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PI3K/AKT signaling and the regulation of the mitochondrial energy-redox axis

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PI3K/AKT signaling and the regulation of the mitochondrial energy-redox axis

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PI3K/AKT SIGNALING AND THE REGULATION OF THE MITOCHONDRIAL
ENERGY-REDOX AXIS

by

Chen Li

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2012

Copyright 2012 Chen Li
ii

DEDICATION

This thesis is dedicated to my parents and grandma for their love and support.
iii

ACKNOWLEDGEMENTS

I would like to express my greatest gratitude to my advisor Enrique Cadenas, M.D, Ph.D
for his guidance, patience and support all through these years. My appreciation for my
current and former laboratory members, Jerome Garcia, Ph.D, Lipeng Yap, Ph.D,
Julianna Hwuang, Pharm.D, Ryan Hamilton, Ph.D, Fei Yan, Harsh Harsheti, Amit
Argawal and Tianying Jiang.

Special acknowledgements to my dissertation committee, Bangyan Stiles, Ph.D and Neil
Kaplowitz, M.D, for their advice and kindness towards me.
iv

TABLE OF CONTENETS

DEDICATION ii

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT x

INTRODUCTION 1

-Hypothesis and Specific Aims 1
-Background and Significance 5

CHAPTER I 21
The PI3K/AKT signaling pathway regulates cellular
bioenergetics in immortalized hepatocytes
21

-Chapter Introduction 21
-Materials and methods 23
-Chapter Results 26
-Chapter Discussion 41

CHAPTER II 46
PI3K/AKT signaling modulates cellular H
2
O
2
homeostasis and
redox status through regulation of mitochondrial H
2
O
2

generation 46

-Chapter Introduction 46
-Materials and methods 48
-Chapter Results 53
-Chapter Discussion
78

v

CHAPTER III

81
Mitochondrial bioenergetics and H
2
O
2
metabolism in control and
Pten
-/-
liver as function of age
81

-Chapter Introduction 81
-Materials and methods 82
-Chapter Results 85
-Chapter Discussion 100

BIBLIOGRAPHY 103

vi

LIST OF TABLES

Table 1. Pten liver-specific knockout model. Pathological changes in
the control and Pten
-/-
mouse liver 20

Table 2. Sequence alignment for ATP synthase subunits 41

Table 3. Steady-state levels of H
2
O
2
([H
2
O
2
]
ss
) in control and Pten
-/-

hepatocytes 57

Table 4. AKT activation enhanced liver mitochondrial respiratory
capacity 61

Table 5. Cellular redox status in control and Pten
-/-
hepatocytes 68

Table 6. LY294002 treatment and redox component 77

vii

LIST OF FIGURES

Fig. 1. PI3K / AKT signaling regulates cellular bioenergetics
4

Fig. 2. Mitochondrial electron transport Chain and the formation of
oxidants 7

Fig. 3. The mitochondrial redox–energy axis 10

Fig. 4. PI3K/AKT Signaling 15

Fig. 5. Effect of IGF-1 on AKT phosphorylation status and cellular
bioenergetics

28

Fig. 6.Effect of PI3K inhibition on AKT and GSK phosphorylation
status and cellular bioenergetics

30

Fig. 7.AKT and GSK3 phosphorylation status in immortalized
hepatocytes from control- and Pten
-/-
mice

32

Fig. 8.Bioenergetics of control- and Pten
-/-
hepatocytes

33

Fig. 9.Mitochondrial localization of AKT

35

Fig. 10. Inhibiting GSK3β with LiCl induces bioenergetics in control-
and Pten
-/-
hepatocytes

38

Fig. 11. AKT phosphorylation targets in mitochondrial fraction
isolated from control- and Pten
-/-
hepatocytes 40

Fig. 12. H
2
O
2
metabolism in control and Pten
-/-
hepatocytes 54

Fig. 13. Steady state cellular H
2
O
2
concentration ([H
2
O
2
]
ss
) in
control and Pten
-/-
hepatocytes 56

Fig. 14. Bioenergetics of control- and Pten
-/-
primary hepatocytes
from 3 month-old mice 59

Fig. 15. AKT activation enhanced liver mitochondrial respiratory
capacity 62
viii

Fig. 16. AKT activation enhanced the expression of mitochondrial
electron transport chain. 63

Fig. 17. AKT activation enhanced the expression of mitochondrial
pyruvate dehydrogenase and tricarboxylic acid (TCA) cycle enzymes 64

Fig. 18. AKT activation enhanced the expression of glucose uptake
transporters and cellular glycolytic enzymes 65

Fig. 19. Cellular redox status in in control and Pten
-/-
immortalized
hepatocytes 67

Fig. 20. AKT activation increased the expression of mitochondrial
isocitrate dehydrogenase-2 (one of the NADPH generating enzymes). 70

Fig. 21. AKT activation enhanced the antioxidant capacity of liver
mitochondria. 71

Fig. 22. AKT activation enhanced the expression of catalase. 72

Fig. 23. AKT and GSK3 phosphorylation status in primary
hepatocytes from control- and Pten
-/-
knockout mice 74

Fig. 24. Effect of PI3K inhibition cellular bioenergetics 75

Fig. 25. Effects of LY294002 on cellular redox status 77

Fig. 26. The mechanisms involved in the regulation of the cellular
steady-state H
2
O
2
concentration ([H
2
O
2
]
ss
) by the AKT signaling. 80

Fig. 27. AKT status in control and Pten
-/-
liver as function of age 87

Fig. 28. GSK3β status in control and Pten
-/-
liver as function of age 88

Fig. 29. Liver mitochondrial respiratory ratio (RCR) as function of
age 90

Fig. 30. Primary hepatocytes basal and maximal oxygen consumption
rate (OCR) as function of age 91

Fig. 31. Robust anaerobic glycolysis in Pten
-/-
liver 93
ix

Fig. 32. H
2
O
2
metabolism of control and Pten
-/-
liver mitochondria as
function of age 95

Fig. 33. Acetyl-CoA carboxylase 1 (ACC1) expression and
phosphorylation status in control and Pten
-/-
mice liver 97

Fig. 34. AMP-activated protein kinase (AMPK) expression and
phosphorylation status in control and Pten
-/-
mice liver 98

Fig. 35. Fatty acid oxidation in control and Pten
-/-
mice liver 99

Fig. 36. AKT signaling, mitochondrial bioenergetics and fatty acid
biosynthesis 102

x

ABSTRACT

The concept of the mitochondrial energy-redox axis integrates the mitochondrial energy-
transduction and redox status as a concerted process with the two components inter-
linked by the reducing equivalents (i.e., NAD(P)
+
/NAD(P)H). Decrease of mitochondrial
energy transduction and pro-oxidant shift of cellular redox status precede the pathological
changes of several diseases (i.e., diabetes) and are key features of aging. Mitochondria
are also recipients of cellular signaling regulations such as MAPKs and PI3K/AKT
pathway of insulin signaling. These studies are aimed at assessing the effect of the
PI3K/AKT signaling pathway in the mitochondrial energy-redox axis and depicting the
molecular mechanisms inherent in the effect. A liver-specific Pten deletion model that
shows a robust insulin signaling was used to study how the PI3K/AKT pathways affect
the mitochondrial energy-redox axis. The hypothesis to be tested is that liver-specific
Pten deletion up-regulates mitochondrial bioenergetics through modulation of PI3K/AKT
signaling pathways, which further affect mitochondrial and cellular H
2
O
2
homeostasis,
redox status, and the intrinsic apoptotic pathway.
These studies revealed that mitochondrial bioenergetics is regulated by PI3K/AKT
signaling through three mechanisms: 1) AKT increases glycolysis and thus, a higher
substrate (pyruvate) supply to mitochondria; 2) AKT, upon activation, translocates to
mitochondria and phosphorylates ATP synthase subunits α/β leading to a higher ATP
synthase activity; 3) AKT phosphorylates / inactivates GSK3β, which is correlated with
the decrease of the phosphorylation (inactivation) of mitochondria PDH-E
1α
at Ser
273
.
xi

These effects translate in a higher bioenergetic capacity of mitochondria and,
consequently, a lower generation of H
2
O
2
by these organelles. This is attributed to: 1) the
highly oxidized state of the mitochondrial respiratory complexes; 2) the higher
expression of mitochondrial and cellular H
2
O
2
removal enzymes; 3) through modulation
of the expression of mitochondrial isocitrate dehydrogenase-2 and consequently the
increased generation of reducing equivalents (NADPH), which are critical for the
mitochondrial H
2
O
2
removal system. The study of AKT activation on mitochondria as a
function of age shows that mitochondria of Pten
-/-
liver have a significantly reduced
H
2
O
2
generation level than control at older age (9-12 month old) and AKT activation
antagonizes the increase of mitochondrial source of H
2
O
2
production caused by aging.
1

INTRODUCTION

Hypothesis and Specific Aims
Hypothesis- The hypothesis to be tested is that liver-specific Pten deletion up-regulates
mitochondrial bioenergetics through modulation of PI3K/AKT signaling pathways, which
further affect mitochondrial and cellular H
2
O
2
homeostasis, redox status, and the
intrinsic apoptotic pathway. This hypothesis is based on the following evidence:
a) Cytosolic regulation- PI3K phosphorylates phosphotidylinositol-4,5-biphosphate (PI-
4,5-P2, PIP
2
) to phosphotidylinositol-3,4,5-biphosphate (PI-3,4,5-P2, PIP
3
) which
recruits and mediates the activation of AKT. PTEN inhibits the activation of AKT
through dephosphorylating PIP
3
to PIP
2
(Stiles, Wang et al. 2004). In addition, PTEN
was reported to inhibit the phosphorylation of IRS-1 and IRS-1/Grb2/Sos complex
formation. IRS-1 complex mediates the activation of PI3K (Weng, Smith et al. 2001).
Therefore, the phosphatase activity of PTEN suppresses the phosphorylation of PIP
3

(PIP
3
PIP
2
) and that of IRS1 (IRS1-PIRS1) leading to the inhibition of PI3K and
AKT activation (Fig.1). Conversely, in the Pten knockout model, PI3K pathway of
insulin signaling is up-regulated and leads to the activation the AKT (Stiles, Wang et al.
2004). In addition, activated AKT phosphorylates and inhibits GSK3β which is reported
to regulate mitochondrial PDH activity (Hoshi, Takashima et al. 1996).
b) Transcriptional Regulation- PGC-1 family proteins are involved in maintaining
glucose, lipid, and energy homeostasis. Among these proteins, PGC1α is a major
2

regulator of oxidative metabolism and mitochondrial biogenesis (Pagel-Langenickel, Bao
et al. 2008). PGC1α activates NRFs expression and both PGC1α and NRFs stimulate
mitochondri al biogenesis and protein expression (Finck and Kelly 2007). Pten knockout
up-regulates PI3K and AKT activation and subsequent phosphorylation and activation of
the transcription factor CREB (Du and Montminy 1998) and activation of mTOR
(Hernando, Charytonowicz et al. 2007). Phosphorylation of CREB further increases
PGC1α expression (Herzig, Long et al. 2001). It was also reported that rapamycin (the
mTOR inhibitor) decreased the expression of PGC1α and nuclear respiratory factors,
which is associated with the decreased mitochondrial proteins expression and energy-
transduction (Cunningham, Rodgers et al. 2007).
c) AKT in mitochondria- AKT rapidly translocates to mitochondria upon PI3K activation
(Bijur and Jope 2003) indicating AKT may also regulate mitochondrial function through
phosphorylation of mitochondrial proteins (Fig.1).
Specific Aims – The above hypothesis will be tested through four specific aims, which
incorporate the following:

• Specific Aim 1: Characterize the effects of Pten deletion on mitochondrial
bioenergetics. i) Determine the effects of Pten deletion on the mitochondrial
energy-transducing capacity of isolated mitochondria and primary hepatocytes; ii)
Identify the expression and activities of mitochondrial enzymes (i.e. ATP
syntahse) in control and liver Pten
-/-
mice.
3

• Specific Aim 2: Investigate PTEN-related cellular signaling pathways that
regulate mitochondrial bioenergetics and glucose homeostasis. Examine the role
of the PI3K/AKT pathway on mitochondrial bioenergetics by experimental
approaches –performed on immortalized hepatocytes– entailing (a) stimulation of
the PI3K/AKT route by IGF1, (b) specific inhibition of PI3K, and (c) genetic
approaches (specific deletion of Pten).
• Specific Aim 3: Characterize the effects of Pten deletion on mitochondrial H
2
O
2

generation and cellular redox status. Determine PI3K/AKT regulation of
mitochondrial and cellular H
2
O
2
homeostasis by 1) measuring cellular and
mitochondrial H
2
O
2
steady-state levels; 2) determining the cellular redox status
based on the GSH/GSSG, NADH/NAD
+
and NADPH/NADP
+
redox couples.
• Specific Aim 4: Determine the effects of Pten deletion on mitochondrial
bioenergetics and H
2
O
2
metabolism as a function of age. Determine the role of
AKT signaling in the process of liver aging by measuring mitochondrial
bioenergetics and H
2
O
2
metabolism in the liver-specific Pten knockout (Pten
-/-
)
model as a function of age.
4

Fig. 1. PI3K / AKT signaling regulates cellular bioenergetics
AKT
PI3K
PDK1
PIP
2
PIP
3
IRS1
PTEN
RTK
C
y
tosol
GSK3
β
PGC1
α
NRF1
,
2
AKT
I-V
e
mTOR
CREB
Cytosolic
regulation
Kinase
activity
Transcriptional
regulation
Mitochondrial
g
enes
Insulin
5

Background and Significance
The energy-transducing capacity of mitochondria
The reducing equivalents (NADH) generated by the tricarboxylic acid (TCA) cycle
donates electrons to the mitochondria electron transport chain (ETC) and form a proton
gradient across the inner mitochondrial membrane, the mitochondrial transmembrane
potential (∆ψ
m
). The protons in the mitochondrial inter-membrane space are channeled
back to the matrix through ATP synthase (complex V) and the energy of the proton
gradient is concurrently utilized to generate ATP which is critical to all aspects of cell
function (Schagger and Pfeiffer 2001; Nicholls 2002; Navarro and Boveris 2007) (Fig. 2).
In 1955, Chance and Williams defined the metabolic states and respiratory control of
mitochondria: State 4 with availability of respiratory substrates (i.e., puruvate, acetyl-
CoA) but without ADP; State 3 with both substrates and ADP (Chance and Williams
1955; Chance and Williams 1955; Chance and Williams 1955; Chance and Williams
1955; Chance, Williams et al. 1955). Under physiological conditions about 65% of
mammalian cell mitochondria are in state 4 and about 35% in the state 3 (Cadenas 2004).
The mitochondrial generation of oxidants
Over 95% of all oxygen in the human body is reduced to H
2
O by cytochrome oxidase
(complex IV) of the mitochondrial electron transport chain (Schagger and Pfeiffer 2001;
Nicholls 2002; Navarro and Boveris 2007). Electron leak accounts for 2-3% in the form
of univalent reduction of O
2
to O
2
-
(by the complex III and complex I)(Boveris, Cadenas
et al. 1976; Cadenas, Boveris et al. 1977; Han, Williams et al. 2001)) which
6

disproportionates to H
2
O
2
, either spontaneously (10
5
M
-1
s
-1
) or in a reaction catalyzed by
Mn-superoxide dismutase (Mn-SOD) (10
9
M
-1
s
-1
) (Han, Antunes et al. 2002). In
physiological conditions, mitochondrial generation of O
2
-
is considered as a major
cellular source of oxidants (Boveris, Cadenas et al. 1976; Cadenas, Boveris et al. 1977;
Han, Williams et al. 2001). In addition, monoamine oxidase at the mitochondrial outer
membrane is another large source of H
2
O
2
(Cadenas and Davies 2000). H
2
O
2
in
mitochondrial matrix is further reduced to H
2
O by glutathione peroxidases and
peroxiredoxins (Antunes, Han et al. 2002; Chen, Na et al. 2008); in the peroxisomes
H
2
O
2
is reduced by catalase (Antunes, Han et al. 2002). The balance between the
generation of H
2
O
2
and its removal establishes the steady-state levels of H
2
O
2
in the
mitochondrial matrix and H
2
O
2
diffuses freely to cytosol, thereby contributing to the
[H
2
O
2
]
ss
in cytosol (Chance, Sies et al. 1979; Naqui, Chance et al. 1986). Disruption of
this balance is associated with oxidative stress, which entails oxidative modifications of
DNA and proteins and peroxidation of membrane lipids (Harman 1973).
7

Fig. 2. Mitochondrial electron transport chain and the formation of oxidants
H
+
H
+
ATP s
y
nthase
III
IV
ADP ATP
C
y
t c
Q
II
I
H
+
H
+
H
+
e
e
e
V
NADH
H
2
O O
2
Acet
y
l CoA
P
y
ruvate
TCA C
y
cle
O
2
Ο
2
.
−
PDH
H
2
O
2
H
2
O
IMM
OMM
Intermembrane s
p
ace
∆Ψ
m
MnSOD
GSH
GSSG
Prx
ox
Prx
red
Trx
red
Trx
ox
GPx
GR TrxR
IDH
2
NADP
+
NADPH
Mitochondrion
NNT
NADH
NAD
+
NAD
+
Mitochondrion
8

The Mitochondrial Energy-Redox Axis
Mitochondrial energy production and redox status are interdependent as reflected in the
mitochondrial energy-redox axis (D.L. Nelson 2008; Yap, Garcia et al. 2009). The energy
component of the axis involves the glycolytic substrates (pyruvate) supply for the
tricarboxylic acid (TCA) cycle and the subsequent production of reducing equivalents
(NADH, FADH
2
) which donate electrons to the mitochondrial electron transport chain. A
proton motive force is generated during electrons transporting along mitochondrial
complexes. The energy of the proton gradient is garnered to produce ATP (Nicholls 2002;
Navarro and Boveris 2007). The redox component of the axis is interlinked by important
mitochondrial redox couples: glutathione (GSH/GSSG), thioredoxin (Trx(-SH)/Trx-SS),
glutaredoxin (Grx), peroxiredoxins (Prx) (Hoshikawa, Sawamura et al. 1998; Miranda,
Espey et al. 2000; Hurd, Costa et al. 2005; Han, Hanawa et al. 2006; Rebrin, Forster et al.
2007; Kemp, Go et al. 2008). As the ultimate reductant to the GSH and thioredoxin
systems, NADPH maintains mitochondrial redox status (Rydstrom 2006; Yankner, Lu et
al. 2008). Two pathways entail in mitochondrial NADPH formation: the NADP
+
-
dependent isocitrate dehydrogenase (IDH) (Yankner, Lu et al. 2008) and nicotinamide
nucleotide transdehydrogenase (NNT) (Ying 2008). Correlated by the reducing
equivalents pool (i.e., NAD(P)
+
/NAD(P)H), the mitochondrial energy component and the
redox component are concerted processes (Yap, Garcia et al. 2009) (Fig. 3).
O
2
-
and H
2
O
2
as signaling metabolites
It has been recognized that both O
2
-
and H
2
O
2
contribute to the regulation of redox
status and subsequently affect redox-sensitive cellular signaling (Cross, Halliwell et al.
9

1987; Boveris and Cadenas 2000). In Jurkat T-cells, the steady-state concentration of
H
2
O
2
determines the transition between proliferation, apoptosis, and necrosis (Antunes
and Cadenas 2001). With the cellular steady-state H
2
O
2
concentration below 0.7 µM,
cells are in the proliferation status. At 1.0-3.0 µM, cells undergo apoptosis and with
concentrations more than 3.0 µM, cells develop necrosis (Antunes and Cadenas 2001). In
addition, several cellular signaling pathways are redox sensitive. Treatment of neurons
with H
2
O
2
activates c-jun N-terminal kinase (JNK) followed by its translocation to
mitochondria, which indicates H
2
O
2
is involved in the redox regulation of mitogen-
activated protein kinases (MAPKs) (Schroeter, Boyd et al. 2003; Zhou, Lam et al. 2008).
Mitochondrion-dependent apoptosis (intrinsic apoptosis) is regulated by MAPK signaling
involving Bcl
2
inactivation, Bax localization to mitochondria, cytochrome c release, and
subsequent activation of caspases (Tsuruta, Sunayama et al. 2004; Letai 2005). Therefore,
H
2
O
2
may regulate the intrinsic apoptosis through the redox-dependent mechanisms.
10

Fig. 3. The mitochondrial redox–energy axis
The mitochondrial energy-redox axis is composed of the energy component and the redox
component. Tricarboxylic acid (TCA) cycle substrates enter the electron transport chain
and ATP is generated through the oxidative phosphorylation. Superoxide is formed
during the electron transfer and is dismutated to H
2
O
2
. The redox component is
interlinked by important mitochondrial redox indicators glutathione and thioredoxins
which are dependent on the steady flux of NADPH. The mitochondrial energy
component and the redox status component are concerted processes linked by inter
convertible reducing equivalent pool NADH/NAD
+
, NADPH/NADP
+
. The perturbation
of either energy component or the redox component modulates the generation of
mitochondrial H
2
O
2
and affects the steady state cellular H
2
O
2
concentration. The steady
state cellular H
2
O
2
concentration modulates cytosolic redox sensitive signaling pathways
among them insulin signaling and GSK3 are what we interested.
NAD
+
NADH
O
2
.-
H
2
O
2
NADPH
NADP
+
RC
IDH2
NNT
GSH
Trx
H
2
O
2
H
2
O
O
2
TCA
H
2
O
2
Domain S
p
eci
f
ic Si
g
nalin
g
ener
gy
mitochondria
c
y
tosol
redox
11

The PI3K/AKT pathway of insulin signaling
Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) phosphorylate the inositol ring of
phosphatidylinositol (PtdIns) at the 3 position hydroxyl group. Insulin and a variety of
growth factors activate PI3K leading to the generation of phosphatidylinositol-3,4,5-
trisphosphate (PIP
3
). PIP
3
, as a second cellular signaling messenger, recruits and
activates the serine/threonine kinase AKT (protein kinase B, PKB), a key signaling
protein for multiple downstream targets (Stiles, Groszer et al. 2004). The
PTEN/PI3K/AKT signaling pathway regulates several biological processes (i.e., survival,
cell growth, and glucose metabolism).
Phosphotidylinositol-3 kinases (PI3Ks)- Phosphotidylinositol-3 kinases (PI3Ks) are lipid
kinases that are activated by the binding of growth factors, insulin, and cytokines to cell
surface receptors and the subsequent activation of the receptor tyrosine kinases (RTKs)
(Wymann and Marone 2005). The activated PI3K phosphorylates the second messenger
phosphotidylinositol-4,5-biphosphate (PI-4,5-P2) to generate phosphotidylinositol-3,4,5-
triphosphate (PI-3,4,5-P3). PI-3,4,5-P3 activates several downstream target molecules
through their pleckstrin-homology (PH) domain (Ingley and Hemmings 1994; Wymann
and Marone 2005). The phosphotidylinositol-3 kinase (PI3K) signaling pathway regulates
glucose and lipid metabolism (Cantley 2002), processes linked to transfer of reducing
equivalents –generated by the tricarboxylic acid cycle– through the electron respiratory
chain and coupled to oxidative phosphorylation. PI3K signaling also plays a key role in
cell survival, partly due to its effect on regulating the release of mitochondrial
12

cytochrome c (Stiles 2009). Insulin signals through PI3K to induce lipogenesis and
glucose uptake/metabolism in both muscle and adipose tissues (Kahn 1985). Activation
of PI3K also leads to lipogenesis by activating the serine/threonine kinase AKT and the
consequential phosphorylation of FoxO that leads to its migration from the nucleus to
cytosol (Stiles, Groszer et al. 2004; He, Hou et al. 2010). In muscle, insulin is a major
regulator for mitochondrial ATP production partly by promoting the synthesis of resident
mitochondrial genes and proteins (Petersen, Befroy et al. 2003; Shelley, Martin-Gronert
et al. 2009). Consistent with a role of PI3K signaling in oxidative phosphorylation,
deletion of forkhead transcriptional factor FoxO1 downstream of PI3K increased electron
transport chain activity and normalized NAD
+
/NADH values in the IRS1/2 double
knockout mouse liver (Cheng, Guo et al. 2009). In mouse models of resistance to insulin
and activation of PI3K, such as the ob/ob mice (Vianna, Huntgeburth et al. 2006; Cheng,
Guo et al. 2009), mitochondrial electron-transfer chain activity and determinants of
NAD
+
/NADH values are downregulated. Oxidative phosphorylation genes are also
deregulated when liver cells fail to respond to insulin, such as in individuals with insulin
resistance (Cheng, Guo et al. 2009).
Pten- Activation of PI3K (Cantley 2002) or inhibition of the negative regulator for the
pathway, Pten (Phosphatase and Tensin Homologue deleted on Chromosome 10) results
in accelerated cell growth and survival (Stiles, Gilman et al. 2002; Stiles, Groszer et al.
2004). Pten was found in 1997 as a tumor suppressor gene which is located on human
chromosome 10q23. It encodes a phosphatase and was named phosphatase and tensin
homolog deleted on chromosome 10. Pten (the protein encoded by gene this gene)
13

dephosphorylates PI-3,4,5-P3 at the 3’ position to generate PI-4,5-P2 and thus inhibits PI-
3-K signaling (Stiles 2009). Pten deficiency was reported to lead to the increased PIP
3

levels and the activation of many signaling proteins including the serine/threonine
kinases AKT, S6 kinases, GSK3 and mTOR (Rodrigues de Amorim, Garcia-Segura et al.
2010). Pten was also reported to regulate of cell migration, cell/organ size, hormone
responses, stem cell function and animal development (Chalhoub and Baker 2009).
AKT- Serine/threonine kinase AKT (protein knase B) is one of the best known PH
domain-containing proteins that is activated by PI3K (Duronio, Scheid et al. 1998). In
response to the activation of the receptor tyrosine kinase (RTK) by growth factors, PI-
3,4,5-P3 level increases in the cytoplasmic membrane. AKT is recruited to the cellular
membrane through binding to PI-3,4,5-P3 and is phosphorylated and activated by PDK1
on Thr
308
and Ser
473
. Active AKT modulates cellular functions through a series of
downstream proteins such as caspase-3 and -9, proapoptotic factor Bad, and forkhead
transcription factor FOXO. Through these downstream factors, AKT regulates cell
growth, cell survival as well as metabolism (Stiles, Groszer et al. 2004; Kok, Geering et
al. 2009; Stiles 2009). AKT was found to regulate the mitochondrial intrinsic cell death
pathway through direct or indirect interactions with pro-apoptotic factors of the Bcl-2
family (i.e. Bad and Bax) and glycogen synthase kinase 3 (Cantley 2002). Bad and GSK3
are the two best characterized substrates that are directly phosphorylated and inhibited by
AKT (Datta, Dudek et al. 1997). How AKT may control mitochondrial respiratory
function is not clear. AKT may phosphorylate hexokinase II and strengthen its
association with the mitochondrial outer membrane voltage-dependent anion channel
14

(VDAC), thus activating the first step in glucose metabolism (Gottlob, Majewski et al.
2001; Pastorino, Hoek et al. 2005). These functions of AKT may have indirect effects on
mitochondrial respiration in terms of increased substrate supply (pyruvate) to
mitochondria as well as direct effects that entail the translocation of AKT to
mitochondria (Bijur and Jope 2003; Antico Arciuch, Galli et al. 2009). The latter is
associated with an increased phosphorylation (inhibition) of a mitochondrial constitutive
form of GSK3. Association of AKT with other mitochondrial proteins, such as ATP
synthase β, was also reported (Bijur and Jope 2003) (Fig.4).
15

Fig. 4. PI3K/AKT Signaling
AKT
TSC BAD
C
y
clinD
GSK3β GLUT4
C
y
clinD
mTOR
Glucose
trans
p
ort
Cell c
y
cle
Growth
Protein translation
Cell size
A
p
o
p
tosis
Proliferation
PI3K
PDK1
PIP
2
PIP
3
IRS1
PTEN
RTK
C
y
tosol
?
ATP
H
2
O
2
Redox Status
Cellular functions
Ener
gy

p
roduction
The Mitochondrial Redox–Ener
gy
Axis
?
Insulin
,
IGF-1 or EGF
16

Mitochondrial dysfunction and diseases
Mitochondrial dysfunction is associated with decreases of ATP production, disturbance
of cellular calcium homeostasis, increases of oxidative stress, and initiation of the
intrinsic apoptotic pathway. Mitochondrial integrity is critical for cell physiology and
mitochondrial dysfunction contributes to several human pathologies, including diabetes,
and cardiovascular diseases (Abdul-Ghani and DeFronzo 2008; Kim, Wei et al. 2008). It
is expected that increasing mitochondrial function can increase insulin sensitivity,
glucose uptake and oxidation as well as affect fatty acid metabolism (Fisher-Wellman
and Neufer 2012). The decline in mitochondrial bioenergetic capacity and the increased
generation of mitochondrial oxidants are observed in mammalian tissues during aging
and age-related disorders (Mattson and Magnus 2006; Navarro and Boveris 2007;
Navarro and Boveris 2010). Therefore, mitochondria may be an important intervention
point for many human diseases, such as type 2 diabetes and age-related disorders (Fresno
Vara, Casado et al. 2004; Cully, You et al. 2006; Lin and Beal 2006). A potential
signaling pathway that regulates mitochondrial bioenergetics is the PI3K/AKT pathway
of insulin signaling. However, the mechanism by which PI3K/AKT signaling regulates
mitochondrial bioenergetics and redox status is not clearly understood.
The new theory of aging
The Free Radical Theory of Aging proposed that the accumulation of oxidants (O
2
-
,
H
2
O
2
) contributes to the structural damage of various macromolecules (mitochondrial
DNA, RNA and cellular proteins) leading to the loss of cellular physiological functions
17

and subsequently the deterioration in the post-reproductive phase of life (Harman 1956).
Although this hypothesis is supported by the observation of the age-dependent increase of
oxidants and oxidation of physiologically critical macromolecules, evidence has emerged
showing that the oxidative damage alone is insufficient in causing the cellular functional
loss and “antioxidant therapies failed to revert oxidative damage associated with aging”
(Agarwal and Sohal 1994; Sohal, Agarwal et al. 1994; Hamilton, Van Remmen et al.
2001; Cadet, Douki et al. 2010). In addition, the enhancement of antioxidant systems
does not increase the lifespan (Sohal 1993; Bayne, Mockett et al. 2005; Mockett, Sohal et
al. 2010; Jackson and McArdle 2011). The oxidants, used to be viewed as solely toxic
and harmful, have been found to be critical in the regulation of cellular signaling
pathways, redox status and cell cycle (Finkel 2000; Cadenas 2004; Garcia, Han et al.
2007). The Free Radical Theory of Aging was later evolved to the oxidative stress
hypothesis defined as the disturbance of the balance of the oxidants and antioxidant
system (Sies and Cadenas 1985; Sohal and Allen 1990; Brandes 2009). However, this
theory is still not consistent with several experimental observations including the
augmentation of the antioxidant systems by enzymatic overexpression in some models, to
the contrary, decreased the lifespan (Sohal 1993; Bayne, Mockett et al. 2005; Mockett,
Sohal et al. 2010; Jackson and McArdle 2011). Recently, a redox stress hypothesis was
brought up indicating the functional loss during aging is caused the pro-oxidization shift
of the redox status (Finkel and Holbrook 2000; Schafer and Buettner 2001; Droge 2002;
Maher 2005; Jones 2006). This theory is supported by the observation of the age-
dependent decrease of GSH/GSSG ratio and increase of mitochondrial H
2
O
2
generation
18

and GSSG content (Sohal, Toy et al. 1987; Sohal and Weindruch 1996; Sohal, Mockett et
al. 2002; Rebrin and Sohal 2008). In addition, the overexpression of antioxidant enzymes
(i.e., Mn-superoxide dismutase) shows little effects on the redox status, which is
consistent with the observation of no effect on lifespan (Sohal 1993; Bayne, Mockett et al.
2005; Mockett, Sohal et al. 2010; Jackson and McArdle 2011). Instead, the
overexpression of enzymes that directly influence cellular redox status (i.e.,
peroxiredoxins) significantly increases the lifespan (Enoksson, Fernandes et al. 2005; Orr,
Radyuk et al. 2005; Radyuk, Michalak et al. 2009; Radyuk, Rebrin et al. 2010).
Oxidative stress and liver diseases
Oxidative stress (the imbalance of pro-oxidants and anti-oxidants(Jones, Lemasters et al.
2010)) and dysregulated redox signaling are involved in several liver diseases.
Accumulation of drugs or their metabolites induces the generation of mitochondrial
oxidants and subsequent alteration of redox sensitive signaling transduction are involved
in the development of drug-induced liver injury (DILI) (Jones, Lemasters et al. 2010). In
addition, Alcoholic liver disease (ALD) is associated with the increased mitochondrial
oxidants formation (induced by alcohol administration) and depletion of antioxidants
(Zhu, Jia et al. 2012). Oxidative stress associated with aging leads to insulin resistance
and contributes to pathogenesis of the type II diabetes (Ceriello and Motz 2004; Meigs,
Larson et al. 2007). Excessive fatty acid load was reported to increase oxidative stress
and exacerbate insulin resistance (Muller, Gardemann et al. 2012). Studies have indicated
that antioxidants (i.e. Vitamin C, E) treatment have shown improvement in the insulin
19

sensitivity (Evans 2007; Hoehn, Salmon et al. 2009; Ban, Rico et al. 2012). The redox
modulator, alpha-lipoic acid, was reported to improve insulin resistance (Konrad 2005).
Therefore, oxidative stress and the alteration of redox status are critical in the
pathogenesis of liver diseases. Modulation of oxidative stress and redox status are key
intervention points for these diseases.
Liver Pten knockout model
Liver-specific Pten knockout model was generated by the Cre-lox system (Lesche,
Groszer et al. 2002; Geoffroy and Raineteau 2007). Breeding of Pten
loxP/loxP
mice with
Alb-Cre mice yields mice with liver specific deletion of Pten (Stiles, Wang et al. 2004).
Table 1 lists pathological phenotype changes in Pten knockout liver as a function age
(Stiles, Wang et al. 2004). This model is an excellent platform to study fatty liver, insulin
signaling, and liver cancer.
20

Ages
(months)
Phenotype Changes in Pten Knockout Liver
1 Onset of steatosis (hepatocytes swollen with minimal lipid deposition).
Increased de novo lipogenesis. Lower plasma leptin levels and circulating
free fatty acids. Normal plasma triglyceride levels.
3 Decreased fasting glucose levels and increased glucose metabolism.
Substantial lipid accumulation in hepatocytes.
6 Decreased fasting glucose levels and increased glucose metabolism. Lipid
accumulation present in the entire liver. Inflammatory cells and fibrosis
infiltration observed in some animals.
9-12 Hyperplasia primarily around ductal region, progression of
hepatocarcinoma and cholagio carcinoma.
Stiles et al, Proc. Nat. Acad. Sci. USA, 2004
Table 1. Pten liver specific knockout model- Pathological changes in the control and
Pten
-/-
mouse liver
21

CHAPTER I
The PI3K/AKT signaling pathway regulates cellular bioenergetics
in immortalized hepatocytes

CHAPTER INTRODUCTION
Mitochondria are major cellular organelles that integrate metabolism, respiration, ATP
production and cell death among other cellular function. The phosphotidylinositol-3
kinase (PI3K) signaling pathway plays a key role in cell survival partially due to its effect
on regulating mitochondrial cytochrome c release (Rountree, Ding et al. 2009). In
addition, PI3K signaling also regulates glucose and lipids metabolisms (Cantley 2002),
processes coupled with mitochondrial oxidative phosphorylation through the TCA cycle
for ATP production. Insulin signals through PI3K to induce lipogenesis and glucose
uptake/metabolism in both muscle and adipose tissues. In muscle, insulin is a major
regulator for mitochondrial ATP production partly by promoting the synthesis of resident
mitochondrial genes and proteins (Petersen, Befroy et al. 2003; Shelley, Martin-Gronert
et al. 2009). Consistent with a role of PI3K signaling in oxidative phosphorylation,
deletion of forkhead transcriptional factor FoxO1 downstream of PI3K increased electron
transport chain activity and normalized NAD
+
/NADH values in the IRS1/2 double
knockout mouse liver (Cheng, Guo et al. 2009). In mouse models of resistance to insulin
and activation of PI3K, such as the ob/ob mice (Vianna, Huntgeburth et al. 2006; Cheng,
Guo et al. 2009), mitochondrial electron-transfer chain activity and determinants of
NAD
+
/NADH values are downregulated. Oxidative phosphorylation genes are also
22

deregulated when liver cells fail to respond to insulin, such as in individuals with insulin
resistance (Cheng, Guo et al. 2009).
A major target of PI3K is the serine/threonine kinase AKT (Franke, Yang et al. 1995;
Kohn, Takeuchi et al. 1996). Activation of PI3K by insulin and other growth factors
leads to phosphorylation and activation of AKT (Franke, Yang et al. 1995; Kohn,
Takeuchi et al. 1996). Inhibition of PTEN (Phosphatase and Tensin Homologue deleted
on Chromosome 10) (Hopkin 1998; Maehama and Dixon 1998), a negative regulator for
the pathway also results in constitutive activation of AKT kinase (Stiles, Gilman et al.
2002; Stiles, Wang et al. 2004; Kurlawalla-Martinez, Stiles et al. 2005; Stanger, Stiles et
al. 2005; Stiles, Kuralwalla-Martinez et al. 2006; Xu, Kobayashi et al. 2006; Rountree,
Ding et al. 2009; Stiles 2009). AKT was found to regulate the mitochondrial intrinsic cell
death signaling through direct or indirect interactions with pro-apoptotic factors of the
Bcl-2 family (i.e., Bad and Bax) and glycogen synthase kinase 3 (Stiles 2009). Bad and
GSK3 are the two best-characterized substrates that are directly phosphorylated and
inhibited by AKT (Datta, Dudek et al. 1997). How AKT may control mitochondrial
respiratory function, however, is not clear. AKT may phosphorylate hexokinase II and
strengthen its association with the outer mitochondrial membrane voltage-dependent
anion channel (VDAC), thus activating the first step in glucose metabolism (Gottlob,
Majewski et al. 2001; Pastorino, Hoek et al. 2005). These functions of AKT suggests that
PI3K/AKT signal may induce mitochondrial respiration. Recent studies reported
localization of AKT in the mitochondrial fraction of the cells (Bijur and Jope 2003;
Antico Arciuch, Galli et al. 2009). This mitochondrial AKT translocation is associated
23

with an increased phosphorylation (inhibition) of a constitutively mitochondrial form of
GSK3β in neuroblastoma cells (Bijur and Jope 2003). However, the biological function
of AKT translocation and phosphoryalation of mitochondrial GSK3β is not clear. This
study examines the role of the PI3K/AKT pathway on mitochondrial bioenergetics by
experimental approaches –performed on immortalized hepatocytes– entailing (a)
stimulation of the PI3K/AKT route by IGF-1, (b) specific inhibition of PI3K, and (c)
genetic approaches (specific deletion of Pten). The results indicate that AKT activation
(phosphorylation) enhances cellular bioenergetics by mechanisms that increase both
substrate supply to- and the catalytic efficiency of mitochondria.
MATERIALS AND METHODS
Cell lines – Immortalized wild type control (Con) and Pten null (Pten
–/–
) hepatocytes
were established from 1 month old Pten
loxP/loxP
; Alb-Cre
-
and Pten
loxP/loxP
; Alb-Cre
+

mouse liver, respectively(Stiles, Wang et al. 2004; Xu, Kobayashi et al. 2006). The cell
lines were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin,
0.1μg/ml insulin, 10 ng/ml EGF in a humidified 5% CO
2
atmosphere.
Chemicals – Oligomycin, Rotenone, and carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP) were purchased from MP Medicals. Lithium chloride, sodium
pyruvate, D-(+)-Glucose, insulin-like growth factor 1 (IGF-1), LY294002, and protease /
phosphatase inhibitor cocktail were from Sigma.
XF24 extracellular metabolic flux analysis – Control and Pten
-/-
hepatocytes were seeded
on XF24 plates at a density of 2×10
4
cells/well. The plate was incubated in a humidified
24

5% CO
2
atmosphere for 24 h. For IGF-1 treatment, cells were further incubated in the
serum free DMEM for 6 h followed by IGF-1 treatment for 1 h. For LY294002 treatment,
cells were treated with 40 μM LY294002 dissolved in unbuffered DMEM for 1 h. In both
IGF1 and LY294002 treatments, unbuffered DMEM was used as control. Four basal
measurements of oxygen consumption rate (OCR) and extracellular acidification rate
(ECAR) were made followed by the addition of mitochondrial inhibitors. Four readings
were made after the injection of each inhibitor. Three mitochondrial inhibitors were used:
oligomycin (1 μM) was added to inhibit the ATP synthase followed by the measurement
of ATP turn over, FCCP (1 μM) was added to uncouple the mitochondria followed by the
measurement of the maximal mitochondrial respiratory capacity, and rotenone (1 μM)
was added to inhibit the complex I followed by the measurement of non-mitochondrial
respiration.
Mitochondria isolation – Mitochondria were isolated from control and Pten
-/-

immortalized hepatocytes by using Percoll gradient with a Thermo Scientific Kit
(Catalog No 89874). Briefly, mitochondria were isolated from control and Pten
-/-

immortalized hepatocytes by using dounce homogenization followed by centrifugation.
Mitochondrial pellet was re-suspended in 15% Percoll and topped on to a pre-formed
discontinuous Percoll gradient. The purified mitochondrial fraction was collected after
centrifugation.
Proteinase K treatment of mitochondria – Mitochondria from control and Pten
-/-

hepatocytes were treated with proteinase K (50 µg/ml) at 4°C for 20 min.
25

Phenylmethylsulfonyl fluoride (2 mM) was added to stop the reaction and mitochondria
were garnered by centrifugation. Mitochondria were then lysed in 2% CHAPS containing
protease and phosphatase inhibitors.
Western blot analysis – Cells from control and Pten
-/-
hepatocytes treated with various
doses of IGF-1, LY294002, or LiCl. Prior to IGF-1 treatment were lysed in RIPA buffer
(50 mM Tris/HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
phosphatase inhibitor and protease inhibitor cocktail). Mitochondrial proteins were
extracted by incubating isolated mitochondria for 30 min at 4°C in 2% CHAPS
containing protease and phosphatase inhibitors. The antibodies used for immunobloting
are anti-pAKT, anti-AKT, anti-pGSK3, and anti-GSK3 from Cell Signaling; anti-pPDH-
E
1α
from EMD Millipore and anti-PDH-E
1α
from MitoSciences. Band intensities were
analyzed by the VersaDoc system (Bio-Rad).
Immunoprecipitation – Mitochondrial proteins prepared from control and Pten
-/-

immortalized hepatocytes were incubated with pAKT-substrate antibody conjugated
beads (Cell Signaling) for 48 h. The beads with the immune complexes were washed 3
times with DPBS, mixed with reducing loading sample buffer (Thermo), and heated for 5
min at 100°C. Proteins were separated by a 12% SDS-PAGE gel. The gel was stained
with Coomassie blue (Biorad).
LC/MS/MS analysis – Proteins were extracted from the Coomassie blue-stained gel
followed by LC/MS/MS analysis at the USC Keck School of Medicine Proteomics Core
Facility. Procedures were carried out as previously described (Garcia, Han et al. 2010).
26

Statistical analysis – Statistical analysis was performed using student’s t test for unpaired
data or ANOVA. P < 0.05 was considered significant.
CHAPTER RESULTS
PI3K/AKT signaling activation induces mitochondrial respiration
Liver is a major organ where lipid and glucose metabolism converge with mitochondrial
respiration. The role of PI3K/AKT signaling in mitochondrial respiration was assessed
with a hepatocyte cell line that we established from mouse liver. Treatment of
hepatocytes with insulin growth factor-1 (IGF-1) –that induces the activation of the
PI3K/AKT pathway– at 50 and 100 ng/ml for 1 h induced a robust activation of AKT as
indicated by the increase in its phosphorylateion (Fig. 5A). A concomitant increase of
the phosphorylated forms of GSK3α and β, indicated increased AKT activity in the IGF-
1-treated samples (Fig. 5A).
IGF-1 treatment of hepatocytes resulted in an increase in oxygen consumption rates
(OCR) and extracellular acidification rates (ECAR), the former reflecting mitochondrial
respiration and the latter anaerobic glycolysis (i.e., lactate formation). IGF-1 pre-
treatment for 1 h did not affect the basal OCR, whereas it elicited a substantial increase of
maximal respiratory capacity (following the addition of the uncoupler FCCP). At an IGF-
1 concentration of 50 ng/ml, the maximal OCR increased by 90.61 ± 2.71 % (compared
to the vehicle-treated cells) (Fig. 5B). A further increase was observed following
treatment of hepatocytes with IGF-1 at a concentration of 100 ng/ml (128.18 ± 7.79 %
higher than vehicle-treated cells) (Fig. 5B). IGF1 treatment also increased ECAR (30.64
27

± 8.64 % and 56.50 ± 5.78 % by 50 ng/ml and 100 ng/ml IGF-1, respectively) indicating
an enhanced anaerobic glycolysis (Fig. 5D). These data suggest that the IGF-
1/PI3K/AKT signaling positively regulates anaerobic glycolysis and mitochondrial
maximal respiratory capacity.
28

Fig. 5. Effect of IGF-1 on AKT phosphorylation status and cellular bioenergetics
(A) Western blots of AKT and GSK3 and their phosphorylated forms. Hepatocytes were
incubated for 1 h with IGF-1. (B) Effect of IGF-1 on oxygen consumption rate (OCR).
Immortalized hepatocytes were pre-incubated with IGF-1 for 1 h before starting the
measurement of basal respiration. Subsequent additions: 1 µM oligomycin; 1 µM FCCP;
1 µM rotenone. Four readings were made after the injection of each inhibitor. ( ) vehicle;
( ) plus 50 ng IGF-1/ml; ( ) plus 100 ng IGF-1/ml. (C) Quantitation of basal and
maximum OCR in vehicle- and IGF-1-treated samples (data from Fig. 5C). Data are the
average of 4 times-points each from 4 independent samples. (D) Effect of IGF-1 on
extracellular acidification rate (ECAR): assay conditions as in Fig. 5B. All experiments
were performed in quadruplicate. Other assay conditions as described in the Materials
and Methods section.
29

Inhibition of PI3K/AKT signaling attenuates mitochondrial respiration
Inhibition of PI3K by LY294002 resulted in a decreased AKT and GSK3
phosphorylation (Fig. 6A), the former indicating a lack of activation and the latter release
of the inhibitory effect exerted upon phosphorylation of GSK3β. Accordingly, basal and
maximal OCR were reduced (92.10 ± 1.10 and 91.90 ± 1.10 %, respectively) by
treatment with LY294002 as compared with vehicle-treated cells (Fig. 6B, C). Decreased
basal and maximal OCR indicates that the inhibition of the PI3K/AKT signaling by
LY294002 treatment leads to the inhibition of ATP generation-coupled (basal) and
uncoupled (maximal) mitochondrial respiration. The inhibition of ECAR by LY294002
treatment implied that anaerobic glycolysis was not compensating for the decrease in
mitochondrion-driven O
2
consumption (Fig. 6D). These data, along with those following
IGF-1 treatment, strengthens the importance of PI3K/AKT signaling on mitochondrial
bioenergetics.
30

Fig. 6. Effect of PI3K inhibition on AKT and GSK phosphorylation status and cellular
bioenergetics
(A) Western blots of AKT and GSK3 and their phosphorylated forms. Cells were
incubated with 40 µM LY294002 for 1 h. (FBS 20%). (B) Effect of PI3K inhibitor on
oxygen consumption rate (OCR). Basal OCR rate is measured 30min after the addition of
LY294002. ( ) vehicle; ( ) plus 40 µM LY294002. (C) Quantitation of basal and
maximal OCR in vehicle- and LY29002-treated samples. (D) Effect of LY294002 on
extracellular acidification rate (ECAR): assay conditions as in Fig. 6B. All experiments
were performed in quadruplicates. Quantitative data in (C) are average of 4 time-points
each from 4 independent samples.
OCR (pmoles/min)
|
400
|
200
20 40 60 80 100
Time (min)
| | | | |
0
oligomycin FCCP rotenone
35
30
20
25
| |
15
|
10
5
| | |
20 40 60 80 100
Time (min)
| | | | |
0
oligomycin FCCP rotenone
p-AKT (Ser
473
)
p-GSK3α (Ser
21
)
p-GSK3β (Ser
9
)
β-Actin
AKT
GSK3α
GSK3β
+FBS -FBS LY294002
400
200
0
OCR (pmoles/min)
**
**
Basal
Respiration
Maximal
Respiratory
Capacity
 Vehicle
 LY294002 40μM
A
B
C
D
31

PTEN
-/-
immortalized hepatocytes have higher mitochondrial respiration
An isogenic murine hepatocyte cell line with constitutive activation of PI3K/AKT
signaling was used in order to confirm the role of PI3K/AKT in mitochondrial respiration.
The Pten null (Pten
–/–
) hepatocyte cell line was established from a 1 month-old mouse
carrying a liver-specific deletion of Pten (Pten
loxP/loxP
; Alb-Cre
+
) (Xu, Kobayashi et al.
2006) (Fig. 7A). Immunostaining analysis revealed that AKT activity –indicated by its
phosphorylation status– was substantially increased in the Pten
–/–
hepatocytes as
compared with the control cell lines with intact PTEN (Fig. 7B). Accordingly, GSK3β
phosphorylation at Ser
9
was decreased (Fig. 7B). Fig. 7C shows a time course of AKT
and GSK3β phosphorylation status following PI3K inhibition by LY294002.
Basal OCR was significantly higher in the Pten
–/–
than in the control hepatocytes (289.76
± 3.74 and 154.88 ± 2.88 pmoles/min, respectively). Following the addition of FCCP,
maximal OCR in the Pten
–/–
hepatocytes (393.32 ± 56.64 pmole/min) remained
significantly higher than that in the controls (213.50 ± 8.14 pmoles/min) (Fig. 8 A,B).
Basal ECAR was higher in the Pten
–/–
hepatocytes (40.48 ± 1.43 mpH/min) than in the
controls (26.99 ± 1.99 mpH/min). The higher anaerobic glycolysis level (ECAR) in Pten
–
/–
hepatocytes indicates a higher substrate supply from glycolysis to mitochondria. Taken
together, these data confirmed that the PTEN-regulated PI3K/AKT pathway controls
bioenergetics by modulating both substrate supply (ECAR) and mitochondrial catalytic /
energy transducing capacity (OCR).
32

Fig. 7. AKT and GSK3 phosphorylation status in immortalized hepatocytes from control
and Pten
-/-
knockout mice
(A) PTEN protein expression in control- and Pten
-/-
hepatocytes. (B) Western blots of
AKT and GSK3 and their phosphorylated forms in cell lysates from control- and Pten
-/-

hepatocytes. Different amounts of proteins (10-, 30-, and 60 µg) were loaded in three sets
of samples. (C) Effect of LY294002 on the time course of p-Akt, p-GSK3α, and p-
GSK3β in control- and Pten
-/-
hepatocytes.
PTEN
β-Actin
__
Control
__ ___
Pten
-/-___
A
p-GSK3β (Ser
9
)
β-Actin
AKT
GSK3α
GSK3β
p-AKT (Ser
473
)
___________
Cell lysate (µg protein)
___________
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
_____
10
_____ _____
30
_____ _____
60
_____
B
C
p-AKT (Ser
473
)
p-GSK3α (Ser
21
)
p-GSK3β (Ser
9
)
β-Actin
Time (min) 60 120 180 5 60 120 180 5 60 120 180 60 120 180 5 60 120 180 5 60 120 180 5
__
Control
__ ______
Pten
-/-________ ______
Pten
-/-________ ______
Pten
-/-________ ___
Control
____ ____
Control
____
_____________
2.5 µM
_____________ _____________
5.0 µM
______________
_______________________________
LY294002
______________________________ ___________
Medium
___________
33

Fig. 8. Bioenergetics of control- and Pten
-/-
hepatocytes
(A) Oxygen consumption rates (OCR) of control- and Pten
-/-
hepatocytes (2 x 10
4
cells/
well). Additions (oligomycin, FCCP, and rotenone) as in Fig. 5A. ( ) control hepatocytes;
( ) Pten
-/-
hepatocytes. (B) Quantitative data of basal and maximal OCR of control ( )-
and Pten
-/-
( ) hepatocytes (data from Fig. 8A). (C) Extracellular acidification rate
(ECAR) of control- and Pten
-/-
hepatocytes (2 x 10
4
cells/ well) (assay conditions as in
Fig. 8A). (D) Quantitative data of basal ECAR of control- and Pten
-/-
hepatocytes. All
experiments were performed in quadruplicate. Data in (B) and (D) are the average of 4
time-points from 4 different samples.
**
**
**
34

Activated AKT translocates to mitochondria
Consistent with previous reports on the translocation of AKT to mitochondria in
NIH/3T3 (Antico Arciuch, Galli et al. 2009) and neuroblastoma (Bijur and Jope 2003)
cells, a small amount of AKT translocated to mitochondria in both control- and Pten
–/–

hepatocytes. Pten
–/–
hepatocytes –with a more robust AKT phosphorylation– showed
significantly more AKT in mitochondria suggesting that the AKT mitochondrial
translocation may depend on its phosphorylation (Fig. 9A). Expectedly, IGF-1 treatment
also induced a higher translocation of AKT to mitochondria. This is more evident in the
Pten
–/–
cell lines, in which the pAKT/AKT ratio is already high without IGF-1 treatment.
When treated with IGF-1, even more AKT translocates to the mitochondria in the Pten
–/–

hepatocytes (Fig. 9B). Fig. 9C shows a time-dependent increase in pAKT in both control
and Pten
–/–
cell lines in response to IGF-1 treatment which results in the translocation of
AKT to mitochondria.
We further evaluated whether AKT is located in the inner membrane of mitochondria or
associated with the outer membrane. Treatment of isolated mitochondria with proteinase
K (that degrades proteins on the outside of the outer mitochondrial membrane) has no
effect on mitochondrial AKT levels (Fig. 9D &E), supported the notion that AKT is
localized in mitochondrial inter-membrane space and mitochondrial matrix.
35

Fig. 9. Mitochondrial localization of AKT
(A) AKT expression in the mitochondrial fraction isolated from control and Pten
-/-

hepatocytes. (B) Time course of AKT expression in the mitochondrial fraction following
incubation of hepatocytes with 50 ng IGF-1/ml. Cells (control and Pten
-/-
hepatocytes)
were incubated for 0, 5, 15, and 30 min previous to the isolation of the mitochondrial
fraction. (C) Time course p-AKT expression in the cell lysate following incubation for
different times with 50 ng IGF-1/ml. L, light exposure; D, dark exposure. (D) Effect of
proteinase K on AKT and p-AKT expression in the mitochondrial fraction isolated from
control and Pten
-/-
hepatocytes. (E) Relative levels of mitochondrial AKT in control and
Pten
-/-
hepatocytes (data from (D)).
______
Mitochondrial fraction
______
0 5 15 30 0 5 15 30
_____
Control
_____ _____
Pten
-/- _____
B
Control
Pten
-/-
Control
Pten
-/-
Control
Pten
-/-
______
Mitochondrial fraction
______
A
L
D
0 5 15 30 0 5 15 30
_____
Control
_____ _____
Pten
-/- _____
AKT
COX IV
Time (min)
AKT
COX IV
AKT
β-Actin
p-AKT (Ser
473
)
p-AKT (Ser
473
)
Time (min)
____________
Cell lysate
_____________
C
AKT
p-AKT(Ser
473
)
COX IV
–
Control
–
Pten
-/-
+
Control
+
Pten
-/-
___________
Proteinase K
___________
D
100
50
0
[AKT]
MITOCHONDRIA
(relative units)
–
Control
–
Pten
-/-
+
Control
+
Pten
-/-
___________
Proteinase K
___________
E
36

AKT regulates mitochondrial respiration through GSK3 and ATP synthase
GSK3β is a well characterized AKT phosphorylation targets in mitochondria (Bijur and
Jope 2003). GSK3β was proposed as a constitutive mitochondrial protein (Bijur and Jope
2003) that was linked to phosphorylation (and inactivation) of the pyruvate
dehydrogenase complex (PDH) (Hoshi, Takashima et al. 1996), a critical mitochondrial
enzyme complex that couples anaerobic glycolysis (in cytosol) to the oxidative
decarboxylation of pyruvate to acetyl-CoA and its entry in the tricarboxylic acid cycle.
Thus, GSK3β is a potential substrate target of mitochondrial AKT that may be
responsible for PI3K/AKT induced mitochondrial respiration. Treatment of hepatocytes
with LiCl, a reagent used to inhibit GSK3β activity (Jope 2003), led to increased
phosphorylation (inactivation) of GSK3β (Fig. 10A) and a dose-dependent reduction in
phosphorylation (inactivation) of PDH-E
1α
(Fig. 10B), indicating activation of the
enzyme complex. Consistent with the role of PDH as a crutial mitochondrial enzyme,
treatment with LiCl increased basal and maximal respiration in both wild type (Fig. 10C)
and Pten
-/-
hepatocytes (Fig. 10D). Thus, mitochondrial respiration may be regulated by
the coordinated balance between GSK3β (inhibitory effect) and AKT (stimulatory effect).
We also explored other potential AKT phosphorylation targets in the mitochondria by
pull-down experiments from mitochondrial lysates prepared from control- and Pten
–/–

hepatocytes. Immunoprecipitation with pAKT substrate antibody identified four bands
at ~100, ~90, ~80, ~50 kDa that were significantly stronger in Pten
–/–
mitochondrial
lysates than in the controls (Fig. 11A). LC/MS/MS analyses revealed several potential
targets for phosphorylation by AKT, including ATP synthase subunits α and β in the 50
37

kDa band (Table 2). Consistent with these observations, ATP synthase activity was
higher in the Pten
–/–
hepatocytes than in control cells (Fig. 11B).
ATP synthase α contains the reverse sequence to the consensus RxRxxS/T sequence for a
potential AKT substrate (Table 1). This sequence is located at a structure characteristic
where the subunit α of ATP synthase may interact with the β subunit (Fig. 11C).
Sequence analysis for ATP synthase α revealed that the consensus sequence segment is
conserved in several species analyzed including human, mouse, rat, and E. coli. ATP
synthase β has similar sequence features as a substrate for AKT (Table 1). These
analyses suggest that the ATP synthase enzymes are likely substrates for AKT and that
their phosphorylation might be responsible for the higher OCR measurements after FCCP
treatment in the Pten
-/-
and IGF-1 treated cultures (Fig. 8).
38

Figure 10:

Fig. 10. Inhibiting GSK3β with LiCl induces bioenergetics in control- and Pten
-/-

hepatocytes
(A) Effect of different concentrations of LiCl on p-GSK3β and GSK3β expression.
Hepatocytes were treated with indicated concentrations of LiCl for 4 h. Total cell lysates
were used to determine p-GSK3β and GSK3β levels. β-actin was detected as loading
control. (B) Effect of LiCl on p-PDH-E
1α
(Ser
273
) and PDH-E
1α
expression.
Hepatocytes were treated with indicated concentrations of LiCl for 4 h. Mitochondrial
lysates were used to determine the levels of PDH and p-PDH. COX IV was detected as
loading control. (C) Effect of LiCl on the oxygen consumption rate (OCR) of control
**
**
**
**
39

Figure 10 continued:
hepatocytes. Cells (2 x 10
4
/well) were incubated for 4 h with 20 mM LiCl before starting
the measurement of basal respiration. Addition of inhibitors (oligomycin, FCCP, and
rotenone) is the same as in Fig. 5A. Right panel: quantitative data indicating the effect of
LiCl on basal- and maximal respiration. (D) Effect of LiCl on the oxygen consumption
rate (OCR) of Pten
-/-
hepatocytes. Assay conditions are the same as in (A). Right panel:
quantitative data indicating the effect of LiCl on basal- and maximal respiration of Pten
-/-

hepatocytes.
40

Fig. 11. AKT phosphorylation targets in mitochondrial fraction isolated from control- and
Pten
-/-
hepatocytes
(A) Commassie blue staining of protein lysates extracted from p-AKT substrate antibody
immunoprecipitation assay. Left lane: control hepatocytes; right lane: Pten
-/-
hepatocytes.
The arrows indicate the bands subjected to LC/MS/MS analyses. (B) ATP synthase
activity of control- and Pten
-/-
hepatocytes (assay conditions as described in the Materials
and Methods section). (C) Structural features of ATP synthase α protein sequence.
Triangles indicate ATP synthase α−β interacting interface. A reverse substrate consensus
sequence for AKT substrate is identified from residue 166-171.
41

_________________________________________________________________________
Subunit α sequence T/SxxRxR
166-171 kgpigSktRrRvglkapgii mouse
166-171 kgpvgSkiRrRvglkap rat
166-171 kgpigSktRrRvglkapg human
247-252 valmgEyfRdRgedaliiy E. coli
_________________________________________________________________________
Subunit β sequence T/SxxRxR
453-458 seedklTvsRaRkiqrfl mouse
399-404 seedklTvsRaRkiqrfl rat
453-458 seedklTvsRaRkiqrfl human
390-406 seedklVvaRaRkiqrfl E. coli
_________________________________________________________________________
Table 2. Sequence alignment for ATP synthase
subunits

CHAPTER DISCUSSION
The PI3K/AKT signaling pathway regulates both growth/survival and metabolism. How
these two functions are integrated is not clear. One potential link between the two
functions may be surmised to be integrated in mitochondria, organelles involved in
energy-transducing process and the regulation of apoptotic and survival pathways. These
functions render mitochondria viable candidates for integrating the various processes
regulated by PI3K/AKT signaling pathway. In this study, we showed that PI3K/AKT
signaling positively regulates mitochondrial bioenergetics in various experimental models
entailing IGF-1-driven stimulation of the PI3K/AKT, inhibition of PI3K (with
42

LY294002), and a Pten
–/–
cell model where PI3K/AKT signal is constitutively activated.
We showed that mitochondrial respiration is significantly enhanced when the PI3K/AKT
pathway is induced. This increased mitochondrial respiratory function is correlated with
the mitochondrial translocation of the activated AKT. In the mitochondria, AKT
phosphorylates and inhibits GSK3β. Inhibition of GSK3β results in robust induction of
mitochondrial respiration. The mitochondrial AKT also phosphorylates a number of
other mitochondrial residence proteins including two isoforms of ATP synthase. The
mitochondrial AKT likely plays a role on respiration directly through these proteins.
Recent evidence suggests that the “Warburg effect” may contribute to the cause of
tumorigenesis (Vander Heiden, Cantley et al. 2009). The “Warburg effects” is
characterized as increased glycolysis simultaneously with enhanced oxidative
phosphorylation. We observed that PI3K/AKT activation leads to the simultaneous
increase of glycolysis (ECAR) and oxidative phosphorylation (OCR). While this
observation does not explain how metabolic changes observed as Warburg effects
contribute to the tumorigenesis process, it suggest that the PI3K/AKT signaling pathway
may be a major candidate for Warburg effects linking growth and metabolism (Cantley
2002). Recent studies have shown that transfection of Rat1a cells with a constitutively
active form of AKT robustly increased the association of hexokinase with outer
mitochondrial membrane VDAC (Gottlob, Majewski et al. 2001), an effect which is
critical for the anti-apoptotic function of AKT (Majewski, Nogueira et al. 2004). The
ability of AKT to inhibit apoptosis is dependent on this association of hexokinase with
the mitochondrial membrane (Gottlob, Majewski et al. 2001; Majewski, Nogueira et al.
43

2004). These studies, thus, suggest that mitochondria are critical for linking the metabolic
and cell survival effects of PI3K/AKT signaling.
While the effect of AKT on Hexokinase and VDAC may explain the enhanced glycolysis
at the presence of oxidative phosphorylation. It does not address how PI3K/AKT
activation induces oxidative phosphorylation. In this study, we observed that the
increased mitochondrial respiratory function observed in cells where PI3K/AKT signal is
activated is correlated with the inhibition of GSK3β. GSK3β is a well-known substrate of
AKT kinase activity (Cross, Alessi et al. 1995). Phosphorylation of GSK3β by AKT
leads to its inactivation. We showed here that the inhibiting GSK3β by LiCl increases
mitochondrial basal and maximal respiration, indicating that GSK3β may negatively
regulates mitochondrial bioenergetics. Thus, inactivation of GSK3β by AKT may at least
partially be responsible for the observed increase of mitochondrial respiration associated
with PI3K/AKT activation. In rat hippocampal neurons, GSK3β was found to be a
mitochondrial resident protein that phosphorylated and inhibited PDH, a key enzyme
controlling the substrate availability for the TCA cycle and mitochondrial respiration
(Hoshi, Takashima et al. 1996). It may be surmised that mitochondrial pAKT
phosphorylates and inhibits GSK3β, thus changing the pPDH
inactive
/ PDH
active
ratio and
shifting the PDH enzyme from inactive to active. We showed that AKT is physically
translocated to the mitochondrial intermembrane space where is can phosphorylate and
inhibit the ability of GSK3β to block PDH activity. This function of AKT may represent
a feed forward reaction that prevents the product of cytosolic glycolysis, pyruvate, from
44

buildup while PI3K/AKT activation induces glucose uptake through interaction with
hexokinase II/VDAC interaction (Gottlob, Majewski et al. 2001). PDK1, the enzyme that
phosphorylates AKT on the second site Thr
308
to achieve full activation, is also
constitutively active in the mitochondrial intermembrane space (Antico Arciuch, Galli et
al. 2009). This observation further supported a role of PDK1-AKT-GSK3β signaling axis
in regulating mitochondrial function.
The increased mitochondrial bioenergetics may also be attributed to the phosphorylation
of a number of other mitochondrial proteins by AKT, including the two isoforms of ATP
synthase, α and β. ATP synthase is a multi-subunit complex composed of an ATPase and
a proton channel and it converts the membrane potential generated by the proton gradient
to ATP. Our data suggest that the ability of mitochondrial to respire without coupling
with glycolysis (after FCCP) is also significantly increased when PI3K/AKT signal is
induced. The phosphorylation of the ATP synthase complex and the accompanying
enhancement in its activity when AKT is activated supported the notion that ATP
synthase phosphorylation and activity is likely regulated by PI3K/AKT. The sequence of
ATP synthase α phosphorylated by AKT is part of the α−β interacting interfaces. These
observations imply that AKT-mediated phosphorylation of ATP synthase α affects its
ability to interact with ATP synthase β and thus may directly affect its function.
Taken together, this study clearly demonstrated that PI3K/AKT activation leads to
increased mitochondrial respiration through both ATP synthase-dependent and
independent signaling pathways; the former may entail a direct phosphorylation of ATP
45

synthase by AKT whereas the latter may involve a stronger metabolism of pyruvate
(PDH activity) by both increasing substrate supply to mitochondria (regulation of
cytosolic processes by AKT) and enhancing the mitochondrial catalytic machinery
(changes in pPDH/PDH ratios) upon inhibition of GSK3β.
46

CHAPTER II
PI3K/AKT signaling modulates cellular H
2
O
2
homeostasis and
redox status through regulation of mitochondrial H
2
O
2
generation

CHAPTER INTRODUCTION
Mitochondria generate energy and are an effective source of oxidants, such as H
2
O
2

which is involved in the regulation of cell proliferation, differentiation and apoptosis
(Giulivi, Poderoso et al. 1998; Cadenas 2004). In 1955, Chance and Williams defined the
metabolic states and respiratory control of mitochondria: State 4 (inactive) with
respiratory substrates (i.e., puruvate, acetyl-CoA) but without ADP; State 3 (active) with
both substrates and ADP (Chance and Williams 1955; Chance and Williams 1955;
Chance and Williams 1955; Chance and Williams 1955; Chance, Williams et al. 1955).
Hence, the classical concept is that mitochondrial respiration is regulated by the
availability of O
2
to cytochrome oxidase (Complex IV) and of ADP to ATP-synthase
(Complex V). It is estimated that in physiological conditions about 65% of mammalian
tissue mitochondria are in state 4 and about 35% in state 3. Electrons leak -between the
rotenone and antimycin sensitive sites- leads to the univalent reduction of O
2
to O
2
•-
and
its prompt dismutation to H
2
O
2
(catalyzed by matrix Mn-SOD). The generation of
mitochondrial O
2
•-
and H
2
O
2
is associated with the metabolic state: mitochondria in state
4 show a relatively high production rate of O
2
•-
and H
2
O
2
; conversely, mitochondria in
respiratory state 3 show a low rate of O
2
•-
and H
2
O
2
production (Boveris and Chance
1973; Boveris, Costa et al. 1999). H
2
O
2
is reduced to H
2
O by a complex array of redox
47

couples (glutathione and thioredoxin) and catalysts (glutathione reductase, peroxidase,
peroxiredoxin and glutathione transferase). The balance between the generation of H
2
O
2

and its removal established the mitochondrial and cellular steady-state levels (Chance,
Sies et al. 1979; Naqui, Chance et al. 1986). H
2
O
2
generated by mitochondria modulates
cytosolic domain-specific signaling by post-translational modulations of cytosolic redox-
sensitive proteins (Yap, Garcia et al. 2009). The cellular H
2
O
2
steady-state concentration
also is known to regulate cell proliferation, apoptosis, and necrosis (Antunes and Cadenas
2001).
The mitochondrial energy-transduction and cellular redox status are concerted processes
embodied by a mitochondrial energy-redox axis. As the effective source of H
2
O
2
,
mitochondria contribute to the regulation of the cellular redox status, which is usually
quantified by the values of glutathinone/glutathione disulfide (GSH/GSSG) and
Trx
red
/Trx
ox
(Trx-(SH)/Trx-SS) (Yap, Garcia et al. 2009). Excessive generation of H
2
O
2

leads to oxidation shift of the redox status and oxidative stress (Jang and Remmen 2009).
Mitochondria are also recipients of signaling regulations such as mitogen-activated
protein kinases (MAPKs) and PI3K/AKT pathway of insulin signaling. Activation of c-
jun N-terminal kinase (JNK) by anisomycin or H
2
O
2
results in its translocation to the
mitochondrial outer membrane where it mediates the phsophorylation of mitochondrial
pyruvate dehydrogenase (mechanism not known) and subsequently inhibition of its
activity and mitochondrial bioenergetics (Schroeter, Boyd et al. 2003; Zhou, Lam et al.
2008; Zhou, Lam et al. 2009). In addition, direct insulin treatment stimulates
48

mitochondrial protein synthesis and respiration (McKee and Grier 1990). Many
downstream proteins of insulin signaling are reported to regulate mitochondrial
bioenergetics: 1) AKT activation enhances mitochondrial energy-transduction in renal
proximal tubular cells (Shaik, Fifer et al. 2008); 2) mTOR transcriptionally controls
mitochondrial oxidative phosphorylation (Cunningham, Rodgers et al. 2007). PTEN is a
negative regulator of PI3K signaling pathway through dephosphorylating PI-3,4,5-P3 at
the 3’ position to PI-4,5-P2 and thus inhibits PI-3-K signaling (Stiles 2009). Pten deletion
in adipose led to increased energy expenditure, thermogenesis and transcription of several
genes that are involved in mitochondrial biogenesis (Komazawa, Matsuda et al. 2004).
All these indicate PI3K.AKT signaling may regulate energy-transduction and
subsequently oxidants metabolism. However, the mechanism that connects cellular
signaling pathway and mitochondrial oxidants metabolism is still elusive.
This study examines the role of the PI3K/AKT pathway on mitochondrial H
2
O
2

homeostasis and cellular redox status by experimental approaches –performed on
immortalized and primary hepatocytes– entailing (a) genetic approaches (specific
deletion of Pten), and (b) specific inhibition of PI3K. The results indicate that AKT
activation (phosphorylation) decreases mitochondrial H
2
O
2
generation.
MATERIALS AND METHODS
Animals- Pten
loxp/loxp
mice were bred with Alb-Cre mice to generate Pten liver specific
deletion mice. Animals of 3-month age were used for experiments.
49

Chemicals- Oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)
were purchased from MP Medicals. Anti-phospho-AKT (Ser
473
), anti-AKT, anti-
phospho-GSK3α/β (Ser
21/9
) and anti-GSK3α/β antibodies were purchased from Cell
Signaling. Anti-complex IV subunit IV (COX IV) antibody and MitoProfile® Total
OXPHOS Rodent WB Antibody Cocktail were purchased from MitoSciences. Anti-
catalase, anti-MnSOD, and anti-peroxinredoxin 3 antibodies were purchased from EMD
Millipore. Sodium pyruvate, D-(+)-Glucose, LY294002, and protease / phosphatase
inhibitor cocktail were from Sigma. Dulbecco's Modified Eagle Medium (DMEM),
Dulbecco's Phosphate Buffered Saline (DPBS) and trypsin were purchased from Cellgro.
Cell lines – Immortalized wild type control (Con) and Pten null (Pten
–/–
) hepatocytes
were established from 1 month old Pten
loxP/loxP
; Alb-Cre
-
and Pten
loxP/loxP
; Alb-Cre
+

mouse liver, respectively(Stiles, Wang et al. 2004; Xu, Kobayashi et al. 2006). The cell
lines were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin,
0.1μg/ml insulin, 10 ng/ml EGF in a humidified 5% CO
2
atmosphere.
Tissue culture- Primary hepatocytes were isolated by collagenase perfusion (Miyazaki
1977). The primary hepatocytes were seeded on XF-24 plates at a density of 10,000
cells/well in 250μl DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin,
0.1 μg/ml insulin, 10 ng/ml EGF in a humidified 5% CO
2
atmosphere for 24 hours
followed by the extracellular flux analysis.
XF24 extracellular metabolic flux analysis – Control and Pten
-/-
hepatocytes were seeded
on XF24 plates at a density of 10
4
cells/well. The plate was incubated in a humidified 5%
50

CO
2
atmosphere for 24 h. For LY294002 treatment, cells were treated with 40 μM
LY294002 dissolved in unbuffered DMEM (DMEM base medium supplemented with
25mM glucose, 1mM sodium pyruvate, 31mM NaCl; PH7.4) for 1 h. Unbuffered DMEM
was used as control. Four basal measurements of oxygen consumption rate (OCR) and
extracellular acidification rate (ECAR) were made followed by the addition of
mitochondrial inhibitors. Four readings were made after the injection of each inhibitor.
Three mitochondrial inhibitors were used: oligomycin (1 μM) was added to inhibit the
ATP synthase followed by the measurement of ATP turn over, FCCP (1 μM) was added
to uncouple the mitochondria followed by the measurement of the maximal
mitochondrial respiratory capacity, and rotenone (1 μM) was added to inhibit the
complex I followed by the measurement of non-mitochondrial respiration.
Isolation of mitochondria - Liver mitochondria were isolated from 3-month old wild type
and Pten
-/-
mice liver by differential centrifugation followed by Percoll discontinuous
gradient purification (Garcia, Han et al. 2010). Liver tissues were homogenized at 4
o
C in
mitochondrial isolation buffer (MIB, containing 230 mM sucrose, 1 mM EDTA, 10 mM
Tris-HCl, 0.5 mM EGTA, 0.1% (w/v) fat-free bovine serum albumin and
protease/phosphatase inhibitor Cocktail, PH 7.4). Liver homogenates were centrifuged at
1500g for 5 minutes. The supernatant containing mitochondria was centrifuged at 21,000
g for 10 minutes. Crude mitochondrial pellet was then re-suspended in 15% Percoll and
topped on to a pre-formed Percoll gradient. The purified mitochondrial pellet was washed
three times, suspended in MIB and used within 4 hours. Mitochondria were isolated from
control and Pten
-/-
immortalized hepatocytes by using Percoll gradient with a Thermo
51

Scientific Kit (Catalog No 89874). Briefly, mitochondria were isolated from control and
Pten
-/-
immortalized hepatocytes by using dounce homogenization followed by
centrifugation. Mitochondrial pellet was re-suspended in 15% Percoll and topped on to a
pre-formed discontinuous Percoll gradient. The purified mitochondrial fraction was
collected after centrifugation.
Mitochondrial respiration- Mitochondria oxygen consumption was measured by a Clark-
type electrode. 200 ug mitochondrial proteins were re-suspended in mitochondrial
respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES, 2 mM MgCl
2
, 1 mM
EGTA, 1% (w/v) bovine album) yielding the concentration of 200 ug/mL. 1 min baseline
oxygen consumption was recorded followed by adding pyruvate (5 mM), glutamate (5
mM) and succinates (5 mM) as substrates respectively. State 4 oxygen consumption was
recorded after adding the substrates (pyruvate, glutamate, or succinate). State 3
respiration is recorded after adding ADP (410 μM). The oxygen consumption was
calculated according to the response of slope after adding the substrates and ADP. The
respiratory control ratio (RCR) is equal to the state 3 (with ADP) oxygen
consumption/min divided by the state 4 (without ADP) oxygen consumption/min.
Hydrogen Peroxide Production- The hydrogen peroxide production from isolated
mitochondria and cultured hepatocytes was measured by the AmplexRed Hydrogen
Peroxide/Peroxidase Assay kit (Invitrogen) according to the manufacturer's instructions.
Hydrogen Peroxide degradation- Wild type control and Pten
-/-
immortalized hepatocytes
were treated with 50 μM H
2
O
2
, the removal of H
2
O
2
was determined by measuring the
52

remaining H
2
O
2
as a function of time. H
2
O
2
concentration

was determined by the
AmplexRed Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) according to the
manufacturer's instructions.
Western blot analysis – Cells from control and Pten
-/-
hepatocytes were lysed in RIPA
buffer (50 mM Tris/HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
phosphatase inhibitor and protease inhibitor cocktail). Mitochondrial proteins were
extracted by incubating isolated mitochondria for 30 min at 4°C in 2% CHAPS
containing protease and phosphatase inhibitors. The antibodies used for immunobloting
are anti-pAKT, anti-AKT, anti-pGSK3, and anti-GSK3 from Cell Signaling;
MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail from MitoSciences and
anti-catalase, anti-MnSOD, and anti-peroxinredoxin 3 from from EMD Millipore. Band
intensities were analyzed by the VersaDoc system (Bio-Rad).
Measurement of NAD(P)H/NAD(P)
+
, GSH/GSSG- NAD(P)H/NAD(P)
+
- GSH/GSSG
were measured by HPLC with electrochemical detection. For NAD(P)H/NAD(P)
+

measurement, wild type and Pten
-/-
immortalized hepatocytes were lysated with a buffer
containing 0.06 M KOH, 0.2 M KCN, and 1 mM bathophenanthroline disulfonic acid
followed by chloroform extraction. Cell lysis with chloroform was centrifuged at
14000rpm at 4 °C for 15min. The supernant was analyzed for NAD(P)H/NAD(P)
+
. The
mobile phase for separating NAD(P)H/NAD(P)
+
consisted 0.2 M ammonium acetate at
pH 5.5 and HPLC grade methanol. For GSH/GSSG measurement, wild type and Pten
-/-

were lysated with 5% o-metaphosphoric acid, or 5% o-metaphosphoric acid with 25 mM
53

ammonium sulfamate and cell lysate was centrifuged at 12000g for 5min and supernatant
was collected for HPLC measurement. The mobile phase consistes 3% acetonitrile,
25 mM sodium monobasic phosphoric acid and 0.5 mM 1-octanesulfanic acid adjusted to
pH 2.7 with o-phosphoric acid. The concentrations of GSH/GSSG, NAD(P)H/NAD(P)
+

were calculated based on injected standards.
Cellular redox status- The cellular redox status was calculated by the Nernst equation
(E
hc
= E
0
+30 log([GSSG]/[GSH]
2
) in which [GSH] and [GSSG] are molar concentrations
and E
0
is taken as −264 mV at pH 7.4.
CHAPTER RESULTS
a. H
2
O
2
metabolism in control and Pten
-/-
hepatocytes
H
2
O
2
generation showed that Pten
-/-
immortalized hepatocytes generate H
2
O
2
at a lower
rate (8.75 ± 0.63 pmol/min/1×10
6
cells) than hepatocytes from control (48.11 ± 2.17
pmol/min/1×10
6
cells) (Fig. 12A). Primary hepatocytes from 3-month old Pten
-/-
mice
liver show a lower H
2
O
2
generation rate (1.64 ± 0.02 nmol/mg/min) than control (3.36 ±
3.64 nmol/mg/min) (Fig.12C). Pten
-/-
hepatocytes have higher H
2
O
2
removal capacity
(immortalized hepatocytes: 3.64 ± 0.31 nmol/min/1×10
6
cells; primary hepatocytes from
3-month old mice: 6.69 ± 1.05 nmol/mg/min) than control (immortalized hepatocytes:
2.44 ± 0.26 nmol/min/1×10
6
cells; primary hepatocytes from 3 month old mice: 5.33 ±
0.24 nmol/mg/min) (Fig.12B,D) (Table 3).
54

Fig. 12. H
2
O
2
metabolism in control and Pten
-/-
hepatocytes
(A) H
2
O
2
production rate of immortalized hepatocytes. ( )control hepatocytes; ( )Pten
-/-

hepatocytes. (B) H
2
O
2
consumption rate of immortalized hepatocytes. ( )control
hepatocytes; ( )Pten
-/-
hepatocytes; (C) Measurement of H
2
O
2
production rate of primary
hepatocytes isolated from 3-month old mice. ( )control hepatocytes; ( )Pten
-/-

hepatocytes; (D) H
2
O
2
consumption rate of primary hepatocytes isolated from 3-month
old mice. ( )control hepatocytes; ( )Pten
-/-
hepatocytes. All experiments were performed
in quadruplicate. Other assay conditions as described in the Materials and Methods
section.
**
56
28
0
4
2
0
**
+ d[H
2
O
2
]/dt
(pmol / min /1×10
6
cells)
– d[H
2
O
2
]/dt
(nmol / min/ 1×10
6
cells )
+ d [H
2
O
2
]/dt
nmol / mg protein / min
5.0
2.5
0
8
4
0
- d [H
2
O
2
]/dt
nmol / mg protein / min
**
*
 Control
 Pten
–/–
A
C D
B
55

b. Steady-state levels of H
2
O
2
([H
2
O
2
]
ss
) in primary and immortalized hepatocytes
To determine whether PI3K/AKT activation affects cellular H
2
O
2
metabolism, [H
2
O
2
]
ss

was determined in primary and immortalized hepatocytes from control and Pten
-/-
mice.
[H
2
O
2
]
ss
in primary and immortalized hepatocytes from Pten
-/-
mice was lower
(immortalized hepatocytes: 0.91 ± 0.07 µM; primary hepatocytes from 3 month old mice:
0.57 ± 0.01 µM) than that from control (immortalized hepatocytes: 4.98 ± 0.22 µM;
primary hepatocytes from 3 month old mice: 1.16 ± 0.10 µM) mice (Fig.13) (Table 3).
[H
2
O
2
]
ss
of control and Pten
-/-
immortalized hepatocytes [H
2
O
2
]
ss
is determined by the
H
2
O
2
generation and degradation rate. The lower [H
2
O
2
]
ss
in Pten
-/-
hepatocytes is in
part due to the low mitochondrial generation rate. This is at variance with the higher
H
2
O
2
content in the Pten
-/-
liver tissues. It is possible that H
2
O
2
from other cells (i.e.
inflammatory cells) in the liver causes higher H
2
O
2
contents in the liver tissues.
56

Fig. 13. Steady state cellular H
2
O
2
concentration ([H
2
O
2
]
ss
) in control and Pten
-/-

hepatocytes
(A) Steady state cellular H
2
O
2
concentration ([H
2
O
2
]
ss
) of primary hepatocytes from 3-
month old mice. ( )control hepatocytes; ( )Pten
-/-
hepatocytes; (B) Steady state cellular
H
2
O
2
concentration ([H
2
O
2
]
ss
) of the immortalized hepatocytes. ( )control hepatocytes;
( )Pten
-/-
hepatocytes. All experiments were performed in quadruplicate. Other assay
conditions as described in the Materials and Methods section.
[H
2
O
2
]
ss
µM
**
 Control
 Pten
–/–
6
3
0
1.6
0.8
0
[H
2
O
2
]
ss
µM
**
Primary
Hepatocytes
Immortalized
Hepatocytes
A B
57

58

c. AKT activation up-regulates mitochondrial energy-transduction

One explanation for the lower rate of H
2
O
2
generation in the Pten
-/-
hepatocytes is that
AKT/PI3K activation yields higher number of bio-energetically competent mitochondria,
i.e., higher ATP turnover and, consequently, lower O
2
•-
/ H
2
O
2
production. Cellular
respiration of primary hepatocytes from 3-month-old control and Pten
-/-
liver was
measured by the XF 24 extracellular flux analyzer. Basal OCR was significantly higher in
the Pten
–/–
hepatocytes from 3 month old animals than in the controls (420.76 ± 21.74 and
360.88 ± 18.88 pmoles/min, respectively). Maximal respiratory capacity, measured after
the addition of FCCP, in the Pten
–/–
hepatocytes from 3-month old animals (620.35 ±
31.64 pmoles/min) remained significantly higher than that in the controls (420.50 ± 21.41
pmoles/min) (Fig.14A,B). Basal ECAR was higher in the Pten
–/–
hepatocytes from 3
month-old animals (40.48 ± 1.43 mpH/min) than in the controls (26.99 ± 1.99 mpH/min).
The higher anaerobic glycolysis level in Pten
–/–
hepatocytes indicates a higher substrate
supply from glycolysis (Fig.14C,D). The higher mitochondrial respiration in Pten
–/–

model correlates with a higher oxidation state of the mitochondrial electron transfer
chain complexes and, consequently, a lower generation of O
2
•-
and

H
2
O
2
.
59

Fig. 14. Bioenergetics of control- and Pten
-/-
primary hepatocytes from 3 month-old mice
(A) Oxygen consumption rates (OCR) of control- and Pten
-/-
hepatocytes from 3 month
mice (2 x 10
4
cells/ well). Subsequent additions: 1 µM oligomycin; 1 µM FCCP; 1 µM
rotenone. Four readings were made after the injection of each inhibitor. ( )control
hepatocytes; ( )Pten
-/-
hepatocytes. (B) Quantitative data of basal and maximal OCR of
control( ) and Pten
-/-
( ) hepatocytes (data from Fig. 14A). (C) Extracellular acidification
rate (ECAR) of control- and Pten
-/-
hepatocytes (2 x 10
4
cells/ well) (assay conditions as
in Fig. 14A). (D) Quantitative data of basal ECAR of control- and Pten
-/-
hepatocytes. All
experiments were performed in quadruplicate. Data in (A) and (C) are the average of 4
time-points from 4 different samples.
0
350
700
0
23
46
0 20 40 60 80 100
0
140
280
420
560
700
0 20 40 60 80 100
OCR (pmoles/min)
oligomycin FCCP rotenone
ECAR (mpH/min)
oligomycin FCCP rotenone
 Control
 Pten
–/–
OCR
ECAR
Time (min)
Time (min)
OCR (pmoles/min)
 Control
 Pten
–/–
**
30
15
0
ECAR
BASAL
(mpH/min)
**
Basal
Respiration
Maximal
Respiratory
Capacity
A
B
C
D
60

Consistent with the data from the cultured hepatocytes, mitochondria from Pten
-/-
liver
(3-month old animals) show an elevated respiratory control ratio (RCR) (energized with
glutamate: 2.01 ± 0.17; pyruvate: 1.88 ± 0.22; succinate: 6.03 ± 0.74) than that of control
(energized with glutamate: 1.64 ± 0.13; pyruvate: 1.60 ± 0.07; succinate: 4.69 ± 0.50)
(Fig. 15) (Table 4), which confirms that the AKT activation is associated with a higher
energy-transducing capacity of mitochondria in part attributed to the increased expression
of mitochondrial electron transport chain complexes (i.e., Complex I, II and V) (Fig. 16).
The production rate of O
2
.-
and H
2
O
2
is associated with the metabolic states. The high
level of metabolic rate and oxidation of electron transport chain members in the Pten
-/-

model are assumed to yield a reduced generation of the superoxide (O
2
.-
) and
subsequently H
2
O
2
(Cadenas and Davies 2000). Mitochondria isolated from Pten
-/-

hepatocytes show an increased expression of pyruvate dehydrogenase and aconitase (Fig.
17); the former increases the substrate supply to the TCA cycle and subsequently,
oxidative phosphorylation. The up-regulated substrate supply in Pten
-/-
model is also
associated with the higher glycolysis which provides pyruvate for mitochondria. Fig. 18
shows that compared with control, Pten
-/-
immortalized hepatocytes have a higher
expression of glycolytic enzymes, which is consistent with the observation of the
increased extracellular acidification rate (ECAR).
61

62

Fig. 15. AKT activation enhanced liver mitochondrial respiratory capacity
Mitochondria oxygen consumption was measured by a Clark-type electrode.
Mitochondria isolated from control and Pten
-/-
liver (3-month old animals) were
energized by adding glutamate (5mM), pyruvate (5mM) and succinate (5mM) as
substrates. State 4 respiration was recorded after adding the substrates. ADP (410 μM)
was added to stimulate the state 3 respiration. The respiratory control ratio (RCR) is
equal to state 3 (with ADP) oxygen consumption/min divided by the state 4 (without
ADP) oxygen consumption/min. ( )control hepatocytes; ( )Pten
-/-
hepatocytes.
2.50
1.25
0
Glutamate
RCR
**
2.40
1.20
0
Pyruvate
RCR
*
8.00
4.00
0
Succinate
RCR
*
 Control
 Pten
–/–
63

Fig. 16. AKT activation enhanced the expression of mitochondrial electron transport
chain
(A) Western blots of mitochondrial electron transport complexes in control- and Pten
-/-

immortalized hepatocytes. Mitochondria were isolated from control- and Pten
-/-

immortalized hepatocytes. 20 µg mitochondrial proteins were loaded in each well; (B)
Density analysis of western blots of Fig.16A. ( )control hepatocytes; ( )Pten
-/-

hepatocytes.
Complex V F1α
Complex III core2
Complex II Ip
Complex I NDUFB8
COX IV
___
Mitochondrial fraction
____
____
Control
___ ____
Pten
-/- ____
70
35
0
160
80
0
50
25
0
140
70
0
**
*
*
Complex V F1α
(Arbitrary units)
Complex III core2
Complex II Ip
(Arbitrary units)
Complex I NDUFB8
A
B
64

Fig. 17. AKT activation enhanced the expression of mitochondrial pyruvate
dehydrogenase and tricarboxylic acid (TCA) cycle enzymes
(A) Western blots of mitochondrial pyruvate dehydrogenase, aconitase, isocitrate
dehydrogenase-2 in control- and Pten
-/-
immortalized hepatocytes. Mitochondria were
isolated from control- and Pten
-/-
immortalized hepatocytes. 20 µg mitochondrial proteins
were loaded in each well; (B) Density analysis of western blots of Fig.17A. ( )control
hepatocytes; ( )Pten
-/-
hepatocytes.
A
B
Pyruvate dehydrogenase-E
1α
COX IV
Control
Pten
-/-
Aconitase
Isocitrate
Dehydrogenase 2
COX IV
Malate
Dehydrogenase
320
160
0
150
75
0
150
75
0
PDH-E
1α
(Arbitrary Units)
Aconitase
IDH-2
Mitochondria isolated from
immortalized hepatocytes
**
*
**
 Control
 Pten
–/–
65

Fig. 18. AKT activation enhanced the expression of glucose uptake transporters and
cellular glycolytic enzymes
(A) Western blots of glucose transporter 1/4, glyceraldehyde 3-phosphate dehydrogenase and
lactate dehydrogenase in control- and Pten
-/-
immortalized hepatocytes. 20 µg proteins were
loaded in each well; (B) Density analysis of western blots of Fig.18A. ( )control
hepatocytes; ( )Pten
-/-
hepatocytes.
β-Actin
Lactate Dehydrogenase
Glyceraldehyde 3-Phosphate
Dehydrogenase
GLUT4
GLUT1
Control Pten
-/-
250
125
0
350
175
0
500
250
0
300
150
0
**
**
**
**
GLUT4
(Arbitrary units)
GAPDH
(Arbitrary units)
GLUT1
LDH
 Control
 Pten
–/–
A
B
Cell lysate
66

d. The effects of AKT signaling on cellular redox status
To determine the cellular redox status was affected by the AKT signaling, cellular
NADPH/NADP
+
, NADH/NAD
+
, GSH/GSSG levels were measured by HPLC. Pten
-/-

immortalized hepatocytes have higher NADPH/NADP
+
and NADH/NAD
+
ratio
([NADPH]/[NADP
+
]: 0.59 ± 0.08; [NADH]/[NAD
+
]: 0.79 ± 0.04) than control
( [NADPH]/[NADP
+
]: 0.39 ± 0.02; [NADH]/[NAD
+
]: 0.37 ± 0.18) (Fig. 19) (Table 5).
The GSH level and GSH/GSSG ratio in Pten
-/-
immortalized hepatocytes also increased
([GSH]: 48.10 ± 3.07; [GSH]/[GSSG]: 205.73 ± 44.56) than control ([GSH]: 33.27 ±
3.28; [GSH]/[GSSG]: 65.38 ± 22.49). In primary hepatocytes isolated from 3- and 6-
month old Pten
-/-
mice, both NAD(P)H/ NAD(P)
+
and GSH/GSSG ratio are higher than
those of age-matched control primary hepatocytes (Table 5). The redox potential of
immortalized hepatocytes was calculated to be -363.35 mV (control) and -383.52 mV
(Pten
-/-
) respectively.
67

Fig. 19. Cellular redox status in in control and Pten
-/-
immortalized hepatocytes
(A) Measurement of cellular NADPH/NADP
+
ratio in control and Pten
-/-
immortalized
hepatocytes by HPLC. ( )control hepatocytes; ( )Pten
-/-
hepatocytes; (B) Measurement
of cellular NADH/NAD
+
ratio in control and Pten
-/-
immortalized hepatocytes by HPLC.
( )control hepatocytes; ( )Pten
-/-
hepatocytes; (C) Measurement of cellular GSH/GSSG
ratio in control and Pten
-/-
immortalized hepatocytes by HPLC. ( )control hepatocytes;
( )Pten
-/-
hepatocytes.
**
0.7
0.35
0
NADPH/NADP
+
**
0.9
0.45
0
NADH/NAD
+
**
300
150
0
GSH/GSSG
 Control
 Pten
–/–
A B C
68

69

As the only reducing source for glutathione and thioredoxin (Trx2) system, NADPH is
critical in the maintenance of mitochondrial redox status. Isocitrate dehydrogenase 2
(IDH-2) is one of the mitochondrial enzymes that generate NADPH. The higher level
expression of IDH-2 (Fig. 20) is consistent with the observation of the higher
NADPH/NADP
+
ratio and contributes to a more reduced redox status in Pten
-/-

hepatocytes. The cellular redox status is correlated with the mitochondrial and cellular
O
2
.–
and H
2
O
2
removal systems. To determine how AKT signaling affects free radical
removal systems, mitochondrial and cellular oxidants removal enzymes were measured
by western blots analysis. Pten
-/-
immortalized hepatocytes have a higher expression of
O
2
.–
and H
2
O
2
removal systems (i.e., Mn-superoxide dismutase and peroxiredoxin 3,
respectively). Both enzymes are compartmentalized in mitochondria (Fig. 21). Pten
-/-

immortalized hepatocytes show a higher expression of catalase, which is
compartmentalized in peroxisomes (Fig. 22). These data suggest that the lower H
2
O
2
production rate in Pten
-/-
hepatocytes may be accounted for 1) the increased oxidation
state of the electron transport chain members; 2) higher capacity of H
2
O
2
removal system
(Mn-superoxide dismutase and Peroxiredoxin 3 in mitochondria; catalase in peroxisome);
3) The modulation of the expression of mitochondrial isocitrate dehydrogenase-2 and
consequently the increased generation of the reducing equivalents NADPH, which is
critical for the mitochondrial H
2
O
2
removal system.
70

Fig. 20. AKT activation increased the expression of mitochondrial isocitrate
dehydrogenase-2 (one of the NADPH generating enzymes)
(A) Western blots of mitochondrial IDH-2 in control- and Pten
-/-
immortalized
hepatocytes. Mitochondria were isolated from control- and Pten
-/-
immortalized
hepatocytes. 20 µg mitochondrial proteins were loaded in each well; (B) Density analysis
of western blots of Fig.19A. ( )control hepatocytes; ( )Pten
-/-
hepatocytes.
160
80
0
IDH-2
(Arbitrary units)
**
B
Isocitrate
Dehydrogenase-2
COX IV
Mitochondria isolated from
immortalized hepatocytes
Control Pten
-/-
A
 Control
 Pten
–/–
71

Fig. 21. AKT activation enhanced the antioxidant capacity of liver mitochondria
(A) Western blots of mitochondrial Mn-superoxide dismutase and Peroxiredoxin 3 in
control- and Pten
-/-
immortalized hepatocytes. Mitochondria were isolated from control-
and Pten
-/-
immortalized hepatocytes. 20 µg mitochondrial proteins were loaded in each
well; (B) Density analysis of western blots of Fig. 21A. ( )control hepatocytes; ( )Pten
-/-

hepatocytes.
Mn-superoxide dismutase
Peroxiredoxin 3
COX IV
Control Pten
-/-
COX IV
Control Pten
-/-
Mitochondria isolated from
immortalized hepatocytes
1.2
0.6
0
Con Pten
-/-
0.7
0.35
0
Con Pten
-/-
Density Analysis
Arbitrary units
MnSOD
Density Analysis
Arbitrary units
Peroxiredoxin 3
* **
B A
 Control
 Pten
–/–
72

Fig. 22. AKT activation enhanced the expression of catalase
(A) Western blots of catalase in cell lysates from control- and Pten
-/-
hepatocytes.
Different amounts of proteins (10-, 30-, and 60 µg) were loaded in three sets of samples;
(B) Density analysis of western blots of Fig.22A. ( )control hepatocytes; ( )Pten
-/-

hepatocytes.
Catalase
Actin
_______________
Cell lysate (µg protein)
_______________
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
_____
10
_____ _____
30
_____ _____
60
_____
A
1.4
0.7
0
Density Analysis
Arbitrary units
Catalase
Con Pten
-/-
*
 Control
 Pten
–/–
B
73

e. The robust mitochondrial energy-transduction in Pten
-/-
primary hepatocytes is partly
PI3K dependent
Primary hepatocytes from 3-month old Pten
-/-
liver show significantly robust
insulin/PI3K signaling than age-matched control hepatocytes (Fig. 23). Maximal and
basal OCR of primary hepatocytes from 3 month wild type control mice were reduced by
15.38 ± 1.10 % and 12.50 ± 2.10 % after treatment with LY294002 (Fig. 24) indicating
the involvement of PI3K signaling in the mitochondrial energy transducing capacity and
consequently, H
2
O
2
generation. Treatment of control and Pten
-/-
hepatocytes by
LY294002 at the concentration of 2.5 µM yields a higher glycolysis which can be
considered as the compensation for the inhibited mitochondrial energy production. These
data demonstrate that higher OCR in Pten
-/-
primary hepatocytes is partly PI3K
dependent. LY294002 may also affect cellular redox status by directly affecting the
production of NADH through glycolysis.
74

Fig. 23. AKT and GSK3 phosphorylation status in primary hepatocytes from control- and
Pten
-/-
knockout mice
(A) Western blots of AKT and GSK3 and their phosphorylated forms in cell lysates from
control- and Pten
-/-
primary hepatocytes. Control- and Pten
-/-
primary hepatocytes were
isolated from 3-month old animals. 20μg cell lysate was loaded in each well ; (B) Density
analysis of western blots of Fig.22A. ( )control; ( )Pten
-/-
.
p AKT(Ser
473
)
p GSK3α(Ser
21
)
p GSK3β(Ser
9
)
AKT
GSK3β
β Actin
2.6
1.3
0
Density Analysis
Arbitrary units
pAKT(Ser
473
)
Con Pten
-/-
*
1.8
0.9
0
Density Analysis
Arbitrary units
pGSK3β(Ser
21
)
Con Pten
-/-
*
 Control
 Pten
–/– A 3 month
Control Pten
-/-
B
75

Fig. 24. Effect of PI3K inhibition cellular bioenergetics
(A) Effect of PI3K inhibitor on oxygen consumption rate (OCR) in primary hepatocytes
from 3 month wild type control mice. Basal OCR rate is measured 30min after the
addition of LY294002. ( )vehicle; ( )plus 2.5 µM LY294002. (B) Quantitation of basal
and maximal OCR in vehicle- and LY29002-treated samples. (C) Effect of LY294002 on
extracellular acidification rate (ECAR): assay conditions as in Fig.24A. All experiments
were performed in quadruplicates. (D) Quantitative data of basal ECAR in vehicle- and
LY29002-treated samples. All experiments were performed in quadruplicate. Data in (A)
and (C) are the average of 4 time-points from 4 different samples.
Basal
Respiration
Maximal
Respiratory
Capacity
76

f. The higher NADH/NAD
+
and NADPH/NADP
+
ratio in Pten
-/-
immortalized
hepatocytes are partially PI3K dependent
To further determine AKT signaling affects the H
2
O
2
metabolism and redox status by the
above mentioned aspects. The control and Pten
-/-
hepatocytes immortalized were treated
with LY294002 and the changes in the levels of NADH/NAD
+
and NADPH/NADP
+
ratio
were observed. The results show that LY294002 treatment decreased the ratio of
NADH/NAD
+
and NADPH/NADP
+
which confirmed that AKT signaling regulates the
cellular redox status (Fig. 25) (Table 6).
77

Fig. 25. Effects of LY294002 on cellular redox status

The control and Pten
-/-
immortalized hepatocytes were treated with LY294002 for 0, 30
and 60 min. NAD(P)H and NAD(P)
+
concentrations were analyzed using HPLC
electrochemical detection.

[NADPH]/[NADP
+
]

[NADH]/[NAD
+
]
Control 0.55 ± 0.17

0.42 ± 0.04
Control + LY 30min 0.60 ± 0.12

0.32 ± 0.08
Control + LY 60min 0.58 ± 0.24

0.36 ± 0.06

Pten
-/-
1.01 ± 0.57

0.91 ± 0.41
Pten
-/-
+ LY 30min 0.93 ± 0.28

0.34 ± 0.14
Pten
-/-
+ LY 60min 0.80 ± 0.28 0.40 ± 0.03

Table 6. LY294002 Treatment and Redox Component
1 2 3 1 2 3
 Control
 Pten
–/–
0
0.6
1.2
0
0.5
1.0
0 30 60 30
NADH/NAD
+
NADPH/NADP
+
0 60
Time (min)
78

CHAPTER DISCUSSION
Mitochondria are major sources of oxidants (O
2
-
and H
2
O
2
) and play a key role in the
regulation of cellular redox status. This study shows that cellular steady-state H
2
O
2

concentration ([H
2
O
2
]
ss
) and redox status are regulated by the PI3K/AKT pathway of
insulin signaling. Compared with control hepatocytes, AKT activation in Pten
-/-
model
yields a decreased cellular [H
2
O
2
]
ss
and consistently a more reduced cellular
environment as shown by the cellular redox potential. The mechanism of these changes
involves the PI3K/AKT regulation of 1) mitochondrial H2O2 generation; 2)
mitochondrial and cellular antioxidant systems; and 3) the level of the enzyme (IDH-2)
responsible for the supply of the reducing equivalents (NADPH) (Fig 26). Our previous
studies have shown that PI3K/AKT activation increases the mitochondrial energy
transduction and consequently a more oxidized status of electron transport chain, which
is assumed to be correlated with a decreased O
2
-
production rate. The AKT activation
also increased the expression of antioxidants systems enzymes (manganese superoxide
dismutase, peroxinredoxin 3 in the mitochondria and catalase in the peroxisome), which
also contributes to a lower [H
2
O
2
]
ss
. Up-regulation of the expression of IDH-2 by AKT
activation is correlated with a higher level of reducing equivalents (NADPH) which is
critical for the antioxidant systems and the maintenance of the reduced redox status. In
addition, the substrate supply level also plays an important role in the regulation of the
redox status. AKT activation increases the glycolysis level and enhances the substrate
availability. In mitochondria from Pten-/- hepatocytes, the up-regulated level of pyruvate
dehydrogenase and its increased active form (unphosphorelated at the Ser
293
) yields a
79

higher substrate catalysis capacity which is critical for the generation of the reducing
equivalents (Data not shown). Treatment of immortalized hepatocytes with LY294002-
an inhibitor of PI3K- results in the decreased mitochondrial energy-transduction and
NAD(P)H/NAD(P)
+
ratio. In the Pten
-/-
hepatocytes, the response of NAD(P)H/NAD(P)
+

ratio to LY294002 treatment is more dramatic compared with control hepatocytes
indicating Pten
-/-
hepatocytes is more sensitive to the PI3K inhibition. This is in variance
with the observation of LY294002 show a lower IC
50
on maximal oxygen consumption
rate in Pten
-/-
hepatocytes than in control hepatocytes (Data not shown). The explanations
for this is that LY294002 affect the NAD(P)H/NAD(P)
+
ratio mostly by the affecting
substrate availability probably through the regulation of the activity of pyruvate
dehydrogenase (30min treatment does not change the pyruvate dehydrogenase level
significantly which involves the transcriptional level of regulation). In sum, PI3K/AKT
pathway of insulin signaling modulates mitochondrial H
2
O
2
metabolism, cellular
[H
2
O
2
]
ss
and subsequently cellular redox status through diverse mechanisms.
80

Fig. 26. The mechanisms involved in the regulation of the cellular steady-state H
2
O
2

concentration ([H
2
O
2
]
ss
) by the AKT signaling
ETC e
Ο
2
.
−
O
2
H
2
O
2
H
2
O
MnSOD
H
2
O
Catalase
GPx
Prx
[
H
2
O
2
]ss
AKT
M itochond
rial Ener
gy
Transduction
H
2
O
2
removal s
y
stems
IDH-2
NADPH
Reducin
g
E
q
uivalent Pool
81

CHAPTER III
The mitochondrial bioenergetics and H
2
O
2
metabolism in control
and Pten
-/-
liver as function of age

CHAPTER INTRODUCTION
The process of aging is featured by the decrease in mitochondrial bioenergetics (Bulos,
Shukla et al. 1975; Wallace, Bohr et al. 1995; Nicholls 2002), which is associated with
redox alterations including increased generation of mitochondrial H
2
O
2
, decreased
GSH/GSSG ratio, and a more oxidized cellular environment (Yap, Garcia et al. 2009).
Instead of being solely toxic, oxidants have been found physiologically critical for the
signaling transduction, transcriptional regulation and redox status modulation (Finkel
2000; Cadenas 2004; Garcia, Han et al. 2007). Being the primary source of O
2
-
and
H
2
O
2
, mitochondria are essential in regulation of oxidative stress and redox status
(Boveris, Cadenas et al. 1976; Cadenas, Boveris et al. 1977; Han, Williams et al. 2001).
The redox stress hypothesis of aging illustrates that the oxidation of the redox status leads
to the malfunction of redox-sensitive proteins associated with functional loss during
aging (Finkel and Holbrook 2000; Schafer and Buettner 2001; Droge 2002; Maher 2005;
Jones 2006). Based on this theory, the maintenance of a reduced redox environment is
critical in preventing the functional loss of proteins during aging. The up-regulation of
the mitochondrial energy-transducing capacity may antagonize the generation of oxidants
and prevent the pro-oxidation shift of the cellular redox status. The activation of
PI3K/AKT pathway of insulin signaling up-regulates mitochondrial bioenergetics and
82

decreases the mitochondrial H
2
O
2
generation. It is interesting to determine whether AKT
activation can antagonize the redox stress associated with oxidative damage during aging
by decreasing oxidants generation and increasing the antioxidant capacity. This study
examines the role of the PI3K/AKT pathway on mitochondrial bioenergetics and H
2
O
2

metabolism in liver as a function of age using a liver specific Pten
-/-
mice model that
exhibits a robust insulin signaling. Liver-specific Pten
-/-
mice

is a strong model that
facilitates the study of the effects insulin signaling on mitochondrial bioenergetics and
H
2
O
2
metabolism with aging.
MATERIALS AND METHODS
Animals- Pten
loxp/loxp
mice were bred with Alb-Cre mice to generate Pten liver specific
deletion mice (Postic and Magnuson 2000; Lesche, Groszer et al. 2002). Animals of 3-,
6-, 9-, and 12 month age were used for experiments.
Chemicals – Oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
(FCCP) were purchased from MP Medicals. Phospho-AKT (Ser
473
), AKT, phospho-
GSK3α/β (Ser
21/9
), GSK3α/β, phospho-AMPKα (Thr
172
), AMPKα, phospho-ACC (Ser
79
)
and ACC1 antibodies were purchased from Cell Signaling. Sodium pyruvate, D-(+)-
Glucose and protease / phosphatase inhibitor cocktail were from Sigma. Dulbecco's
Modified Eagle Medium (DMEM), Dulbecco's Phosphate Buffered Saline (DPBS) and
trypsin were purchased from Cellgro.
Tissue culture- Primary hepatocytes were isolated by collagenase perfusion (Miyazaki
1977). The primary hepatocytes were seeded on XF-24 plates at a density of 1 × 10
4
cells
83

/ well in 250μl DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin,
0.1 μg/ml insulin, 10 ng/ml EGF in a humidified 5% CO
2
atmosphere for 24 hours
followed by the extracellular flux analysis.
XF24 extracellular metabolic flux analysis – Control and Pten
-/-
hepatocytes were seeded
on XF24 plates at a density of 1 × 10
4
cells / well. The plate was incubated in a
humidified 5% CO
2
atmosphere for 24 h. Unbuffered DMEM (DMEM base medium
supplemented with 25mM glucose, 1mM sodium pyruvate, 31mM NaCl; PH7.4) was
used for the metabolic flux analysis. Four basal measurements of oxygen consumption
rate (OCR) and extracellular acidification rate (ECAR) were made followed by the
addition of mitochondrial inhibitors. Four readings were made after the injection of each
inhibitor. Three mitochondrial inhibitors were used: oligomycin (1 μM) was added to
inhibit the ATP synthase followed by the measurement of ATP turn over, FCCP (1 μM)
was added to uncouple the mitochondria followed by the measurement of the maximal
mitochondrial respiratory capacity, and rotenone (1 μM) was added to inhibit the
complex I followed by the measurement of non-mitochondrial respiration. For fatty acid
oxidation assay, oxygen consumption rate (OCR) is measured in the low-buffered Krebs-
Henseleit Buffer (KHB) buffer (110 mM NaCl, 4.7 mM KCl, 2 mM MgSO
4
1.2 mM
Na
2
HPO
4
, 2.5 mM glucose, 0.5mM carnitine, 100nM insulin, pH7.4) with the sodium
palmitate (2 mM) / BSA (0.34 mM, 2.267 g/dL) conjugate as the substrate.
Mitochondrial inhibitors: oligomycin (1 μM), FCCP (1 μM) and rotenone (1 μM) were
used in fatty acid oxidation assay as described previously.
84

Isolation of mitochondrial fraction- Liver mitochondria were isolated from 3-, 6-, 9- and
12-month old control and Pten
-/-
mice liver by differential centrifugation followed by
Percoll discontinuous gradient purification (Garcia, Han et al. 2010). Liver tissues were
homogenized at 4
o
C in mitochondrial isolation buffer (MIB, containing 230 mM sucrose,
1 mM EDTA, 10 mM Tris-HCl, 0.5 mM EGTA, 0.1% (w/v) fat-free bovine serum
albumin and protease/phosphatase inhibitor Cocktail, PH 7.4). Liver homogenates were
centrifuged at 1500g for 5 minutes. The supernatant containing mitochondria was
centrifuged at 21,000 g for 10 minutes. Crude mitochondrial pellet was then re-suspended
in 15% Percoll and topped on to a pre-formed Percoll gradient. The purified
mitochondrial pellet was washed twice, suspended in MIB and used within 4 hours.
Mitochondrial respiration- Mitochondria oxygen consumption was measured by a Clark-
type electrode. 200 ug mitochondrial proteins were re-suspended in mitochondrial
respiratory buffer (130 mM KCl, 2 mM KH
2
PO
4
, 3 mM HEPES, 2 mM MgCl
2
, 1 mM
EGTA, 1% (w/v) bovine album) yielding the concentration of 200 ug/mL. 1 min baseline
oxygen consumption was recorded followed by adding pyruvate (5 mM), glutamate (5
mM) and succinates (5 mM) as substrates respectively. State 4 oxygen consumption was
recorded after adding the substrates (pyruvate, glutamate, or succinate). State 3
respiration is recorded after adding ADP (410 μM). The oxygen consumption was
calculated according to the response of slope after adding the substrates and ADP. The
respiratory control ratio (RCR) is equal to the state 3 (with ADP) oxygen
consumption/min divided by the state 4 (without ADP) oxygen consumption/min.
85

Western blot analysis – Protein was extracted from liver tissues by RIPA buffer (50 mM
Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, phosphatase
inhibitor and protease inhibitor cocktail). The antibodies used for immunobloting are
anti-pAKT, anti-AKT, anti-pGSK3, anti-GSK3, anti-pAMPK, anti-AMPK, anti-pACC
and anti-ACC1 from Cell Signaling. Band intensities were analyzed by the VersaDoc
system (Bio-Rad).
Hydrogen Peroxide Production- The hydrogen peroxide production from isolated
mitochondria was measured by the AmplexRed Hydrogen Peroxide/Peroxidase Assay kit
(Invitrogen) according to the manufacturer's instructions.
Statistical analysis – Statistical analysis was performed using student’s t test for unpaired
data or ANOVA. A P value <0.05 was considered significant.
CHAPTER RESULTS
a AKT activation in Pten
-/-
liver- To study how insulin signaling affects mitochondrial
bioenergetics during aging, we used a liver-specific Pten knockout (Pten
-/-
) model which
exhibits a robust insulin signaling. AKT signaling was determined in wild type and Pten
-/-

liver as a function of age (3-, 6-, 9-, and 12-months old mice). Phosphorylation of AKT
(Ser
473
) is higher in Pten
-/-
liver than wild type control at all ages. Phosphorylation of
AKT reached the highest level in both wild type control and Pten
-/-
liver at 6-month age
with Pten
-/-
liver showing an even stronger AKT phosphorylation level. After 6 month,
the activation of AKT decreased in both wild type and Pten
-/-
liver. With aging, a
decreased AKT protein expression level was observed in wild type control and Pten
-/-

86

liver. At the age of 6 month, Pten
-/-
liver had the highest AKT expression compared with
other ages. Active AKT phosphorylates GSK3β at Ser
9
and inhibits its activity. GSK3β is
proposed to inhibit mitochondrial bioenergetics by direct phosphorylation of
mitochondrial pyruvate dehydrogenase thus leading to its inactivation; higher
phosphorylation of AKT is assumed to increase mitochondrial bioenergetics in part
through inhibition of GSK3β and subsequently releasing the inhibitory effects of the
latter on mitochondrial bioenergetics. At 6- and 9- month age, Pten
-/-
liver shows a higher
pGSK3β(Ser
9
) level which may contribute to the higher mitochondrial bioenergetics.
However, at 3- and 12- month age, Pten
-/-
liver shows a lower pGSK3β(Ser
9
) level. This
may suggest other signaling pathways also regulate GSK3β. At 9- and 12- month age,
both control and Pten
-/-
liver showed a decreased expression of GSK3β compared with 3-
and 6- month age (Fig.27, 28). At the 6 month age, control and Pten
-/-
liver show the
highest level of insulin signaling.
87

Fig. 27. AKT status in control and Pten
-/-
liver as function of age
(A) Western blot of the p-AKT (Ser
473
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice; (B) Density analysis
of western blot of the p-AKT (Ser
473
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice. ( )control; ( )Pten
-/-
.
0
65
130
 Control
 Pten
–/–
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
p-AKT (Ser
473
)
β-Actin
_____
3 month
_____ _____
6 month
____ _____
9 month
____ ____
12 month
_____
AKT
β-Actin
Density
(Arbitrary units)
3 6 9 12
AKT
Density
(Arbitrary units)
**
**
3 6 9 12
_____
Age (Month)
_____
p-AKT (Ser
473
)
0
1
2
0
0.9
1.8
p-AKT (Ser
473
) / AKT
3 6 9 12
**
**
**
_____
Age (Month)
_____
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
p-AKT (Ser
473
)
β-Actin
AKT
β-Actin
_________
12
_________ ________
9
_________ ________
6
_________ ________
3
_________
_________________________________
Age (Months)
______________________________________
A
B
0
1
2
p-AKT (Ser
473
) / AKT
(Arbitrary units)
___________
Age (months)
___________
88

Fig. 28. GSK3β status in control and Pten
-/-
liver as function of age
(A) Western blot of the p-GSK3β (Ser
9
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice; (B) Density analysis
of western blot of the p-GSK3β (Ser
9
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice. ( )control; ( )Pten
-/-
.
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
GSK3β
β-Actin
_____
3 month
_____ _____
6 month
____ _____
9 month
____ ____
12 month
_____
β-Actin
p-GSK3β (Ser
9
)
_____
Age (Month)
_____
0
1.4
2.8
Density
(Arbitrary units)
3 6 9 12
GSK3β
0
60
120
Density
(Arbitrary units)
**
3 6 9 12
p-GSK3β (Ser
9
)
 Control
 Pten
–/–
0
0.7
1.4
p-GSK3β (Ser
9
) / GSK3β
3 6 9 12
**
**
_____
Age (Month)
_____
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
GSK3β
β-Actin
β-Actin
p-GSK3β (Ser
9
)
_________
12
_________ ________
9
_________ ________
6
_________ ________
3
_________
_________________________________
Age (Months)
______________________________________
0
0.7
1.4
3 6 9 12
**
**
p-GSK3β (Ser
9
) / GSK3β
(Arbitrary units)
___________
Age (months)
___________
A
B
89

b AKT activation is associated with higher mitochondrial bioenergetics in Pten
-/-
liver as
function of age- To determine how insulin signaling affects mitochondrial bioenergetics,
mitochondrial respiration was measured in isolated mitochondria and primary
hepatocytes from wild type and Pten
-/-
liver. Consistently with the AKT phosphorylation
level, mitochondria isolated from Pten
-/-
liver showed a higher respiratory control ratio
than control at all ages (Fig. 29). In both control and Pten
-/-
liver, mitochondrial
respiration reached the highest at 6 month age followed by a decrease through aging.
Primary hepatocytes isolated from 3-, 6-, 9-, and 12-months Pten
-/-
liver showed a higher
mitochondrial respiration than control as shown by the higher oxygen consumption rate
(OCR). At 6 month age, control and Pten
-/-
hepatocytes show the highest mitochondrial
respiration (Fig. 30). The data above indicate mitochondrial bioenergetics is associated
with insulin signaling in wild type control and Pten
-/-
liver during aging.
90

Fig. 29. Liver mitochondrial respiratory ratio (RCR) as function of age
Mitochondria oxygen consumption was measured by a Clark-type electrode.
Mitochondria isolated from control and Pten
-/-
liver (3-, 6-, 9- and 12-month old animals)
were energized by adding succinate (5 mM). State 4 oxygen consumption was recorded
after adding the succinate (5mM). ADP (410 μM) was added to stimulate the state 3
respiration. The respiratory control ratio (RCR) is calculated by dividing the state 3 (with
ADP) oxygen consumption/min with the state 4 (without ADP) oxygen consumption/min.
( )liver mitochondria from control mice; ( )mitochondria from Pten
-/-
liver.
0.0
6.5
13.0
RCR
Mitochondrial Respiratory Control Ratio
*
**
**
6 9
 Control
 Pten
–/–
12 3
_____
Age (month)
_____
91

Fig. 30. Primary hepatocytes basal and maximal oxygen consumption rate (OCR) as
function of age
Primary hepatocytes were cultured from control and Pten
-/-
liver (3-, 6-, 9- and 12-month
old animals. Basal and maximal oxygen consumption rates (OCR) were measured by the
XF-24 extracellular flux analyzer. (A) Basal oxygen consumption rates (OCR) of control-
and Pten
-/-
primary hepatocytes as function of age. (B) Maximal OCR of control- and
Pten
-/-
primary hepatocytes as function of age. ( )control hepatocytes; ( )Pten
-/-

hepatocytes.
0
500
1000
6 9 3 12
Basal
_____
Age (month)
_____
**
**
*
*
750
375
0
Basal OCR (pmoles/min)
0
700
1400
6 9 3 12
Maximal
**
**
*
*
0
1050
525
Maximal OCR (pmoles/min)
_____
Age (month)
_____
A
B
92

c Anaerobic glycolysis in Pten
-/-
liver as function of age- Another component of cellular
bioenergetics is glycolysis which is also associated with insulin signaling. Glycolysis
provides substrates for mitochondrial respiration and is critical for the mitochondrial
energy transduction. It may be surmised that the PI3K/AKT pathway affects
mitochondrial bioenergetics in part through regulating substrate supply for mitochondrial
respiration. The effects of PI3K/AKT signaling on glycolysis were determined in
hepatocytes from control and Pten
-/-
mice

as a function of age. Compared with control,
Pten
-/-
hepatocytes show a significantly higher glycolysis (ECAR). Hepatocytes from
wild type control and Pten
-/-
show the highest glycolysis at 6- and 9-month age,
respectively. The highest glycolysis at 9 month age in Pten
-/-
hepatocytes coincide with
the start of tumorigenesis because Pten
-/-
liver develops liver tumor after 9 month age
(Fig. 31). Therefore, the higher insulin signaling in Pten
-/-
liver yields the higher
glycolysis which may in part contribute to the high mitochondrial energy transducing by
increase the substrate supply for mitochondria.
93

Fig. 31. Robust anaerobic glycolysis in Pten
-/-
liver
Extracellular acidification rates (ECAR) of control- and Pten
-/-
primary hepatocytes as
function of age. Primary hepatocytes were cultured from control and Pten
-/-
liver (3-, 6-,
9- and 12-month old animals. ECAR was measured by the XF-24 extracellular flux
analyzer. ( )control hepatocytes; ( )Pten
-/-
hepatocytes.
Control and Pten
-/-
Primary Hepatocytes
Extracellular Acidification Rate (ECAR)
0
35
70
**
**
*
*
6 9 3 12
0
52
26
ECAR (mpH/min)
 Control
 Pten
–/–
_____
Age (month)
_____
94

d Liver mitochondrial state 4 H
2
O
2
generation as function of age- Mitochondria from
control mice liver show an age-dependent increase in H
2
O
2
generation during state 4
respiration. Compared with control, Pten
-/-
liver mitochodnria did not show a dramatic
increase of H
2
O
2
generation with aging. Mitochondria from Pten
-/-
liver show a lower
H
2
O
2
generation at all ages compared with control and at 9- and 12- month age, Pten
-/-

liver mitochondria show a significantly lower H
2
O
2
generation during state 4 respiration
(Fig. 32A). There was observed an age-dependent increase of control and Pten
-/-

mitochondrial H2O2 generation rate ratio (+d[H
2
O
2
]/dt
control
/+d [H
2
O
2
]/dt
pten null
) and
age-dependent decrease of control and Pten
-/-
mitochondrial Respiratory Control Ratio
(RCR
control
/RCR
pten null
) (Fig. 32B), thus supporting the notion that the generation of
H
2
O
2
is reversely correlated with the mitochondrial energy-transduction and in contrast
with Pten null model, control mitochondria show a relatively higher increase of H
2
O
2

production and decrease in mitochondrial bioenergetics as function of age.
95

Fig. 32. H
2
O
2
metabolism of control and Pten
-/-
liver mitochondria as function of age
(A) Measurement of H
2
O
2
production rate of mitochondria isolated from control- and
Pten
-/-
liver as function of age. Mitochondrial state 4 H
2
O
2
generation was measured by
the AmplexRed Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen). Mitochondria
isolated from control and Pten
-/-
liver (3-, 6-, 9- and 12-month old animals) were
energized by adding succinate (5 mM) as substrates. State 4 H
2
O
2
generation was
recorded polarographically after adding the succinate (5 mM). ( )liver mitochondria from
control mice; ( )mitochondria from Pten
-/-
liver; (B) Calculation of the Respiratory
Control Ratio (RCR) and H
2
O
2
generation rate ratio of control- and Pten
-/-
liver
mitochondria as function of age. All experiments were performed in quadruplicate. Other
assay conditions as described in the Materials and Methods section.
0
35
70
3 6 9 12
+d [H
2
O
2
]/dt
pmol / mg protein / min
**
**
**
H
2
O
2
State 4 generation
 Control
 Pten
–/–
+d [H
2
O
2
]/dt
control
/ +d [H
2
O
2
]/dt
pten null
RCR
control
/ RCR
pten null
0
1.25
2.50
2.50
1.25
0
6 9 3 12
_____
Age (month)
_____
A
B
96

e Fatty acid metabolism in control and Pten
-/-
liver- Livers of Pten
-/-
mice were observed
to have increased lipid accumulation (Kurlawalla-Martinez, Stiles et al. 2005). The
expression of acetyl-CoA carboxylase 1 (ACC1), the rate limiting enzyme in the process
of fatty acid synthesis (Tong 2005) was higher in Pten
-/-
liver than control (Fig. 33). ACC
is regulated by AMP-activated protein kinase (AMPK) (Park, Gammon et al. 2002).
AMPK phosphorylates ACC1 on Ser
79
, Ser
1200
, Ser
1215
, and phosphorylates ACC2 on
Ser
218
leading to their inactivation (Hardie 1992). AMPK is also regulated by its
phosphorylation status. When it is phosphorylated on Thr
172
, it is active (Hawley,
Davison et al. 1996; Stein, Woods et al. 2000). In Pten
-/-
liver, the phosphorylation level
of AMPK on Thr
172
is lower (Fig. 34), indicating the inhibitory effects of AMPK on
ACC1 is lower in Pten
-/-
liver. Therefore, the higher lipid accumulation in Pten
-/-
mice
liver compared with control is partially attributed to the higher expression of ACC1 and
its lower inactivation by AMPK.
To further determine the effect of AKT activation on lipid accumulation, fatty acid
oxidation level of the control and Pten
-/-
immortalized hepatocytes was measured by XF-
24 extracellular flux analyzer using palmitate as the substrate. Data show that there were
no significant differences in fatty acid β-oxidation between control and Pten
-/-

immortalized hepatocytes (Fig. 35). These observations indicate that the higher lipid
accumulation in Pten
-/-
liver was attributed to the higher lipid synthesis instead of a lower
β-oxidation rate. In addition, the accumulation of lipid in Pten
-/-
liver is facilitated by the
robust energy supply.
97

Fig. 33. Acetyl-CoA carboxylase 1 (ACC1) expression and phosphorylation status in
control and Pten
-/-
mice liver
(A) Western blot of the p-ACC1 (Ser
79
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice; (B) Density analysis
of western blot of the p-ACC1 (Ser
79
) and its non-phosphorylated form in tissue lysates
from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice. ( )control; ( )Pten
-/-
.
0
100
200
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
p-ACC1 (Ser
79
)
ACC1
β-Actin
 Control
 Pten
–/–
p-ACC1 (Ser
79
)
(Arbitrary units)
ACC1
(Arbitrary units)
0
125
250
**
**
**
**
3 6 9 12 3 6 9 12
p-ACC1 (Ser
79
) ACC1
**
_______________________________________
Age (months)
_______________________________________
_________
12
_________ ________
9
_________ ________
6
_________ _________
3
_________
______________________________________
Age (Months)
__________________________________
A
B
98

Fig. 34. AMP-activated protein kinase (AMPK) expression and phosphorylation status in
control and Pten
-/-
mice liver
(A) Western blot of the p-AMPK (Thr
172
) and its non-phosphorylated form in tissue
lysates from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice; (B) Density
analysis of western blot of the p-AMPK (Thr
172
) and its non-phosphorylated form in
tissue lysates from control- and Pten
-/-
liver of 3-, 6-, 9- and 12- month age mice.
( )control; ( )Pten
-/-
.
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
Control Pten
-/-
p-AMPKα (Thr
172
)
AMPK
β-Actin
 Control
 Pten
–/–
p-AMPKα (Thr
172
)
(Arbitrary units)
AMPK
(Arbitrary units)
0
125
250
0
100
200
**
**
*
3 6 9 12 3 6 9 12
p-AMPKα (Thr
172
) AMPK
_______________________________________
Age (months)
_______________________________________
_________
12
_________ ________
9
_________ ________
6
_________ _________
3
_________
______________________________________
Age (Months)
__________________________________
A
B
99

Fig. 35. Fatty acid oxidation in control and Pten
-/-
mice liver
Measurement of OCR in immortalized control- and Pten
-/-
hepatocytes with sodium
palpitate as the substrate. Oxygen consumption rates (OCR) of control- and Pten
-/-

immortalized

hepatocytes (2 x 10
4
cells/ well) were measured in the low-buffered KHB
buffer. Three basal oxygen consumption readings were made. Subsequent additions: 1
mM Sodium Palmitate/0.17 mM BSA Solution; 1 µM oligomycin; 1 µM FCCP; 1 µM
rotenone. Three readings were made after the injection of each inhibitor. ( )control
hepatocytes; ( )Pten
-/-
hepatocytes.
Palmitate Oligomycin FCCP Rotenone
100

CHAPTER DISCUSSION
The redox hypothesis of aging indicates that the pro-oxidizing shift of the cellular redox-
status and subsequent post-translational modification of the redox-sensitive proteins
contribute to the cellular functional loss associated with aging (Sohal and Orr 2012).
According to this theory, the key to inhibit aging and its related dysfunctions is to
maintain the reduced level of redox status and thus inhibits the malfunction of redox-
sensitive proteins due to the post-translational modifications (i.e., disulfide bond
formation, glutathionylation). Our previous work has shown that AKT activation
modulate cellular steady-state H
2
O
2
concentration ([H
2
O
2
]ss) and redox status through
diverse mechanisms. Therefore, activation of AKT pathway of insulin signaling may
affect the process of aging through regulation of mitochondrial H
2
O
2
metabolism and
cellular redox status. This study shows that in Pten
-/-
model, the age-dependent changes
in the activation level of AKT are correlated with the mitochondrial energy-transduction
capacity (mechanisms involving that the AKT activation releases the inhibitory effects of
GSK3β on mitochondrial pyruvate dehydrogenase and increases its activity). Consistent
with the notion that mitochondrial metabolic state determines the generation rate of H
2
O
2
,
the age-dependent decrease in control and Pten
-/-
liver mitochondrial Respiratory Control
Ratio (RCR
control
/RCR
pten null
) is associated with the age-dependent increase in
mitochondrial H
2
O
2
generation rate ratio (+d[H
2
O
2
]/dt
control
/+d [H
2
O
2
]/dt
pten null
). As
H
2
O
2
modulates the cellular redox status which plays a key role in the process of aging, it
is assumed that AKT signaling may modulate aging process through modulation of
mitochondrial H
2
O
2
production and subsequently the cellular redox status. The fatty acid
101

metabolism in control and Pten
-/-
liver was observed as a function of age. The data show
that at 3- and 6-month age, a key regulator of the fatty acid synthesis (Acetyl-CoA
carboxylase, ACC1) has the highest expression level which is consistent with the fatty
liver phenotype. After 9-month age, the ACC1 expression decreases which may be a
feedback reaction or due to the pathogenesis of cancer. The fatty acid β-oxidation was
found un-changed in control and Pten
-/-
immortalized hepatocytes. Fatty acid
accumulation may decrease insulin signaling and further confound the situation. Taken
together, AKT activation may modulate the aging process through regulating cellular
redox status. The lipid accumulation, however, may antagonize the insulin signaling.
102

Fig. 36. AKT signaling, mitochondrial bioenergetics and fatty acid biosynthesis
ACC
AMPK
Acet
y
l-CoA
Malno
y
l-CoA
Fatt
y
acid
ATP + HCO
3
-
ADP + Pi
Ener
gy,
ATP
Redox Si
g
nal
H
2
O
2
Substrate
su
pp
l
y
Citrate
M itochondria
103

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Abstract (if available)

Abstract The concept of the mitochondrial energy-redox axis integrates the mitochondrial energy-transduction and redox status as a concerted process with the two components inter-linked by the reducing equivalents (i.e., NAD(P)⁺/NAD(P)H). Decrease of mitochondrial energy transduction and pro-oxidant shift of cellular redox status precede the pathological changes of several diseases (i.e., diabetes) and are key features of aging. Mitochondria are also recipients of cellular signaling regulations such as MAPKs and PI3K/AKT pathway of insulin signaling. These studies are aimed at assessing the effect of the PI3K/AKT signaling pathway in the mitochondrial energy-redox axis and depicting the molecular mechanisms inherent in the effect. A liver-specific Pten deletion model that shows a robust insulin signaling was used to study how the PI3K/AKT pathways affect the mitochondrial energy-redox axis. The hypothesis to be tested is that liver-specific Pten deletion up-regulates mitochondrial bioenergetics through modulation of PI3K/AKT signaling pathways, which further affect mitochondrial and cellular H₂O₂ homeostasis, redox status, and the intrinsic apoptotic pathway. ❧ These studies revealed that mitochondrial bioenergetics is regulated by PI3K/AKT signaling through three mechanisms: 1) AKT increases glycolysis and thus, a higher substrate (pyruvate) supply to mitochondria

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Asset Metadata

Creator Li, Chen (author)

Core Title PI3K/AKT signaling and the regulation of the mitochondrial energy-redox axis

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 05/02/2012

Defense Date 03/26/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag aging,AKT,ATP synthase,bioenergetics,GSK3β,hepatocytes,mitochondrial respiration,OAI-PMH Harvest,oxidative stress,PI3K,PTEN,pyruvate dehydrogenase,redox status

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Cadenas, Enrique (committee chair), Kaplowitz, Neil (committee member), Stiles, Bangyan L. (committee member)

Creator Email chenli1062011@gmail.com,cli2@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-23637

Unique identifier UC11290056

Identifier usctheses-c3-23637 (legacy record id)

Legacy Identifier etd-LiChen-718.pdf

Dmrecord 23637

Document Type Dissertation

Rights Li, Chen

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA