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Inhibition of NR3B1 attenuates the progression of NAFLD and NASH in liver-specific Pten knockout mice

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Inhibition of NR3B1 attenuates the progression of NAFLD and NASH in liver-specific Pten knockout mice

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Inhibition of NR3B1 attenuates the progression of NAFLD and NASH in
liver-specific Pten knockout mice

by

Xinwen Zhang

A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

August 2020

Copyright 2020 Xinwen Zhang
ii
Acknowledgements
At the very beginning, I would like to thank my advisor, Dr. Bangyan L. Stiles, for her
insightful mentoring and enthusiastic helping of the research projects. Without her constant
support and guidance, I would not have such interest in pharmacology and complete my
research so smoothly. She is such an amazing scientist and a perfect professor who always
show her genius and diligence in research as well as her patience and kindness in teaching.

I am so glad that I am in such a warm and united lab and I am sincerely grateful for the
support and help of all our lab members. Especially, I am thankful to Dr. Joshua Chen for
patient teaching, brilliant advising, and experience sharing. I am grateful to Lina He, our best
lab manager, for her helpful support and critical suggestion. I also thank Lulu Chen for the
helping of qPCR techniques and data analysis. I am also thankful to Sophia Chen and Taojian
Tu for their helpful advice and encouragement. Also I’d like to thank other master students
for their friendly help and warm-hearted support. Not only during the time in lab had I
mastered a great many lab techniques and gained plenty of knowledge of liver diseases, but
also I harvest incalculable love and encouragement which drive me to keep going and try
harder sometimes when things got tough.

Ultimately, a sincere thank I would like to give to my parents. I would like to thank for their
unconditional love and also thank them for making me a better person. And I also want to
express my gratitude to Dr. Cadenas and Dr. Xie, for their witty remarks and brilliant views
on my thesis.

iii
Table of Contents
Acknowledgements ................................................................................................................ ii
List of Tables ...........................................................................................................................v
List of Figures ....................................................................................................................... vi
Abstract ................................................................................................................................ vii
Chapter 1: Introduction ...........................................................................................................1
1.1.Non-alcoholic fatty liver disease (NAFLD) and its progression ................................1
1.1.1. Etiology of NAFLD .........................................................................................1
1.1.2. Progression of NAFLD ...................................................................................1
1.2. Lipid metabolism in the liver ....................................................................................3
1.2.1. Lipid metabolism in the liver ..........................................................................3
1.2.2. Upstream signals that regulate lipid metabolism in the liver ..........................7
1.3. Pathogenesis of NAFLD/NASH .............................................................................10
1.3.1. Dysfunction of the metabolic pathways ........................................................10
1.3.2. Dysfunction of mitochondria ......................................................................... 11
1.3.2.1. Mitochondria and lipid metabolism ....................................................... 11
1.3.2.2. ROS and lipid metabolism .....................................................................14
1.3.3. ROS, inflammation, cell death and NASH ....................................................14
1.4. Summary and study hypothesis ...............................................................................15
Chapter 2: Inhibition of NR3B1 results in reduced steatosis and NASH .............................17
2.1. Introduction and rationale ......................................................................................17
2.2. Results ....................................................................................................................18
2.2.1. Reduced lipid accumulation in liver tissues treated with NR3B-PA .............18
2.2.2. Inhibition of NR3B1 reduces inflammation associated with NASH .............20
Chapter 3: Inhibition of NR3B1 alleviates fibrosis ..............................................................25
3.1. Introduction and rationale ........................................................................................25
3.2. Results ......................................................................................................................25
3.2.1. Reduced deposition of collagen fibers with NR3B-PA treatment ...................25
3.2.2. Inconsistent change of expression of several fibrotic genes ...........................28
Chapter 4: Inhibition of NR3B1 decreases liver injury and apoptosis .................................31
4.1. Introduction and rationale ........................................................................................31
4.2. Results ......................................................................................................................32
4.2.1. Liver injury is reduced with NR3B-PA treatment ...........................................32
4.2.2. Decreased cell death due to inhibition of NR3B1 ..........................................34
Chapter 5: Discussion ...........................................................................................................37

iv
Chapter 6: Materials and methods ........................................................................................40
Bibliography .........................................................................................................................44

v
List of Tables
Table 1. Primer sequences for qPCR detecting cytokine gene expression 41
Table 2. Primer sequences for qPCR detecting fibrotic gene expression 41

vi
List of Figures

Figure 1. Lipid metabolism in hepatocytes. 6
Figure 2. PI3K/AKT/PTEN signaling pathways regulates metabolism. 9
Figure 3. Lipid metabolism in mitochondria. 13
Figure 4. Reduced liver steatosis with NR3B-PA treatment. 19
Figure 5. Down-regulation of cytokine gene expression with PA-treated. 21
Figure 6. Less recruitment of immune cells due to PA treatment. 24
Figure 7. Less fiber accumulation due to PA treatment. 27
Figure 8. NR3B-PA treatment leads to insignificant change in fibrotic gene expression. 30
Figure 9. Liver injury decreased when the function of NR3B1 is inhibited with NR3B-PA. 33
Figure 10. Decreased cell death induced by loss of function of NR3B1. 35

vii
Abstract
PI3K/AKT signaling regulates many fundamental cellular functions, especially cell survival
and cell proliferation. Abnormal activation of PI3K/AKT signaling is frequent in human
cancer. PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) was identified
as a dual-specificity phosphatase that plays a significant role in antagonizing PI3K/AKT
signaling. A liver-specific deletion of Pten leads to a robust activation of PI3K/AKT
signaling. A liver-specific Pten-null mouse model has been successfully established using
Cre-Lox recombination technology. This mouse model provides us a good observing window
on the progression of chronic liver diseases into liver cancer.

Estrogen-related receptor alpha (ERRα), also known as NR3B1, is an orphan nuclear receptor
that plays an important role in mitochondrial biogenesis and respiration as well as lipid
synthesis. Previous research reported that expression of NR3B1 was upregulated in the
absence of PTEN and that a hairpin pyrrole-imidazole polyamide (PA) was an inhibitor of
NR3B1 (The latter was developed in collaboration with Dr. Peter Dervan’s lab in Caltech).

This research focuses on identifying the pathological effects of NR3B1inhibition on NAFLD
and NASH by using the liver-specific Pten-null mouse model. Two groups were established:
an experiment group with NR3B-PA injection and a vehicle group with PBS+5% DMSO
injection. Characteristics of steatosis, NASH, fibrosis, liver injury and hepatocyte apoptosis
were compared between these two groups. qPCR was performed to assess expression of
several specific cytokine genes following a 2-month PA treatment. Compared with

viii
vehicle-treated group, inhibition of NR3B1 upregulated the expression of cytokine genes.
Moreover, IHC staining with macrophage markers showed less gathering of immune cells in
PA-treated group. In terms of NAFLD, H&E staining revealed less lipid droplets in
PA-treated group. Through Sirius Red staining, it was found that inhibition of NR3B1
alleviated characteristics of fibrosis. Finally, the levels of liver injury (alanine transaminase
(ALT) level in serum) and cell death (TUNEL assay) were also decreased upon NR3B1
inhibition. Taken together, these results suggest that inhibition of NR3B1 can significantly
attenuate the progression of NAFLD and NASH.

1

Chapter 1
Introduction

1.1 Non-alcoholic fatty liver disease (NAFLD) and its progression
1.1.1 Etiology of NAFLD
The liver plays a unique role in detoxifying chemicals [1], synthesizing specific serum
proteins [2], metabolizing nutrients [3], and drugs [4]. Non-alcoholic fatty liver disease
(NAFLD) can broadly be defined as a wide spectrum of liver injury from steatosis,
non-alcoholic steatohepatitis (NASH), fibrosis to cirrhosis in absence of alcohol abuse [5]. In
the US, NAFLD has affected 30% of population in 2016 and is the most common cause of
diverse end-stage liver diseases [6]. Global prevalence of NAFLD was 24% in 2018, and it is
on the rise every year as a result of worldwide spread of obesity. NAFLD is becoming an
increasingly serious worldwide public health problem [7].

The early-stage NAFLD is characterized as simple excessive hepatic fat accumulation
without any significant hepatocellular injury [8]. In clinical practice, it is generally accepted
that steatosis in at least 20%-30% of hepatocytes represents the threshold of NAFLD [9].
Accumulating evidence suggests that the increased concentration of intracellular fatty acids
within hepatocytes resulting from impaired lipid regulation pathways plays a pivotal role in
steatosis [10].

2

1.1.2Progression of NAFLD
Statistically, 30% - 40% of NAFLD patients will develop into non-alcoholic steatohepatitis
(NASH) based on liver biopsy studies of obese individuals. NASH is characterized as severe
steatosis, evident inflammatory activity and noticeable ballooning degeneration within
hepatocytes [11]. As the most severe histological form of NAFLD, NASH is predicted to
become the main cause of liver transplantation in the US in the future [12]. Even though the
detailed mechanisms initiating the progression from simple steatosis to chronic inflammation
still remain unknown, recent evidence demonstrates the significant role of fatty acid toxicity,
oxidant stress and mitochondrial dysfunction in contributing to the pathogenesis of NASH
[10, 13].

Long-lasting NASH and chronic liver damage would induce fibrosis, which is identified as
excessive generation and accumulation of collagen in liver tissues. Fibrosis is a
wound-healing response of the liver that encapsulates ongoing injury with scars [14]. These
fibrous scars can distort the hepatic architecture and affect liver function. Advanced liver
fibrosis often results in cirrhosis in which the fibrous scars develop into the nodules of
regenerating hepatocytes [15]. The hepatocellular dysfunction and increased hepatic
resistance to blood flow observed in cirrhosis often causes hepatic insufficiency and portal
hypertension, respectively [16]. Clinically, there are two stages of cirrhosis, compensated and
decompensated. Patients with decompensated cirrhosis often have complications including
ascites, jaundice or hepatic encephalopathy. As their conditions cannot be reversed, liver
transplant is the only definitive treatment for them [17].
3

Epidemiological evidence suggests that NAFLD-related cirrhosis increases the risk of liver
cancer. In western countries, 4-22% of hepatocellular carcinoma (HCC) cases developed
from NAFLD [18, 19]. In Asia, 1-2% of HCC cases originate from NAFLD [20, 21]. The
prevalence of NAFLD-related HCC worldwide is on the rise every year. However, the
mechanisms underlying the association from NAFLD to HCC are still not well understood
[22].

1.2 Lipid metabolism in the liver
1.2.1 Lipid metabolism in the liver
As the organ involved in lipid metabolism, liver sustains the lipid homeostasis by regulating
lipid acquisition and lipid disposal. The liver achieves this by regulating four key processes
involved in lipid homeostasis: lipid uptake from circulation, de novo lipogenesis, fatty acid
oxidation and lipid export [23] (Fig.1).

The uptake of circulating lipids into hepatocytes is largely dependent on several plasma
membrane transporters, which include fatty acid transport protein 2 (FA TP2), FATP5,
caveolins and CD36. Particularly, CD36 functions as a transporter of long-chain fatty acids
and its expression is regulated by peroxisome proliferator-activated receptorγ(PPARγ) [24].
Following hepatocyte uptake, fatty acids will bind with fatty acid binding protein 1 (FABP1)
and be distributed to different compartments within the cells [25]. Some of them will be
stored as triglyceride (TG, or triacylglycerol, TAG, or triacylglyceride) for future use.
4

Another way to increase hepatic fat is de novo lipogenesis, in which acetyl-CoA units are
built into fatty acids. Up-regulated de novo lipogenesis is a major cause of lipid retention and
excess accumulation [26]. Carbohydrate regulatory element-binding protein (ChREBP) and
sterol regulatory element-binding protein 1c (SREBP1c) are two critical transcription factors
in the regulation of de novo lipogenesisby lipogenic gene expression, including fatty acid
synthase (FAS) and acetyl-CoA carboxylase (ACC). SREBP1c is activated by insulin and
liver X receptor α [23] while ChREBP is activated by carbohydrates and involved in
carbohydrate-induced de novo lipogenesis [27].

Fatty acid oxidation is a process by which hepatocytes degrade cellular fat to generate ATP.
This process mainly occurs in the mitochondria. Entry of fatty acids into mitochondria relies
on Carnitine Palmitoyl Transferase 1 (CPT1) that is located in the outer mitochondrial
membrane. Fatty acids are broken down in the mitochondrial matrix to generate acetyl-CoA
(β-oxidation). Acetyl-CoA enters the TCA cycle to yield reducing equivalents in the form of
NADH and FADH2, which channel electrons through the mitochondrial electron transport
chain to yield ATP at Complex V (ATPase). Some long-chain fatty acids can also be oxidized
in peroxisomes and microsomes through β-oxidation and ω-oxidation, respectively [28].
PPARα controls fatty acid oxidation in all three organelles by regulating the expression of
genes encoding key enzymes in fatty acid oxidation [23, 28].

5

Another pathway of lipid disposal from hepatocytes is export in the form of very low-density
lipoprotein (VLDL). VLDL particles are composed of fatty acids, cholesterol, phospholipids,
and apolipoproteins. In the endoplasmic reticulum, apolipoprotein B100 (apoB100) lipidated
by microsomal triglyceride transfer protein (MTTP) facilitates the formation of VLDL
particles. After further lipidation in the Golgi, mature VLDL particles are exported from
hepatocytes [23, 29].

6

Figure 1

Figure 1. Lipid metabolism in hepatocytes
Four key processes involved in lipid metabolism are regulated in hepatocytes, including lipid
uptake from circulation, de novo synthesis, oxidation and export.

7

1.2.2 Upstream signals that regulate lipid metabolism in the liver
The PI3K/AKT/PTEN signaling pathway regulates metabolism, cell growth and cell survival
[30]. Phosphatidylinositol-3-kinases (PI3Ks) are a family of lipid kinases that catalyze the
production of phosphatidylinositol-3,4,5-trisphosphate (PIP3) on the cell membrane [31].
Protein kinase B (PKB), also known as AKT, is a serine/threonine–specific protein kinase
that can be translocated to the plasma membrane in the presence of PIP3 and activated by
phosphoinositide-dependent kinase 1 (PDK1) and PDK2 [32].

Binding of insulin and insulin-like growth factor (IGF) to their receptors can activate PI3K
directly or phosphorylate insulin receptor substrate (IRS) to recruit and activate PI3K [33].
Following activation of PI3K, PIP3 on the membrane will recruit AKT by binding to its PH
domain. After Ser
473
in the hydrophobic motif of AKT is phosphorylated by mTOR complex
2 (mTOR2), Thr
308
in the activation loop is phosphorylated by PDK1 to achieve full
activation[34]. Upon activation, AKT moves to cytoplasm or nucleus to activate or inhibit a
number of downstream targets, such as GSK3β and transcription factors FoxO [35],
peroxisome proliferator-activated receptor gamma co-activator (PGC-1α) [36], SREBP and
mTOR [33, 37] (Fig. 2).

Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), a dual phosphatase
having both protein and lipid phosphatase activities, acts as a negative regulator of
thePI3K/AKT signaling pathway [33]. PTEN dephosphorylates PIP3 generated by PI3K at
the 3’ position and converts it back into PIP2, thus inhibiting the activity of downstream
8

targets especially AKT[38].

In hepatocytes, PI3K/AKT/PTEN signaling pathway participates in the regulation of hepatic
fat levels mainly via regulating glucose metabolism and de novo lipogenesis. Phosphorylation
of forkhead transcriptional factor (FoxO) by AKT inhibits transcription of
glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which
catalyze two regulatory steps ingluconeogenesis [39]. In addition, phosphorylation of
PGC-1αby AKT can also block the transcription of G6Pase and PEPCK [36].
Phosphorylation and inhibition of GSK3β can activate glycogen synthase that catalyzesthe
synthesis of glycogen [40].

PI3K/AKT/PTEN signaling regulates de novo lipogenesis mainly by mediating the
expression of SREBP. FoxO1, as a downstream target of AKT, can inhibit SREBP
transcription [37]. Another target of AKT, mTORC1, mediates the processing of SREBP that
is controlled by SREBP cleavage-activating protein (SCAP) and insulin induced gene (Insig)
[33] (Fig.2).

9

Figure 2

Figure 2. PI3K/AKT/PTEN signaling pathway regulates metabolism
PI3K/AKT/PTEN signaling regulates cell metabolism by inhibiting or activating several
downstream targets.

10

1.3 Pathogenesis of NAFLD/NASH
1.3.1 Dysfunction of the metabolic pathways
Although plenty of advances have occurred in diagnostics and therapeutics to suppress the
progression of NAFLD/NASH, the underlying mechanisms of pathogenesis of
NAFLD/NASH have not yet been elucidated. Among numerous theories, the “Two-hit”
hypothesis is widely accepted. Accumulation of lipids in hepatocytes is the “first hit”. It
sensitizes the liver to a second metabolic insult by acting as “second hit” and subsequently
activates inflammatory cascades and fibrogenesis [41, 42].

There are multiple factors involving in the “first hit”. Besides high fat diet and obesity,
insulin resistance (IR) plays a pivotal role in steatosis in the liver. Subjects with reduced
insulin sensitivity at the level of muscle and adipose tissue develop hyperinsulinemia. As a
result, the liver is exposed to hyperinsulinemic conditions that drive de novo lipogenesis. IR
also enhances the flux of fatty acids into the liver by impairing inhibition of lipolysis in
adipose tissues [43].

The progression of NAFLD into NASH, which acts as “second hit”, is complicated. It is
believed that lipotoxicity caused by high level of fatty acids, free cholesterol and other lipid
metabolites results in mitochondrial dysfunction and production of reactive oxygen species
(ROS), which consequently, contribute to liver injury and inflammation [44].

11

1.3.2 Mitochondrial dysfunction
1.3.2.1 Mitochondria and lipid metabolism
Each hepatocyte contains around 800 mitochondria, which take up 18% of their entire cell
volume. Mitochondria are the powerhouses of the cell using fat and glucose as substrates to
generate ATP [45]. Fatty acid β-oxidation, occurring primarily in mitochondria, provides
nearly 80% of ATP required for the liver [46].

While the short-chain (chain length < C8) and medium-chain (C8-C12) fatty acids can enter
mitochondria freely as the activated acyl-CoA form, entry of long-chain(C12-C20) fatty acids
is regulated by carnitine palmitoyl transferase 1(CPT-1) that is located on outer
membrane. CPT-1 converts long-chain fatty acids into acylcarnitine in presence of carnitine.
Carnitine-acylcarnitine translocase (CACT) facilitates transport of acylcarnitine across the
inner membrane. At the inner mitochondrial membrane, acylcarnitine is converted to
acyl-CoA mediated by carnitine palmitoyl transferase 2(CPT-2). Subsequently, acyl-CoA is
subjected to a cyclic four-step β-oxidation process: In each cycle of β-oxidation, fatty acids
are converted into one acetyl-CoA and a shortened fatty acid by undergoing dehydrogenation,
hydration, dehydrogenation, and thiolysis. Repeated cycles will split fatty acids into
acetyl-CoA units. Acetyl-CoA enters the citric acid cycle (TCA cycle) to generate electron
equivalents that flow through the respiratory chain to generate a H
+
gradient, which is the
driving force for the generation of ATP at complex V . In prolonged fasting states, acetyl-CoA
moieties are converted into ketone bodies, exported from the liver and used as alternative
sources of energy [45, 47] (Fig. 3).
12

Structural and functional alterations of mitochondria are closely associated with the
pathogenesis of NAFLD/NASH. Specifically, structural alterations include ultrastructural
lesions and depletion of mitochondrial DNA (mtDNA), while functional alterations are
composed of decreased activity of respiratory chain complexes and impaired mitochondrial
β-oxidation [45, 48].

13

Figure 3

Figure 3. Lipid metabolism in mitochondria
Key processes involved in lipid metabolism in mitochondria are shown, including entry of
FFAs, β-oxidation, TCA cycle, ETC, ATP synthesis and ketogenesis.

14

1.3.2.2 ROS and lipid metabolism
Electrons are passed from one member of ETC to another in a series of redox reactions.
Energy generated from these reactions is used to form a proton gradient across the inner
membrane which drives ATP synthesis by chemiosmosis. Together, this process is called
oxidative phosphorylation (OXPHOS). Dysfunction of OXPHOS directly leads to production
of reactive oxygen species (ROS) [49].

In NAFLD/NASH, electron flow across the respiratory chain is increased because of an
enhanced stimulation of FAO and TCA cycle. This leads to an increased electron leak with
subsequent partial reduction of oxygen to form superoxide anion radical [45], whichis
dismutated to hydrogen peroxide by Mn-SOD (SOD2) in the mitochondrial matrix and by
CuZn-SOD (SOD1) in the mitochondrial intermembrane space. Superoxide and hydrogen
peroxide generated in these processes are defined as ROS. Excessive production of ROS
severely damages DNA, lipids, and proteins in the cell. Particularly, ROS-induced damage of
mtDNA can lower mitochondrial number and function significantly, leading to apoptosis [48,
50].

1.3.3 ROS, inflammation, cell death and NASH
NASH is mainly characterized as inflammation and hepatocyte damage. Inflammation is a
critical self-protective response of tissues to infection or injury in which immune cells and
molecular mediators such as cytokines and chemokines are involved [51]. It is widely
believed that ROS play a crucial role in inflammation and cell death in NASH [52].
15

In NASH, excessive production of ROS caused by mitochondrial dysfunction can oxidize
unsaturated lipids, resulting in extensive lipid peroxidation. In turn, reactive aldehydes
generated from lipid peroxidation can damage mtDNA or ETC components, and therefore
lead to increased ROS formation in a vicious cycle. Excessive ROS activates nuclear
factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK), leading to transcription of
adhesion molecules, chemokines, cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 [53].

Particularly, binding of TNF-α to its cognate receptor induces cytochrome c release from
mitochondria to cytosol and activates caspase-9and subsequently caspase-3 that triggers
apoptotic cell death [54]. Furthermore, some reactive aldehydes and IL-8can serve as
chemoattractants for neutrophils [55].

1.4 Summary and study hypothesis
Non-alcoholic fatty liver disease (NAFLD), as a most common metabolic syndrome and a
chronic liver disease, is predicted to be the most frequent indicator for liver transplantation by
2030 [56]. Usually the early-stage simple steatosis will develop into NASH that is considered
as the key factor contributing to hepatocyte apoptosis and liver injury, fibrosis and cirrhosis
and even liver cancer. More importantly, NAFLD is more than a chronic disease confined in
liver. Increasing evidence indicates that it is a multisystem disease that also exerts adverse
effects on extra-liver organs and other regulation pathways, ultimately, cause severe damage
to human health. Thus, investigation on novel therapeutic agents and clinical management are
16

becoming important issues for scientific research.

The goal of this study was examining systemically the role of NR3B1 as a potential target for
treatments of NAFLD and NASH. NR3B1, also known as estrogen-related receptor α (ERRα),
is an orphan nuclear receptor identified in 1988 [57]. It plays a key role in controlling
mitochondria biogenesis, FAO, TCA cycle and OXPHOS by mediating expression of genes
vital for these functions [58].

This study is based on previous work from Dr. Stiles' lab that showed:
1) Novel signal node PTEN/PI3K/AKT-pCREB-PGC-1α/NR3B1 has been established.
PTEN/PI3K signaling controls mitochondria function by mediating expression of ERRα via
AKT-pCREB-PGC-1α axis [59].
2) Pyrrole–imidazole (Py-Im) polyamide (PA)is an inhibitor of NR3B1 [60].

Thus, we hypothesize that inhibition of NR3B1 reverses the progression of NAFLD and
attenuates inflammation associated with NASH as well as the development of fibrosis. This
hypothesis will be tested by investigating: 1) the effects of NR3B-PA treatment on
phenotypes of steatosis and NASH. 2) the role of NR3B-PA treatment in fibrosis
development. 3) the effects of NR3B-PA treatment on apoptosis and liver injury.

17

Chapter 2
Inhibition of NR3B1 results in reduced steatosis and NASH

2.1 Introduction and rationale
NAFLD is identified as a hepatic manifestation of metabolic syndrome characterized by
excess lipid buildup in hepatocytes. NAFLD can progress to chronic inflammation where
inflammatory cells infiltrate liver tissues, resulting in NASH [61].

To address the role of NR3B-PA in NASH development, a NASH model developed in Dr.
Stiles' lab was used. In the liver-specific Pten knockout mouse model, inflammatory cell
infiltration became obvious and progressive after 6 months of age. At 9 months of age, 50-75%
of mice develop HCC phenotypes [62, 63]. To address the role of NR3B-PA on NASH, we
used the 7.5month-old Pten knockout mice that developed a NASH phenotype as a model.

These mice were randomly assigned to two groups (n=5): NR3B-PA and vehicle treated
groups. To assess if inhibition of NR3B1 would lead to reduced inflammation, qPCR was
performed to detect the expression of several cytokine genes including IL-6, TNF-α, IL-1β
and CXCL5. Studies have shown that IL-6 and TNF-α are released by macrophages and
adipocytes. Increased expression of these cytokines are indicative of inflammation in the liver
[64]. Moreover, IL-1β treatment on primary hepatocytes was shown to up-regulate the
expression of fatty acid synthase and promote TG accumulation [65]. CXCL5, a chemokine
which binds to the chemokine receptor CXCL2, plays a crucial role in immune cell
18

recruitment in inflammation [66]. Collectively, increased expression of IL-6, IL-1β, TNF-α
and CXCL5 were used as parameters to indicate the level of liver inflammation to assess
NASH. Immunohistochemistry was also performed to assess the recruitment of immune cells
in liver tissue sections.

2.2 Results
2.2.1 Reduced lipid accumulation in liver tissues treated with NR3B-PA
As mentioned earlier [62, 63], 100% of the liver-specific Pten-deleted mice developed liver
steatosis at 7 months to 9 months of age. In this study, liver tissues taken from the NR3B-PA
and vehicle treated groups and were prepared as paraffin-embedded sections. Hematoxylin
and eosin staining were performed and showed severe lipid accumulation in the livers of the
Pten-null mice without treatment of NR3B-PA. Large-white-rounded hollows that are
indicative of lipid can be seen in hepatocytes. However, in NR3B-PA-treated group, a
compact and tight connection among cells, which resemble normal hepatocytes, can be
observed (Fig. 4).

19

Figure 4

Figure 4. Reduced liver steatosis with NR3B-PA treatment
In the left panel, three representative images of H&E stained liver tissues of three mice in
vehicle-treated group. In the right panel, three representative images of H&E stained liver
tissues of three mice in PA-treated group. In the middle panel, high magnification images of
the cropped areas.

20

2.2.2 Inhibition of NR3B1 reduces inflammation associated with NASH
As the progressive state of NAFLD, NASH can develop further into fibrosis and cirrhosis, as
well as the end stage liver diseases, hepatocyte carcinoma (HCC). In this study, the effects of
NR3B-PA on pro-inflammatory cytokines, which contribute to inflammation associated with
NASH, were assessed. qPCR analysis showed that the expression of inflammatory factors
IL-6, IL-1β, TNF-α and CXCL5 were significantly suppressed by NR3B-PA treatment. A 35%
and 36% expression were observed for IL-6 and TNF-α in PA-treated groups compared to
vehicle groups, respectively (Fig 5A,B). A 31% expression and 41% expression were
observed for IL-1β and CXCL5 in PA-treated groups compared to vehicle groups,
respectively (Fig 5C,D). The down-regulation of these genes suggested that inflammatory
signaling pathways were inhibited by NR3B-PA treatment.

In addition, immuno-fluorescence staining of liver sections using CD68 (red) (a protein
highly expressed by macrophages) and pan-cytokeratin (green) (a marker for bile-duct
structures) revealed a high level of macrophage recruitment around bile ducts in vehicle
groups (Fig 6). However, few macrophages were observed in PA-treated groups (Fig 6). This
staining suggests that less inflammatory cells are accumulating in the liver, consistent with
the qPCR results showing down-regulation of inflammatory cytokines. Together, these data
suggest that NR3B-PA treatment alleviated the inflammation associated with NASH.

21

Figure 5
A.

B.

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Relative expression
Relative expression of IL-6 in
vehicle and PA-treated groups
NR3B-PA
Vehicle
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
relative expression
Relative expression of TNF-a in
vehicle and PA-treated groups
NR3B-PA
Vehicle
22

C.

D.

Figure 5. Down-regulation of cytokine gene expression with PA-treated
The qPCR results show that expression of all four cytokine genes were downregulated in
0
0.2
0.4
0.6
0.8
1
1.2
1.4
relative expression
Relative expression of IL-1b in
vehicle and PA-treated groups
NR3B-PA
Vehicle
*
0
0.2
0.4
0.6
0.8
1
1.2
relative expreesion
Relative expression of CXCL5 in
vehicle and PA-treated groups
NR3B-PA
Vehicle
23

PA-treated mice and expressed in fold change. Blue bar: NR3B-PA treated group. Gray bar:
Vehicle. A. IL-6 (35%), P=0.18; B. TNF-α (36%), P=0.22; C.IL-1β (31%), P=0.04; D.
CXCL5 (41%), P=0.09. All data were analyzed by Excel software and displayed as Mean±
S.E.M. *, P≤ 0.05, n=5.

24

Figure 6

Figure 6. Less recruitment of immune cells due to PA treatment
Immuno-fluorescence was performed to detect the recruitment of immune cells (vehicle
group, left panel; NR3B-PA group, right panel). CD 68 (RED) was used as biomarker of
macrophage while Pan CK (GREEN) was regarded as a marker of biliary epithelial cells.
Nuclei were stained with DAPI (BLUE). With NR3B-PA treatment, less macrophage
25

recruitment can be found.
Chapter 3
Inhibition of NR3B1 alleviates fibrosis

3.1 Introduction and rationale
Approximately 42% of NASH patients will progress to advanced fibrosis, which can be
accompanied cirrhosis, liver failure and portal hypertension [67]. Liver fibrosis is identified
as excessive deposition of extracellular matrix (ECM) proteins, specifically, collagen in the
liver tissue. It occurs as a result of the liver’s response to wound-healing. Persisting liver
injury and failed liver regeneration can lead excessive production of ECM to replace necrotic
and apoptotic hepatocytes [15].

In this chapter, the liver fibrosis phenotype was characterized in the Pten knockout mice with
or without NR3B-PA treatment by two approaches: 1) immunohistochemistry staining for
collagen fibers with Sirius Red; 2) qPCR analysis for expression of genes that are biomarkers
of ECM deposition and HSC activation.

3.2 Results
3.2.1 Reduced deposition of collagen fibers with NR3B-P A treatment
Fibrosis is hallmarked by deposition of ECM such as accumulation of collagen and can be
visualized by Sirius Red staining. Pericellular distribution of fiber matrix was found around
hepatocytes showing fatty change or hydropic swelling in the liver tissues of NASH patients
26

[68]. As shown in Fig. 7, intensive accumulations of red-stained, string-like collagen fibers
were clearly visible in the livers of the Pten-null mice treated with vehicle. These fibers were
closely localized with areas where most lipid droplets were aggregated. However, in
PA-treated group, few white-rounded lipid droplets were found, thus indicating less lipid
deposition. Sirius Red positive stained fibers were barely visible in the PA-treated group (Fig.
7A).

The positively stained areas were quantified using Image J. In the vehicle-treated group, the
percentage of liver tissues stained positive for Sirius Red ranged from 9.75% to 21.04%, with
an average of 15.76%±1.69%. In the PA-treated group, this percentage ranged from 1.32% to
12.16%, with an average of 7.33%±1.58% (Fig. 7B).

Taken together, the Sirius Red analysis showed that inhibiting NR3B1 with NR3B-PA not
only reversed steatosis but also suppressed the development of fibrosis.

27

Figure 7
A.

B.

Vehicle NR3B-PA
NR3B-PA
Vehicle
0
5
10
15
20
25
% Area of Fibrosis in Serius Red Staining
treatments
% area
✱✱
28

Figure 7. Less fiber accumulation due to PA treatment
A. Less collagen stained with Sirius Red in mice treated with PA. (NR3B-PA treated group,
right panel; Vehicle-treated group, left panel) B. Analysis of the percentage of
positive-stained area using Image J software. Each point represents average value of five
different microscopic images from the same mouse. All the data were analyzed by Excel
software and displayed as Mean ±S.E.M. **, P=0.0034 ≤ 0.01, n=7.

3.2.2 Inconsistent change of expression of several fibrotic genes
Previous research from Dr. Stiles' lab showed that Col-1, Timp-1, Desmin, and P75ntr, were
up-regulated concurrent with increased collagen deposition in the Pten- null mice vs. wild
type mice [69]. Hence, it may be hypothesized that NR3B-PA treatment can also reduce the
expression of these genes.

Surprisingly, theresults indicate that NR3B-PA treatment didnot have regulatory effects on the
expression of these fibrotic genes. As shown in Fig. 8, with each circle representing one
mouse, the result of P75ntr and Timp-1 showed significant individual variation. Additionally,
expression of Desmin remained almost unchanged whereas the expression of Col-1 slightly
increased with NR3B-PA treatment. Several issues may account for this observation: First,
the level of fibrosis varied depending on the particular locations within the liver that were
sampled. This is possible as the expressions of these genes are associated with cells that
deposit the fibers. If the sampling occurs in areas that do not contain these cells, the level of
fibrosis will be low and the expression of these fibrotic genes will not change significantly.
29

Variations within the group observed with the Pten knockout samples support this possibility.
Second, sometimes the mRNA levels cannot be directly correlated with protein expression.
Protein post-translational modifications due to extended half-life or failed degradation may
contribute to the observation that the fibrotic proteins increased while the expression of
fibrotic genes did not change. Third, diverse fibrotic proteins are produced due to activation
of hepatic stellate cells (HSCs). Activated HSCs can induce the production of collagens,
fibronectin, undulin, elastin, hyaluronan, laminin, and proteoglycans. For example, three
major types of collagens are abundantly produced during liver fibrosis, including Type I, II
and IV[15]. It is possible that other fibrotic proteins, which are not related with the four genes
upregulated in the Pten knockout mice, are reduced and contributed to fibrosis suppression by
NR3B-PA. Last, the composition and deposition of fibrous matrix varies in different stages of
fibrosis. The activation of signaling pathways regulating production or degradation of
different types of fibrotic proteins are stage-dependent and do not all occur at the same time.
Thus, it is also possible that the expression of these four genes maybe significantly changed if
the treatment duration of NR3B-PA or mouse age were different in the current study.

30

Figure 8

Figure 8. NR3B-PA treatment leads to statistically not-significant changes in fibrotic
gene expression
Expression of fibrotic genes P75ntr, Desmin, Timp-1 and Col-1 were detected using qPCR.
Inconsistent change can be observed among vehicle and PA-treated groups. Each circle
represents one animal.

Relative expression of P75ntr, Desmin, Timp-1, Col-1
in vehicle and PA-treated groups
31

Chapter 4
Inhibition of NR3B1 decreases liver injury and cell death

4.1 Introduction and rationale
Progression of NAFLD is often accompanied with progressive development of liver injury
[70]. This is particularly obvious in NASH, where elevated expression of pro-inflammatory
cytokines and accumulation of ROS can both lead to liver injury. Alanine aminotransferase
(ALT) catalyzes the conversion of alanine to glutamate is normally found in hepatocytes and
it can be released into bloodstream upon hepatocyte injury. Thus, elevated plasma levels of
ALT are regarded as a direct indicator of liver injury [71].

Clinically, increasing cell death of hepatocytes by apoptosis is frequently observed in patients
with NASH [72].Apoptosis can be activated not only by death-receptor mediated extrinsic
pathways but also mitochondrion-driven intrinsic pathways. During apoptosis, genomic DNA
is cleaved and the single strand breaks are exposed. In the terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) assay, terminal deoxynucleotidyl transferase
(TdT) polymerizes fluorescence-labeled nucleotides to free 3’-OH end of DNA breaks. This
is used to label cells that have undergone apoptosis [73].

In this chapter, studies were aimed at investigating the function of NR3B-PA treatment in
liver injury and cell death by detecting plasma ALT level and performing TUNEL assay,
respectively.
32

4.2 Results
4.2.1 Liver injury is reduced with NR3B-P A treatment
Previous research in Dr. Stiles' lab found that plasma ALT level continuously increased as a
function of age in Pten-null mice. By 9-months of age, plasma ALT was230mU/ml, and it
reached more than 250mU/ml at 12-months of age. These values were 5-fold higher than
those in wild type mice[63]. This elevated chronic liver injury was accompanied by the
progression of NAFLD from simple steatosis to NASH and, eventually, liver cancer. Indeed,
the NAFLD/NASH induced liver injury was necessary for cancer development. Fig. 9 shows
that the plasma ALT concentration in PA-treated group was approximately half that of the
vehicle-treated group (167.44mU/ml vs. 356.15mU/ml). This reduced concentration of ALT
in PA-treated group suggested that liver injury was relieved byNR3B-PA treatment.

33

Figure 9

Figure 9. Liver injury decreased when the function of NR3B1 is inhibited with
NR3B-PA
The plasma level of ALT was determined by ALT colorimetric assay. All the data were
analyzed by Excel software and displayed as Mean± S.E.M. **, P = 0.0025≤ 0.01, n=5.

Vehicle
NR3B-PA
0
100
200
300
400
500
Plasma level of ALT in vehicle vs. PA-treated group
treatments
ALT （mU/mL ）
✱✱
34

4.2.2 Decreased cell death due to inhibition of NR3B1
As shown in Fig. 10A, liver tissues were stained with TUNEL to assess cell death. White
arrows pointed to the positive-stained apoptosis events. Considerably less cell death can be
found in the liver tissues of the PA injected mice vs. the vehicle injected mice.

The percentages of dead/dying cells were quantified by counting TUNEL positive and
negative cells in selected view field. As shown in Fig. 10B, the percentage of cells stained
positive for TUNEL in the livers of the vehicle-treated mice, ranged from 2.83% to 13.61%.
These levels were significantly lower in the PA-treated livers where only 0.47% to 2.91%was
observed. These data suggest that inhibition of NR3B1 decreases numbers of dead
hepatocytes, leading to reduced liver injury.

35

Figure 10
A.

B.

DMSO

PA
Vehicle NR3B-PA
NR3B-PA
Vehicle
0
5
10
15
Tunel Assay analysis
treatments
% Tunel
stained
✱
36

Figure 10. Decreased cell death induced by loss of function of NR3B1
A. TUNEL staining of paraffin-embedded liver tissue. (PA-treated group, right panel;
vehicle-treated group, left panel)Co-localized of positive-green with DAPI-blue can be seen
as positive TUNEL stained. B. Analysis of the percentage of positive TUNEL stained cells
among total cells using Image J software. Each point represents the average of % cell among
three different microscopic graphs of one mouse. All the data were analyzed by Excel
software and displayed as Mean± S.E.M. *, P = 0.025≤ 0.05, n=5.

37

Chapter 5
Discussion
Non-alcoholic fatty liver disease (NAFLD) represents a wide spectrum of chronic liver
diseases that ranges from simple hepatic steatosis, NASH, fibrosis and cirrhosis [5]. With the
global prevalence significantly rises in recent years, NAFLD is becoming a potentially major
public health problem worldwide. Various studies have attempted to uncover novel signaling
pathways underlying the pathogenesis or progression of NAFLD to discover potential drug
targets.

Considerable research has been performed to explore the underlying mechanisms of
progression of NAFLD, as well as the intersection of NAFLD with tumor development.
These studies not only collectively broaden our knowledge on the signaling pathways
involved in malignant transformation of NAFLD, but also provided emerging targets for
therapeutics development. The targets of these treatments can be organized into several
categories, including metabolic targets, cell stress and apoptosis targets, immune targets, and
others. For example, Elafibranor (RESOLVE-IT) and Obeticholic acid (REGENERATE) are
PPARγ ligand and FXR ligand, respectively, which are two major metabolic targets of NASH.
Emricasan (ENCORE-NF) is a caspase inhibitor involved in apoptosis. Selonsertib
(STELLAR 3) acts as ASK-1 inhibitor that functions in pro-inflammatory signaling pathways
[74]. Among all ongoing studies on drugs targeting progression of NAFLD, most of them are
targeting NASH. However, metabolic targets involved into steatosis have rarely been studied.

38

The liver-specific Pten knockout mice developed by Dr. Stiles undergo progression of
NAFLD similar with humans [62, 63].The studies progresses to the discovery of a novel
signaling pathways downstream of PI3K/AKT/PTEN that regulates NR3B1, a key gene
involved in mitochondrial oxidative phosphorylation [59]. Furthermore, it was determined
that NR3B1 targeted glycerolipid biosynthesis pathways by regulating the expression of
several glycerolipid genes, therefore, playing an important role in hepatic steatosis.

In this study, 7.5month-old Pten knockout mice that developed a NASH phenotype were used
as a NASH model to investigate the effects of NR3B-PA treatment on NAFLD and NASH.
The goal was to investigate the effect of inhibiting NR3B1 on the development of NASH,
fibrosis, and liver injury.

These studies examined the effects of NR3B-PA treatment on three aspects of progression of
NAFLD/NASH. Firstly, it reduced liver steatosis and inflammation. Down-regulation of
cytokines and less immune cell recruitment indicated that the progression of NASH was
halted due to inhibition of NR3B1. Secondly, NR3B-PA treatment led to less fibrous
materials accumulation, but the changes in fibrotic genes expression were not statistically
significant. Further experiments sampling tissues from different areas or using mice of
different ages, or detecting other fibrotic genes may determine if these factors contribute to
this observation. Thirdly, decreased cell death and liver injury due to NR3B-PA treatment
suggested that NAFLD/NASH was alleviated.

39

In summary, this study demonstrated that inhibition of NR3B1 could efficiently and
significantly attenuate the progression of NAFLD and NASH in the liver-specific Pten
knockout mice. Thus, NR3B1 might be a novel potential target for therapeutics to reduce the
progression of NAFLD.

40

Chapter 6
Materials and methods

Animal and treatment
Pten
loxP/loxP
; Alb-Cre
+
( Pten-null, Pm) mice were reported[62]. Hairpin pyrrole-imidazole
polyamide (PA) was injected through intraperitoneal every three days on mice from 7 month
to 9 month. Control group was given treatment of same dose of PBS+5% DMSO every three
days from 7 month to 9 month. All experimental procedures were conducted based on the
Institutional Animal Care and Use Committee guidelines of the University of Southern
California.

RNA isolation, reverse transcription and quantitative real time-PCR
Total RNA was isolated using TRIzol (Invitrogen) following the manufacturer’s protocol.
Reverse transcription was conducted with M-MLV reverse transcriptase system (Promega).
Quantitative PCR was performed using SYBR Green qPCR Master Mix (Fermentas, Glen
Burnie, MD) and 7900HT Fast Real-Time PCR System (Applied Biosystem, Grand Island,
NY) following the manufacturer’s instructions. The cytokine genes specific primers are listed
as follow in Table 1. (With the 18S as standard) The fibrotic genes specific primers are listed
as follows in Table 2.

Immunohistochemistry
Paraffin-embedded liver sections were stained with hematoxylin and eosin (H&E) for
morphology and Sirius Red to visualize fibrotic fibers. Sirius Red staining is quantified using
Image J. The microscopy services were provided by the Liver Histology Core of the USC
Research Center for Liver Diseases, NIH grant No. P30DK048522.
41

Table 1. Primers used for qPCR detecting cytokine genes

Table 2. Primers used for qPCR detecting fibrotic genes

Gene Forward primer (5’—3’) Reverse primer (5’—3’)
TNF-α AGC CCC CAG TCT GTA TCC
TT
GGT CAC TGT CCC AGC ATC TT
IL-1β AAC CTG CTG GTG TGT GAC
GTT C
CAG CAC GAG GCT TTT TTG TTG T
IL-6 AGA AGG AGT GGC TAA GGA
CCA A
AAC GCA CTA GGT TTG CCG AGT
A
CXCL5 GCA TTT CTG TTG CTG TTC
ACG CTG
CCT CCT TCT GGT TTT TCA GTT
TAG C

Gene Forward primer (5’—3’) Reverse primer (5’—3’)
P75ntr CAG CAG ACC CAC ACA
CAG AC
TCT GTG GGG GCT AGA ACA TC
Col-1 TTT GTG GAC CTC CGG CTC AAG CAG AGC ACT CGC CCT
Timp-1 CAG TAA GGC CTG TAG
CTG TGC
CTC GTT GAT TTC GGG GAA C
Desmin CAG GAC CTG CTC AAT
GTG AA
GTA GCC TCG CTG ACA ACC TC
42

Alanine aminotransferase (ALT) activity colorimetric assay
Alanine aminotrasferase (ALT) activity was detected using Alanine aminotransferase (ALT or
SGPT) Activity Colorimetric /Fluorometric Assay Kit (Bio Vision). Serum samples are
directly diluted in ALT Assay Buffer (1:1). Positive controls are prepared by adding 5µl of
ALT positive control solution to each well and adjusting the final volume to 20µl with ALT
Assay Buffer. After adding 100µl of the Reaction Mix to each well, the orbital shaker was
used to mix solution well for 10 min. Then, incubating at 37°C, the initial measurement after
10 min was recorded as Abinitial at 570 nm. Every 5 min the measurement was taken until the
most active sample value is greater than highest standard. The graph was drafted using Excel.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay)
Apoptosis cells are labeledin situ using In Situ Cell Death Detection Kit, Fluorescein (Roche
Diagnostics GmbH). The paraffin-embedded tissue sections were first running into xylene
and series gradient of ethanol to de-paraffin. Then incubated in Proteinase K working
solution (20µg/ml in 10mM Tris/HCI, pH 7.4-8.0) for 30 min at 37°C in order to antigen
retrival. In positive control, prior to labeling procedures, sections were incubated with DNase
I (3000U/ml) for 15 min at room temperature (25°C) to induce DNA strand breaks. Each test
was treated with 5µl of TUNEL enzyme solution and 45µlof TUNEL labeling solution mixed
well. Two negative control tests are treated with 50µl of TUNEL labeling solution each
without TUNEL enzyme solution. Sections were incubated for 60 min at 37°C in humidified
chamber in dark and coverslips were added to avoid evaporative loss and ensure a equal
spread of solution over tissues.
43

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.

44

Bibliography
1. Grant, D., Detoxification pathways in the liver, in Journal of inherited metabolic disease.
1991, Springer. p. 421-430.
2. McFarlane, A., Measurement of synthesis rates of liver-produced plasma proteins.
Biochemical Journal, 1963. 89(2): p. 277.
3. Danfær, A., Nutrient metabolism and utilization in the liver. Livestock Production
Science, 1994. 39(1): p. 115-127.
4. Remmer, H., The role of the liver in drug metabolism. The American journal of medicine,
1970. 49(5): p. 617-629.
5. Angulo, P., Nonalcoholic fatty liver disease. New England Journal of Medicine, 2002.
346(16): p. 1221-1231.
6. Rinella, M.E. and A.J. Sanyal, Management of NAFLD: a stage-based approach. Nature
reviews Gastroenterology & hepatology, 2016. 13(4): p. 196.
7. Younossi, Z., et al., Global burden of NAFLD and NASH: trends, predictions, risk factors
and prevention. Nature reviews Gastroenterology & hepatology, 2018. 15(1): p. 11.
8. Chalasani, N., et al., The diagnosis and management of non ‐alcoholic fatty liver disease:
Practice Guideline by the American Association for the Study of Liver Diseases, American
College of Gastroenterology, and the American Gastroenterological Association.
Hepatology, 2012. 55(6): p. 2005-2023.
9. Nascimbeni, F., et al., From NAFLD in clinical practice to answers from guidelines.
Journal of hepatology, 2013. 59(4): p. 859-871.
10. Malaguarnera, M., et al., Molecular mechanisms involved in NAFLD progression.
Journal of molecular medicine, 2009. 87(7): p. 679.
11. Yu, J., et al. Obesity, insulin resistance, NASH and hepatocellular carcinoma. in
Seminars in cancer biology. 2013. Elsevier.
12. Wree, A., et al., From NAFLD to NASH to cirrhosis—new insights into disease
mechanisms. Nature reviews Gastroenterology & hepatology, 2013. 10(11): p. 627.
13. Ma, X. and Z. Li, Pathogenesis of nonalcoholic steatohepatitis (NASH). Chinese journal
of digestive diseases, 2006. 7(1): p. 7-11.
14. Albanis, E. and S.L. Friedman, Hepatic fibrosis: pathogenesis and principles of therapy.
Clinics in liver disease, 2001. 5(2): p. 315-334.
45

15. Bataller, R. and D.A. Brenner, Liver fibrosis. The Journal of clinical investigation, 2005.
115(2): p. 209-218.
16. Ginès, P., et al., Management of cirrhosis and ascites. New England Journal of Medicine,
2004. 350(16): p. 1646-1654.
17. Lamichhane Wagle, S., Cirrhosis of the Liver. 2019.
18. Malik, S.M., et al., Liver transplantation in patients with nonalcoholic
steatohepatitis-related hepatocellular carcinoma. Clinical gastroenterology and hepatology,
2009. 7(7): p. 800-806.
19. Yang, J.D., et al. Hepatocellular carcinoma in olmsted county, Minnesota, 1976-2008. in
Mayo Clinic Proceedings. 2012. Elsevier.
20. Kawada, N., et al., Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic
steatohepatitis. Journal of gastroenterology, 2009. 44(12): p. 1190.
21. Tokushige, K., et al., Hepatocellular carcinoma in Japanese patients with nonalcoholic
fatty liver disease, alcoholic liver disease, and chronic liver disease of unknown etiology:
report of the nationwide survey. Journal of gastroenterology, 2011. 46(10): p. 1230.
22. Michelotti, G.A., M.V . Machado, and A.M. Diehl, NAFLD, NASH and liver cancer.
Nature reviews Gastroenterology & hepatology, 2013. 10(11): p. 656-665.
23. Ipsen, D.H., J. Lykkesfeldt, and P. Tveden-Nyborg, Molecular mechanisms of hepatic
lipid accumulation in non-alcoholic fatty liver disease. Cellular and molecular life sciences,
2018. 75(18): p. 3313-3327.
24. Silverstein, R.L. and M. Febbraio, CD36, a scavenger receptor involved in immunity,
metabolism, angiogenesis, and behavior. Science signaling, 2009. 2(72): p. re3-re3.
25. Mashek, D.G., Hepatic fatty acid trafficking: multiple forks in the road. Advances in
nutrition, 2013. 4(6): p. 697-710.
26. Lambert, J.E., et al., Increased de novo lipogenesis is a distinct characteristic of
individuals with nonalcoholic fatty liver disease. Gastroenterology, 2014. 146(3): p.
726-735.
27. Postic, C., et al., ChREBP , a transcriptional regulator of glucose and lipid metabolism.
Annu. Rev. Nutr., 2007. 27: p. 179-192.
28. Reddy, J.K. and M. Sambasiva Rao, Lipid metabolism and liver inflammation. II. Fatty
liver disease and fatty acid oxidation. American Journal of Physiology-Gastrointestinal and
Liver Physiology, 2006. 290(5): p. G852-G858.
46

29. Kawano, Y . and D.E. Cohen, Mechanisms of hepatic triglyceride accumulation in
non-alcoholic fatty liver disease. Journal of gastroenterology, 2013. 48(4): p. 434-441.
30. Sheppard, K., et al., Targeting PI3 kinase/AKT/mTOR signaling in cancer. Critical
Reviews™ in Oncogenesis, 2012. 17(1).
31. Cantley, L.C., The phosphoinositide 3-kinase pathway. Science, 2002. 296(5573): p.
1655-1657.
32. Osaki, M., M.a. Oshimura, and H. Ito, PI3K-Akt pathway: its functions and alterations in
human cancer. Apoptosis, 2004. 9(6): p. 667-676.
33. Chen, C.-Y ., et al., PTEN: tumor suppressor and metabolic regulator. Frontiers in
endocrinology, 2018. 9.
34. Polak, P. and M.N. Hall, mTORC2 caught in a SINful Akt. Developmental cell, 2006.
11(4): p. 433-434.
35. Brunet, A., et al., Akt promotes cell survival by phosphorylating and inhibiting a
Forkhead transcription factor. cell, 1999. 96(6): p. 857-868.
36. Xiong, S., et al., PGC-1α serine 570 phosphorylation and GCN5-mediated acetylation by
angiotensin II drive catalase down-regulation and vascular hypertrophy. Journal of
Biological Chemistry, 2010. 285(4): p. 2474-2487.
37. Haeusler, R.A., et al., Integrated control of hepatic lipogenesis versus glucose production
requires FoxO transcription factors. Nature communications, 2014. 5(1): p. 1-8.
38. Hlobilková, A., et al., The mechanism of action of the tumour suppressor gene PTEN.
Biomedical Papers of the Medical Faculty of the University Palacky, Olomouc,
Czechoslovakia, 2003. 147(1): p. 19-25.
39. Gross, D.N., M. Wan, and M.J. Birnbaum, The role of FOXO in the regulation of
metabolism. Current diabetes reports, 2009. 9(3): p. 208-214.
40. Stiles, B.L., Phosphatase and tensin homologue deleted on chromosome 10: extending its
PTENtacles. The international journal of biochemistry & cell biology, 2009. 41(4): p.
757-761.
41. Day, C.P. and O.F. James, Steatohepatitis: a tale of two “hits”? 1998, Elsevier.
42. Peverill, W., L.W. Powell, and R. Skoien, Evolving concepts in the pathogenesis of
NASH: beyond steatosis and inflammation. International journal of molecular sciences, 2014.
15(5): p. 8591-8638.
43. Bugianesi, E., et al., Insulin resistance in nonalcoholic fatty liver disease. Current
47

pharmaceutical design, 2010. 16(17): p. 1941-1951.
44. Cusi, K., Role of insulin resistance and lipotoxicity in non-alcoholic steatohepatitis.
Clinics in liver disease, 2009. 13(4): p. 545-563.
45. Wei, Y ., et al., Nonalcoholic fatty liver disease and mitochondrial dysfunction. World
journal of gastroenterology: WJG, 2008. 14(2): p. 193.
46. EATON, S., K.B. BARTLETT, and M. POURFARZAM, Mammalian mitochondrial
β-oxidation. Biochemical Journal, 1996. 320(2): p. 345-357.
47. Rogers, G.W., et al., Assessment of fatty acid beta oxidation in cells and isolated
mitochondria. Current protocols in toxicology, 2014. 60(1): p. 25.3. 1-25.3. 19.
48. Begriche, K., et al., Mitochondrial dysfunction in NASH: causes, consequences and
possible means to prevent it. Mitochondrion, 2006. 6(1): p. 1-28.
49. Li, X., et al., Targeting mitochondrial reactive oxygen species as novel therapy for
inflammatory diseases and cancers. Journal of hematology & oncology, 2013. 6(1): p. 19.
50. Demeilliers, C., et al., Impaired adaptive resynthesis and prolonged depletion of hepatic
mitochondrial DNA after repeated alcohol binges in mice. Gastroenterology, 2002. 123(4): p.
1278-1290.
51. Farrell, G.C., et al., NASH is an inflammatory disorder: pathogenic, prognostic and
therapeutic implications. Gut and liver, 2012. 6(2): p. 149.
52. Marra, F., et al., Molecular basis and mechanisms of progression of non-alcoholic
steatohepatitis. Trends in molecular medicine, 2008. 14(2): p. 72-81.
53. Tak, P.P. and G.S. Firestein, NF-κB: a key role in inflammatory diseases. The Journal of
clinical investigation, 2001. 107(1): p. 7-11.
54. Fromenty, B., et al., The ins and outs of mitochondrial dysfunction in NASH. Diabetes &
metabolism, 2004. 30(2): p. 121-138.
55. Pessayre, D., et al. Mitochondria in steatohepatitis. in Seminars in liver disease. 2001.
Copyright© 2001 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New ….
56. Byrne, C.D. and G. Targher, NAFLD: a multisystem disease. Journal of hepatology, 2015.
62(1): p. S47-S64.
57. Villena, J.A. and A. Kralli, ERRα: a metabolic function for the oldest orphan. Trends in
Endocrinology & Metabolism, 2008. 19(8): p. 269-276.

48

58. Eichner, L.J. and V . Giguère, Estrogen related receptors (ERRs): a new dawn in
transcriptional control of mitochondrial gene networks. Mitochondrion, 2011. 11(4): p.
544-552.
59. Li, Y ., et al., Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)
signaling regulates mitochondrial biogenesis and respiration via estrogen-related receptor α
(ERRα). Journal of Biological Chemistry, 2013. 288(35): p. 25007-25024.
60. Chen, C.-y., et al., Repression of the transcriptional activity of ERRα with
sequence-specific DNA-binding polyamides. Medicinal Chemistry Research, 2020: p. 1-10.
61. Hashimoto, E., M. Taniai, and K. Tokushige, Characteristics and diagnosis of
NAFLD/NASH. Journal of gastroenterology and hepatology, 2013. 28: p. 64-70.
62. Stiles, B., et al., Liver-specific deletion of negative regulator Pten results in fatty liver
and insulin hypersensitivity. Proceedings of the National Academy of Sciences, 2004. 101(7):
p. 2082-2087.
63. Galicia, V .A., et al., Expansion of hepatic tumor progenitor cells in Pten-null mice
requires liver injury and is reversed by loss of AKT2. Gastroenterology, 2010. 139(6): p.
2170-2182.
64. Ganz, M. and G. Szabo, Immune and inflammatory pathways in NASH. Hepatology
international, 2013. 7(2): p. 771-781.
65. Negrin, K.A., et al., IL-1 signaling in obesity-induced hepatic lipogenesis and steatosis.
PloS one, 2014. 9(9).
66. Koltsova, E.K. and K. Ley, The mysterious ways of the chemokine CXCL5. Immunity,
2010. 33(1): p. 7-9.
67. Dixon, J.B., P.S. Bhathal, and P.E. O'brien, Nonalcoholic fatty liver disease: predictors of
nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology, 2001.
121(1): p. 91-100.
68. Nakano, M. and T. Fukusato, Histological study on comparison between NASH and ALD.
Hepatology research, 2005. 33(2): p. 110-115.
69. He, L., et al., Activation of hepatic stellate cell in Pten null liver injury model.
Fibrogenesis & tissue repair, 2016. 9(1): p. 8.
70. Browning, J.D. and J.D. Horton, Molecular mediators of hepatic steatosis and liver
injury. The Journal of clinical investigation, 2004. 114(2): p. 147-152.
71. Jaeschke, H., A. Farhood, and C. Smith, Neutrophils contribute to ischemia/reperfusion
injury in rat liver in vivo. The FASEB Journal, 1990. 4(15): p. 3355-3359.
49

72. Alkhouri, N., C. Carter-Kent, and A.E. Feldstein, Apoptosis in nonalcoholic fatty liver
disease: diagnostic and therapeutic implications. Expert review of gastroenterology &
hepatology, 2011. 5(2): p. 201-212.
73. Kyrylkova, K., et al., Detection of apoptosis by TUNEL assay, in Odontogenesis. 2012,
Springer. p. 41-47.
74. Friedman, S.L., et al., Mechanisms of NAFLD development and therapeutic strategies.
Nature medicine, 2018. 24(7): p. 908-922.

Abstract (if available)

Abstract PI3K/AKT signaling regulates many fundamental cellular functions, especially cell survival and cell proliferation. Abnormal activation of PI3K/AKT signaling is frequent in human cancer. PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10) was identified as a dual-specificity phosphatase that plays a significant role in antagonizing PI3K/AKT signaling. A liver-specific deletion of Pten leads to a robust activation of PI3K/AKT signaling. A liver-specific Pten-null mouse model has been successfully established using Cre-Lox recombination technology. This mouse model provides us a good observing window on the progression of chronic liver diseases into liver cancer. ❧ Estrogen-related receptor alpha (ERRα), also known as NR3B1, is an orphan nuclear receptor that plays an important role in mitochondrial biogenesis and respiration as well as lipid synthesis. Previous research reported that expression of NR3B1 was upregulated in the absence of PTEN and that a hairpin pyrrole-imidazole polyamide (PA) was an inhibitor of NR3B1 (The latter was developed in collaboration with Dr. Peter Dervan’s lab in Caltech). ❧ This research focuses on identifying the pathological effects of NR3B1inhibition on NAFLD and NASH by using the liver-specific Pten-null mouse model. Two groups were established: an experiment group with NR3B-PA injection and a vehicle group with PBS+5% DMSO injection. Characteristics of steatosis, NASH, fibrosis, liver injury and hepatocyte apoptosis were compared between these two groups. qPCR was performed to assess expression of several specific cytokine genes following a 2-month PA treatment. Compared with vehicle-treated group, inhibition of NR3B1 upregulated the expression of cytokine genes. Moreover, IHC staining with macrophage markers showed less gathering of immune cells in PA-treated group. In terms of NAFLD, H&E staining revealed less lipid droplets in PA-treated group. Through Sirius Red staining, it was found that inhibition of NR3B1 alleviated characteristics of fibrosis. Finally, the levels of liver injury (alanine transaminase (ALT) level in serum) and cell death (TUNEL assay) were also decreased upon NR3B1 inhibition. Taken together, these results suggest that inhibition of NR3B1 can significantly attenuate the progression of NAFLD and NASH.

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Asset Metadata

Creator Zhang, Xinwen (author)

Core Title Inhibition of NR3B1 attenuates the progression of NAFLD and NASH in liver-specific Pten knockout mice

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Publication Date 07/16/2020

Defense Date 07/14/2020

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

Tag fatty liver,fibrosis,NAFLD,Nash,NR3B1,OAI-PMH Harvest,PTEN,steatosis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stiles, Bangyan (committee chair), Cadenas, Enrique (committee member), Xie, Jianming (committee member)

Creator Email xinwenz@usc.edu,zhang.6641@osu.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-333398

Unique identifier UC11664352

Identifier etd-ZhangXinwe-8673.pdf (filename),usctheses-c89-333398 (legacy record id)

Legacy Identifier etd-ZhangXinwe-8673.pdf

Dmrecord 333398

Document Type Thesis

Rights Zhang, Xinwen

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