University of Southern California Dissertations and Theses

The regulation of fatty acid oxidation by estrogen related receptor alpha

(USC Thesis Other)

The regulation of fatty acid oxidation by estrogen related receptor alpha

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

The Regulation of Fatty Acid Oxidation by Estrogen Related Receptor
Alpha

by

Chenxi Xu

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 2023

ii
Acknowledgements
I would like to thank my parents Jinsong Xu and Lin Chen for financially and
spiritually supporting me during my two-year study at USC.
First, I would like to thank my PI, Dr. Bangyan Stiles for her professional, patient,
and thorough academic mentoring. She guided me on how to become a mature
researcher and inspired my creativity, resilience, and persistent passion in academia.
I also sincerely thank my committee members, Dr. Curtis Okamoto and Dr. Enrique
Cadenas for their help in revising this thesis.
Meanwhile, I would like to thank my lab mates, Lina He, Taojian Tu, Mario Alba,
Qi Tang, Ielyzaveta Slarve, Brittney Hua, Aditi Ashish Datta, Handan Hong, Yunyi Jia,
and Yiren Zhou for their support and encouragement. It was also our collaboration that
contributes to individual progress.
Finally, I would like to thank all my family and specially my friend Xinyu Mao.
Thanks for her always companion and unconditional support these years.

iii
Table of Contents
Acknowledgements ....................................................................................................... ii
List of Figures ............................................................................................................... iv
Abstract ........................................................................................................................... v
Chapter 1 Introduction ................................................................................................... 1
1.1 Introduction of ERRs ....................................................................................... 1
1.1.1 The structure and homology of ERRs ............................................................ 1
1.1.2 The crosstalk between ERRs and ERs .......................................................... 2
1.1.3 The regulation of ERRs and synthetic ligands for ERRs ................................ 3
1.1.3.1 PGC-1a/ERRa axis ................................................................................ 8
1.1.3.2 Regulation of mitochondrial gene transcription by ERRa ....................... 9
1.1.4 Contribution of ERRa to pathophysiology .................................................... 11
1.2 Fatty acid b oxidation .................................................................................... 14
1.2.1 Regulation of fatty acid b oxidation .............................................................. 14
1.2.2 Fatty acid b oxidation and liver diseases ..................................................... 17
1.3 Hypothesis and rationale of the study ......................................................... 18
Chapter 2 Materials and Methods ............................................................................... 20
Chapter 3 ERRa loss leads to reduced mitochondrial respiration and glycolysis 24
3.1 Introduction and rationale ............................................................................ 24
3.2 Results ............................................................................................................ 25
Chapter 4 ERRa loss causes damaged capability of oxidizing exogenous fatty
acids but has no impact on endogenous fatty acids oxidation ............................... 30
4.1 Introduction and rationale ............................................................................ 30
4.2 Results ............................................................................................................ 31
4.2.1 Effect of ERRa knockdown on FAO induced by exogenous FA ........... 31
4.2.2 Effects of ERRa knockdown on FAO induced by endogenous FA ....... 34
Chapter 5 Compound 29 is an effective inhibitor of ERRa in mouse hepatocytes 37
5.1 Introduction and rationale ............................................................................ 37
5.2 Results ............................................................................................................ 37
Chapter 6 Discussion .................................................................................................. 43
Bibliography ................................................................................................................. 48

iv
List of Figures
Figure 1. Structural features of ERRs. ............................................................................ 2
Figure 2. Diagram of ERR coregulators. ......................................................................... 4
Figure 3. Mitochondrial genes regulated by ERRa.. ...................................................... 11
Figure 4. Fatty acids transportation and b oxidation.. .................................................... 16
Figure 5. Seahorse XF Cell Mitochondrial Stress Test assay parameters and kinetic
profile. ............................................................................................................................ 22
Figure 6. Workflow of Seahorse XFe96 FAO Assay. .................................................... 23
Figure 7. Knock-down of ERRa leads to decreased oxidative phosphorylation and
glycolysis.. ...................................................................................................................... 29
Figure 8. Decreased oxidation of exogenous fatty acids but not endogenous fatty acids
was observed in ERRa-knockdown human hepatocytes.. ............................................. 36
Figure 9. C29 is an effective inhibitor of ERRa in mouse hepatocytes.. ....................... 42

v
Abstract
Estrogen related receptors (ERRs) are orphan nuclear receptors involved in the
transcriptional regulation of mitochondrial bioenergetics. ERRa is a dominant isoform in
the liver. As a transcriptional factor, ERRa binds to co-activators like peroxisome
proliferator-activated receptor g coactivator 1-a (PGC-1a) to regulate mitochondrial
respiratory genes such as cytochrome C (Cyt C) and thus plays key roles in oxidative
phosphorylation (OXPHOS). As such, loss of ERRa function is expected to result in lower
OXPHOS and suppress catabolism, leading to metabolic disorders. Given to the
emerging evidence that dysregulated fatty acid oxidation (FAO) exacerbates the
progression of liver diseases, we explored the functional role of ERRa in FAO. Our data
demonstrated that ERRa plays a role in exogenous lipid usage but does not affect FAO
of endogenous lipid oxidation. In addition, previous study from our lab also showed that
the deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)
which activates PI3K/AKT pathway regulates mitochondrial function via ERRa and Pten
deletion increased FAO capacity. Here, we also investigated whether ERRa participates
in the PI3K/AKT pathway regulated FAO.

1
Chapter 1
Introduction
1.1 Introduction of ERRs
1.1.1 The structure and homology of ERRs
Nuclear receptors (NRs) function as major regulators of gene expression. The
orphan estrogen-related receptor (ERR) subfamily of NRs that does not have known
binding ligands include ERRa, ERRb, ERRg. The molecular structure of these ERRs are
almost identical in the DNA-binding domain (DBD) (Fig.1). The central zinc finger at the
DBD binds to TCAAGGTCA, known as the ERR response element (ERRE)
1
. The three
ERRs also share structural similarities in their (N-terminal domain) NTDs
1
that contains
an activation domain-1 (AF-1), typical of ligand-independent transcriptional activation.
The AF-1 domain contains several conserved motifs which enables transcriptional activity
to be regulated by posttranslational modifications. The hinge region provides the protein
flexibility when ERR binds to DNA as a dimer
2
. The ligand-binding domain (LBD) contains
a conserved helix motif activation function-2 (AF-2), which is exposed. LBD, together with
the AF-2, communicates with coregulators in the absence of ligand binding, which is
ascribed to a conformation of LBD that recruits those coactivators
3
. The three ERR
isoforms show distinctive characteristic of their ligand binding pocket
4
, leading to their
individual regulation modes. Interestingly, the LBDs of ERRa and ERRg could be highly
purified from E. coli for crystallization studies, while the ERRb LBD is barely soluble or
stable. Yao et al.
5
identified the residues that contributes the poor solubility of ERRb LBD.
2
A single mutation of tyrosine which is present at Y215 position of ERRb to histidine forms
hydrogen bonds, was found to contribute to its solubility.

1.1.2 The crosstalk between ERRs and ERs
The ERR subfamily are structurally related to the estrogen receptors (ERa and
ERb) and share sequence similarity with these receptors, but they do not bind estrogen,
the endogenous ER ligand. The LBDs of ERRs and ERa share 30-40% homology
6
, while
68% homology are shown in DBDs. ERa and b bind as homodimers to the estrogen
response elements (ERE), AGGTCAXXXTGACCT, where an inverted repeat of the
common NR hexameric half-sites is separated by three nucleotides. On the other hand,
ERRs can bind as monomers to both ERREs (TCAAGGTCA) and the closely related half
ERE sites (AGGTCA) embedded within an ERRE sequence on DNA. In addition, ERRa
can form either homodimers or heterodimers with ERa. Meanwhile, ERRg has been
reported to form homodimer
7
. Chromosome- and genome-wide location analysis
indicated that the binding sites of ERRs located within the promoter regions of target

Figure 1. Structural features of ERRs. Each ERR contains an NH2-terminal region that holds a
ligand-independent transcriptional activation function (AF-1) (A/B); a DNA-binding domain (DBD)
containing two highly conserved zinc finger motifs (C); a hinge region (D); a ligand-binding domain
(LBD) containing a conserved AF-2 helix motif (E, F).

3
genes are proportionally broader than those of ERa
8
. This functional distinction benefits
ERRs in identifying their target genes.

1.1.3 The regulation of ERRs and synthetic ligands for ERRs
Instead of ligand binding, the transcriptional activity of ERRs depends on the
presence of either coactivating or co-repressing proteins for its activation
9
. Serving as the
co-activators, steroid receptor coactivator (SRC)-1,2,3, PPARg coactivator (PGC)-1a and
PGC-1b positively regulate the expression and activity of these ERRs (Fig. 2). In lung
type II cells, SRC-2, ERRa, and protein kinase A catalytic subunit (PKAcat) interacted at
the ERRE
10
, which was enhanced by cAMP and inhibited by the PKA inhibitor, H89.
Brown et al.
11
found that overexpression of PGC-1a and PGC-1b increases protein
synthesis and myotube hypertrophy in C2C12 myotubes and this effect is dependent on
ERRa. Meanwhile, receptor interacting protein 140 (RIP140), also termed nuclear
receptor interacting protein 1 (NRIP1) was found to compete with PGC-1a for interaction
with ERR binding, leading to inhibition of ERR regulated transcriptional activity
9
. The
interaction between RIP40 and ERRa is demonstrated in both in vitro and in vivo studies.
In these studies, ERRa activates RIP140 gene transcription, directly via ERRE binding or
indirectly dependent on specificity factor 1 (Sp1) binding sites in the proximal promoter.
Meanwhile, increased RIP140 gene expression in response to ERRa serves as a
negative regulator of glucose uptake in mice
12
. Guanine-nucleotide binding protein 3-like
(GNL3L), as the closest homologue of a stem cell-enriched factor nucleostemin,
specifically binds to ERRg through the interaction between its intermediate (I) domain and
4
the AF2 domain of ERRg
13
. GNL3L competes with SRC1 and SRC2 for ERRg binding
which depends on the I-domain of GNL3L.

As demonstrated from the cellular mechanism of transcriptional modulation, ERRa
is an acetylated protein whose dynamic acetylation/deacetylation switch controls its
transcriptional activity
14
. DBD of ERRa is acetylated by p300 coactivator associated factor
(PCAF), which reduces its transcriptional function. On the other hand, histone
deacetylase 8 (HDAC8) and sirtuin 1 homolog (Sirt1) are identified as activators for ERR
transcriptional activity. Further, Parkin, an E3 ubiquitin ligase, interacts with and
ubiquitinates ERRa, increasing the degradation rate of ERRa
15
. However, the effect of
Parkin on ERRa gene transcription varies among tissues. Shires et al.
15
determined the
presence of nuclear Parkin increases the transcriptional activity of endogenous ERRa in
HeLa cells as indicated by the elevated ERRa transcript (ESRRA) levels. In neuronal
cultures, however, Ren et al.
16
proposed that the absence of Parkin benefits ERRa

Figure 2. Diagram of ERR coregulators. Steroid receptor coactivator (SRC)-1, 2, 3 and PPARg
coactivator (PGC)-1a and PGC-1b positively regulate the expression and activity of ERRs. Receptor
interacting protein 140 (RIP140) negatively regulates ERR activity.

5
transcription revealed by increased target genes Monoamine Oxidase A and B in brain
sections. Additionally, F-box and leucine-rich repeat protein 10 (FBXL10), acting as a
ubiquitin ligase, was discovered as a positive regulator of ERRa transcriptional activity
that enables the enrichment of ERRa at the promoter region of its target genes
17
.
Furthermore, enhancer of zeste homolog 2 (EZH2), a methyltransferase was
shown to specifically and negatively regulates the histone methylation of ERRs in breast
carcinoma
18
.
ERRs do not respond to natural estrogens such as ER agonist 17b-estradiol and
no endogenous ligands for ERRs have been identified. A list of compounds (Table.1)
have been reported to modulate the transcriptional activities of ERRs, serving as potential
synthetic ligands. Diethylstilbestrol (DES) is a synthetic nonsteroidal form of the female
hormone estrogen. DES acts as an inverse agonist for all ERR subtypes
19
. Recently, it
was shown that DES treatment induces discrete cluster formation and reduces
intranuclear mobility of ERRs. Among all three subtypes, ERRg uniquely displays nuclear
export and relatively high mobility in response to DES
20
. Previously, Coward et al.
19

showed that DES specifically binds to ERRg with submicromolar affinities, while low
affinity binding with ERRa was observed. Of note, DES treatment results in colocalization
of all three ERR isoforms with scaffold attachment factor B1 (SAFB1) in the nuclear matrix,
explaining DES-mediated transrepression of ERRs
20
.
4-hydroxytamoxifen (4-OHT) is a metabolite of the antiestrogen, tamoxifen (TAM).
Computational docking study revealed that the phenol part of 4-OHT has intact hydrogen
bonding with Arg316 and Asp275 at the binding pocket of LBDs of ERRb and ERRg
21
.
The residue located at the bottom of helix 3 in ligand binding pocket of ERRb and ERRg
6
is an alanine, but a phenylalanine in ERRa, which accounts for the selectivity of 4-OHT
towards ERRb/g. A single mutation of phenylalanine at ERRa position 232 to alanine was
sufficient to successfully bind ERRa with 4-OHT at similar concentration to that of ERRg
19
.
Given that 4-OHT possesses a poor inverse agonist profile, Yu et al.
22
aimed at
developing higher potency antagonists based on Z-4-OHT analogs which binds to the
LBD of ERRb and ERRg but not ERRa. Hydroxyl group (4-OH) was either removed or
replaced with small acetyl and alkyl substituents. Their effort identified compound DY40
as the most potent ERRg selective inverse agonist and compound DY181 as an isoform-
specific repressor for ERRb.
Bisphenol A (BPA) is a chemical primarily used in the production of polycarbonate
plastics. It is known as an endocrine disruptor that weakly binds ERa and ERb compared
to natural hormone 17b-estradiol. Interestingly, BPA binds strongly to ERRg. However,
Matsushima et al.
23
uncovered that BPA somehow does not affect the transcriptional
activity of ERRg. One of the two phenol-hydroxyl groups in BPA is essential for
incorporation in the ligand-binding pocket of ERRg but causing little conformational
change. Meanwhile, a chlorine-containing BPA derivative, bisphenol C was recently
discovered to bind preferentially to ERRa as an agonist and ERRb as an antagonist
24, 25
.
Several ERRa exclusively targeted compounds have been reported. XCT790,
thiadiazoleacrylamide is an ERRa inverse agonist, initially discovered by Busch et al.
26
.
It was reported that XCT790 were able to increase mitochondrial reactive oxygen species
(ROS) production by elevating membrane potential and down-regulating superoxide
dismutase (SOD) expression
27
. C29, an analogue of diary ether-based thiazolidinedione,
is another ERRa inverse agonist. In vivo animal study indicated that oral administration
7
of C29 maintained homeostasis of insulin and circulating triglyceride levels
28
.
Furthermore, the statins were recently identified as ERRa agonists in a screening of the
Tox21 compound library
29
. Transfected cell lines were developed in high-throughput
screening. These cell lines, express either ERRa multiple hormone response element
reporter (ERR) alone or both ERR and lentiviral PGC-1a (PGC/ERR)
30
. Notably, these
statins are active in the ERR reporter assay but not PGC/ERR assay, implying that they
modulate activation through the receptor and not the coactivator PGC-1. A small synthetic
agonist compound named JND003 which targets the liver-specific ERRa, was shown to
improve insulin sensitivity and enhance FAO and mitochondrial function
31
. Py-Im
polyamides represents a group of synthetic DNA minor groove-binding ligands with
sequence selectivity. Polyamide sequence designed to bind TCAAGGTCA is able to
block the binding of ERRa to the consensus ERRE and reduce the transcriptional activity
and OXPHOS function regulated by ERRa
32
. This compound was also shown to block
the development of diet and genetic driven NAFLD and reverse NASH in a genetic
model
33
.

Table 1. Synthetic compounds binding to ERRs.
8
1.1.3.1 PGC-1a/ERRa axis
It’s worthwhile to take an in-depth look into the physiological impact of ERRa/PGC-
1a on metabolism as they are abundantly reported and the findings are often
contradictory. The mRNA and protein levels of PGC-1a and ERRa were both significantly
higher in the high fat diet (HFD)-induced obesity mouse models than HFD-induced
obesity resistant ones
34
. Previous study also reported that Esrra deletion mice with
impaired mitochondrial function are prevented from HFD-induced insulin resistance
33
.
However, Rinnankoski-Tuikka et al.
35
pointed out that instead of decreased qualitative or
quantitative properties of mitochondria, insulin resistance are supposed to be a
consequence of the inhibition of pyruvate dehydrogenase by pyruvate dehydrogenase
kinase-4 (PDK4) via continuing activation of PGC-1a/ ERRa under chronic high fatty acid
availability. Chronic hyperactivation of AMPK/PGC-1a/ERRa axis is also credited for the
protected effects from HFD-induced obesity in adipose-specific Folliculin knockout mice
36
.
PGC-1a itself is identified as a potent activator for hepatic gluconeogenesis,
specifically activating the transcription of gluconeogenic enzyme phosphoenolpyruvate
carboxykinase (PEPCK) gene. In contrast to the acknowledged co-stimulating effects of
ERRa and PGC1a on mitochondrial respiration, ERRa acts as a repressor of the PEPCK
gene. Mechanistically, ERRa functions to antagonize PGC-1a at this gene promoter. This
finding provides an alternative explanation for the benefiting effects on diabetes via
enhancing ERRa activity, where hepatic glucose production is suppressed
37
. PGC-
1a/ERRa axis is also a key effector of metabolic reprogramming in cancer. ERRa, in
parallel with PGC-1a was found to negatively regulate one-carbon metabolism where the
9
biosynthesis of purine is perturbated, which sensitize breast cancer cells to the anti-folate
drug methotrxate
38
.
Other investigations further establish a link between ERRa/PGC-1a axis and
disease progression. It was reported that ERRa mediates PGC-1a’s induction of
CYP11A1, a key rate-limiting enzyme involved in the initialization of steroidogenesis
39
.
AMPK-PGC1a/ERRa signaling pathway activated by dihydromyricetin, increases SIRT3
expression which ameliorates mitochondrial dysfunction in HFD-induced NAFLD
40
.
Mehlem et al.
41
identified Vegfb as a downstream target of PGC-1a/ERRa axis. It was
uncovered that ablation of Vegfb in HFD-fed mice overexpressing PGC-1a reversed
glucose intolerance, insulin resistance, and dyslipidemia.

1.1.3.2 Regulation of mitochondrial gene transcription by ERRa
ERRa activation impacts other genes involved in mitochondrial system such as
nuclear respiratory factor 1/2 (NRF1/2), cytochrome C (Cyt c), PPARa and medium-chain
acyl-CoA dehydrogenase (MCAD) etc (Fig. 3). These mitochondrial target genes are
associated with a series of energy production pathways including glycolysis and
cholesterol metabolism, fatty acid uptake, fatty acid oxidation, and mitochondrial electron
transporter chain. Like ERRs, NRF-1 is a major transcriptional factor in mitochondrial
biogenesis. However, overexpression of NRF-1 itself failed to induce such biogenesis in
skeletal muscle
42
. Mootha et al.
43
proposed that NRF-1 induced by PGC-1a participates
downstream of the ERRa-mediated to regulate mitochondrial events. In other words,
NRF-1 acts as a relatively late mediator of the PGC-1a induced mitochondrial
transcriptional program compared to ERRa. Nonetheless, in brown adipose tissue, levels
10
of NRF1/2 levels are not altered by ERRa knockout, suggesting potential tissue specific
relationships of ERRs and NRFs
44
. Cyt c functions in the respiratory chain by collecting
electrons from complex I, II and III and donating electrons to complex IV. Huss et al.
45

demonstrated that ERRa induced the expression of Cyt c in cardiac myocytes, though
less robust than PGC-1a mediated regulation. In the same study, ERRa was found to
activate mitochondrial fatty acid oxidation by directly binding to the PPARa gene promoter,
acting within the transcriptional complex of the PGC-1a regulatory network
45
. MCAD is
an enzyme involved in mitochondrial FAO. A consensus ERRE sequence is present in
the promoter nuclear receptor response element 1 (NRRE-1) of MCAD. ERRa thus
controls MCAD expression through NRRE-1 as indicated by the study where a VP16-
ERRa chimera activates both natural and synthetic NRRE-1 promoters
46
.
Glycerophosphate acyltransferase (GPAT) remains the rate-limiting enzyme in the de
novo pathway of glycerolipid synthesis. Among its isoforms, GPAT1 and GPAT4 have
been identified as transcriptional targets of ERRa
33
.

11

1.1.4 Contribution of ERRa to pathophysiology
The three ERR isoforms are differentially distributed in various tissues. They are
associated with various metabolic events such as glucose and glutamine metabolism,
lipid disposal, mitochondrial activity, and energy sensing. ERRb is highly expressed in
embryonic tissues, regulating placental development and stem cell maintenance. ERRa,
together with ERRg, are found abundantly expressed in high-energy consuming organs
including the heart, skeletal muscle, and rich in the cortex, hippocampus, etc. Of note,
Figure 3. Mitochondrial genes regulated by ERRa. PGC-1a binds to ERR as a coactivator,
entering subsequent process. It further impacts mitochondrial biogenesis transcriptional genes such
as NRF1/2. Meanwhile, genes encode proteins involved in mitochondrial oxidative phosphorylation,
b oxidation, including Cyt c, PPARa, and MCAD.

12
ERRa is the dominant isoform in the liver, upregulated during liver steatosis compared to
healthy state
33
.
Given the role of ERRs on the transcriptional regulation of mitochondrial and
metabolic genes, studies have focused on the roles of ERRs in the pathogenesis of
metabolic diseases. However, the pathological and physiological consequence of these
ERR-regulated functions vary depending on the nutritional state
47
. In the liver, compared
to the prevention of NAFLD in HFD-treated ERRa-null mice
47
, ERRa ablation showed no
impact on fasting-induced NAFLD
47
. Of note, ERRa is responsible for the acute
refeeding-mediated hepatic triglyceride (TG) accumulation reversal in the fasting
condition, indicated by adipose TG lipolysis and impaired hepatic mitochondrial oxidative
activity. In addition, ERRa-mediated very low-density lipoprotein (VLDL) secretion was
also found to contribute to the sex disparity in NAFLD development
48
.
Additionally, excessive production of reactive oxygen species (ROS) serves as a
contributing factor to the pathogenesis of cancer, inflammatory diseases, and
neurodegeneration, etc. ERRa plays a significant role in the control of ROS-related
metabolism. Inhibiting ERRa decreases total cellular ROS levels, which can be ascribed
to the metabolic rewiring and shifting of glutamine flux away from TCA cycle entry and
towards its utilization for glutathione production
49
. Xia et al.
50
showed that knockdown of
ERRa enhanced the downregulation of Sirt3, a primary mitochondrial deacetylase
expression upon LPS stimulation, restricting mitochondrial-derived ROS production. The
PGC-1a stimulation of mouse Sirt3 gene expression in hepatocytes is also mediated by
ERRE
51
. ERRa, together with PGC-1a, co-localizes to the mouse Sirt3 promoter to
upregulate its transcription. As a result, Sirt3 promotes the induction of ROS-detoxifying
13
enzymes and reduction of ROS level by PGC-1a aside from suppressing the production
of basal ROS in cells. In Lapatinib-resistant breast cancer cells, ERRa expression and
protein levels were found significantly increased
52
. In these cells, ERRa knockdown led
to down-regulated glutathione to glutathione disulfide (GSH/GSSG) ratio and elevated
ROS production, indicating suppressed cell detoxification and enhanced oxidative
damage. In summary, ERRa induction may contribute to the occurrence of ROS-
associated diseases.
Furthermore, concurrent upregulation of PGC-1a/ERRa and pyruvate
dehydrogenase kinase, isozyme 4 (PDK4) was observed in chronic non-suppurative
destructive cholangitis (CNSDC) of primary biliary cirrhosis (PBC). The pathogenesis of
CNSDC is ascribed to the switching of cellular energy systems from glycolytic dependent
to dependence on mitochondrial FAO
53
. In mitochondria, pyruvate was oxidized to acetyl-
CoA by pyruvate dehydrogenase complex (PDC). The PGC-1a/ERRa axis interrupts
glycolysis via upregulating the expression of PDK4. PDK3 and PDH complex occur in
mitochondria. Regulation of glycolysis (glucose to pyruvate) occurs in cytosol. This means
that pyruvate accumulates because of the mitochondrial activity of PDK4. Accumulation
of pyruvate inhibits glycolysis and increases mitochondrial FAO
54
. ERRa inhibition can
also prevent pyruvate from entering mitochondria by means of inhibiting mitochondrial
pyruvate carrier 1 (MPC1) expression, resulting in enhancement of glutamine oxidation
and pentose phosphate pathway in breast cancer
55
. In other words, ERRa is essential for
PGC-1a-mediated stimulation of MPC1 transcription, leading to pyruvate-dependent
mitochondrial oxygen consumption
56
.

14
1.2 Fatty acid b oxidation
1.2.1 Regulation of fatty acid b oxidation
For catabolism of fatty acids, medium and long chain fatty acids (LCFA) stored in
the cytosol are transported into the mitochondria matrix where they can be oxidized. The
enzymes responsible for the transfer of fatty acids into the mitochondria are termed L-
carnitine acetyltransferases that catalyze the reversible transfer of acyl groups between
CoA and L-carnitine, converting acyl-CoA esters into acyl-carnitine esters and vice versa.
This exchange step between CoA and carnitine is essential for oxidation of LCFA to occur
in the mitochondrial matrix as the mitochondrial inner membrane is impermeable to LCFA.
Human L-carnitine acyltransferases include carnitine acetyl-transferase (CRAT),
carnitine-octanolytransferase (CROT), CPT1, and CPT2. Each of these enzymes has
preferences for specific chain length esters and specific subcellular localizations. The
medium and long acyl-CoA chains only use CPT1 and CPT2 to enter the mitochondrial
matrix and for further metabolism through the b-oxidation cycles. After being convert to
Acyl-CoA via the actions of Acyl-CoA synthases (ACS), the acyl-CoA groups (12-18
carbons chain lengths) are transferred to L-carnitine to form acyl-carnitine esters via the
actions of CPT1 located in the outer mitochondrial membrane. These newly made acyl-
carnitines are then translocated into the mitochondria matrix in exchange for free carnitine.
This translocation step is mediated by the enzyme carnitine acylcarnitine translocase
(CAT). Once inside the mitochondrial matrix, acyl groups are converted back to CoA by
the CPT2 enzyme. Fatty acid b-oxidation then takes place in the mitochondria, which
sequentially removes two-carbon units from the acyl chain each cycle and produces final
the product acetyl-CoA. Acetyl-CoA enters the tricarboxylic acid (TCA) cycle to generate
15
the electron equivalent NADH/FADH2, to support ATP production via electron transport
chain (Fig. 4).
PGC-1a plays a dominant role in organizing the transcriptional regulation of FAO.
Upregulated PGC-1a activates FAO transcriptional regulatory genes ERRa and NOR1,
and also induces the expression of the fatty acid transport gene CD36 and the three
mitochondrial b-oxidation genes CPT1, MCAD, ACO
57, 58
. During the development of liver
steatosis, hypomethylated PGC-1a decreases its binding with PPARa, ERRa, and HNF-
4a. This reduced PGC-1a binding to the transcriptional factors led to impaired
mitochondrial FAO, accounting for liver steatosis
59
. Reduced FAO is also associated with
the progression of clear cell renal cell carcinoma (ccRCC) progression as repressing
CPT1A was shown to be essential for reducing fatty acid transport into the mitochondria
and resulting in lipid deposition
60
. However, accelerated FAO is also related to tumor
progression. In tamoxifen (TAM)-resistant breast cancer cells (TRC) derived from the ER+
MCF7 cell lines, both mRNA and protein expression level of ERRa and PGC-1b increased
compared to the parental MCF7 cells
61
. TRC displayed increased fatty acid oxidation,
which could be attributed to AMPK-mediated expression of ERRa and PGC-1b and
subsequent lipid metabolism gene expression of MCAD/CPT1. In H-ras oncogene
transfected MCF10A human breast epithelial cells, downregulation of c-Myc/PGC-
1b/ERRa pathway led to advanced cancer progression. Inhibited FAO protein expression,
including PPARa, MCAD and CPT1C, as well as restriction of OXPHOS complexes are
observed in these cells
62
.
16

Figure 4. Fatty acids transportation and b oxidation. Fatty acids are stored as triglycerides in
adipose tissue. Acyl-CoA synthases (ACS) convert LCFA into Acyl-CoA, which is a substrate for the
CPT1 enzyme. The enzymes that catalyze the transfer of fatty acids into the mitochondria for
oxidation are called L-carnitine acetyltransferases and they catalyze the reversible transfer of acyl
groups between CoA and L-carnitine, converting acyl-CoA esters into acyl-carnitine esters and vice
versa. The medium and long acyl-CoA chains need to use CPT1 and CPT2 to enter the mitochondrial
matrix and proceed to the b-oxidation cycles. The CPT1 enzyme catalyzes the reversible transfer of
acyl-CoA groups to L-carnitine to form acyl-carnitine esters. These newly made acyl-carnitines are
then translocated into the mitochondria matrix in exchange for free carnitine. This translocation step
is mediated by the enzyme carnitine acylcarnitine translocase (CAT). Once inside the mitochondrial
matrix, acyl groups are transferred back to CoA by the CPT2 enzyme. The products of b-oxidation
are acetyl-CoA, NADH/FADH2. NADH/FADH2 can further enters electron transport chain to support
ATP production.

17
1.2.2 Fatty acid b oxidation and liver diseases
Non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease
(ALD) are two major chronic liver diseases worldwide. Both NAFLD and ALD are
pathological conditions that include simple steatosis as well as more severe
steatohepatitis. These conditions can further progress to irreversible cirrhosis and even
hepatocellular carcinoma (HCC). Hepatic steatosis serves as an early sign of both NAFLD
and ALD, typical of lipid droplets accumulation in hepatocytes. The development of
hepatic steatosis can be attributed to several mechanisms such as increased free fatty
acid uptake, de novo lipogenesis, triglyceride (TAG) synthesis, as well as decreased fatty
acid b oxidation and very low-density lipoprotein (VLDL) secretion. Hepatic triglyceride
hydrolysis and FAO are interdependent processes and their discordance leads to ultimate
liver disease
63
. Studies show that loss of either Cpt2 or adipose triglyceride lipase (Atgl)
resulted in steatosis and loss of both caused significant inflammation and fibrosis
characteristic of steatohepatitis
63
. Chaveroux et al.
64
proposed that ERRa is a key
determinant of rapamycin-induced NAFLD. They found that ERRa-deficient mice showed
an increase in p-acetyl CoA carboxylase (ACC), suggesting a decrease in the conversion
of acetyl-CoA to malonyl-CoA catalyzed by ACC. Such observation suggests reduced
fatty acids biosynthesis followed by activation of FAO. Furthermore, rapamycin treatment
leads to the degradation of ERRa and administration of rapamycin to ERRa-null mice
exacerbated NAFLD compared to WT mice. Similarly, Chen et al.
33
also showed that
inhibiting ERRa dramatically suppressed the development of NAFLD and nonalcoholic
steatohepatitis (NASH). Meanwhile, glycerolipid synthesis in addition to de novo
18
lipogenesis was found to be the alternative mechanism accounting for ERRa-regulated
NAFLD/NASH development.
Liver metabolism impaired in human patients can be reproduced in mice injected
with lipopolysaccharide (LPS). LPS generates potent inflammatory cytokine response,
which induces reduced serum high density lipoprotein (HDL) and impairs FAO
65
.

1.3 Hypothesis and rationale of the study
The preservation of liver function plays a crucial role in maintaining overall health,
as it serves as a vital metabolic organ. Nevertheless, unhealthy behaviors like
uncontrolled consumption of caloric rich foods or alcohol contribute to the development
of fatty liver disease, characterized by the accumulation of fat deposits. If left untreated,
the impaired liver undergoes a perilous pathological progression, wherein liver fibrosis,
cirrhosis, and ultimately malignant liver cancer manifest sequentially. Emerging evidence
points out the importance of dysregulated fatty acid oxidation in the development and
progression of liver diseases
66
. As a dominant ERR isoform in the liver, loss of ERRa is
expected to result in lowered OXPHOS and suppressed catabolism, and lead to metabolic
disorders. However, in vivo animal studies indicate that mice lacking ERRa were resistant
to metabolic disorders including HFD-induced obesity and NAFLD
67
. The contradictory
evidence demands detailed analysis of the diverse functions regulated by ERRa and its
context dependency. Accordingly, we proposed to explore the functional role of ERRa in
FAO. As ERRa have been shown to transcriptionally regulate MCAD, we propose that
ERRa downregulation will result in decreased FAO in hepatocytes. Furthermore, our lab
previously showed that the deletion of phosphatase and tensin homolog deleted on
19
chromosome 10 (PTEN) which negatively regulates the insulin responsive PI3K/AKT
signaling pathway regulates mitochondrial function via ERRa
68
. Our data also showed
that Pten deletion increased FAO capacity
69
. Thus, we will also address whether ERRa
participates in the regulation of FAO by PI3K/AKT pathway.

20
Chapter 2
Materials and Methods
Cell Culture
Human hepatocyte Huh-7 cell line and ERRa knockdown (sh-ERRa) Huh-7 cell
line were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum (FBS). Mouse hepatocytes cell lines wild-type (WT) and Pten-
null (PM) were cultured in DMEM supplemented with 10% FBS, 5µg/ml insulin and
10ng/ml epidermal growth factor (EGF). The hepatocytes were then incubated in 37°C,
5% CO2 incubator.

Western Blot
Cells were harvested and lysed in 10% sodium dodecyl sulfate (SDS). Lysates
with equal amount of protein were then loaded on the SDS-PAGE and transferred to
PVDF membranes. The membranes were further incubated with ERRa antibody.

C29 treatment
Different concentrations (500nM, 1µM and 5µM) of C29 were tested first to
determine the most efficient inhibition concentration. 5µM C29 was added into each 96
well and cells were treated for 24 hours before starvation and analyzed of oxygen
consumption on Seahorse XFe 96 Analyzer.

RNA isolation, reverse transcription and quantitative real-time PCR
21
Total RNA of mouse hepatocytes was extracted using Trizol. RNA was then
reverse transcribed using M-MLV reverse transcriptase system. Synthesized cDNA was
amplified by quantitative real-time PCR with SYBR Green Master Mix on a instrument
following the manufacture’s instructions. Gene-specific primers are listed below. GAPDH
was used as a standard.

Seahorse XFe96 Cell Mitochondrial Stress Test
Hepatocytes were transferred from T75 cell culture flasks to XF-96 Cell Culture
Microplates (1,000 cells per well) and cultured overnight in growth medium. Meanwhile,
a sensor cartridge was hydrated using Seahorse XF Calibrant at 37°C in a non-CO2
incubator overnight. On the day of assay, wash cell twice with XF assay medium. Assay
medium used was Seahorse XF DMEM supplemented with 1mM pyruvate, 2mM
glutamine, and 10mM glucose. A final volume of 180µL assay medium. 20µL 15µM
Oligomycin and 22µL 10µM FCCP were added into certain drug loading ports on sensor
cartridge, respectively. Oxygen consumption rate (OCR) and Extracellular acidification
rate (ECAR) were measured simultaneously in Seahorse XFe96 Analyzer.

22

Starvation Treatment
After overnight culture in XF-96 plates to allow attachment, cells were washed
twice with substrate-limited medium in place of growth medium. Substrate-limited medium
contains Dulbecco’s Modified Eagle’s Medium without glucose, glutamine, sodium
pyruvate, or HEPES supplemented with 0.5mM glucose, 1.0mM GlutaMax, 0.5mM L-
carnitine, and 1%FBS. A final volume of 100µL substrate-limited medium was added to
each well. The cell plates were put back to 37°C, 5% CO2 incubator and starved 24hrs.

Seahorse XFe96 Fatty Acid Oxidation Assay
After 24hrs starvation, cell plates were retrieved from the incubator. Cells were
washed twice with assay medium which was XF DMEM supplemented with 2mM glucose
and 0.5mM L-carnitine. A final volume of 135µL assay medium per well was used for
assay. 90 minutes prior to the assay, 15µL 400µM etomoxir or vehicle was added to the
cells. After incubation in non-CO2 incubator for 30 minutes, 30µL palmitic acid or BSA
was added to certain well for an additional 1hr incubation. Cell plate was then placed on
the Seahorse XFe96 Analyzer, following the running protocol as cell mitochondrial stress
test. 8µM Oligomycin and 1µM FCCP were sequentially injected into each well to measure
Figure 5. Seahorse XF Cell Mitochondrial Stress Test assay parameters and kinetic profile.
Basal respiration: Oxygen consumption used to meet cellular ATP demand resulting from
mitochondrial proton leak. Shows energetic demand of the cell under baseline conditions. ATP
Production: The decrease in oxygen consumption rate upon injection of the ATP synthase inhibitor
oligomycin represents the portion of basal respiration that was being used to drive ATP production.
Shows ATP produced by the mitochondria that contributes to meeting the energetic
needs of the cell. Proton leak: Remaining basal respiration not coupled to ATP production. Maximal
respiration: The maximal oxygen consumption rate obtained by adding the uncoupler FCCP, which
causes rapid oxidation of substrates (sugars, fats, and amino acids) to meet this metabolic challenge.
Shows the maximum rate of respiration that the cell can achieve.
23
OCR. Proteins were extracted using 1%SDS to normalize cell numbers. Data were
analyzed by XF software and displayed as an average value of replicate wells +/- S.E.M.

Figure 6. Workflow of Seahorse XFe96 FAO Assay. Human hepatocyte cell lines are cultured in
T75 until the confluence reaches 70-90%. Cells are then transferred into Seahorse cell culture
microplates and cultured overnight. On the next day, change the cell growth medium into substrate-
limit medium for starvation. After 24 hours starvation, cells are ready for FAO assay. Cell numbers
are normalized at protein level via BCA assay.
24
Chapter 3
ERRa loss leads to reduced mitochondrial respiration and glycolysis
3.1 Introduction and rationale
Oxidative phosphorylation (OXPHOS) takes place in the mitochondria. OXPHOS
generates high-energy phosphate bonds by means of oxygen reduction, serving as the
connection between the tricarboxylic acid (TCA) cycle and the production of adenosine
triphosphate (ATP). A series of oxidation-reduction reactions facilitate transfer of
electrons from reduced nicotinamide adenine dinucleotide (NADH) or reduced flavin
adenine dinucleotide (FADH2) to oxygen throughout the electron transporter chain (ETC).
Five main protein complexes involved in OXPHOS are NADH-coenzyme Q
oxidoreductase (Complex I), Succniate-Q oxidoreductase (Complex II), Q-cytochrome c
oxidoreductase (Complex III), Cytochrome c oxidase (Complex IV), and ATP synthase.
The integrity of these enzymes ensures the final energy production.
Many chemicals have been developed to target one or the other components of
these ETC complexes in an effort to study mitochondrial function. Oligomycin reduces
respiration by inhibiting ATP synthase. This causes an accumulation of protons outside
the mitochondria, disturbing proton transport and reducing oxygen uptake. However, the
electron flow is not completely stopped due to proton leak, a process where protons can
still migrate to the mitochondrial matrix independent of ATP synthase due to the leaky
mitochondrial membrane. Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP)
is a mitochondrial OXPHOS uncoupler, penetrating the inner mitochondrial membrane for
electron flux. The use of FCCP allows for the measurement of maximum capacity of the
mitochondria to respire as respiration will no longer be dependent on ATP production.
25
Antimycin A/ Rotenone is an inhibitor of both Complex I and III, consequently completely
disabling the function of ETC and mitochondrial respiration. The use of these compounds
allows us to understand how mitochondrial ETC is affected by our experimental conditions.
Here, we validated the functional knocking down of ERRa in Huh7 hepatocyte cell line
expressing shERRa vs. those expressing scrambled control shRNA. Oxygen
consumption rate (OCR) with the presence of one or the other of these compounds are
used to study mitochondrial respiration in these cells.
3.2 Results
To address the function of ERRa on lipid metabolism, we first verified that ERRa
was successfully knockdown in Huh7 hepatocytes expressing shRNA for ERRa. Our data
showed that ERRa protein levels are completely diminished when shERRa is expressed
in Huh7 cells (Fig. 7A). To validate that this loss of ERRa protein is indeed functional, we
measured OCR as well as extracellular acidification rate (ECAR) in Huh7 cells with or
without shRNA knockdown of ERRa. At steady state basal condition, the ERRa knock-
down group showed overall lower levels of both OCR and ECAR compared to the control
(Fig. 7B, D). It was calculated at the baseline level that ERRa knockdown led to
40pmol/minute lower O2 consumption (OCR) compared with the control Huh7 cells (Fig.
7C i). In addition, a 20mPH/minute decreased lactate production (ECAR) was also
observed (Fig. 7E). Together, these observed downregulation on OCR and ECAR
demonstrate that ERRa loss indeed led to significantly reduced mitochondrial respiration
and glycolysis under basic energy demand.
Additionally, mitochondrial respiration was measured as maximal and ATP
production linked OCR as well (Fig. 7C iii, iv). Maximal OCR is measured after FCCP
26
addition as FCCP uncouples oxygen consumption from ATP production need.
Interestingly, we did not observe significant differences between shERRa expressing
Huh7 cells vs. control Huh7 cells after the addition of FCCP. Thus, while chronic
knockdown of ERRa reduces cellular respiration when ATP is being produced, it does
not appear to alter the overall capacity of the mitochondria to respirate. This data is
surprising as previous experiments from our laboratory using siERRa indeed reported a
significant decrease in maximal OCR
32
. We also confirmed that siERRa reduces
maximum OCR in WT mouse hepatocytes (Fig. 7G, F, right). The reason for this
divergence is unclear but may result from adaptation after chronic loss of ERRa in the
shERRa expressing cell line. Thus, while ERRa knockdown is capable of reducing
OXPHOS and mitochondrial respiration as indicated with basal OCR, hepatocytes appear
to adapt to the lack of ERRa and induce mitochondrial respiration to compensate for ATP
production, at least when glucose is used as the energy source.
In shERRa expressing H295R adrenocortical cancer cells, a significant increase
in proton leak was observed compared to the control H295R cells
70
. Proton leak is
indicative of defective mitochondria and ERRa is characterized as a regulator of
mitochondria integrity
71
. Our Seahorse data also indicated that there was also no change
in ATP production coupled respiration with or without ERRa (Fig. 7C iv). We thus
measured the proton leak in the shERRa vs. shScr expressing Huh7 cells. Our data
shows that ERRa knockdown led to a significant decreased proton leak coupled cellular
respiration by 16pmol/minute on OCR measurement, suggesting that lower ERRa may
preserve the mitochondrial integrity in hepatocytes (Fig. 7C ii). Together with the lack of
difference observed for ATP production associated OCR between shERRa expressing
27
Huh7 cells and controls, these data suggest that the differences observed for basal OCR
between the two cell lines are likely contributed by the effects on ERRa on proton leak.
Thus, while previous work from our lab showed that transient inhibition of ERRa reduces
OXPHOS associated OCR, the long-term effect of ERRa inhibition is primarily on proton
leakage when glucose is provided as substrates.
While some adaptation may have contributed to the unexpected effects observed
with knockdown of ERRa, our immunoblotting data and the reduced basal OCR confirms
the functional knockdown of ERRa in the shERRa expressing Huh7 cells.

28

29

Figure 7. Knock-down of ERRa leads to decreased oxidative phosphorylation and glycolysis.
(A) Immunoblot of ERRa protein level. (B) OCR was measured (Seahorse XFe96 Analyzer) in human
hepatocyte cells. (C i-iv) Mitochondrial respiration related measurements including base-line, proton
leak, maximum, and ATP production linked OCR were quantified. (D) ECAR was simultaneously
measured with OCR in human hepatocyte cells. (E) Quantification of base-line ECAR. (F-G) OCR
was measured in siERRa transfected WT hepatocytes. Mitochondrial respiration related
measurements including base-line and maximum OCR were quantified.
a Last rate measurement before Oligomycin injection. b Minimum rate measurement after Oligomycin
injection. c Maximum rate measurement after FCCP injection. d Minimum rate measurement after
Rotenone/Antimycin A injection.
30
Chapter 4
ERRa loss causes damaged capability of oxidizing exogenous fatty
acids but has no impact on endogenous fatty acids oxidation
4.1 Introduction and rationale
Lipids display in structure of simple short hydrocarbon chains to more advanced
complexes including triacylglycerols (TAGs), phospholipids (PLs) and sterols and their
esters
72
. These complexes consist of fatty acids (FA) that are carboxylic acids each with
a chain of commonly even numbers of carbon atoms, either saturated or unsaturated.
Saturated FAs such as stearic acid and palmitic acid consist of all single carbon bonds,
while unsaturated FAs contain one or more double carbon bonds, e.g. oleic acid with 18
carbons and one double bond. FAs are also classified by their length: short-chain (five or
fewer carbons), medium-chain (6-12 carbons), long-chain (13-21 carbons), and very long
chain (22 or more carbons). The existing forms of FAs in organisms can be found either
in their standard acid form or esters: triglycerides, phospholipids, and cholesteryl esters.
While circulating in plasma, FAs can exist in non-esterified form, bound to a
transport protein such as albumin and consequently named as free fatty acids (FFAs).
FAs serve as major substrate group for energy production similar to carbohydrates. Lipids
are also synthesized as endogenous form within cells via de novo lipogenesis (DNL),
while exogenous lipids from dietary fat also contribute to circulating and tissue FA
contents. These FAs (both endogenously generated and from exogenously uptake) are
oxidized in the mitochondria via a series of catabolic reactions, providing hepatocytes
with ATP and NADH to facilitate gluconeogenesis. In the liver, this process generates
acetyl-CoA, the carbon substrate for ketogenesis as well. The liver is consequently able
31
to buffer blood glucose and fuel highly oxidative tissues with ketone bodies during food
deprivation.
Besides fueling cell growth and survival, both endogenous and exogenous FAs
are acknowledged to relate with disease progression. In rat islets, endogenous FAs act
as essential signaling factors for b-cell activity and insulin secretion
73,74
. Lowering
endogenous FA levels in insulinoma cell line MIN6 and mouse primary b-cells led to
diminished insulin secretion. In triple-negative breast cancer cells, incorporation of PA
and DHA results in the remodeling of lipid composition of the endoplasmic reticulum
membrane
75
. Lung cancer cells also preferentially rely on exogenous PA for proliferation
over de novo synthesis
76
. On the other hand, overloaded PA can lead to lipotoxicity and
apoptosis. Chronic exposure to exogenous lipids attenuated the ability of b-cells to
proliferate
76
. In rat hepatoma H4IIE liver cells, PA also induced apoptosis, by altering
calcium flux
77
.
Palmitic acid (PA) is the most commonly found saturated fatty acid in human
tissues, accounting for 20-30% of total fatty acids in membrane phospholipids. PA can be
derived from the diet or generated endogenously from other fatty acids via DNL. In this
chapter, we investigated the effects of inhibiting ERRa on the rate of cellular respiration
in the presence of PA to address the effect of ERRa on cellular respiration/mitochondrial
oxidation induced by exogenous PA exposure.
4.2 Results
4.2.1 Effect of ERRa knockdown on FAO induced by exogenous
FA
32
In this study, hepatocytes are starved to exclude the utilization of other substrates
such as glucose, glutamine which consume oxygen well. Thus, the OCR measured in this
assay serves as surrogate for the measurement of fatty acid oxidation (FAO) induced by
the exposure to exogenous fatty acids such as PA. We performed Seahorse fatty acid
oxidation assay using BSA conjugated Palmitate in hepatocyte cell line Huh7 cells with
or without shERRa introduced. The exogenous palmitate is water insoluble. BSA acts as
a carrier, creating an aqueous-soluble reagent that can be absorbed by cells.
We quantified oxygen respiration rate in the presence of either BSA or PA. Basal
level respiration rate is calculated using the last time point measurement before the
addition of Oligomycin is used. The maximal level respiration is deducted from the
maximum rate measurement after FCCP is injected. As expected, the addition of
palmitate caused elevated OCR at both basal and maximal levels compared to cells
provided with BSA as controls in either control group or shERRa groups (Fig. 8A). At
basal condition without the addition of exogenous fatty acid and limited substrate, OCR
rate are the same between ERRa knockdown and control cells. When PA is added, OCR
increased by 83 pmol/min/µg/L in Huh7 control cells and 47 pmol/min/µg/L in shERRa
Huh7 cells (Fig. 8B i). This data suggested a significant reduced ability for the Huh7 cells
to oxidize exogenous fatty acid when ERRa was lost and mitochondrial respiration is
coupled to ATP production. Comparing the PA added conditions, knockdown of ERRa
led to a 42 pmol/min/µg/L reduction OCR before the addition of Oligomycin (Fig. 8A).
These data indicated reduced fatty acid utilization for ATP production with knockdown of
ERRa when exogenous fatty acid was used as substrate.

33
After the addition of FCCP to uncouple the mitochondria respiration from ATP
synthesis, the exogenous palmitate contributed to an extra 39 pmol/min/µg/L and 14
pmol/min/µg/L oxygen consumption in Huh7 control and shERRa Huh7 cells respectively
(Fig. 8B ii). This result contrasts the lack of effects of ERRa knockdown on maximal OCR
when glucose is used as substrate (Fig. 7C iii). Thus, while hepatocytes adapt to the lack
of ERRa to induce mitochondrial respiratory capacity for glucose handling, the ability for
ERRa to regulate fatty acid usage in both ATP synthesis coupled and uncoupled manner
are still intact after chronic adaptation to ERRa loss.
Compared to the control Huh7 cells, ERRa knockdown also reduced PA induced
OCR when mitochondria is uncoupled. However, this difference (18 pmol/min/µg/L
reduction) is significantly diminished when compared to the baseline OCR reduction
caused by knockdown of ERRa (42 pmol/min/µg/L reduction) (Fig. 8A). Thus, while ERRa
loss appears to impair the ability of hepatocytes to utilize fatty acids as energy source
through inhibiting FAO, its ability to attenuate the ATP synthesis-uncoupled oxidation of
fatty acids is not as significantly affected by ERRa knockdown.
Meanwhile, ATP synthesis induced by exogenous palmitate is measured by
subtracting proton leak associated OCR from basal level OCR. Consistent with the
observed difference in maximal OCR, ERRa knockdown significantly reduces ATP
synthesis associated OCR by 14 pmol/min/µg/L (Fig. 8B iv). This observation is in
contrast to the lack of any effect in the OXPHOS experiment when glucose was provided
as substrate. Thus, ERRa appears to more preferentially regulates ATP production when
lipid (and not glucose) is being used as substrate. Altogether, our analysis revealed an
obvious decrease in exogenous FA utilization when ERRa is knockdown.
34
When Oligomycin is added to inhibit ATP synthesis, some ETC activities are
retained due to proton leakage. This remaining OCR after the addition of Oligomycin is
therefore independent of ATP synthesis. It was reported that due to chronic exposure to
elevated circulating FFAs, proton leak in the mitochondria of beta cell increased, leading
to inadequate insulin secretion at high blood glucose
78
. We quantified the proton leak
using the minimum rate measurement after Oligomycin is injected. ERRa loss led to a 23
pmol/min/µg/L reduction in proton-leak associated OCR (Fig. 8B iii). This observation is
consistent with the data reported above when glucose is provided as substrate (Fig. 7C
ii). With either substrate, chronic knockdown of ERRa leads to reduced proton-leak in
hepatocytes, further establishing a more preserved mitochondrial integrity associated
with lower ERRa levels.

4.2.2 Effects of ERRa knockdown on FAO induced by endogenous FA
Our analysis using BSA as substrate also suggests that ERRa knockdown did not
affect FAO when no exogenous lipid is added (Fig. 8A) When only BSA is used as
substrate, 118 pmol/min/µg/L OCR and 111 pmol/min/µg/L OCR were observed in Huh7
and shERRa Huh7 cells respectively (Fig. 8A). To specifically investigate the role of
ERRa on endogenous FAO regulation, we performed FAO analysis with the presence of
Etomoxir (Eto). Eto is an inhibitor of CPT-1a. It prevents both endogenous and exogenous
long-chain FAs in the cell cytosol from entering mitochondrial matrix, where they are
oxidized. FAs are consequently unable to be oxidized. In the ERRa knockdown
hepatocytes, both basal and maximal levels of respiration decreased due to Eto treatment
as expected (Fig. 8C). Specifically, Eto treatment induced a 20 pmol/min/A and 30
35
pmol/min/A reduction of OCR at baseline (before Oligomycin addition) and maximum
capacity (after FCCP addition) in Huh7 cells (Fig. 8D i, ii). In shERRa expressing
hepatocytes, a 16 pmol/min/A and 31 pmol/min/A OCR reduction is observed for baseline
and maximal respiration with the addition of Eto (Fig. 8D i, ii). These changes induced by
Eto are comparable between the two cell lines with no statistically difference. The data
suggests there is no difference in the usage of endogenous FAs as substrates for
respiration between the two cell lines. Altogether, the results indicate that ERRa has no
impact on endogenous FAO.

36

36

Figure 8. Decreased oxidation of exogenous fatty acids but not endogenous fatty acids was
observed in ERRa-knockdown human hepatocytes. (A) ERRa loss in human hepatocyte cells
leads to a lower capacity to oxidize exogenous fatty acids. (B i-iv) Mitochondrial respiration related
measurements due to oxidizing exogenous fatty acid including base-line, maximum, proton leak, and
ATP synthesis linked OCR were quantified in both control and shERRa human hepatocytes. (C)
ERRa loss in human hepatocyte cells doesn’t affect endogenous fatty acids oxidation. (D i-ii)
Quantification of base-line (left) and maximum (right) OCR in both control and shERRa human
hepatocytes due to oxidizing endogenous fatty acids.
37
Chapter 5
Compound 29 is an effective inhibitor of ERRa in mouse hepatocytes
5.1 Introduction and rationale
Previous studies in our lab indicated that PTEN regulates mitochondrial biogenesis
and respiration via ERRa
68
. Using transient transfection, inhibiting ERRa function with
siERRa displayed decreased mitochondrial respiration in the Pten-null mouse
hepatocytes compared to the siScrambled transfected cells
68
. We also observed that Pten
deletion which activates PI3K/AKT pathway increased exogenous FAO
69
. Given the
observation that ERRa loss decreased exogenous FAO (Chapter 4), we intend to
investigate whether ERRa also participates in the induction of FAO due to activation of
PI3K/AKT pathway in mouse hepatocytes lacking PTEN. In this chapter, we explored the
potential of pharmaceutical intervention of ERRa on FAO induced due to activation of
PI3K/AKT pathway in mouse hepatocytes.
Pharmacological inhibition of ERRa activity has been used in several studies to
investigate the regulation of ERRa
3
. In lapatinib resistant breast cancer mouse model,
C29, a highly specific ERRa inverse agonist
28
was found to impair glutamine metabolism
and ROS detoxification in tumor cell
79
. In ERRa phospho-mutant (ERRa
3SA
) mice where
ERRa protein is stabilized due to reduced degradation, C29 administration gradually
reduced body mass and alleviated hyperinsulinemia, and hypertriglyceridemia despite
HFD
80
. Thus, we used C29 as a pharmacological approach to inhibit ERRa and address
if ERRa is responsible for the PTEN loss and PI3K/AKT activation induced FAO.
5.2 Results
38
Few studies have reported the in vitro usage of C29 treatment, particularly in
hepatocytes. Here, we first performed dosage experiment to determine the effective dose
of C29 using mouse hepatocyte cell lines with or without intact PTEN. ERRa is a
transcription target of itself
81
. Our qPCR analysis showed that as the dose of C29
increases from 500nM to 5µM, a steady decrease of ERRa mRNA expression was
observed. This dose-dependent inhibition of C29 on ERRa expression is observed in both
WT and Pten-null hepatocytes (Fig. 9A). Of note, a significant (p<0.05) decrease of ERRa
mRNA was observed in Pten-null hepatocytes when the dose of C29 reached 1µM. A
more profound 80% (p<0.001) decreased was observed at the 5µM dose (Fig. 9A, right).
However, a higher dose of C29 appears to be necessary to inhibit the expression of ERRa
mRNA in WT hepatocytes. While 40% reduction of ERRa mRNA was observed at the
high dose of 5µM C29, 1µM C29 was not able to induce any inhibition (Fig. 9A, left). Thus,
C29 is maybe more efficient at blocking the induced transcriptional activity of ERRa rather
than the unstimulated state. Such an effect is consistent with the mechanism of action for
C29 which directly targets at LBD of ERRa that interacts with co-activators and covalently
modifies Cys325 of helix H3
28
.
Consistent with the inhibition of ERRa transcriptional activity, Seahorse analysis
shows an inhibitory effect of C29 on OXPHOS in hepatocytes with or without PTEN (Fig.
9D). When WT and Pten deleted hepatocytes were treated with C29 (5µM) for 48 hours,
our data shows that C29 is able to inhibit baseline mitochondrial respiration in WT
hepatocytes (Fig. 9E i, right) but not that in the Pten deleted hepatocytes (Fig. 9E ii, left).
Since previous study from our lab showed that siERRa treatment inhibited basal
respiration in both cell lines
68
, a higher dose of C29 might be needed to achieve inhibition
39
in the Pten deleted cells that exhibit higher baseline OCR. When FCCP was added to
uncouple mitochondrial respiration from ATP production, 5µM dose of C29 is sufficient to
inhibit OCR in the Pten deleted hepatocytes (Fig. 9E ii, right) but not WT cells (Fig. 9E i,
left). Such observation suggests that the non-ATP synthesis coupled OCR induced by
PTEN loss is regulated by ERRa. Specifically, what particular process is regulated by this
PTEN-ERRa axis is unknown but likely involves ERRa and its interaction with PGC-1a.
Finally, the lack of effects of C29 on maximal OCR after FCCP treatment in WT
hepatocytes differs from that of siERRa treatment (Fig. 7F) which indeed attenuated OCR
following FCCP treatment (Fig. 7G, right). This observation suggests that C29 while is
able to attenuate ERRa regulated OCR, may also have additional effects on metabolism
independent of its effects on ERRa regulated OXPHOS.
To address the effects of ERRa on fatty acid oxidation, we determined the mRNA
level of MCAD, a target gene of ERRa that encodes for the rate-limiting enzyme for FAO.
qPCR analysis indicated that C29 treatment at 500nM already induced a 30% decrease
of MCAD mRNA expression in WT hepatocytes (Fig. 9B i, left). No further reduction was
observed with increased dose of C29. When PTEN is lost, no appreciative reduction of
MCAD mRNA was observed even at the 5µM dose of C29 (Fig. 9B i, right). Thus, while
ERRa inhibition maybe able to inhibit FAO, the PTEN loss induced FAO may not be
dependent on ERRa. We also compared MCAD mRNA expression level between WT
and Pten-null hepatocytes under different doses of C29 treatment conditions (Fig. 9B ii).
When no C29 was added, Pten-null hepatocytes displayed a significant (p<0.001) higher
MCAD mRNA level than WT ones (Fig. 9B ii). Treatment with C29 did not alter this
40
difference between Pten-null and WT hepatocytes. The differences in the expression of
MCAD mRNA between PM and control groups remain highly significant (p<0.01) (Fig. 9B
ii). PPARa is a well characterized transcriptional factor that regulates mitochondrial b-
oxidation. It was proposed that ERRa activates the mitochondrial fatty acid oxidation by
directly binding to the PPARa gene promoter and induces its transcription
45
. We thus
explored the mRNA level of PPARa as well. In WT hepatocytes, 1µM C29 is needed to
induce a significant (p<0.05) decrease of PPARa mRNA expression (Fig. 9C i, left). When
PTEN is lost, a more significant (p<0.01) reduction was already observed at 500nM dose
of C29 (Fig. 9C i, right). A more profound impact (p<0.001) was induced when the dose
of C29 reached 1µM. Similar to the upregulation of MCAD, Pten deletion also induced the
expression of PPARa mRNA transcription (p<0.001) (Fig. 9C ii). Comparing to the WT
hepatocytes, expression of PPARa is 2.3 fold higher in the Pten-null hepatocytes when
no C29 was added (Fig. 9C ii). As C29 treatment impaired PPARa mRNA expression,
this difference became less profound with increased dose of C29. At 5µM C29 condition,
no difference of PPARa mRNA was observed between WT and Pten-null hepatocytes
(Fig. 9C ii). Taken together, these data suggest that regulation of PPARa but not MCAD
by ERRa may participate in PTEN loss induced FAO. The specific mechanisms by which
this signaling axis is regulated needs further investigation.

41

42

Figure 9. C29 is an effective inhibitor of ERRa in mouse hepatocytes. (A-C) Quantitative real-
time PCR analysis was used to measure the mRNA expression levels of the genes that we were
interested. (A) mRNA levels of ERRa in WT hepatocytes, left. mRNA levels of ERRa in Pten-null
hepatocytes, right. The inhibition of C29 on ERRa is dose-dependent. (B i) mRNA levels of MCAD in
WT hepatocytes, left. mRNA levels of ERRa in Pten-null hepatocytes, right. 500nM C29 already
induced a decrease of MCAD mRNA expression in WT hepatocytes but no further reduction was
observed. No reduction of MCAD mRNA was observed in C29 treated Pten-null hepatocytes. (B ii)
MCAD expression between WT and Pten-null hepatocytes under different doses of C29 treatment
conditions. (C i) mRNA levels of PPARa in WT hepatocytes, left. mRNA levels of PPARa in Pten-null
hepatocytes, right. The inhibition of C29 on PPARa is dose-dependent. (C ii) PPARa expression
between WT and Pten-null hepatocytes under different doses of C29 treatment conditions. (D-E)
OCR was measured (Seahorse XFe96 Analyzer) in C29 treated WT and Pten-null hepatocytes.
Mitochondrial respiration related measurements including base-line and maximum OCR were
quantified.
43
Chapter 6
Discussion
In this study, we explored the role of orphan nuclear receptor ERRa for its role in
regulating fatty acid oxidation. Measuring oxygen consumption rate in the presence of
palmitate and not glucose or glutamine as a surrogate for the rate of mitochondrial fatty
acid oxidation, our results showed that ERRa indeed regulates the fatty acid utilization
from exogenous fatty acid sources. Our data demonstrate significantly different oxygen
consumption rate in ERRa knockdown vs. control hepatocytes. We also report here that
ERRa does not regulate the metabolism of endogenously produced fatty acid.
Additionally, we found that the ERRa regulated PPARa expression but not MCAD
participate in the PI3K/AKT regulated FAO.
ERRa plays an important role in maintaining regular mitochondrial fatty acid
oxidation. Mitochondria are dynamic double membrane-bound organelles, playing critical
roles in energy production, integration of various metabolic pathways, and apoptosis
regulation etc. The structure of a mitochondrion includes an inner and outer membrane,
intermembrane space, and the matrix. The mitochondrial matrix contains the
mitochondrial genome, a 16.5kb double-strand circular DNA. Mitochondrial fusion and
fission are two critical events responsible for mitochondrial integrity and homeostasis. The
fusion proteins include Mitofusion 1/2 (MFN1/2) and Optic atrophy protein 1 (OPA1).
Fission in mammals is mediated by a dynamin-like GTPase called Dynamin-related
protein 1 (DRP1), as well as Dynamin 2 (DNM2), human mitochondrial dynamics proteins
49/51 (MID49/51), mitochondrial fission 1 protein (FIS1) and mitochondrial fission factor
(MFF). It was reported that thyroid hormone (TH) promotes DRP1-mediated mitochondrial
44
fission through ERRa
82
. In HepG2 cells, increased mitochondrial fission induced by ERRa
and TH is necessary for maintaining mitochondrial homeostasis and activities such as
OXPHOS and FAO. In the liver, such impaired mitochondrial fission was shown to drive
non-alcoholic steatohepatitis (NASH)
83
. Mice with liver-specific deletion of MFF (MffLiKO)
displayed aberrant mitochondrial morphologies, along with upregulated genes related to
endoplasmic reticulum (ER) stress and downregulation of triacylglycerol secretion genes.
Consequently, the MffLiKO mice was prone to develop NASH when fed high-fat diet
(HFD). Additional studies reported that the deletion of fusion proteins MFN2 leads to
increased proton leak and consequently reduced FAO in mammalian cells, indicating
reduced mitochondrial intergrity
84
. However, knockdown of Mfn1 in Proopiomelanocortin
(POMC) neurons resulted in increased OXPHOS, suggesting enhanced mitochondrial
function
85
. These seemingly contradictory data indicated that MFNs may have functions
other than mitochondrial fusion
85
. Here, our data indicated that ERRa loss leads to
reduced proton-leak in hepatocytes when either glucose or palmitic acid is provided as
substrate, suggesting that loss of ERRa preserves the mitochondrial integrity. Meanwhile,
a decrease of overall FAO was also displayed when ERRa was knockdown, which can
be potentially ascribed to impaired mitochondrial fission. These data seem to be contrary
to the enhanced integrity of mitochondria when ERRa was lost. Thus, these seemingly
contradictory data may indicate that ERRa knockdown interacts with one of the fission
proteins, which needs further investigation.
Although the presence of ERRa is critical for exogenous FAO, it has no impact on
endogenously generated FAO. The preferences for and dysfunction of exogenous or
endogenous metabolism have been studied and shown to contribute to pathology of
45
diseases, as acknowledged by several studies. Exogenous fatty acids are mainly
obtained from diets and once transported into the cytoplasm and then transported into
the mitochondria for oxidation or stored as triacylglycerol (TAG). The balance between
exogenous fatty acid metabolism and storage is essential for health. In the case of
diabetes, despite a high concentration of circulating plasma fatty acids, a dramatically
high myocardial TAG storage is observed as well
86
. Such imbalance of lipid homeostasis
was attributed to be a casual factor for cardiomyopathy. A decrease of endogenous TAG
oxidation was observed in early cardiac failure regardless of an increased ATP synthesis
demand of the failing heart, leading to more severe cardiac failure
87
. Schweitzer et al.
88

investigated the link between lipid availability and immune response. They found that the
limit availability of exogenous lipids causes decreased lysosomal acidity and major
histocompatibility (MHC) expression while inhibition of endogenous fatty acids synthesis
only leads to reduced MHC expression but not lysosomal acidity. The preference of usage
for specific resource of fatty acids suggests a potentially feasible diet or pharmacologic
intervention in inflammatory diseases. Our result showed that ERRa loss results in
reduced exogenous fatty acids utilization but not endogenous FAO. Thus, ERRa
inhibition (and potentially PPARa but not MCAD targeting) may serve as an approach to
improve dysregulated FAO contributed to diet.
A significantly higher MCAD and PPARa mRNA expression level displayed in
Pten-null hepatocytes suggests that PTEN deletion which activates PI3K/AKT signaling
pathway elevates FAO capacity. Inhibiting ERRa activity by C29 revealed a more
profound decrease of PPARa mRNA expression in the Pten-null hepatocytes compared
to WT control. However, MCAD mRNA expression in general is not affected by C29. Since
46
ERRa activates the mitochondrial FAO by directly binding to the promoter of the PPARa
gene
45
, the PPARa regulated induction of FAO due to Pten deletion may be dependent
on ERRa. Since PPARa also directly controls the transcription of MCAD
89
, the induction
of MCAD observed in the Pten deleted hepatocytes is likely not ERRa dependent but is
dependent on PPARa and other unknown factors. Of note, it was found that in PPARa
null fibroblasts, ERRa overexpression has no effect on MCAD expression level while
ERRa activates MCAD in PPARa-expressing cells
45
. In other words, only co-expression
of PPARa and ERRa controls certain gene involved in FAO. Further investigations are
needed to figure out whether inhibiting both ERRa and PPARa will reduce PTEN loss
induced FAO.
Several pharmacological targets regarding treating metabolic liver diseases have
been proposed. Methylation-Controlled J protein (MCJ), as an endogenous negative
regulator of the electron transporter chain Complex I, has emerged as a therapeutic target
to treat NAFLD
90
. Attenuating MCJ expression enhances the hepatocytes’ ability to
undergo FAO and minimizes lipid accumulation, resulting in reducing hepatocyte damage
and fibrosis. The bioactive compound aurantio-obtusin (AO) promoted autophagy flux and
triggered transcriptional factor EB (TFEB) activation in a hepatic steatosis mouse model.
It consequently accelerated FAO via inducing AMPK phosphorylation
91
, implying AO as
a potential treatment for NAFLD. A mitochondria-targeted anti-oxidant (AntiOxCIN4)
decreased lipid droplets number/size in Western diet (WD)-fed mice model and human
HepG2 cells by means of upregulating anti-oxidant defense systems and increasing FAO
activity, ameliorating early NAFLD
92
. MicroRNA-376b-3p was uncovered to regulate lipid
oxidation by targeting fibroblast growth factor receptor 1 (FGFR1), ameliorating hepatic
47
lipid accumulation
93
. It may serve as a promising therapeutic target for NAFLD. Here, our
study demonstrated that ERRa positively regulates exogenous FAO. Consequently, the
inducing of ERRa can be a promising solution in treating metabolic liver diseases. In
addition, we also suggest that the regulation of PPARa but not MCAD by ERRa may
participate in PTEN loss induced FAO. Thus, a better understanding of the regulation of
PPARa by ERRa needs to be done in the future.

48
Bibliography
1. Xia, H.; Dufour, C. R.; Giguere, V., ERRalpha as a Bridge Between Transcription
and Function: Role in Liver Metabolism and Disease. Front Endocrinol (Lausanne) 2019,
10, 206.

2. Helsen, C.; Claessens, F., Looking at nuclear receptors from a new angle. Mol Cell
Endocrinol 2014, 382 (1), 97-106.

3. Huss, J. M.; Garbacz, W. G.; Xie, W., Constitutive activities of estrogen-related
receptors: Transcriptional regulation of metabolism by the ERR pathways in health and
disease. Biochim Biophys Acta 2015, 1852 (9), 1912-27.

4. Kallen, J.; Schlaeppi, J. M.; Bitsch, F.; Filipuzzi, I.; Schilb, A.; Riou, V.; Graham,
A.; Strauss, A.; Geiser, M.; Fournier, B., Evidence for ligand-independent transcriptional
activation of the human estrogen-related receptor alpha (ERRalpha): crystal structure of
ERRalpha ligand binding domain in complex with peroxisome proliferator-activated
receptor coactivator-1alpha. J Biol Chem 2004, 279 (47), 49330-7.

5. Yao, B.; Zhang, S.; Wei, Y.; Tian, S.; Lu, Z.; Jin, L.; He, Y.; Xie, W.; Li, Y.,
Structural Insights into the Specificity of Ligand Binding and Coactivator Assembly by
Estrogen-Related Receptor beta. J Mol Biol 2020, 432 (19), 5460-5472.

6. Tang, J.; Liu, T.; Wen, X.; Zhou, Z.; Yan, J.; Gao, J.; Zuo, J., Estrogen-related
receptors: novel potential regulators of osteoarthritis pathogenesis. Mol Med 2021, 27 (1),
5.

7. Huppunen, J.; Aarnisalo, P., Dimerization modulates the activity of the orphan
nuclear receptor ERRgamma. Biochem Biophys Res Commun 2004, 314 (4), 964-70.

8. Deblois, G.; Giguere, V., Functional and physiological genomics of estrogen-
related receptors (ERRs) in health and disease. Biochim Biophys Acta 2011, 1812 (8),
1032-40.

9. Misawa, A.; Inoue, S., Estrogen-Related Receptors in Breast Cancer and Prostate
Cancer. Front Endocrinol (Lausanne) 2015, 6, 83.

10. Liu, D.; Benlhabib, H.; Mendelson, C. R., cAMP enhances estrogen-related
receptor alpha (ERRalpha) transcriptional activity at the SP-A promoter by increasing its
interaction with protein kinase A and steroid receptor coactivator 2 (SRC-2). Mol
Endocrinol 2009, 23 (6), 772-83.

11. Brown, E. L.; Foletta, V. C.; Wright, C. R.; Sepulveda, P. V.; Konstantopoulos,
N.; Sanigorski, A.; Della Gatta, P.; Cameron-Smith, D.; Kralli, A.; Russell, A. P., PGC-
1alpha and PGC-1beta Increase Protein Synthesis via ERRalpha in C2C12 Myotubes.
Front Physiol 2018, 9, 1336.
49

12. Nichol, D.; Christian, M.; Steel, J. H.; White, R.; Parker, M. G., RIP140 expression
is stimulated by estrogen-related receptor alpha during adipogenesis. J Biol Chem 2006,
281 (43), 32140-7.

13. Yasumoto, H.; Meng, L.; Lin, T.; Zhu, Q.; Tsai, R. Y., GNL3L inhibits activity of
estrogen-related receptor gamma by competing for coactivator binding. J Cell Sci 2007,
120 (Pt 15), 2532-43.

14. Wilson, B. J.; Tremblay, A. M.; Deblois, G.; Sylvain-Drolet, G.; Giguere, V., An
acetylation switch modulates the transcriptional activity of estrogen-related receptor alpha.
Mol Endocrinol 2010, 24 (7), 1349-58.

15. Shires, S. E.; Quiles, J. M.; Najor, R. H.; Leon, L. J.; Cortez, M. Q.; Lampert, M.
A.; Mark, A.; Gustafsson, A. B., Nuclear Parkin Activates the ERRalpha Transcriptional
Program and Drives Widespread Changes in Gene Expression Following Hypoxia. Sci
Rep 2020, 10 (1), 8499.

16. Ren, Y.; Jiang, H.; Ma, D.; Nakaso, K.; Feng, J., Parkin degrades estrogen-
related receptors to limit the expression of monoamine oxidases. Hum Mol Genet 2011,
20 (6), 1074-83.

17. Yang, Y.; Li, S.; Li, B.; Li, Y.; Xia, K.; Aman, S.; Yang, Y.; Ahmad, B.; Zhao,
B.; Wu, H., FBXL10 promotes ERRalpha protein stability and proliferation of breast
cancer cells by enhancing the mono-ubiquitylation of ERRalpha. Cancer Lett 2021, 502,
108-119.

18. Kumari, K.; Adhya, A. K.; Rath, A. K.; Reddy, P. B.; Mishra, S. K., Estrogen-
related receptors alpha, beta and gamma expression and function is associated with
transcriptional repressor EZH2 in breast carcinoma. BMC Cancer 2018, 18 (1), 690.

19. Coward, P.; Lee, D.; Hull, M. V.; Lehmann, J. M., 4-Hydroxytamoxifen binds to
and deactivates the estrogen-related receptor gamma. Proc Natl Acad Sci U S A 2001,
98 (15), 8880-4.

20. Tanida, T.; Matsuda, K. I.; Uemura, T.; Yamaguchi, T.; Hashimoto, T.; Kawata,
M.; Tanaka, M., Subcellular dynamics of estrogen-related receptors involved in
transrepression through interactions with scaffold attachment factor B1. Histochem Cell
Biol 2021, 156 (3), 239-251.

21. Kim, Y.; Koh, M.; Kim, D. K.; Choi, H. S.; Park, S. B., Efficient discovery of
selective small molecule agonists of estrogen-related receptor gamma using
combinatorial approach. J Comb Chem 2009, 11 (5), 928-37.

50
22. Yu, D. D.; Huss, J. M.; Li, H.; Forman, B. M., Identification of novel inverse
agonists of estrogen-related receptors ERRgamma and ERRbeta. Bioorg Med Chem
2017, 25 (5), 1585-1599.

23. Matsushima, A.; Teramoto, T.; Kakuta, Y., Crystal structure of endocrine-
disrupting chemical bisphenol A and estrogen-related receptor gamma. J Biochem 2022,
171 (1), 23-25.

24. Iwamoto, M.; Masuya, T.; Hosose, M.; Tagawa, K.; Ishibashi, T.; Suyama, K.;
Nose, T.; Yoshihara, E.; Downes, M.; Evans, R. M.; Matsushima, A., Bisphenol A
derivatives act as novel coactivator-binding inhibitors for estrogen receptor beta. J Biol
Chem 2021, 297 (5), 101173.

25. Masuya, T.; Iwamoto, M.; Liu, X.; Matsushima, A., Discovery of novel oestrogen
receptor alpha agonists and antagonists by screening a revisited privileged structure
moiety for nuclear receptors. Sci Rep 2019, 9 (1), 9954.

26. Busch, B. B.; Stevens, W. C., Jr.; Martin, R.; Ordentlich, P.; Zhou, S.; Sapp, D.
W.; Horlick, R. A.; Mohan, R., Identification of a selective inverse agonist for the orphan
nuclear receptor estrogen-related receptor alpha. J Med Chem 2004, 47 (23), 5593-6.

27. Wang, J.; Wang, Y.; Wong, C., Oestrogen-related receptor alpha inverse agonist
XCT-790 arrests A549 lung cancer cell population growth by inducing mitochondrial
reactive oxygen species production. Cell Prolif 2010, 43 (2), 103-13.

28. Patch, R. J.; Searle, L. L.; Kim, A. J.; De, D.; Zhu, X.; Askari, H. B.; O'Neill, J.
C.; Abad, M. C.; Rentzeperis, D.; Liu, J.; Kemmerer, M.; Lin, L.; Kasturi, J.; Geisler,
J. G.; Lenhard, J. M.; Player, M. R.; Gaul, M. D., Identification of diaryl ether-based
ligands for estrogen-related receptor alpha as potential antidiabetic agents. J Med Chem
2011, 54 (3), 788-808.

29. Lynch, C.; Zhao, J.; Huang, R.; Kanaya, N.; Bernal, L.; Hsieh, J. H.; Auerbach,
S. S.; Witt, K. L.; Merrick, B. A.; Chen, S.; Teng, C. T.; Xia, M., Identification of Estrogen-
Related Receptor alpha Agonists in the Tox21 Compound Library. Endocrinology 2018,
159 (2), 744-753.

30. Teng, C. T.; Hsieh, J. H.; Zhao, J.; Huang, R.; Xia, M.; Martin, N.; Gao, X.;
Dixon, D.; Auerbach, S. S.; Witt, K. L.; Merrick, B. A., Development of Novel Cell Lines
for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators.
SLAS Discov 2017, 22 (6), 720-731.

31. Mao, L.; Peng, L.; Ren, X.; Chu, Y.; Nie, T.; Lin, W.; Zhao, X.; Libby, A.; Xu,
Y.; Chang, Y.; Lei, C.; Loomes, K.; Wang, N.; Liu, J.; Levi, M.; Wu, D.; Hui, X.; Ding,
K., Discovery of JND003 as a New Selective Estrogen-Related Receptor alpha Agonist
Alleviating Nonalcoholic Fatty Liver Disease and Insulin Resistance. ACS Bio Med Chem
Au 2022, 2 (3), 282-296.
51

32. Chen, C. Y.; Li, Y.; Jia, T.; He, L.; Hare, A. A.; Silberstein, A.; Gallagher, J.;
Martinez, T. F.; Stiles, J. W.; Olenyuk, B.; Dervan, P. B.; Stiles, B. L., Repression of the
transcriptional activity of ERRalpha with sequence-specific DNA-binding polyamides.
Med Chem Res 2020, 29 (4), 607-616.

33. Chen, C. Y.; Li, Y.; Zeng, N.; He, L.; Zhang, X.; Tu, T.; Tang, Q.; Alba, M.; Mir,
S.; Stiles, E. X.; Hong, H.; Cadenas, E.; Stolz, A. A.; Li, G.; Stiles, B. L., Inhibition of
Estrogen-Related Receptor alpha Blocks Liver Steatosis and Steatohepatitis and
Attenuates Triglyceride Biosynthesis. Am J Pathol 2021, 191 (7), 1240-1254.

34. Sun, J.; Huang, T.; Qi, Z.; You, S.; Dong, J.; Zhang, C.; Qin, L.; Zhou, Y.; Ding,
S., Early Mitochondrial Adaptations in Skeletal Muscle to Obesity and Obesity Resistance
Differentially Regulated by High-Fat Diet. Exp Clin Endocrinol Diabetes 2017, 125 (8),
538-546.

35. Rinnankoski-Tuikka, R.; Silvennoinen, M.; Torvinen, S.; Hulmi, J. J.; Lehti, M.;
Kivela, R.; Reunanen, H.; Kainulainen, H., Effects of high-fat diet and physical activity on
pyruvate dehydrogenase kinase-4 in mouse skeletal muscle. Nutr Metab (Lond) 2012, 9
(1), 53.

36. Yan, M.; Audet-Walsh, E.; Manteghi, S.; Dufour, C. R.; Walker, B.; Baba, M.;
St-Pierre, J.; Giguere, V.; Pause, A., Chronic AMPK activation via loss of FLCN induces
functional beige adipose tissue through PGC-1alpha/ERRalpha. Genes Dev 2016, 30 (9),
1034-46.

37. Herzog, B.; Cardenas, J.; Hall, R. K.; Villena, J. A.; Budge, P. J.; Giguere, V.;
Granner, D. K.; Kralli, A., Estrogen-related receptor alpha is a repressor of
phosphoenolpyruvate carboxykinase gene transcription. J Biol Chem 2006, 281 (1), 99-
106.

38. Audet-Walsh, E.; Papadopoli, D. J.; Gravel, S. P.; Yee, T.; Bridon, G.; Caron,
M.; Bourque, G.; Giguere, V.; St-Pierre, J., The PGC-1alpha/ERRalpha Axis Represses
One-Carbon Metabolism and Promotes Sensitivity to Anti-folate Therapy in Breast
Cancer. Cell Rep 2016, 14 (4), 920-931.

39. Grasfeder, L. L.; Gaillard, S.; Hammes, S. R.; Ilkayeva, O.; Newgard, C. B.;
Hochberg, R. B.; Dwyer, M. A.; Chang, C. Y.; McDonnell, D. P., Fasting-induced hepatic
production of DHEA is regulated by PGC-1alpha, ERRalpha, and HNF4alpha. Mol
Endocrinol 2009, 23 (8), 1171-82.

40. Zeng, X.; Yang, J.; Hu, O.; Huang, J.; Ran, L.; Chen, M.; Zhang, Y.; Zhou, X.;
Zhu, J.; Zhang, Q.; Yi, L.; Mi, M., Dihydromyricetin Ameliorates Nonalcoholic Fatty Liver
Disease by Improving Mitochondrial Respiratory Capacity and Redox Homeostasis
Through Modulation of SIRT3 Signaling. Antioxid Redox Signal 2019, 30 (2), 163-183.

52
41. Mehlem, A.; Palombo, I.; Wang, X.; Hagberg, C. E.; Eriksson, U.; Falkevall, A.,
PGC-1alpha Coordinates Mitochondrial Respiratory Capacity and Muscular Fatty Acid
Uptake via Regulation of VEGF-B. Diabetes 2016, 65 (4), 861-73.

42. Baar, K.; Song, Z.; Semenkovich, C. F.; Jones, T. E.; Han, D. H.; Nolte, L. A.;
Ojuka, E. O.; Chen, M.; Holloszy, J. O., Skeletal muscle overexpression of nuclear
respiratory factor 1 increases glucose transport capacity. FASEB J 2003, 17 (12), 1666-
73.

43. Mootha, V. K.; Handschin, C.; Arlow, D.; Xie, X.; St Pierre, J.; Sihag, S.; Yang,
W.; Altshuler, D.; Puigserver, P.; Patterson, N.; Willy, P. J.; Schulman, I. G.; Heyman,
R. A.; Lander, E. S.; Spiegelman, B. M., Erralpha and Gabpa/b specify PGC-1alpha-
dependent oxidative phosphorylation gene expression that is altered in diabetic muscle.
Proc Natl Acad Sci U S A 2004, 101 (17), 6570-5.

44. Villena, J. A.; Hock, M. B.; Chang, W. Y.; Barcas, J. E.; Giguere, V.; Kralli, A.,
Orphan nuclear receptor estrogen-related receptor a is essential for adaptive
thermogenesis. Proc Natl Acad Sci U S A 2007, 104 (4), 1418-1423.

45. Huss, J. M.; Torra, I. P.; Staels, B.; Giguere, V.; Kelly, D. P., Estrogen-related
receptor alpha directs peroxisome proliferator-activated receptor alpha signaling in the
transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol
2004, 24 (20), 9079-91.

46. Sladek, R.; Bader, J. A.; Giguere, V., The orphan nuclear receptor estrogen-
related receptor alpha is a transcriptional regulator of the human medium-chain acyl
coenzyme A dehydrogenase gene. Mol Cell Biol 1997, 17 (9), 5400-9.

47. B'Chir, W.; Dufour, C. R.; Ouellet, C.; Yan, M.; Tam, I. S.; Andrzejewski, S.;
Xia, H.; Nabata, K.; St-Pierre, J.; Giguere, V., Divergent Role of Estrogen-Related
Receptor alpha in Lipid- and Fasting-Induced Hepatic Steatosis in Mice. Endocrinology
2018, 159 (5), 2153-2164.

48. Yang, M.; Liu, Q.; Huang, T.; Tan, W.; Qu, L.; Chen, T.; Pan, H.; Chen, L.; Liu,
J.; Wong, C. W.; Lu, W. W.; Guan, M., Dysfunction of estrogen-related receptor alpha-
dependent hepatic VLDL secretion contributes to sex disparity in NAFLD/NASH
development. Theranostics 2020, 10 (24), 10874-10891.

49. Vernier, M.; Dufour, C. R.; McGuirk, S.; Scholtes, C.; Li, X.; Bourmeau, G.;
Kuasne, H.; Park, M.; St-Pierre, J.; Audet-Walsh, E.; Giguere, V., Estrogen-related
receptors are targetable ROS sensors. Genes Dev 2020, 34 (7-8), 544-559.

50. Xia, W.; Pan, Z.; Zhang, H.; Zhou, Q.; Liu, Y., Inhibition of ERRalpha Aggravates
Sepsis-Induced Acute Lung Injury in Rats via Provoking Inflammation and Oxidative
Stress. Oxid Med Cell Longev 2020, 2020, 2048632.

53
51. Kong, X.; Wang, R.; Xue, Y.; Liu, X.; Zhang, H.; Chen, Y.; Fang, F.; Chang, Y.,
Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS
and mitochondrial biogenesis. PLoS One 2010, 5 (7), e11707.

52. Li, X.; Zhang, K.; Hu, Y.; Luo, N., ERRalpha activates SHMT2 transcription to
enhance the resistance of breast cancer to lapatinib via modulating the mitochondrial
metabolic adaption. Biosci Rep 2020, 40 (1).

53. Harada, K.; Kakuda, Y.; Sato, Y.; Ikeda, H.; Shimoda, S.; Yamamoto, Y.; Inoue,
H.; Ohta, H.; Kasashima, S.; Kawashima, A.; Nakanuma, Y., Alteration of energy
metabolism in the pathogenesis of bile duct lesions in primary biliary cirrhosis. J Clin
Pathol 2014, 67 (5), 396-402.

54. Wu, P.; Blair, P. V.; Sato, J.; Jaskiewicz, J.; Popov, K. M.; Harris, R. A.,
Starvation increases the amount of pyruvate dehydrogenase kinase in several
mammalian tissues. Arch Biochem Biophys 2000, 381 (1), 1-7.

55. Park, S.; Safi, R.; Liu, X.; Baldi, R.; Liu, W.; Liu, J.; Locasale, J. W.; Chang, C.
Y.; McDonnell, D. P., Inhibition of ERRalpha Prevents Mitochondrial Pyruvate Uptake
Exposing NADPH-Generating Pathways as Targetable Vulnerabilities in Breast Cancer.
Cell Rep 2019, 27 (12), 3587-3601 e4.

56. Koh, E.; Kim, Y. K.; Shin, D.; Kim, K. S., MPC1 is essential for PGC-1alpha-
induced mitochondrial respiration and biogenesis. Biochem J 2018, 475 (10), 1687-1699.

57. He, F.; Jin, J. Q.; Qin, Q. Q.; Zheng, Y. Q.; Li, T. T.; Zhang, Y.; He, J. D., Resistin
Regulates Fatty Acid Beta Oxidation by Suppressing Expression of Peroxisome
Proliferator Activator Receptor Gamma-Coactivator 1alpha (PGC-1alpha). Cell Physiol
Biochem 2018, 46 (5), 2165-2172.

58. Wang, B.; Zhu, L.; Sui, S.; Sun, C.; Jiang, H.; Ren, D., Cilostazol induces
mitochondrial fatty acid beta-oxidation in C2C12 myotubes. Biochem Biophys Res
Commun 2014, 447 (3), 441-5.

59. Pooya, S.; Blaise, S.; Moreno Garcia, M.; Giudicelli, J.; Alberto, J. M.; Gueant-
Rodriguez, R. M.; Jeannesson, E.; Gueguen, N.; Bressenot, A.; Nicolas, B.; Malthiery,
Y.; Daval, J. L.; Peyrin-Biroulet, L.; Bronowicki, J. P.; Gueant, J. L., Methyl donor
deficiency impairs fatty acid oxidation through PGC-1alpha hypomethylation and
decreased ER-alpha, ERR-alpha, and HNF-4alpha in the rat liver. J Hepatol 2012, 57 (2),
344-51.

60. Du, W.; Zhang, L.; Brett-Morris, A.; Aguila, B.; Kerner, J.; Hoppel, C. L.;
Puchowicz, M.; Serra, D.; Herrero, L.; Rini, B. I.; Campbell, S.; Welford, S. M., HIF
drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism. Nat
Commun 2017, 8 (1), 1769.
54
61. Duan, L.; Calhoun, S.; Shim, D.; Perez, R. E.; Blatter, L. A.; Maki, C. G., Fatty
acid oxidation and autophagy promote endoxifen resistance and counter the effect of AKT
inhibition in ER-positive breast cancer cells. J Mol Cell Biol 2021, 13 (6), 433-444.

62. Yan, X.; Zhang, G.; Bie, F.; Lv, Y.; Ma, Y.; Ma, M.; Wang, Y.; Hao, X.; Yuan,
N.; Jiang, X., Eugenol inhibits oxidative phosphorylation and fatty acid oxidation via
downregulation of c-Myc/PGC-1beta/ERRalpha signaling pathway in MCF10A-ras cells.
Sci Rep 2017, 7 (1), 12920.

63. Selen, E. S.; Choi, J.; Wolfgang, M. J., Discordant hepatic fatty acid oxidation and
triglyceride hydrolysis leads to liver disease. JCI Insight 2021, 6 (2).

64. Chaveroux, C.; Eichner, L. J.; Dufour, C. R.; Shatnawi, A.; Khoutorsky, A.;
Bourque, G.; Sonenberg, N.; Giguere, V., Molecular and genetic crosstalks between
mTOR and ERRalpha are key determinants of rapamycin-induced nonalcoholic fatty liver.
Cell Metab 2013, 17 (4), 586-98.

65. El Kebbaj, R.; Andreoletti, P.; El Hajj, H. I.; El Kharrassi, Y.; Vamecq, J.;
Mandard, S.; Saih, F. E.; Latruffe, N.; El Kebbaj, M. S.; Lizard, G.; Nasser, B.;
Cherkaoui-Malki, M., Argan oil prevents down-regulation induced by endotoxin on liver
fatty acid oxidation and gluconeogenesis and on peroxisome proliferator-activated
receptor gamma coactivator-1alpha, (PGC-1alpha), peroxisome proliferator-activated
receptor alpha (PPARalpha) and estrogen related receptor alpha (ERRalpha). Biochim
Open 2015, 1, 51-59.

66. Pei, K.; Gui, T.; Kan, D.; Feng, H.; Jin, Y.; Yang, Y.; Zhang, Q.; Du, Z.; Gai,
Z.; Wu, J.; Li, Y., An Overview of Lipid Metabolism and Nonalcoholic Fatty Liver Disease.
Biomed Res Int 2020, 2020, 4020249.

67. Luo, J.; Sladek, R.; Carrier, J.; Bader, J. A.; Richard, D.; Giguere, V., Reduced
fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha. Mol
Cell Biol 2003, 23 (22), 7947-56.

68. Li, Y.; He, L.; Zeng, N.; Sahu, D.; Cadenas, E.; Shearn, C.; Li, W.; Stiles, B. L.,
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates
mitochondrial biogenesis and respiration via estrogen-related receptor alpha (ERRalpha).
J Biol Chem 2013, 288 (35), 25007-25024.

69. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling
regulates fatty acid beta-oxidation.

70. Avena, P.; De Luca, A.; Chimento, A.; Nocito, M. C.; Sculco, S.; La Padula, D.;
Zavaglia, L.; Giulietti, M.; Hantel, C.; Sirianni, R.; Casaburi, I.; Pezzi, V., Estrogen
Related Receptor Alpha (ERRalpha) a Bridge between Metabolism and Adrenocortical
Cancer Progression. Cancers (Basel) 2022, 14 (16).

55
71. Audet-Walsh, E.; Giguere, V., The multiple universes of estrogen-related receptor
alpha and gamma in metabolic control and related diseases. Acta Pharmacol Sin 2015,
36 (1), 51-61.

72. Burdge, G. C.; Calder, P. C., Introduction to fatty acids and lipids. World Rev Nutr
Diet 2015, 112, 1-16.

73. Hosokawa, H.; Corkey, B. E.; Leahy, J. L., Beta-cell hypersensitivity to glucose
following 24-h exposure of rat islets to fatty acids. Diabetologia 1997, 40 (4), 392-7.

74. Hauke, S.; Keutler, K.; Phapale, P.; Yushchenko, D. A.; Schultz, C., Endogenous
Fatty Acids Are Essential Signaling Factors of Pancreatic beta-Cells and Insulin Secretion.
Diabetes 2018, 67 (10), 1986-1998.

75. Rizzo, A. M.; Colombo, I.; Montorfano, G.; Zava, S.; Corsetto, P. A., Exogenous
Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells. Cells
2021, 10 (1).

76. Yao, C. H.; Fowle-Grider, R.; Mahieu, N. G.; Liu, G. Y.; Chen, Y. J.; Wang, R.;
Singh, M.; Potter, G. S.; Gross, R. W.; Schaefer, J.; Johnson, S. L.; Patti, G. J.,
Exogenous Fatty Acids Are the Preferred Source of Membrane Lipids in Proliferating
Fibroblasts. Cell Chem Biol 2016, 23 (4), 483-93.

77. Zhang, Y.; Xue, R.; Zhang, Z.; Yang, X.; Shi, H., Palmitic and linoleic acids induce
ER stress and apoptosis in hepatoma cells. Lipids Health Dis 2012, 11, 1.

78. Grubelnik, V.; Zmazek, J.; Zavrsnik, M.; Marhl, M., Lipotoxicity in a Vicious Cycle
of Pancreatic Beta Cell Exhaustion. Biomedicines 2022, 10 (7).

79. Deblois, G.; Smith, H. W.; Tam, I. S.; Gravel, S. P.; Caron, M.; Savage, P.;
Labbe, D. P.; Begin, L. R.; Tremblay, M. L.; Park, M.; Bourque, G.; St-Pierre, J.; Muller,
W. J.; Giguere, V., ERRalpha mediates metabolic adaptations driving lapatinib resistance
in breast cancer. Nat Commun 2016, 7, 12156.

80. Xia, H.; Scholtes, C.; Dufour, C. R.; Ouellet, C.; Ghahremani, M.; Giguere, V.,
Insulin action and resistance are dependent on a GSK3beta-FBXW7-ERRalpha
transcriptional axis. Nat Commun 2022, 13 (1), 2105.

81. Handschin, C.; Mootha, V. K., Estrogen-related receptor α (ERRα): A novel target
in type 2 diabetes. Drug Discovery Today: Therapeutic Strategies 2005, 2 (2), 151-156.

82. Singh, B. K.; Sinha, R. A.; Tripathi, M.; Mendoza, A.; Ohba, K.; Sy, J. A. C.;
Xie, S. Y.; Zhou, J.; Ho, J. P.; Chang, C. Y.; Wu, Y.; Giguere, V.; Bay, B. H.; Vanacker,
J. M.; Ghosh, S.; Gauthier, K.; Hollenberg, A. N.; McDonnell, D. P.; Yen, P. M., Thyroid
hormone receptor and ERRalpha coordinately regulate mitochondrial fission, mitophagy,
biogenesis, and function. Sci Signal 2018, 11 (536).
56

83. Takeichi, Y.; Miyazawa, T.; Sakamoto, S.; Hanada, Y.; Wang, L.; Gotoh, K.;
Uchida, K.; Katsuhara, S.; Sakamoto, R.; Ishihara, T.; Masuda, K.; Ishihara, N.;
Nomura, M.; Ogawa, Y., Non-alcoholic fatty liver disease in mice with hepatocyte-specific
deletion of mitochondrial fission factor. Diabetologia 2021, 64 (9), 2092-2107.

84. Liu, Y. J.; McIntyre, R. L.; Janssens, G. E.; Houtkooper, R. H., Mitochondrial
fission and fusion: A dynamic role in aging and potential target for age-related disease.
Mech Ageing Dev 2020, 186, 111212.

85. Ramirez, S.; Gomez-Valades, A. G.; Schneeberger, M.; Varela, L.; Haddad-
Tovolli, R.; Altirriba, J.; Noguera, E.; Drougard, A.; Flores-Martinez, A.; Imbernon, M.;
Chivite, I.; Pozo, M.; Vidal-Itriago, A.; Garcia, A.; Cervantes, S.; Gasa, R.; Nogueiras,
R.; Gama-Perez, P.; Garcia-Roves, P. M.; Cano, D. A.; Knauf, C.; Servitja, J. M.;
Horvath, T. L.; Gomis, R.; Zorzano, A.; Claret, M., Mitochondrial Dynamics Mediated by
Mitofusin 1 Is Required for POMC Neuron Glucose-Sensing and Insulin Release Control.
Cell Metab 2017, 25 (6), 1390-1399 e6.

86. Stanley, W. C.; Lopaschuk, G. D.; McCormack, J. G., Regulation of energy
substrate metabolism in the diabetic heart. Cardiovasc Res 1997, 34 (1), 25-33.

87. O'Donnell, J. M.; Fields, A. D.; Sorokina, N.; Lewandowski, E. D., The absence
of endogenous lipid oxidation in early stage heart failure exposes limits in lipid storage
and turnover. J Mol Cell Cardiol 2008, 44 (2), 315-22.

88. Schweitzer, S. C.; Reding, A. M.; Patton, H. M.; Sullivan, T. P.; Stubbs, C. E.;
Villalobos-Menuey, E.; Huber, S. A.; Newell, M. K., Endogenous versus exogenous fatty
acid availability affects lysosomal acidity and MHC class II expression. J Lipid Res 2006,
47 (11), 2525-37.

89. Pawlak, M.; Lefebvre, P.; Staels, B., Molecular mechanism of PPARalpha action
and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver
disease. J Hepatol 2015, 62 (3), 720-33.

90. Barbier-Torres, L.; Fortner, K. A.; Iruzubieta, P.; Delgado, T. C.; Giddings, E.;
Chen, Y.; Champagne, D.; Fernandez-Ramos, D.; Mestre, D.; Gomez-Santos, B.;
Varela-Rey, M.; de Juan, V. G.; Fernandez-Tussy, P.; Zubiete-Franco, I.; Garcia-
Monzon, C.; Gonzalez-Rodriguez, A.; Oza, D.; Valenca-Pereira, F.; Fang, Q.; Crespo,
J.; Aspichueta, P.; Tremblay, F.; Christensen, B. C.; Anguita, J.; Martinez-Chantar, M.
L.; Rincon, M., Silencing hepatic MCJ attenuates non-alcoholic fatty liver disease (NAFLD)
by increasing mitochondrial fatty acid oxidation. Nat Commun 2020, 11 (1), 3360.

91. Zhou, F.; Ding, M.; Gu, Y.; Fan, G.; Liu, C.; Li, Y.; Sun, R.; Wu, J.; Li, J.; Xue,
X.; Li, H.; Li, X., Aurantio-Obtusin Attenuates Non-Alcoholic Fatty Liver Disease Through
AMPK-Mediated Autophagy and Fatty Acid Oxidation Pathways. Front Pharmacol 2021,
12, 826628.
57

92. Amorim, R.; Simoes, I. C. M.; Teixeira, J.; Cagide, F.; Potes, Y.; Soares, P.;
Carvalho, A.; Tavares, L. C.; Benfeito, S.; Pereira, S. P.; Simoes, R. F.; Karkucinska-
Wieckowska, A.; Viegas, I.; Szymanska, S.; Dabrowski, M.; Janikiewicz, J.; Cunha-
Oliveira, T.; Dobrzyn, A.; Jones, J. G.; Borges, F.; Wieckowski, M. R.; Oliveira, P. J.,
Mitochondria-targeted anti-oxidant AntiOxCIN(4) improved liver steatosis in Western diet-
fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation,
quality control mechanism and antioxidant defense systems. Redox Biol 2022, 55,
102400.

93. Wang, X. Y.; Lu, L. J.; Li, Y. M.; Xu, C. F., MicroRNA-376b-3p ameliorates
nonalcoholic fatty liver disease by targeting FGFR1 and regulating lipid oxidation in
hepatocytes. Life Sci 2022, 308, 120925.

Abstract (if available)

Abstract Estrogen related receptors (ERRs) are orphan nuclear receptors involved in the transcriptional regulation of mitochondrial bioenergetics. ERRa is a dominant isoform in the liver. As a transcriptional factor, ERRa binds to co-activators like peroxisome proliferator-activated receptor gamma coactivator 1-a (PGC-1a) to regulate mitochondrial respiratory genes such as cytochrome C (Cyt C) and thus plays key roles in oxidative phosphorylation (OXPHOS). As such, loss of ERRa function is expected to result in lower OXPHOS and suppress catabolism, leading to metabolic disorders. Given to the emerging evidence that dysregulated fatty acid oxidation (FAO) exacerbates the progression of liver diseases, we explored the functional role of ERRa in FAO. Our data demonstrated that ERRa plays a role in exogenous lipid usage but does not affect FAO of endogenous lipid oxidation. In addition, previous study from our lab also showed that the deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) which activates PI3K/AKT pathway regulates mitochondrial function via ERRa and Pten deletion increased FAO capacity. Here, we also investigated whether ERRa participates in the PI3K/AKT pathway regulated FAO.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

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

PDF

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates fatty acid beta-oxidation

PDF

Regulation of mitochondrial bioenergetics via PTEN (phosphatase and tensin homolog deleted on chromosome 10)/estrogen-related receptor alpha (ERRα) signaling

PDF

Transcriptional regulation of lipid metabolism and exploring the specific roles of AKT isoforms

PDF

The role of estrogen-related receptor alpha (NR3B1) in nonalcoholic fatty liver diseases

PDF

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

PDF

PTEN loss antagonizes aging through promoting regeneration and prevents oxidative stress induced cell death

PDF

Neuronal and glial metabolic alterations in the liver-specific PTEN knockout mouse model

PDF

Protein kinase Bα (AKT1) but not protein kinase Bβ (AKT2) controls pancreatic β cell growth and survival

PDF

Steatosis induces Wnt/β-catenin pathway to stimulate proliferation of hepatic tumor initiating cells and promote liver cancer development

PDF

Eicosanoids in the progression of liver steatosis to cancer

PDF

Ultra-sensitive ethanol receptors as novel tools for alcohol and brain research: optimizing loop 2 mutations in α2 glycine receptors, γ2 and α1 γ-aminobutyric acid type A receptors

PDF

AKT exhibits isoform-specific function in hepatocyte transformation and response to cellular stress

PDF

Akt1 deletion decrease proliferation in aged pancreatic beta-cells by arresting cell cycle in S phase

PDF

The regulation of the expression of MRP2 in vivo

PDF

Energy metabolism and inflammation in brain aging: significance of age-dependent astrocyte metabolic-redox profile

PDF

Nuclear respiratory factor-1 regulation by PTEN signaling

PDF

Pten regulates beta-cell regeneration intrinsically and independently of development

PDF

Insulin sensitivity in cognition, Alzheimer's disease and brain aging

PDF

Age dependent modulation of synaptic plasticity and insulin mimetic effect of lipoic acid on a 3xTg-AD mouse model of Alzheimer's disease: implications as a therapeutic/nutraceutical agent

Asset Metadata

Creator Xu, Chenxi (author)

Core Title The regulation of fatty acid oxidation by estrogen related receptor alpha

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2023-08

Publication Date 07/31/2023

Defense Date 07/31/2023

Publisher University of Southern California. Libraries (digital)

Tag estrogen related receptors,fatty acid oxidation,metabolic disease,mitochondrial genes,OAI-PMH Harvest,PI3K/Akt

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stiles, Bangyan (committee chair), Cadenas, Enrique (committee member), Okamoto, Curtis (committee member)

Creator Email chenxixu@usc.edu,x2571689729@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113291581

Unique identifier UC113291581

Identifier etd-XuChenxi-12171.pdf (filename)

Legacy Identifier etd-XuChenxi-12171

Document Type Thesis

Rights Xu, Chenxi

Internet Media Type application/pdf

Type texts

Source 20230731-usctheses-batch-1076 (batch), 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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA

Repository Email cisadmin@lib.usc.edu