Effect of TRB3 on Skeletal Muscle Mass Regulation and

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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.

iii

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

iv

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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4.4 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


5.4 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

xii

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

xiii

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

1

CHAPTER 1

INTRODUCTION

2

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

3

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

4

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

5

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

6

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.

7

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.

8

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

9

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

10

Proposed working model

11

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.

12

CHAPTER 2

LITERATURE REVIEW

13

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,

14

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

15

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

16

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.

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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

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**

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<250

250-7

50

750-1

500

1500

-2000

2000

-2500

2500

-3000

3000

-3500

3500

-4000

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00

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CSA (µm2)

Prop

ortio

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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

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WT TG WT TG WT TG WT TGPonceau S Anti-Puromycin

250150

75100

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kDa

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puro

myc

in

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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

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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

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0.5

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S473

/Akt

(A.U

.)

pAktS473

Akt

WT KO WT KO

250150

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kDa

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in *

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20

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Mus

cle

wei

ght (

mg)

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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

**

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1.0

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XO3a

S253

/FO

XO3a

(A.U

.)

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FOXO3a

WT KO WT KO

P=0.06

Atrogin-1

MuRF-1

Cathep

sin L

0.0

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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.


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

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.U.) P=0.058

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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

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pAkt

S473

/Akt

(A.U

.)pAktS473

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.)

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0.5

1.0

1.5

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/Akt

(A.U

.)

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A B C

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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

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/Akt

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.)FedFD

#

**

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1

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/Akt

(A.U

.)

**

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Fed FD Fed FD

WT KO0.0

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WT KO0.0

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gin-

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.U.) * *

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Fed FD Fed FD

WT KO0.0

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.)

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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

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25

1 20

50

100

150

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au (A

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(A.U

.)

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AB

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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.

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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).

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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


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).

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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.

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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


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


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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