Effect of TRB3 on Skeletal Muscle Mass Regulation and ...
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Theses and Dissertations
Spring 2019
Effect of TRB3 on Skeletal Muscle MassRegulation and Exercise-Induced AdaptationRan Hee Choi
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EFFECT OF TRB3 ON SKELETAL MUSCLE MASS REGULATION AND EXERCISE-INDUCED ADAPTATION
by
Ran Hee Choi
Bachelor of Science Ewha Womans University, 2010
Master of Science
University of Texas at Austin, 2013
Submitted in Partial Fulfillment of the Requirements
For the Degree of Doctor of Philosophy in
Exercise Science
Norman J. Arnold School of Public Health
University of South Carolina
2019
Accepted by:
Ho-Jin Koh, Major Professor
Xuewen Wang, Committee Member
Ashley J. Smuder, Committee Member
E. Angela Murphy, Committee Member
Cheryl L. Addy, Vice Provost and Dean of the Graduate School
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© Copyright by Ran Hee Choi, 2019 All Rights Reserved.
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DEDICATION
To my dearest grandmother, late Nak Sung An, and my parents, Doo Joo and Hye
Ryun, for their unconditional love, endless support, and constant encouragement
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ACKNOWLEDGEMENTS
First and foremost, praises and thanks to the almighty God for establishing me to
complete this work successfully. I would like to express immense gratitude to my
beloved and inspiring family for their endless love, constant encouragement, prayer
throughout my life and time in graduate school: this would not have been happened
without their support.
I am grateful to my advisor, Dr. Ho-Jin Koh, for his patient, support, and
guidance throughout this journey. Also, I wish to thank my committee members, Dr.
Xuewen Wang, Dr. Ashley Smuder, and Dr. Angela Murphy for their time and effort to
provide me constant motivation and guidance toward the completion of this research.
Furthermore, I would like to express sincere thanks to Dr. James Carson for his
invaluable encouragement and support throughout the doctoral training.
Lastly, my genuine gratitude goes to Abigail McConahay and João Silvestre for
their assistance and constant support. This work would not have been able to complete
without their help. I also wish to thank all my colleagues in the Department of Exercise
Science for their friendship and empathy.
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ABSTRACT
Skeletal muscle, which composes over 40% of body mass, is responsible for daily
locomotion and energy metabolism. It is a malleable tissue that can adapt its structure and
function in response to internal and external environmental stimuli. Changing muscle
mass and energy substrate utilization is a common skeletal muscle adaptation in response
to various pathological conditions, including type 2 diabetes and obesity. Failure to
maintain skeletal muscle mass and function has been correlated with increasing morbidity
and mortality, as well as poor quality of life. Hence, maintenance of skeletal muscle
integrity is a recommended strategy in achieving better quality of life. TRB3 is a
pseudokinase that is known to negatively regulate Akt phosphorylation, a key protein
kinase in regulating protein turnover and energy metabolism in skeletal muscle. Although
TRB3 is found in skeletal muscle and its expression is associated with Akt signaling, no
previous studies have elucidated the effects of TRB3 on skeletal muscle mass regulation.
The purpose of this dissertation was to determine the role of TRB3 in skeletal muscle
mass regulation and exercise-induced adaptation. We hypothesized that TRB3 expression
would regulate protein turnover by regulating Akt and its downstream proteins, mTOR
and FOXO, at the basal state and under atrophic conditions. We also expected TRB3
expression to blunt exercise-induced skeletal muscle adaptation. In Aim 1, we tested
whether TRB3 expression in mouse skeletal muscle regulated protein turnover through
the Akt/mTOR/FOXO pathway at the basal state. We found that skeletal muscle protein
turnover was regulated by TRB3 expression. In Aim 2, we examined whether TRB3
expression in mouse skeletal muscle affected food deprivation (FD)-induced skeletal
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muscle atrophy. We observed that muscle-specific TRB3 overexpression worsened FD-
induced atrophy via increasing proteolysis systems, while TRB3 knockout prevented the
atrophy by preserving protein synthesis. In Aim 3, we tested whether TRB3 expression
was involved in exercise-induced skeletal muscle adaptation. Here, we found that muscle
specific TRB3 overexpression blunted the benefits of exercise training in glucose uptake
and mitochondrial adaptation. These findings provide evidence that TRB3 is a potential
target to improve skeletal muscle integrity and quality in both healthy conditions and
atrophic conditions.
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TABLE OF CONTENTS
DEDICATION ....................................................................................................................... iii
ACKNOWLEDGEMENTS ........................................................................................................ iv
ABSTRACT ............................................................................................................................v
LIST OF TABLES .................................................................................................................. ix
LIST OF FIGURES ...................................................................................................................x
LIST OF ABBREVIATIONS .................................................................................................... xii
CHAPTER 1: INTRODUCTION ..................................................................................................1
CHAPTER 2: LITERATURE REVIEW .......................................................................................12
2.1 TRIBBLES HOMOLOG 3 (TRB3) .........................................................................13
2.2 SKELETAL MUSCLE MASS REGULATION .............................................................25
2.3 EXERCISE-INDUCED SKELETAL MUSCLE ADAPTATION .......................................32
CHAPTER 3: TRIBBLES 3 REGULATES PROTEIN TURNOVER IN MOUSE SKELETAL MUSCLE ....38
3.1 ABSTRACT .........................................................................................................39
3.2 INTRODUCTION ..................................................................................................39
3.3 MATERIAL AND METHODS .................................................................................41
3.4 RESULTS ...........................................................................................................44
3.5 DISCUSSION ......................................................................................................47
CHAPTER 4: TRB3 REGULATES SKELETAL MUSCLE MASS IN FOOD DEPRIVATION-INDUCED ATROPHY .............................................................................................................................54
4.1 ABSTRACT .........................................................................................................55
4.2 INTRODUCTION ..................................................................................................55
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4.3 MATERIAL AND METHODS .................................................................................59
4.4 RESULTS ...........................................................................................................62
4.5 DISCUSSION ......................................................................................................70
CHAPTER 5: EFFECTS OF TRB3 ON EXERCISE-INDUCED SKELETAL MUSCLE ADAPTATION ..88
5.1 ABSTRACT .........................................................................................................89
5.2 INTRODUCTION ..................................................................................................90
5.3 MATERIAL AND METHODS .................................................................................93
5.4 RESULTS ...........................................................................................................96
5.5 DISCUSSION ....................................................................................................103
CHAPTER 6: OVERALL DISCUSSION ...................................................................................120
REFERENCES .....................................................................................................................128
APPENDIX A: REPRINT PERMISSIONS FROM PUBLISHERS ...................................................145
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LIST OF TABLES
Table 4.1 Primer sequences for RT-PCR ...........................................................................77
Table 5.1 Primer sequences for RT-PCR .........................................................................108
Table 5.2 Characteristics of TG mice in voluntary wheel cage exercise .........................109
Table 5.3 Characteristics of TG mice in treadmill exercise .............................................111
Table 5.4 Characteristics of KO mice in voluntary wheel cage exercise ........................114
Table 5.5 Characteristics of KO mice in treadmill exercise ............................................117
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LIST OF FIGURES
Figure 1.1 Overall working model for proposed aims .......................................................10
Figure 3.1 Effects of TRB3 on muscle mass and function in mice ...................................50
Figure 3.2 Effects of TRB3 overexpression on proteins synthesis in mouse skeletal muscle ................................................................................................................................51 Figure 3.3 Effects of TRB3 knockout on protein synthesis and muscle mass ...................52
Figure 3.4 Effects of TRB3 in skeletal muscle on protein degradation signaling and E3 ubiquitin ligases .................................................................................................................53 Figure 4.1 Food deprivation increases TRB3 expression and decreases Akt and FOXOs phosphorylation ..................................................................................................................78 Figure 4.2 ER stress is responsible for food deprivation-induced TRB3 expression ........79
Figure 4.3 Muscle-specific TRB3 overexpression increases food deprivation-induced skeletal muscle atrophy ......................................................................................................80 Figure 4.4 TRB3 overexpression in skeletal muscle facilitates the protein degradation pathways, including ubiquitin-proteasomal and autophagy-lysosomal systems after 48 hr food deprivation .................................................................................................................81 Figure 4.5 Muscle-specific TRB3 overexpression does not augment the effect of food deprivation on protein synthesis rate and signaling pathway ............................................82 Figure 4.6 TRB3 knockout mice were protected from food deprivation-induced atrophy .............................................................................83 Figure 4.7 TRB3 knockout maintains protein synthesis rate after 48hr food deprivation ................................................................................................84 Figure 5.1 Muscle-specific TRB3 overexpression impaired exercise capacity measured by 6-week voluntary wheel running .....................................................................................110 Figure 5.2 Muscle-specific TRB3 overexpression deteriorates glucose uptake in response to 6-week treadmill training .............................................................................................112 Figure 5.3 Muscle-specific TRB3 overexpression represses gene expressions of mitochondrial biogenesis markers ...................................................................................113
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Figure 5.4 TRB3 knockout improves glucose tolerance but does not augment the benefits of exercise on glucose uptake ..........................................................................................115 Figure 5.5 TRB3 knockout does not facilitate mitochondrial adaptation in response to VW training .....................................................................................................................116 Figure 5.6 TRB3 knockout does not further improve molecular markers for glucose uptake in response to involuntary exercise ......................................................................118 Figure 5.7 TRB3 knockout does not promote mitochondrial biogenesis in response to involuntary treadmill exercise ..........................................................................................119
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LIST OF ABBREVIATIONS
4EBP1 ....................................... Eukaryotic translation initiation factor 4E-binding protein
AMPK ................................................. Adenosine monophosphate-activated protein kinase
APPL1 .................................................................................... Endosomal adaptor protein 1
AS160 ......................................... Rab GTPase-activating protein Akt substrate of 160 kDa
ATF4 ................................................................................. Activating transcription factor 4
ATF6 ................................................................................. Activating transcription factor 6
Atrogin-1 ............................................................................................ Muscle atrophy F box
C/EBPs .......................................................................... CCAAT-enhancer binding proteins
CHOP ............................................ CCAAT/enhancer-binding protein homologous protein
EDL ............................................................................................. Extensor digitorum longus
eIF2α ........................................................... Eukaryotic translation initiation factor 2 alpha
ER .................................................................................................... Endoplasmic reticulum
FOXOs ....................................................................... Forkhead box O transcription factors
GAS .............................................................................................................. Gastrocnemius
GLUT4 ................................................................................................ Glucose transporter 4
GSK3 ........................................................................................ Glycogen synthase kinase 3
IGF-1 ........................................................................................ Insulin-like growth factor-1
IRE1 ......................................................................................... Inositol requiring enzyme 1
IRS .............................................................................................. Insulin receptor substrates
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LC3B ......................................................... Microtubule-associated protein 1 light chain 3B
mTOR ............................................................................... Mechanistic target of rapamycin
MuRF-1 ............................................................................................ Muscle RING finger-1
NRF ............................................................................................. Nuclear respiratory factor
PEPCK ..................................................................... Phosphoenolpyruvate carboxylkinase
PERK ................................ Double-stranded RNA-activated protein kinase-like ER kinase
PGC1α .................. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha
PI3K ....................................................................................... Phosphatidylinositol 3-kinase
RNAi ......................................................................................................... RNA interference
S6K1 ................................................................................................. Ribosomal S6 kinase 1
siRNA ............................................................................................... Small interfering RNA
SOL ............................................................................................................................ Soleus
T2DM .......................................................................................................... Type 2 diabetes
TA ................................................................................................................ Tibialis anterior
TG ........................................................................................................................ Transgenic
TRB3 ..................................................................................................... Tribbles homolog 3
UPR ............................................................................................. Unfolded protein response
WT ........................................................................................................................ Wild-type
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CHAPTER 1
INTRODUCTION
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Skeletal muscle is a highly malleable tissue that responds to internal and external
environmental stimuli. It is able to alter muscle fiber size, function, and metabolism
depending on whether the stimuli are anabolic or catabolic. Moreover, skeletal muscle
supports daily locomotion, force generation, and energy storage, which are essentials for
daily life. However, pathological stimuli can disrupt the regulation of skeletal muscle
integrity, resulting in loss of muscle mass and mechanical and metabolic function. The
loss of skeletal muscle mass, also known as muscle atrophy or wasting, is a common
phenomenon caused by physical inactivity; fasting; aging and several diseases, including
cancer, diabetes, and heart failure. Muscle atrophy is also associated with high morbidity
and mortality, and poor quality of life. Therefore, the maintenance of skeletal muscle
mass has been perceived as a critical component in pursuing a better quality of life.
Although researchers have made efforts to prevent or delay skeletal muscle atrophy, they
have not fully uncovered the molecular mechanism(s) involved in the progress of the
disease. Hence, there is a critical need to develop new and more effective treatments for
muscle atrophy.
Skeletal muscle mass is determined by the balance between protein synthesis and
degradation. When the rate of protein synthesis exceeds the rate of protein breakdown, it
causes an increase in muscle mass and fiber size, known as muscle hypertrophy. By
contrast, if the overall rate of protein degradation surpasses protein synthesis, it induces
muscle atrophy, leading to a decrease in muscle mass and fiber size (Schiaffino, Dyar,
Ciciliot, Blaauw, & Sandri, 2013). Anabolic stimuli, such as insulin, insulin-like growth
factor-1 (IGF-1), and other growth factors, are required to increase or preserve skeletal
muscle mass. These stimuli activate phosphatidylinositol 3-kinase (PI3K) and Akt, which
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are considered as pivotal signaling molecules in regulating protein balance. For example,
IGF-1 binds to its receptor and phosphorylates insulin receptor substrates (IRS) to recruit
and activate PI3K, leading to subsequent phosphorylation and activation of Akt (Glass,
2005). Akt is a chief regulator of protein synthesis and degradation because its activation
can phosphorylate the mechanistic target of rapamycin (mTOR) for the activation of
protein synthesis via the regulation of ribosomal S6 kinase 1 (S6K1) and eukaryotic
translation initiation factor 4E-binding protein 1 (4EBP1), while it can inhibit the
translocation of forkhead box O transcription factors (FOXOs) into the nucleus to
inactivate protein degradation. Research has demonstrated that constitutively active Akt
in C2C12 cells and in mouse tibialis anterior muscle is sufficient to increase muscle in
cross-sectional area and fiber size (Bodine, Stitt, et al., 2001; Rommel et al., 2001). Akt-
induced hypertrophy is attenuated by treatment with rapamycin, an mTOR inhibitor,
suggesting the Akt/mTOR pathway is necessary for muscle hypertrophy (Bodine, Stitt, et
al., 2001; Rommel et al., 2001). In addition, IGF-1 treatment in dexamethasone-treated
C2C12 myotubes prevents dexamethasone-induced myotube atrophy by phosphorylating
Akt and FOXOs, resulting in decreased skeletal muscle-specific E3 ubiquitin ligases,
atrogin-1 and muscle RING finger-1 (MuRF-1) expression (Sandri et al., 2004; Stitt et
al., 2004). This effect has been further demonstrated under in vivo atrophic conditions,
using glucocorticoids and denervation, that increasing Akt phosphorylation by IGF-1
supplement significantly blunts skeletal muscle atrophy associated with increasing the
phosphorylation of FOXOs and reducing the expression of atrogenes (Stitt et al., 2004).
Taken together, the IGF-1/PI3K/Akt pathway is a central mechanism in regulating
skeletal muscle mass in basal and atrophic conditions. However, it has not been fully
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understood what mechanism(s) manage Akt activity to create a balance between
protein synthesis and degradation.
Tribbles homolog 3 (TRB3) is a mammalian homolog of Drosophila Tribbles. It
is a pseudokinase, meaning it contains a kinase-like domain without an ATP-binding site
or catalytic activity (Grosshans & Wieschaus, 2000; Hegedus, Czibula, & Kiss-Toth,
2006). Research has shown that, due to its lack of catalytic activity, TRB3 acts like a
scaffold or a mediator protein by binding to target proteins to control their functions
(Boudeau, Miranda-Saavedra, Barton, & Alessi, 2006). TRB3 is known as a negative
regulator of Akt because it disrupts the phosphorylation in liver and skeletal muscle (Du,
Herzig, Kulkarni, & Montminy, 2003; Koh et al., 2013; J. Liu et al., 2010). Studies have
demonstrated that in the liver, hepatic TRB3 expression increases under stressful
conditions, including fasting and insulin resistance; and that it inhibits Akt
phosphorylation at both T308 and S473 phosphorylation sites (Du et al., 2003). The liver
of a mouse with overexpressed TRB3 displays impaired glucose tolerance, increased
hepatic glucose output, and reduced glycogen content in comparison with the liver of a
control mouse (Du et al., 2003). Previous studies have reported that higher TRB3
expression is found in muscle samples from obese and type 2 diabetic patients than in
samples from healthy controls (Koh et al., 2013; J. Liu et al., 2010). High expression of
TRB3 is positively correlated with fasting blood glucose and is inversely related to
glucose disposal rate (J. Liu et al., 2010). Moreover, endoplasmic reticulum (ER) stress-
induced insulin resistance in mouse skeletal muscle relates to increasing TRB3
expression (Koh et al., 2013). TRB3 knockdown via RNA interference (RNAi) in C2C12
myotubes ameliorates the impairment of insulin-stimulated phosphorylation of IRS-1 and
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Akt under ER stress conditions (Koh et al., 2013). In addition, TRB3 knockout mice do
not show high-fat diet-induced weight gain, but do show improved glucose tolerance and
enhanced insulin-stimulated phosphorylation of IRS-1 and Akt when compared to wild
type mice (Koh et al., 2013). These data suggest that TRB3 expression is associated with
impaired insulin signaling under stressful conditions such as obesity, diabetes, and ER
stress. However, it has not been fully understood whether TRB3 plays a role in skeletal
muscle mass regulation mediated by the IGF-1/PI3K/Akt pathway. Given the function
of TRB3 in the regulation of Akt activity under metabolically stressful conditions,
TRB3 would be a potent candidate for coordinating protein turnover in skeletal
muscle by managing Akt and its downstream proteins. Currently, little is known
about the roles of TRB3 in skeletal muscle, especially in muscle mass regulation.
Therefore, it is important to investigate whether TRB3 is a key molecule in
mediating protein synthesis and breakdown in basal and atrophic conditions.
Increasing physical activity is another strategy for retaining skeletal muscle
integrity in order to produce better outcomes following metabolic deterioration and
chronic disease. It is well-established that exercise not only improves metabolic
conditions, such as glycemic control, whole-body glucose, and fat metabolism, but also
maintains the integrity of skeletal muscle, including its mass and function, resulting in
better quality of life (Egan & Zierath, 2013). Metabolic benefits of regular physical
activity on skeletal muscle include elevated enzyme activity involved in glucose and fat
metabolism, as well as enhanced glucose uptake and insulin sensitivity (Egan & Zierath,
2013). These allow skeletal muscle to produce and utilize energy sources more
efficiently. It has been found that short- and long-term use of moderate endurance
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exercise increases the expression and translocation of glucose transporter 4 (GLUT4)
(Goodyear et al., 1990; Ploug et al., 1990), glucose uptake (Dela, Mikines, von Linstow,
Secher, & Galbo, 2006; Richter, Mikines, Galbo, & Kiens, 1989), and the expression of
hexokinase II (Jorgensen, Jensen, & Richter, 2007; O'Doherty, Bracy, Osawa,
Wasserman, & Granner, 1994) in rodent and human skeletal muscle. Moreover, skeletal
muscle fibers are directly influenced by aerobic exercise, resulting in an alteration of
specific contractile proteins, such as myosin heavy chain isoforms, and an increase in
oxidative capacity (Gollnick et al., 1973; J. O. Holloszy & Booth, 1976; Rockl et al.,
2007). Overall, these exercise-induced alterations in skeletal muscle contribute to
improved glucose metabolism, insulin sensitivity, and oxidative capacity.
Elevated TRB3 expression has been found in obese and diabetic in mice and in
humans (Koh et al., 2013; J. Liu et al., 2010).Previous studies have also shown that
TRB3 is responsive to exercise training in obese and diabetic rodent models. Acute 2 h-
swimming exercise in ob/ob mice improves insulin-simulated IRS-1 and Akt signaling
with decreased TRB3 expression in skeletal muscle (Matos et al., 2010). Exercise also
diminishes TRB3/Akt association in exercised skeletal muscle from ob/ob mice
compared to sedentary groups (Matos et al., 2010). Another recently published study has
demonstrated that TRB3 expression may assist exercise capacity in mouse models.
Muscle-specific TRB3 transgenic mice have displayed a greater time to exhaustion
exercise capacity and a higher portion of oxidative muscle fiber types in soleus and
tibialis anterior muscles (An et al., 2014). Although the role of TRB3 in the regulation
of the PI3K/Akt pathway has been demonstrated, little is known about the effects of
TRB3 in overall exercise-induced metabolic and skeletal muscle adaptations.
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Overall premise
Skeletal muscle is a highly plastic tissue, meaning that it can adjust its function
and formation depending on internal and external stimuli. Chronic diseases, which need
long-term care and cost tremendous amounts in health-related billing, often arouse
skeletal muscle wasting and impair skeletal muscle function, thus leading to high
mortality and poor quality of life. Therefore, preserving skeletal muscle integrity is an
essential approach to enhancing quality of life among patients with pathologic conditions.
To preserve better muscle quality, maintaining skeletal muscle mass via nourishing diet
and regular physical activity is critical. However, current knowledge is limited regarding
the protection of muscle quality under catabolic conditions. The IGF-1/PI3K/Akt
pathway has been explored as a major molecular mechanism in regulating skeletal muscle
mass. The activity of Akt controls protein synthesis and breakdown through the
regulation of mTOR and FOXOs activation. Although the significance of Akt signaling is
well known, it has not been completely elucidated the manner in which molecule(s)
switch Akt signaling on and off to regulate the balance of protein synthesis and
degradation. TRB3 has been known as a negative regulator of Akt in multiple tissues,
including skeletal muscle. Its function in skeletal muscle has been limited with regard to
insulin resistance and glucose homeostasis. However, with regard to maintaining skeletal
muscle integrity through mass regulation and exercise-mediated adaptation, TRB3 has
great potential to be a direct regulator of the Akt signaling process which manages
protein balance.
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Overall purpose and hypothesis
The overall purpose of this study is to determine the roles of TRB3 in muscle
mass regulation and exercise-induced adaptation in mouse skeletal muscle. The central
hypothesis is that TRB3 expression will decrease protein synthesis and increase
degradation by regulating Akt signaling at basal state and under atrophic conditions. In
addition, TRB3 will play a pivotal role in exercise-induced skeletal muscle adaptation
with respect to glucose metabolism and oxidative capacity. In order to test this
hypothesis, we will determine whether TRB3 regulates skeletal muscle mass through
regulation of Akt and its downstream proteins at basal state. Next, we will examine the
effects of TRB3 on skeletal muscle mass under atrophic conditions. Lastly, we will
determine whether TRB3 plays a significant role in exercise-induced skeletal muscle
adaptation. We will utilize muscle-specific TRB3 transgenic and whole-body TRB3
knockout mice models to determine the roles of TRB3 in skeletal muscle mass regulation
and exercise adaptation. Atrophy will be induced by fasting, a method which is well
established and confirmed by other researchers (Garlick, Millward, James, & Waterlow,
1975; Gomes, Lecker, Jagoe, Navon, & Goldberg, 2001; Lecker et al., 2004; Li &
Goldberg, 1976). Exercise training will be induced via two different procedures,
voluntary wheel cage and forced treadmill training.
Specific aims
To investigate proposed hypotheses, we set three specific aims, including sub-
aims:
Specific Aim 1: To determine whether TRB3 regulates skeletal muscle mass through
Akt and its downstream proteins, mTOR and FOXOs at the basal state
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Aim 1.1: To determine whether muscle-specific TRB3 overexpression disrupts Akt and
its downstream signaling proteins
Aim 1.2: To determine whether global deletion of TRB3 benefits skeletal muscle mass
and function through the Akt signaling pathway
Specific Aim 2: To examine the role of TRB3 in skeletal muscle mass regulation
under atrophic conditions
Aim 2.1: To test whether fasting-induced ER stress regulates TRB3 expression in C2C12
mouse myoblasts
Aim 2.2: To determine whether muscle-specific TRB3 overexpression aggravates fasting-
induced atrophy
Aim 2.3: To determine whether the knockout of TRB3 ameliorates fasting-induced
atrophy
Specific Aim 3: To examine whether TRB3 affects exercise-induced skeletal muscle
adaptation
Aim 3.1: To determine whether muscle-specific TRB3 overexpression disrupts exercise-
induced skeletal muscle adaptation through inactivation of Akt and its downstream
proteins
Aim 3.2: To determine whether TRB3 is required for exercise-induced metabolic and
morphological skeletal muscle adaptation
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Proposed working model
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Figure 1.1 Overall working model for proposed aims. The overall purpose of the current study is to determine the roles of TRB3 in skeletal muscle in terms of mass regulation and exercise-induced adaptation. Our central hypothesis is that increasing TRB3 expression in mouse skeletal muscle will interfere with Akt and its downstream signaling pathways, including mTOR and FOXOs, to disrupt the balance between protein synthesis and degradation at basal state and under atrophic conditions. Specific aim 1 will investigate the effects of TRB3 in skeletal muscle mass regulation at basal state, using muscle-specific TRB3 overexpressed and whole-body TRB3 knockout mouse models. Specific aim 2 will examine the roles of TRB3 in fasting-induced skeletal muscle atrophy with our mouse models. In addition, we will directly examine an upstream mechanism to induce TRB3 expression under fasting-induced atrophy conditions in C2C12 mouse myoblasts. Specific aim 3 will determine the roles of TRB3 in exercise-induced skeletal muscle adaptation with regard to glucose metabolism and oxidative capacity.
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CHAPTER 2
LITERATURE REVIEW
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2.1 Tribbles homolog 3 (TRB3)
Discovery of tribbles protein
A mammalian homolog of Drosophila tribbles was first discovered in Drosophila
melanogaster, and as a protein it is highly conserved from Drosophila to humans (Mata,
Curado, Ephrussi, & Rorth, 2000; Seher & Leptin, 2000). The tribbles protein inhibits
mitosis by arresting the cell cycle in the G2 phase and degrading two Drosophila cell
cycle regulators, string and twine (homologs of mammalian cyclin-dependent kinase 25)
via proteasomal breakdown (Mata et al., 2000). Moreover, the tribbles protein has been
reported to alter the ubiquitin-proteasome in a way that degrades slow border cells, the
mammalian homolog of CCAAT-enhancer binding proteins (C/EBPs) (Masoner et al.,
2013; Rorth, Szabo, & Texido, 2000). In mammals, there are three isoforms of the
tribbles homolog protein, including TRB1, TRB2, and TRB3. All these isoforms contain
a conserved protein kinase domain lacking kinase capacity, called a pseudokinase, and
have similar functional domains. Although they share analogous domains, each isoform
is expressed in different tissues and shows distinct roles in various cellular processes.
First, TRB1 is preferentially found in the nucleus which has been shown to increase
metabolic dysfunction in vascular tissues and to regulate Akt in retinoic acid receptor and
endothelial cells (Ashton-Chess et al., 2008; Imajo & Nishida, 2010; Mondal, Mathur, &
Chandra, 2016). In smooth muscle cells, TRB1 overexpression mediates the mitogen-
activated protein kinase pathway (Sung et al., 2007). Recently, studies have shown that
M2 macrophage differentiation is affected by TRB1 expression, suggesting an
association with inflammation (Y. H. Liu, Tan, Morrison, Lamb, & Argyle, 2013).
Research has connected TRB2 to tumorigenesis in acute myelogenous leukemia,
melanoma, and hepatocellular carcinoma (Keeshan et al., 2006; Keeshan, Shestova,
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Ussin, & Pear, 2008; J. Wang et al., 2013; Zanella et al., 2010). Meanwhile, TRB3 has
been found in both nucleus and cytoplasm, and is globally associated with several
different diseases, including type 2 diabetes (T2DM), cardiovascular disease, obesity, and
cancer (Avery et al., 2010; Hua et al., 2015; Oberkofler et al., 2010; Ti et al., 2011). In
addition, studies have revealed that TRB3 regulates stress-related metabolic responses,
such as glucose and lipid metabolism, apoptosis, tumorigenesis, and endoplasmic
reticulum (ER) stress (Koh et al., 2013; J. Liu et al., 2010; Ord & Ord, 2003, 2005;
Saleem & Biswas, 2017; Schwarzer, Dames, Tondera, Klippel, & Kaufmann, 2006;
Yacoub Wasef, Robinson, Berkaw, & Buse, 2006).
Functions of TRB3
TRB3 is the most-studied isoform among tribbles homolog proteins because it is
ubiquitously expressed in multiple tissues, including liver, pancreas, adipose tissue,
kidney, and skeletal muscle. Also, TRB3 is known to be a cellular stress-responsible gene
in various conditions such as fasting (Du et al., 2003; Koo et al., 2004), cancer (Bowers,
Scully, & Boylan, 2003; Xu et al., 2007), T2DM (J. Liu et al., 2010; Zhang et al., 2016),
obesity (Koh et al., 2013; Marinho et al., 2012), and ER stress (Koh et al., 2013; Ord &
Ord, 2005). It has been shown that, to maintain cellular homeostasis in response to these
stresses, TRB3 is involved in several cellular processes, including insulin signaling (Du
et al., 2003; Koh et al., 2006; Koh et al., 2013; J. Liu et al., 2010), glucose and lipid
metabolism (Bi et al., 2008; Qi et al., 2006), autophagy (Salazar et al., 2009; Saleem &
Biswas, 2017), and cell differentiation (Kato & Du, 2007). Research has continuously
discovered the functions of TRB3 and it premise to be a potential target to ameliorate the
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consequences of various cellular stress circumstances. Therefore, it is important to
discuss what is currently known and how to determine further roles of TRB3.
TRB3 and Insulin signaling
TRB3 is well known as a negative regulator of Akt which works by interfering
with the phosphorylation sites Thr308 and Ser473, which are responsible for a critical
insulin signaling pathway (Du et al., 2003). Several studies have demonstrated that
insulin resistance condition in rodents and humans leads to an increase in TRB3
expression, and that this is associated with an impairment of insulin signaling, especially
the inhibition of Akt phosphorylation (Fischer et al., 2017; Prudente & Trischitta, 2015).
A number of in vitro and in vivo studies have already demonstrated that TRB3 plays a
pivotal role in Akt activation and is associated with insulin signaling pathways. In
cultured cell studies, it has been reported that high-fat conditional media incubation,
including palmitic acids, on HepG2 hepatocytes, human umbilical vein endothelial cells,
and C2C12 myoblasts, increases TRB3 expression and decreases insulin-stimulated
phosphorylation of Akt (Geng et al., 2013; Koh et al., 2013; Liang et al., 2015).
Moreover, knockdown of TRB3 expression in HepG2 hepatocytes by RNAi increases
Akt phosphorylation at Ser473, resulting in the deactivation of glycogen synthase kinase
3 (GSK3) and FOXO1 (Du et al., 2003). Another study confirms these findings in the
same HepG2 cells transfected by small interfering RNA (siRNA) against TRB3, and
further demonstrates that inhibition of TRB3 expression increases the phosphorylation of
S6K1 and 4E-BP1, which are downstream proteins of the Akt signaling pathway
(Matsushima, Harada, Webster, Tsutsumi, & Nakaya, 2006). On the other hand,
adenoviral injection-induced TRB3 overexpression in primary mouse hepatocytes
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reverses these results, diminishing the levels of phosphorylated S6K1 and 4E-BP1 and
suggesting that Akt activation would be impaired (Matsushima et al., 2006). In C2C12
myoblasts, the overexpression of TRB3 has been shown to decrease insulin-stimulated
Akt and IRS1 phosphorylation (Koh et al., 2006; Koh et al., 2013). In addition, the cells
treated with ER stress inducers, including tunicamycin and thapsigargin, greatly increase
TRB3 expression and decrease the activation of insulin-stimulated IRS1 and Akt.
However, deletion of TRB3 by RNAi in tunicamycin-treated C2C12 myoblasts recovers
insulin-stimulated phosphorylation of IRS1 and Akt (Koh et al., 2013). Overexpression of
TRB3 in rat L6 skeletal muscle cells also significantly reduces the phosphorylation of
Akt and extracellular signal-regulated kinases 1 and 2 with suppressed GLUT4
translocation after insulin stimulation (J. Liu et al., 2010). In in vivo studies, TRB3
expression has been demonstrated to play a significant role in insulin signaling pathways.
Recently, it has been reported that higher TRB3 expression is found in the skeletal
muscle of obese and diabetic populations than in the skeletal muscle of healthy, normal-
weight people (Koh et al., 2013; J. Liu et al., 2010). Consistent with the findings in obese
and diabetic human subjects, elevated TRB3 expression is evident in various
metabolically dysfunctional animal models, including db/db (lack of leptin receptor) and
ob/ob (lack of leptin hormone) mice, Zucker fatty rats, and diet-induced obesity rodent
models (Du et al., 2003; Koh et al., 2013; Lima et al., 2009; J. Liu et al., 2010; Marinho
et al., 2012; Sun et al., 2017). Subsequently, these models have demonstrated that obese
and diabetic pathophysiological conditions induce TRB3 expression and also impair
insulin-stimulated Akt activation (Koh et al., 2013; Lima et al., 2009; J. Liu et al., 2010;
Marinho et al., 2012). In addition, hepatic TRB3 overexpression via adenoviral tail vein
injection disrupts glucose tolerance and gluconeogenesis in test mice as compared to
17
control mice (Du et al., 2003). Although TRB3 overexpression in rodent models could be
responsible for insulin insensitivity, the knockout of TRB3 in mice has not demonstrated
a clear effect on insulin signaling. First, a study knocking out TRB3 in the mouse showed
no change in insulin-stimulated Akt activation and other downstream proteins, glucose
tolerance, and insulin sensitivity (Okamoto et al., 2007). However, whole-body TRB3
knockout (KO) mice prevent diet-induced obesity over the course of 8 weeks of high-fat
feeding. And these KO mice has shown improved insulin signaling demonstrated by
increasing insulin-stimulated phosphorylation of IRS1, Akt, and FOXO (Koh et al.,
2013). It should be noted that in this study, liver weight and triglyceride contents after 8
weeks of high-fat diet were also decreased in KO (Koh et al., 2013). In summary, several
in vivo and in vitro studies have demonstrated that TRB3 is an important regulator of the
insulin signaling pathway through Akt under metabolically abnormal conditions.
TRB3 and Metabolism
Since TRB3 is known as a negative regulator of Akt phosphorylation, the primary
focus in determining its roles has been Akt-related insulin signaling, including glucose
metabolism and insulin resistance. The relationships between these topics and roles of
TRB3 have been demonstrated in multiple tissues, including liver, heart, fat, and skeletal
muscle, using diverse animal models and cell cultured studies. In the liver, an organ
essential in controlling whole-body glucose and lipid metabolism, mice overexpressing
TRB3 via adenoviral tail vein injection present impaired glucose tolerance and glucose
output – which suggests developed insulin insensitivity. Meanwhile, knocking down
TRB3 in FAO hepatocytes decreases insulin-response glucose output and increases
glycogen synthesis, as confirmed by the inhibition of insulin-stimulated GSK3b (Du et
18
al., 2003). In line with results showing that TRB3 overexpression disrupts glucose
metabolism, another study using a tail vein injection of TRB3 expressed adenovirus to
C57BL/6 mice supports the hypothesis that TRB3 overexpression in the liver debilitates
glucose and insulin tolerance without changing the body weight or serum insulin level
(Matsushima et al., 2006). On the other hand, in adipose tissue, a chief area responsible
for the lipid metabolism which supplies energy for the demands of daily life, fat-specific
TRB3 transgenic mice demonstrate increased fatty acid oxidation, which prevents diet-
induced obesity (Qi et al., 2006). This study has further determined that fat-specific
TRB3 transgenic mice show little or no body weight gain after 5 weeks of high-fat diet
when compared to wild type, and they also show decreased white adipose tissue mass
after high-fat diet feeding (Qi et al., 2006). Furthermore, studies have shown that TRB3
can be associated with E3 ubiquitin ligase, called COP1, in adipocytes to degrade acetyl-
coenzyme A carboxylase, which is a rate limiting enzyme of fatty acid synthesis, in order
to inhibit lipogenesis (Qi et al., 2006). In the heart, cardiac-specific TRB3
overexpression in mice decreases glucose oxidation rates (though not fatty acid
oxidation) (Avery et al., 2010). In accordance with the role of TRB3 in glucose
metabolism and insulin signaling in liver and heart, a number of studies have reported
that TRB3 expression plays a significant role in metabolism in skeletal muscle as well.
Earlier studies (using human subjects and genetically modified animal models, including
db/db and ob/ob mice and Zucker fatty rats) have proven that the expression of TRB3 in
skeletal muscle is associated with metabolic dysfunctions such as diabetes and
obesity(Koh et al., 2013; J. Liu et al., 2010). Other animal models with deleted liver
kinase B 1 in skeletal muscle show improved glucose tolerance, resulting from a decrease
in TRB3 expression in skeletal muscle (Koh et al., 2006). In addition, TRB3
19
overexpression in rat L6 myoblasts tagged with GLUT4myc decreases insulin-stimulated
2-deoxy-D-glucose uptake and GLUT4 translocation (J. Liu et al., 2010). Furthermore,
research has demonstrated a relationship between TRB3 and ER stress-induced insulin
resistance (Koh et al., 2013). Treatments of tunicamycin and thapsigargin, ER stress
inducers, induce TRB3 expression and result in a reduction of glucose uptake in mouse
C2C12 myoblasts and skeletal muscle (Koh et al., 2013). However, TRB3 knockout mice
treated with tunicamycin blunt the reduction of insulin-stimulated glucose uptake and
resist high-fat diet-induced obesity, showing improved glucose tolerance and increased
insulin-stimulated glucose uptake in soleus muscle compared to wild type (Koh et al.,
2013). Also, TRB3 expression in skeletal muscle is induced by glucose-induced insulin
resistance or glucose toxicity. It is associated with increase in O-linked glycosylation,
which is already linked to insulin resistance (Zhang et al., 2013; Zhang et al., 2016).
Zhang et al. (2013) reported that glucose toxicity, induced by high (>25 mmol/L) and low
(0 mmol/L) glucose, dramatically elevates TRB3 expression in rat L6 myoblasts, along
with increasing O-linked glycosylation (Zhang et al., 2013). Recently, they created
muscle-specific TRB3 overexpressed mice (using human TRB3 cDNA) and knockout
mice (using the flippase recombinase target flanked-sequence in TRB3) to determine
whether TRB3 mediates glucose toxicity in skeletal muscle (Zhang et al., 2016). Muscle-
specific TRB3 overexpression in mice impairs glucose tolerance and phosphorylation of
insulin-stimulated Akt, Akt substrate 160kD, and glucose synthase after 6 weeks of
diabetic condition induced by 5 days of streptozotocin injections (Zhang et al., 2016).
High-fat diet feeding of these mice significantly increases body weight and fasting blood
glucose compared to wild type, and mice with muscle-specific TRB3 overexpression
shows highly increased pro-inflammatory markers, including NF-κB and TNF-α; and
20
activated stress-responsible markers, such as activating transcription factor 4 (ATF4) and
CCAAT/enhancer-binding protein homologous protein (CHOP). However, muscle-
specific TRB3 knockout in mice abolishes these negative effects of hyperglycemia and
diet-induced obesity (Zhang et al., 2016).
TRB3 and ER stress
As stated above, TRB3 is a gene responsible for stress, particularly ER stress
(Jousse et al., 2007; Koh et al., 2013; Ohoka, Yoshii, Hattori, Onozaki, & Hayashi, 2005;
Ord & Ord, 2003, 2005). Cellular stresses, like nutrient excess or deprivation, can disrupt
homeostasis. This increases the protein synthesis load to ER in order to coordinate
metabolic alteration and cellular transformation, resulting in an increase in unfolded
protein response (UPR). UPR is a regulatory response to cellular stress; however, it can
also initiate stress-signaling pathways that attenuate stress-adaptive reactions and
promote uncontrolled UPR in ER. These phenomena, called ER stress, can hinder cell
survival mechanisms and facilitate cell death, resulting in metabolic diseases and cancer
(Dicks, Gutierrez, Michalak, Bordignon, & Agellon, 2015; Guerrero-Hernandez, Leon-
Aparicio, Chavez-Reyes, Olivares-Reyes, & DeJesus, 2014; S. Wang & Kaufman, 2012).
Excessive UPR responses can induce ER stress responses by activating three different
arms: activating transcription factor 6 (ATF6), inositol requiring enzyme 1 pathway
(IRE1), and double-stranded RNA-activated protein kinase-like ER kinase (PERK)
pathway (Walter & Ron, 2011). It has been demonstrated that TRB3 is particularly
associated with the PERK pathway that exerts ER stress-mediated responses through the
phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α), leading to an
arrest of global translation (Bromati et al., 2011; Fang et al., 2014; Ohoka et al., 2005;
21
Qian et al., 2008). Although this process reduces the burden of protein folding load to the
ER lumen, phosphorylated eIF2α increases ATF4 translation, which can regulate many
genes related to metabolism and amino acid transport in response to oxidative stress
(Harding et al., 2000). In addition, CHOP, a transcription factor, is activated by ATF4
and induces encoding of genes involved in apoptosis (Sok et al., 1999; X. Z. Wang et al.,
1998; Zinszner et al., 1998). Therefore, the activation of the PERK arm is considered a
powerful protective mechanism of ER stress-induced UPR responses.
A number of reports describe TRB3 as a regulatory protein for ATF4 and CHOP
(Corcoran et al., 2005; Jousse et al., 2007; Ohoka et al., 2005; Ord & Ord, 2003, 2005).
ER stress induced by treatment with tunicamycin or thapsigargin can induce TRB3
mRNA and protein expression in several in vitro settings, such as HepG2, C2C12, GT1-7
cells (Koh et al., 2013; Ohoka et al., 2005; Ord & Ord, 2003, 2005). Increased TRB3
expression by an ER stress inducer follows elevation of ATF4 and CHOP (Ohoka et al.,
2005; Ord & Ord, 2003). Moreover, it has been demonstrated that overexpression of
ATF4 and/or CHOP can induce TRB3 promoter activity, whereas the deletion of
ATF4/CHOP by siRNA decreases ER stress-induced TRB3 promoter activity (Ohoka et
al., 2005). Interestingly, TRB3 co-expression with either ATF4 or CHOP overexpression
inhibits its own expression, suggesting that TRB3 regulates its expression by suppressing
ATF4 and CHOP in a negative feedback loop (Jousse et al., 2007; Ohoka et al., 2005). In
addition, another study has reported that ATF4 and TRB3 co-expression in HEK293 cells
suppresses ATF4-induced apoptosis, indicating that TRB3 plays an inhibitory role in
ATF4 transcriptional activity (Ord, Meerits, & Ord, 2007). Nutrient deprivation
conditions, such as leucine deprivation, induce TRB3 expression as well. The increment
of TRB3 is shown to be associated with stress-induced ATF4 activation and to regulate
22
ATF4-related transcription activity (Jousse et al., 2007). Furthermore, overexpression or
inhibition of TRB3 regulates CHOP promoter activity by leucine starvation, confirming
the negative feedback loop between ATF4/CHOP and TRB3 (Jousse et al., 2007). Given
the findings regarding the interaction of TRB3 with ATF4 and CHOP, ER stress shows
potential as a regulator of TRB3 in stress conditions.
TRB3 and Exercise
Regular exercise is important not only in maintaining general health but also in
preventing many diseases, including cardiovascular disease, obesity, and diabetes (Arena
et al., 2017; Katzmarzyk, Lee, Martin, & Blair, 2017). Exercise also provides numerous
beneficial effects on skeletal muscle with regard to insulin sensitivity in animals and
humans. However, the question of whether insulin sensitivity in skeletal muscle can be
restored by physical activity remains unanswered.
Uncontrolled hepatic glucose production by insulin stimulation is an essential
indicator of whole-body insulin insensitivity, but exercise has been shown to suppress the
process, and to improve insulin signaling and tolerance (De Souza et al., 2010). Since the
liver is the main organ that expresses TRB3 (Du et al., 2003; Koo et al., 2004; Ord & Ord,
2005), previous research has focused on the effects of TRB3 in the liver on the regulation
of hepatic insulin signaling with physical activity. One study, which applied acute
swimming exercises (four sets of 30-minute swimming sessions with 5-minute rest
periods for a total duration of 2 hours of exercise) to high-fat diet induced obesity mice
and to ob/ob mice, demonstrates that hepatic TRB3 expression is significantly reduced
after insulin stimulation; however, the level of TRB3 binding to Akt decreases
dramatically in both exercise groups (Lima et al., 2009). Consequently, exercised obese
23
groups increase insulin sensitivity and glycogen content while they decrease
gluconeogenesis proteins, such as phosphoenolpyruvate carboxylkinase (PEPCK),
through FOXO1 inactivation compared to obese groups (Lima et al., 2009). Furthermore,
another research group has reported that exercise-reduced TRB3 expression is due to an
increased expression of endosomal adaptor protein (APPL1) (Marinho et al., 2012).
APPL1 is an adaptor protein known to regulate Akt activity via direct binding, and
research has shown that this protein competes with TRB3 in order to bind to Akt (Cheng
et al., 2009; Hosch, Olefsky, & Kim, 2006). Endurance training using 60minute
swimming protocol (5 days/week for 8 weeks) significantly decreases hepatic TRB3
expression in high-fat diet-induced obese mice when compared to lean mice; this
reduction is accompanied by an increase in APPL1. Elevated hepatic APPL1 has been
shown to increase Akt activity, thus suppressing the expression of gluconeogenesis
proteins, including FOXO1, peroxisome proliferator-activated receptor gamma
coactivator 1 alpha (PGC1α), PEPCK, and glucose 6-phosphatase, in the livers of
exercise-trained high-fat diet-induced obese mice (Marinho et al., 2012). These studies
have confirmed that hepatic TRB3 expression responds to acute and chronic exercise
protocols. Moreover, the reduction in hepatic TRB3 expression in obese mice shows
beneficial effects on insulin sensitivity under obesity-induced insulin resistance
conditions.
Skeletal muscle is the primary site where insulin-stimulated glucose is taken for
energy storage and utilization. Given the function of TRB3 that inhibits insulin-
stimulated Akt phosphorylation, it rises attentions to study whether the expression of
TRB3 in skeletal muscle is regulated by exercise and whether TRB3 affects the
adaptation of exercise-induced skeletal muscle to glucose metabolism. The first study,
24
which measured TRB3 expression in skeletal muscle in response to exercise,
demonstrates that acute swimming exercise (four sets of 30-minute swimming sessions
with 5-minute rest periods for a total duration of 2 hours of exercise) reduces TRB3
expression in skeletal muscle from exercised leptin-deficient ob/ob mice in comparison
with sedentary ob/ob mice (Matos et al., 2010). The decreased TRB3 expression in the
skeletal muscle of exercised ob/ob mice restores insulin-stimulated phosphorylation of
IRS1 and Akt in comparison with sedentary ob/ob mice, leading to higher GLUT4
expression in the plasma membrane, greater glucose disappearance during insulin
tolerance test, and increased muscle glycogen content (Matos et al., 2010). However, one
recent study reports that TRB3 mRNA and protein expression increases in skeletal
muscle from healthy wild-type mice following 4 weeks of wheel cage exercise (An et al.,
2014). These conflicting results may be due to the use of different species of mice and
experimental settings, for instance, leptin-deficient ob/ob mice versus healthy wild-type
mice and swimming versus wheel cage exercises.
The effect of TRB3 in skeletal muscle on exercise capacity has also been
investigated using muscle-specific TRB3 transgenic mice. There appears to be no
difference in insulin-stimulated IRS1 and Akt phosphorylation, or glucose and insulin
tolerance, in the muscle-specific TRB3 transgenic mice when compared to the wild type
(An et al., 2014). Interestingly, muscle-specific TRB3 transgenic mice show a
significantly greater maximal exercise capacity as measured by time-to-exhaustion tests;
and they also show a greater amount of oxidative fibers than wild-type mice (An et al.,
2014). These data suggest that TRB3 may play a critical role in exercise-induced skeletal
muscle adaptation and fiber type shifting.
25
2.2 Skeletal muscle mass regulation
Skeletal muscle shows high plasticity, meaning that it is able to alter and adapt its
compositions and functions in response to different stimuli, including nutrient and energy
status, contraction, and growth hormones (Schiaffino et al., 2013). Muscle mass changing
is one of the most common responses used to demonstrate skeletal muscle flexibility. The
term hypertrophy refers to increasing muscle mass caused by weight-bearing, balanced
nutrients, and anabolic hormones; while atrophy refers to reducing skeletal muscle mass
caused by catabolic signals, including inactivity and malnutrition (Schiaffino et al., 2013;
Schiaffino & Mammucari, 2011). These conditions of skeletal muscle can be determined
by measuring the balance between protein synthesis and degradation (Schiaffino et al.,
2013). A positive balance occurs when the protein synthesis rate in skeletal muscle
exceeds protein breakdown; when this happens, the muscle fibers will increase in size
(hypertrophy). However, when skeletal muscle maintains a prolonged negative balance,
the rate of protein degradation is likely to exceed the rate of protein synthesis, resulting in
atrophic status. The IGF-1)/PI3K/Akt signaling cascade is considered a key mechanism
in controlling protein turnover in skeletal muscle (Bodine, Stitt, et al., 2001; Latres et al.,
2005; Rommel et al., 2001; Sacheck, Ohtsuka, McLary, & Goldberg, 2004; Schiaffino &
Mammucari, 2011; Velloso, 2008). Akt (or protein kinase B) is thought to be the master
regulator of protein turnover in skeletal muscle protein because it stimulates protein
synthesis via mTOR and protein degradation through the FOXO family (Blaauw et al.,
2009; Rommel et al., 2001; Schiaffino & Mammucari, 2011; Vander Haar, Lee,
Bandhakavi, Griffin, & Kim, 2007). Therefore, in order to maintain protein turnover and
skeletal muscle mass in animal and human subjects, it is important to understand how the
26
IGF-1/PI3K/Akt pathway in skeletal muscle regulates protein synthesis and degradation
and alters skeletal muscle mass.
Protein synthesis via the IGF-1/PI3K/Akt/mTOR pathway
IGF-1 is an essential growth hormone that induces muscle growth by activating
the PI3K/Akt pathway (Bodine, Stitt, et al., 2001; Latres et al., 2005; Rommel et al.,
2001; Sacheck et al., 2004; Schiaffino & Mammucari, 2011; Velloso, 2008). Insulin,
another anabolic hormone, also stimulates the growth of muscle fibers by binding to
insulin receptors, which activate the same downstream signaling as IGF-1(Bolster,
Jefferson, & Kimball, 2004; Kitamura et al., 1998). After IGF-1 and insulin bind to their
receptors, IRS1 is recruited in order to phosphorylate its downstream molecules, PI3K
and Akt, for activation (Bolster et al., 2004; Latres et al., 2005; Rommel et al., 2001).
Activated (phosphorylated) Akt can regulate protein synthesis with its downstream
proteins, mTOR and GSK3b (Glass, 2005; Rommel et al., 2001). First, Akt activation
indirectly regulates mTOR phosphorylation for increasing protein synthesis in
consequence of the phosphorylation of tuberous sclerosis complex 2 and proline-rich Akt
substrate 40 kD (Inoki, Li, Zhu, Wu, & Guan, 2002; Manning, Tee, Logsdon, Blenis, &
Cantley, 2002; Vander Haar et al., 2007). The phosphorylated mTOR further activates
and inhibits S6K1 and 4EBP1 by phosphorylation. These processes increase translation
initiation and elongation, resulting in upregulating protein synthesis (Fingar & Blenis,
2004; Hara et al., 1997; Rommel et al., 2001). Moreover, the inhibition of mTOR
activation by rapamycin treatment, a pharmacological compound used as an mTOR
inhibitor, in cultured myotubes decreases IGF-1-induced phosphorylation of S6K to
suppress protein synthesis (Rommel et al., 2001; Rommel et al., 1999). In vivo study has
27
also demonstrated that the inhibition of mTOR by rapamycin treatment blocks
compensatory hypertrophy-induced phosphorylation of Akt, S6K, and 4EBP1 (Bodine,
Stitt, et al., 2001). Furthermore, skeletal muscle hypertrophy induced by a constitutively
activated form of Akt to mouse TA muscle is abolished by rapamycin treatment (Bodine,
Stitt, et al., 2001). In line with this result, Akt transgenic mice display large muscle fiber
size and enhanced muscle function, including strength (Blaauw et al., 2009; Izumiya et
al., 2008; Sartori et al., 2009). These findings suggest that the activation of Akt and
downstream molecules are critical to the stimulation of protein synthesis in response to
anabolic stimuli, for instance IGF-1 and insulin.
Protein degradation through the IGF-1/PI3K/Akt pathway
The IGF-1/PI3K/Akt pathway also plays a pivotal role in the regulation of protein
degradation. Previously, it has been found that muscle-specific IGF-1 overexpression
promotes hypertrophy and protects against age-related atrophy (Musaro et al., 2001).
Insulin and/or IGF-1 treatment activates protein synthesis while it suppresses protein
breakdown, and these processes are mediated by the PI3K/Akt pathway (Monier, Le
Cam, & Le Marchand-Brustel, 1983; Rommel et al., 2001; Sacheck et al., 2004). In
atrophic conditions, the PI3K/Akt signaling is suppressed, leading to the inhibition of
mTOR and the activation of FOXOs (Bodine, Stitt, et al., 2001; Sandri et al., 2004; Stitt
et al., 2004). FOXOs are a family of forkhead box O transcription factors containing
three members, FOXO1, FOXO3a, and FOXO4 which are abundant in mammalian cells
(Tran, Brunet, Griffith, & Greenberg, 2003). As the activation of Akt facilitates protein
synthesis through the phosphorylation of mTOR in skeletal muscle under anabolic status,
it can also govern FOXOs activity via phosphorylation in order to inactivate protein
28
degradation. A dephosphorylated form of FOXOs can translocate into the nucleus, where
it works as a transcription factor, whereas a phosphorylated form is excluded from the
cytoplasm to be inactivated. The activation of FOXOs, increasing dephosphorylated
nuclear FOXOs, is required to express muscle-specific E3 ubiquitin ligases such as
muscle atrophy F box (atrogin-1) and muscle RING finger-1 (MuRF-1). These genes
encode ubiquitin ligases, which cause ubiquitination to designated protein substrate and
degradation in skeletal muscle. In addition to the ubiquitin-proteasome system, FOXOs
can also regulate muscle autophagy-lysosomal protein breakdown (Sandri, 2010).
Therefore, the regulation of FOXOs by the IGF-1/PI3K/Akt is an important way to
maintain skeletal muscle mass under atrophic conditions.
Ubiquitin-proteasome system
Ubiquitination is the process of tagging proteins that are designated for
degradation with ubiquitin proteins (Finley & Varshavsky, 1985). Protein conjugated
with ubiquitination undergoes degradation through the 26S proteasome system (Coux,
Tanaka, & Goldberg, 1996). A few ubiquitin ligases have been identified in the striated
muscles, where they are responsive to muscle atrophic conditions, including fasting,
denervation, disuse, and cancer cachexia (Bodine, Latres, et al., 2001; Gomes et al., 2001;
Lecker et al., 2004; Sacheck et al., 2007). Atrogin-1 and MuRF-1 are muscle-specific
ubiquitin ligases that increase under muscle wasting conditions, leading to skeletal
muscle atrophy. Knocking out atrogin-1 in mice causes them to become resistant to
denervation-induced atrophy (Bodine, Latres, et al., 2001) and fasting-induced muscle
atrophy (Cong, Sun, Liu, & Tien, 2011), while deletion of MuRF-1 abolishes
dexamethasone-induced muscle wasting (Baehr, Furlow, & Bodine, 2011). The activation
29
of FOXOs is required for the upregulation of both muscle-specific ubiquitin ligases
during muscle atrophic conditions (Sandri et al., 2004; Stitt et al., 2004). FOXO1 has
been identified as necessary for transcription of atrogin-1 and MuRF-1, but it is not alone
sufficient to induce the transcription of both genes (Stitt et al., 2004). IGF-1 treatment
can abolish atrogin-1 and MuRF-1 transcriptional activity in response to dexamethasone
via the blocking of FOXO1 activation (Stitt et al., 2004). Another study has demonstrated
that FOXO3a is a key regulator of atrogin-1 among downstream proteins of Akt, and that
constitutively activated FOXO3a in C2C12 myotubes and mouse skeletal muscle
promotes muscle atrophy through the activation of atrogin-1 promoter (Sandri et al.,
2004). Furthermore, FOXO1 is found to be a main transcription factor in activating
MuRF-1 promoter activity under dexamethasone-induced atrophy in C2C12 myotubes
(Waddell et al., 2008). This effect of FOXO1 on MuRF-1 expression is blocked by IGF-1
treatment via the activation of the PI3K/Akt pathway (Sacheck et al., 2004). Recently,
triple FOXOs muscle-specific knockout mice, the deletion of FOXO1, 3a, and FOXO4,
have demonstrated that FOXOs are required to induce the expression of atrophy-related
ubiquitin ligase genes; and these mice prevent loss of skeletal muscle mass and function
from fasting- and denervation-induced skeletal muscle atrophy (Milan et al., 2015).
Altogether, these data suggest that FOXOs activity is a major player in the regulation of
muscle-specific ubiquitin ligase expression under atrophic conditions; and that this
expression can be prevented by the activation of Akt signaling.
Autophagy-lysosomal system
Autophagy is a highly conserved homeostatic process that breaks down and
recycles cell components through lysosomal machinery in response to cellular stress
30
(Sandri, 2010). Increased rates of autophagy have been observed in skeletal muscle under
various stimuli, including cancer (Penna et al., 2013), aging (Sandri, 2010, 2013),
nutrient restriction (Mizushima, Yamamoto, Matsui, Yoshimori, & Ohsumi, 2004),
muscle disuse and denervation (Brocca et al., 2012; Mammucari et al., 2007; O'Leary,
Vainshtein, Carter, Zhang, & Hood, 2012; Zhao et al., 2007). Skeletal muscle that has
undergone fasting or denervation induces the expressions of autophagy markers, such as
microtubule-associated protein 1 light chain 3B (LC3B), Bnip3, and Atg4b; while the
upregulation of autophagy genes is completely abolished in mice expressing the
constitutively active form of Akt (Mammucari et al., 2007). Moreover, like ubiquitin-
proteasome genes, FOXO3a has shown involvement in the regulation of autophagic
markers. Constitutively activated FOXO3a promotes LC3B transcriptional activity to
increase the formation of autophagosome, which then increases lysosomal proteolysis;
while the inhibition of FOXO3a by RNAi blocks fasting-induced autophagy in mouse
skeletal muscle (Mammucari et al., 2007). The triple FOXOs muscle-specific knockout
mice have decreased fasting- and denervation-induced lipidation and autophagosome
vesicle formation via LC3B and p62 expression (Milan et al., 2015). Taken together,
these results imply that FOXOs play a pivotal role in lysosomal autophagosome
proteolysis system; and the activity of Akt is critical to the regulation of the proteolytic
system in skeletal muscle via management of FOXOs activity.
Fasting-induced skeletal muscle atrophy
Prolonged starvation destroys the balance between anabolism and catabolism in
the system, resulting in increasing degradation of tissues that store energy sources,
including fat, liver, and skeletal muscle. Skeletal muscle contains a great number of
31
proteins which can be utilized to maintain blood glucose levels via gluconeogenesis in
the liver when the body undergoes severe catabolism due to fasting or diseases. It has
been demonstrated that fasting over 12 hours reduces ~ 20% of body weight and skeletal
muscle mass in rodent models (Garlick et al., 1975; Li & Goldberg, 1976). In addition to
mass changes, the rate of protein degradation is critically elevated while the rate of
protein synthesis is suppressed (Garlick et al., 1975; Li & Goldberg, 1976). Fasting has
been associated with an increase in E3 ubiquitin ligases, such as atrogin-1 and MuRF-1,
which are regulated by suppressed Akt and activated FOXOs signaling (Dehoux et al.,
2004; Gomes et al., 2001; Jagoe, Lecker, Gomes, & Goldberg, 2002; Sandri et al., 2004).
In vitro experiments have confirmed that the incubation of PBS for 6 hours, which
mimics fasting conditions, impairs phosphorylation of Akt and S6K1 while FOXOs is
activated (Sandri et al., 2004). Furthermore, the activation of FOXOs links to the
activation of atrogin-1 and MuRF-1 in C2C12 myotubes and mouse skeletal muscle
(Gomes et al., 2001; Sandri et al., 2004). Starvation has also been shown to induce
lysosomal-autophagosome proteolysis in rodent models. One-day food deprivation
disrupts Akt and its downstream proteins, mTOR and FOXO, signaling to accelerate
protein degradation; while autophagy-related genes, including LC3B and Bnip3, are
simultaneously activated to a high degree(Mammucari et al., 2007). This fasting-induced
autophagy is inhibited in mouse skeletal muscle with overexpressed Akt, whereas the
dominant negative form of FOXO3a completely abolishes fasting-induced
autophagosome formation in mouse skeletal muscle (Mammucari et al., 2007). These
findings support the hypothesis that food deprivation over 12 hours is sufficient to
provoke skeletal muscle atrophy through the activation of ubiquitin-proteasome and
lysosomal-autophagosome proteolysis.
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2.3 Exercise-induced skeletal muscle adaptation
Skeletal muscle plasticity responds not only to the balance between anabolism
and catabolism, but also to contractile activity. Regular physical activity is associated
with increasing repeated sporadic muscle contraction, which is considered a foundation in
the prevention, management, and treatment of chronic metabolic diseases, such as
T2DM, obesity, and cancer (Haskell et al., 2007). Physical activity has been successful
in delaying complications and improving symptoms of diseases, which in turn reduces
the individual’s burden of healthcare cost. Exercise has also been shown to contribute to
skeletal muscle physiological adaptation, for example, maintaining or gaining muscle
mass, increasing the shift in fiber type from glycolytic to oxidative, improving glucose
metabolism, and increasing the efficiency of oxidative phosphorylation (Egan & Zierath,
2013; Phillips, Green, Tarnopolsky, Heigenhauser, & Grant, 1996) . These beneficial
outcomes vary depending on the type (endurance vs. resistance), duration (acute vs.
chronic), and intensity (low to high) of the exercise sessions (Ferraro, Giammarioli,
Chiandotto, Spoletini, & Rosano, 2014; Nader & Esser, 2001). In general, exercise-
induced adaptations originate from alterations of gene expression, protein contents, and
enzymatic activities. These active changes are involved in myogenesis, carbohydrate and
fat utilization and metabolism, mitochondrial oxidative metabolism, and mitochondrial
biogenesis (Booth & Thomason, 1991; Mahoney et al., 2005; Pilegaard, Saltin, & Neufer,
2003). As a result, these progressions enable working muscle to efficiently utilize and
deliver substrates and to maximize mitochondrial respiratory capacity, resulting in
optimal performance and enhanced homeostatic maintenance to resist fatigue (Egan &
Zierath, 2013).
33
Exercise-induced glucose homeostasis and insulin sensitivity
Continuous contractile activity, which exercise can elicit, has been associated
with improved glucose metabolism in human and rodent models (Booth & Thomason,
1991; Dela et al., 2006; Ferraro et al., 2014). Exercise-induced glucose uptake was first
explored in animal models using a single bout of swimming exercise or electrical
stimulation. Contracting rat hindlimb muscles elevates the rate of glucose uptake in
comparison with control muscle, both in the absence and presence of insulin; but
stimulated muscles are more likely to be sensitive to insulin infusion (Ivy & Holloszy,
1981; Richter, Garetto, Goodman, & Ruderman, 1984). This result has been confirmed in
human subjects as well (Mikines, Sonne, Farrell, Tronier, & Galbo, 1988; Richter et al.,
1989). In this regard, exercise has proven its role as a medicine by effectively improving
glucose tolerance and increasing insulin sensitivity in normal, insulin resistance, and
aging skeletal muscle (Castorena, Arias, Sharma, & Cartee, 2014; Ivy, 1997; Kahn et al.,
1990; Kjobsted et al., 2017).
Exercise-induced improvements in glucose metabolism and insulin sensitivity are
associated with increasing expressions and functions of glucose transporters and
metabolic enzymes in skeletal muscle. It has been demonstrated that a single bout of
exercise can increase GLUT4 mRNA expression and protein expression in rat skeletal
muscle (Kuo, Hunt, Ding, & Ivy, 1999; Neufer, Shinebarger, & Dohm, 1992); however,
not all studies demonstrate the same effects (Funai & Cartee, 2009; Hansen, Nolte, Chen,
& Holloszy, 1998). In human skeletal muscle, GLUT4 mRNA expression is increased
immediately post-exercise (Kraniou, Cameron-Smith, & Hargreaves, 2006; McGee &
Hargreaves, 2004), although varied results of GLUT4 protein expression have been
observed (Frosig, Pehmoller, Birk, Richter, & Wojtaszewski, 2010; Kraniou et al., 2006;
34
Leick et al., 2010). Exercise-increased glucose uptake occurs after exercise in both
insulin-dependent and independent conditions. Acute and chronic exercise induces
insulin-stimulated glucose uptake (G. D. Cartee et al., 1989; Frosig et al., 2007; Gulve,
Cartee, Zierath, Corpus, & Holloszy, 1990) by increasing insulin-stimulated GLUT4
translocation (Hansen et al., 1998). Insulin-mediated GLUT4 translocation has been
found to be associated with the Rab GTPase-activating protein Akt substrate of 160 kDa
(AS160; also known as TBC1D4) (G. D. Cartee & Wojtaszewski, 2007; Kramer et al.,
2006). In the absence of insulin, phosphorylated AS160 is higher in the exercised group
and it remains higher than in the sedentary group for up to 14 hours (Arias, Kim, Funai,
& Cartee, 2007; Frosig et al., 2007). Furthermore, insulin treatment subsequently
stimulates the phosphorylation of AS160 via Akt phosphorylation (Arias et al., 2007;
Funai, Schweitzer, Sharma, Kanzaki, & Cartee, 2009). This insulin-dependent exercise-
induced glucose uptake can be completely inhibited by a PI3K inhibitor, suggesting that
the PI3K/Akt pathway is a key mechanism in regulating exercise-induced glucose
disposal and metabolism (Funai & Cartee, 2009; Wojtaszewski, Hansen, Kiens, &
Richter, 1997).
Another mechanism of exercise-induced glucose uptake is the elevation of the
ADP/ATP ratio and Ca2+ influxes in working muscle fibers, which results in activated
adenosine monophosphate-activated protein kinase (AMPK) (Egan et al., 2010; Merrill,
Kurth, Hardie, & Winder, 1997; Wojtaszewski et al., 2003). A paralog Rab GAP of
AS160, TBC1D1, has shown involvement in exercise-induced glucose transport and is
activated by insulin, muscle contraction, and the AMPK-activator AICAR (Funai &
Cartee, 2009; Taylor et al., 2008). Furthermore, AMPK has shown to be important in
mediating contraction-induced phosphorylation of TBC1D1 and glucose transport (Funai
35
& Cartee, 2009). Therefore, the activation of AMPK through contractile activity assists in
exercise-induced glucose uptake and metabolism.
In addition to exercise-induced GLUT4 expression and translocation, the elevated
expression and function of metabolic enzymes contributes to improved glucose
homeostasis after exercise (Egan & Zierath, 2013; Ferraro et al., 2014). Hexokinase II is
a rate-limiting enzyme used in glycolysis to convert glucose to glucose-6-phosphate,
which is a suitable form for transportation into the cytoplasm, where it can be further
metabolized (Wilson, 2003). It has been reported that even a single bout of exercise can
increase hexokinase II mRNA expression and enzymatic activity in rat skeletal muscle
(O'Doherty, Bracy, Granner, & Wasserman, 1996; O'Doherty et al., 1994). Previous
studies using muscle-specific hexokinase II transgenic mouse models demonstrate that
hexokinase II overexpression in skeletal muscle improves exercise-induced and insulin-
stimulated glucose uptake (Fueger et al., 2004; Halseth, Bracy, & Wasserman, 1999).
Furthermore, hexokinase II expression and activity are a determinants of exercise-
induced glucose uptake. It has been shown that skeletal muscle glucose uptake, glucose
oxidation rate, and exercise capacity are impaired in hexokinase II knockout mice(Fueger
et al., 2003; Fueger et al., 2005).
Exercise-induced oxidative capacity and muscle fiber type shifting
Endurance exercise training improves oxidative capacity and metabolic efficiency
in skeletal muscle. This benefit is elicited by increased mitochondrial content resulting
from the regulation of protein expressions and transcriptional activities pertaining to
transport and oxidation of energy substrates (Baar et al., 2002; Pilegaard et al., 2003).
Mitochondrial adaptation begins with mitochondrial biogenesis defined by an elevation in
36
mitochondria number and size, and an increase in efficiency of mitochondrial capacity.
Nuclear respiratory factor (NRF) -1 and NRF-2 have been identified as promoting gene
expression associated with components of the electron transport chain and mitochondrial
biogenesis (Scarpulla, 2002). Moreover, PGC-1a is highlighted as a key regulator of
mitochondrial biogenesis in skeletal muscle (Baar et al., 2002; Pilegaard et al., 2003). It
has been demonstrated that acute and chronic endurance exercise induces PGC-1a
mRNA and protein expression in animal and human models, with the level of expression
dependent on exercise intensity (Baar et al., 2002; Edgett et al., 2013; Egan et al., 2010;
Meirhaeghe et al., 2003; Pilegaard et al., 2003).
In addition to exercise-induced oxidative capacity via the alteration of these
transcriptional factors, the transformation of skeletal muscle fiber permits adaptation in
oxidative capacity. Skeletal muscle fibers vary in structural and metabolic characteristics
(Qaisar, Bhaskaran, & Van Remmen, 2016). Depending on myosin heavy chain isoforms
diversity, muscle fiber types in rodents can be divided into four group: slow-twitch
oxidative type I, fast-twitch and oxidative type IIa, fast-twitch and glycolytic type IIb/x,
and fast-twitch and highly glycolytic type IIb (Rivero, Talmadge, & Edgerton, 1998,
1999; Termin, Staron, & Pette, 1989). Humans express all the same isoforms as rodents
except type IIb (Ennion, Sant'ana Pereira, Sargeant, Young, & Goldspink, 1995).
Research has shown that endurance exercise promotes fiber type shifting among fast-
twitch fibers from glycolytic type IIb and IIb/x fiber to oxidative type IIa fiber (Green,
Thomson, Daub, Houston, & Ranney, 1979). PGC-1a plays an essential role in
transforming muscle fiber types induced by exercise. Studies of mice with muscle-
specific PGC-1a overexpression show that the mice improve in exercise capacity and
contain more slow-twitch and oxidative fibers (Calvo et al., 2008; Lin et al., 2002), while
37
whole-body or muscle-specific PGC-1a knockout mice demonstrate decreased oxidative
fiber types and properties in skeletal muscle (Handschin et al., 2007; Leone et al., 2005).
However, it has been reported that muscle-specific PGC-1a mice retain the capability for
exercise-induced fiber type shifting just as wild-type mice do, suggesting that the roles of
PGC-1a in skeletal muscle and exercise-induced fiber type transformation are not yet
completely understood (Geng et al., 2010)
38
CHAPTER 3
TRIBBLES 3 REGULATES PROTEIN TURNOVER IN MOUSE SKELETAL
MUSCLE1
1 Choi RH, McConahay A, Jeong HW, McClellan JL, Hardee JP, Carson JA, Hirshman MF, Goodyear LJ, Koh HJ. 2017. Biochemical and Biophysical Research Communications. 493:1236-1242. Reprinted here with permission of publisher.
39
3.1 ABSTRACT
Skeletal muscle atrophy is associated with a disruption in protein turnover
involving increased protein degradation and suppressed protein synthesis. Although it has
been well studied that the IGF-1/PI3K/Akt pathway plays an essential role in the
regulation of the protein turnover, molecule(s) that triggers the change in protein turnover
still remains to be elucidated. TRB3 has been shown to inhibit Akt through direct
binding. In this study, we hypothesized that TRB3 in mouse skeletal muscle negatively
regulates protein turnover via the disruption of Akt and its downstream molecules.
Muscle-specific TRB3 transgenic (TRB3TG) mice had decreased muscle mass and fiber
size, resulting in impaired muscle function. We also found that protein synthesis rate and
signaling molecules, mTOR and S6K1, were significantly reduced in TRB3TG mice,
whereas the protein breakdown pathway was significantly activated. In contrast, TRB3
knockout mice showed increased muscle mass and had an increase in protein synthesis
rate, but decreases in FoxOs, atrogin-1, and MuRF-1. These findings indicate that TRB3
regulates protein synthesis and breakdown via the Akt/mTOR/FOXOs pathways.
Key words: TRB3, protein degradation, protein synthesis, atrophy
3.2 INTRODUCTION
Skeletal muscle plays many important roles in general health, including whole-
body metabolism and mobility not only in the elderly but also in young individuals
(Goodpaster et al., 2006). Moreover, reduced muscle mass resulting from several critical
conditions, such as diabetes, cancer, and obesity, has substantially contributed to poor
quality of life and increased mortality (Martin et al., 2013; Rantanen et al., 2000;
Wannamethee, Shaper, Lennon, & Whincup, 2007). Therefore, a better understanding of
40
the signaling molecules that regulate muscle mass could enhance future treatments and
significantly reduce a healthcare burden.
The maintenance of muscle mass is a direct consequence of muscle protein
turnover, involving the homeostatic regulation of protein accretion and breakdown and it
directly impacts muscle growth and atrophy. Insulin and insulin-like growth factor-1
(IGF-1) are key anabolic hormones that regulate muscle protein homeostasis through the
PI3K/Akt signaling pathway (Latres et al., 2005; Rommel et al., 2001; Velloso, 2008).
Particularly, Akt controls both anabolic and catabolic signals in skeletal muscle via its
downstream proteins, mammalian target of rapamycin (mTOR) and forkhead box O
families (FOXOs), respectively (Bodine, Stitt, et al., 2001; Rommel et al., 2001; Sandri et
al., 2004; Stitt et al., 2004). mTOR mediates protein synthesis through the
phosphorylation of ribosomal S6 kinase 1 (S6K1) and 4EBP1, and it promotes cell
proliferation and growth in response to anabolic stimuli, including insulin, IGF-1, and
nutrients (Proud, 2004; Rommel et al., 2001). On the other hand, FOXOs regulate
catabolic processes and their activities are suppressed by the activation of Akt (Brunet et
al., 1999). FOXOs activates protein breakdown in skeletal muscle through the ubiquitin-
proteasome system by upregulating the expression of two muscle-specific E3 ubiquitin
ligases, atrogin-1 and MuRF-1, also called atrogenes (Sandri et al., 2004; Stitt et al.,
2004). However, signaling molecules that regulate protein turnover in the IGF-
1/PI3K/Akt pathway have not been fully described.
A mammalian Drosophila tribbles homolog 3 (TRB3) is a pseudokinase that lacks
an enzymatic domain of protein kinase (Du et al., 2003). Multiple tissues including
skeletal muscle have displayed an increase in expression of TRB3 under various stressful
conditions, such as fasting, ER stress, and insulin resistance (Du et al., 2003; Kato & Du,
41
2007; Koh et al., 2013; Liew et al., 2010; Qi et al., 2006). In mouse myoblast C2C12
cells, TRB3 overexpression directly disrupts Akt signaling and delays muscle cell
differentiation (Kato & Du, 2007). In vivo studies have established that TRB3 regulates
glucose homeostasis and ER stress-induced insulin resistance in skeletal muscle by
inhibiting Akt activity (Koh et al., 2013). Although numerous studies have demonstrated
that TRB3 negatively regulates the Akt signaling pathway to influence metabolism,
TRB3’s effects on downstream pathways of Akt, such as protein synthesis and
breakdown, in skeletal muscle have not been elucidated.
In the present study, we hypothesized that overexpression of TRB3 would disrupt
protein turnover through the suppression of Akt activation in skeletal muscle. Our
findings show that muscle-specific TRB3 transgenic (TRB3TG) mice exhibited impaired
muscle function and decreased muscle mass, resulting from suppressed mTOR/S6K1
signaling and increased FOXO1 and FOXO3a activation. Further, the mRNA expression
of atrogin-1 and MuRF-1 was elevated in TRB3TG mice. In contrast, the TRB3 knockout
mice tended to have increased muscle mass and protein synthesis rate, while the mRNA
expression of atrogenes was significantly suppressed. These results suggest that TRB3
plays a prominent role in the regulation of skeletal muscle protein balance and function.
3.3 MATERIALS AND METHODS
Animal Care and Use
Female (10-11-week-old) C57BL/6 wild type (WT), TRB3TG (An et al., 2014),
and whole-body TRB3 knockout (TRB3KO) mice (Koh et al., 2013; Okamoto et al.,
2007) were used for all experiments. All animals were maintained on a standard chow
diet (no. 8604, Harlan Teklad Diet, Madison, WI, USA) and allowed ad libitum access to
42
food and water. They were housed under 12 h (7AM – 7PM) of light per day in
temperature-controlled environment. All protocols were approved by the Institutional
Animal Care and Use Committee of the University of South Carolina.
Western Blot Analysis
Gastrocnemius (GAS) muscles were collected and protein lysates were prepared
for Western blot analysis as previously described (Fujii et al., 2004; Koh et al., 2013).
Primary and secondary antibodies were purchased from commercial sources, including p-
Akt Thr308 (9275) and Ser473 (9271), Akt (9272), p-mTOR Ser2448 (2971), mTOR (2972),
p-S6K1 Thr389 (9205), S6K1 (9202), p-FOXO1 Ser256 (9461), FOXO1 (2880), p-
FOXO3a Ser253 (13129), FOXO3a (12829), GAPDH (2118) from Cell Signaling
Technology (Danvers, MA, USA), Atrogin-1 (AP2041), MuRF-1 (MP3401) from ECM
(Versailles, KY, USA), TRB3 (ST1032; Calbiochem, San Diego, CA, USA), anti-
puromycin (MABE343; EDM Millipore Billerica, MA, USA), anti-rabbit (NA934; GE
healthcare, Pittsburgh, PA, USA) IgG- and anti-mouse (61-0220; Invitrogen, Carlsbad,
CA, USA) IgG2a-horesradish peroxidase conjugated secondary antibodies. The specific
bands were visualized using ECL reagents (NEL105001EA; PerkinElmer, Waltham, MA,
USA), and the intensity of bands was quantified using Image Studio Lite (LI-COR
Biosciences, Lincoln, NE, USA).
Real-Time Polymerase Chain Reaction Analysis
Tibialis anterior (TA) muscles were used for real-time PCR analysis as previously
described (Koh et al., 2013). Total RNA was extracted using Trizol reagent (15596018;
Life Technologies, Carlsbad, CA, USA) and cDNA was synthesized from 4 μg of total
RNA by High Capacity cDNA kit (Life Technologies, Carlsbad, CA, USA). SYBR green
43
PCR master mix (4309155; Life Technologies, Carlsbad, CA, USA) carried cDNA and
primers. Primer sequences were as follows: TBP forward, 5’-
ACCCTTCACCAATGACTCCTATG-3’, and TBP reverse, 5’-
TGACTGCAGCAAATCGCTTGG-3’; TRB3 forward, 5’-
TCTCCTCCGCAAGGAACCT-3’, and TRB3 reverse, 5’-
TCTCAACCAGGGATGCAAGAG-3’; Atrogin-1 forward, 5’-
GCAGAGAGTCGGCAAGTC-3’, and Atrogin-1 reverse, 5’-
CAGGTCGGTGATCGTGAG-3’; MuRF-1 forward, 5’-
GGAACCTGCTGGTGGAAAACATC-3’, and MuRF-1 reverse, 5’-
CGTCTTCGTGTTCCTTGCACATC-3’; Cathepsin-L forward, 5’-
GTGGACTGTTCTCACGCTCAAGG-3’, and Cathepsin-L reverse, 5’-
TCCGTCCTTCGCTTCATAGGT-3’. TBP expression served as an internal control and
relative levels of mRNA expression were normalized to its expression.
In vivo Protein Synthesis Measurement
The rate of in vivo protein synthesis was determined by measuring puromycin
incorporation into a nascent peptide as previously described (Goodman et al., 2011).
Briefly, mice received an intraperitoneal injection of 0.04 μmol/g body weight with
puromycin (540411; EDM Millipore, Billerica, MA, USA) dissolved in 100 μl of PBS 30
minutes before euthanasia. Tissues were collected and stored at -80 °C for future
analysis. Western blot was used to determine the amount of puromycin incorporation.
Muscle Cross-Sectional Area Analysis
Frozen TA muscles were sectioned (10-μm) on a cryostat microtome and affixed
to slides. Sections were stained with hematoxylin and eosin according to a previous study
(Hardee et al., 2016; McClung, Davis, Wilson, Goldsmith, & Carson, 2006). Fibers were
44
analyzed by ImageJ 1.48v (National Institutes of Health, Bethesda, MD, USA).
Approximately 200 fibers were traced per sample, as determined by a plateau in standard
deviation in order to determine the proximate fiber number and cross-sectional area.
Statistical Analysis
Data are expressed as means ± standard error of the mean. Student t-test and two-
way ANOVA followed by the Bonferroni Post hoc analysis were used to determine
significance. P < 0.05 was considered statistically significant.
3.4 RESULTS
Muscle-specific overexpression of TRB3 decreases skeletal muscle mass and impairs
muscle function
To determine the effects of TRB3 on skeletal muscle mass and function, we
studied TRB3TG mice that have previously been examined (An et al., 2014). The
expression of TRB3 mRNA and protein was significantly elevated in TA and GAS,
respectively, confirming the overexpression in mouse skeletal muscle (Fig. 3.1A and B).
Body weights were similar between WT and TRB3TG mice (Fig. 3.1C). However,
TRB3TG mice showed a significant decrease in muscle mass (Fig. 3.1D). TRB3TG mice
also displayed a significantly lower (16%) grip strength compared to WT (Fig. 3.1E).
Furthermore, we individually housed WT and TRB3TG mice in wheel cages for 8 weeks
to examine the capacity of voluntary wheel exercise. TRB3TG mice performed poorly
with a 46% decrease in total running distance compared to WT (Fig. 3.1F). Taken
together, these data suggest that TRB3 plays important roles in skeletal muscle mass
regulation and function.
45
TRB3 decreases muscle cross-sectional area in mouse skeletal muscle
To investigate if the decreased muscle mass in TRB3TG mice is associated with
an alteration in fiber size, we measured the cross-sectional area (CSA) in WT and
TRB3TG mice. Consistent with decreased muscle weight (Fig. 3.1D), TRB3TG mice
showed a marked decrease (23%) in mean CSA compared to WT (Fig. 3.1G and H).
Furthermore, the percentage of fibers less than 1000 μm2 was increased in TRB3TG
mice, while WT littermates were likely to have larger fibers (Fig. 3.1I). These data
indicate that TRB3 adversely affects myofiber size in mouse skeletal muscle.
TRB3TG mice display a decrease in protein synthesis
Given that TRB3TG mice had a decrease in muscle mass compared to WT, we
next determined if TRB3 overexpression affects signaling molecules involved in protein
synthesis. We have previously shown that overexpression of TRB3 in C2C12 myotubes
decreases insulin-stimulated Akt phosphorylation through direct binding to Akt (Koh et
al., 2006; Koh et al., 2013). To determine the effect of TRB3 on protein synthesis in
skeletal muscle, we studied Akt-regulated downstream proteins, mTOR/S6K1.
Overexpression of TRB3 significantly decreased mTOR phosphorylation at Ser2448
compared to WT (Fig. 3.2A). Consistent with reduced mTOR phosphorylation, the
phosphorylation of S6K1 was substantially suppressed (60%) in TRB3TG mice (Fig.
3.2B). Basal Akt phosphorylation at Thr308 and Ser473 was not different between groups
(Fig. 3.2C and D). To determine how decreased mTOR/S6K1 signaling affects the rate of
protein synthesis, WT and TRB3TG mice were given an intraperitoneal injection of
puromycin, a structural analog of tyrosyl-tRNA, to measure the rate of protein synthesis
as previously described (Goodman et al., 2011). The protein synthesis rate was reduced in
EDL (20%) and SOL (60%) muscles from TRB3TG mice compared to the muscles from
46
WT (Fig. 3.2E–G). We further tested the signaling molecules involved in protein
synthesis and measured the rate of protein synthesis in TRB3KO mice. Although the
level of phosphorylated mTOR was slightly higher in the TRB3KO group (Fig. 3.3A), we
did not observe a significant difference in the Akt/mTOR/S6K1 signaling in TRB3KO
mice compared to WT (Fig. 3.3A–D). The rate of protein synthesis in SOL muscle in
TRB3KO mice was higher than that of WT (Fig. 3.3G), although there was no difference
in protein synthesis rate in EDL muscle (Fig. 3.3F). Moreover, the muscle weights of
TRB3KO mice were increased (Fig. 3.3H and I) compared to WT, without a change in
body weight (unpublished observations by RH Choi and HJ Koh). These results
demonstrate that TRB3 regulates the anabolic signaling pathway and the rate of protein
synthesis in mouse skeletal muscle.
TRB3TG mice have activated protein degradation and increased atrogin-1 and MuRF-
1 mRNA expression
We next determined if TRB3 affects the protein degradation pathway, including
the FOXOs and the muscle-specific E3 ubiquitin ligases, atrogin-1 and MuRF-1.
TRB3TG mice showed a decrease in phosphorylation of FOXO1 and FOXO3a compared
to WT, indicating activated FOXO signaling (Fig. 3.4A and B). Interestingly, the mRNA
expression of MuRF-1 was significantly elevated, and there was a tendency (P=0.07) to
increase the atrogin-1 expression in TRB3TG mice without a change in cathepsin L
mRNA expression, a marker for lysosomal protease (Fig. 3.4C). FOXO1 and FOXO3a
activities were inhibited in TRB3KO mice compared with WT (Fig. 3.4D and E). These
results also coincided with a significant reduction in atrogin-1 and MuRF-1 mRNA
expression, whereas cathepsin L was not altered (Fig. 3.4F). Taken together, these data
indicate that TRB3 regulates FOXOs and protein degradation pathway.
47
3.5 DISCUSSION
The balance between anabolic and catabolic signal is critical to maintain muscle
mass. Akt, a key protein kinase in insulin signaling, regulates both protein synthesis and
degradation pathways. However, it has not yet been clearly elucidated what molecule(s)
may regulate Akt signaling. In the current study, we found that TRB3TG mice had
reduced muscle mass, decreased fiber size, and impaired muscle function. Protein
synthesis rate and signaling molecules, mTOR and S6K1, were significantly decreased in
TRB3TG mice. Furthermore, atrogin-1 and MuRF-1 were highly increased in TRB3TG
mice compared to WT, indicating an activation of protein degradation. In addition,
TRB3KO mice displayed increased muscle mass and also elevated protein synthesis rate,
while the protein degradation pathway was significantly suppressed. Taken together,
these findings suggest that TRB3 plays a prominent role in the regulation of protein
turnover.
Given the proposed role of TRB3 in the inhibition of Akt signaling (Du et al.,
2003; Koh et al., 2006; Koo et al., 2004), our findings demonstrate that TRB3 expression
in skeletal muscle affects Akt downstream proteins, including mTOR and FOXOs, in
order to regulate protein balance. The decreased mTOR/S6K1 phosphorylation and
protein synthesis rate in TRB3TG mice were associated with increased FOXOs activation
and atrogenes expression, which may influence the reduced muscle mass. We believe that
the decreased muscle mass and function in TRB3TG mice is due, at least in part, to the
negative regulation of the Akt/mTOR/FOXOs signaling. Earlier studies have reported
that the effect of TRB3 on the inhibition of Akt is related to metabolic dysfunctions, such
as insulin resistance and glucose intolerance (Du et al., 2003; Koh et al., 2013). However,
the regulation of downstream pathways of Akt by TRB3 has not been directly tested. Our
48
findings support the hypothesis that TRB3 negatively regulates Akt downstream
signaling in mouse skeletal muscle at basal state to disrupt protein balance, leading to a
suppression of protein synthesis and an activation of protein breakdown. In the present
study, we examined the basal Akt phosphorylation and did not detect a decreased Akt
phosphorylation in TRB3TG (Fig 3.2C and D). Given that Akt is activated by anabolic
stimulants, such as insulin and IGF-1 (Downward, 1998), it is possible that the marginal
difference of basal Akt phosphorylation in TRB3TG mice was undetectable by the
Western blot analysis. Our previous study and others have used insulin treatment to
demonstrate that Akt activation is impaired by TRB3 expression while the level of Akt
phosphorylation was not different at basal level (Koh et al., 2013; Zhang et al., 2013). For
future studies, it will be necessary to determine Akt activation and its downstream
signaling molecules in TRB3TG mice at both basal and insulin-stimulated conditions to
find a link between Akt activity and protein turnover pathways.
In contrast to our findings, An et al. have reported that overexpression of TRB3 in
skeletal muscle results in greater muscle mass in TA, SOL, and GAS and contains more
oxidative muscle fibers (An et al., 2014). The reasons for the discrepancy are not clear. It
is possible that different genetic backgrounds resulted in different phenotypes. The
TRB3TG mice were generated in the embryos with mixed background (Qi et al., 2006).
Mice carrying the TRB3 transgene on the mixed background were backcrossed three to
four times to C57BL/6 WT mice before used for the previous experiments (An et al.,
2014). We continued to backcross the TRB3TG mice with C57BL/6 WT mice additional
three times (six to seven times in total) for the present experiments. In addition, the
inconsistent phenotypes might also result from different diet between the previous study
(Harlan Teklad Diet #8604) and current study (LabDiet Mouse Diet 9F) as different diet
49
may affect study outcomes, including phenotypes of animal models (Reliene & Schiestl,
2006). Another contradict to previous report is exercise capacity in TRB3TG mice. An et
al. has revealed TRB3 transgenic mice increased maximal running capacity compared to
WT whereas ours showed significantly decreased total running distance on 8 weeks of
voluntary wheel exercise (An et al., 2014). In addition to the different genetic
backgrounds and diets, different exercise capacity results may be due to the different
exercise testing protocol. We utilized voluntary wheel cage running for 8 weeks and
measured total running distance, but maximal time-to-exhaustion treadmill protocol was
applied in the previous experiment and they analyzed maximal exercise capacity in total
work. Therefore, depending on the purpose of testing, these two different measurements
would produce different results, and it is possible that the same animal model could
respond differently as previously described (Y. F. Liu et al., 2009; Noble et al., 1999).
In summary, our results demonstrate that TRB3 overexpression in skeletal muscle
decreased muscle weight and impaired muscle function that were associated with
decreased protein synthesis and increased protein breakdown. Our results suggest that
TRB3 plays a key role in muscle mass balance through regulating protein synthesis and
breakdown pathways.
ACKNOLEDGEMENTS
We thank M. Montminy (Salk Institute) for providing the muscle-specific TRB3
transgenic mice, and Regeneron for TRB3 knockout mice. This work was supported by
National Institutes of Health grants to H.J.K. (R03AR066825 and P20GM10909), L.J.G.
(R01DK099511) and a departmental start-up grant to H.J.K.
50
Figure 3.1 Effects of TRB3 on muscle mass and function in mice. (A-B) Ten- to eleven-week-old female wild type (WT) and muscle-specific TRB3 transgenic (TG) mice were used to determine TRB3 mRNA and protein expression from tibialis anterior and gastrocnemius, respectively. (C) Body weight was similar between WT and TG. (D) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS) muscles were measured. (E-F) Muscle function was evaluated by measuring grip strength and voluntary wheel exercise capacity for 8 weeks. (G) Frozen TA muscles were sectioned in 10-μm-thickness by a cryostat microtome and stained with hematoxylin and eosin. Original magnification: x400; scale bar: 50 μm. (H) Fibers were traced and analyzed by ImageJ in order to determine the mean CSA. (I) The frequency distribution of the fibers in wild type and TRB3TG mice was determined. Data are the means ± S.E.M (n = 5-6 per group). * P < 0.05, ** P < 0.01 vs. WT
WT TG0
200
400
600
800TR
B3
mR
NA
expr
essi
on (A
.U.)
***
WT TG0.0
0.5
1.0
1.5
2.0
2.5
Grip strength
Forc
e (N
) *
WT TG0
50
100
150
Running distance
Run
ning
Dis
tanc
e (k
m)
**
WT TG
0
5
10
15
20
25
Bod
y w
eigh
t (g)
WT TG0
500
1000
1500
2000
Mea
n C
SA (µm
2 ) *
TA EDLSOL
GAS0
20
40
60
80
100
Muscle weight
Mus
cle
wei
ght (
mg)
WTTG
*
**
*
<250
250-7
50
750-1
500
1500
-2000
2000
-2500
2500
-3000
3000
-3500
3500
-4000
4000
-4500
>450
00
10
20
30
40
50
CSA (µm2)
Prop
ortio
n of
fibe
rs (%
) WT
TG
A B C D
E F
GWT
TG
H
I
51
Figure 3.2 Effect of TRB3 overexpression on protein synthesis in mouse skeletal muscle. GAS muscles were collected from WT and TRB3TG mice. (A-D) The activations of signaling molecules related to protein synthesis, including mTOR (A), S6K1 (B), and Akt (C and D), were analyzed by Western blot. (E-G) After 5 hours of fasting, mice received an intraperitoneal injection of 0.04 μmol/g body weight with puromycin dissolved in 100 μl of PBS 30 min before euthanasia to measure the rate of protein synthesis. Western blot was used to measure the amount of puromycin incorporation into nascent peptides. (E) Ponceau S staining of puromycin analysis from SOL muscle showed that proteins were equally loaded. EDL (F) and SOL (G) muscles were analyzed. Data are the means ± S.E.M. (n = 5-6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. WT
WT TG0.0
0.5
1.0
1.5
pmTO
RS2
448 /m
TOR
(A.U
.)pmTORS2448
mTOR
WT TG WT TG
***
WT TG0.0
0.5
1.0
1.5
pS6K
1T389
/S6K
1 (A
.U.)
pS6K1T389
S6K1
WT TG WT TG
**
WT TG0.0
0.5
1.0
1.5
pAkt
T308
/Akt
(A.U
.)
pAktT308
Akt
WT TG WT TG
WT TG0.0
0.5
1.0
1.5
pAkt
S473
/Akt
(A.U
.)
pAktS473
Akt
WT TG WT TG
0.0
0.5
1.0
1.5
WT TG WT TG WT TG WT TGPonceau S Anti-Puromycin
250150
75100
50
37
2515
kDa
WT TG0.0
0.5
1.0
1.5
EDL
puro
myc
in
WT TG0.0
0.5
1.0
1.5
SOL
puro
myc
in
*
A B C
D E F
G
52
Figure 3.3 Effects of TRB3 knockout on protein synthesis and muscle mass. Ten- to eleven-week-old female wild type (WT) and whole-body TRB3 knockout (TRB3KO) mice were euthanized and GAS muscles were collected for analyses. (A-D) The levels of phosphorylation of signaling molecules, including mTOR (A), S6K (B), and Akt (C and D) were determined by Western blot. (E) Equally amount of protein lysates was analyzed for puromycin incorporation from SOL muscle. (F-G) The amount of puromycin incursion was determined in EDL (F) and SOL (G) muscles. (H-I) Muscle weights in TRB3KO mice were determined for TA (H) and GAS (I) muscles. Data are the means ± S.E.M. (n = 5-6 per group). * P < 0.05 vs. WT
WT KO0.0
0.5
1.0
1.5
2.0
pmTO
RS2
448 /m
TOR
(A.U
.)
pmTORS2448
mTOR
WT KO WT KO
WT KO0.0
0.5
1.0
1.5
pS6K
1T389
/S6K
1 (A
.U.)
pS6K1T389
S6K1
WT KO WT KO
WT TG0.0
0.5
1.0
1.5
pAkt
T308
/Akt
(A.U
.)
pAktT308
Akt
WT TG WT TG
WT KO0.0
0.5
1.0
1.5
pAkt
S473
/Akt
(A.U
.)
pAktS473
Akt
WT KO WT KO
250150
75100
50
37
2515
kDa
0.0
0.5
1.0
1.5
2.0
WT KO WT KO WT KO WT KOPonceau S Anti-Puromycin
WT KO0.0
0.5
1.0
1.5
2.0
SOL
puro
myc
in *
WT KO0.0
0.5
1.0
1.5
EDL
puro
myc
in
WT KO0
10
20
30
40
50
Tibialis anterior
Mus
cle
wei
ght (
mg)
*
WT KO0
50
100
150
Gastrocnemius
Mus
cle
wei
ght (
mg)
P=0.08
A B
C D
E
F
G
H
I
53
Figure 3.4 Effects of TRB3 in skeletal muscle on protein degradation signaling and E3 ubiquitin ligases. (A-B) Phosphorylated FOXO1 (A) and 3a (B) were measured in GAS muscles from wild type (WT) and TRB3TG (TG) mice. (C) The mRNA expression of atrogin-1, MuRF-1, and cathepsin L was determined in TA muscle. (D-E) The levels of phosphorylation of FOXO1 (D) and 3a (E) were measured in GAS muscles from WT and TRB3KO (KO) mice. (F) Atrogin-1, MuRF-1, and cathepsin L mRNA expressions were determined in TA muscle. Data are the means ± S.E.M. (n = 5-6 per group). * P < 0.05, ** P < 0.01 vs. WT
WT TG0.0
0.5
1.0
1.5
pFO
XO1S2
56/F
OXO
1 (A
.U.)
pFOXO1S256
FOXO1
WT TG WT TG
*
WT TG0.0
0.5
1.0
1.5
pFO
XO3a
S253
/FO
XO3a
(A.U
.)
pFOXO3aS253
FOXO3a
WT TG WT TG
*
Atrogin-1
MuRF-1
Cathep
sin L
0.0
0.5
1.0
1.5
2.0
mR
AN
exp
ress
ion
(A.U
.)
WTTGP=0.07 **
WT KO0.0
0.5
1.0
1.5
pFO
XO1S2
56/F
OXO
1 (A
.U.)
pFOXO1S256
FOXO1
WT KO WT KO
**
WT KO0.0
0.5
1.0
1.5
2.0
pFO
XO3a
S253
/FO
XO3a
(A.U
.)
pFOXO3aS253
FOXO3a
WT KO WT KO
P=0.06
Atrogin-1
MuRF-1
Cathep
sin L
0.0
0.5
1.0
1.5
mR
AN
exp
ress
ion
(A.U
.)
WTKO
** **
A B
C
D E
F
54
CHAPTER 4
TRB3 REGULATES SKELETAL MUSCLE MASS IN FOOD DEPRIVATION-
INDUCED ATROPHY1
1 Choi RH, McConahay A, Silvestre JG, Moriscot AS, Carson JA, Koh HJ. 2019. The FASEB Journal. 33. Reprinted here with permission of publisher.
55
4.1 ABSTRACT
Tribbles 3 (TRB3) is a pseudokinase that has been found in multiple tissues in
response to various stress stimuli, such as nutrient deprivation and endoplasmic reticulum
(ER) stress. We recently found that TRB3 has the potential to regulate skeletal muscle
mass at the basal state. However, it has not yet been explored whether TRB3 regulates
skeletal muscle mass under atrophic conditions. Here, we report that food deprivation for
48 hr in mice significantly reduces muscle mass by ~15% and increases TRB3
expression, which is associated with increased ER stress. Interestingly, inhibition of ER
stress in C2C12 myotubes reduces food deprivation-induced expression of TRB3 and
muscle specific E3-ubiquitin ligases. In further in vivo experiments, muscle-specific
TRB3 transgenic mice increase food deprivation-induced muscle atrophy compared to
WT littermates presumably by the increased proteolysis. On the other hand, TRB3
knockout mice ameliorate food deprivation-induced atrophy compared to WT littermates
by preserving a higher protein synthesis rate. These results indicate that TRB3 plays a
pivotal role in skeletal muscle mass regulation under food deprivation-induced muscle
atrophy and TRB3 could be a pharmaceutical target to prevent skeletal muscle atrophy.
Key Words: protein synthesis, protein degradation, Akt, mTOR, FOXO
4.2 INTRODUCTION
Skeletal muscle is a highly malleable tissue, which can transform its
characteristics depending on anabolic or catabolic stimuli. Anabolic signals, including
insulin or insulin-like growth factor-1 (IGF-1), activate protein synthesis, leading to
56
muscle hypertrophy; whereas catabolic stimuli, such as energy imbalance and
inflammation, activate protein degradation, resulting in muscle atrophy. These
phenomena are important for regulation of skeletal muscle mass in order to maintain
proper muscle function and strength for a population undergoing aging or diseases,
including obesity, type 2 diabetes, and cancer.
Muscle atrophy occurs in various pathophysiological conditions, such as
starvation, inactivity, denervation, and sepsis, leading to poor quality of life among
patients (Cohen, Nathan, & Goldberg, 2015). Multiple mechanisms can induce muscle
wasting, including an imbalance between the rate of protein synthesis and breakdown
(Schiaffino et al., 2013). The IGF-1/phosphoinositide 3 kinase (PI3K)/Akt pathway has
been considered as a main mechanism to manage muscle protein homeostasis (Latres et
al., 2005; Stitt et al., 2004). Phosphorylation/activation of Akt provokes protein synthesis
through the activation of mechanistic target of rapamycin (mTOR) and inhibits protein
breakdown via the phosphorylation of forkhead box O families (FOXOs) (Glass, 2005).
Phosphorylation of mTOR mediates a subsequent phosphorylation of ribosomal S6
kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)
to induce protein synthesis (Bodine, Stitt, et al., 2001; Gingras, Kennedy, O'Leary,
Sonenberg, & Hay, 1998; Rommel et al., 2001). FOXOs, another downstream target of
Akt, are transcription factors that induce transcription of the genes involved in protein
degradation, including muscle-specific E3 ubiquitin ligases, muscle atrophy F-box
(atrogin-1) and muscle RING finger-1 (MuRF-1) (Sandri et al., 2004; Waddell et al.,
2008). In addition to the activation of the ubiquitin-proteasome proteolysis system by
FOXOs, dephosphorylation of FOXOs has been associated with the upregulation of
autophagy-lysosomal protein degradation (Mammucari et al., 2007; Milan et al., 2015;
57
Zhao et al., 2007). These studies have established the idea that the regulation of Akt
signaling is a significant target to control protein synthesis and degradation in skeletal
muscle. However, the mechanism(s) that manages the activation of Akt to regulate
skeletal muscle mass has not been fully described.
Tribbles 3 (TRB3), a mammalian Drosophila tribbles homolog 3, is a
pseudokinase which contains a kinase domain without catalytic function (Grosshans &
Wieschaus, 2000). Its expression has been associated with several stress conditions, such
as endoplasmic reticulum (ER) stress, insulin resistance, glucose toxicity, and
tumorigenesis (Koh et al., 2013; J. Liu et al., 2010; Ohoka et al., 2005; Xu et al., 2007;
Zhang et al., 2013). Functionally, TRB3 has been known as a negative regulator of Akt in
metabolic tissues, including liver, fat, and skeletal muscle, through disrupting
phosphorylation of Akt (Du et al., 2003; Koh et al., 2013). In skeletal muscle, increased
TRB3 has been associated with insulin resistance. A study that utilized TRB3 knockout
mice fed a high-fat diet found that the knockout mice had improved glucose tolerance and
insulin-stimulated Akt activation, resulting in the prevention of high-fat diet-induced
obesity (Koh et al., 2013). Previously, we found that TRB3 in mouse skeletal muscle
plays a critical role in the regulation of protein turnover at the basal state using both
muscle-specific TRB3 transgenic (TG) and whole-body TRB3 knockout (KO) mice. TG
mice had an inhibited protein synthesis rate confirmed by decreased phosphorylation of
mTOR and S6K1 and puromycin incorporation, while the KO mice had an enhanced
protein synthesis rate in soleus muscle (R. H. Choi et al., 2017). TG mice also showed an
increase in the protein degradation pathway, evidenced by upregulation of atrogin-1 and
MuRF-1, whereas KO mice had a decreased expression of atrogin-1 and MuRF-1 (R. H.
Choi et al., 2017). Although skeletal muscle TRB3 has the potential to regulate skeletal
58
muscle mass at basal levels, the effect of TRB3 on skeletal muscle mass regulation under
atrophic conditions has not yet been defined.
Atrophic conditions, such as starvation and denervation, can prompt ER stress and
the unfolded protein response (UPR) (Paul et al., 2012; Yu et al., 2011). These processes
have been associated with the regulation of skeletal muscle mass and function (Bohnert,
McMillan, & Kumar, 2018), however the mechanism(s) is not completely understood.
TRB3 interacts with activating transcription factor 4 (ATF4) and C/EBP homologous
protein (CHOP), which are ER stress markers, under ER stress conditions and nutrient
deprivation conditions (Jousse et al., 2007; Ohoka et al., 2005; Ord & Ord, 2003).
Moreover, TRB3 has been known to respond to ER stress in skeletal muscle (Koh et al.,
2013). The activation of ER stress via ER stress inducers, such as tunicamycin or
thapsigargin, increases TRB3 expression (Koh et al., 2013; Ohoka et al., 2005; Ord &
Ord, 2005). The ER stress-induced TRB3 has been related to the activation of ATF4 and
CHOP, which is an important arm among three UPR pathways to regulate global
translation (Jousse et al., 2007; Ohoka et al., 2005; Ord & Ord, 2005; Walter & Ron,
2011). Although a direct mechanism(s) of stress-induced TRB3 expression in skeletal
muscle has not been revealed, based on previous findings, it is promising that ER stress
could upregulate TRB3 expression in response to food deprivation. Therefore, in this
study, we withdrew food to induce muscle atrophy. In addition to the protein turnover,
we also investigated whether food deprivation-induced ER stress mediates TRB3
expression.
Here we show that 48 hr food deprivation increased TRB3 expression in mouse
skeletal muscle and blunted the phosphorylation of Akt and its downstream signaling
molecules, resulting in an upregulation of muscle-specific E3 ubiquitin ligases.
59
Furthermore, food deprivation induced ER stress markers in vivo and in vitro. Blocking
of ER stress using 4-phenylbutric acid attenuated ER stress and TRB3 expression in
C2C12 cells after 6 hr of PBS-induced starvation. TG mice had significantly higher levels
of food deprivation-induced muscle atrophy along with greater activation of FOXOs and
atrogenes. In contrast, the knockout of TRB3 prevented mice from food deprivation-
induced atrophy, resulting from maintenance of protein synthesis rate after 48 hr
starvation with higher activation of the protein synthesis pathway. Therefore, our study
reveals that TRB3 plays an important role in food deprivation-induced skeletal muscle
atrophy and may be a potentials target strategy for the maintenance of skeletal muscle
mass during atrophic conditions.
4.3 MATERIALS AND METHODS
Animal care and food deprivation procedure
Ten- to thirteen-week-old C57BL/6 wild type, muscle-specific TRB3 transgenic
(TG), and whole-body TRB3 knockout (KO) mice were used for the current study. All
animals were maintained in the animal facility at the University of South Carolina and
were housed in an environmentally controlled room on a 12 hr light-dark cycle. During
the 48 hr food deprivation experiment, standard chow diet (Harlan Teklad Diet,
Madision, WI, USA) was removed from the food deprivation group with ad libitum
access to water. For the fed group, mice had free access to food and water. All protocols
were approved by the Institutional Animal Care and Use Committee of the University of
South Carolina.
60
Cell culture
C2C12 myoblasts were grown in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% fetal bovine serum. When the cells reached ~ 90 % of confluence,
differentiation was induced by replacing the growth media with media containing 2%
horse serum for 4 days as described previously (Koh et al., 2013). For cell starvation
experiments, myotubes were incubated in sterile phosphate-buffered saline (PBS) for 6
hr. To block ER stress during cell starvation, additional myotubes were pre-incubated
with 5 mM of 4-phenylbutric acid (4-PBA) (P21005; Sigma, St. Louis, MO) 30 min prior
to PBS starvation. Cells were then starved in PBS in the presence of 4-PBA for 6 hr.
Protein extraction and western blot analysis
Frozen tibialis anterior (TA) muscles were grinded by a homogenizer (Next
Advandce, Inc., Troy, NY, USA) and lysed in a buffer containing 20 mM Tris-HCl
pH7.4, 5 mM EDTA, 10 mM Na3PO4, 100 mM NaF, 2 mM Na3VO4, 1% NP-40, and
HaltTM Protease and Phosphatase Inhibitor Single-Use Cocktail. To collect proteins from
C2C12 myotubes, media from each well was removed and washed twice with ice-cold
PBS. Then, the lysis buffer was added and the myotubes were scrapped by using a sterile
cell scrapper. Tissue and cell lysates (30 – 60 mg) were separated by 15 – 8% SDS-
PAGE and transferred to PVDF membrane. The membranes were incubated in 5%
bovine serum albumin blocking buffer for 1 h then primary antibodies were applied for
overnight incubation at 4 ºC. Blots were washed three times for 10 min each with TBST,
then incubated with a specific secondary antibody (1:2000 – 5000). Blots were visualized
with chemiluminescent substrates (PerkinElmer, Waltham, MA, USA) and quantified by
Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA). All primary and secondary
61
antibodies used in this study were commercially available, including pAktT308 (#9275),
Akt (#9271), pmTORS2448 (#2971), mTOR (#2972), pFOXO1S256 (#9461), FOXO1
(#2880), pFOXO3aS253 (#13129), FOXO3a (#12829), GAPDH (#2118) from Cell
Signaling Technology (Danvers, MA, USA), atrogin-1 (AP2041), MuRF-1 (MP3401)
from ECM (Versailles, KY, USA), TRB3 (ST1032; Calbiochem, San Diego, CA, USA),
anti-puromycin (MABE343; ECM Millipore, Billerica, MA, USA), horseradish
peroxidase-lined anti-rabbit secondary antibody (NA934; GE healthcare, Pittsburgh, PA,
USA), and IgG2a-horseradish peroxidase conjugate anti-mouse secondary antibody (61-
0220; Invitrogen, Carlsbad, CA, USA).
RNA extraction and real-time polymerase chain reaction
Total RNA was extracted from TA muscles and C2C12 myotubes using a Trizol
reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s
instructions. cDNA was synthesized from 4 µg of total RNA with the High Capacity
cDNA kit (Life Technologies, Carlsbad, CA, USA). cDNA and primers for real-time
polymerase chain reaction (RT-PCR) were carried by SYBR green PCR master mix (Life
Technologies, Carlsbad, CA, USA). Primer sequences are as shown in Table 1. All genes
were normalized to the level of TBP for in vivo tissues and the level of 18S for in vitro
samples.
In vivo protein synthesis measurement
In vivo protein synthesis rate was measured with a nonradioactive technique using
puromycin incorporation into a nascent peptide as previously described (Goodman et al.,
2011). Briefly, mice were given an intraperitoneal injection of 0.04 µmol/g puromycin
(EDM Millipore, Billerica, MA, USA) dissolved in 100 µl of PBS 30 min before
62
euthanasia. After 30 min of the injection, tissues were collected and frozen immediately
in liquid nitrogen then stored at – 80 ºC until the analysis. Details of puromycin
immunoblotting can be found in the Protein extraction and western blot analysis section.
Statistical analysis
All results are presented as mean ± SEM. Statistical analysis was performed using
GraphPad Prism 7 (La Jolla, CA, USA). Student’s two-tailed unpaired t tests were used
for comparisons between two groups. One- or two-way ANOVA was conducted to
determine significance between more than two group followed by Tukey’s or Sidak Post
hoc analysis. P < 0.05 was considered statistically significant.
4.4 RESULTS
48 hr food deprivation increases TRB3 expression in mouse skeletal muscle
To determine the role of TRB3 in food deprivation-induced muscle atrophy, we
first determined TRB3 expression in response to 48 hr food deprivation. C57BL/6 wild-
type mice were divided into two groups. One group freely consumed standard chow diet
(Fed) while the other group was withdrawn food for 48 hr. Water was accessible ad
libitum for both groups. After 48 hr, body weight was measured before euthanasia. Mice
that had undergone 48 hr food deprivation had significantly decreased body weight more
than 20% compared to Fed controls (Fig. 4.1A). Consistently, multiple skeletal muscles,
including tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and
gastrocnemius (GAS), from the food deprivation group showed a 15% decrease in muscle
weight compared to those from Fed (Fig. 4.1B). There was no significant difference in
63
the percentage of muscle atrophy among four muscles after 48 hr of food deprivation.
Our method, food deprivation for 48 hr, was sufficient to induce skeletal muscle atrophy
in mice. Interestingly, 48 hr food deprivation significantly increased TRB3 mRNA and
protein expression in mouse skeletal muscle by 300% and 30%, respectively (Fig. 4.1C
and D). These data demonstrate that food deprivation for 48 hr induces skeletal muscle
atrophy and increases TRB3 expression in mouse skeletal muscle.
48 hr food deprivation inhibits Akt phosphorylation and activates protein degradation
Muscle wasting is regulated by the balance between protein synthesis and protein
degradation (Schiaffino et al., 2013). The IGF-1/PI3K/Akt pathway is critical for the
control of protein turnover through mTOR and FOXOs signaling (Latres et al., 2005; Stitt
et al., 2004). Since our method was sufficient to decrease muscle mass (Fig. 4.1B), we
next examined if Akt and FOXOs are involved in food deprivation-induced skeletal
muscle atrophy. After 48 hr food deprivation, phosphorylation of Akt at Thr308 in the
food deprivation group was significantly decreased compared to the Fed group (Fig.
4.1E). Phosphorylation of Akt at Ser473 did not respond to 48 hr food deprivation
(Supplementary Fig. S4.1A). Further, phosphorylation of FOXO1 and FOXO3a was
dramatically decreased in the food deprivation group, suggesting the activation of
FOXOs (Fig. 4.1F and G). Dephosphorylated FOXOs are required for upregulation of
atrogin-1 and MuRF-1, skeletal muscle-specific E3 ubiquitin ligases (Sacheck et al.,
2004; Sandri et al., 2004; Waddell et al., 2008). In line with this, we also found that 48 hr
food deprivation significantly increased atrogin-1 and MuRF-1 mRNA expression in
mouse skeletal muscle (Fig. 4.1H). Together, these results demonstrate that 48 hr food
deprivation inhibits Akt signaling but activates FOXOs and muscle-specific E3 ubiquitin
64
ligases, which may be responsible for 48 hr food deprivation-induced skeletal muscle
atrophy.
Food deprivation induces ER stress in vivo and in vitro and ER stress meditates food
deprivation-induced TRB3 expression
It has been previously determined that TRB3 interacts with ATF4 and CHOP,
which are ER stress markers and one of the UPR pathways, and coordinates ATF4-
related apoptosis during ER stress (Jousse et al., 2007; Ohoka et al., 2005; Ord & Ord,
2003). Therefore, we next hypothesized that food deprivation-induced TRB3 expression
is mediated by increased ER stress. We first analyzed ER stress markers in skeletal
muscles from food deprived wild-type mice. The expression of ATF4 and XBP1 was
significantly increased in skeletal muscles from the food deprivation group (Fig. 4.2A).
The mRNA expression of ATF6 and GRP78 tended to be higher in response to food
deprivation (Fig. 4.2A). To further determine if ER stress triggers the upregulation of
TRB3 in response to food deprivation, we incubated C2C12 myotubes with PBS, which
can mimic food deprivation-induced muscle atrophy (Paul et al., 2012; Sandri et al.,
2004). After 6 hr of PBS incubation, the expression of ER stress markers, including
ATF4, CHOP, XBP1, and GPR78/Bip, and atrogin-1 was robustly elevated (Fig. 4.2B).
Interestingly, TRB3 mRNA expression was also increased by more than 2.6-fold (Fig.
4.2B). To test if ER stress is required to increase TRB3 expression in response to food
deprivation, C2C12 myotubes were treated with 4-PBA, an ER stress blocker, and TRB3
and ER stress markers were determined. The 4-PBA treatment significantly blunted ER
stress responses in starved cells (Fig. 4.2B). Interestingly, PBS incubated C2C12
myotubes with 4-PBA treatment had significantly reduced expression of TRB3 and
muscle-specific E3 ubiquitin ligases (Fig. 4.2B). These data suggest that ER stress is
65
necessary to induce TRB3 expression in response to food deprivation and TRB3 may
play a regulatory role in the expression of atrogenes.
Overexpression of TRB3 aggravates food deprivation-induced skeletal muscle atrophy
To further determine the effect of TRB3 in skeletal muscle atrophy, we used a
muscle-specific TRB3 transgenic (TG) mouse model (Fig. 4.3A). In our previous studies,
we found that TG mice displayed smaller muscle weight and cross-sectional area with
suppressed protein synthesis and accelerated protein degradation compared to wild-type
(WT) littermates at the basal state (R. H. Choi et al., 2017). Therefore, we hypothesized
that TG mice are more prone to food deprivation-induced atrophy compared to WT. WT
and TG mice were undergone food deprivation for 48 hr and body weight was measured.
Forty-eight hours of food deprivation significantly decreased body weight in both WT
and TG mice and there was no difference between genotypes (Fig. 4.3B). Interestingly,
TG mice had markedly increased muscle atrophy by ~22% in various muscles compared
to wild type (Fig. 4.3C). These findings suggest that overexpression of TRB3 in skeletal
muscle exacerbates skeletal muscle atrophy in response to 48 hr food deprivation.
TRB3 overexpression activates FOXOs and ubiquitin-proteasome proteolysis
Based on our data indicating that overexpression of TRB3 aggravated food
deprivation-induced muscle atrophy (Fig. 4.3C), we next investigated whether muscle-
specific TRB3 overexpression may influence the Akt-FOXOs-ubiquitin-proteasome
protein degradation pathway. The phosphorylation of Akt (Thr308) and subsequently
FOXO1 (S256) phosphorylation was significantly diminished after 48 hr food deprivation
(Fig. 4.4A and B), whereas Akt phosphorylation at Ser 473 was not altered
(Supplementary Fig. S4.1B). Additionally, there was a trend for activation and
66
dephosphorylation of FOXO3a in WT after 48 hr food deprivation, whereas TG mice had
significantly activated FOXO3a (Fig. 4.4C). In agreement with increased FOXOs
activation, atrogin-1 and MuRF-1 mRNA expression was highly elevated independently
of genotype (Fig. 4.4D). In protein expression, however, TG mice displayed a greater
increase in atrogin-1 and MuRF-1 after 48 hr food deprivation compared to WT (Fig.
4.4E and F). These data suggest that TRB3 may accelerate the ubiquitin-proteasome
proteolysis system.
It has been well established that autophagy plays an important role in skeletal
muscle mass regulation under atrophic conditions (Mammucari et al., 2007; Ogata, Oishi,
Higuchi, & Muraoka, 2010). To determine the effect of TRB3 on food deprivation-
induced autophagy, we measured autophagic markers, including polyubiquitin-binding
protein (p62) and microtubule-associated protein 1 light chain-3B (LC3B), which have
been previously reported to increase in skeletal muscle in response to food deprivation
(Mammucari et al., 2007; Paul et al., 2012). Recently, it has been demonstrated that
TRB3 interacts with p62 to induce an accumulation in the human branchial epithelial cell
line (Hua et al., 2015). In our current study, 48 hr food deprivation greatly increased p62
in both genotypes, but TG mice displayed a further increase in p62 protein expression
(Fig. 4.4G). In addition to p62, conversion of LC3B-I to LC3B-II is a critical marker of
autophagosome formation (Levine & Kroemer, 2008). We measured LC3B by Western
blot and found that 48 hr food withdrawal dramatically increased LC3B conversion in
both genotypes with a trend to be higher in TG mice (Fig. 4.4H). These results suggest
that TRB3 plays a role in the upregulation of food deprivation-induced autophagy.
67
Muscle-specific TRB3 overexpression does not augment the effect of food deprivation
on protein synthesis rate and signaling pathway
Loss of muscle mass is stimulated by not only increased protein degradation, but
also decreased protein synthesis (Schiaffino et al., 2013). Our previous study has reported
that TRB3 overexpression in mouse skeletal muscle results in a decrease in protein
synthesis rate at the basal condition (R. H. Choi et al., 2017). In our current study, we
observed that TRB3 overexpression in mouse skeletal muscle promoted atrogin-1 and
MuRF-1 expression after 48 hr food deprivation compared to WT (Fig. 4.4E and F).
Therefore, we further determined whether TRB3 overexpression would have an effect on
Akt downstream molecules related to protein synthesis under atrophic conditions. Forty-
eight hours of food deprivation decreased the protein synthesis rate by 60 – 65% in WT
(Fig. 4.5A and B). Consistent with our previous study (R. H. Choi et al., 2017),
overexpression of TRB3 decreased protein synthesis rate at fed state (Fig. 4.5A and B).
Although TG mice showed significantly suppressed protein synthesis rate in fed state, TG
mice had no further effect on protein synthesis rate in response to 48 hr food derivation
(Fig. 4.5B). There was no significant difference in protein synthesis rate between
genotypes after 48 hr food deprivation. Next, we determined the effect of TRB3 on
signaling molecules related to protein synthesis. Forty-eight hours of food withdrawal did
not affect mTOR phosphorylation in either genotype (Fig. 4.5C). Although a statistical
significance was not found, phosphorylation of mTOR in TG mice at the basal state was
50% less than WT (Fig. 4.5C). To understand what factors may influence the decreased
protein synthesis rate in TG mice, we examined the proteins upstream and downstream of
mTOR, including PRAS40, TSC2, and 4EBP1. Forty-eight hours of food deprivation
resulted in a significant increase in dephosphorylation of 4EBP1 (S65) in both genotypes,
68
suggesting diminished initiation of protein synthesis (Fig. 4.5D). It is known that Akt
regulates mTOR activity by phosphorylating PRAS40 and TSC2 (Inoki et al., 2002;
Vander Haar et al., 2007). Therefore, we tested both signaling molecules to evaluate
whether TRB3 affects them to regulate mTOR activity. Phosphorylated PRAS40 (T246)
was significantly decreased only in TG mice after 48 hr food deprivation (Fig. 4.5E).
Phosphorylation of TSC2 (T1462) was significantly decreased in TG mice at fed state
like mTOR (Fig. 4.5F). Forty-eight hours of food deprivation decreased TSC2
phosphorylation in both WT and TG mice, and there was no difference in TSC2
phosphorylation between genotypes (Fig. 4.5F). These data support the result that TG
mice showed the decreased protein synthesis rate under fed condition but did not
augment the rate in response to 48 hr food deprivation. Taken together, these findings
demonstrate that the worsening food deprivation-induced skeletal muscle atrophy in TG
mice is not due to the change in protein synthesis rate.
TRB3 knockout prevents 48 hr food deprivation-induced muscle atrophy
We found that TRB3 overexpression in mouse skeletal muscle notably decreased
muscle mass in response to 48 hr food deprivation (Fig. 4.3C). This augmented muscle
atrophy in TG mice could result more likely from an increased expression of muscle-
specific E3 ubiquitin ligases (Fig. 4.4E and F) but less likely from protein synthesis rate
and activity of signaling molecules (Fig. 4.5A – F). To determine whether TRB3 has the
potential to be a pharmaceutical target to ameliorate skeletal muscle wasting, we next
examined TRB3 knockout (KO) mice with the 48 hr food deprivation We first confirmed
that KO mice expressed significantly lower amount of TRB3 in TA muscle compared to
WT littermates (Fig. 4.6A). Next, WT and KO mice underwent 48 hr food deprivation,
and displayed reduced body weight more than 20 % after food withdrawal (Fig. 4.6B).
69
Intriguingly, we observed a smaller degree of muscle wasting in some muscles from KO
mice compared to WT (Fig. 4.6C). These findings indicate that the knockout of TRB3 in
skeletal muscle prevents food deprivation-induced muscle atrophy.
TRB3 knockout blunts a reduction of protein synthesis after 48 hr food deprivation
The preventive effect of TRB3 knockout on food deprivation-induced muscle loss
may be associated with increased protein synthesis based on our previous report (R. H.
Choi et al., 2017). To examine the mechanism(s) by which knockout of TRB3 prevented
food deprivation-induced muscle atrophy, we measured the protein synthesis rate and
signaling pathway in TA muscle. As previously shown, protein synthesis rate was
dramatically reduced after 48 hr food withdrawal in both WT and KO mice (Fig. 4.7A
and B). However, the knockout of TRB3 ameliorated the food deprivation-induced
reduction in the protein synthesis rate (Fig. 4.7B). This phenomenon could promote
maintenance of muscle mass in KO mice and protect the KO mice from food deprivation-
induced atrophy compared to WT. Next, we examined signaling molecules involved in
protein synthesis, including mTOR, 4EBP1, PRAS40 and TSC2. However, there was no
significant differences between genotypes (Fig. 4.7C – F). The same results were
observed in EDL muscle (Supplementary Fig. S4.2 and S4.3). This may be due to a flaw
in our methodology. We may not be able to capture the correct time point for detection of
alterations in signaling pathways since we sacrifice mice 30 min after puromycin
injection. On the basis of the increased protein synthesis rate in KO mice, there is the
possibility that the protein synthesis pathway may be affected by genotype. These data
suggest that increased muscle mass in KO mice could be due to preserving protein
synthesis rate.
70
4.5 DISCUSSION
TRB3 has been known as a negative regulator of the insulin signaling pathway
focused on Akt phosphorylation (Du et al., 2003; Koh et al., 2006). Its expression is
involved in pathophysiology of multiple diseases, including diabetes and obesity,
resulting in insulin resistance (Koh et al., 2013; Zhang et al., 2013). Recently, we
demonstrated that the overexpression of TRB3 in mouse skeletal muscle decreases
protein synthesis and increases protein degradation through the disruption of Akt and its
downstream molecules at the basal state (R. H. Choi et al., 2017). However, it has not yet
been elucidated whether TRB3 affects skeletal muscle mass regulation under atrophic
conditions. Therefore, in this study we examined the role of TRB3 in the regulation of
skeletal muscle mass under 48 hr food deprivation-induced skeletal muscle atrophy.
This study demonstrated that TRB3 mRNA and protein expressions were
significantly elevated in mouse skeletal muscle after 48 hr food deprivation (Fig. 4.1C
and D). Previously, it has been established that food deprivation for 24 hr can induce
TRB3 expression in liver and fat (Du et al., 2003; Qi et al., 2006). Our data is the first
report showing that 48 hr food deprivation is sufficient to induce TRB3 expression in
mouse skeletal muscle (Fig. 4.1C and D). In addition, our study and others demonstrated
that an incremental increase of TRB3 expression in multiple tissues impairs the insulin-
stimulated Akt (Avery et al., 2010; Koh et al., 2013). Here, we showed that 48 hr food
deprivation significantly reduced skeletal muscle mass along with a decrease in
phosphorylation of Akt and FOXOs, leading to upregulation of atrogin-1 and MuRF-1
(Fig. 4.2A - D). Taken together, food deprivation-induced TRB3 expression in mouse
skeletal muscle may decrease Akt phosphorylation and activate FOXOs and muscle-
specific E3 ubiquitin ligases.
71
A significant reduction in the phosphorylation of Akt in response to food
withdrawal (Fig. 4.2A) has been observed in other studies (Paul et al., 2012; Sandri et al.,
2004). Blunted Akt phosphorylation subsequently leads to the dephosphorylation of
FOXO1 and 3a, which translocate to the nucleus to promote the transcriptions of protein
degradation genes, such as atrogin-1 and MuRF-1 (Sandri et al., 2004; Stitt et al., 2004).
In line with this, we observed that 48 hr food deprivation significantly reduced the
phosphorylation of FOXO1, but not of FOXO3a (Fig. 4.1F and G), leading to increased
muscle-specific E3 ubiquitin ligases (Fig. 4.1H). This phenomenon has previously been
observed (Ogata et al., 2010) and there has been evidence that different isoforms of
FOXOs can coordinately assist their activation (Sandri et al., 2004).
Our finding that ER stress mediates TRB3 and atrogenes expression in response
to starvation suggest that ER stress is an important regulator of TRB3 expression in
skeletal muscle (Fig. 4.2B). Although earlier studies have shown a tight relationship
between ER stress and TRB3 in regulatory mechanisms, it has not been determined in
skeletal muscle and food deprivation conditions. For example, tunicamycin treated
HepG2 cells have an increase in TRB3 expression following an elevation of ATF4 and
CHOP (Ohoka et al., 2005), whereas ER stress-induced TRB3 expression is completely
blocked in ATF4 knockdown mouse embryonic fibroblasts (Jousse et al., 2007). While
these studies suggest that ER stress may play a critical role in TRB3 expression, our
results indicate that ER stress may be required to induce TRB3 expression in skeletal
muscle under food deprivation conditions. Furthermore, our data suggest that food
deprivation-induced atrophy may be mediated by ER stress-induced TRB3 expression. In
the current experiment, however, we could not determine if TRB3 is required for food
deprivation-induced and/or ER stress-mediated atrophy. To examine a precise
72
mechanism, it will be necessary to utilize TRB3 knockdown in C2C12 cells or isolated
primary cells from skeletal muscle of KO mice to demonstrate if food deprivation-
induced atrophy is prevented in the absence of TRB3. These experiments will be
performed in our future studies.
In this study, we demonstrated that muscle-specific TRB3 overexpression resulted
in increased 48 hr food deprivation-induced muscle atrophy compared to WT (Fig. 4.3C).
Previously, we have reported that TG mice present smaller muscle weights and a reduced
rate of protein synthesis compare to WT at the basal condition (R. H. Choi et al., 2017).
Our previous and current studies consistently demonstrate that muscle-specific TRB3
overexpression significantly increased atrogin-1 and MuRF-1 at basal and under food
deprivation-induced atrophic conditions (Fig. 4.4E and F) (R. H. Choi et al., 2017). In
contrast, An et al. reported that the same mice display larger muscle mass in SOL and
GAS compared to WT littermates (An et al., 2014). This may be due to differences in the
number of TG mice backcrossing and diet as previously reported (R. H. Choi et al.,
2017). Although the reason for the discrepancy is not clear, our current results were
consistent with our previous study. Therefore, based on our findings, TRB3 may have the
potential to be a critical regulator of skeletal muscle mass at basal and also under food
deprivation-induced atrophy conditions.
Given that TRB3 impairs Akt signaling in skeletal muscle and other tissues (Du et
al., 2003; Koh et al., 2013; Zhang et al., 2016), we anticipated the decreased Akt
phosphorylation in TG mice after 48 hr food deprivation. However, we did not observe
any differences in Akt phosphorylation between WT and TG mice. The discrepancy is
likely due to the different experimental settings. Our previous study and others have
examined Akt phosphorylation after insulin stimulation (Du et al., 2003; Koh et al., 2013;
73
Zhang et al., 2016). In our current study, we measured the basal level of Akt
phosphorylation in order to precisely determine the role of TRB3 in food deprivation-
induced skeletal muscle atrophy since insulin can initiate anabolic signaling pathways to
alter protein turnover (Schiaffino et al., 2013). Although we did not detect differences in
Akt phosphorylation between genotypes, our data demonstrate that TRB3 overexpression
in skeletal muscle could affect Akt-downstream proteins associated with protein turnover.
For future studies, it will be important to determine Akt phosphorylation in insulin-
stimulated condition. In addition, more sensitive methodology to detect definite changes
in Akt activity will be necessary, such as Akt kinase assay.
In addition to upregulating the ubiquitin-proteasome system in response to food
withdrawal, autophagy-lysosomal protein degradation is activated under atrophic
conditions (Mammucari et al., 2007; Paul et al., 2012). In this study, TG mice displayed
significantly increased p62 accumulation compared to WT (Fig. 4.4G). Recently, it has
been reported that the overexpression of TRB3 in human bronchial epithelial cells and
diabetic mice results in the accumulation of p62 (Hua et al., 2015). Although the current
and previous findings have been confirmed in different tissues and experimental
conditions, TRB3 expression seems to play an essential role in the autophagic process.
The activation of FOXOs has been considered as an important regulator of not only
ubiquitin-proteasome proteolysis (Sandri et al., 2004; Waddell et al., 2008) but also
autophagy-lysosomal protein degradation (Mammucari et al., 2007; Milan et al., 2015;
Zhao et al., 2007). However, TG mice did not show any differences in FOXO1 and
FOXO3a activation in response to food deprivation compared to WT (Fig. 4.4B and C).
These results represent only a small piece of the process. Although we could not find any
differences in the activation of FOXOs between WT and TG mice in response to food
74
deprivation, we cannot fully exclude the possibility that the overexpression of TRB3 may
affect the activation of FOXOs to induce ubiquitin-proteasome and autophagy-lysosomal
protein degradations.
Along with protein degradation, skeletal muscle mass is also regulated by protein
synthesis. Forty-eight hours of food deprivation diminished protein synthesis rate by
more than 60% in WT mice (Fig. 4.5A – B). In accordance with our previous study (R.
H. Choi et al., 2017), TG mice showed significantly suppressed protein synthesis rate at
fed state (Fig. 4.5B). However, we did not detect further reduction in protein synthesis
rate in TG mice after 48 hr food deprivation. This may be due to the fact that the level of
protein synthesis was already decreased in TG mice in fed state (60 % of fed WT mice)
and no further decrease could be made by 48 hr food deprivation. To determine how
TRB3 overexpression hinders protein synthesis, we further investigated expression of
signaling molecules related to mTOR regulation, such as 4EBP1, PRAS40, and TSC2.
TRB3 overexpression in mouse skeletal muscle significantly reduced the phosphorylation
of PRAS40 and TSC2 during food deprivation and at fed conditions, respectively (Fig.
4.5E and F). Both PRAS40 and TSC2 are known to be negative regulators of mTOR and
are inhibited by Akt phosphorylation (Inoki et al., 2002; Vander Haar et al., 2007). In line
with our results, TRB3 overexpression in primary mouse hepatocytes has been shown to
downregulate Akt-TSC2-mTOR pathway and activate 4EBP1, resulting in an inhibition
of nutrient- and insulin-induced S6K1 activity (Matsushima et al., 2006). These findings
suggest that TRB3 plays a critical role in the regulation of not only the Akt but also Akt-
associated proteins, including PRAS40 and TSC2. Taken together, our data indicates that
the worsening skeletal muscle atrophy in response to 48 hr food deprivation may be due
to the increased proteolysis pathways rather than changing protein synthesis rate.
75
We utilized our whole-body TRB3 knockout mouse model to determine the role
of TRB3 in skeletal muscle mass regulation under atrophic conditions. We collected
multiple hindlimb muscles and measured their weights to study whether there were
variations of food deprivation-induced muscle atrophy in a muscle fiber-type specific
manner. In contrast to TG mice, the knockout of TRB3 protected mice from food
deprivation-induced muscle atrophy, especially in TA and EDL (Fig. 4.6C). This
variation may be caused by various compositions of muscle fiber-type in specific
muscles. For instance, TA and EDL contains more fast-glycolytic type IIb fiber than
SOL, which mainly contains slow-oxidative type I and fast-oxidative type IIa
(Kammoun, Cassar-Malek, Meunier, & Picard, 2014). In addition, it has been revealed
that food deprivation could preferentially affect protein turnover in fast-glycolytic fibers
(Ciciliot, Rossi, Dyar, Blaauw, & Schiaffino, 2013; Li & Goldberg, 1976). Based on our
previous study, KO mice have larger muscle mass and higher protein synthesis rate
compared to WT mice (R. H. Choi et al., 2017). Therefore, we measured the protein
synthesis rate in the skeletal muscle from food deprived mice to examine whether KO
mice can maintain a higher protein synthesis rate. Puromycin incorporation was highly
preserved in KO mice compared to WT after 48 hr food deprivation (Fig. 4.7B). This
may support the result that food deprivation-induced muscle atrophy was inhibited in KO
mice. However, in this study, we did not find any differences in the expression of
signaling molecules related to protein synthesis pathways (Fig. 4.7C – F). Furthermore,
there were no differences in protein degradation pathways, including FOXO1 and 3a, and
atrogenes expression (data not shown). It is possible that changes in signaling activities at
the molecular level may have already occurred prior to the sacrifice, which occurred 30
min after puromycin injection. In addition, unlike TG mice, the KO mice were a global
76
knockout, meaning that TRB3 expression is downregulated in a whole-body manner.
Therefore, it is difficult to observe muscle-specific roles of TRB3 in the regulation of
skeletal muscle mass under food deprivation conditions. Nonetheless, our data suggest a
possibility that TRB3 has the potential to be a therapeutic target for skeletal muscle
atrophy.
In summary, 48 hr food deprivation induced the expression of TRB3 in skeletal
muscle and resulted in muscle atrophy. ER stress was required to induce TRB3
expression in response to food deprivation. Overexpression of TRB3 in mouse skeletal
muscle increased food deprivation-induced atrophy via activating ubiquitin-proteasomal
and autophagy-lysosomal protein degradation. TRB3 knockout mice were protected from
food deprivation-induced atrophy via the maintenance of proteins synthesis rate. We
conclude that TRB3 plays a pivotal role in food deprivation-induced skeletal muscle
atrophy. Our data also suggest that TRB3 may be a potential target for maintaining
skeletal muscle mass under food deprivation-induced atrophic condition.
ACKNOWLEDGEMENTS
We thank M. Montminy (Salk Institute) for providing the muscle-specific TRB3
transgenic mice, and Regeneron for TRB3 knockout mice. This work was supported by
NIH grants to H.J.K. (R03AR066825 and P20GM10909) and a departmental start-up
grant to H.J.K.
77
Table 4.1 Primer sequences for RT-PCR
TBP, TATA-box binding protein; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; ATF6, activating transcription factor 6; XBP1s, spliced X-box binding protein 1, XBP1t, total X-box binding protein 1; GRP78, 78 kDa glucose-regulated protein; eIF2α, eukaryotic translation initiation factor 2α
78
Figure 4.1 Food deprivation increases TRB3 expression and decreases Akt and FOXOs phosphorylation. Ten- to thirteen-week-old female C57BL/6 wild-type mice underwent either free access to food (Fed) or food deprivation (FD) for 48 hr. Body weight was measured before and after the procedure. (A) Percentage difference in body weight before and after 48 hr of each treatment was calculated. (B) Percentage of skeletal muscle atrophy after 48 hr food deprivation was determined in tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL), and gastrocnemius (GAS). (C-D) TRB3 mRNA (C) and protein expression (D) were determined in TA muscles. (E-G) Phosphorylation and total expression of Akt (E), FOXO1 (F), and FOXO3a (G) were determined by Western blot analysis. (H) mRNA expression of muscle-specific E3 ubiquitin ligases was determined by RT-PCR. Data are the means ± S.E.M. (n=4 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed.
Fed FD-30
-20
-10
0
10
Cha
nge
in b
ody
wei
ght (
%)
***
TA EDLSOL
GAS0
5
10
15
20
25
Mus
cle
atro
phy
(%)
**** **
***
Fed FD0
1
2
3
4
TRB
3 m
RN
A ex
pres
sion
(A.U
.)
**
Fed FD0.0
0.5
1.0
1.5
TRB
3/G
APD
H (A
.U.)
**
TRB3
GAPDH
Fed FD Fed FD
Fed FD0.0
0.5
1.0
1.5
pAkt
T308
/Akt
(A.U
.)
*
pAktT308
Akt
Fed FD Fed FD
Fed FD0.0
0.5
1.0
1.5
pFO
XO1S2
56/F
OXO
1 (A
.U.)
pFOXO1S256
FOXO1
***
Fed FD Fed FD
Fed FD0.0
0.5
1.0
1.5
pFO
XO3a
S253
/FO
XO3a
(A.U
.)
pFOXO3aS253
FOXO3a
Fed FD Fed FD
Atrogin-1
MuRF-10
5
10
15
mR
NA
expr
essi
on (A
.U.) Fed
FD
***
***
A B
CD E
F G
H
79
Figure 4.2 ER stress is responsible for food deprivation-induced TRB3 expression. (A) mRNA expression of multiple ER stress markers was determined in TA muscles by RT-PCR after 48 hr starvation. (B) mRNA expression of several ER stress markers was analyzed in C2C12 cells after 6 hr PBS and/or 4-PBA treatment. ATF6, activating transcription factor 6; ATF4, activating transcription factor 4; CHOP, C/EBP homologous protein; XBP1, X-box binding protein 1; GRP78/Bip, glucose-regulated protein 78/Binding immunoglobulin protein; eIF2α, eukaryotic translation initiation factor 2α. Data are the means ± S.E.M. (n=4 per group for in vivo, n=9 per group for in vitro). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed or Control + DMSO.
ATF6ATF4
CHOPXBP1
GRP78/B
ipeIF
2α0
1
2
3
mR
NA
expr
essi
on (A
.U.) Fed
FD*
**
**
**
A B
TRB3ATF6
ATF4CHOP
XBP1
GPR78/B
ipeIF
2α
Atrogin-1
MuRF10123458
10121416
mR
NA
expr
essi
on (A
.U.) Control + DMSO
PBS + DMSOPBS + 4PBA
****
******
***
*
**********
80
Figure 4.3 Muscle-specific TRB3 overexpression increases food deprivation-induced skeletal muscle atrophy. Ten- to thirteen-week-old female muscle-specific TRB3 transgenic (TG) and their wild-type littermate control (WT) mice were used to determine the role of TRB3 in skeletal muscle mass regulation under food deprivation-induced atrophy conditions. (A) TRB3 overexpression was confirmed by RT-PCR with increased TRB3 mRNA expression in TA muscles. TG and WT mice were divided into Fed and FD groups and underwent starvation for 48 hr. (B) Percentage change in body weight between Fed and FD groups in both WT and TG mice was determined. (C) Percentage of food deprivation-induced atrophy was determined in TA, EDL, SOL and GAS muscles. Data are the means ± S.E.M. (n=6 per group). *** P < 0.001 vs. Fed, # P < 0.05, ## P < 0.01 vs. WT.
WT TG-30
-20
-10
0
10
Cha
nge
in b
ody
wei
ght (
%)
FedFD
*** ***
TA EDLSOL
GAS0
10
20
30
40
Mus
cle
atro
phy
(%)
WTTG
#
#
##
A B C
WT TG0
5
10
15TR
B3
mR
NA
expr
essi
on (A
.U.)
##
81
Figure 4.4 TRB3 overexpression in skeletal muscle facilitates the protein degradation pathways, including ubiquitin-proteasomal and autophagy-lysosomal systems after 48 hr food deprivation. TA muscles from Fed and FD groups in both genotypes were used for analysis. (A-C) The phosphorylation of Akt (A), FOXO1 (B), and FOXO3a (C) were determined by Western blot analysis. (D) mRNA expression of atrogenes was measured by RT-PCR in both genotypes. (E-F) Protein expression of atrogin-1 (E) and MuRF-1 (F) was determined by Western blot analysis. (G-H) The markers of autophagy-lysosomal protein degradation, including p62 (G) and LC3B (H), were analyzed after 48 hr food deprivation. Data are the means ± S.E.M. (n=6 per group). P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed. # P < 0.05, ## P < 0.01 vs. WT.
WT TG0.0
0.5
1.0
1.5
pAkt
T308
/Akt
(A.U
.)
FedFD
***
WT TG
pAktT308
Akt
**
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
pFO
XO1S2
56/F
OXO
1 (A
.U.)
** **
WT TG
pFOXO1S256
FOXO1
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
pFO
XO3a
S253
/FO
XO3a
(A.U
.)
WT TG
pFOXO3aS253
FOXO3a
*
P=0.07
Fed FD Fed FD
Fed FDFed FD
0
5
10
15
mR
NA
expr
essi
on (A
.U.) WT
TG
***
***
***
***
Atrogin-1 MuRF-1 WT TG0
1
2
3
4
Atro
gin-
1/G
APD
H (A
.U.)
*
** #
WT TG
Atrogin-1
GAPDH
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
2.0
MuR
F-1/
GA
PDH
(A.U
.)
** ##
WT TG
MuRF-1
GAPDH
Fed FD Fed FD
WT TG0
1
2
3
p62/
GA
PDH
(A.U
.)
**
*** #
WT TG
p62
GAPDH
Fed FD Fed FD
WT TG0
2
4
6
8
10
LC3B
-II/L
C3B
-I (A
.U.)
**
***
WT TG
LC3B-I
GAPDH
LC3B-II
Fed FD Fed FD
A B C
DE F
G H
82
Figure 4.5 Muscle-specific TRB3 overexpression does not augment the effect of food deprivation on protein synthesis rate and signaling pathway. TA muscles from fed and food deprived muscle-specific TRB3 transgenic (TG) and wild-type littermate control (WT) mice were processed for analysis. (A) Western blot with anti-puromycin antibody was used to analyze puromycin incorporation into nascent peptides in WT and TG mice. Ponceau S staining was used to demonstrate an equal loading of protein. Methodology details can be found in Materials and Methods. (B) The amount of puromycin incorporation was normalized by ponceau S staining. (C-F) The phosphorylation and total expression of mTOR (C), 4EBP1 (D), PRAS40 (E), TSC2 (F) were measured by Western blot analysis. Data are the means ± S.E.M. (n=6 – 9 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed. # P < 0.05, ## P < 0.01 vs. WT.
WT TG0.0
0.5
1.0
1.5
Puro
myc
in/p
once
au (A
.U.) Fed
FD
##
***
250150
75
100
50
37
25
Fed FD Fed FD
WT TGPonceau S
Fed FD Fed FD
WT TGPuromycin
kDa
WT TG0.0
0.5
1.0
1.5
pmTO
RS2
448 /m
TOR
(A.U
.)
WT TG
pmTORS2448
mTOR
P=0.07
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
p4EB
P1S6
5 /4EB
P1 (A
.U.)
******
WT TG
p4EBP1S65
4EBP1
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
pPR
AS4
0T246
/PR
AS4
0 (A
.U.)
* ##
WT TG
pPRAS40T246
PRAS40
Fed FD Fed FD
WT TG0.0
0.5
1.0
1.5
pTSC
2T146
2 /TSC
2 (A
.U.)
*** ###
WT TG
pTSC2T1462
TSC2
P=0.08
Fed FD Fed FD
AB
C D
E F
83
Figure 4.6 TRB3 knockout mice were protected from food deprivation-induced atrophy. Ten- to thirteen-week-old female whole-body TRB3 knockout (KO) and wild-type littermate (WT) mice were used to demonstrate whether TRB3 is a key molecule to induce food deprivation-induced atrophy. (A) TRB3 mRNA expression in TA muscle was determined to confirm the knockout of TRB3. KO and WT mice were divided into Fed and FD groups. (B) Percentage change in body weight between Fed and FD groups in both WT and KO mice was determined. (C) Percentage of food deprivation-induced atrophy was determined in TA, EDL, SOL and GAS muscles. Data are the means ± S.E.M. (n=4 – 6 per group). *** P < 0.001 vs. Fed, # P < 0.05, ### P < 0.001 vs. WT.
WT KO0.0
0.5
1.0
1.5TR
B3
mR
NA
expr
essi
on (A
.U.)
###
WT KO-30
-20
-10
0
10
Cha
nge
in b
ody
wei
ght (
%) Fed
FD
*** ***
TA EDLSOL
GAS0
10
20
30
40
Mus
cle
atro
phy
(%)
WTKO
##
A B C
84
Figure 4.7 TRB3 knockout maintains protein synthesis rate after 48 hr food deprivation. Frozen TA muscles were processed for analysis. (A) Protein synthesis rate was measured by Western blot and ponceau S staining represented that an equal amount of proteins was loaded for each sample. (B) All puromycin blots were normalized by ponceau S staining. (C-F) Protein synthesis pathway factors, including mTOR, 4EBP1, PRAS40, and TSC2, was analyzed by Western blot. Data are the means ± S.E.M. (n=6 per group). * P < 0.05, *** P < 0.001 vs. Fed, # P < 0.05 vs. WT.
250150
75
100
50
37
25
Fed FD Fed FD
WT KOPonceau S
Fed FD Fed FD
WT KOPuromycin
kDa
WT KO0.0
0.5
1.0
1.5
Puro
myc
in/p
once
au (A
.U.)
FedFD
***
* #
WT KO0.0
0.5
1.0
1.5
pmTO
RS2
448 /m
TOR
(A.U
.)
WT KO
pmTORS2448
mTOR
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
2.0
p4EB
P1S6
5 /4EB
P1 (A
.U.) P=0.058
WT KO
p4EBP1S65
4EBP1
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
pPR
AS4
0T246
/PR
AS4
0 (A
.U.)
*
WT KO
pPRAS40T246
PRAS40
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
2.0
pTSC
2T146
2 /TSC
2 (A
.U.)
WT KO
pTSCT1462
TSC2
Fed FD Fed FD
AB
C D
E F
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Supplementary Figure S4.1 Forty-eight hours of food deprivation did not change Akt phosphorylation at Ser 473. Ten- to thirteen-week-old female C57BL/6 wild-type (WT), muscle-specific TRB3 transgenic (TG), TRB3 knockout (KO) mice were underwent either free access to food (Fed) or food deprivation (FD) for 48 hr. Tibialis anterior (TA) muscles were analyzed to examine food deprivation-induced changes in Akt phosphorylation at Ser 473. (A) Two days of food deprivation did not alter the level of phosphorylated Akt at Ser 473. (B-C) Phosphorylation of Akt at Ser 473 was not affected by 48 hr food deprivation in both TG (B) and KO (C) mice. Data are the means ± S.E.M. (n=4 – 6 per group).
Fed FD0.0
0.5
1.0
1.5
pAkt
S473
/Akt
(A.U
.)pAktS473
Akt
Fed FD Fed FD
FedFD
WT TG0.0
0.5
1.0
1.5
pAkt
S473
/Akt
(A.U
.)
Fed FD Fed FDWT TG
pAktS473
Akt
WT KO0.0
0.5
1.0
1.5
pAkt
S473
/Akt
(A.U
.)
Fed FD Fed FDWT KO
pAktS473
Akt
A B C
86
Supplementary Figure S4.2 The effect of 48 hr food deprivation on extensor digitorum longus (EDL) muscle from KO mice. Ten- to thirteen-week-old female TRB3 knockout (KO) and wild-type littermate (WT) mice were underwent either free access to food (Fed) or food deprivation (FD) for 48 hr. EDL muscle were used to demonstrate whether knockout of TRB3 prevents food deprivation-induced atrophy. (A-D) The phosphorylation of Akt (A-B), FOXO1(C), and FOXO3a (D) were determined by Western blot analysis. (E-F) Protein expression of atrogin-1 (E) and MuRF-1(F) was determined by Western blot analysis. Data are the means ± S.E.M. (n=4 – 6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed. # P < 0.05 vs. WT.
WT KO0
1
2
3
pAkt
T308
/Akt
(A.U
.)FedFD
#
**
WT KO
pAktT308
Akt
Fed FD Fed FD
WT KO0
1
2
3
pAkt
S473
/Akt
(A.U
.)
**
WT KO
pAktS473
Akt
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
pFO
XO1S2
56/F
OXO
1 (A
.U.)
*** ***
WT KO
pFOXO1S256
FOXO1
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
pFO
XO3a
S253
/FO
XO3a
(A.U
.)
*** **
WT KO
pFOXO3aS253
FOXO3a
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
Atro
gin-
1/G
APD
H (A
.U.) * *
WT KO
Atrogin-1
GAPDH
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
2.0
MuR
F-1/
GA
PDH
(A.U
.)
**
WT KO
MuRF-1
GAPDH
Fed FD Fed FD
A B
C D
E F
87
Supplementary Figure S4.3 The effect of 48 hr food deprivation on EDL from KO mice was not different from TA muscles. Ten- to thirteen-week-old female TRB3 knockout (KO) and wild-type littermate (WT) mice were underwent either free access to food (Fed) or food deprivation (FD) for 48 hr. (A-B) Protein synthesis rate was measured by Western blot and ponceau S straining represented that an equal amount of proteins was loaded for each sample. (B) All puromycin blots were normalized by Ponceau S staining. (C-F) Proteins involved in the protein synthesis pathway, including mTOR (C), 4EBP1 (D), PRAS40 (E), and TSC2 (F), were analyzed by Western blot. Data are the means ± S.E.M. (n=4 – 6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Fed. # P < 0.05 vs. WT.
250150
75
100
50
37
25
1 20
50
100
150
WT KOPonceau S
WT KOPuromycin
Fed FD Fed FD Fed FD Fed FDkDa
WT KO0.0
0.5
1.0
1.5
Puro
myc
in/p
once
au (A
.U.) Fed
FD
***
*** #
WT KO0.0
0.5
1.0
1.5
2.0
pmTO
RS2
448 /m
TOR
(A.U
.)
WT KO
pmTORS2448
mTOR
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
2.0
p4EB
P1S6
5 /4EB
P1 (A
.U.)
WT KO
p4EBP1S65
4EBP1
****
Fed FD Fed FD
WT KO0.0
0.5
1.0
1.5
pPR
AS4
0T246
/PR
AS4
0 (A
.U.)
WT KO
pPRAS40T246
PRAS40
Fed FD Fed FD
WT KO0
1
2
3
4
5
pTSC
2T146
2 /TSC
2 (A
.U.)
*
WT KO
pTSC2T1462
TSC2
Fed FD Fed FD
AB
C D
E F
88
CHAPTER 5
EFFECTS OF TRB3 ON EXERCISE-INDUCED SKELETAL MUSCLE ADAPTATION1
1 Choi RH, McConahay A, Johnson M, Koh HJ. To be submitted to Journal of Applied Physiology
89
5.1 ABSTRACT
Tribbles 3 (TRB3) is a pseudokinase and its expression has been shown to disrupt
glucose metabolism through the inhibition of Akt under obese and diabetic conditions.
Previously, we found that TRB3 overexpression in mouse skeletal muscle decreased
skeletal muscle mass and function, including running capacity. Here, we examined
whether TRB3 affects exercise training-induced skeletal muscle adaptation. We trained
muscle-specific TRB3 transgenic (TG) and wild-type (WT) littermates using a voluntary
wheel running protocol for 6 weeks and observed that TG mice ran significantly shorter
weekly distances than WT littermates. To eliminate the possibility that different skeletal
muscle adaptations would be produced due to different training intensities, involuntary
treadmill exercise (TM) was used as a training regimen. At the 5th week of training, we
measured glucose tolerance and found that trained TG mice showed glucose intolerance
compared to WT littermates. Furthermore, TRB3 overexpression significantly suppressed
the expressions of genes needed for glucose uptake and mitochondrial biogenesis,
independent of training status. To further determine the role of TRB3 in exercise-induced
adaptation, TRB3 knockout mice were trained by voluntary and involuntary exercise
protocols. KO mice presented improved glucose tolerance compared to WT littermates
independent of training status. However, we did not observe significant change in
markers of glucose uptake and mitochondrial biogenesis. Taken together, our results
indicate that TRB3 expression in skeletal muscle may blunt the benefits of exercise-
induced skeletal muscle adaptation.
90
5.2 INTRODUCTION
Tribbles homolog 3 (TRB3), a mammalian homolog of Drosophila tribbles, is a
pseudokinse, which lacks catalytic function, thus it serves as a molecular scaffold
(Boudeau et al., 2006). It is responsible for various cellular events including glucose
regulation, endoplasmic reticulum stress response, differentiation of skeletal muscle cells
and adipocytes, and cell cycle regulation (Du et al., 2003; Kato & Du, 2007; Ohoka et al.,
2005; Ord et al., 2007; Takahashi, Ohoka, Hayashi, & Sato, 2008).
For decades, many studies have investigated and confirmed the finding that TRB3
has a negative effect on Akt phosphorylation, which is a major process for glucose and
lipid metabolism in metabolic regulatory tissues, such as liver and skeletal muscle (Du et
al., 2003; Zhang et al., 2013). For instance, hepatic TRB3 expression decreases
phosphorylation of Akt and impairs glucose tolerance, while TRB3 knockdown improves
hepatic glucose regulation (Du et al., 2003). In skeletal muscle, muscle-specific TRB3
overexpression worsens high-fat diet-induced obesity and decreases phosphorylation of
Akt and glycogen synthase kinase 3 (GSK3), while muscle-specific TRB3 knockout
improved glucose homeostasis with high-fat diet (HFD) feeding (Zhang et al., 2013). Our
lab has also recently demonstrated that muscle-specific TRB3 overexpression in healthy
conditions and under food deprivation-induced atrophic states can regulate skeletal
muscle mass by altering protein turnover through the regulation of Akt and its
downstream molecules, for instance, mechanistic target of rapamycin (mTOR) and
forkhead box O transcription factors (FOXO) (R. H. Choi et al., 2017; R. H. Choi et al.,
2019). Given the findings that TRB3 could be an important regulator of skeletal muscle
mass and integrity, we hypothesized that TRB3 may significantly alter the exercise-
induced adaptation to skeletal muscle.
91
Skeletal muscle, which composes 40-50% of body mass, is a major organ that
regulates whole body energy metabolism because it is responsible for ~80% of
postprandial glucose uptake (DeFronzo & Tripathy, 2009). Continuous contractile
activity, such as that elicited by exercise, has been associated with improved glucose and
oxidative metabolism in skeletal muscle (Arena et al., 2017; Baar et al., 2002; Dela et al.,
2006). Exercise-induced improvements in glucose metabolism are associated with
increased expressions and functions of glucose transporters and metabolic enzymes in
skeletal muscle. Previous studies have found that glucose transporter 4 (GLUT4) mRNA
expression is increased after exercise (Kraniou et al., 2006; Kuo et al., 1999), but protein
expression after exercise can vary (Frosig et al., 2010; Leick et al., 2010). In addition to
increased GLUT4, it has been reported that a single bout of exercise can increase
hexokinase II (HXKII) expression and enzymatic activity in skeletal muscle, which
improves glucose metabolism (O'Doherty et al., 1996; O'Doherty et al., 1994). Exercise-
induced oxidative capacity and metabolic efficiency are elicited by increased
mitochondrial biogenesis (Pilegaard et al., 2003). Peroxisome proliferator-activated
receptor gamma co-activator 1α (PGC1α) is highlighted as a key regulator of
mitochondrial biogenesis in skeletal muscle in response to endurance exercise (Baar et
al., 2002; J.O. Holloszy, 1967). PGC1α is a transcription factor that can coordinate the
expression of genes related to mitochondrial biogenesis, such as mitochondrial
transcription factor A (TFAM), cytochrome oxidase (COX) 1 and 4, and cytochrome c
(Cyt c) (Yan, Okutsu, Akhtar, & Lira, 2011). Previously, muscle-specific PGC1α
knockout mice showed impaired muscle function, locomotion, and oxidative capacity,
but muscle-specific PGC1α knockout did not prevent exercise-induced COX and Cyt c
expression (Geng et al., 2010; Handschin et al., 2007).
92
Increased TRB3 expression has been found in the liver and skeletal muscle from
obese and diabetic rodent models, including ob/ob and HFD-induced obese mice, and this
increased TRB3 was decreased in response to acute and chronic endurance exercise
(Lima et al., 2009; Marinho et al., 2012; Matos et al., 2010). In addition, reduced TRB3
expression is associated with increased phosphorylation of Akt and improved glucose
metabolism (Lima et al., 2009; Marinho et al., 2012; Matos et al., 2010). One recent
study has shown that muscle-specific TRB3 transgenic mice display increased exercise
capacity with greater amount of oxidative fibers (An et al., 2014). Although there is some
evidence that TRB3 is regulated by exercise, the effect of TRB3 on exercise training-
induced skeletal muscle adaptation has not been elucidated.
In the current study, we investigate whether TRB3 expression in mouse skeletal
muscle will disrupt exercise-induced skeletal muscle adaptation, including glucose uptake
and mitochondrial biogenesis. Our findings show that muscle-specific TRB3 transgenic
(TG) mice run significantly shorter distances than wild-type (WT) littermates when they
are trained by voluntary wheel running. TG mice display significantly suppressed gene
expressions of GLUT4 and HXKII compared to WT littermates. Furthermore, TRB3
overexpression represses the expression of genes responsible for mitochondrial contents,
such as PGC1α, COX4, and Cyt c in both sedentary and trained groups. TRB3 knockout
(KO) mice have better glucose tolerance than WT littermates independent of training
status, but there is no significant improvement in exercise-induced skeletal muscle
adaptation at the molecular level. Taken together, these results indicate that TRB3
expression in skeletal muscle may blunt the benefits of exercise in glucose uptake and
mitochondrial biogenesis.
93
5.3 MATERIALS AND METHODS
Animal care
Ten- to fourteen-week-old muscle-specific TRB3 transgenic (TG) and whole-
body TRB3 knockout (KO) mice, and their wild-type littermates were used for the
current study. All animals were fed standard chow diet (Harlan Teklad Diet, Madision,
WI, USA) and accessed water ad libitum. Mice were maintained in the animal facility at
the University of South Carolina and were housed in an environmentally controlled room
on a 12 hr light-dark cycle. All protocols were approved by the Institutional Animal Care
and Use Committee of the University of South Carolina.
Voluntary wheel cage exercise
Mice were randomly assigned to housing in individual cages with or without
running wheels (9.5 inch in diameter) for 6 weeks. Mice freely accessed to wheel (Mini
Mitter company, Sunriver, OR, USA). Daily activities, including running time and wheel
revolutions, were monitored by computer monitoring software Vital View (Starr Life
Science Corp., Oakmont, PA, USA). Average daily running distance and 6-week total
running distance were calculated from the monitored data using Microsoft excel. Before
sacrifice, the wheels were removed from the cage for 48 h to minimize acute exercise
effect.
Involuntary treadmill running protocol
Separated ten- to fourteen-week old TG and WT mice were randomly assigned to
treadmill exercise groups. All mice in exercise groups had an acclimation period for 3
days on the treadmill (Columbus instruments treadmill simplex II, Columbus, OH, USA)
94
for 10 min at 8 meters/minute speed. For the 1st week of the training, mice ran for 1 h/day
at 12 meters/minutes at 5 degrees incline for 5 days/week. From the 2nd week to last
week, running speed was increased up to 16 meters/minutes and remained the same for 6
weeks.
Glucose tolerance test
At week 5, mice were transported from wheel cages to normal cages and
underwent overnight 16 h fasting to determine glucose tolerance. After 16 h fasting,
fasting blood glucose was determined. Glucose (2 g/kg body weight) was
intraperitoneally injected. Blood glucose was measured by a glucometer (Bayer,
Leverkusen, Germany) at 15, 30, 45, 60, 90, and 120 minutes after the injection.
Muscle glycogen content
Frozen gastrocnemius muscles were pulverized and used to detect glycogen
content after exercise training. The content was determined by hydrochloric acid
hydrolysis for 2 h at 95 ℃ followed by sodium hydroxide neutralization. Then, the
concentration was measured spectrophotometrically using a hexokinase-based assay kit
(Sigma, St. Louis, MO).
Citrate synthase activity
Citrate synthase activity was determined spectrophotometically in gastrocnemius
muscle as previously described (Srere, 1969). Gastrocnemius muscles were pulverized
and used 5 – 7 mg of muscle to measure the enzyme activity. Homogenates were
incubated with substrate and cofactors at room temperature and the working wavelength
is 412 nm to detect the reaction product thionitrobenzoic acid.
95
Body weight, food intake, blood glucose and body composition
Body weight, food intake, and blood glucose were monitored weekly at the same
time. Body composition was assessed at week 5. Mice were briefly anesthetized by
isoflurane inhalation and assessed for percentage fat and lean mass by dual-energy X-ray
absorptiometry (DEXA; Lunar PIXmus, Madison, WI).
Western blot analysis
Frozen gastrocnemius muscles was used to prepare protein lysates for Western
blot analysis as previously described (R. H. Choi et al., 2019). Protein lysates (30 µg)
were separated on 7 – 15% of SDS-polyacrylamide gels and transferred to PVDF
membranes. Primary and secondary antibodies purchased from commercial sources,
including GLUT4 (07-1404; Millipore, Billerica, MA, USA), hexokinase II (sc-3521;
Santa Cruz Biothechnology Inc., Dallas, TX, USA), PGC1α (ab5441; Abcam,
Cambridge, MA, USA), COX4 (4844), Cyt c (4280) from Cell Signaling Technology
(Danvers, MA, USA), TRB3 (ST1032; Calbiochem, San Diego, CA, USA), anti-rabbit
(NA934; GE healthcare, Pittsburgh, PA, USA) and anti-goat (31400; Thermo Fisher
Scientific, Waltham, MA, USA) IgG-horesradish peroxidase conjugated secondary
antibodies. The specific bands were visualized using ECL reagents (NEL105001EA;
PerkinElmer, Waltham, MA, USA), and the intensity of bands was quantified using
Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA).
RNA extraction and RT-PCR
Total RNA was extracted from TA muscles using Trizol reagent (Life
Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction. cDNA
was synthesized from 4 mg of total RNA by High Capacity cDNA kit (Life
96
Technologies, Carlsbad, CA, USA). cDNA and primers for real-time polymerase chain
reaction (RT-PCR) was carried by SYBR green PCR master mix (Life Technologies,
Carlsbad, CA, USA). Primer sequences used to analyze exercise-induced glucose
metabolism and oxidative capacity can be found in Table 5.1. All genes were normalized
to the level of 18S, a house keeping gene.
Statistical analysis
Data were expressed as mean ± SEM. Statistical analysis was performed using
GraphPad Prism 7 software (La Jolla, CA, USA). Student’s two-tailed unpaired t tests
were used for comparisons between two groups. One- or two-way ANOVA was
conducted to determine significance between more than two group followed by Tukey’s
or Bonferroni’s Post hoc analysis. P < 0.05 was considered statistically significant.
5.4 RESULTS
Muscle-specific TRB3 transgenic mice run shorter distances
We report in a previous study that increased TRB3 in skeletal muscle under stress
conditions, including ER stress and HFD, impairs insulin-stimulated Akt phosphorylation
and whole-body metabolism, and TRB3 knockout abolishes the development of HFD-
induced insulin resistance (Koh et al., 2013). Furthermore, our recent study demonstrates
that TRB3 overexpression in mouse skeletal muscle disrupts protein turnover, which in
turn worsens food deprivation-induced atrophy (R. H. Choi et al., 2019). Based on these
findings, we hypothesized that TRB3 expression would play a significant role in
exercise-induced skeletal muscle adaptation. In order to test our hypothesis, we
97
individually housed wild-type (WT) littermates and muscle-specific TRB3 transgenic
(TG) mice in cages with or without wheels for 6 weeks. We weekly monitored body
weight, food intake, and random blood glucose. No significant difference was observed
in any parameter between the groups (Table 2). The percentage of fat and lean mass was
determined by DEXA, and no changes were observed between sedentary and voluntary
wheel (VW) groups (Table 2). After 6 weeks of training, gastrocnemius muscles were
removed and used for analysis. TRB3 mRNA expression was not significantly altered by
VW in either WT or TG mice (Fig. 1A). Intriguingly, we observed that TG mice ran
significantly shorter distances per week than WT littermates (Fig. 1B). These results
suggest that muscle-specific TRB3 overexpression may reduce exercise capacity.
TRB3 overexpression in mouse skeletal muscle causes deterioration of glucose
metabolism in response to 6-week treadmill training
Since we observed that TG mice ran significantly shorter distances compared to
WT littermates, we speculated that since the mice received different training intensities,
this could result in different adaptation effects between genotypes. Thus, we utilized an
involuntary exercise protocol, forced treadmill running (TM), to give all mice the same
amount of exercise. WT littermates and TG mice were encouraged to run on the treadmill
for 6 weeks (details of this procedure can be found in Materials and Methods section).
During the 6-week TM training, body weight did not differ between the groups (Table 3).
However, we observed that TM groups showed significantly reduced body fat and
increased lean mass compared to sedentary groups (Table 3). To investigate whether
TRB3 was responsive to forced involuntary exercise, we measured TRB3 mRNA
expression. TM running did not alter TRB3 mRNA expression in WT mice, but TG mice
showed a greater increase in the mRNA expression in response to the training (Fig. 2A).
98
Next, to determine whether TRB3 overexpression regulates glucose metabolism in
response to involuntary exercise, we tested glucose tolerance 5 weeks after the training.
In trained WT mice, glucose tolerance was slightly improved, but not by a statistically
significance amount (Fig. 2B and C). TG mice in both sedentary and trained groups
tended to maintain higher blood glucose during 120-min test periods and showed a higher
area under the curve, indicating impaired glucose tolerance (Fig. 2B and C). We further
analyzed molecular markers associated with exercise-induced skeletal muscle glucose
uptake. Trained WT mice showed no alteration of GLUT4 protein or mRNA expression,
while hexokinase II (HXKII) was highly elevated (Fig. 2D – F). Interestingly, TG mice
showed slightly increased GLUT4 and HXKII protein expression in response to TM
training; however, we found that the mRNA expression of both markers was notably
suppressed in both sedentary and trained TG mice (Fig. 2D – F). The exercise-induced
adaption in glucose uptake and enzymatic activity has been associated with increased
post-exercise glycogen storage (Ivy & Kuo, 1998). Therefore, we measured muscle
glycogen contents. No significant differences were detected among groups; however, TG
mice tended to show lower amounts of glycogen after training compared to WT
littermates (Fig. 2G). These results indicate that TRB3 overexpression in mouse skeletal
muscle impairs the benefits of exercise on glucose metabolism by suppressing gene
expression.
Muscle-specific TRB3 overexpression represses exercise-induced mitochondrial
biogenesis
Endurance exercise training is known to increase oxidative metabolism in skeletal
muscle by increasing the number and size of mitochondria (Baar et al., 2002; J.O.
Holloszy, 1967). This process is mediated by a transcription coactivator peroxisome
99
proliferator-activated receptor-gamma coactivator 1α (PGC1α). It has been reported that
both a single bout of exercise and prolonged training can activate PGC1α, resulting in the
regulation of other transcription factors related to mitochondrial protein and function
(Baar et al., 2002; J.O. Holloszy, 1967). Previous research has demonstrated that TRB3 is
a mediator of PGC1α-induced insulin resistance in mouse liver (Koo et al., 2004).
Furthermore, muscle-specific PGC1α transgenic mice have shown a paradoxical effect
with regard to glucose metabolism, and have displayed a two-fold increase in TRB3
expression in their skeletal muscle (C. S. Choi et al., 2008). The previous findings
suggest a close relationship between TRB3 and PGC1α in skeletal muscle; however, no
research has investigated whether TRB3 expression affects exercise training-induced
mitochondrial biogenesis. Therefore, we next tested markers associated with
mitochondrial biogenesis and oxidative metabolism. Six-week treadmill training
increased the protein expression of mitochondrial biogenesis markers, such as PGC1α,
COX4, and Cyt c, in WT mice without altering mRNA expression. (Fig. 3A – D).
Furthermore, muscle-specific TRB3 overexpression slightly increased PGC1α and Cyt c
protein expression, but significantly reduced COX4 protein expression in trained TG
mice compared to trained WT mice (Fig 3. A – C). Intriguingly, TG mice showed
dramatically suppressed gene expressions of mitochondrial content markers, such as
PGC1α, COX4, and citrate synthase (CS), compared to WT littermates in both sedentary
and training conditions (Fig. 3D). Therefore, we tested CS activity to examine whether
repressed CS gene expression would affect physiological enzymatic activity. We found
that TM training slightly increased CS activity, but it did not reach statistical significance
independent of genotypes (Fig. 3E). These results indicate that muscle-specific TRB3
100
overexpression may debilitate exercise-induced mitochondrial adaptation by suppressing
the expression of genes necessary for mitochondrial biogenesis.
TRB3 knockout improves glucose tolerance but does not augment the benefits of
exercise in glucose metabolism
In the present study, we found that TRB3 overexpression in mouse skeletal
muscle blunted exercise training-induced skeletal muscle adaptation by repressing gene
expression. Therefore, we considered that a deletion of TRB3 in skeletal muscle might
enhance exercise-induced skeletal muscle adaptation. To examine the hypothesis, we next
recruited TRB3 knockout (KO) mice and WT littermates for exercise training with
voluntary wheel running (details found in the Materials and Methods section). During 6-
week VW training, there were no significant changes in body weight, food intake, blood
glucose, or body composition among the groups (Table 4). KO mice ran slightly more
often than WT littermates, but the increase did not reach to a statistical difference (Table
4). To examine the effect of TRB3 knockout on exercise-induced improvement in
glucose metabolism, we measured glucose tolerance after overnight fasting at the 5th
week of the training. Exercise training likely improved glucose tolerance in WT and KO
mice during the120-min test period (Fig. 4A). Interestingly, trained KO mice showed
significantly lower blood glucose than trained WT mice at the 60-min time (Fig. 4A).
These results were clearly reflected in the area under the curve: TRB3 knockout tended to
improve glucose tolerance in sedentary animals and after training (Fig. 4B). To
investigate the possible mechanism for improved glucose tolerance in KO mice, we
measured GLUT4 and HXKII expressions. In WT littermates, there was no change in
GLUT4 protein and mRNA expression, but HXKII was greatly increased in response to
VW exercise (Fig 4C – E). VW running did not alter GLUT4 expression in KO mice but
101
did slightly increase HXKII protein expression (Fig 4C and D). Furthermore, KO mice
displayed significantly reduced mRNA expression of GLUT4 and HXKII in response to
VW training (Fig. 4E). Muscle glycogen content was slightly increased in WT mice after
training. KO mice tended to show more glycogen content in sedentary status, but exercise
training produced no other effects (Fig. 4F). These results suggest that TRB3 knockout
improves glucose tolerance in sedentary and trained conditions, but that the improved
glucose tolerance is not due to changes in GLUT4 or HXKII expressions.
TRB3 knockout does not facilitate mitochondrial adaptation in response to VW
exercise training
We observed that TRB3 overexpression in mouse skeletal muscle significantly
abrogated the expression of genes responsible for mitochondrial biogenesis (Fig. 3D). To
determine whether TRB3 knockout accelerates the regulation of exercise-induced
mitochondrial adaptation, we examined mitochondrial markers at the protein and mRNA
levels. Six-week VW training increased mitochondrial markers, including PGC1α,
COX4, and Cyt c, in both trained WT and KO mice compared to sedentary groups (Fig.
5A – C). Consistent with protein expression, trained WT mice showed greatly increased
mRNA expression of mitochondrial biogenesis markers, such as PGC1α, COX1 and 4,
and CS (Fig. 5D). Surprisingly, TRB3 knockout mice did not show any significant
changes in the expression of genes needed for mitochondrial biogenesis in response to
VW (Fig. 5D). To further examine whether TRB3 knockout affects mitochondrial
enzymatic activity, we measured CS activity and found a notable increase in the activity
after VW running in both genotypes (Fig. 5E). These results indicate that TRB3 knockout
does not further promote mitochondrial biogenesis markers after VW exercise training.
102
TRB3 knockout does not benefit exercise training-induced metabolic adaptations in
response to involuntary running
In the present study, we have found that TRB3 knockout did not further assist
exercise-induced skeletal muscle adaptation even though KO mice were likely to show a
better glucose tolerance than WT mice (Fig. 4A and B). To eliminate the possibility of
the mice receiving different intensities of exercise due to the voluntary exercise protocol,
we repeated the experiment using an involuntary exercise protocol, forced treadmill
running (see Materials and Methods section for details). There were no differences in
body weight, food intake, or blood glucose among the groups during the 6-week training
period (Table 5). However, we did observe a significant reduction in fat mass in KO mice
after TM training compared to WT mice (Table 5). Next, the mice underwent a glucose
tolerance test at the 5th week of the training. There was no noticeable difference in
glucose tolerance among the groups (Fig. 6A). In KO mice, both sedentary and trained
mice showed a lower area under the curve compared to WT mice, suggesting improved
glucose tolerance (Fig. 6B). We also analyzed GLUT4 and HXKII to determine possible
mechanisms for improved glucose tolerance. GLUT4 and HXKII were increased in both
WT and KO mice in response to the involuntary running protocol (Fig. 6D – E).
Furthermore, TM running slightly depleted muscle glycogen in both genotypes, but we
did not find any statistical difference (Fig. 6F). Mitochondrial contents at the protein and
mRNA levels showed a tendency to respond to TM running, but there were no
remarkable differences between genotypes (Fig. 7A – D). In CS activity, TRB3 knockout
increased CS activity in response to TM exercise while WT littermates did not show any
response to the training (Fig. 7E). Taken together, these data suggest that TRB3 knockout
103
improves glucose tolerance regardless of training status or types of training, but TRB3
knockout does not benefit exercise-induced skeletal muscle adaptation.
5.5 DISCUSSION
TRB3 is known to inhibit Akt phosphorylation, and increased TRB3 expression is
associated with metabolic dysfunction in multiple tissues, including the liver and skeletal
muscle (Koo et al., 2004; Zhang et al., 2016). In addition, a reduction of TRB3
expression in obese and diabetic mouse models has been observed in response to acute
and chronic exercise, resulting in amelioration of metabolic stress (Lima et al., 2009;
Marinho et al., 2012; Matos et al., 2010). Although research has demonstrated that TRB3
is strongly associated with metabolic dysregulation in the liver under obese and diabetic
conditions, the role of TRB3 in skeletal muscle has not been clearly understood. Our
previous studies demonstrated that TRB3 plays an important role in the regulation of
protein turnover in skeletal muscle at the basal level and under food deprivation-induced
atrophy (R. H. Choi et al., 2017; R. H. Choi et al., 2019). To further determine the role of
TRB3 in skeletal muscle, we examined the effects of TRB3 on exercise training-induced
skeletal muscle adaptation with regard to glucose uptake and mitochondrial biogenesis.
Our findings indicate that muscle-specific TRB3 overexpression cause deterioration in
glucose tolerance and also suppresses the expression of genes responsible for glucose
uptake and mitochondrial biogenesis regardless of exercise training. By contrast with the
detrimental effects of TRB3 overexpression, TRB3 knockout improves glucose tolerance
in both sedentary and trained groups; however, there were no further improvements in
glucose uptake or mitochondrial biogenesis between genotypes in response to exercise
104
training. These findings suggest a possible role for TRB3 in exercise-induced skeletal
muscle adaptation: the regulation of whole-body metabolism and oxidative capacity.
In the current study, we observed that voluntary wheel running did not alter TRB3
expression in either WT or TG mice, and involuntary treadmill training greatly increased
TRB3 expression in TG mice only (Fig. 1A and 2A). Previously, An et al. reported that
4-week voluntary exercise increases TRB3 mRNA and protein expression in C57BL/6
WT mice (An et al., 2014). However, we did not observe an increase of TRB3 expression
in WT littermates in response to either exercise training protocols. This inconsistency
may be due to different types of training procedures, intensity, and duration. Our mice
ran about 20 km/week for 6 weeks compared to about 46 km/week for 4 weeks based on
An et al.’s report. This difference in running distance raises the possibility of different
responses. Future study may be required using similar intensity, duration, and training
protocols to produce comparable results. Furthermore, we found increased TRB3
expression in TG mice only after involuntary exercise (Fig. 2A). This discrepancy could
be due to sex difference in the mice that we used for each study and/or to different
training procedures. There are several reports of female and male mice showing different
consequences after exercise training (De Bono, Adlam, Paterson, & Channon, 2006;
Konhilas et al., 2004). As the relationship between TRB3 expression and sex different is
unknown, further study would be necessary to reveal any relationship involving sex
difference.
Exercise training promotes glucose uptake in skeletal muscle with increasing
GLUT4 expression and HXKII enzymatic activity (Cusi et al., 2001; Derave et al., 1999;
Rodnick, Henriksen, James, & Holloszy, 1992). Several previous studies have reported
that GLUT4 protein and mRNA expression can vary due to different experiment settings
105
and exercise intensities (Frosig et al., 2010; Kuo et al., 1999; Leick et al., 2010). In the
current study, we found that GLUT4 protein expression was not altered by exercise
training in either genotype (Fig. 2D), but TRB3 overexpression significantly decreased
gene expression of GLUT4 in sedentary and trained groups (Fig. 2F). TRB3
overexpression repressed HXKII mRNA expression as well. HXKII is a rate-limiting
enzyme in glycolysis known to be responsible for exercise-induced glucose uptake
(O'Doherty et al., 1996; O'Doherty et al., 1994). Furthermore, impaired glucose uptake
and exercise capacity have been observed in HXKII knockout mice in response to
exercise training (Fueger et al., 2003; Fueger et al., 2005). Given the importance of
GLUT4 and HXKII in glucose uptake, suppressed GLUT4 and HXKII gene expressions
in mouse skeletal muscle may induce impaired glucose tolerance in TG mice (Fig. 2B
and C). Moreover, the decreased gene expressions in TG mice possibly impairs exercise
capacity, as we observed shorter distances in voluntary running training caused by a
disruption of energy substrate utilization (Fig. 1B). The mechanism(s) by which TRB3
regulates the expressions of genes responsible for metabolism remains to be answered.
Endurance exercise training also stimulates mitochondrial biogenesis. PGC1α is
considered to be a main regulator of exercise-induced mitochondrial biogenesis (Baar et
al., 2002). Muscle-specific overexpression of PGC1α increases slow-type fibers and
improves exercise capacity (Lin et al., 2002; Wende et al., 2007), while muscle-specific
PGC1α knockout mice show decreased daily locomotion and attenuated exercise-induced
mitochondrial biogenesis (Handschin et al., 2007). We observed that TG mice displayed
significantly lower PGC1α mRNA expression with no further increment after exercise
training, and that they also show remarkably reduced the expressions of genes necessary
for mitochondrial biogenesis independent of training status (Fig. 3D). In addition, it has
106
been demonstrated that PGC1α overexpression in the liver and skeletal muscle impair
glucose homeostasis by levating hepatic TRB3 expression (C. S. Choi et al., 2008; Koo et
al., 2004). Although the role of TRB3 in regulating PGC1α expression has not been
elucidated, we believe our results offer at least the possibility that TRB3 overexpression
impairs PGC1α expression and disrupts the regulation of exercise-induced mitochondrial
biogenesis.
In the present study, TRB3 knockout mice seem to demonstrate improved glucose
tolerance compared to WT littermates, independent of training status (Fig. 5B and 6B).
Previously, our lab has shown that TRB3 knockout prevents HFD-induced development
of insulin resistance (Koh et al., 2013). This would be the first observation of TRB3
knockout possibly benefiting whole-body glucose tolerance under normal healthy
conditions, regardless of exercise training. However, we did not detect any further
changes in protein or mRNA expressions of GLUT4 or HXKII (Fig. 4C – E, 5C – E).
These results suggest that improved glucose tolerance in KO mice is possibly caused by
other mechanisms than GLUT4 and HXKII expression, which can regulate glucose
metabolism, involve in the improved glucose tolerance in KO mice.
Taken together, our data suggest that muscle-specific TRB3 overexpression might
impair exercise capacity and glucose tolerance in response to exercise training via the
regulation of transcriptions of genes responsible for glucose uptake and mitochondrial
biogenesis. We have also demonstrated that the lack of TRB3 might improve glucose
tolerance, independent of training status. The current study elucidates the possibility that
TRB3 expression could affect glucose metabolism and mitochondrial biogenesis in
response to exercise training. Additional works are necessary to find the molecular
107
mechanism(s) by which TRB3 regulates transcriptional activity of the genes responsible
for glucose uptake and mitochondrial biogenesis.
108
Table 5.1 Primer sequences for RT-PCR
GLUT4, glucose transporter 4; PGC1α, Peroxisome proliferator-activated receptor gamma co-activator 1 α; TFAM, mitochondrial transcription factor A; COX1, cytochrome oxidase 1; COX4, cytochrome oxidase 4; Cyt c, cytochrome c
109
Table 5.2 Characteristics of TG mice in voluntary wheel cage exercise
Ten- to fourteen-week old male female wild-type (WT) littermates and muscle-specific TRB3 transgenic (TG) mice were randomly assigned in two groups; sedentary (Sed) and voluntary wheel (VW) running for 6 weeks. Data are means ±SEM (n=4/group).
110
Figure 5.1 Muscle-specific TRB3 overexpression impairs exercise capacity as measured by 6-week voluntary wheel running. Ten- to fourteen-week old female wild-type (WT) littermates and muscle-specific TRB3 transgenic (TG) mice were separated in sedentary (Sed) and voluntary wheel (VW) running group for 6 weeks. (A) TRB3 expression was determined in gastrocnemius. (B) Wheel activity was monitored by the software and weekly average distance was determined. Data are means ±SEM (n=4/group). ** P<0.01 vs. WT
WT TG0
5
10
15
20
25
Aver
age
dist
ance
(km
/wee
k)
**
WT TG
0
100
200
300
TRB
3 m
RN
A e
xpre
ssio
n (A
.U.)
Sed
VW
Genotype main effect p=0.04A B
111
Table 5.3 Characteristics of TG mice in treadmill exercise
Ten- to fourteen-week old male wild-type (WT) littermates and muscle-specific TRB3 transgenic (TG) mice were randomly assigned in two groups; sedentary (Sed) and involuntary treadmill (TM) training for 6 weeks. Data are means ±SEM (n=4/group). * P<0.05 vs. Sed.
112
Figure 5.2 Muscle-specific TRB3 overexpression deteriorates glucose uptake in response to 6-week treadmill training. Ten- to fourteen-week old male wild-type (WT) littermates and muscle-specific TRB3 transgenic (TG) mice were randomly assigned in sedentary (Sed) and involuntary treadmill (TM) training groups for 6 weeks. (A) After 6 weeks of training, TRB3 mRNA expression was measured in gastrocnemius muscle. (B-C) At 5th week, glucose tolerance (B) was tested in sedentary (Sed) and treadmill (TM) trained mice and the results were represented as area under the curve (C). (D-E) Protein expressions of glucose transporter 4 (GLUT4; D) and hexokinase II (HXKII; E) were analyzed by Western blot. (F) GLUT4 and HXKII mRNA expression was determined by RT-PCR. (G) Muscle glycogen content were measured in gastrocnemius muscle. Data are means ±SEM (n=4/group). * P<0.05, ** P<0.01 vs. Sed; # P <0.05, ## P<0.01, ### P<0.001 vs. WT.
WT TG0
100
200
300
400
500
TRB
3 m
RN
A ex
pres
sion
(A.U
.)
Sed
TM
Genotype main effect p<0.05
*
0 15 30 45 60 90 120
0
100
200
300
400
500
Time (min)
Blo
od g
luco
se (m
g/dl
)WT SedWT TMTG SedTG TM
WT TG0
10000
20000
30000
40000
Are
a un
der t
he c
urve
(A.U
.)
SedTM
#
WT TG0.0
0.5
1.0
1.5
2.0
GLU
T4/G
APD
H (A
.U.)
Sed
TM
Sed TM Sed TM
WT TG
GLUT4
GAPDH
WT TG0.0
0.5
1.0
1.5
2.0
2.5
Hex
okia
nse
II/G
APD
H (A
.U.)
**
Sed TM Sed TM
WT TG
HXKII
GAPDH
WT TG WT TG0.0
0.5
1.0
1.5
mR
NA
expr
essi
on (A
.U.)
Sed
GLUT4 HXKII
######
TM
###
WT TG
0
2
4
6
8
10
Gly
coge
n (µ
g/m
g tis
sue) Sed
TM
A
B C
D E
F G
113
Figure 5.3 Muscle-specific TRB3 overexpression represses gene expressions of mitochondrial biogenesis markers. Ten- to fourteen-week old male wild-type (WT) littermates and muscle-specific TRB3 transgenic (TG) mice were randomly assigned in sedentary (Sed) and involuntary treadmill (TM) training groups for 6 weeks. (A-C) Protein expression of PGC1α (A), COX4 (B), and Cyt c (C) were determined by Western blot. (D) The expression of genes for mitochondrial contents were measured by RT-PCR. (E) Citrate synthase activity was measured in gastrocnemius to determine oxidative function in response to exercise training. Data are means ±SEM (n=4/group). * P<0.05, ** P<0.01 vs. Sed; # P <0.05, ## P<0.01, ### P<0.001 vs WT.
WT TG0.0
0.5
1.0
1.5
2.0
2.5
PGC
1α/G
APD
H (A
.U.)
Sed
TM*
Sed TM Sed TM
WT TG
PGC1α
GAPDH
WT TG0.0
0.5
1.0
1.5
2.0
Cyt
c/G
APD
H (A
.U.)
Sed
TM
Sed TM Sed TM
WT TG
Cyt c
GAPDH
WT TG0.0
0.5
1.0
1.5
CO
X4/G
APD
H (A
.U.)
SedTM##
Sed TM Sed TMWT TG
COX4
GAPDH
WT TG WT TG WT TG WT TG WT TG WT TG0.0
0.5
1.0
1.5
mR
NA
expr
essi
on (A
.U.)
SedTM
PGC1α TFAM COX1 COX4 Cyt c
### ####
##
####
CS
## ##
WT TG0
50
100
150
200
Citr
ate
synt
hase
act
ivity Sed
TM
A B C
D E
114
Table 5.3 Characteristics of KO mice in voluntary wheel exercise
Ten- to fourteen-week old female wild-type (WT) littermates and TRB3 knockout (KO) mice were randomly assigned in two groups; sedentary (Sed) and voluntary wheel (VW) training for 6 weeks. Data are means ±SEM (n=4-5/group).
115
Figure 5.4 TRB3 knockout improves glucose tolerance but does not augment the benefits of exercise in glucose uptake. Ten- to fourteen-week old female wild-type (WT) littermates and TRB3 knockout (KO) mice were divided into sedentary (Sed) and voluntary wheel (VW) training groups for 6 weeks. (A-B) At 5th week of training, glucose tolerance (A) was measured to determine glucose metabolism and the results were represented as area under the curve (B). (C-D) Protein expressions of glucose transporter 4 (GLUT4; C) and hexokinase II (HXKII; D) were analyzed by Western blot. (E) GLUT4 and HXKII mRNA expression was determined by RT-PCR. (F) Muscle glycogen content were measured in gastrocnemius muscle. Data are means ±SEM (n=4-5/group). * P<0.05 vs. Sed; # P <0.05, ## P<0.01 vs. WT.
0 15 30 45 60 90 120
0
100
200
300
400
500
Time (min)B
lood
glu
cose
(mg/
dl)
WT SedWT VWKO SedKO VW
* ##
WT KO0
5000
10000
15000
Are
a un
der t
he c
urve
(A.U
.)
SedVW
Genotype main effec P<0.05
WT KO0.0
0.5
1.0
1.5
GLU
T4/G
APD
H (A
.U.)
SedVW
Sed VW Sed VWWT KO
GLUT4
GAPDH
WT KO0
1
2
3
4
Hex
okia
nse
II/G
APD
H (A
.U.)
SedVW*
Sed VW Sed VWWT KO
HXKII
GAPDH
WT KO WT KO0.0
0.5
1.0
1.5
2.0
2.5
mR
NA
expr
essi
on (A
.U.) Sed
VW
##
*
GLUT4 HXKII
WT KO0
5
10
15
20G
lyco
gen
(µg/
mg
tissu
e) SedVW
A B
C D
E F
116
Figure 5.5 TRB3 knockout dose not facilitate mitochondrial adaptation in response to VW training. Ten- to fourteen-week old female wild-type (WT) littermates and TRB3 knockout (KO) mice were divided into sedentary (Sed) and voluntary wheel (VW) training groups for 6 weeks. After 6 weeks of training, gastrocnemius muscles were analyzed. (A-C) Protein expression of PGC1α (A), COX4 (B), and Cyt c (C) were determined by Western blot. (D) The expression of genes for mitochondrial contents were measured by RT-PCR. (E) Citrate synthase activity was measured to determine oxidative function in response to exercise training. Data are means ±SEM (n=4-5/group). * P<0.05, ** P<0.01 vs. Sed; # P <0.05, ## P<0.01, ### P<0.001 vs WT.
WT KO0
1
2
3
4
PGC
1α/G
APD
H (A
.U.)
SedVW
Sed VW Sed VWWT KO
PGC1α
GAPDH
Exercise main effect p<0.05
WT KO0
2
4
6
8
CO
X4/G
APD
H (A
.U.)
SedVW
Sed VW Sed VWWT KO
COX4
GAPDH
Exercise effect p<0.01
WT KO0
1
2
3
Cyt
c/G
APD
H (A
.U.)
SedVW
Sed VW Sed VWWT KO
Cyt c
GAPDH
Exercise effect p<0.01
WT KO WT KO WT KO WT KO WT KO WT KO0
1
2
3
mR
NA
expr
essi
on (A
.U.)
SedVW
PGC1α TFAM COX1 COX4 Cyt c
P=0.08
*
**
#
CS
*
#
WT KO0
50
100
150
200
Citr
ate
synt
hase
act
ivity Sed
VW**
*
A B C
D E
117
Table 5.4 Characteristics of KO mice in treadmill exercise
Ten- to fourteen-week old male wild-type (WT) littermates and TRB3 knockout (KO) mice were randomly assigned in two groups; sedentary (Sed) and involuntary treadmill (TM) training for 6 weeks. Data are means ±SEM (n=4/group). # P<0.05 vs. WT
118
Figure 5.6 TRB3 knockout does not further improve molecular markers for glucose uptake in response to involuntary exercise. Ten- to fourteen-week old male wild-type (WT) littermates and TRB3 knockout (KO) mice were divided into sedentary (Sed) and involuntary treadmill (TM) training groups for 6 weeks. (A-B) At 5th week of TM training, glucose tolerance (A) was measured to determine glucose metabolism and the results were represented as area under the curve (B). (C-D) Protein expressions of glucose transporter 4 (GLUT4; C) and hexokinase II (HXKII; D) were analyzed by Western blot. (E) GLUT4 and HXKII mRNA expression was determined by RT-PCR. (F) Muscle glycogen content were measured in gastrocnemius muscle. Data are means ±SEM (n=4/group). * P<0.05 vs. Sed.
0 15 30 45 60 90 120
0
100
200
300
400
500
Time (min)B
lood
glu
cose
(mg/
dl)
WT SedWT TMKO SedKO TM
WT KO0
5000
10000
15000
20000
Are
a un
der t
he c
urve
(A.U
.)
SedTM
Genotype main effect P<0.01
WT KO0.0
0.5
1.0
1.5
2.0
GLU
T4/G
APD
H (A
.U.)
SedTM*
Sed TM Sed TMWT KO
GLUT4
GAPDH
WT KO0.0
0.5
1.0
1.5
2.0
2.5
Hex
okia
nse
II/G
APD
H (A
.U.) Sed
TM
Sed TM Sed TMWT KO
HXKII
GAPDH
WT KO WT KO0.0
0.5
1.0
1.5
2.0
mR
NA
expr
essi
on (A
.U.)
GLUT4 HXKII
SedTM
WT KO0
2
4
6
8
10G
lyco
gen
(µg/
mg
tissu
e) SedTM
A B
C D
E F
119
Figure 5.7 TRB3 knockout does not promote mitochondrial biogenesis in response to involuntary treadmill exercise. Ten- to fourteen-week old female wild-type (WT) littermates and TRB3 knockout (KO) mice were divided into sedentary (Sed) and forced treadmill (TM) training groups for 6 weeks. After 6 weeks of training, gastrocnemius muscles were analyzed. (A-C) Protein expression of PGC1α (A), COX4 (B), and Cyt c (C) were determined by Western blot. (D) The expression of genes for mitochondrial contents were measured by RT-PCR. (E) Citrate synthase activity was measured to determine oxidative function in response to exercise training. Data are means ±SEM (n=4-5/group). * P<0.05, ** P<0.01 vs. Sed; # P <0.05 vs WT.
WT KO0.0
0.5
1.0
1.5
PGC
1α/G
APD
H (A
.U.)
SedTM
Sed TM Sed TMWT KO
PGC1α
GAPDH
WT KO0.0
0.5
1.0
1.5
2.0
CO
X4/G
APD
H (A
.U.)
SedTM
*
Sed TM Sed TMWT KO
COX4
GAPDH
WT KO0.0
0.5
1.0
1.5
2.0
Cyt
c/G
APD
H (A
.U.)
SedTM
#
**
Sed TM Sed TMWT KO
Cyt c
GAPDH
WT KO WT KO WT KO WT KO WT KO WT KO0.0
0.5
1.0
1.5
2.0
mR
NA
expr
essi
on (A
.U.)
SedTM
PGC1α TFAM COX1 COX4 Cyt c
Exercise main effect P<0.05
CS WT KO0
50
100
150
Citr
ate
synt
hase
act
ivity Sed
TM*
A B C
D E
120
CHAPTER 6
OVERALL DISCUSSION
121
Skeletal muscle displays high plasticity, adapting its function and structure in
response to various stimuli, such as nutrients, hormones, and muscle contractions. The
loss of muscle mass and integrity has been associated with an increasing prevalence of
chronic diseases, including hypertension, type 2 diabetes, obesity, and cancer. Therefore,
maintenance of skeletal muscle mass and function is considered as a powerful strategy in
preventing and overcoming pathological conditions. The IGF-1/PI3K/Akt pathway is
known to be a major signaling cascade that regulates energy metabolism and protein
turnover in skeletal muscle. Although the role of Akt in energy metabolism and protein
turnover has been studied for decades, only a few studies have defined a direct regulator
of Akt activity. TRB3 has been identified as a negative regulator of Akt via a direct
binding to phosphorylation sites (Du et al., 2003). Its expression is found in multiple
tissues, including the liver and skeletal muscle. Since TRB3 is strongly expressed in the
liver and its function is associated with the inhibition of Akt, resulting in metabolic
dysregulation, previous research has focused on the effects of TRB3 on metabolic
dysregulation, such as insulin resistance (Du et al., 2003; Koo et al., 2004). As previously
stated, Akt is a key protein kinase that regulates not only energy metabolism but also
protein turnover in skeletal muscle. However, only a few recent studies have revealed the
role of TRB3 in skeletal muscle (Koh et al., 2013; J. Liu et al., 2010; Zhang et al., 2013),
and no study has focused on muscle protein turnover with TRB3 expression. Therefore,
the overall purpose of this dissertation was to determine the role of TRB3 in skeletal
muscle mass regulation and exercise-induced adaptation. To investigate this purpose, we
first hypothesized that TRB3 expression would regulate protein synthesis and degradation
by affecting Akt and its downstream proteins, mTOR and FOXO, in both basal states and
atrophic conditions. We also hypothesized that TRB3 expression would influence
122
exercise-induced skeletal muscle adaptation in glucose uptake and mitochondrial
biogenesis. The major findings from the experiments conducted in this dissertation
demonstrates that (1) muscle-specific TRB3 overexpression decreases protein synthesis
and increases protein degradation, while TRB3 knockout improves protein synthesis and
reduces protein degradation, (2) under food-deprivation induced atrophic conditions,
muscle-specific TRB3 overexpression worsens atrophy by accelerating protein
degradation systems, whereas TRB3 knockout prevents atrophy by preserving the protein
synthesis rate, and (3) muscle-specific TRB3 overexpression impairs exercise capacity
and suppresses gene expressions related to glucose uptake and mitochondrial biogenesis,
independent of training status, while TRB3 knockout improves glucose tolerance in both
the absence and the presence of training. These findings provide preliminary insights into
an important role that TRB3 might play in skeletal muscle plasticity.
TRB3 regulates protein turnover through mTOR and FOXO at the basal status and
under atrophic conditions
Skeletal muscle mass is determined by the net dynamic balance between the rates
of protein synthesis and protein degradation (Schiaffino et al., 2013; Schiaffino &
Mammucari, 2011). TRB3 expression is mainly known as an inhibitor of Akt
phosphorylation, an important cellular process not only in activating protein synthesis
through phosphorylation of mTOR, but also in inactivating protein degradation through
phosphorylation of FOXO (Blaauw et al., 2009; Schiaffino & Mammucari, 2011).
Previously, hepatic and muscle-specific TRB3 overexpression has been shown to
decrease insulin-stimulated Akt phosphorylation, resulting in impaired glucose tolerance
and the development of insulin resistance (Du et al., 2003; Zhang et al., 2013). In Aim 1,
we found that TG mice showed decreased hindlimb muscle weights compared to WT
123
littermates. This phenomenon could be due to suppressed phosphorylation of mTOR and
S6K1 with decreased phosphorylation of FOXO in TG mice. In line with activated
FOXO, atrogin-1 and MuRF-1 mRNA expression was greatly increased in TG mice with
no changes to cathepsin L. However, we did not observe basal-level changes in Akt
phosphorylation at either sites (T308 and S473) in TG mice. This could be due to our
method, Western blot, which was perhaps not sufficient or not sensitive enough to detect
the basal change in Akt phosphorylation. These results may suggest that other sensitive
methods, including immunoprecipitation or Akt activity assay, are required to detect
changes in Akt phosphorylation. In contrast to our results, a study from another group
reveals that muscle-specific TRB3 overexpressed mice showed increased muscle mass
and improved exercise capacity, along with more oxidative fibers (An et al., 2014). This
discrepancy may be due to mouse background crossing, sex, and food differences; and/or
to the distinct experimental settings. We have also demonstrated that TRB3 knockout
increases muscle weights and protein synthesis rates, and that it decreases protein
degradation. Although inconsistent data exists, our results are in line with our conclusion
that TRB3 overexpression impairs the protein turnover needed to reduce muscle mass and
function.
In Aim 2, we observed that muscle-specific TRB3 overexpression worsened 48 hr
food deprivation-induced atrophy, while TRB3 knockout protected loss of muscle mass
in response to 48 hr food deprivation. Although TRB3 is known to be responsive to
starvation in the liver (Du et al., 2003), we further demonstrated that food deprivation
could increase TRB3 expression in skeletal muscle. We also reported that the detrimental
effects of muscle-specific TRB3 overexpression on food deprivation-induced skeletal
muscle atrophy could result from aggravated ubiquitin-proteasomal and autophagy-
124
lysosomal proteolysis. Consistent with our previous finding, TG mice displayed
significantly suppressed protein synthesis rates in fed state, but this finding was not
further exacerbated by 48 hr food deprivation. On the other hand, TRB3 knockout did
decrease the protein synthesis rates after 48 hr food deprivation, but KO mice preserved a
higher protein synthesis rate than WT littermates. This could explain how KO mice
ameliorated skeletal muscle atrophy under 48 hr food deprivation condition. Taken
together, these findings strongly support the hypothesis that TRB3 is a major regulator of
protein turnover in skeletal muscle at the basal level and under food deprivation-induced
atrophic conditions.
ER stress controls TRB3 expression in skeletal muscle in response to 48 hr food-
deprivation atrophic condition
TRB3 is known to be responsive to various cellular stresses, including starvation
and insulin resistance (Ord et al., 2007; Ord & Ord, 2003, 2005). Endoplasmic reticulum
(ER) is an organelle responsible for protein synthesis in cells. Cellular stress increases
burdens of protein synthesis in ER and can activates unfolded protein response (UPR) to
manage protein synthesis. Uncontrolled UPR can induce ER stress, which can not only
inhibit cell survival mechanisms but also facilitate apoptosis (Dicks et al., 2015;
Guerrero-Hernandez et al., 2014; J. Wang et al., 2013). Previously, ER stress markers,
such as ATF4 and CHOP, showed the possibility of regulating TRB3 expression in
multiple cell lines (Ohoka et al., 2005; Ord et al., 2007; Ord & Ord, 2003, 2005). In
addition, our lab has found that TRB3 expression mediates ER stress-induced insulin
resistance in mouse skeletal muscle (Koh et al., 2013). However, no prior studies had
investigated the means by which TRB3 expression was controlled in skeletal muscle
under stressful conditions. Our second study provided an evidence that ER stress could be
125
an upstream regulator of TRB3 expression in skeletal muscle. When we measured ER
stress markers in skeletal muscle, we observed that various ER stress markers, including
XBP1 and ATF4, were greatly increased after 48 hr food deprivation. Furthermore, we
treated C2C12 myotubes with 4-PBA, a chemical chaperon to block ER stress, in PBS for
6 hr to mimic starvation. Intriguingly, we found that the blocking of ER stress completely
abolished TRB3 expression and expression of muscle-specific ubiquitin ligases.
Collectively, these data provide evidence that ER stress can be a regulator of TRB3
expression in skeletal muscle and that TRB3 is possibly associated with regulating
muscle-specific ubiquitin ligases under atrophic conditions. Future studies are required to
determine the mechanism(s) by which ER stress regulates TRB3 expression and muscle-
specific ubiquitin ligases in skeletal muscle.
TRB3 expression in healthy skeletal muscle influence exercise-induced adaptation in
glucose tolerance and mitochondrial biogenesis
Endurance exercise training induces skeletal muscle adaptations in both structure
and function (Baar et al., 2002; J.O. Holloszy, Oscai, Mole, & Don, 1970; J.O. Holloszy,
Rennie, Hickson, Conlee, & Hagberg, 1977). Established skeletal muscle adaptation is
largely categorized into metabolic and structural adaptations (e.g., improved glucose
uptake and increased mitochondrial biogenesis, respectively) (G.D. Cartee & Holloszy,
1990; Hansen et al., 1998; J.O. Holloszy, 1988; J. O. Holloszy & Booth, 1976; J.O.
Holloszy & Coyle, 1984; Ivy, Young, McLane, Fell, & Holloszy, 1983). Increased TRB3
expression found in obese and diabetic rodent models is abolished after both acute and
chronic endurance exercise (Lima et al., 2009; Marinho et al., 2012; Matos et al., 2010).
This reduction in TRB3 expression results in decreased interaction between Akt and
TRB3 and increased glucose uptake in the liver and skeletal muscle (Lima et al., 2009;
126
Marinho et al., 2012; Matos et al., 2010). However, no studies have investigated whether
TRB3 expression in skeletal muscle affects exercise-induced skeletal adaptation at the
basal conditions. In Aim 3, we observed that muscle-specific TRB3 overexpression
impaired running capacity as measured by 6-week voluntary wheel exercise. Previously,
another group tested the same TG mice for exercise capacity as assessed by time-to-
exhaustion, and they reported that muscle-specific TRB3 overexpression improved
exercise capacity, along with containing more oxidative fibers (An et al., 2014). This
variation could have multiple explanations, including a different number of backcrossing,
food, sex, and age. Furthermore, we demonstrated that muscle-specific TRB3
overexpression sstrongly suppressed the expressions of genes responsible for glucose
uptake, such as GLUT4 and HXKII; and mitochondrial contents, including PGC1α,
COX4, Cyt c, and CS. This could explain why muscle-specific TRB3 overexpression
impaired glucose tolerance after 6-week treadmill running. Reduced HXKII expression in
skeletal muscle has been associated with impaired glucose uptake and exercise capacity
(Fueger et al., 2003; Fueger et al., 2005). Additionally, it has been demonstrated that
PGC1α-deficient mice show decreased muscle glycogen and GLUT4 expression post-
exercise (Wende et al., 2007). Furthermore, PGC1α expression activates GLUT4 gene
expression through myocyte enhance factor 2C (MEF2C) (Michael et al., 2001).
Unfortunately, it is currently unknown whether muscle-specific TRB3 overexpression
could be associated with MEF2C in regulating the expression of GLUT4 and PGC1α.
Future studies are recommended to examine the mechanism(s) by which TRB3
expression controls transcription of genes involved in exercise-induced skeletal muscle
adaptation.
127
SUMMARY
In summary, muscle-specific TRB3 overexpression decreased muscle mass while
TRB3 knockout increased muscle weight at the basal state. Muscle-specific TRB3
overexpression suppressed protein synthesis and promoted protein degradation via
regulation of the Akt/mTOR/FOXO pathway. TRB3 knockout increased the protein
synthesis rate in healthy skeletal muscle. Under 48 hr food deprivation, muscle-specific
TRB3 overexpression worsened atrophy by stimulating proteolysis systems, whereas
TRB3 knockout prevented atrophy by sustaining a higher protein synthesis rate.
Furthermore, in C2C12 myotubes, food deprivation-induced ER stress could regulate
TRB3 expression and muscle-specific ubiquitin E3 ligases. Lastly, muscle-specific TRB3
overexpression impaired exercise capacity and suppressed the expressions of genes
needed for glucose uptake and mitochondrial biogenesis, while TRB3 knockout improved
glucose tolerance independent of training status without altering the expressions of genes
responsible for glucose uptake or mitochondrial biogenesis. In conclusion, these data
suggest that TRB3 plays a critical role in regulating skeletal muscle plasticity.
128
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