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Neuronal and glial metabolic alterations in the liver-specific PTEN knockout mouse model
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Neuronal and glial metabolic alterations in the liver-specific PTEN knockout mouse model
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i
NEURONAL AND GLIAL METABOLIC ALTERATIONS
IN THE LIVER-SPECIFIC PTEN KNOCKOUT MOUSE
MODEL
by
ISHAN PATIL
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
June 2014
Copyright 2014 Ishan Patil
ii
ACKNOWLEDGEMENTS
I would like to express my gratitude to my advisor Prof. Enrique Cadenas for giving me
an opportunity to carry out research in his laboratory and also for providing me with constant
guidance and inspiration. I want to thank Dr. Bangyan Stiles and Dr. Curtis Okamoto for serving
as a member of my Thesis Committee.
I specially thank my mentor, Dr. Harsh Sancheti, for teaching me experimental skills and
for his everyday support and for constantly motivating me to aim higher. I am grateful for the
help and support of my lab members Zhigang Liu, Dr. Fei Yin, Dr. Tianyi Jiang and my
colleague Saranya Sankar.
I owe a debt of gratitude to my parents for their blessings and support. I owe all the
accomplishments and success in my life to my parents who have always put my needs before
theirs. I would like to thank all my friends, especially Aditi Ghirnikar for her endless
encouragement and confidence in my abilities.
iii
TABLE OF CONTENTS
Acknowledgements ii
List of Tables iv
List of Figures v
Abstract 1
Chapter 1: Introduction 2
1.1 PTEN: A negative regulator of the insulin signaling pathway 2
1.2 Insulin resistance and neurodegeneration 2
1.3 Impaired liver function affects brain function 3
Chapter 2: Materials and Methods 5
Chapter 3: Results 12
Chapter 4: Discussion 26
Chapter 5: Future Studies 28
References 29
iv
LIST OF TABLES
Table 1 Concentrations of different isotopomers of
13
C Glu, Gln, Asp, NAA,
GABA, and MI
20
v
LIST OF FIGURES
Figure 1
Phenotype of the PTEN
loxP/loxP
mice
13
Figure 2 Tail vein infusion 15
Figure 3 Representative NMR spectrum after [1-
13
C]glucose and [1. 2-
13
C]acetate infusion
16
Figure 4 Typical labelling pattern after [1-
13
C]glucose and [1. 2-
13
C]acetate infusion
17
Figure 5 Concentrations
13
C labelled isotopomers after [1-
13
C]glucose +
[1,2-
13
C]acetate infusion
22
Figure 6
Metabolic ratios calculated after [1-
13
C]glucose + [1,2-
13
C]acetate infusion
22
Figure 7 Brain glucose uptake and rate of glucose uptake 24
1
Abstract:
Phosphatase and Tensin Homologue (PTEN) is a negative regulator of the
phosphatidylinositol 3-kinase/AKT pathway. Liver-specific deletion of PTEN results in increased
fatty acid synthesis, accompanied by hepatomegaly and a fatty liver phenotype. Interestingly,
deletion of PTEN in the liver also causes an enhanced liver insulin action with improved systemic
glucose tolerance. Liver plays a major role in glucose metabolism and adequate distribution of
glucose to the brain, whereas, insulin controls both glucose and lipid metabolism in the liver. Thus,
a liver-specific deletion of PTEN provides a valuable model to directly probe the effect of liver
glycolytic metabolism on brain function. Recent studies have reported hepatic inflammation and
metabolic dysfunction as extrinsic risk factors for neurodegeneration mediated by brain
insulin/IGF resistance and deficiency.
In this study, we have used the liver-specific PTEN knockout mouse model
(PTEN
loxP/loxP
;Alb-Cre
+
) at the age of 4.5 months to assess neuronal and astrocytic metabolism
after [1-
13
C]glucose and [1,2-
13
C]acetate infusion using
13
C Nuclear Magnetic Resonance (NMR)
spectroscopy. Specifically, we studied the glycolytic and tricarboxylic acid cycle metabolism and
the exchange of neurotransmitters between neurons and astrocytes in PTEN
loxP/loxP
;Alb-Cre
+
mice.
Preliminary results show an increased flux of metabolites from glucose and acetate, resulting in
increased flux of the major neurotransmitter in the brain, i.e., glutamate. Additional studies are
underway to study brain glucose uptake using in vivo PET-CT imaging. The functional outcome
of PTEN KO on the brain will be determined by measuring synaptic plasticity through
electrophysiological techniques measuring long term potentiation and input-output relationships.
2
CHAPTER 1: Introduction
1.1 PTEN: A negative regulator of the insulin signaling pathway
Insulin controls glucose and lipid homoeostasis through modulation of multiple organs
such as the liver, muscle and fat. It is responsible for promoting glycolysis, glycogen synthesis and
fatty acid (FA) synthesis in the liver (Baudry et al., 2002; Saltiel and Kahn, 2001). Insulin controls
this metabolic homeostasis via the insulin signaling pathway, i.e. the IRS-PI3K-AKT signaling
pathway (Cheng et al., 2010). Action of insulin on the insulin receptor (IR) leads to the activation
of PI3K and its downstream target AKT. PTEN (phosphatase and tensin homologue deleted on
chromosome 10) is one of the negative regulators of the insulin signaling pathway. PTEN has been
the focus of many studies as a tumor suppressor (Mester and Eng, 2013). Recent studies have
extended the reach of PTEN to include diabetes and neurological diseases such as Parkinson’s,
autism, mental retardation, and epilepsy (Garcia-Junco-Clemente and Golshani, 2014). Previous
studies have demonstrated that inhibiting PTEN can improve glycemic control and sensitize mice
to insulin (Butler et al., 2002). Overexpression of PTEN has shown to increase energy expenditure,
decrease body weight, improve lifespan and preserve insulin sensitivity in mice (Garcia-Cao et al.,
2012; Ortega-Molina et al., 2012; Ortega-Molina and Serrano, 2013). These discoveries of the
effect of PTEN on metabolism have opened new avenues for exploration relevant to diabetes as
well as aging and neurodegeneration.
1.2 Insulin resistance and neurodegeneration
The human brain has the highest consumption of glucose, about 60% of the body’s resting
state glucose. The energy generated from glucose metabolism directly affects synaptic
3
transmission; thus it can also be said that synaptic plasticity is susceptible to the bioenergetic state
of the brain (Schubert, 2005). The insulin-stimulated brain glucose uptake is mostly dependent on
the insulin-sensitive glucose transporter GLUT4 (Bingham et al., 2002). Activation of the insulin
signaling pathway results in AKT-dependent phosphorylation of many substrates, and also results
in translocation of GLUT4 from the intracellular storage compartment to the plasma membrane
(Rowland et al., 2011). Several clinical studies have shown decreased brain glucose uptake to be
a common condition in patients with Alzheimer’s disease (AD) and mild cognitive impairment
(MCI) (Mosconi, 2005; Mosconi et al., 2009). Insulin has been shown to influence synaptic
transmission by modulating the cell membrane expression of NMDA (N-methyl-D-aspartic acid)
receptors, affecting long-term potentiation (LTP) (Grillo et al., 2009). Hence, insulin resistance
can affect cognition and brain function through disruption of brain glucose uptake and metabolism
and ‘treating brain insulin resistance’ is being widely considered as a therapeutic approach for
slowing the process inherent in neurodegeneration (Stefanelli et al., 2014).
1.3 Impaired liver function affects brain function
The liver plays a prime role in carbohydrate metabolism and one of its main functions is to
provide the body with glucose or store glucose in form of glycogen for later use. Non-alcoholic
fatty liver disease (NAFLD) negatively affects the liver’s capacity to carry out its functions
properly. Insulin resistance is regarded as a major contributor to the pathogenesis of NAFLD.
Oxidative stress and mitochondrial dysfunction are observed in fatty liver, thus leading to further
decline in liver function (Birkenfeld and Shulman, 2014; Chang et al., 2013). It is now becoming
increasingly evident that chronic liver diseases negatively impact brain function. The diseased
4
liver is known to communicate with the brain via three major inflammatory cytokines: TNFα, IL-
1β and IL-6, which induce their respective signaling pathways and effect brain dysfunction
(D'Mello and Swain, 2011). Also, steatohepatitis has been known to induce production of
ceramides (cytotoxic lipid signaling molecules), which can exacerbate pro-inflammatory
responses and can also cross the blood brain barrier and exacerbate the metabolic impairments in
the brain and advance the cascade of neurodegeneration (de la Monte, 2012).
The liver-specific deletion of PTEN has been shown to improve liver insulin sensitivity,
improve overall glucose tolerance, decrease body fat content and decrease plasma non-esterified
fatty acid (NEFA) levels in mice (Stiles et al., 2004) (Figure 1). Thus, the liver-specific PTEN
knockout mouse model (PTEN
loxP/loxP
;Alb-Cre
+
) is a valuable model to study insulin action in the
liver, and more importantly, to understand the complex interaction among insulin-sensitive organs
such as the brain.
This study is aimed at understanding the effect of modulating liver insulin signaling on
brain function using the PTEN
loxP/loxP
;Alb-Cre
+
(PT) mouse model. The hypothesis is that a robust
insulin signaling pathway should improve the capacity of the liver to provide energy to the brain
and prevent metabolic impairments by maintaining insulin sensitivity.
5
CHAPTER 2: MATERIALS AND METHODS
2.1 Materials
[1-
13
C]glucose (99%) was purchased from Sigma-Aldrich (St Louis, MO, USA). [1, 2-
13
C]acetate (99%) and deuterium oxide (99.9%) were obtained from Cambridge Isotope
Laboratories (Andover, MA, USA). All other chemicals were the purest grade available from
Sigma-Aldrich. 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).
2.2 Animals
PTEN
loxP/loxP
mice were bred with Alb-Cre mice to generate mice with a liver specific
deletion(Stiles et al., 2004) and maintained at the University of Southern California (Los Angeles,
CA) following National Institutes of Health guidelines on use of laboratory animals and an
approved protocol by the University of Southern California Institutional Animal Care and Use
Committee. Mice were housed on 12-h light/dark cycles and provided ad libitum access to food
and water. Animals of 4 and a half months were used for the experiments.
2.3 Glucose Tolerance Test (GTT)
A GTT was performed on animals that were fasted for 16 hours. For glucose measurement,
tail veins were punctured and a small amount of blood was released and applied onto a OneTouch
glucometer. For GTT, the mice were given a single dose (2 g/kg of body weight) of D-Dextrose
6
(Sigma) by i.p injection after a baseline glucose check. Circulating glucose levels were then
measured at 15, 30, 60 and 120 mins after glucose injection.
2.4 Intravenous Glucose and Acetate Infusion
The mouse was first restrained using a rotating tail vein restrainer. Anesthesia was not used
during the entire procedure; which allowed us to measure metabolism in an awake non-
anesthetized mouse. Awake animals were used to avoid the effect of anesthesia on cerebral glucose
utilization.(Ori et al., 1986) After restraining the mice, the basal glucose levels were tested as
described in the GTT procedure. The puncture made for testing the basal blood glucose levels was
also used for inserting a tail vein catheter in the mouse tail (Braintree Scientific, Inc, MA, USA).
The catheter was inserted using the manufacturer’s instructions and was tested for bulges by
pushing some saline solution 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 the procedure was
performed again at a more proximal point in either the same vein or the next vein. The glucose and
acetate infusion protocol was carried out as described earlier.(Fitzpatrick et al., 1990; Marin-
Valencia et al., 2012) Animals first received a bolus of [1-
13
C]glucose and [1, 2-
13
C]acetate
solution (0.6M) to raise the blood glucose levels to normoglycemic range, followed by
exponentially decreasing amount of glucose for 8 min. Finally infusion at a constant rate was
performed for 150 mins to achieve steady state concentration of labelled metabolites. Mice were
kept in a quiet and warm environment to avoid too much stress during the glucose and acetate
infusion. The constant infusion was carried out using a pump from Harvard Apparatus (Figure 2).
7
2.5 Tissue Collection and Extraction Procedure
At the end of the 150 min infusion, the catheter was removed and the final blood glucose
levels were measured as described earlier. The mouse brain was immediately frozen in liquid
nitrogen, and stored at -85
o
C. The entire procedure, from the end of the infusion to snap freezing
the brain, was ensured to be less than one minute for each mouse to avoid post-mortem metabolic
changes.
After freezing of the brain, it was weighed and perchloric acid extraction was performed
as previously described.(Farrow et al., 1990; Sancheti et al., 2014) Tissue samples were extracted
in such a way as to maximize the concentration of metabolites for NMR analysis. The brain was
first grounded in a mortar to a fine powder using a pestle under liquid nitrogen and then transferred
to a weighed centrifuge tube. Ice-cold 20% (w/v) perchloric acid (2 ml/g of tissue) was added to
the powdered sample while ensuring that no thawing occurs. The mixture was then homogenized
and allowed to thaw on ice. The precipitate was removed by centrifugation at 22000 g for 20 mins
at 2
o
C. The supernatant was then neutralized to pH 7.4 using potassium hydroxide and the mixture
was allowed to stand on ice for 20 mins. The supernatant was then collected after centrifugation
at 22000 g for 20 mins at 2
o
C and this final brain extract supernatant was stored at -85
o
C until used
for NMR analysis.
2.6 NMR Spectroscopy
The stored brain extracts were thawed and mixed in appropriate proportion with D 2O,
sodium azide (preservative), and 1.5 µL of 1, 4 Dioxane (chemical shift reference and internal
standard).
13
C NMR analysis was carried out on a Varian VNMRS 600MHz instrument at 150.86
8
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 delay
of 5 sec, 251ppm spectral width with 32,768 spectral points. A total of 7312 scans were acquired
at 25 °C to obtain a good signal to noise ratio. The chemical shift reference peak of 1, 4 dioxane
was set to exactly 67.4ppm, followed by peak identification using chemical shift values from
previous literature.(Blüml et al., 2000; Preece and Cerdán, 1996)
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 as follows:
13
C spectra of Glu, Gln, Asp,
NAA, lactate and GABA at natural abundance of
13
C were acquired in a single solution at different
concentrations to construct a standard curve of peak area vs.
13
C concentration for each carbon of
the compound. This standard curve was used to convert the observed peak area of each
13
C-
metabolite (after normalization using the peak area of 1, 4-dioxane as internal standard) to
13
C
concentration.
2.7
13
C Labeling Patterns and Interpretation
The Labelling pattern of brain metabolites from [1-
13
C]glucose and [1, 2-
13
C]acetate has
been well described previously by Dr. Sonnewald(Nilsen et al., 2013). The
13
C labelling pattern
has been shown in Figure 4. Shortly after [1-
13
C]glucose is converted to [3-
13
C]pyruvate via
glycolysis, [3-
13
C]pyruvate can be either reduced to [3-
13
C]lactate, carboxylated to oxaloacetate
(OAA) (in astrocytes), transaminated to [3-
13
C]alanine, or decarboxylated to [2-
13
C]acetyl CoA
and enter the triacrboxylic acid (TCA) cycle. Once [1-
13
C]glucose enters the TCA cycle, it
9
undergoes several steps to form [4-
13
C] α-ketoglutarate that can be transaminated to form [4-
13
C]glutamate. In the GABAergic neurons, [4-
13
C]glutamate is subsequently converted to [2-
13
C]GABA via the enzyme glutamic acid decarboxylase or to [4-
13
C]glutamine in astrocytes via
the astrocyte-specific enzyme glutamine synthetase. [4-
13
C]glutamate released by the
glutamatergic neurons during neurotransmission is removed from the synaptic cleft via
transporters, followed by conversion to [4-
13
C]glutamine or [4-
13
C] α-ketoglutarate. [2-
13
C]-/[3-
13
C]OAA can be derived from [4-
13
C] α-ketoglutarate, which can be 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 astrocytes, [1, 2-
13
C]acetate is converted to [1, 2-
13
C]acetyl CoA. After entry into the
TCA cycle, [4, 5-
13
C] α-ketoglutarate is formed after several steps, which is then converted to [4,5-
13
C]glutamate and [4,5-
13
C]glutamine. After [4, 5-
13
C]glutamine is transferred to neurons, it is
converted to [4, 5-
13
C]glutamate (by phosphate activated glutaminase) in the glutamatergic
neurons. It can also be converted to [1, 2-
13
C]GABA in GABAergic neurons. If [4, 5-
13
C] α-
ketoglutarate is metabolized in ]the TCA cycle, it might form [3-
13
C]/[1, 2-
13
C]glutamate and
glutamine and [3-
13
C]/[4-
13
C]GABA after the second turn of the cycle.
2.8 Metabolic Ratios
The cycling ratio gives us an idea of how long the label stays in the TCA cycle before
getting converted to glutamate or glutamine. The cycling ratio for
13
C from [1-
13
C]glucose was
calculated as follows: ([3-
13
C]glutamate (glutamine) – [1,2-
13
C]glutamate (glutamine))/[4-
10
13
C]glutamate. The cycling ratio for
13
C from [1, 2-
13
C]acetate was calculated as follows: [1, 2-
13
C] glutamate (glutamine)/[4, 5-
13
C]glutamate (glutamine).
The acetate vs glucose utilization ratio provides an estimate of the relative contribution
from neurons and astrocytes to glutamate, glutamine and GABA formation. The acetate vs glucose
ratios are expressed as [4, 5-
13
C]glutamate (glutamine)/[4-
13
C]glutamate (glutamine) and [1, 2-
13
C]GABA/ [2-
13
C]GABA (Kondziella et al., 2006).
Glycolytic activity was calculated by calculating the % change in levels of [3-
13
C]alanine
(Morken et al., 2013)
2.9 Brain Glucose Uptake
Positron emission tomography utilizing the radiotracer fluoro-2-deoxy-2-[
18
F]-fluoro-D-
glucose (FDG-PET) was used in a clinical setting to measure the brain glucose uptake. Brain
glucose uptake was measured by standard uptake value (SUV), 40 mins post-injection of [
18
F]-
FDG-PET as a tracer using a MicroPET scanner. SUV represents the standardized uptake value
after taking into consideration the actual radioactivity concentration found in the brain at a specific
time and the concentration of radioactivity, assuming an even distribution of the injected
radioactivity across the whole body. Mice were fasted overnight and then sedated using 2%
isoflurane by inhalation, followed by i.v administration of the radiotracer 2-deoxy-2-[
18
F]-fluoro-
D-glucose. Mice were then immobilized on the scanner bed with a warming bed (to maintain body
temperature) and were scanned 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 mins. Baseline blood
11
glucose levels were measured before administration of the tracer to ensure that abnormalities in
glucose metabolism during FDG-PET imaging are due to the phenotype of the mice and not due
to differences in the baseline blood levels. Immediately after the completion of the FDG-PET scan,
the animals underwent CT scanning with intravenous contrast material, providing information of
the brain structure and anatomical data.
PET data were reconstructed using the 2D-OSEM algorithm supplied by the MicroPET
manager (Siemens Medical Solutions USA, Inc., Knoxville, TN) into 128x128x63 images with
0.084 mm x 0.084 mm x 1.21 mm resolution. Two bed positions were used to obtain the CT scans
using the following settings: 80 kVp, 500µA, 100 ms/180 steps covering 360 degrees and finally
reconstructed into 768x768x923 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. SUV were calculated by drawing the regions of interest.(Sancheti et
al., 2013)
2.10 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 figure (*p ≤ 0.05, **p ≤
0.01).
12
CHAPTER 3: RESULTS
3.1 Glucose Tolerance in the PTEN
loxP/loxP
mice
Similar to what has been reported previously (Stiles et al., 2004), deletion of PTEN
improved the glucose clearance in animals. This phenomenon can be attributed to increased insulin
sensitivity due to decreased plasma NEFA levels, resulting in a significant decrease in the fasting
plasma insulin levels.
When administered and i.p glucose load, the mutant mice demonstrated a significantly
lower peak glucose concentration at 15 min and a faster decline of plasma glucose levels
throughout the entire duration of the experiment (Figure 1, **P<0.01) as compared to the Wild
type (WT) control mice.
3.2
13
C Metabolite concentrations
Well resolved peaks obtained from NMR spectrum were used to measure concentrations
of
13
C metabolites. Figure 3 shows a representative
13
C NMR spectrum.
13
C labelled isotopomers
of lactate, glutamate (Glu), glutamine (Gln), aspartate (Asp), γ-aminobutyric acid (GABA), N-
acetyl-aspartate (NAA), myoinositol (MI), and glucose (C1α and β) were observed. An earlier time
course study has shown that a 60 min infusion of [1-
13
C]glucose was enough to ensure a steady
state
13
C enrichment of cerebral metabolite pools (Sancheti et al., 2014). Hence, a 2.5h infusion
may be deemed sufficient to examine possible differences between the WT and the
PTEN
loxP/loxP
;Alb-Cre
+
mice.
13
The concentrations of different isotopomers of Glu, Gln, Asp, NAA, GABA and MI in the
4.5 month old WT and PTEN
loxP/loxP
;Alb-Cre
+
are shown in Table 1 (expressed in mM ± SEM).
An overall increase in labeling was observed in the PTEN
loxP/loxP
;Alb-Cre
+
mice in comparison to
the age-matched WT mice. The highest labeling was of [3-
13
C]lactate, followed by [4-
13
C]glutamate, [3-
13
C]glutamate, [2-
13
C]glutamate and the lowest labeling being of [1-
13
C]glutamate.
PTEN
loxP/loxP
;Alb-Cre
+
Liver
0
100
200
300
400
500
600
-50 0 50 100 150
Glucose level (mg/dl)
Time (mins)
WT
A
.
B
.
C
.
D
.
Figure 1: Phenotype of the PTEN
loxP/loxP
;Alb-Cre
+
mice. (A) Robust insulin signaling in the PTEN
loxP/loxP
;Alb-Cre
+
mice, (B) Fasting plasma insulin concentrations, (C) Body fat content, (D) Glucose tolerance test (GTT) in the 4.5
month old PTEN
loxP/loxP
;Alb-Cre
+
mice. (Figures B and C adopted from Stiles, Wang et al. 2004)
*
*
WT PTEN
loxP/loxP
WT PTEN
loxP/loxP
WT PTEN
loxP/loxP
WT PTEN
loxP/loxP
14
3.3 Comparison of neuronal and glial metabolism after co-infusion of [1-
13
C]glucose+[1,2-
13
C]acetate
To assess the metabolic state of the brain, these studies were carried out by infusing [1-
13
C]glucose and [1,2-
13
C]acetate for a period of 150 mins. The typical NMR trace (Figure 3) and
the typical labelling pattern for metabolites (Figure 4) after the co-infusion are shown. An overall
hypermetabolic state was observed in both neurons and astrocytes of the PTEN
loxP/loxP
;Alb-Cre
+
mice, evidenced by an increase in the levels of the
13
C metabolite isotopomers at the end of the
150 min infusion.
The most significant differences were observed in the levels of glutamate isotopomers. All
glutamate isotopomers labelled in the 1
st
, 2
nd
, and 3
rd
turns showed an average increase of 25% in
comparison to those in the WT mice (except [4, 5-
13
C]glutamate). Similar trends of increase in the
labelled isotopomers for alanine, lactate, glutamine, GABA, and aspartate were observed, although
not statistically significant.
The levels of [4, 5-
13
C]glutamine and [2, 3-
13
C]glutamate reflect astrocytic metabolism and
were found to be significantly increased in the PTEN
loxP/loxP
;Alb-Cre
+
mice. Also, an increase in
the levels of [1, 2-
13
C]GABA and [2, 3-
13
C]aspartate was seen (not statistically significant). No
difference was observed in the levels of [4, 5-
13
C]glutamate.
These results suggest a possible hypermetabolic state in the brain of the PTEN
loxP/loxP
;Alb-
Cre
+
mice.
15
Figure 2: Tail vein infusion
16
Figure 3: Representative NMR spectrum after [1-
13
C]glucose and [1. 2-
13
C]acetate
infusion(Sancheti, 2014).
17
Figure 4: Typical labelling pattern after [1-
13
C]glucose and [1. 2-
13
C]acetate infusion (Nilsen et
al., 2013).
18
19
20
Figure 5: Concentrations
13
C labelled isotopomers after [1-
13
C]glucose + [1,2-
13
C]acetate
infusion - (A) Glutamate isotopomers, (B) Aspartate isotopomers, (C) Glutamine isotopomers,
and (D) GABA isotopomers.
Table 1: Concentrations of different isotopomers of
13
C Glu, Gln, Asp, NAA, GABA, and MI
Metabolite WT PTEN
loxP/loxP
;Alb-Cre
+
WT vs PTEN
loxP/loxP
;Alb-Cre
+
P value
[4-
13
C]Glu 1.489 ± 0.17 1.840 ± 0.09 0.007 (*)
[3-
13
C]Glu 1.147 ± 0.07 1.381 ± 0.05 0.009 (*)
[2-
13
C]Glu 1.128 ± 0.09 1.384 ± 0.12 0.042 (*)
[1-
13
C]Glu 0.630 ± 0.04 0.717 ± 0.01 0.010 (*)
[4,5-
13
C]Glu 0.427 ± 0.04 0.517 ± 0.03 0.118
[2,3-
13
C]Glu 0.525 ± 0.08 0.839 ± 0.07 0.003 (*)
[3,4-
13
C]Glu 0.784 ± 0.13 1.073 ± 0.105 0.028 (*)
[1,2-
13
C]Glu 0.378 ± 0.06 0.507 ± 0.08 0.065
[4-
13
C]Gln 0.506 ± 0.07 0.550 ± 0.008 0.307
[3-
13
C]Gln 0.521 ± 0.05 0.594 ± 0.01 0.057
[2-
13
C]Gln 0.503 ± 0.02 0.597 ± 0.04 0.010 (*)
[1-
13
C]Gln 0.338 ± 0.04 0.384 ± 0.02 0.116
[4,5-
13
C]Gln 0.339 ± 0.06 0.380 ± 0.06 0.382
[2,3-
13
C]Gln 0.220 ± 0.04 0.278 ± 0.03 0.077
[3,4-
13
C]Gln 0.210 ± 0.25 0.445 ± 0.008 0.166
[1,2-
13
C]Gln 0.222 ± 0.05 0.258 ± 0.07 0.430
[4-
13
C]Asp 0.296 ± 0.07 0.298 ± 0.03 0.960
[3-
13
C]Asp 0.403 ± 0.03 0.433 ± 0.02 0.212
[2-
13
C]Asp 0.283 ± 0.02 0.315 ± 0.01 0.058
[1-
13
C]Asp 0.281 ± 0.09 0.236 ± 0.04 0.470
[3,4-
13
C]Asp 0.110 ± 0.03 0.113 ± 0.04 0.879
[2,3-
13
C]Asp 0.157 ± 0.03 0.204 ± 0.06 0.527
[1,2-
13
C]Asp 0.064 ± 0.02 0.111 ± 0.002 0.020 (*)
[3-
13
C]NAA 0.123 ± 0.02 0.132 ± 0.06 0.764
[2-
13
C]NAA 0.082 ± 0.02 0.089 ± 0.001 0.614
[6-
13
C]NAA 0.115 ± 0.02 0.111 ± 0.02 0.813
[4-
13
C]GABA 0.280 ± 0.03 0.342 ± 0.08 0.192
21
[3-
13
C]GABA 0.347 ± 0.02 0.370 ± 0.06 0.470
[2-
13
C]GABA 0.337 ± 0.04 0.380 ± 0.03 0.209
[1-
13
C]GABA 0.209 ± 0.03 0.342 ± 0.08 0.821
[3,4-
13
C]GABA 0.280 ± 0.001 0.040 ± 0.02 0.195
[2,3-
13
C]GABA 0.132 ± 0.008 0.118 ± 0.04 0.522
[1,2-
13
C]GABA 0.099 ± 0.04 0.095 ± 0.08 0.931
[4,6-
13
C]MI 0.097 ± 0.006 0.117 ± 0.02 0.098
[2-
13
C]MI 0.040 ± 0.01 0.035 ± 0.001 0.567
[1,3-
13
C]MI 0.105 ± 0.02 0.117 ± 0.02 0.493
[5-
13
C]MI 0.056 ± 0.02 0.070 ± 0.001 0.416
Table 1: ASP, aspartate; GABA, gamma-aminobutyric acid; Gln, glutamine; Glu, glutamate; MI,
myo-inositol; NAA, N-acetylaspartate. Concentrations of the different isotopomers of
13
C Glu,
Gln, Asp, NAA, GABA, and MI in 4.5 month old PTEN
loxP/loxP
;Alb-Cre
+
and nonTg mice after
150 min infusion. Results in column 2 and 3 are presented as average mmol/L ± SD. Results in
column 4 are P values obtained from a two-tailed student t-test after comparing between the two
groups. *P<0.05, **P<0.01 (indicated in parenthesis); n=4 per group.
3.4 Metabolic Ratios
A 50% increase was found in the glycolytic activity (calculated as the % change in the
concentration of [3-
13
C]alanine). A slight increase was observed in the
13
C cycling ratios for both
glucose and acetate metabolism and in the glucose versus acetate utilization index for Glu, Gln
and GABA (not statistically significant).
22
23
Figure 6: Metabolic ratios calculated after [1-
13
C]glucose + [1,2-
13
C]acetate infusion:
Relevant metabolic ratios, calculated as described in the materials and methods section, after [1-
13
C]glucose + [1,2-
13
C]acetate infusion for 150 min are shown in the Graphs A-D. % Glycolytic
activity based on the levels of [3-
13
C]alanine (A), glucose versus acetate utilization for formation
of Glu, Gln and GABA (B),
13
C glucose cycling ratio for glutamate and glutamine respectively
(Ci,ii),
13
C Acetate cycling ratio for glutamate and glutamine respectively (Di,ii).
3.5 Brain glucose uptake
[
18
F]-FDG-PET imaging revealed a slight increase in the brain glucose uptake (Figure 7)
in the standard uptake values (SUV) in the PTEN
loxP/loxP
;Alb-Cre
+
mice compared to the WT mice,
although not statistically significant.
24
Figure 7: Brain glucose uptake and rate of glucose uptake: Standard uptake value (SUV) was
calculated after [
18
F]-FDG injection followed by dynamic PET and CT scanning as described in
25
the Materials and Methods section. Representative image of the [
18
F]-FDG-PET technique and
image reconstruction (A) (Sancheti et al., 2013), average rate of uptake of glucose with error bar
indicating ±SEM (B), representative combined images from PET-CT scanning of nonTg and
PTEN
loxP/loxP
;Alb-Cre
+
mice at 180 and 3300 sec (C).
26
CHAPTER 4: DISCUSSION
This study characterized the effect of modulation of liver insulin signaling on brain glucose
metabolism through deletion of PTEN in the liver. Nuclear magnetic resonance allows for
quantification of the different glucose metabolites as they proceed through the TCA cycle. The ex
vivo approach used in this study is advantageous compared to the in vivo approach as it facilitates
measurement of glucose metabolites in nonanesthetized mice, thus avoiding interferences due to
anesthesia. Also, the ex vivo approach provides well resolved
13
C metabolite peaks by high-
resolution NMR as compared to the in vivo approach. The disadvantages of using the ex vivo
approach are possible postmortem changes in metabolite levels and the inability to monitor
metabolite levels dynamically for calculation of metabolic rates (Sancheti et al., 2014).
The concentrations of
13
C Glu, Gln, Asp, NAA, MI isotopomers in Table 1 show the extent
of label transferred from the infused [1-
13
C]glucose and [1, 2-
13
C]acetate to the downstream TCA
cycle related metabolites. The prominent increase in the concentrations of
13
C isotopomers of Glu
and Gln shows that more label was transferred to these metabolites in the PTEN
loxP/loxP
;Alb-Cre
+
mice as compared to the WT mice. Although not statistically significant, almost all of the
13
C
isotopomers of Gln, Asp, NAA, MI, alanine and lactate showed an increased transfer of label in
the PTEN
loxP/loxP
;Alb-Cre
+
mice as compared to the WT mice. Infusion of [1-
13
C]glucose with [1,
2-
13
C]acetate allows us to observe the metabolic state in neurons as well as astrocytes, and the
concentration of
13
C metabolites at 150 min revealed a general increase in the PTEN
loxP/loxP
;Alb-
Cre
+
mice. Dynamic [
18
F]-FDG-PET scanning revealed a slight increase in the rate of glucose
uptake which is mostly likely due to the overall insulin sensitive state of the PTEN
loxP/loxP
;Alb-
Cre
+
mice. These results point towards an enhanced glucose metabolism in the brain which can
27
be attributed to the increased insulin sensitivity observed in the PTEN
loxP/loxP
;Alb-Cre
+
mice as a
result of robust insulin signaling in the liver.
Mounting evidence supports the concept that insulin resistance and metabolic impairments
are associated with and probably cause cognitive impairment and neurodegeneration. Furthermore,
the increase incidence rates of neurodegenerative diseases like AD and insulin resistance disease
states such as type 2 diabetes, obesity, non-alcoholic fatty liver disease, and metabolic syndrome
underline the importance of preserving insulin sensitivity and the integrity of the insulin signaling
pathway (de la Monte and Tong, 2014). Independent studies have shown that steatophepatitis and
hepatic insulin resistance caused by various etiologies including Hepatitis C infection, alcohol
abuse, obesity, and nitrosamine exposures are associated with cognitive impairment and
neuropsychiatric disorders (Perry et al., 2008; Tong et al., 2010; Tong et al., 2009; Weiss and
Gorman, 2006). In fact, cognitive impairment and neuropsychiatric disorders correlate more with
steatohepatitis and insulin resistance rather than with T2DM or obesity (Kopelman et al., 2009;
Schmidt et al., 2005). Mechanistically, hepatic insulin resistance leads to dysregulation of lipid
metabolism leading to lipolysis (Kao et al., 1999), which increases production of toxic lipid such
as ceramides, which further impairs insulin signaling and impair mitochondrial function, thus
further exacerbating insulin resistance (de la Monte, 2012; Holland and Summers, 2008; Kraegen
and Cooney, 2008; Langeveld and Aerts, 2009). NAFLD, with T2DM and visceral obesity has
been shown to be associated with cognitive impairment and neurodegeneration (de la Monte et al.,
2006; Lyn-Cook et al., 2009; Moroz et al., 2008; Tong et al., 2010; Tong et al., 2009). Humans
with NASH have been shown to be more at risk for developing cognitive impairment (Elwing et
al., 2006). This it is important to consider the role of hepatic insulin resistance as a mediator of
neurodegeneration.
28
The fatty liver phenotype in PTEN
loxP/loxP
;Alb-Cre
+
is different from that observed in other
models of fatty liver (i.e., hyperinsulinemic and insulin-resistant) and is associated with
hyoinsulinemia, enhanced insulin action and low plasma NEFA (Stiles et al., 2004). One must note
the importance of PTEN as the negative regulator of the insulin signaling pathway, and the effect
of inhibiting its activity could have in preserving insulin sensitivity of insulin-sensitive organs, not
only the brain. Pharmacological inhibition of PTEN could prove as a useful strategy to prevent
and possibly even reverse the effects of the cluster of diseases that form the metabolic syndrome.
CHAPTER 5: FUTURE STUDIES
1. It is important to observe the physiological effect of increased rate of glucose metabolism
using LTP assay.
2.
13
C enrichment values of the isotopomers with respect to total metabolite levels need to be
measured to further help understand metabolic differences. Total levels of metabolites can
be measured using HPLC.
3. The expression and levels of inflammatory markers (TNFα, IL-1β and IL-6), and levels of
ceramides need to be measured to confirm cross-talk between the liver and the brain.
4. Protein levels of insulin receptor and components of the insulin signaling pathway need to
be measured to verify modulation of insulin sensitivity.
5. Testing the hypothesis on a mouse model of Alzheimer’s disease (e.g.: 3xTgAD mouse
model for AD) and/or mouse model for obesity using experimental molecules that inhibit
PTEN activity would provide valuable information if the hypothesis is a viable therapeutic
approach against neurodegenerative disease and implications of the metabolic syndrome.
29
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Abstract (if available)
Abstract
Phosphatase and Tensin Homologue (PTEN) is a negative regulator of the phosphatidylinositol 3‐kinase/AKT pathway. Liver‐specific deletion of PTEN results in increased fatty acid synthesis, accompanied by hepatomegaly and a fatty liver phenotype. Interestingly, deletion of PTEN in the liver also causes an enhanced liver insulin action with improved systemic glucose tolerance. Liver plays a major role in glucose metabolism and adequate distribution of glucose to the brain, whereas, insulin controls both glucose and lipid metabolism in the liver. Thus, a liver‐specific deletion of PTEN provides a valuable model to directly probe the effect of liver glycolytic metabolism on brain function. Recent studies have reported hepatic inflammation and metabolic dysfunction as extrinsic risk factors for neurodegeneration mediated by brain insulin/IGF resistance and deficiency. ❧ In this study, we have used the liver‐specific PTEN knockout mouse model (PTENˡᵒˣᴾ/ˡᵒˣᴾ
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Patil, Ishan Y.
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Neuronal and glial metabolic alterations in the liver-specific PTEN knockout mouse model
School
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Degree
Master of Science
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Molecular Pharmacology and Toxicology
Publication Date
07/25/2014
Defense Date
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Cadenas, Enrique (
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