lipoic acid treatment restored the concentrations of these
13
C-metabolites to levels
close to those in the nonTg mice. These results demonstrate a substantial effect of lipoic acid
towards increasing metabolite labeling in 3xTg-AD mice. Conversely, lipoic acid treatment on
nonTg mice did not result in statistically significant differences in the Glu, Gln, Asp, GABA, and
NAA
13
C isotopomers. A possible reason is that glucose uptake and TCA flux are already
maximal in nonTg mice and, thus, lipoic acid has no effect. Also, there was no difference in the
levels of myoinositol isotopomers among the different groups. It must be noted that 1 h of [1-
13
C]glucose infusion would not necessarily result in any
13
C-enrichment of myoinositol; hence its
concentrations listed in Table 1 may mainly reflect natural-abundance
13
C.
13
C Enrichment of Glu, Gln and Asp – The total concentrations (
12
C +
13
C) of Glu, Gln, and
Asp in various mice groups (Fig. 3) showed only minor differences that did not reach statistical
difference (except for increase of total Asp in the 3xTg-AD mice fed lipoic acid compared with
81
the 3xTgAD mice not fed lipoic acid). However, the % enrichments of Glu, Gln, and Asp
isotopomers, calculated from the total concentrations and the concentrations of
13
C-metabolites,
were significantly decreased by ~50% in the 3xTg-AD mice compared to the nonTg mice (Figs.
4-6). Because this was accompanied with relatively minor differences in the total levels (
12
C +
13
C) of Glu, Gln, and Asp, it may be speculated that the glucose TCA cycle-related
metabolites flux is impaired in the 3xTg-AD mice. Thus, an alternate metabolic source in the AD
brain may be supplying acetyl-CoA for TCA cycle-related metabolites like Glu, Gln, and Asp,
thus ensuring their homeostatic levels.
In summary, the total levels of
13
C label transferred from glucose are decreased in the 3xTg-
AD mice and similarly, the %
13
C enrichments are also decreased. These results strongly suggest
that either (1) there was a decrease of glucose uptake by the brain and/or (2) the utilization of
that glucose for conversion to TCA cycle-related metabolites was impaired. If only the levels of
13
C labeling are considered, it is tempting to speculate that an impairment of glucose uptake
might be the causal reason of decrease in labeling as shown by the ~30-40% decrease in the total
brain glucose uptake in the 13 month-old 3xTg-AD mice.[128] However, taking the %
enrichment and the total (
12
C +
13
C) levels into consideration, it seems unlikely that only low
glucose uptake could be contributing to the lower
13
C labeling and lower %
13
C enrichment. If
only lower glucose uptake were contributing the decrease of
13
C labeling and %
13
C enrichments,
then it should have also affected the total levels of metabolites (
12
C +
13
C) proportionately.
However, data in this study show that the decrease in total metabolite levels is rather minimal in
the 3xTg-AD mice (in contrast to the substantially decreased
13
C labeling and %
13
C
enrichments). Thus, both substrate supply and metabolic rate alterations are likely to be present
in the 3xTg-AD mice that lead to a lower amount of glucose being converted to TCA cycle-
82
related metabolites. These results further substantiate the metabolic alterations present in the
different Alzheimer’s disease rodent models[144,145,146,147]. Magnetic resonance studies
conducted in the APP-PS1 model of Alzheimer’s disease show decreased NAA and Glu[144],
along with an increase of myoinositol.[148] A systematic evaluation on the APP-PS1 model
found the ratio of choline to creatine (Cr) (in the cortical and subcortical areas) as a non-invasive
biomarker in these mice but did not find the utility of Glu, Gln, and MI as biomarkers.[149]
Longitudinal monitoring of the APP-PS1 mice showed lower glutamate, NAA, and taurine with
more apparent changes of NAA in the female mice.[150] Decrease of NAA, Glu, and glutathione
was found in the APP
Tg2576
mice.[145] Similarly, results of decrease in Glu, Gln, NAA, and
GABA, along with metabolic perturbations in the other metabolites, was found by a
metabolomic study of the CRND8 transgenic mouse model of Alzheimer’s disease.[146] Using
1
H-[
13
C]-NMR on the APP-PS1 mouse model of Alzheimer’s disease, it was found that the
levels of [4-
13
C]Glu/Gln and [2-
13
C]GABA were reduced after [1,6-
13
C
2
]glucose infusion at an
early time point but not at isotopic steady state levels.[1] In summary, metabolic analysis of the
different mouse models of Alzheimer’s disease as referenced above (along with several not
referenced here), have shown metabolic perturbations. However, selecting the most relevant
Alzheimer’s disease mouse model based on the hypothesis and the type of study should be an
important consideration. Mouse models like the APP-PS1 are very aggressive in the display of
Aβ pathology and would serve useful in monitoring of a potential therapeutic in reducing Aβ
pathology. Additionally, metabolic alterations are well characterized in the APP-PS1 mouse
model by several studies and have helped to further our understanding of this widely used mouse
model of Alzheimer’s disease. However, the lesser aggressive 3xTg-AD mouse model has
several advantages over the APP-PS1 mouse model in terms of having age-dependent
83
appearance of pathology and synaptic impairments in addition to the close mimicking of the
pathology type (plaques and tangles). Moreover, a very important advantage of the 3xTg-AD
mouse model is the development of one hallmark pathology (plaques) leading to the
development of other signature lesion (tau pathology) – a feature missing in all the previous
mouse models of Alzheimer’s disease.[133] Thus, understanding the metabolic differences in
glucose utilization by the brain (in the 3xTg-AD mouse model) would further our understanding
of this advanced pre-clinical mouse model that is regularly used to assess potential therapeutics
for Alzheimer’s disease. To our knowledge, this is the first study that documents the metabolic
alterations in the 3xTg-AD mouse model of Alzheimer’s disease and complements well with the
earlier studies in other transgenic mouse models but shows a much steeper decline of brain
glucose metabolism. Moreover, the ability of lipoic acid to restore glucose metabolism and the
subsequent TCA-related metabolites specifically in the 13 month-old 3xTg-AD mice with little
effect in the nonTg mice point to the specificity of the restorative effect.
It must be noted that exogenous lipoic acid equilibrates among different intracellular and
extracellular compartments but cannot substitute for covalently bound lipoic acid (as the cofactor
-ketoglutarate
dehydrogenase). It is likely that thiol/disulfide exchange reactions facilitated by lipoic acid are
involved in activation or stimulation of cysteine-rich member of insulin signaling, such as the
insulin receptor itself and insulin receptor substrate (IRS).[45,46] This, in turn, leads to a positive
feedback loop that stimulates greater uptake, utilization, and metabolism of glucose and the
subsequent TCA cycle-related metabolites. These effects may be viewed as an insulin-like effect
of lipoic acid, providing further support to the role of insulin resistance and Alzheimer's
disease.[151,152]
84
Overall, the brain of 13 month-old 3xTg-AD mice show a hypometabolic state that was
encompassed by prominently lower glucose TCA cycle-related metabolites flux (Glu, Gln,
Asp, and NAA and accompanying shift of substrate supply); administration of lipoic acid was
successful in overcoming this hypometabolic state.
85
Table 1. Concentrations of the different isotopomers of
13
C Glu, Gln, Asp, NAA, GABA, and MI
Metabolite nonTg
(A)
nonTg + LA
(B)
3xTg-AD
(C)
3xTg-AD + LA
(D)
A vs. B
A vs. C C vs. D
------------------- p value-----------------
[4-
13
C]Glu 1.59 ± 0.15 1.66 ± 0.19 0.83 ± 0.08 1.47 ± 0.12 0.776 0.001(**) 0.001(**)
[3-
13
C]Glu 1.09 ± 0.17 1.12 ± 0.11 0.56 ± 0.05 0.95 ± 0.10 0.897 0.007(**) 0.005(**)
[2-
13
C]Glu 0.98 ± 0.14 0.92 ± 0.08 0.44 ± 0.05 0.81 ± 0.10 0.72 0.002(**) 0.006(**)
[1-
13
C]Glu 0.33 ± 0.04 0.34 ± 0.04 0.14 ± 0.04 0.25 ± 0.03 0.8 0.004(**) 0.037(*)
[4-
13
C]Gln 0.53 ± 0.10 0.60 ± 0.11 0.27 ± 0.04 0.51 ± 0.10 0.662 0.022(*) 0.034(*)
[3-
13
C]Gln 0.48 ± 0.08 0.42 ± 0.06 0.18 ± 0.03 0.43 ± 0.08 0.583 0.003(**) 0.015(*)
[2-
13
C]Gln 0.38 ± 0.07 0.40 ± 0.06 0.19 ± 0.02 0.37 ± 0.08 0.778 0.018(*) 0.034(*)
[1-
13
C]Gln 0.14 ± 0.02 0.11 ± 0.02 0.04 ± 0.03 0.14 ± 0.04 0.302 0.008(**) 0.046(*)
[4-
13
C]Asp 0.12 ± 0.02 0.19 ± 0.07 0.07 ± 0.02 0.08 ± 0.02 0.369 0.076 0.722
[3-
13
C]Asp 0.32 ± 0.01 0.31 ± 0.01 0.37 ± 0.06 0.32 ± 0.01 0.616 0.445 0.443
[2-
13
C]Asp 0.25 ± 0.05 0.28 ± 0.06 0.13 ± 0.01 0.21 ± 0.02 0.748 0.019(*) 0.013(**)
[1-
13
C]Asp 0.13 ± 0.02 0.28 ± 0.11 0.05 ± 0.01 0.12 ± 0.02 0.198 0.007(**) 0.012(**)
[3-
13
C]NAA 0.12 ± 0.02 0.24 ± 0.10 0.07 ± 0.01 0.11 ± 0.01 0.296 0.012(**) 0.021(*)
[2-
13
C]NAA 0.08 ± 0.01 0.16 ± 0.08 0.05 ± 0.01 0.07 ± 0.01 0.344 0.047(*) 0.281
[4-
13
C]GABA 0.25 ± 0.02 0.27 ± 0.07 0.13 ± 0.01 0.21 ± 0.02 0.781 0.001 (**) 0.010 (**)
[3-
13
C]GABA 0.20 ± 0.03 0.25 ± 0.06 0.12 ± 0.01 0.20 ± 0.02 0.558 0.005 (**) 0.003 (**)
[2-
13
C]GABA 0.42 ± 0.04 0.51 ± 0.10 0.20 ± 0.01 0.45 ± 0.08 0.609 0.000(**) 0.008 (**)
[1-
13
C]GABA 0.12 ± 0.02 0.21 ± 0.05 0.10 ± 0.01 0.14 ± 0.01 0.219 0.128 0.071
[4,6-
13
C]MI 0.03 ± 0.00 0.04 ± 0.00 0.03 ± 0.01 0.04 ± 0.00 0.056 0.034 0.162
[2-
13
C]MI 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.01 0.02 ± 0.00 0.423 0.270 0.410
[1,3-
13
C]MI 0.04 ± 0.00 0.05 ± 0.00 0.04 ± 0.00 0.05 ± 0.01 0.022 (*) 0.276 0.051
[5-
13
C]MI 0.02 ± 0.00 0.03 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.051 0.315 0.237
Concentrations of the different isotopomers of
13
C Glu, Gln, Asp, NAA, GABA, and MI in 13 month- old nonTg
and 3xTg-AD mice plus/minus lipoic acid, after 1 h of [1-
13
C]glucose infusion are shown in the Table 1. Results
in the column 2-5 are presented as average mM ± SEM; results in the columns 6-8 are the p values obtained from
a two-tailed student t-test after comparing between the groups as indicated. *p < 0.05, **p < 0.01 (indicated in
parenthesis); total n = 28 and n ≥ 6/group. The results for Glu, Gln, and Asp are corrected for natural abundance
(the results for NAA, GABA, and MI are not corrected for natural abundance).
86
FIGURES
Fig. 1. A representative
13
C NMR spectrum of brain extract
A proton-decoupled NOE-enhanced
13
C spectrum (150.86 MHz) of typical perchloric
acid brain extract after [1-
13
C]glucose infusion showing the different isotopomers of Glu, Gln,
Asp, GABA, NAA, glucose and MI. The chemical shift and internal standard, 1,4 dioxane is at
67.4 ppm and isopropyl alcohol with three equivalent methyl carbons (solvent in the pH
indicator) at 24.6 ppm. For [4-
13
C]Glu and [3-
13
C]Glu, doublets derived from contiguously
13
C
labeled isotopomers are observed in addition to the singlets.
87
Fig. 2. Time-course of
13
C enrichment in awake nonTg and 3xTg-AD mouse brain
The concentration (mM) of different isotopomers for (A) nonTg glutamate (B) nonTg
glutamine (C) 3xTg-AD glutamate (D) 3xTg-AD glutamine after infusion for 8, 30, 60, and 120
min ( , [4-
13
C]Glu/Gln; , [3-
13
C]Glu/Gln; , [2-
13
C]Glu/Gln; , [1-
13
C]Glu/Gln); for the
nonTg mice, n ≥ 3/time point, for the 3xTg-AD mice, n ≥ 2/time point.
88
Fig. 3. Total concentrations (
12
C +
13
C) in 13 month-old nonTg and 3xTg-AD mice plus/minus
lipoic acid feeding
Total concentrations for (A) Glu, (B) Gln, and (C) Asp are presented as average mM ±
SEM; p values obtained from a two-tailed student t-test comparing the specific groups are
indicated under those groups. *p < 0.05, **p < 0.01; total n = 28 and n ≥ 6/group.
89
Fig. 4.
13
C enrichment percentage of the different isotopomers of glutamate in 13 month-old
nonTg and 3xTg-AD mice plus/minus lipoic acid feeding
Enrichment percentage for (A) [4-
13
C]Glu, (B) [3-
13
C]Glu, (C) [2-
13
C]Glu, and (D) 1-
13
C]Glu are shown as average percentage ± SEM; p values obtained from a two-tailed student t-
90
test comparing the specific groups are indicated under those groups. *p < 0.05, **p < 0.01; total
n = 28 and n ≥ 6/group.
91
Fig. 5.
13
C enrichment percentage of the different isotopomers of glutamine in 13 month-old
nonTg and 3xTg-AD mice plus/minus lipoic acid feeding
92
Enrichment percentage for (A) [4-
13
C]Gln, (B) [3-
13
C]Gln, (C) [2-
13
C]Gln, (D) [1-
13
C]Gln are shown as average percentage ± SEM; p values obtained from a two-tailed student t-
test comparing the specific groups are indicated under those groups. *p < 0.05, **p < 0.01; total
n = 28 and n ≥ 6/group.
93
Figure 6
Fig. 6.
13
C enrichment percentage of the different isotopomers of aspartate in 13 month-old
nonTg and 3xTg-AD mice plus/minus lipoic acid feeding
94
Enrichment percentage for (A) [4-
13
C]Asp, (B) [3-
13
C]Asp, (C) [2-
13
C]Asp, (D) [1-
13
C]Asp are shown as average percentage ± SEM; p values obtained from a two-tailed student t-
test comparing the specific groups are indicated under those groups. *p < 0.05, **p < 0.01; total
n = 28 and n ≥ 6/group.
95
CHAPTER 4: Glucose hypermetabolism in the young 3xTg-AD mouse model and the effect
of lipoic acid
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
TABLE
FIGURES
96
ABSTRACT
Alzheimer’s disease is characterized by age-dependent biochemical, metabolic, and
physiological changes. These age-dependent changes ultimately converge to impair cognitive
functions. This study was carried out to examine the metabolic changes by probing glucose and
TCA cycle metabolism in a 7-month-old triple transgenic mouse model of Alzheimer’s disease
(3xTg-AD). The effect of lipoic acid, an insulin mimetic agent, was also investigated to examine
its ability in modulating age-dependent metabolic changes.
7 month-old 3xTg-AD mice were given intravenous infusion of [1-
13
C]glucose followed by
an ex vivo
13
C NMR to determine the concentrations of
13
C labeled isotopomers of glutamate,
glutamine, aspartate, GABA, and N-acetylaspartate. An intravenous infusion of [1-
13
C]glucose +
[1,2-
13
C]acetate was given for different periods of time to distinguish neuronal and astrocytic
metabolism. Enrichments of glutamate, glutamine, and aspartate were calculated after
quantifying the total (
12
C +
13
C) concentrations by HPLC.
A hypermetabolic state was clearly evident in 7-month-old 3xTg-AD mice in contrast to the
hypometabolic state reported earlier in 13-month-old mice.[128] Hypermetabolism was
evidenced by prominent increase of
13
C labeling and enrichment in the 3xTg-AD mice. Lipoic
acid feeding to the hypermetabolic 3xTg-AD mice brought the metabolic parameters to the
nonTg mice.
97
INTRODUCTION
Alzheimer’s disease (AD) is a complicated neurodegenerative disease with several gaps in
our understanding about its etiology and a marked absence (to date) of drug therapies targeting
the underlying mechanisms of neurodegeneration. However, hallmarks of AD include the
widespread presence of β-amyloid plaques and neurofibrillary tangles; their detection in post-
mortem tissue validates a definite diagnosis. Additionally, there is a clear association of AD with
biochemical,[153] metabolic,[73] and physiological changes.[154] These three factors also show
age-dependent changes that ultimately converge to impair cognitive abilities. Research from
several pre-clinical and clinical studies have established the presence of multiple metabolic
changes in brain and disturbances of the primary brain energy source i.e., glucose.[73] These
disturbances are not limited to brain glucose uptake but also extend to glycolytic metabolism,
TCA cycle, and the subsequent formation of neurotransmitters and neurochemicals like Glu,
GABA, Gln, Asp, and several others. Ultimately, the impaired metabolic pathways may become
incapable of supporting the highly intricate supply of energy and metabolites required for
neuronal function, leading to a synaptic failure that impinges on cognitive abilities.
Several mouse models with one or multiple transgenes have been designed to further our
understanding about AD and test potential therapeutics.[155] Each mouse model highlights one
or multiple facets of AD, i.e., metabolic abnormalities, cognitive impairment, Aβ plaques,
hyperphosphorylated tau and/or tangles; thus, choosing the correct mouse model according to the
hypothesis being addressed is critical. In that regards, the 3xTg-AD mouse model shows age-
dependent appearance of pathology type (plaques and tangles), pathology location (restricted to
hippocampus, amygdala, and cerebral cortex), and synaptic impairments. Additionally, it shows
a sequence of development of one hallmark pathology (plaques) leading to the development of
other signature lesions (tau pathology) – a feature missing in all the previous mouse models of
AD.[133] In an earlier study, we characterized the brain glucose utilization of 13-month-old
3xTg-AD mice and the insulin mimetic effect of lipoic acid. The important finding of that study
98
was the occurrence of a glucose hypometabolic state and an insulin-mimetic effect by lipoic acid
leading to partial restoration of glycolytic metabolism.[156] It is worth noting that 13-month-old
3xTg-AD mice show extracellular -amyloid deposits in cortex and human tau
immunoreactivity.[133] Because a hypometabolic state was predominant in the 13-month-old
3xTg-AD mice, it was critical to examine its preceding metabolic status that leads to
hypometabolism (at an earlier time point with less pathology). Thus, we selected the 7-month-
old 3xTg-AD mice that are characterized by ‘diffusion of -amyloid plaques in neocortex but no
human tau immunoreactivity’ as the appropriate age for further investigation of brain
metabolism.
The current study was conducted to examine metabolic differences in the brain of 7-month-
old 3xTg-AD and nonTg mice and assess the therapeutic effect of insulin mimetic, lipoic acid. In
addition to [1-
13
C]glucose infusion, the metabolic state was also probed with [1-
13
C]glucose +
[1,2-
13
C]acetate co-infusion to assess neuronal and astrocytic metabolism. It must be noted that,
glucose is metabolized by both neurons and astrocytes, however, it is a preferential fuel for
neurons, whereas, acetate is exclusively metabolized by astrocytes. Thus, neuronal and astrocytic
metabolism can be studied simultaneously by a co-infusion of
13
C labeled glucose and
acetate.[157]
99
MATERIALS AND METHODS
Materials – Deuterium Oxide (99.9%) and [1,2-
13
C]acetate (99%) were obtained from
Cambridge Isotope Laboratories, Inc (MA, USA). [1-
13
C]glucose (99%) was purchased from
Sigma-Aldrich (MO, USA) and all other chemicals were the purest grade available from Sigma-
Aldrich (MO, USA). Rodent tail vein catheter and restraining apparatus were obtained from
Braintree Scientific, Inc (MO, USA). The constant infusion of [1-
13
C]glucose and [1,2-
13
C]acetate was carried out by using a pump from Bio-Rad Laboratories Inc (CA, USA).
Mice colonies and lipoic acid feeding – Colonies of 3xTg-AD and nonTg mouse strain
(C57BL6/129S; Gift from Dr. Frank Laferla, University of California, Irvine) 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. The triple
transgenic mouse model of AD (3xTg-AD) was first developed by Oddo et al [133]. Mice were
housed on 12-h light/dark cycles and provided ad libitum access to food and water. 3xTg-AD
and nonTg mice were either fed with water containing 0.23% R-sodium lipoic acid (gift from
Geronova Research, Inc.) or normal water for 4 weeks. Thus, at the time of glucose infusion,
mice were ~7 months old.
Intravenous Glucose and Acetate Infusion – The mouse to be infused was first restrained
using a rotating tail vein injection restrainer. Anesthesia was not used during the entire
procedure; this allowed us to measure metabolism in awake non-anesthetized mice. After
restraining the mice, we tested the basal blood glucose levels as described[156]. The puncture
made for testing the basal blood glucose levels was also used for inserting a vein catheter in the
mouse tail (Braintree Scientific, Inc, MA, USA). The catheter was inserted following the
manufacturer’s instructions and was ensured to be in the tail vein by pushing some saline
100
through the catheter. Any bulge at the bottom of the tail, resistance, or back flow of saline was
considered as an improper insertion of catheter and performed again at a more proximal point in
the same vein or the next vein was used. The glucose and acetate infusion protocol was carried
out as described earlier.[134,158] Briefly, 1-
13
C]glucose (0.3M) + [1,2-
13
C]acetate (0.6M)
solution was prepared. A bolus injection to raise the blood glucose levels to normoglycemic
range was followed by exponentially decreasing amount of glucose for 5 min. Finally infusion at
a constant rate was performed for different durations as specified (20, 60, and 150 min). Mice
were kept in a quiet and warm environment to avoid too much stress during the glucose and
acetate infusion. The labeling pattern for [1-
13
C]glucose and [1,2-
13
C]glucose has been well
described earlier.[135,158]
Tissue Collection and Extraction – The infusion was stopped after the specified time and
catheter removed, followed by testing for final blood glucose levels as described below. The
mouse brain was then immediately snap frozen using liquid nitrogen. It was ensured that the total
time taken for snap freezing of the brain (from the end of infusion) was less than one minute for
all mice to minimize the post-mortem metabolic changes. Subsequently, weighing and perchloric
acid extraction was carried out as described previously.[137,156] Briefly, frozen brain was
powdered using a mortar and pestle (while keeping the brain cold by constantly adding liquid
nitrogen) followed by addition of perchloric acid and subsequent centrifugations. A final
centrifugation was carried out to remove precipitation and the final brain extract supernatant was
stored in -80 °C freezer until used for NMR or HPLC analysis.
NMR – The stored brain extracts were thawed and mixed in appropriate proportion with D
2
O,
1.5 L 1, 4 Dioxane (chemical shift reference and internal standard) and a preservative (sodium
azide).
13
C NMR analysis was carried out on a Varian VNMRS 600MHz instrument at 150.86
MHz.
13
C spectra were acquired with proton-decoupling and nuclear Overhauser enhancement
with the following parameters: pulse angle of 45°, acquisition time of 1 sec and a relaxation
101
delay of 5 sec, 251ppm spectral width with 32,768 spectral points. A total of 7312 scans were
acquired at 25 °C. The chemical shift reference peak of 1, 4 dioxane was set to exactly 67.4ppm.
This was followed by peak identification using chemical shift values from previous
literature.[106,137]
The peak areas were normalized by using the peak area of 1, 4-dioxane as the
internal standard. MestRenova software from Mestrelab Research (CA, USA) was used to
integrate relevant peaks after normalization of peak areas. The quantification of each peak was
carried out by acquiring natural-abundance
13
C spectra of Glu, Gln, Asp, NAA, and GABA in a
single solution at different concentrations and constructing a standard curve of peak area vs.
13
C
concentration for each isotopomer of these metabolites.
13
C Labeling Patterns and Interpretation – Labeling of brain metabolites from [1-
13
C]glucose and [1,2-
13
C]acetate is well described in several earlier publications by Dr.
Sonnewald.[147] Briefly, after the formation of [3-
13
C]pyruvate through glycolysis, it can be
either decarboxylated to [2-
13
C]acetyl CoA, or transaminated to [3-
13
C]alanine, or reduced to [3-
13
C]lactate or caboxylated to oxaloacetate (OAA) (in astrocytes). However, once the
13
C label
originating from [1-
13
C]glucose enters the TCA cycle, it undergoes several steps to form [4-
13
C] α-ketoglutarate that can be transaminated to form [4-
13
C]glu. In GABAergic neurons, [4-
13
C]glu can be further decarboxylated to [2-
13
C]GABA. Astrocytes remove [4-
13
C]glu from the
synaptic cleft (to prevent excitotoxicity), followed by its conversion to [4-
13
C]gln (via astrocytes
specific glutamine synthetase) or to [4-
13
C] α-ketoglutarate, which can enter TCA cycle and form
[2-
13
C]-/[3-
13
C]OAA and can be further transaminated to [2-
13
C]-/[3-
13
C]aspartate. If the
13
C
label is not released in the 1
st
turn of TCA cycle, it would form [2-
13
C]-/[3-
13
C]glutamate and
glutamine and [4-
13
C]-/[3-
13
C]GABA can be formed after several steps if OAA labeled from the
1
st
turn of the cycle condenses with unlabeled acetyl CoA. In regards to the exclusive metabolism
of [1,2-
13
C]acetate in astrocytes, it is first converted to [1,2-
13
C]acetyl CoA in astrocytes. Once
[1,2-
13
C]acetyl CoA enters the astrocytic TCA cycle, it can form, after several steps, [4,5-
13
C] α-
102
ketoglutarate which is the precursor of [4,5-
13
C]glutamate and [4,5-
13
C]glutamine. After transfer
of [4,5-
13
C]glutamine to the neurons it is converted to [4,5-
13
C]glutamate (by phosphate
activated glutaminase) in glutamatergic neurons. It can also be converted to [1,2-
13
C]GABA in
GABAergic neurons.
The
13
C labelling pattern can be influenced by pyruvate carboxylation, which occurs only in
astrocytes, and in the brain of normal anesthetized rat given i.v. infusion of
13
C-glucose with the
same protocol as used by us, comprised only 19-26% of the total glutamine synthesis.[159]
Similarly, in our mice, the
13
C labelling of Glu and Gln are determined mainly by the pyruvate
dehydrogenase pathway, which labels C4 in the 1
st
turn and C3 and C2 equally in the 2nd turn.
Metabolic Ratios – Relevant metabolic ratios have been calculated according to several
publications by Dr. Sonnewald.[160,161,162] We calculated the glycolytic activity,[161] TCA
cycle activity,[160]
13
C glucose and acetate cycling ratio in terms of glutamate and glutamine,[162] and transfer ratio
from astrocytes to neurons.[161]
Regional analysis of brain glucose uptake – The PET images acquired earlier[128] of the 7-
month-old nonTg and 3xTg-AD were re-analyzed to study brain region-specific differences since
only the whole brain glucose uptake was measured earlier. A structural MRI Rapid Acquisition
with Refocusing Echoes (RARE) T2 image of a normal 5-month-old C57BL6 mouse brain
(dimensions 128 x 128 x 14; voxel size 0.1 x 0.1 x 1 mm
3
) was used as a template for FDG-PET
analysis. FDG images (dimensions 128 x 128 x 63; voxel size 0.845 x 0.845 x 1.212 mm
3
) were
first preprocessed using AMIDE (Andreas Loening, http://.amide.sourceforge.net) and Mango
(Research Imaging Institute, University of Texas Health Science Center at San Antonio, Texas)
by converting them to nifti files followed by rotation to match MRI template’s orientation, and
re-sliced in Z to MRI voxel size. Then they were registered to the MRI template using Mango
103
point-matching co-registration tool with mutual information cost function for transformation, 12
degree of full search, and tri-linear cost function for interpolation. In the end the mean value of
FDG image intensity for each animal was extracted on bilateral hippocampus, motor
somatosensory cortex, as well as the whole brain regions, which were first drawn on the high
resolution MRI image. All FDG images were done in the same batch and the image groups were
blinded to the image analyst.
HPLC – HPLC analysis was carried out to measure the total (
12
C +
13
C) Glu, Gln, Asp and
GABA concentrations in the brain extracts as described previously.[156]
,[138]
Briefly, precolumn
derivatization with o-phthaldehyde (OPA) and 2-mercaptoethanol and separation on a reverse-
phase column with fluorometric detection was carried out with the following modification in the
program for gradient elution with methanol and 0.05 M phosphate buffer (pH 5.29); the
percentage of methanol was increased from 25% to 40% in 29.5 min, then to 84% in 22.3 min to
resolve GABA from adjacent peaks. The peak areas of the pure chemicals (standards) were used
to quantify the metabolites. The percentage
13
C enrichment of [4-
13
C]Glu for example, was
calculated from the concentration of [4-
13
C]Glu (after correction for natural-abundance
13
C) and
the total Glu (
12
C +
13
C) concentration in each mouse.
Data analysis – Student's two-tailed t-test was used for statistical analysis of paired data. The
level of statistical significance and the values of n are indicated in the respective figures. *p ≤ 0.05,
**p ≤ 0.01.
104
RESULTS
Comparison of
13
C-metabolite concentrations among different mouse groups after [1-
13
C]glucose infusion – To compare results with our earlier studies performed in 13-month-old
mice, we carried out [1-
13
C]glucose infusion in 7-month-old nonTg and 3xTg-AD mice. Well-
resolved peaks of
13
C labeled isotopomers of alanine, lactate, Glu, Gln, Asp, GABA, NAA, MI,
and glucose (C1α and β) were observed as shown previously.[156] The concentrations of the
different isotopomers of Glu, Gln, Asp, GABA, and NAA in 7 month-old nonTg and 3xTg-AD
mice plus/minus lipoic acid feeding are shown in the Table 1. In all the four groups of mice, the
highest labeling was of [4-
13
C]Glu followed by [3-
13
C]Glu and [2-
13
C]Glu (almost identical),
with the lowest labeling of [1-
13
C]Glu. Overall, labeling pattern was in good agreement with the
known
13
C labeling pattern of brain metabolites after [1-
13
C]glucose infusion.[140] Slightly
lower labeling was found in the 7-month-old nonTg mice compared to previous results in 13-
month-old nonTg mice[156]. Surprisingly, a prominent increase of labeling was observed in the
7-month-old 3xTg-AD mice in comparison to the age matched nonTg mice.
13
C isotopomers of Glu showed a marked ‘increase’ in the 3xTg-AD mice compared with
nonTg mice; (values in parenthesis represents the % increase in 3xTg-AD compared to nonTg
mice): [4-
13
C]Glu (~69%), [3-
13
C]Glu (~64%), [2-
13
C]Glu (~40%), [1-
13
C]Glu (~100%).
Similarly,
13
C isotopomers of Gln also showed the trend of marked increase in the 3xTg-AD
mice compared with nonTg mice: [4-
13
C]Gln (~58%), [3-
13
C]Gln (~77%), [2-
13
C]Gln (~61%),
[1-
13
C]Gln (~100%). Overall, more than 70% increase in the levels of
13
C Glu and Gln labeling
was observed in the 3xTg-AD mice compared with the nonTg mice. Similar trends were also
observed in the labeling for the different isotopomers of Asp, NAA and GABA (Table 1). Lipoic
acid feeding to the nonTg mice had almost no effect but it normalized the metabolite levels by
bringing it down to the levels of nonTg mice. (Table 1).
105
Fractional
13
C (%) enrichment of metabolites after glucose infusion - comparison among
different mouse groups – The total [
12
C +
13
C] concentrations of Glu, Gln and Asp after glucose
infusion were measured by HPLC and represented as mean ± SEM for each group (Fig. 1 (Ai, B
i, and Ci). Similar to the results in 13-month-old mice,[156] there was no statistically significant
difference in total [
12
C +
13
C] Glu, Gln, and Asp concentrations among different groups of mice
at 7-months of age (except the effect of lipoic acid in increasing total Gln of the 3xTg-AD mice).
The enrichment for each of the
13
C labeled metabolites was calculated in order to assess the
flux of
13
C label. The calculated %
13
C enrichment levels for the different isotopomers of Glu,
Gln, and Asp are represented in Fig. 1 as mean ± SEM. The trend of increase in
13
C labeled
metabolites levels was also replicated in the % enrichment levels (values in parenthesis
represents the % increase in the 3xTg-AD mice compared to nonTg mice); [4-
13
C]Glu (~92%)
(Fig. 1 Aii), [3-
13
C]Glu (~84%) (Fig. 1 Aiii), and [2-
13
C]Glu (~60%) (Fig. 1 Aiv). The
13
C
isotopomers of Gln also showed a similar trend of marked increase in the 3xTg-AD mice
compared with nonTg mice: [4-
13
C]Gln (~94%) (Fig. 1 Bii), [3-
13
C]Gln (~114%) (Fig. 1 Biii),
[2-
13
C]Gln (~90%) (Fig. 1 Biv). Similarly, the
13
C isotopomers of Asp also showed a similar
trend of marked increase in the 3xTg-AD mice compared with nonTg mice: [4-
13
C]Asp (~101%)
(Fig. 1 Cii), [3-
13
C]Asp (~8%) (Fig. 1 Ciii), [2-
13
C]Asp (~69%) (Fig. 1 Civ). As seen in the
metabolite isotopomer concentration, the lipoic acid-fed 3xTg-AD mice showed a prominent
decrease and the levels in these mice were close to the nonTg mice (Fig. 1). However, no effect
of lipoic acid was seen in the nonTg mice.
Regional brain glucose uptake in 7 month-old nonTg and 3xTg-AD mice plus/minus lipoic
acid feeding – The re-sliced [
18
F]FDG-PET images were used to calculated the region specific
brain glucose uptake in the hippocampus and motor and somatosensory cortex (Fig. 1Di): no
regional differences were observed amongst the different groups (Fig. 1Dii and iii), although
glucose uptake in the whole brain was slightly lower in the 7-months old.
7
106
Comparison of glial and neuronal metabolism -
13
C-metabolite concentration after [1-
13
C]glucose + [1,2-
13
C]acetate infusion – These studies were carried out by infusing [1-
13
C]glucose + [1,2-
13
C]acetate for different periods of time (5 min, 20 min, 60 min, and 150 min)
to assess time-dependent changes in
13
C flux. Co-infusion of [1-
13
C]glucose + [1,2-
13
C]acetate
leads to a typical pattern of labeling (Fig. 2) and NMR trace (Fig. 3). A hypermetabolic state in
both neurons and astrocytes was evidenced by the absolute levels of different
13
C metabolite
isotopomers at 60 min (data not shown) and 150 min (Table 2). However, a clear trend of
increase in the
13
C labeling of 3xTg-AD mice was not seen at 5 min and 20 min (data not
shown). This is not unexpected because, at 5 min, mainly the metabolites labeled from 1
st
cycle
of TCA cycle are detected. Although several of them showed an increase in the 3xTg-AD
compared to nonTg a uniform trend was not seen.
The calculated
13
C metabolite isotopomers levels at 150 min infusion from 7-month-old
nonTg and 3xTg-AD mice are shown in the Table 2. The 3xTg-AD mice show no increase of the
major neuronal metabolite from the 1
st
turn of TCA cycle from [1-
13
C]glucose i.e., [4-
13
C]Glu;
similarly, there was no increase in the major astrocytic metabolite from the 1
st
turn TCA cycle of
[1,2-
13
C]acetate i.e., [4,5-
13
C]Glu. However, metabolites labeled in the 2
nd
and 3
rd
turns of TCA
cycle showed a clear trend of increase in the 3xTg-AD mice compared to the nonTg mice (Table
2). The glutamate isotopomers labeled in the 2
nd
and 3
rd
turns showed an average increase of
>60% in comparison to age matched nonTg mice. Similarly, the glutamine isotopomers labeled
in the 2
nd
and 3
rd
turns showed an average increase of greater than 40% in comparison to the age
matched nonTg mice. Similar trends of increase in the 3xTg-AD for
13
C labeled metabolites for
Asp, GABA, and MI were observed. No differences were seen in the NAA isotopomers after 150
min infusion of [1-
13
C]glucose + [1,2-
13
C]acetate. Mice fed lipoic acid showed similar trend as
seen with just [1-
13
C]glucose infusion, i.e., there were almost no changes in the nonTg mice fed
lipoic acid but the 3xTg-AD mice fed lipoic acid showed a decrease in the metabolite
107
isotopomers concentration and almost brought the levels of metabolite isotopomer concentration
to those of nonTg mice (Table 2).
Comparison of glial and neuronal metabolism - Fractional
13
C (%) enrichment of
metabolites after [1-
13
C]glucose + [1,2-
13
C]acetate infusion – The fractional enrichment of the
different metabolite isotopomers for glutamate and glutamine was calculated at the different time
points (Fig. 4). No statistically significant differences were observed for enrichment of glutamate
isotopomers from the 1
st
turn of TCA cycle i.e., [4-
13
C]Glu and [4,5-
13
C]Glu from [1-
13
C]glucose
and [1,2-
13
C]acetate respectively at 150 min (Fig. 4 Ai and Aiv). However, [4-
13
C]Glu
enrichment at 60 min was increased by ~50% (Fig. 4 Ai). Albeit, not statistically significant,
trends of increasing [4-
13
C]Glu and [4,5-
13
C]Glu enrichment were also observed in the 3xTg-AD
mice after 5 min and 20 min (magnitude varied across metabolites). Similar trends were also
seen in the enrichment of glutamine isotopomers labeled from 1
st
turn of TCA cycle, i.e., [4-
13
C]Gln and [4,5-
13
C]Gln from [1-
13
C]glucose and [1,2-
13
C]acetate respectively (Fig. 4Bi,iv).
Lipoic acid treatment had no statistically significant effect on these metabolites in both nonTg
and 3xTg mice (Fig. 4Ai,iv,Bi,iv).
Interestingly, the metabolites labeled in the subsequent turns of TCA cycle at 60 min and
150 min show a general trend of increasing enrichment in the 7-month-old 3xTg-AD compared
with age matched nonTg mice (Fig. 4Aii,iii,v,vi, Bii,iii,v,vi). For several of the metabolite
isotopomers, the enrichment was increased by 100% and thus clearly showing a state of
hypermetabolism in the 7-month-old 3xTg-AD mice. Lipoic acid feeding to the nonTg mice did
not result in statistically significant differences but lipoic acid feeding to the 3xTg-AD mice
showed similar decrease of enrichment as seen in the metabolite isotopomer concentrations (Fig.
4Aii,iii,v,vi, Bii,iii,v,vi).
108
Metabolic ratios in 7 month-old nonTg and 3xTg-AD mice plus/minus lipoic acid feeding –
As shown in Fig. 5Ai,ii, TCA cycle activity and % glycolytic activity were increased by 2-fold in
the 3xTg-AD mice as compared with the age-matched nonTg mice. Lipoic acid had almost no
effect in the nonTg mice but substantially decreased the TCA cycle activity and % glycolytic
activity in the 3xTg-AD mice, bringing it close to the levels of nonTg mice (Fig. 5Ai,ii). Looking
at the
13
C glucose cycling ratio in terms of glutamate and glutamine, a very similar trend was
seen (Fig. 5Bi,ii).
13
C acetate cycling ratio in terms of glutamate also showed a similar increase
in the 3xTg-AD mice and statistically significant decrease upon lipoic acid treatment (Fig. 5Ci).
The
13
C acetate cycling ratio in terms of glutamine, showed a minor, statistically not significant,
increase in the 3xTg-AD mice; herein lipoic acid induced a statistically non significant decrease
in the nonTg mice and 3xTg-AD mice (Fig. 5Cii).
Notably, glutamine transfer from astrocytes to glutamatergic neurons was decreased in the
3xTg-AD mice and a statistically insignificant increase was seen with lipoic acid feeding to the
3xTg-AD mice (Fig. 5Di). In terms of substrate transfer from astrocytes to GABAergic neurons,
there was almost no difference among the nonTg and 3xTg-AD mice but a minor, statistically
not significant decrease with lipoic acid feeding in the nonTg and 3xTg-AD mice (Fig. 5Dii). It
should be noted that the transfer ratio between astrocytes to glutamatergic or GABAergic
neurons represents the substrate transfer (glutamine) from astrocytes to the specific neurons.
109
DISCUSSION
This study was aimed at understanding the age-related metabolic changes in the 7-month-old
mouse model of Alzheimer's disease (3xTg-AD) and the effect of lipoic acid. The study used
13
C
ex vivo NMR after supplying a single (glucose) or multiple (glucose and acetate) brain substrates
to dissect neuronal and astrocytic metabolism. The flux of
13
C from glucose and acetate through
the TCA cycle, synthesis of metabolic and neurotransmitter pools of Glu and GABA, their
precursor Gln and of several other metabolites, have been calculated. The 7-month-old 3xTg-AD
mice show a clear hypermetabolic state evidenced by an increase (> 50%) in the concentration of
several [
13
C]-metabolites. Treatment with lipoic acid brought the levels of these
13
C-metabolites
close to those observed in the nonTg mice, thus hinting at its ability to regulate glucose uptake
and metabolism.
[1-
13
C]Glucose metabolism – The concentrations of
13
C labeled Glu, Gln, Asp, GABA, and
NAA isotopomers in Table 1 show a clear increase of
13
C flux in the 3xTg-AD mice by ~50% in
key metabolites. This signals greater transfer of
13
C label from glucose and thus faster turnover
of TCA cycle-related metabolites. A simple explanation of these results would be an increase in
brain glucose uptake; however, whole brain glucose uptake in the 7-month-old 3xTg-AD mice
was slightly decreased.[128] Whole brain FDG-PET can mask regional differences due to spill-
over effects (strong near facial glands) and the generally low FDG-PET sensitivity. Accordingly,
previous data were reanalyzed in order to calculate regional glucose uptake in hippocampus and
motor and somatosensory cortex (Fig. 1Di). No regional glucose uptake differences were found
among the four groups. Hence, the hypermetabolic state in the 3xTg-AD mice cannot be
explained by increased glucose uptake in these regions. That, FDG-PET data should complement
[1-
13
C]glucose-
13
C NMR results is debatable. In FDG-PET experiments, the amount of glucose
injected is very low due to concerns about radioactive toxicity. In fact, the amount of glucose
injected during FDG-PET is so low that the blood glucose levels actually go down slightly after a
110
typical FDG injection to a fasting mouse (data not shown), most likely due to insulin activation.
By contrast, [1-
13
C]glucose-
13
C NMR experiments involve a bolus dose followed by constant
infusion of glucose that ensures physiological levels of blood glucose levels (raised 3-4 times the
fasting blood glucose levels). Another interesting observation during the FDG-PET experiments
was ~100% increase of standard uptake values (SUV) in the heart of 7-month-old 3xTg-AD mice
compared to age-matched nonTg (data not shown). Hence, it may be speculated whether the
actual brain glucose uptake in the 3xTg-AD mice was being masked by a ~100% increase in
heart glucose uptake.
[1-
13
C]Glucose + [1,2-
13
C]acetate metabolism – The question of whether the
hypermetabolism observed with [1-
13
C]glucose infusion (single substrate) can be ascribed to
mainly hypermetabolic neurons or a similar hypermetabolic state also exists in astrocytes was
addressed with co-infusion of [1-
13
C]glucose and [1,2-
13
C]acetate over four different time
periods (with 150 min being the last time period); it must be noted that by 150 min of [1-
13
C]glucose + [1,2-
13
C]acetate infusion, the fractional amounts of
13
C multiplets in glutamate and
glutamine reach ~steady state levels.[158] The concentration of [
13
C]metabolites at 150 min
(Table 2) and the enrichment of different metabolites (Fig. 4) revealed a general increase in the
3xTg-AD mice, especially at 60 min and 150 min. There was almost no difference among the
four groups in metabolite and enrichment levels of [4-
13
C]glu/gln, [4,5-
13
C]glu/gln at 150 min
after infusion. These are the metabolites that are derived mainly from the 1
st
turn of the TCA
cycle. It is possible that after the 150-min infusion, inter-group differences in the rate of
formation of the TCA cycle-related metabolites from its 1
st
turn are masked. However, the
metabolites labeled in the 2
nd
and subsequent turns of TCA i.e., [3/2-
13
C]glu/gln and [1,2-
13
C]glu/gln show an almost generalized increase of enrichment at 60 min and 150 min (Fig. 4).
111
Metabolic Ratios – Hypermetabolism was also evidenced by a 100% increase in (a)
glycolytic activity (measured from alanine levels) and (b) TCA cycle activity (based on the
glutamate from [1-
13
C]glucose in the 2nd turn relative to the 1st turn of TCA cycle) (Fig. 5);
these increases were based on comparisons with the nonTg mice. Similar increases of
13
C
glucose cycling ratio in terms of glutamate and glutamine were also observed. The
13
C acetate
cycling ratio of the 3xTg-AD mice in terms of glutamate also increased by ~100% but not in
terms of glutamine (Fig. 5). It must be noted that the
13
C glucose or acetate cycling ratio gives
information about how long the
13
C label derived from [1-
13
C]glucose and [1,2-
13
C]acetate stays
in the TCA cycle before being incorporated into glutamate or glutamine. The results in this study
suggest that the
13
C label turns over faster in the TCA cycle of 7-month-old 3xTg-AD mice.
Astrocytes and neurons interact actively, with astrocytes supporting neuronal metabolic
requirements and clearing excess glutamate from extracellular space to prevent neuronal
excitotoxicity. A large fraction of the glutamate and GABA present in the neurons is synthesized
from glutamine transferred from astrocytes. Using the data of glutamine labeled specifically in
the astrocytes (from [1,2-
13
C]acetate) and comparing it to the similarly labeled glutamate, the
transfer ratio between astrocytes and neurons may be calculated. Interestingly, the transfer of
glutamine from astrocytes to neurons for glutamate was decreased in the 3xTg-AD mice (Fig. 5).
This indicates that the hypermetabolic state in the 7-month-old 3xTg-AD mice does not have a
strong astrocytic support system to fulfill the neuronal metabolic demands. It is possible that the
reduction in supply of metabolites from astrocytes would render the neurons energy deficient and
thus force the neurons into a hypermetabolic state to meet their energy demands; that might
result in greater flux of glutamate. Clinical studies showing less supportive astrocytic system in
normal healthy elderly individuals has also been documented earlier.[163]
Effect of Lipoic Acid – The data shown in this study indicates that lipoic acid treatment
brought the high metabolite levels observed in the 3xTg-AD mice close to those of nonTg mice.
112
Exogenous lipoic acid is expected to equilibrate among the different intracellular and
extracellular compartments but cannot substitute for covalently bound lipoic acid (as the cofactor
of mitochondrial complexes such as pyruvate- and -ketoglutarate dehydrogenases). It is likely
that thiol/disulfide exchange reactions facilitated by lipoic acid are involved in activation or
stimulation of cysteine-rich members of insulin signaling, such as the insulin receptor itself and
insulin receptor substrate (IRS).[45,46] This, in turn, regulates metabolism of glucose and the
subsequent TCA cycle-related metabolites. Lipoic acid fed to 7-month-old 3xTg-AD mice did
not improve the reduced LTP but was effective in restoring the reduced LTP in 13-month-old
3xTg-AD mice.[128] The functional effects of lipoic acid in the 7- (this study) and 13-month
old[156] 3xTg-AD mice seems to be related or determined by the cellular redox environment.
y , β q , y y z D –
Of key importance in understanding the hypermetabolic state reported in the present study is the
stage of pathology in the 3xTg-AD mice at 7 months of age i.e., diffusion of -amyloid plaques
in neocortex is present but immunoreactivity to human tau is absent.[133] Thus, at 7-months of
age, the 3xTg-AD mice are comparable to several amyloid mouse models of AD (e.g., Tg2576
and APP/PS1) in terms of the presence of plaque pathology and the absence of tau pathology.
The 7-month-old Tg2576 mice show an increased cerebral glucose uptake[164] and the APP/PS1
mouse model revealed increased glucose uptake only close to plaques but not in amyloid-free
cerebral tissues[165] and increased basal metabolic rate of cells surrounding amyloid
plaques.[166] Studies carried out in the APP23xPS45 mouse model of AD found that the
synchronized neuronal firing could well increase the risk for seizure like activity.[167] This is
correlated with increased epileptic seizures in AD patients[168] and also reflected in a hAPP
mouse model of AD by occurrence of spontaneous nonconvulsive seizures.[169] Additionally,
the risk of epileptic activity is 87-fold greater in AD patients with early-onset dementia (that is
typically characterized by Aβ pathology) and the relationship between AD and seizures is even
113
tighter in autosomal early-onset familial AD.[170,171] Altered calcium homeostatic mechanisms
have been implicated in numerous models of epilepsy to be associated with hyperactivity near
plaques[172] and leads to decrease in synaptic inhibition.[167] This raises an important question
of whether the extent of subclinical epileptic activity in AD has been underestimated.
The fact that total metabolite levels (
12
C +
13
C - Glu, Gln, Asp) did not change (Fig. 1) but
was accompanied by major changes in the flux of
13
C from [1-
13
C]glucose and [1,2-
13
C]acetate
to metabolites strengthens the possibility of a shift in metabolic pathways. More importantly,
whether the sudden increase of labeled glutamate after [1-
13
C]glucose and [1,2-
13
C]acetate
infusion leads to excitotoxic damage remains to be determined. It may be speculated whether this
excitotoxic damage (that might be initiated at 7-months or earlier) may be partially responsible
for the hypometabolic state seen in the 13-month-old 3xTg-AD mice.[156] One of the few drugs
approved to treat AD is memantine, a drug that acts against glutamate-mediated hyperactivity by
partially blocking the NMDAR and in turn hypothesized to control excitotoxicity. It would be
interesting to assess the effect of memantine and lipoic acid given as a two-hit combination
therapy wherein the former targets the glutamate mediated excitotoxicity and the latter addresses
the regulation of glucose metabolism.
In addition to our observation of ~100% increase in heart glucose uptake, there are other
reports showing physiological changes in the 3xTg-AD mice evidenced by increased food
consumption, changes in weight, increased oxygen consumption, carbon dioxide production,
defective gut-brain signaling, changes in core body temperature; thus, showing physiological
hyperactivity.[173,174,175] Other possible causes of modifications in metabolism are modified
vasculature (amyloid angiopathy), altered 3-D structure of the brain vasculature, alterations in
cerebral flood flow, and changes in blood brain barrier.[176,177,178,179] The changes in
vasculature and blood brain barrier could lead to a modification of substrate supply to the brain.
In addition to the association of hypermetabolism with plaques, a metabolic study in the tau
transgenic mouse model of AD showed hypermetabolism in cerebral cortex.[147]
114
Concluding remarks – The major finding of this study is the hypermetabolic state in the 7-
month-old 3xTg-AD mice as compared to age-matched nonTg mice and in contrast to the
hypometabolism observed in 13-month-old 3xTg-AD mice. Importantly, the specific role of
metabolism in the coordinated picture of hyperactivity (electrophysiological and physiological),
previously reported in multiple mouse models of AD was highlighted. In view of the studies
referenced in this section an important question emerges, i.e., does cerebral glucose metabolism
in very early stage of AD patients show an increase that has not been examined closely enough?
Although, whole brain glucose uptake measured by PET-CT imaging may show a decrease
associated with MCI and/or AD, this does not necessarily mean a decreased cerebral glucose
metabolism. Finally, the effects of lipoic acid in stabilizing glucose metabolism emphasize its
utility as an insulin-mimetic agent with multidimensional effects that need to be assessed in a
major clinical study.
ACKNOWLEDGEMENTS – We thank Dr. Pratip Bhattacharya (MD Anderson Cancer Center) for
his insights about NMR studies, Dr. David Carlson (Geronova, Inc) for providing the lipoic acid
used in this study, and Dr. Henry Chan for helping with the intravenous catheter infusion in
mice.
115
Table 1. Concentrations of the different isotopomers of
13
C Glu, Gln, Asp, GABA, and NAA after
[1-
13
C]glucose infusion
nonTg
(A)
nonTg + LA
(B)
3xTg-AD
(C)
3xTg-AD + LA
(D)
A vs. B
A vs. C C vs. D
------------------- p value-----------------
Metabolite
[4-
13
C]Glu 1.18 ± 0.10 1.56 ± 0.11 1.99 ± 0.32 1.31 ± 0.18 0.010(**) 0.010(**) 0.046(*)
[3-
13
C]Glu 0.76 ± 0.08 0.94 ± 0.11 1.25 ± 0.25 0.73 ± 0.08 0.197 0.041(*) 0.034(*)
[2-
13
C]Glu 0.72 ± 0.04 0.83 ± 0.04 1.01 ± 0.23 0.58 ± 0.08 0.040(*) 0.158 0.050
[1-
13
C]Glu 0.23 ± 0.02 0.28 ± 0.03 0.46 ± 0.10 0.22 ± 0.05 0.160 0.020(*) 0.027(*)
[4-
13
C]Gln 0.43 ± 0.07 0.54 ± 0.08 0.68 ± 0.09 0.41 ± 0.05 0.205 0.019(*) 0.008(**)
[3-
13
C]Gln 0.31 ± 0.04 0.45 ± 0.07 0.55 ± 0.06 0.30 ± 0.05 0.060 0.004(**) 0.004(**)
[2-
13
C]Gln 0.28 ± 0.03 0.37 ± 0.06 0.45 ± 0.07 0.26 ± 0.02 0.144 0.028(*) 0.012(*)
[1-
13
C]Gln 0.10 ± 0.02 0.14 ± 0.01 0.20 ± 0.04 0.10 ± 0.01 0.029(*) 0.014(*) 0.012(*)
[4-
13
C]Asp 0.10 ± 0.02 0.09 ± 0.03 0.18 ± 0.06 0.10 ± 0.02 0.716 0.139 0.141
[3-
13
C]Asp 0.30 ± 0.01 0.29 ± 0.01 0.32 ± 0.01 0.32 ± 0.01 0.410 0.103 0.498
[2-
13
C]Asp 0.17 ± 0.01 0.20 ± 0.02 0.28 ± 0.04 0.16 ± 0.02 0.078 0.015(*) 0.015(*)
[1-
13
C]Asp 0.11 ± 0.01 0.12 ± 0.03 0.18 ± 0.04 0.09 ± 0.01 0.864 0.078 0.022(*)
[4-
13
C]GABA 0.18 ± 0.02 0.21 ± 0.02 0.26 ± 0.14 0.14 ± 0.02 0.310 0.034(*) 0.009(**)
[3-
13
C]GABA 0.18 ± 0.02 0.18 ± 0.02 0.25 ± 0.03 0.16 ± 0.02 0.872 0.070 0.019(*)
[2-
13
C]GABA 0.25 ± 0.06 0.37 ± 0.03 0.43 ± 0.06 0.27 ± 0.02 0.124 0.075 0.033(*)
[1-
13
C]GABA 0.16 ± 0.02 0.18 ± 0.03 0.24 ± 0.03 0.14 ± 0.02 0.645 0.048(*) 0.017(*)
[3-
13
C]NAA 0.09 ± 0.01 0.10 ± 0.01 0.13 ± 0.03 0.09 ± 0.02 0.109 0.121 0.197
[2-
13
C]NAA 0.07 ± 0.01 0.08 ± 0.01 0.09 ± 0.02 0.06 ± 0.00 0.189 0.295 0.116
Concentrations of the different isotopomers of
13
C Glu, Gln, Asp, GABA, and NAA in 7 months
old nonTg and 3xTg-AD mice plus/minus lipoic acid, after 60 min of [1-
13
C]glucose infusion.
Results in the column 2-5 are presented as average mM ± SEM; results in the columns 6-8 are
the p values obtained from a two-tailed student t-test after comparing between the groups as
indicated. * p ≤ 0.05, ** p ≤ 0.01 (indicated in parenthesis); total n = 22 and n ≥ 5/group. The
116
results for Glu, Gln, and Asp are corrected for natural abundance (the results for NAA and
GABA are not corrected for natural abundance).
117
Table 2. Concentrations of the different isotopomers of
13
C Glu, Gln, Asp, GABA, MI, and NAA
after [1-
13
C]glucose + [1,2-
13
C] acetate infusion
nonTg
(A)
nonTg + LA
(B)
3xTg-AD
(C)
3xTg-AD + LA
(D)
A vs. B
A vs. C C vs. D
------------------- p value-----------------
Metabolite
[4-
13
C]Glu 1.55 ± 0.08 1.55 ± 0.15 1.48 ± 0.15 1.90 ± 0.15 0.991 0.681 0.096
[3-
13
C]Glu 1.21 ± 0.15 1.13 ± 0.10 2.06 ± 0.21 1.38 ± 0.10 0.686 0.010(**) 0.026(*)
[2-
13
C]Glu 1.09 ± 0.10 1.09 ± 0.12 2.11 ± 0.29 1.28 ± 0.09 0.998 0.009(**) 0.036(*)
[4,5-
13
C]Glu 0.41 ± 0.05 0.45 ± 0.06 0.38 ± 0.04 0.49 ± 0.09 0.571 0.712 0.282
[1,2-
13
C]Glu 0.31 ± 0.05 0.21 ± 0.03 0.51 ± 0.05 0.34 ± 0.02 0.969 0.029(*) 0.016(*)
[2,3-
13
C]Glu 0.40 ± 0.07 0.46 ± 0.06 0.69 ± 0.06 0.47 ± 0.06 0.543 0.019(*) 0.037(*)
[2,3-
13
C]Glu∞ 0.87 ± 0.15 0.91 ± 0.06 1.35 ± 0.12 0.93 ± 0.06 0.830 0.046(*) 0.037(*)
[4-
13
C]Gln 0.44 ± 0.04 0.39 ± 0.02 0.60 ± 0.15 0.49 ± 0.03 0.391 0.292 0.490
[3-
13
C]Gln 0.48 ± 0.04 0.43 ± 0.04 0.92 ± 0.07 0.59 ± 0.04 0.401 0.001(**) 0.008(**)
[2-
13
C]Gln 0.43 ± 0.04 0.40 ± 0.05 0.58 ± 0.12 0.46 ± 0.11 0.655 0.250 0.487
[4,5-
13
C]Gln 0.46 ± 0.11 0.61 ± 0.05 0.56 ± 0.10 0.48 ± 0.10 0.298 0.540 0.593
[1,2-
13
C]Gln 0.18 ± 0.04 0.16 ± 0.01 0.28 ± 0.01 0.19 ± 0.03 0.775 0.039(*) 0.029(*)
[2,3-
13
C]Gln 0.18 ± 0.03 0.17 ± 0.03 0.22 ± 0.04 0.16 ± 0.03 0.906 0.375 0.301
[2,3-
13
C]Gln∞ 0.43 ± 0.06 0.41 ± 0.04 0.66 ± 0.06 0.48 ± 0.05 0.787 0.034(*) 0.065
[4-
13
C]Asp 0.36 ± 0.02 0.33 ± 0.04 0.65 ± 0.10 0.44 ± 0.05 0.516 0.015(*) 0.102
[3-
13
C]Asp 0.43 ± 0.04 0.39 ± 0.04 0.80 ± 0.08 0.46 ± 0.03 0.528 0.003(**) 0.006(**)
[2-
13
C]Asp 0.25 ± 0.02 0.23 ± 0.04 0.38 ± 0.09 0.27 ± 0.07 0.657 0.177 0.366
[1-
13
C]Asp 0.27 ± 0.08 0.23 ± 0.03 0.63 ± 0.15 0.47 ± 0.15 0.716 0.065 0.485
[3,4-
13
C]Asp 0.08 ± 0.02 0.08 ± 0.01 0.14 ± 0.03 0.10 ± 0.03 0.804 0.116 0.323
[2,3-
13
C]Asp 0.09 ± 0.01 0.08 ± 0.01 0.15 ± 0.02 0.08 ± 0.02 0.232 0.030(*) 0.035(*)
[4-
13
C]GABA 0.37 ± 0.10 0.39 ± 0.03 0.59 ± 0.07 0.40 ± 0.03 0.793 0.058 0.057
[3-
13
C]GABA 0.32 ± 0.04 0.30 ± 0.06 0.51 ± 0.05 0.29 ± 0.09 0.789 0.004(**) 0.074(*)
[2-
13
C]GABA 0.47 ± 0.04 0.40 ± 0.03 0.65 ± 0.08 0.41 ± 0.12 0.090 0.023(*) 0.156
[1-
13
C]GABA 0.19 ± 0.03 0.12 ± 0.03 0.27 ± 0.03 0.17 ± 0.04 0.078 0.060 0.120
[1,2-
13
C]GABA 0.12 ± 0.03 0.13 ± 0.02 0.16 ± 0.02 0.11 ± 0.02 0.709 0.304 0.174
[2,3-
13
C]GABA 0.17 ± 0.04 0.17 ± 0.01 0.24 ± 0.04 0.17 ± 0.02 0.916 0.172 0.182
[3,4-
13
C]GABA 0.06 ± 0.02 0.08 ± 0.01 0.09 ± 0.02 0.06 ± 0.01 0.359 0.201 0.130
[4,6-
13
C]MI 0.13 ± 0.01 0.11 ± 0.01 0.17 ± 0.02 0.12 ± 0.03 0.108 0.100 0.190
[2-
13
C]MI 0.05 ± 0.01 0.04 ± 0.01 0.12 ± 0.02 0.06 ± 0.01 0.387 0.023(*) 0.043(*)
[1,3-
13
C]MI 0.14 ± 0.01 0.11 ± 0.02 0.17 ± 0.02 0.11 ± 0.02 0.203 0.146 0.112
[5-
13
C]MI 0.08 ± 0.00 0.07 ± 0.01 0.13 ± 0.01 0.09 ± 0.01 0.085 0.001(**) 0.009(**)
[6-
13
C]NAA 0.14 ± 0.03 0.14 ± 0.02 0.14 ± 0.01 0.13 ± 0.02 0.902 0.992 0.543
[3-
13
C]NAA 0.31 ± 0.07 0.17 ± 0.01 0.32 ± 0.06 0.26 ± 0.04 0.132 0.917 0.413
[2-
13
C]NAA 0.12 ± 0.01 0.10 ± 0.01 0.13 ± 0.01 0.08 ± 0.02 0.148 0.686 0.114
118
Concentrations of the different isotopomers of
13
C Glu, Gln, Asp,GABA,MI, and NAA in 7
months old nonTg and 3xTg-AD mice plus/minus lipoic acid, after 150 min of [1-
13
C]glucose +
[1,2-
13
C] acetate infusion. Results in the column 2-5 are presented as average mM ± SEM;
results in the columns 6-8 are the p values obtained from a two-tailed student t-test after
comparing between the groups as indicated. * p ≤ 0.05, ** p ≤ 0.01 (indicated in parenthesis);
total n = 17 and n ≥ 4/group. The results for Glu, Gln, and Asp are corrected for natural
abundance (the results for GABA, MI, and NAA are not corrected for natural abundance). ∞[2,3
and 3,4-
13
C]Glu/Gln
119
FIGURES
Fig. 1. Total metabolite levels and percent
13
C enrichment of the different metabolite
isotopomers after [1-
13
C]glucose infusion and regional brain glucose uptake
120
Total metabolite levels (
12
C +
13
C) of Glu (Ai), Gln (Bi), and Asp (Ci) in 7 month-old
nonTg and 3xTg-AD mice plus/minus lipoic acid feeding shown as mean ± SEM. Enrichment
percentage for glutamate isotopomers (Aii-iv), glutamine isotopomers (Bii-iv), and aspartate
isotopomers (Cii-iv) shown as mean percentage ± SEM; p values obtained from a two-tailed
student t-test comparing the specific groups is indicated under those groups. *p ≤ 0.05, ** p ≤
0.01; total n = 22 and n ≥ 5/group. FDG-PET images after [
18
F]-FDG injection were co-
registered to a high resolution MRI image to obtain region specific glucose uptake as described
in the materials and methods section. (Di) Representation of how the regions of interest were
drawn to calculate the glucose uptake for hippocampus and motor and somatosensory cortex.
The mean intensity values of glucose uptake calculated in regions of (Dii) hippocampus and
(Diii) motor and somatosensory cortex. As shown, no statistically significant differences were
seen among the different groups; total n = 24, n ≥ 5/group.
121
Fig. 2. Typical labeling pattern after [1-
13
C]glucose + [1,2-
13
C]acetate infusion
Labeling pattern after co-infusion of [1-
13
C]glucose + [1,2-
13
C]acetate as described in the
materials and methods section (Adapted from [180]).
122
123
Fig. 3. A representative
13
C NMR spectrum of brain extract
A proton-decoupled NOE-enhanced representative
13
C spectrum (150.86 MHz) of typical
perchloric acid brain extract after [1-
13
C]glucose and [1,2-
13
C]acetate infusion for 150 min
showing several metabolite isotopomers; parts of the spectra have been zoomed to clearly show
all peaks (A) and spectra focusing on glutamate/glutamine isotopomers between 27 and 35ppm
and changes in the spectra as a function of time after [1-
13
C]glucose + [1,2-
13
C]acetate infusion
for 5 min (bolus), 20 min, 60 min, and 150 min (B). The chemical shift and internal standard, 1,
4 dioxane is at 67.4 ppm and isopropyl alcohol with three equivalent methyl carbons (solvent in
the pH indicator) at 24.6 ppm.
124
Fig. 4.
13
C enrichment percentage of the different metabolite isotopomers after [1-
13
C]glucose
and [1,2-
13
C]acetate infusion for different time periods
The different graphs show the labeled glutamate/glutamine isotopomers after the
specified period of [1-
13
C]glucose + [1,2-
13
C]acetate infusion for either 5 min, 20 min, 60 min,
or 150 min. Left Panel (A) shows glutamate isotopomers generated primarily from glucose
125
metabolism (single labeled) (Fig. Ai-iii) and acetate metabolism (double labeled) (Fig. Aiv-vi).
Right Panel (B) shows glutamine isotopomers generated primarily from glucose metabolism
(single labeled) (Fig. Bi-iii) and acetate metabolism (double labeled) (Fig. Biv-vi). *p ≤ 0.05, **p ≤ 0.01; total n = 62 and n ≥ 15/time point. Note that [2,3 and 3,4-
13
C]glu/gln doublets at 60
and 180 min also contain those derived from [1-
13
C]glucose.
126
127
Fig. 5. Metabolic ratios calculated after [1-
13
C]glucose + [1,2-
13
C]acetate infusion for 150 min
Relevant metabolic ratios calculated after [1-
13
C]glucose + [1,2-
13
C]acetate infusion for
150 min are shown in the Graphs A-D. These metabolic ratios have been calculated as described
in the materials and methods section. % Glycolytic activity (A i), TCA cycle activity based on
glutamate formation from [1-
13
C]glucose (A ii),
13
C glucose cycling ratio for glutamate and
glutamine respectively (B i and ii),
13
C Acetate cycling ratio for glutamate and glutamine
respectively (C i and ii), [transfer of glutamate from astrocytes to neurons (D i), and transfer of
GABA from astrocytes to neurons (D ii)]
128
CHAPTER 5:
Conclusions
This is a comprehensive study aimed at establishing the effects of dietary lipoic acid on brain
function in the 3xTg-AD mouse model of Alzheimer's disease. It addresses effects on substrate
supply assessed by measuring brain glucose uptake and glucose transporters translocation to the
plasma membrane, the modulation of cell signaling and mitochondrial bioenergetics assessed by
the PI3K/Akt pathway of insulin signaling, mitochondrial oxidative metabolism capacity, the
modulation of brain metabolism assessed by studying glycolytic and astrocytic metabolism, and
functional outcome assessed by measuring synaptic plasticity.
The 3xTg-AD mice showed decreased substrate supply that was demonstrated by a decrease
of whole brain glucose uptake (assessed by FDG-PET imaging) and glucose transporters
(assessed by measuring the levels of total and active GLUT3 and GLUT4). The cell-signaling
pathways supporting energy metabolism i.e., IRS/PI3K/Akt were found to be less active in the
3xTg-AD mice in an age-dependent manner. However, the young 3xTg-AD (that showed a slight
decrease in whole brain glucose uptake) showed almost no regional glucose uptake differences.
Besides, a state of hypermetabolism was seen in the young 3xTg-AD mice, demonstrated by
increased metabolism of glucose and acetate to form greater amounts of neurotransmitters and
neurochemicals like Glu, GABA, Asp, Gln, NAA, and MI. Whereas, the old 3xTg-AD mice
showed a hypometabolic state demonstrated by decreased glycolytic metabolism and decrease in
formation of neurotransmitters and neurochemicals like Glu, GABA, Asp, Gln, NAA, and MI. In
terms of the synaptic plasticity, the 3xTg-AD mice showed an age-dependent decrease in
synaptic plasticity measured by input/output ratios and long term potentiation.
129
Lipoic acid successfully reversed the age-associated decrease of glucose uptake and glucose
transporters by increasing whole brain glucose uptake, total and active levels of GLUT3 and
GLUT4 in the 3xTg-AD mice. It stimulated the PI3K/Akt pathway of insulin signaling in the
3xTg-AD mice by stimulating IRS and Akt and by inhibiting GSK-3β. Moreover, the effects of
lipoic acid are PI3K dependent because the stimulatory effect of lipoic acid on mitochondrial
bioenergetics was abolished in presence of a PI3K inhibitor. Substrate metabolism (glucose and
acetate) in neurons and astrocytes was also regulated by lipoic acid – 3xTg-AD mice fed lipoic
acid had metabolite levels similar to that of age matched nonTg mice. The synaptic deficits
associated with the old 3xTg-AD mice were reversed by lipoic acid feeding. This was
demonstrated by an increase in input/output and long term potentiation in the CA1 region of
hippocampus of the old 3xTg-AD mice. Although, no effects of lipoic acid were seen in the
young 3xTg-AD mice in terms of restoring synaptic deficits present at that age.
To our knowledge, this is the first study that bioenergetically and functionally characterizes
the 3xTg-AD mouse models in an age-dependent manner. We demonstrate age-dependent
correlation between impairment of glucose uptake in vivo assessed by PET-CT imaging along
with decrease in insulin signaling and concomitant decrease of synaptic plasticity in the 3xTg-
AD mouse model of Alzheimer’s disease. Moreover, the NMR studies provide an important
metabolic dimension to these studies. Surprisingly, a hypermetabolic state was found in the
young 3xTg-AD mice. However, this hypermetabolic state progressed to a hypometabolic state
in the old 3xTg-AD mice; thus, clearly establishing age-dependent metabolic changes in these
mice. Albeit, this study does not provide causal relationship between impairment of glucose
uptake/metabolism and impaired synaptic plasticity, but it hints at a plausible hypothesis that
impairments in glucose uptake and metabolism ultimately affects the high energy-demanding
130
synaptic transmission, leading to impaired synaptic plasticity. Importantly, administration of
lipoic acid reversed several of these impaired states and ultimately restored synaptic plasticity in
the old 3xTg-AD mice. Previous behavioral studies have shown the positive effects of lipoic acid
on memory in different mouse models of aging [117,118,119] and Alzheimer’s disease
[115,120]. Keeping cognizance of the studies presented here and several other studies conducted
on lipoic acid (referenced), it warrants for a thorough investigation of lipoic acid in double-
blinded multicenter clinical trials.
131
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Abstract (if available)
Abstract
Alzheimer’s disease is a type of dementia that causes problems with memory, thinking and behavior. Currently, there are no approved drugs to treat Alzheimer’s disease
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Sancheti, Harshkumar
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Core Title
Age dependent modulation of synaptic plasticity and insulin mimetic effect of lipoic acid on a 3xTg-AD mouse model of Alzheimer's disease: implications as a therapeutic/nutraceutical agent
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Molecular Pharmacology and Toxicology
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03/03/2014
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Alzheimer's disease,brain,glucose metabolism,glucose uptake,glutamate,hypermetabolism,hypometabolism,IRS,lipoic acid,long term potentiation,OAI-PMH Harvest,PI3K/Akt,synaptic plasticity,triple transgenic mice (3xTg-AD)
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Tags
Alzheimer's disease
brain
glucose metabolism
glucose uptake
glutamate
hypermetabolism
hypometabolism
IRS
lipoic acid
long term potentiation
PI3K/Akt
synaptic plasticity
triple transgenic mice (3xTg-AD)