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Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates fatty acid beta-oxidation
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Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates fatty acid beta-oxidation
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Content
Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN)
Signaling Regulates Fatty Acid Beta-oxidation
by
Jingyu Chen
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2015
i
Dedication
To
my family and friends
for their unconditional love and support
ii
Acknowledgements
First of all, I want to thank my advisor, Dr. Bangyan Stiles, for her constant guidance
during my study at USC School of Pharmacy. It is she who helps me to develop the
ability of critical thinking and independently conducting my research. I am very grateful
for the opportunities that she gave me. The passion for science and the dedication to work
I learned from her would benefit me for a lifelong time.
My sincere thanks also go to my committee members, Dr. Curtis Okamoto and Dr.
Enrique Cadenas, for their help in revising this thesis.
Their support and suggestions
allowed me to complete my master studies at USC smoothly.
In addition, I would like to thank all the members in Dr. Stiles lab, especially Dr. Lina He,
Dr. Ni Zeng, Dr. Yang Li, Dr. Ankeste Debebe, Dr. Indra Mahajan, Dr. Chengyou Jia,
Zhechu Peng, Richa Aggarwal, Chien-Yu Chen, Yating Guo, Fan Fei, Mengchen Li.
Especially, I want to thank Dr. Yang Li who taught me a lot when I first joined the lab
and helped me get started with my own research.
Finally, my sincere thanks also go to my parents. Without their unconditional love and
constant support, I would never have made it so far.
iii
Table of Contents
Dedication ........................................................................................................................... i
Acknowledgements ........................................................................................................... ii
List of Tables ..................................................................................................................... v
List of Figures ................................................................................................................... vi
Abstract ............................................................................................................................ vii
Chapter 1 Introduction ..................................................................................................... 1
1.1 Introduction of PTEN ........................................................................................... 1
1.1.1 PTEN antagonizes PI3K/AKT signaling pathway ..................................... 1
1.1.2 The role of PTEN in non-alcoholic fatty liver disease .............................. 5
1.1.2.1 Introduction of non-alcoholic fatty liver disease ............................ 5
1.1.2.2 PTEN alterations and loss of function in non-alcoholic fatty
liver disease ................................................................................................ 8
1.2 Fatty Acid β-oxidation .......................................................................................... 8
1.3 Uncoupling of Mitochondria by Fatty Acids ...................................................... 12
1.4 The Production of Reactive Oxygen Species in Mitochondria ........................... 12
1.5 Rationale of the Study ......................................................................................... 13
Chapter 2 Materials and Methods ................................................................................. 17
Chapter 3 PTEN Loss in Immortalized Hepatocytes Leads to Increased Fatty
Acid β-oxidation .............................................................................................................. 25
3.1 Introduction and Rationale .................................................................................. 25
3.2 Results ................................................................................................................. 29
3.2.1 Mitochondrial respiration is elevated during starvation in Pten-null
hepatocytes ........................................................................................................ 29
3.2.2 PTEN regulates exogenous fatty acid β-oxidation and the
uncoupling of mitochondria caused by fatty acids ........................................... 32
3.2.3 PTEN regulates the β-oxidation of endogenous fatty acids ..................... 38
iv
Chapter 4 Increased Fatty Acid Oxidation in Pten-null Immortalized
Hepatocytes Leads to Increased Cellular Reactive Oxygen Species (ROS)
Production ....................................................................................................................... 43
4.1 Introduction and Rationale .................................................................................. 43
4.2 Results ................................................................................................................. 47
4.2.1 Increased exogenous fatty acid β-oxidation and increased fatty acid
uncoupling of mitochondria upon PTEN loss aggravate ROS production
in immortalized hepatocytes ............................................................................. 48
4.2.2 Inhibition of endogenous fatty acid β-oxidation by etomoxir
elevates ROS level in Pten-deficient immortalized hepatocytes ...................... 52
Chapter 5 Molecular Mechanisms Underlying Increased Fatty Acid
Oxidation in Pten-null Immortalized Hepatocytes ...................................................... 55
5.1 Introduction and Rationale .................................................................................. 55
5.2 Results ................................................................................................................. 56
Chapter 6 Overall Discussion ........................................................................................ 60
Bibliography .................................................................................................................... 66
v
List of Tables
Table 1. XF-24 analyzer run protocol.
Table 2. Primer
sequences
for
qPCR.
Table 3. XF FAO Assay parameters and their corresponding calculations.
vi
List of Figures
Figure 1. Signaling Pathways Controlled by PTEN.
Figure 2. Diagram of Fatty Acid Transport and Fatty Acid β-oxidation.
Figure 3. Pten deletion leads to increased mitochondrial respiration in both fed and
starving state.
Figure 4. Increased exogenous fatty acid oxidation and mitochondrial uncoupling in
Pten-deficient immortalized hepatocytes.
Figure 5. Glycolytic capacity is increased along with elevated mitochondrial respiration
in Pten-deficient immortalized hepatocytes.
Figure 6. Increased endogenous fatty acid oxidation in Pten-deficient immortalized
hepatocytes.
Figure 7. Glycolytic capacity is slightly decreased with suppressed mitochondrial
respiration in Pten-deficient immortalized hepatocytes.
Figure 8. Reactive oxygen species (ROS) production in the cells.
Figure 9. Reactive oxygen species (ROS) level is up-regulated by increased exogenous
fatty acid oxidation and fatty acid uncoupling in Pten-null immortalized hepatocytes.
Figure 10. Reactive oxygen species (ROS) level is up-regulated by etomoxir (ETO) in
Pten-null immortalized hepatocytes.
Figure 11. mRNA expression levels of several representative genes involved in fatty acid
oxidation and lipogenesis in Pten wild-type and Pten-null immortalized hepatocytes.
vii
Abstract
PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) is a
dual-specificity phosphatase that can antagonize PI3K/AKT signaling pathway. Emerging
evidences have indicated that alterations of PTEN expression in hepatocytes are common
and recurrent events associated with liver disorders of various etiologies, suggesting an
important role for PTEN in liver diseases. Previous studies from our lab demonstrated
that hepatic Pten deletion in mice can lead to liver steatosis at an early stage, and
enhanced de novo lipogenesis might partially contribute to the observed accumulation of
fat. However, the dysregulated lipid metabolism in liver could be multifactorial, and
altered fatty acid β-oxidation in hepatocytes might be another reason.
Hence, the main purpose of this study is to investigate the role of fatty acid β-oxidation in
liver steatosis induced by the loss of PTEN. By analyzing the immortalized hepatocytes
that we established from the hepatic Pten deletion mouse model, we found that the ability
of the cell to oxidize both exogenous and endogenous fatty acids was enhanced when
Pten was deleted in hepatocytes, and this increased fatty acid β-oxidation was very likely
the strategy used by Pten-null hepatocytes to protect themselves from the lipotoxicity
caused by the increased de novo lipogenesis in these cells. Meanwhile, Pten-null
hepatocytes were more susceptible to the mitochondrial uncoupling effect of the fatty
acids. Moreover, we also found that the increased fatty acid β-oxidation might partially
viii
be responsible for the elevated oxidative stress condition that we observed in Pten-null
liver since increased fatty acid β-oxidation can lead to an increased oxidative
phosphorylation (OXPHOS) flux. Finally, we also looked into the molecular mechanisms
underlying the elevated fatty acid β-oxidation in Pten-null hepatocytes, and the result
suggested that the augmented ability to oxidize fatty acids was most likely due to the
increased levels of the enzymes involved in β-oxidation including CPT1a, CACT and
MCAD, and an increased level of intracellular fatty acid transporter, FABP1, upon Pten
deletion.
1
Chapter 1
Introduction
1.1 Introduction of PTEN
1.1.1 PTEN antagonizes PI3K/AKT signaling pathway
PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10), a dual-specificity
phosphatase encoded by a tumor suppressor gene located on human chromosome 10q23,
is a negative regulator for PI3K/AKT signaling pathway. Phosphatidylinositol 3-kinases
(PI3Ks) are a family of lipid kinases that integrate signals from growth factors, cytokines
and other environmental cues, translating them into intracellular signals that regulate
multiple signaling pathways (Thorpe et al., 2015). These pathways control many cellular
processes, which include cell growth, proliferation, survival, motility, as well as cellular
glucose and lipid metabolism (Vivanco and Sawyers, 2002). As suggested by their name,
PI3Ks can phosphorylate the hydroxyl group on the 3
rd
carbon of the inositol group of
phosphatidylinositol (Yuan and Cantley, 2008). Signaling through the PI3K pathway can
be initiated by the binding of growth factors to their receptors (Sansal and Sellers, 2004).
Upon ligand activation, receptor tyrosine kinases (RTKs) auto-phosphorylates at their
tyrosine residues, and the phosphorylated tyrosine residues on the receptor can
subsequently serve as a docking site for the p85 subunit of the heterodimeric PI3K.
However, in some cases such as the activation of insulin receptor, the phosphorylation of
2
receptor can alternatively mediate the recruitment of an adaptor protein, i.e. insulin
receptor substrate protein (IRS), to facilitate the binding of PI3K (Yonezawa et al., 1992).
In this way, the resting PI3Ks that exist in the cytoplasm can be brought to the cell
membrane where various PI species are located.
Moreover, the binding of p85 to the activated RTK can induce the catalytic activity of
PI3K by inducing a transition from an inhibited state to a disinhibited state, and
consequently an increased intracellular levels of PI(3,4)P
2
(phosphatidylinositol
(3,4)-biphosphate) and PI(3,4,5)P
3
(phosphatidylinositol (3,4,5)-triphosphate) (Yu et al.,
1998). A large number of proteins have a Pleckstrin Homology (PH) domain that allows
them to bind to the PI(3,4)P
2
or PI(3,4,5)P
3
at the inner surface of plasma membrane
(Park et al., 2008). Among all of these proteins, protein kinase B (PKB/AKT) is a major
downstream target of the PI3Ks. Following the activation of PI3K, AKT is recruited to
the plasma membrane via its PH domain, and meanwhile another PH domain-containing
protein, phosphoinositide-dependent kinase 1 (PDK1), can also be recruited to the same
area where PDK1 phosphorylates AKT at Thr 308 (Matsuda et al., 2013). Of note, the
initial activation of AKT requires the phosphorylation of AKT at Ser473 by mTORC2
before the subsequent phosphorylation by PDK1 (Jacinto et al., 2006; Sarbassov et al.,
2005). Once activated, AKT moves to the cytoplasm and nucleus where it phosphorylates
and activates/inhibits many downstream targets, which includes but not limits to the
3
inhibition of pro-apoptotic protein BAD (Datta et al., 1997) and pro-apoptotic
transcription factor FOXO (Zhang et al., 2011), and the activation of NF-κB via
regulating IκB kinase, IKKα, for cell survival (Bai et al., 2009); the inhibition of cell
cycle inhibitor p21 (Lawlor and Rotwein, 2000) and p27 (Liang et al., 2002) for
proliferation; the inhibition of GSK3β for increased glycogen synthesis to maintain
homeostasis (Embi et al., 1980; Pap and Cooper, 1998). The signaling pathways
controlled by PTEN are showed in Figure 1.
4
Figure 1
Figure 1. Signaling pathways controlled by PTEN.
5
As a PIP
3
phosphatase, PTEN negatively regulates the activity of PI3K/AKT signaling by
removing the phosphate from the 3’ position of the inositol ring, converting PIP
3
back to
PIP
2
, and therefore down-regulate the signaling downstream of PI3K (Downes et al.,
2007). The tumor suppression activity of PTEN is most likely due to its antagonistic
effects on the anti-apoptotic and proliferative activity of PI3K. However, recent studies
have also demonstrated that PTEN can shuttle between cytoplasm and nucleus to
maintain chromosomal stability and participate in DNA repair (Peyrou et al., 2010; Shen
et al., 2007). For the past almost 20 years, more than 7,800 publications have described
different aspects in regards to the tumor-suppressor function of PTEN and its relationship
to various human cancers, making it a promising therapeutic target for the treatment of
human cancer (Worby and Dixon, 2014).
1.1.2 The role of PTEN in non-alcoholic fatty liver disease
1.1.2.1 Introduction of non-alcoholic fatty liver disease
Non-alcoholic fatty liver disease (NAFLD) represents a hepatic metabolic syndrome,
which is commonly associated with obesity and diabetes conditions with
hyperinsulinemia and insulin resistance (Clark and Diehl, 2002; Matsuda et al., 2013).
NAFLD ranges from non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH),
which often precedes liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma.
Accumulating evidences have indicated that alterations of PTEN expression in
6
hepatocytes are common and recurrent events associated with liver disorders, suggesting
an important role for PTEN in liver diseases (Peyrou et al., 2010). However, to date, the
molecular pathogenesis of NAFLD is still unclear (Matsuda et al., 2013), but insulin
resistance, deregulated cytokine signaling and mitochondrial dysfunction are likely
critical events in the progression of NAFLD (Choudhury and Sanyal, 2005).
First of all, because the physiological function of PTEN is to dephosphorylate the second
messengers, PIP3, generated by PI3K, PTEN can down-regulate or terminate the insulin
signaling downstream of PI3K and cause insulin resistance (IR) (Matsuda et al., 2013).
IR in muscle and white adipose tissue (WAT) is suggested to play a central role in the
pathogenesis of fatty liver (Cohen et al., 2011; Rabol et al., 2011). Particularly, IR in
WAT favors the lipolysis of triacylglycerol, thus leading to the uncontrolled free fatty
acids (FFAs) release into circulation (Begriche et al., 2013). Since the uptake of FFAs by
hepatocytes is concentration-dependent, IR can largely enhance the influx of FFAs to the
liver (Tamura and Shimomura, 2005), and the excessive fat can then in turn cause IR in
this organ as well (Begriche et al., 2013). It is suggested that hyperinsulinemia can
overactivate sterol regulatory element-binding protein-1c (SREBP-1c), causing fatty
acids to be synthesized more actively in liver during IR (Begriche et al., 2006; Tamura
and Shimomura, 2005). Based on these observations, the potential role of PTEN has been
implicated in the development of NAFLD. Study from our lab also prove that PTEN
regulated PI3K signals directly control the de novo lipogenesis signals in the liver.
7
Moreover, in addition to an excess of circulating FFAs, deregulated production of
inflammatory cytokines by immune cells or by the adipose tissue have been clearly
associated with NAFLD (Tilg and Moschen, 2008). In fact, inflammation is believed to
be the main reason causing the diseases, and may also be responsible for the progression
to fibrosis and the subsequent cirrhosis via aberrant activation of hepatic stellate and
Kupffer cells (Garcia-Ruiz et al., 2011; Gressner and Weiskirchen, 2006). Because
PI3K/AKT/mTOR signaling pathway has been suggested to activate immune cells by
controlling key inflammatory cytokines, it is postulated that the deregulation of PTEN
may have specific therapeutic effects on NAFLD through its regulation of PI3K/AKT
signaling pathway (Matsuda et al., 2013; Weichhart and Saemann, 2008).
Finally, reactive oxygen species (ROS) accumulation induced by mitochondrial
dysfunction is believed to be another key factor in the pathogenesis of NAFLD. ROS can
be generated during the cellular processes including mitochondrial respiration,
inflammation and bacterial invasion (Hulsmans et al., 2012; Rocha et al., 2012). When
excessive ROS are produced in the cells, the subsequently induced oxidative stress can
cause hepatocyte apoptosis, a key component which is believed to play a role in the
progression of simple steatosis to NASH (Gambino et al., 2011; Mantena et al., 2009). In
addition, oxidative stress activates a series of stress pathways including stress related
serine/threonine kinases such as AKT, and thereby alters the insulin signaling
consequently (Blandino-Rosano et al., 2012; Matsuda et al., 2013).
8
1.1.2.2 PTEN alterations and loss of function in non-alcoholic fatty liver disease
Hepatic PTEN down-regulation has been observed in obese rats and in humans with
steatosis (Vinciguerra et al., 2008). Studies have showed that unsaturated fatty acids can
down-regulate PTEN through a mechanism involving the up-regulation of microRNA-21
following the interaction between mTOR and NF-κB. The degradation of PTEN can be
induced by the binding of microRNA-21 to PTEN mRNA 3’-UTR (Vinciguerra et al.,
2009; Vinciguerra et al., 2008). Furthermore, it has been demonstrated that the hepatic
deletion of Pten can cause increased insulin sensitivity in the liver and improved overall
glucose tolerance (Stiles et al., 2004). However, paradoxically, fatty liver was also
observed in mouse model that Pten is specifically deleted in liver (Stiles et al., 2004).
Although the mechanism for this paradox is yet to be clarified, it is suggested that the
up-regulated de novo lipogenesis in Pten-null liver might play a role in it (Byrne, 2010;
Matsuda et al., 2013; Stiles et al., 2004).
1.2 Fatty Acid β-oxidation
Fatty acid β-oxidation is a major pathway of energy metabolism and occurs primarily in
mitochondria, providing nearly 80% of ATP required for the liver and heart (Eaton et al.,
1996; Rogers et al., 2014). Liver fatty acid β-oxidation is high in the fasted state while
relatively low in the fed state (Rui, 2014). ATP production is coupled to the oxidation of
fatty acids by the electron transfer through the respiratory chain of mitochondria, which is
9
unique because electrons can be fed into the Q-pool through respiratory complex I and
the electron-transferring flavoprotein dehydrogenase (ETFDH) as well (Rogers et al.,
2014).
Unlike short-chain (chain length < C
6
) and medium-chain (chain length between C
6
and
C
12
) fatty acids which can get into mitochondria matrix directly, long-chain (chain length
between C
14
and C
18
) fatty acids in their activated form, acyl-CoA, cannot cross the inner
mitochondrial membrane (Aon et al., 2014). Instead, long-chain acyl-CoA species first
needs to be converted to acylcarnitine derivatives by carnitine palmitoyl transferase 1
(CPT1), a rate-limiting enzyme in the β-oxidation pathway, at the outer mitochondrial
membrane in the presence of carnitine. The acylcarnitine derivatives can then be
transported across the inner mitochondrial membrane by carnitine:acylcarnitine
translocase (CACT) (Rogers et al., 2014). Finally, at the inner mitochondrial membrane,
the acylcarnitine derivatives are converted back to acyl-CoA species by carnitine
palmitoyl transferase 2 (CPT-2), and are then subjected to the cyclic four-step fatty acid
oxidation process (Rinaldo et al., 2002). β-oxidation of activated fatty acids occurs in the
mitochondrial matrix and is catalyzed by the cyclic function of four enzyme families
(acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase,
and 3-ketoacyl-CoA thiolase), with acyl-CoA dehydrogenase exhibiting substrate
specificity for fatty acids of different chain lengths (Aon et al., 2014; Kerner and Hoppel,
10
2000). For each cycle of β-oxidation, fatty acids are shortened by two carbons with acetyl
CoA, NADH and FADH
2
being produced per cycle (Rinaldo et al., 2002), and the
shortened fatty acids can then subsequently enter the next round of β-oxidation.
Therefore, dozens of NADH and FADH
2
molecules can be produced during the
β-oxidation of a single long acyl chain, and are used to generate ATP through the process
of oxidative phosphorylation (OXPHOS). In addition, the acetyl CoA produced from the
β-oxidation can also enter the citric acid cycle (TCA cycle) to generate additional
electron donors, NADH and FADH
2
, for energy synthesis (Aon et al., 2014). A schematic
diagram illustrating fatty acid transport and utilization is showed in Figure 2.
11
Figure 2
Figure 2. Diagram of fatty acid transport and fatty acid β-oxidation.
12
1.3 Uncoupling of Mitochondria by Fatty Acids
In addition to their metabolic role as a fuel, fatty acids can also serve as uncouplers for
mitochondria. As indicated in the fatty acid cycling model (Skulachev, 1991),
undissociated fatty acid molecules can undergo a spontaneous transbilayer movement
(flip-flop) from the outer leaflet to the inner leaflet of the inner mitochondrial membrane
where they would release the protons due to the alkaline milieu of the mitochondrial
matrix; In their anionic form, fatty acids can then be transported back to the cytosolic side
by adenine nucleotide translocator (ANT) (Aon et al., 2014). Therefore, in this way, one
proton is transferred from the cytosol to the mitochondrial matrix by each fatty acid
molecule per cycle, leading to energy dissipation through a selective protonophoric action
mediated by several mitochondrial carriers (Aon et al., 2014; Rial et al., 2010). With a
decreased proton-motive force, mitochondrial respiration and ATP production can thus be
affected by the uncoupling effect of fatty acids.
1.4 The Production of Reactive Oxygen Species in Mitochondria
Mitochondria are often referred as the essential powerhouse of the cell because they are
able to generate energy currency, i.e. ATP, for the cell through the process of oxidative
phosphorylation (OXPHOS). Although most of the transferred electrons during OXPHOS
end up in water, complexes I and III of the respiratory chain are not perfectly insolated
from the oxygen (Fromenty and Pessayre, 1995). Therefore, reactive oxygen species
13
(ROS) can be generated through the reduction of oxygen to superoxide at these two sites
in mitochondria. About 1~2% of mitochondrial oxygen consumption leads to ROS
production under normal condition (Boveris and Chance, 1973). However, the production
of ROS can be up-regulated when increased electron transfer is not coupled with a
corresponding increase in ATP production, a condition in which excessive electrons get
accumulated at respiratory chain and can cause the leakage of electrons in mitochondria
eventually. It has been known for a long time that the increased mitochondrial OXPHOS
flux can cause increased formation of ROS (Brownlee, 2001; Osmundsen et al., 1991).
Once ROS production exceeds antioxidant defenses of the cell, components such as lipids,
proteins and nucleic acids (in particular mtDNA) can be damaged severely, leading to
oxidative stress and ultimately apoptosis of the cell, a process that has been observed in
NASH when there is an increased oxidation of free fatty acids in the liver (Begriche et al.,
2006).
1.5 Rationale of the Study
Liver is a key metabolic organ, the activity of which is tightly controlled by insulin and
other metabolic hormones (Rui, 2014). Recently, obesity has been identified as a major
cause for the increased incidence of metabolic disorders including non-alcoholic fatty
liver disease (NAFLD). As a hepatic manifestation of metabolic syndrome, NAFLD is
not only associated with other metabolic diseases such as diabetes, hyperlipidemia and
14
hyperglycemia (Nassir and Ibdah, 2014b), but also can give rise to more severe liver
diseases including non-alcoholic steatohepatitis (NASH), hepatic cirrhosis and
hepatocellular carcinoma (HCC). Emerging evidences have suggested a critical role for
PTEN in liver diseases as the alterations or loss of PTEN have been frequently observed
(Peyrou et al., 2010).
By utilizing liver-specific Pten knockout mice model, our laboratory found that hepatic
Pten deletion can result in lipid accumulation and liver steatosis followed by
hepatocellular carcinoma, a process which closely resembles the progression of human
liver diseases (Galicia et al., 2010). Moreover, previous study using this liver-specific
Pten-null mice model already demonstrated that the enhanced de novo lipogenesis in
mutated hepatocytes is one factor leading to the observed liver steatosis (Stiles et al.,
2004). However, the accumulation of fat in liver could also result from other factors as
well, and impaired fatty acid β-oxidation could be one of them. Therefore, in this study,
specifically in chapter 3, I attempt to investigate whether fatty acid β-oxidation is
impaired in Pten-null hepatocytes, and evaluate the role of PTEN in regulating fatty acid
β-oxidation in PTEN loss-induced fatty liver by using the immortalized hepatocytes that
our laboratory established from the hepatic Pten deletion mouse model.
15
Fatty acid β-oxidation occurs primarily in mitochondria which also are the main cellular
source of reactive oxygen species (ROS). As suggested by some studies, mitochondrial
ROS can oxidize hepatic fat deposits in patients with steatosis and subsequently trigger
lipid peroxidation, the products of which may then further impair the flow of electrons
along the respiratory chain and cause overreduction of the components on the chain,
leading to a vicious circle that can eventually aggravates ROS production and lipid
peroxidation (Pessayre et al., 2004). Previous studies in our laboratory observed an
elevated oxidative stress condition in Pten-null liver (Galicia et al., 2010; Li et al., 2013b),
and we proposed that the increased mitochondrial biogenesis controlled by the
up-regulated PI3K/AKT-pCREB-PGC-1α/ERRα signal node in response to the loss of
PTEN could partially contribute to the elevated ROS in Pten-null liver (Li et al., 2013b).
Here, I intended to investigate whether the altered fatty acid β-oxidation in Pten-null
hepatocytes could be another reason accounting for the increased ROS production that we
observed in Pten-null liver, since it is known that the rate of fatty acid β-oxidation can
influence mitochondrial OXPHOS flux and therefore the dysregulation of fatty acid
oxidation can consequently affect ROS formation in mitochondria (Brownlee, 2001;
Osmundsen et al., 1991).
Finally, in chapter 5, I evaluated the mRNA expression levels of selective enzymes and
transcriptional factors involved in fatty acid β-oxidation and de novo lipogenesis.
16
Through this study, we aim to gain some insights into the molecular mechanisms
underlying the altered lipid metabolism in Pten-null hepatocytes, and further our
understanding of the role played by PTEN in NAFLD.
17
Chapter 2
Materials and Methods
Strains and Growth Condition
Immortalized hepatocyte cell lines were established by our lab from livers of Control
(Con) and Pten-null (Pm) mice (Zeng et al., 2011), and were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM, Mediatech, Manassas, VA) supplemented with 10%
fetal bovine serum (Atlas Biologicals, Fort Collins, CO), 5µg/ml insulin (Sigma-Aldrich,
St. Louis, MO) and 10ng/ml epidermal growth factor (EGF, Invitrogen, Carlsbad, CA).
The immortalized hepatocytes were incubated at 37°C and 5% CO
2
atmosphere.
Starvation Treatment
After immortalized hepatocytes were cultured in plates overnight to allow attachment, the
growth medium was then replaced with substrate-limited Dulbecco’s Modified Eagle’s
Medium (DMEM, Life Technologies, Grand Island, NY) supplemented with 0.5mM
glucose (EMD Millipore, Temecula, CA), 1mM GlutaMAX (Life Technologies, Grand
Island, NY), 0.5mM L-carnitine hydrochloride (Sigma-Aldrich, St. Louis, MO) and 1%
fetal bovine serum (Atlas Biologicals, Fort Collins, CO) for an additional 24 hours.
Carnitine was added fresh on the day of the media change. Sterile filtration was needed
for the substrate-limited medium following the adjustment of pH to 7.4.
18
ROS Staining and Flow Cytometry
Immortalized hepatocytes were seeded in 60mm cell culture dishes (Sigma-Aldrich, St.
Louis, MO) overnight in growth medium, followed by another 24 hours of starvation. On
the day of the assay, cells were grown to 80% confluence. 90 minutes prior to starting the
assay, etomoxir (Sigma-Aldrich, St. Louis, MO) or vehicle was added to the cells and the
final concentration in each plate would be 40 µM. After incubating for 30 minutes, XF
Palmitate-BSA FAO Substrate or BSA control (Seahorse Bioscience, North Billerica,
MA) was added to the appropriate plates with a final concentration of 1%. After an
additional 1 hour incubation, cells were stained with 1µM H
2
DCFDA
(5-(and-6)-chloromethyl-2’,7’-dichlorodihydro- fluorescein diacetate acetyl ester,
Invitrogen, Carlsbad, CA) for 20 minutes at 37 °C. Cells were then washed in PBS
(Mediatech, Manassas, VA) and trypsinized. Once cells were gently resuspended in PBS,
each sample was analyzed with a flow cytometer (BD Bioscience, San Jose, CA). The
fluorescence intensity was determined from a total number of 10,000 cells from each
sample, and the data was analyzed with the software FlowJo.
19
Seahorse XF-24 Fatty Acid Oxidation (FAO) Assay
Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
were used to monitor fatty acid oxidation and glycolysis in real time when the appropriate
substrates and modulators were added to the cells as indicated below. Immortalized
hepatocytes were seeded in an XF-24 Cell Culture Microplate (Seahorse Bioscience,
North Billerica, MA) overnight in growth medium, followed by another 24 hours of
starvation. The FAO assay medium used for this experiment was 1×KHB (111mM NaCl
(VWR International, Visalia, CA), 4.7mM KCl (EMD Millipore, Temecula, CA),
1.25mM CaCl
2
(Sigma-Aldrich, St. Louis, MO), 2mM MgSO
4
(Sigma-Aldrich, St. Louis,
MO), 1.3mM Na
2
HPO
4
(EMD Millipore, Temecula, CA)) supplemented with 2.5 mM
glucose (EMD Millipore, Temecula, CA), 0.5 mM L-carnitine hydrochloride
(Sigma-Aldrich, St. Louis, MO) and 5mM HEPES (Life Technologies, Grand Island, NY)
on the day of the assay. In addition, sterile filtration was needed following pH adjustment
to 7.4. 90 minutes prior to starting the assay, etomoxir (Sigma-Aldrich, St. Louis, MO) or
vehicle was added to the cells and the final concentration in each plate would be 40 µM.
After incubating for 30 minutes at 37°C in a non-CO
2
incubator, XF Palmitate-BSA FAO
Substrate or BSA control (Seahorse Bioscience, North Billerica, MA) was added to the
appropriate wells for an additional 1 hour incubation. At time 0, XF-24 Cell Culture
Microplate was inserted into XF-24 Analyzer, and the XF Cell Mito Stress Test was
conducted with the command protocol in Table 1. Modulators of electron transport chain
20
(ETC) were purchased as XF Cell Mito Stress Test Kit from Seahorse Bioscience, and
oligomycin (1µM), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 1µM)
and a mix of rotenone and antimycin A (1µM) were added sequentially after 4
measurements of basal OCR and ECAR. Protein concentration was measured after the
FAO assay, and was used as the normalization values for each well. OCR and ECAR
were measured simultaneously, and all the data were analyzed by XF software and
displayed as average value of replicate wells +/− S.E.M.
21
Table 1
Command Number of Cycles Time (min)
Calibrate - -
Equilibrate auto auto
Mix, Wait, Measure 4 3,2,3
Inject Oligomycin - -
Mix, Wait, Measure 3 3,2,3
Inject FCCP - -
Mix, Wait, Measure 3 3,2,3
Inject Antimycin A and Rotenone - -
Mix, Wait, Measure 3 3,2,3
Table 1. XF-24 analyzer run protocol.
22
RNA Isolation, Reverse Transcription and Quantitative Real-time PCR
Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) following the
manufacturer’s instructions. Reverse transcription was conducted with M-MLV reverse
transcriptase system (Promega, Madison, WI). Quantitative PCR was performed using
MaximaTM SYBR Green qPCR Master Mix (Fermentas, Glen Burnie, MD) and 7900
HT Fast Real-Time PCR System (Applied Biosystem, Grand Island, NY) following the
manufacture’s instructions. Gene-specific primers are listed below in Table 2. Relative
expression of mRNA levels was determined by the delta-delta method, and 18S was used
as a standard.
23
Table 2
Symbol Full Name Primer Sequence
(5’-3’)
Fabp1 Fatty acid binding protein 1
F – GCAGAGCCAGGAGAACTTTG
R – GGGTCCATAGGTGATGGTGAG
Slc27a2 Solute carrier family 27 (fatty acid transporter),
member 2
F – GAAAGGCGACATCTACTTCAACAGC
R – TTTCCACCGGAAAGTATCTCCAACC
Cpt1a carnitine palmitoyltransferase 1a
F – CCATGAAGCCCTCAAACAGATC
R – ATCACACCCACCACCACGATA
Slc25a20 Solute carrier family 25 (mitochondrial
carnitine/acylcarnitine translocase), member 20
F – TGGACACTGTTGCTGAGAGG
R – TTGGCCAAAGGTATCGAGTC
Acadm Acyl-Coenzyme A dehydrogenase, medium
chain
F – TGTGGAGGTCTTGGACTTGGA
R – TCCTCAGTCATTCTCCCCAAA
Ppara Peroxisome proliferator activated receptor
alpha
F – ACACTGCCAAGGAGTCGAG
R – AGGCATCTACCACCATGTCCATAA
Fasn Fatty acid synthase
F – AGCGGCCATTTCCATTGCCC
R – CCATGCCCAGAGGGTGGTTG
Acaca Acetyl-Coenzyme A carboxylase alpha
F – ACAGTGGAGCTAGAATTGGAC
R – ACTTCCCGACCAAGGACTTTG
Srebf1 Sterol regulatory element binding transcription
factor 1
F – AACGTCACTTCCAGCTAGAC
R – CCACTAAGGTGCCTACAGAGC
Pparg Peroxisome proliferator activated receptor
gamma
F – TTTTCAAGGGTGCCAGTT T
R – GGAGGCCAGCATCGTGTAG
Rn18s 18S ribosomal RNA F – AAACGGCTACCACATCCAAG
R – CAATTACAGGGCCTCGAAAG
Table 2. Primer sequences for qPCR.
F: Forward primer; R: Reverse primer
24
Western Blot
Cells were harvested and lysed in cell lysis buffer. Lysates with equal amount of protein
were then subjected to SDS-PAGE and transferred to PVDF membranes for
immunoblotting. Antibody against PTEN was purchased from Cell Signaling Technology
(Danvers, MA). Anti-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO).
Statistical Analysis
Data in this study are presented as mean ± the standard error of the mean (SEM).
Differences between individual groups were analyzed by Student’s t test, and two-tailed p
values less than 0.05 was considered as statistically significant.
25
Chapter 3
PTEN Loss in Immortalized Hepatocytes Leads to Increased
Fatty Acid β-oxidation
3.1 Introduction and Rationale
Hepatocytes are rich in mitochondria given the fact that each hepatocyte contains about
800 mitochondria, occupying approximately 18% of the entire cell volume (Nassir and
Ibdah, 2014a). Importantly, mitochondria in the liver have a more distinct role compared
with other organs as they actively participate in the metabolism of glucose, lipids and
proteins. Recently, non-alcoholic fatty liver disease (NAFLD) has increased
tremendously in incidence with the abundances of modern life (Yeh and Brunt, 2014). As
multistage disease, the progressions of NAFLD are characterized by the following 3
stages: a relatively mild and reversible steatosis with simple accumulation of triglyceride
and cholesterol to begin with; followed by nonalcoholic steatohepatitis (NASH), an
intermediate stage with the combination of steatosis, hepatocyte injury and hepatic
inflammation that may further progress to fibrosis; cirrhosis, the irreversible stage in
which normal liver tissue has been replaced by fibrotic scar tissue and may end up
ultimately with hepatocellular carcinoma (HCC). The elevated lipid accumulation during
the early stage of NAFLD can result from several factors. First, increased lipolysis of
adipose tissue or increased intake of dietary fat, followed by the enhancement of free
26
fatty acids in the liver, can explain this phenomenon (Koo, 2013). Moreover, impaired
β-oxidation of fatty acids can contribute to the lipid accumulation as well (Fromenty et al.,
2004). In addition, de novo lipogenesis is also largely involved in the hepatic steatosis
(Postic and Girard, 2008). Finally, decreased lipid clearance which is often associated
with insulin resistance can also exacerbate the condition (Koo, 2013).
Previous studies in our laboratory have found that liver-specific deletion of tumor
suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) in mice
can ultimately lead to HCC accompanied by a phenotype of fatty liver at an early stage
(Galicia et al., 2010; Stiles et al., 2004), which is very similar to the progression of
NAFLD in human. Hence in this study, we attempted to utilize the immortalized
hepatocytes established from this hepatic Pten deletion mouse model in order to
understand the role played by PTEN in the regulation of fatty acid β-oxidation in PTEN
loss-induced fatty liver.
In the experiment, key parameters of mitochondrial function were determined directly by
measuring the oxygen consumption rate (OCR) of the cells. Additionally, modulators that
can specifically target the components of the electron transport chain (ETC) of the
mitochondria were serially injected to the cells to allow the measurement of these
parameters. All the calculations used in this experiment are illustrated in Table 3.
27
Oligomycin can inhibit ATP synthase (complex V), and the decrease of OCR following
the injection of this modulator correlates to the mitochondrial respiration associated with
the ATP production. In addition, FCCP can uncouple respiration from the production of
ATP. As a result, electrons can flow through the ETC uninhibitedly, and oxygen can be
maximally consumed by complex IV. Finally, the last injection is a mixture of rotenone
and antimycin A, which are the inhibitors for complex I and III respectively. With this
injection, mitochondrial respiration can be shut down completely.
28
Table 3
Table 3. XF FAO Assay parameters and their corresponding calculations.
Parameters
Calculations
Oxygen Consumption due to Uncoupling
by Free Fatty Acids
Oligomycin Palm-ETO rate minus Oligomycin
BSA-ETO rate.
Basal Respiration due to Utilization of
Exogenous Fatty Acids
Basal Palm-ETO rate minus Basal BSA-ETO rate
minus OCR due to uncoupling by free fatty acids.
Basal Respiration due to Utilization of
Endogenous Fatty Acids
Basal BSA-ETO rate minus Basal BSA+ETO rate.
29
3.2 Results
3.2.1 Mitochondrial respiration is elevated during starvation in Pten-null
hepatocytes
To begin with, we first wanted to see the effect of serum starvation on overall oxygen
consumption. In order to prime the cells to oxidize fatty acids, we starved the cells with
substrate-limited medium for 24 hours to eliminate the influence that other substrates,
such as glucose present in the medium, might have on the cellular respiration of the cells.
As showed in Figure 3-B, in the fed state, Pten-null immortalized hepatocytes exhibited
remarkably elevated OCR with approximately 35% elevation of basal OCR when
compared with Pten wild-type cells (Figure 3-C), and this elevation has also been
confirmed by our previous study too (Li et al., 2013b). Moreover, despite the basal OCR
of both cell types were decreased during starvation, Pten-null immortalized hepatocytes
still had a higher OCR during both basal respiration and maximal respiration stage
compared with starved Pten wild-type immortalized hepatocytes (Figure 3-B).
Approximately 2-fold increase of basal OCR has been observed in Pten-null
immortalized hepatocytes during starvation (Figure 3-D), indicating that mitochondrial
respiration is still higher under starving condition when Pten is deleted in immortalized
hepatocytes.
30
Figure 3
31
Figure 3. Pten deletion leads to increased mitochondrial respiration in both fed and
starving state.
(A) PTEN level is down-regulated in Pten-mutated immortalized hepatocytes compared
with Pten wild-type immortalized hepatocytes. (B) In fed state, both basal (the first four
measurements) and maximal (the three measurements following the injection of FCCP)
oxygen consumption rate (OCR) are higher in Pten-null immortalized hepatocytes
compared to Pten wild-type cells. In starving state, although the basal and maximal OCR
are decreased in both cell types compared with their own fed cells respectively, Pten-null
immortalized hepatocytes still have an elevated OCR compared to starved Pten wild-type
immortalized hepatocytes at both basal and maximal respiration stages. (C)
Quantification of baseline OCR under fed condition in Pten-null and wild-type
immortalized hepatocytes. (D) Quantification of baseline OCR under starving condition
in Pten-null and wild-type immortalized hepatocytes. Oligomycin (1µM), FCCP (1µM)
and a mix of rotenone and antimycin A (1µM) were added sequentially after measuring
basal OCR in an aim to inhibit ATP synthesis, uncouple electron transport chain of the
mitochondria and completely shut down mitochondrial respiration respectively. All the
data were analyzed by XF software and displayed as average value of replicate wells +/−
S.E.M. *, P≤ 0.05, different from Pten wild-type immortalized hepatocytes. n=5.
32
3.2.2 PTEN regulates exogenous fatty acid β-oxidation and the uncoupling of
mitochondria caused by fatty acids
Palmitate-BSA has been used extensively to investigate free fatty acid oxidation (Huynh
et al., 2014; Pike et al., 2011; Sebastian et al., 2009). In XF FAO Assay, the ability of the
cells to oxidize exogenously added fatty acids is indicated by an increase of OCR. In
order to determine the intrinsic rate and the capacity of a cell to oxidize fatty acids,
starvation is needed ahead of time to rule out or limit the influence that other exogenous
substrates such as glucose present in the medium, and stores of endogenous substrates
such as glycogen and triglycerides can have on OCR. Moreover, since free fatty acids are
weak lipophilic acids that can mildly uncouple mitochondria, the exogenously added free
fatty acids can contribute additionally to an increase of OCR but not due to fatty acid
oxidation. Therefore, by using XF FAO Assay, we attempted to drive cells to oxidize
exogenous fatty acids and meanwhile reveal the portion of OCR signal coming from three
different sources: endogenous fatty acid oxidation, exogenous fatty acid oxidation and
uncoupling by fatty acids as well.
In Pten-null immortalized hepatocytes, both basal and maximal respiration were
increased when palmitate was added to the cells exogenously (Figure 4-A). Similar result
has also been observed for Pten wild-type hepatocytes provided with palmitate compared
to the ones provided with the BSA carrier (Figure 4-A). However, when we calculated
33
the difference in proton leak between the Pten-null cells provided with palmitate and
Pten-null cells provided with BSA control, we found that uncoupling contributed 65
pmole O
2
/min to the increase of basal respiration rate in Pten-null hepatocytes whereas
oxygen consumption due to the uncoupling for wild-type hepatocytes was 33 pmole
O
2
/min (Figure 4-B). Moreover, when the portion of OCR due to uncoupling was
deducted from the basal OCR difference, we can get the baseline OCR resulted from the
oxidation of exogenous fatty acids. The quantification of OCR due to exogenous fatty
acid oxidation during the stage of basal respiration was showed in Figure 4-C. Exogenous
fatty acid oxidation contributed 33 pmole O
2
/min to the basal OCR in Pten-null
hepatocytes while contributing 5 pmole O
2
/min in wild-type hepatocytes (Figure 4-C).
Therefore, our result may suggest that the capacity of hepatocytes to oxidize exogenous
fatty acids is higher when Pten is deleted, but at the same time Pten-null hepatocytes
could also be more susceptible to the uncoupling caused by fatty acids when compared
with Pten wild-type hepatocytes.
34
Figure
4
35
Figure 4. Increased exogenous fatty acid oxidation and mitochondrial uncoupling in
Pten-deficient immortalized hepatocytes.
(A) PTEN loss in immortalized hepatocytes leads to a higher capacity to oxidize
exogenous fatty acids, and can cause free fatty acids to uncouple mitochondria to a much
larger degree. (B) Quantification of OCR due to uncoupling by free fatty acids in both
Pten-null immortalized hepatocytes and Pten wild-type immortalized hepatocytes. (C)
Quantification of basal respiration rate resulted from oxidation of exogenous fatty acids
in both Pten-null immortalized hepatocytes and Pten wild-type immortalized hepatocytes.
All the data were analyzed by XF software and displayed as average value of replicate
wells +/− S.E.M. *, P≤ 0.05, different from Pten wild-type immortalized hepatocytes.
n=5.
36
In addition to oxidative phosphorylation, glycolysis is another major energy-producing
pathway in the cell. The conversion of glucose to pyruvate, and subsequently lactate, can
result in a net production and extrusion of protons into the culture medium surrounding
the cells. In this experiment, extracellular acidification rate (ECAR) was measured
simultaneously along with OCR as an indicator of glycolytic function. Due to the
inhibition of mitochondrial activity, energy production shifted to glycolysis following the
injection of oligomycin. Consistent with this notion, a subsequent increase of ECAR
was observed in both Pten-null and Pten wild-type hepatocytes when oligomycin was
added (Figure 5). This result indicated that glycolysis and oxidative phosphorylation
remained coupled in response to metabolic alterations in hepatocytes regardless of their
PTEN status (Li et al., 2013b). Interestingly, following the injection of oligomycin,
compared to the Pten-null hepatocytes without palmitate, ECAR was increased to a
higher degree when palmitate was added to the Pten-null cells, whereas for the Pten
wild-type cells provided with or without palmitate, ECAR was enhanced to almost the
same level after the addition of oligomycin (Figure 5). This observation suggested that
the glycolytic capacity of the Pten-null hepatocytes was enhanced by the addition of fatty
acids ,but the exogenously added fatty acids had no effect on the Pten wild-type
hepatocytes.
37
Figure 5
Figure 5. Glycolytic capacity is increased along with elevated mitochondrial
respiration in Pten-deficient immortalized hepatocytes.
Fatty acids caused a constantly higher extracellular acidification rate (ECAR) in
Pten-null immortalized hepatocytes throughout the experiment, whereas the ECAR in
Pten wild-type hepatocytes was almost not affected by the exogenously added fatty acids.
All the data were analyzed by XF software and displayed as average value of replicate
wells +/− S.E.M. n=5.
38
3.2.3 PTEN regulates the β-oxidation of endogenous fatty acids
After we investigated the role of PTEN status in exogenous fatty acid oxidation in
immortalized hepatocytes, we next wanted to know whether endogenous fatty acid
oxidation is also affected by the loss of PTEN. In order to analyze the utilization of
endogenous fatty acids, etomoxir (ETO), a carnitine palmitoyl transferase-1 (CPT-1)
inhibitor, was given to the hepatocytes that were not exposed to the exogenously added
fatty acids. With the treatment of ETO, long chain fatty acids that already exist in the
cytosol can no longer be transported into mitochondria to get oxidized, and therefore the
endogenous fatty acid oxidation can thus be blocked.
In Pten-null hepatocytes, both basal and maximal respiration were decreased with the
treatment of ETO (Figure 6-A). Similar result has also been observed for Pten wild-type
hepatocytes when comparing the ones treated with ETO with the ones without (Figure
6-A). However, when we calculated the difference in basal respiration rate between the
Pten-null cells treated with ETO and Pten-null cells with no ETO treatment, we found
that oxidation of endogenous fatty acids contributed 74 pmole O
2
/min to the basal
respiration in Pten-null cells, whereas oxygen consumption due to endogenous fatty acid
oxidation in wild-type hepatocytes was 24 pmole O
2
/min (Figure 6-B). Based on these
observations, our result suggested that endogenous fatty acid oxidation was increased
when Pten was deleted in hepatocytes.
39
Figure 6
40
Figure 6. Increased endogenous fatty acid oxidation in Pten-deficient immortalized
hepatocytes.
(A) PTEN loss in immortalized hepatocytes leads to a higher capacity to oxidize
endogenous fatty acids. (B) Quantification of basal respiration rate contributed by the
oxidation of endogenous fatty acids in both Pten-null immortalized hepatocytes and Pten
wild-type immortalized hepatocytes. All the data were analyzed by XF software and
displayed as average value of replicate wells +/− S.E.M. *, P≤ 0.05, different from Pten
wild-type immortalized hepatocytes. n=5.
41
Moreover, when mitochondrial respiration was suppressed by ETO, we observed an
increased basal ECAR in Pten wild-type immortalized hepatocytes (Figure 7). This
observation was consistent with our earlier observation that mitochondrial respiration and
glycolysis were coupled in both cell lines. However, contrary to the wild-type
hepatocytes, basal ECAR almost did not change in Pten-null hepatocytes when we
inhibited mitochondrial respiration of the cells (Figure 7). In addition, it should be noted
that the glycolytic activity of Pten-null cells was higher than that of wild-type cells even
when their glycolytic capacity reached maximum. Taken together, these observations
indicated that PTEN may control mitochondrial respiration and glycolysis independent of
their metabolic coupling in hepatocytes.
42
Figure 7
Figure 7. The level of basal glycolysis did not change when mitochondrial
respiration was inhibited in Pten-deficient immortalized hepatocytes.
Etomoxir (ETO) nearly had no effect on basal extracellular acidification rate (ECAR)
level in Pten-null immortalized hepatocytes, whereas the ECAR in Pten wild-type
hepatocytes was constantly increased when ETO was added to the wild-type cells to
inhibit mitochondrial respiration. All the data were analyzed by XF software and
displayed as average value of replicate wells +/− S.E.M. n=5.
43
Chapter 4
Increased Fatty Acid Oxidation in Pten-null Immortalized Hepatocytes
Leads to Increased Cellular Reactive Oxygen Species (ROS) Production
4.1 Introduction and Rationale
Mitochondria are the oxygen-consuming powerhouse of the cells (Speijer et al., 2014),
which can provide a critical milieu for the synthesis of many essential molecules and
allow for the highly efficient energy production through oxidative phosphorylation
(OXPHOS) (Scheibye-Knudsen et al., 2015). However, there is a price to pay (Harman,
1981) because the use of oxygen can inevitably lead to the formation of damaging
reactive oxygen species (ROS). On the one hand, increased mitochondrial function can
cause elevated ROS production because enhanced electron flow can inevitably lead to
increased electron leak (Brownlee, 2001; Osmundsen et al., 1991). On the other hand,
defects in respiratory complexes, with consequent decrease of electron transfer and
energy output, can also lead to further production of ROS as the electrons frequently leak
from complex I and III of the electron transport chain (Carreras et al., 2004; Genova et al.,
2004; Jastroch et al., 2010; Lenaz, 2001) (Figure 8-A). Although ROS play a critical role
in normal cellular signaling, increased levels or prolonged exposure to ROS can impair a
variety of cellular components including DNA, RNA, carbohydrates, proteins and lipids
(Frohnert and Bernlohr, 2013). Free fatty acids can supply mitochondrial OXPHOS with
44
electrons via fatty acid β-oxidation (Nakamura et al., 2009). In addition, NADH and
FADH
2
can be further generated when the final metabolite from fatty acid β-oxidation,
acetyl-CoA, is metabolized in trichloroacetic acid (TCA) cycle (Nakamura et al., 2009),
providing even more electrons for OXPHOS.
In the mouse models where Pten is specifically deleted in liver, we observed increased
oxidative stress conditions as indicated by the elevated H
2
O
2
and
trans-4-hydroxy-2-nonenal (4-HNE) production (Galicia et al., 2010). Moreover, the
increased oxidative stress present in Pten-null mouse has also been confirmed by our
previous observations that the levels of oxidized form of glutathione (GSSG) in Pten-null
liver was 2-fold higher than that of control liver at 18 weeks, and the expressions of
glutathione peroxidase (GPx) and glutathione S-transferase (GST) were significantly
higher in Pten-null liver at different ages (Li et al., 2013b). Since ROS has been known to
be able to induce the expressions of antioxidant enzymes (Hayes and Pulford, 1995), the
increased levels of GPx and GST in Pten-null liver therefore was an indication that the
liver was under oxidative stress in Pten-null mice. For another, the elevated expressions
of these antioxidant enzymes at later stages could also suggest that cellular ROS may
have been controlled at a relatively low level since antioxidant enzymes can scavenge
them (Figure 8-B). Therefore, depending on the different stages of the phenotype
development in Pten-null mice as well as the level of oxidative stress, the up-regulated
expression of antioxidant enzymes can imply either a high level or a low level of cellular
45
ROS. However, even with increased level of scavengers, the antioxidant defenses of the
Pten-null hepatocytes eventually were still not sufficient to overcome the massive cellular
H
2
O
2
production (Figure 8-B). In fact, despite ROS can be eliminated by antioxidant
enzymes to some degree, the observation of increased 4-HNE level in Pten–null liver
demonstrated the fact that excessive levels of ROS already had done certain damage to
the hepatocytes. We have previously showed that PTEN status is a determining factor for
the ROS levels in vitro (Li et al., 2013b), and the up-regulated mitochondrial biogenesis
through the activated PI3K/AKT-pCREB-PGC-1α/ERRα signaling pathways upon PTEN
loss has been proposed as one molecular mechanism underlying the elevated ROS
production in Pten-null hepatocytes by our laboratory (Li et al., 2013b). In this study, we
thus want to further investigate whether the alterations we see with β-oxidation can also
exacerbate ROS production in Pten-null hepatocytes. It has been known for a long time
that the increased mitochondrial OXPHOS flux can cause increased formation of ROS
(Brownlee, 2001; Osmundsen et al., 1991). Therefore, it is reasonable for us to
hypothesize that the increased fatty acid β-oxidation as we observed in Pten-mutated
hepatocytes could further enhance the increased ROS production in these cells.
46
Figure 8
47
Figure 8. Reactive oxygen species (ROS) production in the cells.
(A) Both increased and decreased mitochondrial function can lead to dysregulated ROS
production in the cells. SOD, superoxide dismutase; GPx, glutathione peroxidase; GST,
glutathione S-transferase. (B) i) In the healthy conditions, cells can maintain a balance
between ROS production and their clearance by antioxidant enzymes. ii) When mild
oxidative stress occurs, antioxidant enzymes can be induced and, to some degree, relieve
ROS generation. iii) When ROS production eventually exceeds antioxidant defenses of
the cells, severe oxidative stress can be induced and may cause extensive damage to the
cells.
48
4.2 Results
4.2.1 Increased exogenous fatty acid β-oxidation and increased fatty acid uncoupling
of mitochondria upon PTEN loss aggravate ROS production in immortalized
hepatocytes
Before we subjected the cells to ROS staining and FACS analysis, immortalized
hepatocytes were prepared in the same condition as we used for Seahorse XF-24 Fatty
Acid Oxidation (FAO) Assay. In chapter 3, we have demonstrated that the exogenous
fatty acid oxidation and mitochondrial uncoupling in Pten-null hepatocytes were
significantly increased compared with the Pten wild-type hepatocytes, and here we
attempted to investigate whether the increased exogenous fatty acid oxidation and
mitochondrial uncoupling can subsequently lead to the elevated ROS production within
the cells. As indicated by the FACS result, ROS production was significantly elevated in
the Pten-null hepatocytes provided with palmitate when compared with the ones only
provided with BSA control (Figure 9-B). However, palmitate showed almost no effect on
the induction of ROS in Pten wild-type hepatocytes (Figure 9-A). Quantification of
median fluorescence intensity for ROS staining in Pten wild-type and Pten-null
immortalized hepatocytes were showed respectively in Figure 9-C and Figure 9-D.
Finally, quantification data also showed that exogenously added palmitate can increase
ROS production in Pten-null immortalized hepatocytes by 9 folds compared to the Pten
wild-type cells (Figure 9-E). Taken together, these observations may suggest that PTEN
49
loss can induce ROS production in immortalized hepatocytes partially via increased
exogenous fatty acid oxidation as well as increased mitochondrial uncoupling induced by
fatty acids.
50
Figure 9
51
Figure 9. Reactive oxygen species (ROS) level is up-regulated by increased
exogenous fatty acid oxidation and fatty acid uncoupling in Pten-null immortalized
hepatocytes.
(A) and (B) Immortalized hepatocytes were first starved for 24 hours, and then stained
with H
2
DCFDA and subjected to a flow cytometer to measure ROS level in the cells.
Exogenously added palmitate can induce ROS production to a much larger degree in
Pten-null immortalized hepatocytes than Pten wild-type immortalized hepatocytes when
compared with their corresponding BSA control group. (C) and (D) Quantification of
median fluorescence intensity. *, P≤ 0.05, different from BSA control group. n=3. (E)
Quantification of ROS induced by the palmitate. *, P≤ 0.05, different from Pten
wild-type immortalized hepatocytes. n=3.
52
4.2.2 Inhibition of endogenous fatty acid β-oxidation by etomoxir elevates ROS level
in Pten-deficient immortalized hepatocytes
In order to evaluate whether endogenous fatty acid oxidation also plays a role in ROS
formation, we added etomoxir (ETO) to the starved immortalized hepatocytes for the
same period of time as we did for the Seahorse XF-24 Fatty Acid Oxidation (FAO) Assay.
If increased endogenous fatty acid oxidation in Pten-null immortalized hepatocytes
indeed contribute to an increase of ROS level, we would expect a larger reduction in ROS
when endogenous fatty acid oxidation is inhibited by ETO in these cells compared with
the wild-type hepatocytes. Interestingly, contrary to our expectation, the treatment of
etomoxir consistently induced a higher level of ROS in both Pten-null and Pten wild-type
immortalized hepatocytes (Figure 10-A and Figure 10-B). Quantification of median
fluorescence intensity for ROS staining in wild-type and Pten-null immortalized
hepatocytes were showed respectively in Figure 10-C and Figure 10-D, and it is showed
that the ROS induction by ETO in Pten-null hepatocytes was 5 folds higher than that of
wild-type hepatocytes (Figure 10-E). As indicated by other studies, one possible reason
for the increased ROS production that we observed within immortalized hepatocytes in
this experiment could be the off-target effect of ETO in immortalized hepatocytes
(Merrill et al., 2002) ,which will be further addressed in the later discussion part.
53
Figure 10
54
Figure 10. Reactive oxygen species (ROS) level is up-regulated by etomoxir (ETO)
in Pten-null immortalized hepatocytes.
(A) and (B) Immortalized hepatocytes were first starved for 24 hours, and then stained
with H
2
DCFDA and subjected to a flow cytometer to measure ROS level in the cells.
ETO can induce ROS production to a much larger degree in Pten-null immortalized
hepatocytes than wild-type hepatocytes when compared with their corresponding no ETO
treatment group. (C) and (D) Quantification of median fluorescence intensity. *, P≤ 0.05,
different from no ETO treatment group. n=3. (E) Quantification of ROS induced by the
ETO. *, P≤ 0.05, different from wild-type immortalized hepatocytes. n=3.
55
Chapter 5
Molecular Mechanisms Underlying Increased Fatty Acid Oxidation in
Pten-null Immortalized Hepatocytes
5.1 Introduction and Rationale
We have demonstrated that both exogenous and endogenous fatty acid oxidation were
significantly increased in Pten-null immortalized hepatocytes (see chapter 3). In order to
investigate the possible underlying molecular mechanisms, we analyzed the mRNA levels
of several representative genes involved in fatty acid oxidation by qPCR. Carrier proteins
are required for the transport of long chain fatty acids (LCFAs) into the cells (Aon et al.,
2014). Fatty acid transport protein 2 (FATP2) is a plasma membrane-associated LCFA
transporter that is predominantly expressed in liver, and has been known to play an
important role in hepatic LCFA uptake (Falcon et al., 2010). Fatty acid-binding protein 1
(FABP1), also known as liver-type fatty acid-binding protein (L-FABP), is an
intracellular transporter that can enhance cellular LCFA uptake and facilitate the delivery
of LCFAs to different cellular organelles including mitochondria (Atshaves et al., 2010).
Moreover, liver isoform carnitine palmitoyltransferase 1 (CPT1a) is an outer
mitochondrial membrane associated enzyme that can catalyze the conversion of
long-chain acyl CoA to long-chain acylcarnitine, which is the form used to translocate
across the inner mitochondrial membrane by carnitine: acylcarnitine translocase (CACT)
56
(Lopaschuk et al., 2010). Furthermore, we also examined the mRNA expression levels of
genes encoding medium chain acyl-coenzyme A dehydrogenase (MCAD), a key fatty
acid β-oxidation enzyme (Barger and Kelly, 1999), and peroxisome proliferator activated
receptor alpha (PPAR-α), a transcriptional factor that has been showed to regulate the
expression of many genes involved in fatty acid oxidation (van Raalte et al., 2004).
In addition, it is known that lipotoxicity can be induced by an imbalance between de novo
lipogenesis and fatty acid oxidation (Lelliott and Vidal-Puig, 2004). Since we already
observed the formation of fatty liver in hepatic Pten deletion mouse at an early stage, we
therefore will determine the mRNA expression levels of representative genes involved in
de novo lipogenesis. Acetyl-CoA carboxylase1 (ACC1) is a critical enzyme for the
formation of malonyl-CoA from acetyl-CoA, and fatty acid synthase (FAS) is an
important enzyme for the synthesis of palmitic acid by using precursors such as
malonyl-CoA and NADPH as substrates (Rui, 2014). Finally, two lipogenic
transcriptional factors, sterol regulatory element-binding protein-1c (SREBP-1c) and
proliferator activated receptor gamma (PPAR-γ) were also analyzed in this study.
5.2 Results
As for the genes involved in fatty acid β-oxidation, qPCR analysis showed that the
mRNA expression levels of genes encoding FABP1, CPT1a, CACT and MCAD were
57
significantly increased in Pten-null immortalized hepatocytes compared with Pten
wild-type hepatocytes (Figure 11-A). The increased transcription of these genes may
partially explain the higher fatty acid oxidation in Pten-null hepatocytes since FABP1 can
facilitate the process of transporting fatty acids to the mitochondria, and CPT1a, CACT
and MCAD are the key enzymes directly involved in fatty acid β-oxidation. However,
less mRNA levels of FATP2 and PPAR-α were found in Pten-null hepatocytes, but with
no significant change for FATP2 expression (Figure 11-A). This result may imply that
the increased fatty acid β-oxidation in Pten-null hepatocytes was not due to an enhanced
uptake of exogenous fatty acids since FATP2 is a plasma membrane-associated
transporter for long-chain fatty acids. As a matter of fact, no significant differences have
been found in regards to the total fatty acid uptake in Pten-null and wild-type hepatocytes
(Stiles et al., 2004). Interestingly, although we observed a decreased transcription for
gene encoding PPAR-α in Pten-null hepatocytes, the activity of this transcriptional factor
was actually increased in Pten-null cells as indicated by the elevated transcription of its
targeted genes including CPT1a, CACT and MCAD. The possible reasons will be further
discussed in chapter 6. Furthermore, as for the genes involved in de novo lipogenesis,
Pten-null hepatocytes had higher expression levels for mRNA of ACC1, FAS, SREBP-1c
and PPAR-γ than wild-type hepatocytes (Figure 11-B), which may shed some light on the
mechanisms underlying the liver steatosis that we observed in Pten-null mice.
58
Figure 11
59
Figure 11. mRNA expression levels of several representative genes involved in fatty
acid oxidation and lipogenesis in wild-type and Pten-null immortalized hepatocytes.
Quantitative real-time PCR analysis was used to measure the mRNA expression levels of
the genes that we were interested. (A) mRNA levels of FABP1, CPT1a, CACT and
MCAD were significantly increased in Pten-null immortalized hepatocytes. However, the
mRNA levels of FATP2 and PPAR-α were decreased in Pten-null immortalized
hepatocytes despite the decrease of FATP2 mRNA was not significant. (B) mRNA levels
of FAS, ACC1, SREBP-1c and PPAR-γ were all elevated in Pten-null immortalized
hepatocytes. *, P≤ 0.05, different from wild-type immortalized hepatocytes. n=3.
60
Chapter 6
Overall Discussion
Functional impairments associated with increased circulating levels of lipids and their
induced metabolic alterations in fatty acids utilization and intracellular signaling, have
been broadly termed “lipotoxicity” (Aon et al., 2014). In the mouse model that Pten is
specifically deleted in the liver, we observed the accumulation of lipid and a phenotype of
fatty liver at the early stage of these mice, and it has been proposed that the increased de
novo lipogenesis is responsible for the observed liver steatosis in Pten-null liver (Stiles et
al., 2004). Given the multiple factors that liver steatosis can stem from, we focused on
hepatic fatty acid β-oxidation in order to understand the dysregulated lipid metabolism in
fatty liver induced by the loss of PTEN. Contrary to our expectation that fatty acid
β-oxidation may be impaired in Pten-null hepatocytes, our result showed that Pten-null
hepatocytes had a higher capacity to oxidize both exogenous fatty acids and endogenous
fatty acids, and meanwhile they were more susceptible to the uncoupling of fatty acids
when compared with Pten wild-type hepatocytes. This result may seem to be a little
unexpected at first sight. However, cells need to protect themselves from lipotoxicity by
either sequestering them as triacylglycerol within lipid droplets, which has been
confirmed by our H&E staining and Oil Red O staining in Pten-null liver section (data
not shown), or by actively oxidizing fatty acids. Thus, increased mitochondrial fatty acid
oxidation observed here is likely one of the several metabolic adaptations that develops
61
during the early stages of NAFLD in an attempt to restrain hepatic fat accumulation
(Begriche et al., 2013). So far, the mechanisms responsible for the higher mitochondrial
fatty acid oxidation in NAFLD are still not quite understood yet.
In this study, we observed that the mRNA expression levels of the genes that encode the
enzymes directly involved in fatty acid β-oxidation including CPT1a, CACT and MCAD
were significantly increased in Pten-null hepatocytes. Consistently, the mRNA level of
the gene encoding FABP1, an intracellular transport protein that can facilitate the
delivery of fatty acids to the mitochondria, was also dramatically increased in Pten-null
hepatocytes. Therefore, these observations may explain the higher fatty acid oxidation
capacity in the liver steatosis caused by the hepatic deletion of Pten. However, it is
noteworthy that the mRNA level of the gene encoding PPAR-α, a transcriptional factor
that can target many genes involved in fatty acid oxidation including CPT1a, CACT and
MCAD (Rakhshandehroo et al., 2010) was actually decreased in Pten-null hepatocytes.
The reason why we observed a higher activity of PPAR-α, as indicated by the increased
transcription of CPT1a, CACT and MCAD in Pten-deficient hepatocytes may be several
folds. First, an increases in PGC-1α, a transcription co-factor of PPAR-α (Vega et al.,
2000), may explain such enhanced transcription of PPAR-α target genes. Previous study
in our lab has demonstrated that both mRNA and protein levels of PGC-1α were
significantly induced by the activated PI3K/AKT-pCREB-PGC-1α signal node in
62
Pten-deficient immortalized hepatocytes (Li et al., 2013b). Therefore, even with a
decreased transcription level of PPAR-α, the activity of this transcription factor can still
be up-regulated as a result of its increased co-activator. Second, increased levels of
PPAR-α ligands may induce its transactivation ability. PPAR-α is a nuclear receptor
family member that can be activated by a subtype of LCFA (Chakravarthy et al., 2009),
and products of FAS appear to be the endogenous PPAR-α ligands in liver (Chakravarthy
et al., 2009; Chakravarthy et al., 2005). In consistent with our earlier study that Pten
deletion can enhance the fatty acid synthesis in mice liver (Stiles et al., 2004), data from
the current study suggests an increased de novo lipogenesis in Pten-null hepatocytes as
indicated by the elevated transcription levels of genes involved in fatty acid synthesis
including ACC1, FAS, SCREBP-1c and PPAR-γ. Thus, the activity of PPAR-α can be
enhanced by the increased amount of ligands. Furthermore, the transcription of MCAD
may also be regulated by an orphan nuclear receptor ERRα, which is known to be
up-regulated in Pten-null hepatocytes (Li et al., 2013b).
Despite the moderately decreased mRNA level of gene encoding FATP2, a plasma
membrane-associated LCFA transporter, in Pten-null hepatocytes, we observed an
increased oxidation of exogenous fatty acids in those cells. In fact, it is found that the
total fatty acid uptake by Pten-null hepatocytes was not significantly altered compared
with wild-type hepatocytes, and no significances were observed in the primary Pten-null
63
hepatocytes in regards to the protein levels of FATP2 and FATP5 (Stiles et al., 2004).
Based on these observations, with the similar mean uptake of fatty acids for both mutated
and wild-type hepatocytes, the observed increase of exogenous fatty acid oxidation in
Pten-null hepatocytes is very likely due to the enhanced levels of fatty acid oxidation
enzyme such as FABP1, CPT1a, CACT and MCAD, other than an elevated uptake of
exogenous fatty acids.
Previous study in our laboratory already found that PTEN loss was able to simultaneously
induce a higher mitochondrial respiration (OCR) and a higher glycolysis (ECAR) (Li et
al., 2013b). In this study, we found that the glycolytic capacity of the Pten-null
hepatocytes can be further enhanced by the addition of fatty acids while the exogenously
added fatty acids had no effect on the wild-type hepatocytes. Moreover, no significant
ECAR change has been observed in Pten-null hepatocytes when mitochondrial
respiration was inhibited by ETO. However, as for the wild-type hepatocytes, a
significantly increased ECAR was observed due to the inhibition of their coupled
energy-producing pathway, i.e. mitochondrial respiration. Therefore, contrary to the
Pten-null hepatocytes whose elevated glycolysis may already meet the energy demand of
the cells, wild-type hepatocytes have to further enhance their glycolysis when
mitochondrial respiration was suppressed, so that a sufficient ATP supply can be
guaranteed. Of note, even with increased ECAR in wild-type hepatocytes when
64
suppressing mitochondrial respiration, the glycolytic activity of the wild-type cells was
still lower than that of Pten-null hepatocytes. Taken together, it seems that Pten-null
hepatocytes have the ability to maintain a high level of both mitochondrial respiration and
glycolysis at the same time, a phenomenon which closely resembles the metabolic profile
exhibited by cancer cells, termed as “Warburg effect”. In fact, the elevation of the two
coupled energy-producing pathways in Pten-null hepatocytes can be a result due to the
activated PI3K/AKT signaling pathway in these cells, because the pro-survival kinase
AKT has been suggested to be able to up-regulate many proteins involved in both
respiration and glycolysis through multiple mechanisms (Berwick et al., 2002; Deprez et
al., 1997; Gottlob et al., 2001; Li et al., 2013a; Zaid et al., 2008; Zhang et al., 2006).
Finally, we observed an increased capacity of oxidizing fatty acids in Pten-null
hepatocytes. Thus, it is reasonable for us to hypothesize that the enhanced fatty acid
β-oxidation in Pten-null hepatocytes may be one of the many reasons responsible for the
elevated ROS level in those cells. An increased mitochondrial OXPHOS flux can lead
to more cellular ROS formation. Indeed, when we provided fatty acids to the
hepatocytes to promote exogenous fatty acid oxidation and fatty acid uncoupling in the
cells, an enhanced ROS production was observed in Pten-null hepatocytes. However,
when we tried to investigate the effect that endogenous fatty acid oxidation can have on
cellular ROS generation, it seems that the etomoxir (ETO) that we added to the cells had
65
some off-target effects in addition to inhibiting fatty acid oxidation, and therefore we
were unable to specifically test whether the enhanced β-oxidation had resulted in more
ROS production. ETO had been found to significantly modulate the transcription of
several redox-related genes in HepG2 cells. These include genes encoding proteins such
as Mn
+
superoxide dismutase precursor (SOD2), γ-glutamate-cysteine ligase modifier
subunit (GCLM) and thioredoxin reductase (TXNRD1) (Merrill et al., 2002). However,
there is one study also suggesting that ETO can indeed reduce palmitate-induced ROS
production by 80% in rat hepatoma cell line H4IIEC3 (Nakamura et al., 2009). Therefore,
to better address this question, genetic manipulation of Cpt1a in hepatocytes may be
needed in the future to avoid the off-target effects of ETO.
In summary, our study demonstrated that PTEN loss in hepatocyte can enhance the
ability of the cell to oxidize fatty acids, serving as a protective mechanism for the
hepatocytes to protect themselves from lipotoxicity due to the increased de novo
lipogenesis in the cells. Moreover, the elevated fatty acid oxidation in Pten-null
hepatocytes was most likely due to the increased levels of the enzymes involved in the
β-oxidation. To our knowledge, at present, only increased de novo lipogenesis has been
reported in Pten-null liver, therefore our study in regards to the regulation of fatty acid
β-oxidation in hepatocytes may lay a foundation for a better understanding of PTEN-loss
induced fatty liver in the future.
66
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Abstract (if available)
Abstract
PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) is a dual‐specificity phosphatase that can antagonize PI3K/AKT signaling pathway. Emerging evidences have indicated that alterations of PTEN expression in hepatocytes are common and recurrent events associated with liver disorders of various etiologies, suggesting an important role for PTEN in liver diseases. Previous studies from our lab demonstrated that hepatic Pten deletion in mice can lead to liver steatosis at an early stage, and enhanced de novo lipogenesis might partially contribute to the observed accumulation of fat. However, the dysregulated lipid metabolism in liver could be multifactorial, and altered fatty acid β-oxidation in hepatocytes might be another reason. ❧ Hence, the main purpose of this study is to investigate the role of fatty acid β-oxidation in liver steatosis induced by the loss of PTEN. By analyzing the immortalized hepatocytes that we established from the hepatic Pten deletion mouse model, we found that the ability of the cell to oxidize both exogenous and endogenous fatty acids was enhanced when Pten was deleted in hepatocytes, and this increased fatty acid β-oxidation was very likely the strategy used by Pten‐null hepatocytes to protect themselves from the lipotoxicity caused by the increased de novo lipogenesis in these cells. Meanwhile, Pten‐null hepatocytes were more susceptible to the mitochondrial uncoupling effect of the fatty acids. Moreover, we also found that the increased fatty acid β-oxidation might partially be responsible for the elevated oxidative stress condition that we observed in Pten‐null liver since increased fatty acid β-oxidation can lead to an increased oxidative phosphorylation (OXPHOS) flux. Finally, we also looked into the molecular mechanisms underlying the elevated fatty acid β-oxidation in Pten‐null hepatocytes, and the result suggested that the augmented ability to oxidize fatty acids was most likely due to the increased levels of the enzymes involved in β-oxidation including CPT1a, CACT and MCAD, and an increased level of intracellular fatty acid transporter, FABP1, upon Pten deletion.
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Creator
Chen, Jingyu
(author)
Core Title
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates fatty acid beta-oxidation
School
School of Pharmacy
Degree
Master of Science
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
07/16/2015
Defense Date
07/15/2015
Publisher
University of Southern California
(original),
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Tag
fatty acid β-oxidation,non‐alcoholic fatty liver disease,OAI-PMH Harvest,PTEN,reactive oxygen species,ROS,uncoupling of mitochondria
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English
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Stiles, Bangyan (
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), Cadenas, Enrique (
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), Okamoto, Curtis (
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jingyuch@usc.edu,sophiacjy1990@hotmail.com
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Tags
fatty acid β-oxidation
non‐alcoholic fatty liver disease
PTEN
reactive oxygen species
ROS
uncoupling of mitochondria