The Pennsylvania State University
The Graduate School
College of Medicine
THE REGULATION OF MTORC1 SIGNALING IN IMMOBILIZED RAT
HINDLIMB SKELETAL MUSCLE
A Dissertation in
Physiology
by
Andrew R. Kelleher
© 2014 Andrew R. Kelleher
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
August 2014
The dissertation of Andrew R. Kelleher was reviewed and approved* by the following:
Leonard S. Jefferson, Jr. Evan Pugh Professor of Physiology Dissertation Adviser Chair of Committee
Scot R. Kimball Professor of Cellular and Molecular Physiology
Christopher R. Yengo Associate Professor of Cellular and Molecular Physiology
Lisa Shantz Associate Professor of Cellular and Molecular Physiology
Ian A. Simpson Professor of Neural and Behavioral Sciences
Donald Gill Professor and Chair of Cellular and Molecular Physiology
*Signatures are on file in the Graduate School.
! ii! ABSTRACT Limb immobilization, limb suspension, and bed rest cause substantial loss of skeletal muscle mass, a phenomenon termed disuse atrophy. Disuse atrophy is attributed to a depression in the rates of protein synthesis in a fasted state, and a resistance to stimulation by nutrients and other anabolic stimuli. Skeletal muscle protein synthesis is modulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling, which is repressed during hindlimb immobilization. The overall goal of this research was to understand the molecular mechanisms responsible for repression of mTORC1 signaling in immobilized rat hindlimb soleus muscle. The overall hypothesis was that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction in the mRNA expression of two repressors of mTORC1 signaling, regulated in development and DNA damage responses (REDD) 1 and REDD2. The studies show that REDD1 and REDD2 mRNA expression are induced in association with repression of mTORC1 signaling after 1-3 days of hindlimb immobilization. Hindlimb immobilization repressed mTORC1 signaling in a fasted state and blunted the stimulation of mTORC1 signaling in response to a bolus of leucine, a potent nutrient stimulator of mTORC1 signaling. Fixed muscle length was identified as a physiological trigger for the expression of genes associated with disuse atrophy, particularly REDD1 and REDD2. These genes were induced in soleus muscle immobilized for 3 days in a shortened position, but not in a stretched position. Aging was also associated with repression of mTORC1 signaling and induction in the mRNA expression of REDD2, while 7 days of remobilization was associated with augmented mTORC1 signaling and repression in the mRNA expression of REDD2. Collectively, these findings indicate that the mRNA expression of REDD1, and primarily REDD2 at time points longer than 3 days, is associated with changes in mTORC1 signaling under conditions of hindlimb immobilization, aging, and remobilization. REDD1 and REDD2 act as repressors and governors of the capacity for mTORC1 signaling in fasted and fed states. Furthermore, impaired PDK1 signaling to 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) and induction of REDD1 and REDD2 are associated with resistance to anabolic stimulation of mTORC1 signaling. The results support the overall hypothesis that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction of REDD1 and REDD2 mRNA expression.
! iii! TABLE OF CONTENTS
List of Tables ...... vi List of Figures ...... vii List of Abbreviations ...... ix Acknowledgements ...... x
Chapter 1. INTRODUCTION ...... 1 Disuse Atrophy ...... 1 Protein Turnover ...... 3 mTORC1 Signaling ...... 6 REDD1 and REDD2 ...... 9 Disuse and mTORC1 Signaling ...... 11 Fixed Muscle Length ...... 13 Aging and Remobilization ...... 14 Figures ...... 17
Chapter 2. MATERIALS AND METHODS ...... 20 Animals ...... 20 Hindlimb Immobilization ...... 20 Administration of Puromycin and Sample Collection ...... 21 SDS-PAGE and Immunoblot Procedure ...... 23 Measurement of p70S6K1 and 4E-BP1 Hyperphosphorylation ...... 25 Measurement of Skeletal Muscle Protein Synthesis ...... 25 Measurement of mRNA Expression ...... 26 Statistical Analysis ...... 27
Chapter 3. THE REPRESSORS OF MTORC1 SIGNALING REDD1/2 ARE RAPIDLY INDUCED AND ACTIVATION OF P70S6K1 BY A NUTRIENT STIMULUS IS DEFECTIVE IN SKELETAL MUSCLE OF AN IMMOBILIZED RAT HINDLIMB ..29 Introduction ...... 29 Methods ...... 30 Results ...... 31 Discussion ...... 36 Figures and Tables ...... 40
Chapter 4. CHANGES IN REDD1, REDD2, AND ATROGENE MRNA EXPRESSION ARE PREVENTED IN SKELETAL MUSCLE FIXED IN A STRETCHED POSITION DURING HINDLIMB IMMOBILIZATION ...... 51 Introduction ...... 51 Methods ...... 51 Results ...... 52 Discussion ...... 55 Figures ...... 59
! iv!
Chapter 5. AGE-RELATED RESPONSES OF MTORC1 SIGNALING IN RAT HINDLIMB SKELETAL MUSCLE TO IMMOBILIZATION AND REMOBILIZATION ...... 64 Introduction ...... 64 Methods ...... 65 Results ...... 66 Discussion ...... 72 Figures ...... 78
Chapter 6. CONCLUSIONS AND FUTURE DIRECTIONS ...... 87 mTORC1 Signaling and the Genesis of Disuse Atrophy ...... 88 Anabolic Resistance ...... 91 Limitations ...... 98 Muscle Length and Disuse Atrophy ...... 103 Conclusion ...... 106 Figure ...... 108
REFERENCES ...... 109
! v! TABLES
Table 3.1 Protein concentration of soleus muscle from immobilized and control hindlimbs after 1-3 days of hindlimb immobilization………………………44
Table 3.2 End points not associated with immobilization-induced repression of mTORC1 signaling………………………………………………………….47
! vi! FIGURES
Figure 1.1. Our current understanding of the genesis of disuse atrophy of skeletal muscle ...... 17
Figure 1.2. mTORC1 signaling modulates the rates of protein synthesis in response to stimulation by growth factors and nutrients ...... 18
Figure 1.3. Growth factors and nutrients are both necessary for full activation of mTORC1 ...... 19
Figure 3.1. Time course changes in muscle weight following immobilization ...... 43
Figure 3.2. Effect of immobilization on rates of protein synthesis in soleus muscle .....45
Figure 3.3. Effect of immobilization on p70S6K1 hyperphosphorylation and 4E-BP1 phosphorylation in the soleus muscle ...... 46
Figure 3.4. Effect of immobilization on site-specific p70S6K1 phosphorylation in the soleus muscle ...... 48
Figure 3.5. Effect of immobilization on REDD mRNA expression in soleus muscle ....49
Figure 3.6. Regulation of mTORC1 signaling under conditions of 1-3 days of hindlimb immobilization ...... 50
Figure 4.1. Mass and rates of protein synthesis in the soleus are reduced only when the muscle is immobilized in a shortened position ...... 59
Figure 4.2. Phosphorylation of p70S6K1 at Thr389 in the soleus is reduced only when the muscle is immobilized in a shortened position ...... 60
Figure 4.3. REDD1 and REDD2 mRNA expression in the soleus is induced only when the muscle is immobilized in a shortened position ...... 61
Figure 4.4. Atrogene expression is induced in the soleus only when the muscle is immobilized in a shortened position ...... 62
Figure 4.5. Phosphorylation of Akt at Ser473 and FoxO3a at Ser253 are reduced only when the soleus muscle is immobilized in a shortened position ...... 63
Figure 5.1. Young rats are more susceptible to hindlimb immobilization-induced loss of muscle mass than older rats ...... 78
! vii! Figure 5.2. Nutrient-induced activation of mTORC1 signaling declines dramatically during aging and in older rats the pathway is not responsive to immobilization, but is augmented following remobilization ...... 79
Figure 5.3. Feeding-induced activation of Akt is blunted with age and hindlimb immobilization in skeletal muscle from young vs. older rats, and is augmented following remobilization ...... 80
Figure 5.4. REDD2, but not REDD1, mRNA expression increases with age and becomes unresponsive to immobilization, but is repressed following remobilization ...... 81
Figure 5.5. REDD2 mRNA expression is inversely proportional to phosphorylation of p70S6K1 at Thr389 in rat soleus muscle ...... 82
Figure 5.6. Nutrient-induced activation of PDK1 signaling declines during 7 days of hindlimb immobilization and aging, but in older rats the pathway is not responsive to immobilization ...... 83
Figure 5.7. MAFbx mRNA increases with age and 7 days of immobilization, while MuRF1 increases with immobilization only in 2-month old animals and decreases in response to remobilization ...... 84
Figure 5.8. Gastrocnemius muscle RNA content decreases with aging ...... 85
Figure 5.9. Regulation of mTORC1 signaling under conditions of aging and 7 days of hindlimb immobilization ...... 86
Figure 6.1. Phosphorylation of p70S6K1 at Thr389 was not attenuated despite reduced muscle mass-to-body mass ratio in soleus muscle from the immobilized hindlimb of REDD1 knockout ...... 108
! viii! ABBREVIATIONS
4E-BP1, eukaryotic initiation factor 4E-binding protein 1 AMPK, AMP-activated protein kinase ATF4, activating transcription factor 4 Deptor, DEP domain containing mTOR-interacting protein eIF, eukaryotic initiation factor eIF2α, alpha subunit of eukaryotic initiation factor 2 ER, endoplasmic reticulum ERK, extracellular signal-regulated kinase FoxO, forkhead box O Grb10, growth factor receptor-bound protein 10 GAP, GTPase activating protein HIF1-α, hypoxia-inducing factor 1 alpha subunit IGF-1, insulin-like growth factor LKB1, liver kinase B1 MAFbx, Muscle Atrophy F-box MEFs, murine embryonic fibroblasts mTORC1, mechanistic target of rapamycin complex 1 MuRF1, Muscle Ring Finger 1 p70S6K1, 70 kilodalton ribosomal protein S6 kinase 1 PDCD4, programmed cell death 4 PI(3)K, phosphatidylinositol-3-OH kinase PP2A, protein phosphatase 2A Rag, Ras-related GTP binding REDD, regulated in development and DNA damage responses Rheb, Ras-homology enriched in brain SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis TBC1D7, Tre2-Bub2-Cdc16-1 domain family member 7 Tbp, TATA-binding protein TBS-T, Tris-buffered saline + Tween 20 TSC, tuberous sclerosis complex
! ix! ACKNOWLEDGMENTS
I would like to thank my mentors, Dr. Leonard Jefferson and Dr. Scot Kimball, for their guidance, patience, support, generosity, and trust throughout my doctoral training. I would also like to thank my doctoral committee members, Dr. Christopher Yengo, Dr. Lisa Shantz and Dr. Ian Simpson, for their guidance, respect and confidence in my abilities to complete this doctoral dissertation. A special thank you to all members of the Jefferson Laboratory for their time, effort, technical expertise, positive attitude and teamwork during all experiments over the last five years of research. Specifically, I would like to thank Sharon Rannels, Holly Lacko, Dr. Bradley Gordon, Dr. Alex Tuckow, Dr. Michael Dennis, Dr. Ruud Schilder, Lydia Kutzler, Emily Sun, Lauren Luongo, Tony Martin, Chen Yang, Joel Coble, Suhana Ravi and Abid Kazi. In addition, I acknowledge the National Institutes of Health (NIH DK-15658, L.S.J), the Pennsylvania Department of Health (Tobacco Settlement Funds), and the Abbott Nutrition Company (S.R.K.) for their financial support of my doctoral dissertation experiments. I recognize that my accomplishments are products of the love and generous support of friends and family. I would be remiss if I did not thank my wonderful friends for their friendship and sharing in so many joyous occasions. I would especially like to thank friends like Darshan Trivedi, Rachel Drew, Jimmy McGhee, Andrew Winslow, Julian Joseph, Clayton Belcher, Matt and Annie Muller, Mariano Russo, Dan and Lynley Lapp, and Jacob and Katy Serfass. A special thanks to my former biology and exercise physiology mentors who inspired me to pursue a scientific career including Dr. Mary Constant Byrne and Dr. Paul Meier from Muhlenberg College, and Dr. Stefan Keslacy and Dr. Lori Ploutz-Snyder from Syracuse University. Thank you to my loving family, especially Jason, Colin, Eddie, Ave, Mott, and Dr. Stephen and Polly Newsholme. A special thank you to my parents, Cheryl and Kevin, for all their love, faith, support and confidence in me. Sadly, my father passed away while I was writing this doctoral dissertation. Finally, I would like to dedicate this dissertation and all Ph.D. work to my loving wife and mother-to-be, Rosalind, who is my source of inspiration and strength.
! x! Chapter 1. INTRODUCTION
Disuse Atrophy
Skeletal muscle is a remarkably plastic tissue that is essential for health and survival. Comprising 40% of body weight, it is important for locomotion, energy consumption, fuel for other tissues and amino acid storage (51). Plasticity allows skeletal muscle to adapt its size as well as metabolic, structural and contractile properties in response to altered activity (24, 34, 60). Increased mechanical loading and workload lead to gains in skeletal muscle mass, (also known as hypertrophy). In contrast, physical inactivity, mechanical unloading, hypodynamia and hypokinesia lead to losses in skeletal muscle mass (also known as atrophy). Disuse is an expansive label for low mechanical load or unloading of muscle with physical inactivity (24). This thesis investigates some of the molecular mechanisms associated with skeletal muscle atrophy in response to disuse.
The loss of skeletal muscle mass due to disuse (e.g. limb immobilization, limb unloading/suspension and bed rest) that occurs in the absence of local or systemic inflammation is a phenomenon known as ‘disuse atrophy’ (143, 162). Disuse atrophy is a major healthcare problem that can lead to physical frailty, loss of independent living, prolonged periods of rehabilitation, increased risks of falls and fractures and reduced overall prognosis (139, 143, 200). Exercise and physical training, nutritional aids, growth factors, ergogenic supplements and drugs have been implemented in an attempt to prevent or reverse disuse atrophy (for review, see (34). Unfortunately, these approaches have not led to effective therapeutic interventions that prevent disuse atrophy in humans.
The development of such therapeutic interventions will require an understanding of the
1 molecular mechanisms responsible for disuse atrophy. To this end, the objective of my thesis research is to understand the molecular mechanism(s) responsible for disuse atrophy.
Rodent models of disuse atrophy are often used in studies aimed at understanding the molecular mechanisms that regulate skeletal muscle mass. Immobilization of a limb
(also known as limb immobilization) in humans is equivalent to hindlimb immobilization in rodents (82). Hindlimb immobilization is a procedure that involves fixation of an animal’s ankle joint (123) and sometimes knee joint (62) with a cast or material that prevents joint mobility. The prevention of joint mobility reduces mechanical loading on skeletal muscles in an immobilized hindlimb and leads to disuse atrophy, particularly in muscles fixed in a shortened position (21). Unloading of a limb (also known as limb unloading or limb suspension) in humans is used to simulate the effects of extended periods of microgravity and spaceflight on human tissues (85). An equivalent model in rodents is hindlimb unloading; a procedure that involves the fixation of a rodent’s tail to a cage ceiling in order to lift the rodent’s hindlimbs off the cage floor for extended periods
(154). This procedure reduces mechanical loading on the anti-gravity muscles of rodent hindlimbs, which leads to disuse atrophy. Finally, denervation is used to reduce muscle contraction muscles of the lower hindlimbs of rodents by damaging or removing a portion of the sciatic nerve (76). Denervation prevents neuromuscular communication between the spine and hindlimb muscles. Consequently, disuse atrophy results from a lack of muscular activation and loading (178).
Compared to humans, rodent models exhibit disuse atrophy quite rapidly. For example, 2 weeks of limb immobilization reduced vastus lateralis muscle fiber diameter
2 (cross-sectional area or CSA) 5-20% in human subjects (72, 201). Similarly, 2 weeks of bed rest and limb unloading reduced human leg lean mass 4% and vastus lateralis muscle
CSA 5%, respectively (2, 50, 61). In contrast, rat soleus muscle mass was reduced 56% in the immobilized hindlimb after 10 days of hindlimb immobilization (220). Moreover,
2 weeks of hindlimb unloading caused a 37-60% reduction in rat soleus muscle CSA (44,
66). Differences in the extent of disuse atrophy between humans and rodents may be explained by the high rates of metabolism and protein turnover in rodents (162). As a benefit, the molecular mechanisms that cause disuse atrophy may be easier to identify in rapid-atrophying rodents because the processes responsible for atrophy are likely to be amplified. Other benefits of using rodent models to study disuse atrophy include more control over experimental characteristics, short-lifespan, ease of sample collection, and manipulation of gene expression (1). With this in mind, I characterized a rodent model of unilateral hindlimb immobilization and used this model to investigate the molecular mechanisms responsible for disuse atrophy.
Protein Turnover
Disuse atrophy is the result of an imbalance in skeletal muscle protein turnover.
The turnover of skeletal muscle proteins involves the ongoing processes of protein synthesis and protein degradation (51). Skeletal muscle atrophy occurs under conditions wherein the rates of protein degradation exceed the rates of protein synthesis resulting in a negative net protein balance. This can result from either a reduction in the rates of protein synthesis, a rise in the rates of protein degradation, or a combination of both processes (80). In human skeletal muscle, the rates of protein synthesis are depressed
3 during limb immobilization (69, 70), unloading (40) and bed rest (191). This depression in the rates of protein synthesis is observed in both fasted (post-absorptive) and fed
(postprandial) states (50, 72, 127, 162). In the fed state, the depression in protein synthesis is attributed to a resistance to stimulation by nutrients and other anabolic stimuli termed ‘anabolic resistance’ (169-172). On average, the rates of protein synthesis in fasted and fed states are depressed by ~60% and ~50%, respectively, resulting in a rate of loss of protein equal to ~0.5%/day (162). This calculated rate of loss is in agreement with the observed loss of muscle mass. In addition, biomarkers such as 13C-labelled amino acid appearance and 3-methylhistidine excretion measured in human blood and urine before and after bed rest (16, 61) and spaceflight (197, 198) indicate that the rates of protein degradation do not increase in response to disuse. Therefore, it would seem that the depressions in rates of protein synthesis in the fasted and fed states are primarily responsible for the observed disuse atrophy in humans.
In rodent skeletal muscle, disuse leads to depressed rates of protein synthesis in addition to augmented rates of protein degradation (74-76, 121). The observed changes in rodent skeletal muscle protein turnover occur rapidly with disuse whereby 6 hours of hindlimb immobilization depressed the rates of protein synthesis (22). Similar to humans, rates of protein synthesis are depressed ~50% in rat soleus muscle during hindlimb immobilization (75). Moreover, hindlimb immobilization causes resistance to nutrient-induced stimulation of protein synthesis in rodent gastrocnemius muscle (127).
If rodents and humans exhibit the same manifestations of depressed protein synthesis in response to disuse, then rodent models are suitable for studying human-relevant mechanisms of disuse atrophy. Since disuse atrophy in humans is the result of
4 depressions in skeletal muscle protein synthesis and not increases in skeletal muscle protein degradation, my thesis research is focused on understanding the molecular mechanisms responsible for depressions in rates of protein synthesis.
Unlike humans, the rates of protein degradation increase in rat soleus muscle during hindlimb immobilization (74, 75), hindlimb suspension (137) and denervation (74,
76). Increases in the rates of protein degradation during disuse in rodents have been attributed to increases in skeletal muscle inflammatory signaling (12, 13), metabolic disturbances and mitochondrial dysfunction (48, 164, 165), muscle remodeling, and activation of apoptosis (144, 165). The rates of protein degradation are modulated in part through the ATP-dependent ubiquitin proteasomal pathway (for review see (182)). In response to disuse, the proteasome-mediated degradation of skeletal muscle protein is regulated by the expression of ‘atrogenes’; two muscle-specific E3 ubiquitin ligases known as MAFbx/atrogin-1 (Muscle Atrophy F-box) and MuRF1 (Muscle Ring Finger 1)
(18). A critical role for these two ligases in disuse atrophy was demonstrated in atrogene knockout animals that exhibited resistance to denervation-induced atrophy in gastrocnemius muscle (18). Hindlimb immobilization and unloading induce both
MAFbx and MuRF1 gene expression in skeletal muscle (11, 189). However, the role of atrogenes and other mediators of protein degradation in human disuse atrophy are unclear, because the rates of protein degradation do not increase during disuse (143, 162,
171). While the precise role of atrogenes in disuse atrophy is unclear, they can be used to assess gene expression patterns associated with disuse atrophy.
5 mTORC1 Signaling
The rates of protein synthesis in skeletal muscle are modulated, in part, by the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. Nutrients, growth factors such as insulin and insulin-like growth factor (IGF-1), and exercise activate mTORC1 signaling, while hypoxia, stress, DNA damage and low energy levels repress it (118, 129). Upon activation, mTORC1 phosphorylates protein substrates at specific residues including p70 ribosomal protein S6 kinase 1 (p70S6K1) at Thr389, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) at Ser65, and uncoordinated-
51 like autophagy activating kinase 1 (ULK1) at Ser757 (8, 27, 79, 114, 146). When phosphorylated, these substrates promote cap-dependent mRNA translation and repress autophagy (114, 118). Phosphorylation of 4E-BP1 prevents its association with eukaryotic initiation factor (eIF) 4E and thereby permits eIF4E to associate with eIF4G and eIF4A in the formation of the active mRNA cap-binding complex, eIF4F (118). Cap- dependent mRNA translation is also bolstered by phosphorylation of p70S6K1, which subsequently phosphorylates two proteins that participate in cap-dependent translation initiation and another that participates in translation elongation. Activated p70S6K1 phosphorylates programmed cell death 4 (PDCD4), which interacts with eIF4A and prevents its association with eIF4G (218). Once phosphorylated, PDCD4 is released from eIF4A, allowing eIF4A to interact with eIF4G (80). Activated p70S6K1 also phosphorylates eIF4B, which enhances the RNA helicase activity of eIF4A (176).
Finally, activated p70S6K1 phosphorylates and inactivates eukaryotic elongation factor
(eEF) 2 kinase, which reduces phosphorylation of eEF2 and allows eEF2 to participate in mRNA translation elongation (212). Measuring the phosphorylation state of mTORC1
6 substrates provides a readout of mTORC1 signaling and the activation state of critical participants in protein synthesis.
Nutrients, particularly amino acids such as leucine, are necessary but not sufficient for full activation of mTORC1 (88, 129, 149). Nutrients induce the movement of mTORC1 to lysosomal membranes where Ras-related GTP binding (Rag) proteins are located (179, 180). On the lysosomal surface, Rag proteins interact with a protein complex called ‘Ragulator’ that is essential for activation of mTORC1 in response to amino acids (179). Rag proteins form a GTPase heteroduplex consisting of either RagA or RagB in association with either RagC or RagD (115, 180). When RagA or RagB is bound to GTP and RagC or RagD is bound to GDP, the heterodimer promotes mTORC1 signaling. In contrast, when RagA or RagB is bound to GDP and RagC or RagD is bound to GTP, the heterodimer does not promote mTORC1 signaling. Localizing mTORC1 to the lysosome brings it into close proximity with a small GTPase called ras homolog enriched in brain (Rheb) (129). Rheb activates mTORC1 when Rheb is associated with GTP, while Rheb•GDP does not (136). Thus, the activation of mTORC1 requires nutrients to signal through the Rag and Ragulator proteins and localizes mTORC1 to the lysosome where it can interact with Rheb•GTP (149).
Rheb GTP/GDP-loading state is regulated by growth factors such as insulin and
IGF-1 (46, 149). Growth factors and exercise promote mTORC1 signaling through activation of Akt and extracellular signal-regulated kinase (ERK) signaling pathways, respectively. These lead to phosphorylation and repression of tuberous sclerosis complex
(TSC) function (102, 138, 142). The TSC complex is composed of the TSC tumor suppressors, TSC1 and TSC2, and Tre2-Bub2-Cdc16-1 domain family member 7
7 (TBC1D7) (46). TSC2 is a GTPase activating protein (GAP) for Rheb, which promotes
GTP hydrolysis by Rheb, while TSC1 and TBC1D7 are required for the stability and full
GAP activity of TSC2 towards Rheb (101, 149). The TSC complex negatively regulates mTORC1 signaling through its Rheb-GAP activity whereby mTORC1-activating
Rheb•GTP gets converted into its inactive Rheb•GDP state (129). Like nutrients, growth factors are also necessary but not sufficient for full activation of mTORC1 (88, 129, 149).
Perhaps the interdependence of nutrient and growth factor signaling for activation of mTORC1 can be explained by mTORC1 localization and Rheb GTP/GDP-loading state.
According to a recent unifying model for integrated coregulation of mTORC1 by nutrients and growth factors, nutrients localize mTORC1 to the lysosome and bring it into close proximity with its essential activator Rheb. At the same time, growth factor signaling represses TSC complex function to promote Rheb-dependent activation of mTORC1 (149).
Many repressors of mTORC1 signaling act on the TSC complex (80). Hypoxia, stress, DNA damage and low energy increase TSC2 GAP activity towards Rheb by phosphorylation on alternative residues from Akt and ERK that stabilize the TSC complex (45, 103, 196). Energy stress activates Liver kinase B1 (LKB1), which leads to the phosphorylation and activation of AMP-activated protein kinase (AMPK) (193).
Activated AMPK phosphorylates TSC2 on distinct residues that promote TSC2 GAP activity toward Rheb and repression of mTORC1 signaling (20, 192). Genotoxic and oxidative stress repress mTORC1 signaling through the p53-dependent genes, Sestrin 1 and Sestrin2, which activate AMPK (26). Moreover, stress to the endoplasmic reticulum
(ER stress) is known to induce activating transcription factor 4 (ATF4) and increase the
8 expression of Sestrin 2 and another known repressor of mTORC1 signaling, REDD1
(regulated in development and DNA damage responses) (25).
REDD1 and REDD2
Although the molecular mechanisms are still unclear, REDD1 and REDD2 repress mTORC1 signaling through the TSC complex (152, 196). REDD1 and REDD2 share only 34% DNA and 61% amino acid sequence homology, but seem to repress mTORC1 signaling by a similar mechanism upstream of Rheb that requires TSC2 (152).
One mechanism that has been accepted by the field involves the release of TSC2 from growth factor-induced association with inhibitory 14-3-3 proteins (45, 152). According to a model proposed by DeYoung et al (45), insulin and growth factors lead to Akt- mediated phosphorylation of TSC2. This promotes TSC2/14-3-3 association and thereby inhibits TSC function. In the presence of REDD1 and REDD2, 14-3-3 proteins dissociate from TSC2 and bind REDD proteins (45, 152). Knockdown of REDD1 expression in muscle cells leads to disruption of the TSC complex (211). Stabilization of
TSC2 in the TSC complex is believed to promote GTP hydrolysis by Rheb, leading to repression of mTORC1 signaling through its Rheb-GAP activity whereby mTORC1- activating Rheb•GTP gets converted into its inactive Rheb•GDP state (129). In contrast, structural-based docking and functional analyses suggest that REDD1 does not directly bind to 14-3-3 proteins (209). Some recent evidence from our lab suggests that REDD1 may act as a targeting protein for the catalytic subunit of protein phosphatase 2 (PP2A).
According to this model, REDD1 recruits PP2A to dephosphorylate Akt at Thr308, which reduces phosphorylation of TSC2, Rheb-GTP loading and mTORC1 signaling (42).
9 Since REDD1 and REDD2 share a conserved mTORC1-inhibitory motif, it is assumed that they repress mTORC1 signaling by a similar mechanism with few exceptions (150).
For example, differences in the N-terminus of REDD1 allow it, and not REDD2, to be sequestered to the plasma membrane where it does not repress mTORC1 signaling in response to G-protein-coupled receptor signaling. Also, REDD2 contains a distinct loop region that allows for REDD2 to interact with interferon-regulatory factor-1, which may allow for cross-talk between mTORC1 signaling and cytokine pathways during the regulation of cell growth (84).
REDD1 and REDD2 expression is induced under conditions of stress. In animal models, REDD1 is induced during obesity (217), starvation (145), and conditions of hypercortisolemia including stroke, type 2 diabetes, and stress induced by confinement
(125). In vitro, REDD1 is induced in response to DNA damage (58), oxidative stress, energy stress (196), hypoxia (45), hypertonicity (160) glucocorticoids (211) and ER stress (214). The mechanism in which REDD1 is induced during ER stress is well- characterized. ER stress activates PKR-like ER kinase (PERK), which phosphorylates eIF2alpha and leads to an increases the mRNA and protein expression of a REDD1 transcription factor, ATF4 (119, 214). ATF4 induces REDD1 expression in response to both ER stress and growth factor deprivation (i.e. serum starvation) (43, 214). The induction in REDD1 expression seems to be regulated at the level of mRNA transcription
(i.e. serum starvation-induced ER stress caused an increase in the transcription rate of
REDD1 mRNA in Rat 2 fibroblasts) (43). REDD2 is induced in response to hypoxia
(152, 168), hypertonicity (160), artherosclerotic lesions (100) and hindlimb unloading
(163), and it is currently unknown if ER stress, ATF4 or growth factor deprivation are
10 also inducers. Growth factor signaling may play a role in the repression of both REDD1 and REDD2 mRNA expression. REDD1 mRNA and protein expression was induced in rat gastrocnemius muscle under conditions of low serum insulin (e.g. starvation and diabetes) and reduced after refeeding (145). IGF-1 treatment reduced REDD2 mRNA expression in myotubes and rat gastrocnemius muscle following IGF-1 treatment (63).
Thus, a relationship exists between reduced or impaired growth factor signaling and induction of REDD1 and/or REDD2 mRNA expression. However, the precise mechanisms responsible for regulation of REDD1 and REDD2 mRNA transcription under such conditions is currently unknown.
Disuse and mTORC1 Signaling
Depressed rates of protein synthesis during disuse are associated with attenuated mTORC1 signaling. Ten days of hindlimb immobilization reduced p70S6K1 phosphorylation in soleus muscle from immobilized hindlimbs of adult rats (220).
Similarly, 7 days of hindlimb immobilization attenuated mTORC1 signaling in gastrocnemius muscle from immobilized hindlimbs of young mice (127) and rats (126).
Attenuated mTORC1 signaling has also been observed in other models of disuse.
Compared to control conditions, phosphorylation of p70S6K1 was reduced in rat soleus muscle after just 12 hours of hindlimb unloading (10, 94). Moreover, reduced p70S6K1 phosphorylation has consistently been observed in rat soleus, gastrocnemius, and quadriceps muscles after 1 and up to 21 days of hindlimb unloading (10, 86, 94, 135).
An important role for mTORC1 signaling in the regulation of skeletal muscle mass during disuse is further supported by gene knockdown studies in mice. Genetic deletion
11 of the mTORC1 obligatory component, raptor, inactivated mTORC1, prevented skeletal muscle growth and enhanced atrophy (15). In the same study, deletion of the mTORC1 suppressor, TSC1, induced skeletal muscle hypertrophy and made mouse soleus muscle resistant to denervation-induced atrophy. Furthermore, gene silencing of another mTORC1 inhibitory protein, DEP domain containing mTOR-interacting protein (Deptor), rescued rates of protein synthesis and preserved skeletal muscle mass in gastrocnemius muscle during hindlimb immobilization (109). Despite strong evidence implicating attenuated mTORC1 signaling in depressed rates of protein synthesis and disuse atrophy, the molecular mechanism responsible for repression of mTORC1 signaling during disuse is unknown. Moreover, the majority of these studies have assessed mTORC1 signaling in a fasted condition and not in a fed condition. As a result, the role of mTORC1 signaling in anabolic resistance to nutrient stimulation is also unknown. Hindlimb unloading has been found to induce skeletal muscle p53 (194) and REDD2 (163) expression. However, no study has characterized the expression patterns of these repressors with mTORC1 signaling during disuse atrophy. Thus, the first goal of my research was to understand the molecular mechanisms responsible for repression of mTORC1 signaling in skeletal muscle during hindlimb immobilization.
Some evidence suggests that ATF4 and impaired growth factor signaling may be responsible for disuse atrophy. A prototypical ER stress response was not exhibited in rat soleus muscle following 7 days of hindlimb unloading (97). However, this does not preclude ATF4 from playing a role in disuse atrophy. Interestingly, mice that had the ER stress-induced protein ATF4 knocked out were resistant to muscle atrophy after 24 hours of starvation and 3 days of hindlimb immobilization (56). Moreover, silencing of growth
12 arrest and DNA damage-inducible 45a protein, a gene induced by ATF4, protected mouse tibialis anterior muscle from atrophy induced by 1 day of fasting, and 7 days of denervation and hindlimb immobilization. Physical inactivity and disuse are also associated with insulin resistance and impaired growth factor signaling through the
Akt/TSC pathway (159, 187). Impaired growth factor (i.e. insulin) signaling has been attributed to the attenuated phosphorylation of Akt at Ser473 observed in muscles during hindlimb immobilization and unloading (54, 87, 159, 220). As introduced in 'REDD1 and REDD2', ATF4 and reduced or impaired growth factor signaling are associated with induction of REDD1 and REDD2. This has led me to speculate that REDD1 and REDD2 are induced in skeletal muscle during disuse due to induction of ATF4 and impaired growth factor signaling.
Fixed Muscle Length
Fixed muscle length during hindlimb immobilization plays a key role in the regulation of skeletal muscle mass and protein synthesis. Prior investigations by two separate laboratories show differences in the amount of disuse atrophy when hindlimb muscles were immobilized in either a shortened or stretched position (21, 75). Disuse atrophy of the soleus muscle was observed when a rat ankle joint was immobilized for 7 days in full plantarflexion (soleus muscle placed in a shortened position). In contrast, no disuse atrophy of the soleus muscle was observed when a rat ankle joint was immobilized for 7 days in full dorsiflexion (soleus muscle placed in a stretched position). In association with the observed patterns for muscle mass, rates of protein synthesis were depressed in the soleus muscle immobilized in plantarflexion, but not in soleus
13 immobilized in dorsiflexion (75). The same phenomenon has been observed in other disuse models including hindlimb unloading and denervation (76, 137). However, the molecular mechanism by which fixed muscle length regulates skeletal muscle mass and protein synthesis is unknown.
Depressed rates of protein synthesis in skeletal muscle fixed in a shortened position may be the result of repressed mTORC1 signaling. Upon close analyses of the studies that have assessed mTORC1 signaling during limb immobilization, there is an association between repression of mTORC1 signaling and fixed limb position that is under-recognized. The evidence that indicates mTORC1 signaling is attenuated during disuse comes from studies where skeletal muscle is placed in a shortened position (94,
126, 127, 220). Interestingly, those studies that have not observed attenuated mTORC1 signaling used immobilization techniques where the muscle of interest was fixed in a neutral (not stretched or shortened) position (40, 72). No study has measured mTORC1 signaling in skeletal muscle maintained in a stretched position during disuse. If skeletal muscle protein synthesis is not depressed when muscles are fixed in stretched positions during disuse, and mTORC1 signaling modulates the rates of protein synthesis, then mTORC1 signaling is not likely to be attenuated in these muscles. Understanding how fixed muscle length influences mTORC1 signaling during disuse will elucidate the mechanism responsible for disuse atrophy during hindlimb immobilization. Therefore, the second goal of my research was to understand the role of fixed muscle length during hindlimb immobilization in the regulation of mTORC1 signaling.
14 Aging and Remobilization
Aging has a profound influence on skeletal muscle mass. In humans and rodents, skeletal muscle hypertrophies during the juvenile and adolescent stages prior to adulthood, and atrophies during old age. In the fasted state, initial studies observed higher rates of protein synthesis in skeletal muscle from young adults compared to elderly (213, 219). However, more recent studies have not observed differences in the fasted rates of protein synthesis between young and old adults (64, 65, 167, 206). Rather, the negative protein balance that causes age-induced skeletal muscle atrophy likely results from anabolic resistance to exercise (55), amino acid infusion (83) and feeding
(38, 39, 174). Such studies have not observed differences in mTORC1 signaling between young and old adult humans (38, 83) or rats (166) in either fasted or fed states. However, some have observed blunted nutrient-induced stimulation of mTORC1 signaling in muscles from old subjects (64, 83, 98). Unlike juveniles and adolescents, the muscles of young adult subjects are not actively growing. As a result, none of these studies have compared the nutrient-induced stimulation of mTORC1 signaling between skeletal muscles from actively growing young subjects and non-growing older subjects. Since disuse- and age-induced skeletal muscle atrophy are linked in the blunted stimulation of protein synthesis and mTORC1 signaling, I speculate that disuse and aging share a common mechanism responsible for regulating mTORC1 signaling.
Remobilization following hindlimb immobilization rescues the muscle mass lost during disuse in young (23, 33), but not old (140, 155) subjects. Recovery of lost muscle mass during remobilization involves multiple stress signaling pathways and various transcriptional and hypertrophic mechanisms (33, 82, 139). Among these mechanisms,
15 remobilization increases the rates of protein synthesis and activates mTORC1 signaling in skeletal muscle (98). However, the mechanism responsible for remobilization-induced activation of mTORC1 signaling is unknown. It is important that this mechanism be understood because it will help us to understand the regulation of skeletal muscle mass in response to changes in mechanical loading and physical activity. I speculate that the same mechanism responsible for repression of mTORC1 signaling during hindlimb immobilization is involved (only in a reversed manner) in the activation of mTORC1 signaling during remobilization. Therefore, the third goal of my research was to understand and compare the mechanisms responsible for modulation of mTORC1 signaling with aging, hindlimb immobilization and remobilization.
The overall objective of my dissertation research project was to understand the molecular mechanism(s) responsible for the repression of mTORC1 signaling in immobilized rat hindlimb skeletal muscle. I hypothesized that the molecular mechanism(s) responsible for the repression of mTORC1 signaling with hindlimb immobilization are also responsible for the modulation of mTORC1 signaling with changes in fixed muscle length, aging, and remobilization.
16
17 Figure 1.2. mTORC1 signaling modulates the rates of protein synthesis in response to stimulation by growth factors and nutrients. Growth factors activate PI(3)K, which in turn leads to the activa- tion of both PDK1 and Akt. Once activated, PDK1 promotes Akt and p70S6K1 signaling by phos- phorylation of these proteins. Akt and activated ERK phosphorylate and inhibit TSC GAP activity towards Rheb. This reduces the conversion of Rheb-GTP into Rheb-GDP. The presence of nutri- ents stimulates the formation of RagA/B-GTP RagC/D-GDP heterodimers. These heterodimers, along with Rheb-GTP lead to the full activation of mTORC1. When activated, mTORC1 phos- phorylates p70S6K1, ULK1, and 4E-BP1. This, in turn, promotes protein synthesis through the activation of eukayotic initiation factors (eIFs) and release of eIF4E from 4E-BP1.
18 A) Growth Factor and Amino Acid Deprivation - mTORC1 OFF
mTORC1
TSC GDP GDP RagA Rheb Ragulator RagC GTP cytosol Lysosome lumen
B) Growth Factor and Amino Acid Stimulation - mTORC1 ON
Growth Factors
Nutrients
TSC p p p p p GTP GTP Rheb RagA mTORC1 Ragulator RagC GDP cytosol Lysosome lumen
Figure 1.3. Growth factors and nutrients are both necessary for full activation of mTORC1. A) In the absence of growth factors, TSC promotes the conversion of mTORC1-activating Rheb GTP into inactive Rheb GDP. In the absence of nutrients (amino acids), mTORC1 does not bind Rag proteins on the lysosomal surface. B) Stimulation by growth factors leads to the phosphorylation of TSC2 on inhibitory residues that reduce TSC2 GAP activity towards Rheb and prevent GTP hydrolysis of Rheb GTP. In the presence of nutrients, RagA/B GDP and RagC/D GTP gets con- verted into RagA/B GTP and RagC/D GDP that is capable of binding mTORC1 on the lysosomal surface. Recruitment of mTORC1 to the lysosome places it in close proximity to Rheb GTP for activation.
19 Chapter 2. Methods
Animals
Male Sprague-Dawley rats aged 2, 9, and 18 months [Charles River Laboratories,
Wilmington, MD; and Harlan Laboratories, Indianapolis, IN] were housed in wire cages in a temperature- (25°C) and light-controlled environment. Rats were provided rodent chow (Harlan-Teklad 8604, Indianapolis, IN; and AIN-93M, Research Diets, New
Brunswick, NJ) and water ad libitum. Before hindlimb immobilization, rats were adapted to a reversed 12:12-hr light-dark cycle (lights off at 0700 hrs) for one week. Animal facilities and experimental protocols were approved by the Institutional Animal Care and
Use Committee of The Pennsylvania State University College of Medicine.
Hindlimb Immobilization
Rats were anesthetized using isoflurane inhalation (2.5%) and subjected to unilateral hindlimb immobilization as described previously (123). The left hindlimb was shaved, taped (1/2” Curity Kendall Standard Porous Tape, Walpole, MA) and wrapped in four layers of protective cast padding (Specialist brand; Johnson and Johnson, Raynham,
MA). Fiberglass casting tape (VetCast Plus veterinary casting tape; 3M, St. Paul, MN) was activated by hot water, and five layers were wrapped over cast padding and allowed to harden. Multiple layers of casting tape were used to wrap around the leg and over the toes in order to construct a strong smooth cast. Except for a subset of rats that were immobilized in ankle dorsiflexion (Chapter 4), all rat hindlimbs were immobilized with the foot positioned in full plantarflexion so as to place the soleus muscle placed in a
20 shortened position. When the casting material had hardened, rats were removed from the anesthesia apparatus and allowed to recover in their cages.
Rats were anesthetized using isoflurane during cast removal. Casts were removed by making two lateral incisions using a Stryker Cast Cutter (Kalamazoo, MI). Incisions were connected across the boot of the cast and the cast halves were pulled apart by hand.
Cast padding and tape were cut using scissors and peeled off the rat leg.
Two controls were included in initial hindlimb immobilization experiments
(Chapter 3). Controls consisted of non-immobilized rats and the contralateral, non- immobilized hindlimb of rats subjected to unilateral hindlimb immobilization. Non- immobilized rats were included to determine if changes in biomechanical gait patterns or circulating stress hormones due to the hindlimb immobilization procedure influenced skeletal muscle in the non-immobilized hindlimb of rats subjected to hindlimb immobilization. As described in the Results section of Chapter 3, no differences in muscle mass-to-body mass ratio, rates of protein synthesis, mTORC1 signaling, or
REDD1 and REDD2 gene expression were observed between controls. Since hindlimb immobilization did not influence the variables of interest in skeletal muscle from the non- immobilized hindlimb, follow-up experiments (Chapters 4 and 5) did not include non- immobilized rats as additional controls.
Administration of Puromycin and Sample Collection
On the day of the experiment, rats were anesthetized using isoflurane inhalation
(2.5%). After 5 minutes of anesthesia, rats were placed on a heating pad covered with sterile absorbent pads to maintain body temperature for the remainder of the procedure.
21 A dose (0.040 µmol/g body wt) of puromycin dihydrochloride (AG Scientific, San Diego,
CA) in a solution of 10 mg/mL saline was administered via tail vein injection for the measurement of protein synthesis. Ten minutes following administration of puromycin
(30 min post-gavage), muscles were individually excised, cleared of visible fascia, weighed, and either homogenized or frozen using freeze clamps in liquid nitrogen.
Soleus muscles were homogenized in 10 volumes of homogenization buffer consisting of
20 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH 7.4), 100 mmol/L
KCl, 0.2 mmol/L EDTA, 2 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid, 1 mmol/L dithiothreitol, 50 mmol/L sodium fluoride, 50 mmol/L β- glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 0.5 mmol/L sodium vanadate on ice using a Polytron homogenizer (37). An aliquot
(0.5 mL) of the homogenate was reserved for the measurement of mRNA expression as described in ‘Measurement of mRNA Expression’ below. The remainder of the homogenate was immediately centrifuged at 2,000 x g for 3 minutes at 4°C. An aliquot
(200 µL) of the supernatant fraction was added to an equal volume of 2X Laemmli buffer consisting of 0.0625 M Tris·HCl (pH 6.8), 12.5% (vol/vol) glycerol, 1.25% sodium dodecyl sulfate, 1.25% (vol/vol) β-mercaptoethanol, and 0.1% bromophenol blue and boiled at 100°C for 5 minutes. A separate aliquot (10 µL) of supernatant was used to measure protein concentration by BioRad Protein Assay. While under isoflurane anesthesia, rats were euthanized by opening the chest cavity.
22 SDS-PAGE and Immunoblot Procedure
Soleus muscle samples in 2X Laemmli sample buffer were diluted with 1X
Laemmli sample buffer (2X Laemmli sample buffer diluted with an equal volume of water) to equal protein concentrations as assessed in triplicate by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) using crystalline BSA as a standard. Equal volumes of soleus muscle sample (20 µg) in sample buffer were loaded onto Criterion precast 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Bio-Rad) (208). Following SDS-PAGE, resolved proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Pall Life Sciences) for 1.5 hours.
Membranes were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline +
Tween 20 (TBS-T) for 1 hour at room temperature. PVDF membranes were carefully cut into strips using a razor and each strip was incubated overnight at 4°C with one of the following primary antibodies recognizing proteins phosphorylated on specific residues including: p70S6K1 Thr389 (1:1000 dilution), 4E-BP1 Thr37/46 (1:1000 dilution), 4E-BP1
Ser65 (1:1000 dilution), AMPK Thr172 (1:1000 dilution), eukaryotic initiation factor 2 alpha (eIF2α) Ser51 (1:3000 dilution), p42/44 (ERK 1/2) Thr202Tyr204 (1:1000 dilution),
ULK1 Ser757 (1:1000 dilution), TSC2 Ser939 (1:1000 dilution), Akt Ser473 (1:2000 dilution), Akt Thr308 (1:1000 dilution), or FoxO3a Ser253 (1:1000 dilution), all of which were purchased from Cell Signaling Technology, Inc (Danvers, MA). Alternatively, blots were probed with antibodies against AMPK (1:1000 dilution), ERK1/2 (1:1000 dilution), ULK1 (1:1000 dilution) or Akt (1:1000 dilution) purchased from Cell Signaling
Technology; p70S6K1 (1:10,000 dilution) or 4E-BP1 (1:10,000 dilution) purchased from
Bethyl Laboratories, Inc (Montgomery, TX); α-tubulin purchased from Santa Cruz
23 Biotechnology (Santa Cruz, CA) (1:1000 dilution); hypoxia-inducing factor 1 alpha subunit (HIF1-α) (1:1000 dilution) purchased from Novus Biologicals (Littleton, CO); or eIF2α (mouse monoclonal antibody; 1:5000 dilution; hybridoma cells provided by the late Dr. Edgar Henshaw) which was produced in house. Activating transcription factor 4
(ATF4) antibody (1:200 dilution) was generously provided by Dr. Michael Kilberg
(University of Florida). In a similar manner, equal volumes of soleus muscle sample (90
µg) in sample buffer were subjected to SDS-PAGE, transferred to a PVDF membrane, blocked, and probed with an antibody recognizing anti-phospho-p70S6K1 Thr229 (1
µg/µL dilution) purchased from Abcam (Cambridge, MA). After overnight incubation with primary antibody, membranes were washed with TBST (3 times, 5 minutes each) and probed with appropriate horseradish peroxidase-conjugated secondary antibodies
(Bethyl; 1:10,000 dilution in TBS-T with 5% nonfat dry milk) for 1 hour at room temperature. Blots were washed again with TBS-T (2 times, 5 minutes each), and incubated with enhanced chemiluminescence (ECL) (Pierce; Thermo Scientific,
Rockford, IL) or ECL Plus (GE Healthcare, Fairfield, CT) for 1 or 5 minutes, respectively. Blots were developed using a FluorChem M Multifluor System
(ProteinSimple, San Jose, CA) and band densities were quantified using AlphaView
(ProteinSimple) and Genetools (Syngene, Cambridge, MA) software. For determination of protein phosphorylation status, membranes were initially immunoblotted with phosphospecific antibodies and then stripped (using buffer containing 62.5 mM Tris·HCl,
69.4 mM SDS, and 18.3 µM β-mercaptoethanol, pH 6.7) and reprobed with antibodies directed against the respective total protein.
24
Measurement of p70S6K1 and 4E-BP1 Hyperphosphorylation
The phosphorylation of p70S6K1 and 4E-BP1 causes a decrease in the electrophoretic mobility of the proteins during SDS-PAGE (68, 89, 131). As a result, p70S6K1 and 4E-BP1 in tissue extracts gets separated into multiple electrophoretic forms during SDS-PAGE with the more slowly migrating forms representing more highly phosphorylated proteins (5). Phosphorylation of p70S6K1 and 4E-BP1 was assessed by immunoblot analysis using 7.5% and 15% polyacrylamide gels, respectively, with 0.19% bisacrylamide to permit resolution of p70S6K1 and 4E-BP1 into multiple electrophoretic forms (5, 120). Polyclonal antibodies for p70S6K1 (1:10,000 dilution) or 4E-BP1
(1:10,000 dilution) purchased from Bethyl Laboratories, Inc (Montgomery, TX) recognized both phosphorylated and unphosphorylated forms of the proteins. Following
SDS-PAGE, p70S6K1 separated into 4 distinct bands, while 4E-BP1 separated into 3 bands. Hyperphosphorylation of p70S6K1 was calculated as percentage of the protein in the top 3 bands relative to all 4 bands. Hyperphosphorylation of 4E-BP1 was calculated as percentage of the protein in the top band (γ-form) relative to all 3 bands (37).
Measurement of Skeletal Muscle Protein Synthesis
Skeletal muscle protein synthesis was measured by the incorporation of puromycin into peptide chains (78). Equivalent amounts of soleus muscle protein (40
µg) were dissolved in Laemmli buffer and subjected to immunoblot analysis as described in ‘SDS-PAGE and Immunoblot Procedure’ with a few exceptions. Electrophoresis was terminated when the dye front reached the bottom of the gel. Proteins were transferred to
25 a PVDF membrane for 2.5 hours to ensure transfer of all proteins. Membranes were incubated and rocked overnight at 4°C with a mouse monoclonal anti-puromycin antibody generated in-house (1 µg/mL in Tris-buffered saline). Following development of these immunoblots, puromycin incorporation was assessed by summating the immunoblot intensity of all protein bands in a sample lane. Soleus muscle samples from rats not injected with puromycin were included in immunoblot analysis and values obtained using these samples were subtracted from values obtained for the other samples.
Measurement of mRNA Expression
Total RNA was isolated from 500 µL of skeletal muscle homogenate in 1 mL
TRIzol reagent (Invitrogen, Grand Island, NY) following a standard TRIzol protocol according to the manufacturer’s instructions. The RNA pellet was reconstituted in 40 µL
RNase-free water, and the total RNA (1 µL) concentration was determined (NanoDrop
2000; Thermo Fischer Scientific, Waltham, MA). Total RNA was reverse transcribed using an ABI High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster
City, CA). For quantitation of specific mRNA, reverse transcriptase polymerase chain reaction was performed using Taqman gene expression assays and the gene expression master mix (Applied Biosystems, Foster City, CA) (109). Samples were loaded in triplicate on a 384-well plate and 2 µL (100 ng) cDNA was placed in each well with 0.5
µL gene expression assay, 5 µL gene expression master mix and 2.5 µL water. The cycling parameters as described (109), were an initial 50°C for 1 min followed by 40 cycles at 95°C for 15 s followed by 60°C for 1 min. Real-time PCR quantitation was calculated on the basis of Ct values, where Ct is defined as the PCR cycle number that
26 crosses an arbitrary signal threshold on the amplification plot. The comparative quantitation method 2−ΔΔCt was used in presenting gene expression of target genes in reference to an endogenous control, and 2−ΔCt was used in presenting expression of each
C housekeeping gene in validating the use of the endogenous control gene. Δ t is expressed as the difference between the target and control samples (Ct target – Ct control). Primers were purchased from Applied Biosystems including: Ddit4 (REDD1) (Assay ID:
Rn01433735_g1); Ddit4l (REDD2) (Rn00589659_g1); Sesn1 (Rn01440906_m1);
RGD1566319 (Sesn2) (Rn01520467_m1); ATF4 (Rn00824644_g1); Trim63 (MuRF1)
(Rn00590197_m1); Fbxo32 (MAFbx) (Rn00591730_m1); β-actin (Rn00667869_m1); and Tbp (Rn01455646_m1). β-actin and Tbp mRNA expression were compared against the gene expression of other common housekeeping genes including Rpo2-1 (Polr2a)
(Rn01752026_m1), Rpl32 (Rn00820748_g1) and Hprt (Rn01527838_g1). β-actin and
Tbp mRNA expression were used as internal controls as their expression did not change substantially with aging or in response to hindlimb immobilization or remobilization.
Statistical Analysis
For Chapters 3 and 4, results from individual experiments (n = 5 per group) were replicated in two or more independent experiments. Results are presented as means ± SE calculated from pooled data in Chapters 3 and 5. Results from the immobilized hindlimb soleus are presented as a percentage of the non-immobilized hindlimb soleus ± SEM in
Chapter 4. For each of the variables measured, no differences were observed in soleus muscles from the non-immobilized hindlimbs between dorsiflexed and plantarflexed immobilization techniques. Outliers were determined using Grubb’s test (alpha level
27 0.05) and excluded from further analysis. Paired t tests were used to compare differences between soleus muscles from immobilized and non-immobilized hindlimbs within the same rat. Unpaired Student’s t-test was used to compare differences between results obtained from different groups and conditions. All comparisons were analyzed using
GraphPad Software. Differences between groups were considered significant at p < 0.05.
28 Chapter 3. THE REPRESSORS OF MTORC1 SIGNALING REDD1/2
ARE RAPIDLY INDUCED AND ACTIVATION OF P70S6K1 BY A
NUTRIENT STIMULUS IS DEFECTIVE IN SKELETAL MUSCLE
OF AN IMMOBILIZED RAT HINDLIMB
Introduction
In humans, disuse atrophy results from a depression in skeletal muscle protein synthesis (80, 162). The depression is observed in both the fasted and fed state (anabolic resistance) (50, 72, 127, 162). The rates of protein synthesis in skeletal muscle are modulated by mTORC1 signaling (118). Despite evidence of attenuated mTORC1 signaling in skeletal muscle during hindlimb immobilization (155, 220), hindlimb unloading (10, 94), and bed rest (50), the mechanism responsible for repression of mTORC1 signaling during disuse is unknown. Furthermore, anabolic resistance to nutrient-induced stimulation of protein synthesis is associated with attenuated nutrient- induced stimulation of mTORC1 signaling during hindlimb immobilization (127, 140) and bed rest (50). However, the molecular mechanism responsible for resistance to nutrient stimulation is unknown. The goal of the research presented in this chapter was to
1) characterize a model of immobilization-induced disuse atrophy that exhibits depressed rates of protein synthesis and attenuated mTORC1 signaling, and 2) assess known repressors of mTORC1 signaling in order to gain a better understanding of the molecular mechanism(s) responsible for immobilization-induced disuse atrophy. I hypothesized that hindlimb immobilization would depress rates of protein synthesis in rat hindlimb skeletal muscle due to increased expression and/or activation of one or more repressors of the mTORC1 signaling pathway. Furthermore, I hypothesized that hindlimb
29 immobilization would blunt nutrient-induced stimulation of mTORC1 signaling due to increased expression and/or activation of one or more repressors of the mTORC1 signaling pathway.
Methods
Experimental design
After a 1-week adaptation period, rats (8-9 weeks of age; 230-350 g) were anesthetized with isoflurane inhalation (2.5%) and subjected to unilateral hindlimb immobilization for 1, 2, 3, or 7 days prior to the day of the experiment. Control rats were prepared for immobilization but were not immobilized. An equal number of rats from each experimental group were processed on experimental days. All rats were fasted overnight (21h), but allowed free access to water. On the day of the experiment, all rats
(except for those immobilized for 7 days) were randomly divided into groups that received either saline (0.155 M) or 1.35 g L-leucine/kg body wt by oral gavage as a nutrient stimulus as described previously (6). Rats subjected to 7 days of hindlimb immobilization received saline by oral gavage. Fifteen minutes after oral gavage, the rats were anesthetized using isoflurane and remained anesthetized for the remainder of the experiment. Thirty minutes after oral gavage, puromycin was administered by injection.
At 40 minutes post-gavage, the soleus, gastrocnemius, and plantaris muscles were individually excised, weighed, and prepared for analysis as described in Chapter 2,
‘Administration of Puromycin and Sample Collection’. The experiment was repeated once and the figures presented include data from both experiments.
30 Results
Effect of immobilization on muscle mass
Initially, experiments were designed to examine the time course of changes in muscle mass and protein synthesis following unilateral hindlimb immobilization.
Illustrated in Figure 3.1 is the muscle mass-to-body mass ratio of three muscles of the plantarflexor group, the soleus, gastrocnemius, and plantaris. Following 1 and 2 days of immobilization, there was no detectable change in mass for any of the three muscles. By
3 days, however, the mass for each muscle was significantly (p < 0.05) reduced and the reduction in mass was greater 7 days following immobilization. No differences in muscle mass-to-body mass ratio were observed between control (non-immobilized) rats and the control (contralateral, non-immobilized) hindlimbs of immobilized rats. Because the reduction in mass was greatest for the soleus muscle it was selected for further investigation into the mechanism(s) contributing to disuse atrophy. No differences in protein content (mg/g) were observed in muscles from immobilized compared to control hindlimbs (Table 3.1).
Effect of immobilization on muscle protein synthesis
To investigate the cause of the observed loss of muscle mass, rates of protein synthesis were measured in the soleus muscle after 1, 2, 3, and 7 days of immobilization.
As illustrated in Figure 3.2, hindlimb immobilization depressed rates of protein synthesis approximately 40-50% on each of the days studied compared to contralateral non- immobilized (control) hindlimbs. The effect was maximal after only a single day of
31 immobilization. No differences in rates of protein synthesis were observed between control rats and the control hindlimbs of immobilized rats.
Effect of immobilization on mTORC1 signaling
To gain an understanding of potential events that cause depressed protein synthesis following hindlimb immobilization, mTORC1 signaling was assessed across the first three days of immobilization in both a fasted condition (saline gavage) and in response to an oral leucine gavage as a nutrient stimulus. An analysis of p70S6K1
(Figure 3.3A) and 4E-BP1 (Figure 3.3B) hyperphosphorylation demonstrated attenuated mTORC1 signaling in soleus muscle of the immobilized compared to the control hindlimb across the 3-day time period. As described in Chapter 2 (‘Measurement of p70S6K1 and 4E-BP1 Hyperphosphorylation’), the phosphorylation of p70S6K1 and 4E-
BP1 causes a decrease in the electrophoretic mobility of the proteins during SDS-PAGE and the proteins separate into multiple bands. During SDS-PAGE, the more slowly migrating forms of p70S6K1 and 4E-BP1 represent more highly phosphorylated proteins.
As depicted in Figures 3.3A and 3.3B, respectively, p70S6K1 and 4E-BP1 migrated more quickly during SDS-PAGE in soleus muscle from an immobilized hindlimb compared to a control hindlimb. This is indicative of a lower phosphorylation state of these proteins during hindlimb immobilization, because phosphorylation retards gel migration. In addition, soleus muscles from saline-gavaged rats exhibited more quickly-migrating proteins during SDS-PAGE compared to leucine-gavaged rats. This analysis showed that mTORC1 signaling was elevated in response to the nutrient stimulus in the soleus muscle of both the immobilized and control hindlimbs. While the relative magnitude of increase
32 in the phosphorylation state of p70S6K1 and 4E-BP1 were similar between hindlimbs, the maximal response was attenuated in the soleus muscle from the immobilized hindlimb. Similar results were obtained when the relative phosphorylation of 4E-BP1 at
Ser65 (i.e., a site phosphorylated by mTORC1) was assessed. Figure 3.3C shows that relative phosphorylation of 4E-BP1 Ser65 was attenuated in the soleus muscle of the immobilized compared to the control hindlimb across the 3-day time period. While the nutrient stimulus produced an elevation in 4E-BP1 Ser65 phosphorylation in the soleus muscle of both the immobilized and control hindlimbs, the maximal response was attenuated in the immobilized hindlimb. No differences in the phosphorylation patterns for p70S6K1 or 4E-BP1 were observed between control rats and the control hindlimbs of immobilized rats. Overall, these results point to an immobilization-induced attenuation of mTORC1 signaling that is nonetheless responsive to stimulation by leucine administration.
Effect of immobilization on repressors of mTORC1 signaling
A number of upstream regulatory inputs were investigated as potential mediators of the attenuated mTORC1 signaling observed in soleus muscle of immobilized compared to control hindlimbs (Table 3.2). Sestrin 1 and Sestrin 2, whose expression is regulated by the transcription factor p53, activate LKB1 to phosphorylate AMPK at
Thr172 (26, 134). Phosphorylation at this site is required for AMPK activation, which in turn activates TSC2 and represses mTORC1 signaling (20, 134). However, examination of mRNA expression for Sestrin 1 and Sestrin 2 revealed no differences in soleus muscle between immobilized and control hindlimbs. Moreover, no differences were observed in
33 protein expression of Sestrin 1 or p53 in soleus muscle between immobilized and control hindlimbs. Finally, immobilization did not alter AMPK phosphorylation at Thr172.
In contrast, two regulatory inputs were observed to change in parallel with the attenuation of mTORC1 signaling following immobilization. As illustrated in Figure 3.4, expression of mRNA for REDD1 and REDD2, both of which repress mTORC1 signaling through activation of TSC1/2 (152, 196), was induced as early as 1 day following immobilization and their expression increased further following 2 and 3 days. REDD1 and REDD2 mRNA expression was lowered by leucine gavage in the soleus muscle of rats subjected to hindlimb immobilization for 2 and 3 days, however, with the exception of REDD1 mRNA expression in control soleus muscle of rats immobilized for 2 days, the decrease was not statistically significant. No differences in either REDD1 or REDD2 mRNA expression were observed between control rats and the control hindlimbs of rats subjected to hindlimb immobilization.
Hypoxia was considered as a potential mediator of the induced expression of
REDD1 and REDD2 mRNA. We next examined protein expression of HIF1-α, a known inducer of REDD1 mRNA expression (45, 105), as a potential mediator of the responses of REDD1 and REDD2 mRNA to immobilization. However, its expression was not elevated with immobilization (Table 3.2). Stress in the endoplasmic reticulum (ER stress) was also considered as being responsible for the elevated expression of REDD1 and REDD2 (214). In particular, ATF4 has been shown to increase in expression and induce REDD1 mRNA expression in response to ER stress. Moreover, ATF4 has been shown to play a critical role in disuse atrophy (56). However, no significant differences were observed in eIF2α phosphorylation at Ser51 (a marker of ER stress (122)) or ATF4
34 mRNA and protein expression (a known inducer of REDD1) (214) in soleus muscle between immobilized and control hindlimbs (Table 3.2). Finally, blood borne regulatory inputs such as glucocorticoids, which are known to induce REDD1 expression in muscle
(145, 211), were also considered. However, these were not pursued as a likely cause of
REDD1 mRNA induction due to the lack of a systemic effect on mTORC1 signaling in the control hindlimb of the same rat. In other words, the induction in REDD1 mRNA expression was localized to soleus muscle from the immobilized hindlimb. If circulating glucocorticoids were responsible for the induction in REDD1 mRNA expression, then
REDD1 would be induced in soleus muscle from both hindlimbs of a rats subjected to hindlimb immobilization when compared to control rats. Thus, glucocorticoids were ruled out as a likely cause of the localized induction in REDD1 mRNA expression during hindlimb immobilization.
Effect of immobilization on p70S6K1 phosphorylation at Thr389
Intriguingly, although leucine-induced phosphorylation of 4E-BP1 at Ser65 appeared to be unaffected by immobilization, a different pattern of response was observed when relative phosphorylation of p70S6K1 at Thr389 was assessed as a marker of mTORC1 signaling (Figure 3.5A). Thus, although relative phosphorylation at this site was likewise attenuated across the 3-day time period, the response to the leucine-induced stimulus was markedly different between the two conditions. In the soleus muscle of the immobilized hindlimb, phosphorylation at this site was elevated 3-4–fold following leucine administration whereas in the control hindlimb the elevation was 7-14–fold compared to the saline-administered control. Given that phosphorylation of p70S6K at
35 Thr229 (a PDK1 targeted site) is a prerequisite for mTORC1-mediated phosphorylation of Thr389 (112), its responses to immobilization and a nutrient stimulus, respectively, were assessed. As illustrated in Figure 3.5B, phosphorylation of p70S6K1 at Thr229 was of similar magnitude in the soleus muscle of both the immobilized and control hindlimb and for the former did not respond to the leucine-induced stimulus. In contrast, leucine administration produced a robust elevation in Thr229 phosphorylation in the soleus of the control hindlimb. Thus, analysis of p70S6K1 phosphorylation sites Thr389 and Thr229 supports an immobilization-induced state of “anabolic resistance” that has been described in other models of disuse atrophy (50, 72, 172).
Discussion
The first goal of the studies presented in this chapter was achieved having characterized a model of immobilization-induced disuse atrophy that exhibited depressed rates of protein synthesis and attenuated mTORC1 signaling. Protein synthesis was maximally depressed in the soleus muscle within 24 hours of immobilization and the depression was sustained for at least 7 days compared to the soleus muscle from the contralateral control hindlimb. In contrast, protein synthesis was unaltered in the soleus muscle of the control limb compared to control rats. This finding is in agreement with previous studies showing that immobilization-induced depression of protein synthesis begins to manifest as early as 6 hours after application of a cast, and is maintained throughout a 7-day period (22, 75). The magnitude of depression in response to immobilization observed in the present study (reduction to approximately 50% of either the control hindlimb or control rat) also agrees with previous studies utilizing animal
36 models of immobilization [reduction of 30-70% compared to control (22, 75)] as well as in human studies using limb unloading (40) and bed rest (61) in which protein synthesis was reduced to 53% and 50% of control values, respectively.
The depression of protein synthesis was accompanied by attenuated signaling through mTORC1 in the soleus muscle of immobilized compared to that of the control hindlimbs. The attenuation of mTORC1 signaling occurred within 24 hours of immobilization, as assessed by a reduction in the proportion of p70S6K1 and 4E-BP1 present in hyperphosphorylated forms, as well as reduced phosphorylation of p70S6K1 at
Thr389 and 4E-BP1 at Ser65. Attenuated mTORC1 signaling has been observed in skeletal muscle of rats and mice subjected to hindlimb unloading (19, 94), immobilization
(127), or denervation (94), but was not observed in one study in rats following 5 days of unilateral hindlimb immobilization (123). Studies in humans have failed to detect a change in mTORC1 signaling in skeletal muscle following 10 or 21 days of limb unloading, despite significant reductions in the rate of myofibrillar protein synthesis (40).
The basis for these disparate results in mTORC1 signaling are not clear, but could be due to different muscles being studied, e.g. unloading attenuated mTORC1 signaling in soleus muscle (19), but not in vastus lateralis (40). Differences in age group might also explain disparate results, e.g. juvenile, rapid-growing rats vs. adult, non-growing humans.
Finally, disparate results could be due to the model being employed, e.g. immobilization vs. unloading, species, or to the feeding status of the animals.
The second goal of the studies presented in this chapter was also achieved through assessing known repressors of mTORC1 signaling and identifying an induction in the mRNA expression of REDD1 and REDD2 with hindlimb immobilization (Figure 3.6).
37 The immobilization-induced attenuation of mTORC1 signaling likely involves upstream regulatory inputs to the kinase. The TSC complex acts to integrate signals from several upstream pathways, including those emanating from Akt and ERK (138, 190). Both of these kinases phosphorylate, and thereby inactivate the TSC complex, leading to a stimulation of mTORC1 signaling. In the studies presented here, immobilization did not reduce phosphorylation of Akt at Ser473 or TSC2 at Ser939, a site directly phosphorylated by Akt (177). The possibility that immobilization promoted reduced phosphorylation of TSC2 by ERK1/2 could not be tested because of the lack of an antibody that reliably detects rat TSC2 phosphorylated at Ser644, a site phosphorylated by ERK1/2 (138). However, no difference in ERK1/2 Thr202/Tyr204 phosphorylation was observed, suggesting that ERK1/2 signaling did not mediate the immobilization- induced attenuation of mTORC1 signaling.
In addition to Akt and ERK, the TSC1•TSC2 complex is also regulated by the p53–AMPK signaling pathway. Previous studies have shown that p53 protein expression is induced in gastrocnemius muscle in response to 14 days of hindlimb unloading (194), and chronic activation of p53 in muscle promotes atrophy (186). Moreover, two downstream targets of p53 action, Sestrins 1 and 2, have been shown to repress mTORC1 signaling through activation of AMPK (26). However, neither Sestrin 1 or Sestrin 2 mRNA, Sestrin 1 protein expression, nor AMPK phosphorylation on the activating residue, Thr172, were observed, suggesting that the attenuation of mTORC1 signaling was not mediated by the p53–Sestrin–AMPK signaling pathway.
Like AMPK, REDD1 and REDD2 also act to attenuate mTORC1 signaling through a mechanism involving TSC1/2 (152, 196). REDD1 and REDD2 mRNA
38 expression was induced (100% and 400%, respectively) after one day of immobilization followed by a larger induction (250-350% and 400-600%) after 2 and 3 days: changes that paralleled the greater attenuation of mTORC1 signaling at the later time points.
Enhanced protein expression of either REDD1 or REDD2 could not be assessed because of the lack of antibodies that reliably detect the rat proteins. Nonetheless, previous studies have demonstrated concomitant responses of REDD1 mRNA and protein expression to ER stress (214), starvation (145), and dexamethasone treatment (211).
Thus, it seems reasonable to conclude that the increases in REDD1 and REDD2 mRNA expression observed in the studies presented here are indicative of similar responses in expression of the respective proteins.
REDD1 and REDD2 mRNA expression are potently induced in response to a variety of stresses. One potential mechanism for the observed induction of REDD1/2 expression with hindlimb immobilization could be development of hypoxia (45) due to a reduction in blood flow to the immobilized hindlimb. However, HIF-1α expression was not elevated in soleus muscle from the immobilized hindlimb compared to the contralateral, control hindlimb. In addition, the lack of change in either eIF2α phosphorylation on Ser51 or ATF4 expression suggest that ER stress was not involved in the induction of REDD1 expression (214). REDD1 expression is also upregulated in response to other stresses, e.g. conditions that cause DNA damage (58) or decrease ATP concentrations (196), or in response to oxidative stress (58). However, the mechanism(s) through which such stresses lead to upregulation of REDD1 expression are presently undefined.
39 In addition to depressed rates of protein synthesis in the fasted condition, both immobilization (72) and bed rest (16, 50) are associated with development of resistance to nutrient-induced stimulation of skeletal muscle protein synthesis in humans.
Moreover, two studies showed that skeletal muscle protein synthesis is resistant to nutrient-induced stimulation during hindlimb immobilization in old rats (140) and in mice (127). The studies showed that global rates of protein synthesis in skeletal muscle were unresponsive to chow feeding or oral leucine administration, respectively, in an immobilized hindlimb compared to fasted controls. In the studies presented here, the leucine-induced stimulus produced an elevation of similar magnitude in mTORC1 signaling in soleus muscle from both the immobilized and control hindlimbs, as assessed by gel-shift (hyperphosphorylation) analysis of the phosphorylation state of p70S6K1 and
4E-BP1. Assessment of the phosphorylation of 4E-BP1 at Ser65, a site of mTORC1- mediated phosphorylation (71), produced results similar to those obtained with the gel- shift analysis. Substrates of mTORC1 vary greatly in their response to both pharmacological (e.g. rapamycin and Torin) and natural (e.g. amino acids and growth factors) regulators of mTORC1 signaling (106). For example, the phosphorylation of p70S6K1 at Thr389 is sensitive to inhibition by rapamycin, while the phosphorylation of growth factor receptor-bound protein 10 (Grb10) at Ser150, another mTORC1 phosphorylation site, is resistant to rapamycin. In addition, when murine embryonic fibroblasts (MEFs) were placed in media containing 100, 20, and 0% of normal levels of amino acids, phosphorylation of p70S6K1 at Thr389 was strongly reduced when cells were placed in medium containing 20% amino acids. In contrast, Grb10 phosphorylation at Ser150 was reduced only when placed in medium containing 0% amino acids. Similar
40 results were obtained when MEFs were placed in medium containing 10, 2, or 0% fetal bovine serum (a potent growth factor). Interestingly, phosphorylation of p70S6K1 at
Thr389 was more resistant to rapamycin treatment and reductions in amino acids when
Thr389 was mutated to Ser389. It was concluded from these experiments that the sequence composition of an mTORC1 phosphorylation site, particularly the presence of serine or threonine as the phosphoacceptor, is a mechanism for allowing downstream effectors of mTORC1 to respond differently to regulators of mTORC1 signaling. It is noteworthy that all of the mTORC1 phosphorylation sites on 4E-BP1 as well as the rapamycin-resistant site in the turn motif of p70S6K1 (Ser371) are followed by a Pro residue (112, 156), providing a possible explanation for the coordinated changes in 4E-
BP1 phosphorylation on Ser65 and hyperphosphorylation of 4E-BP1 and p70S6K1. In contrast, p70S6K1 Thr389 is not followed by Pro, but instead is followed by a Tyr residue (106, 112), suggesting that its phosphorylation by mTORC1 might be differentially regulated compared to 4E-BP1. Indeed, in contrast to 4E-BP1 phosphorylation on Ser65 or hyperphosphorylation of either 4E-BP1 or p70S6K1, phosphorylation of p70S6K1 on both Thr229 and Thr389 in response to the nutrient stimulus was severely blunted in soleus muscle from immobilized compared to control hindlimbs. Since phosphorylation of Thr229 by PDK1 is a prerequisite for phosphorylation of Thr389 by mTORC1 (112), the results are consistent with a model in which PDK1 activation is rapidly impaired in response to immobilization. In conclusion, the results presented here demonstrate an immobilization-induced attenuation of mTORC1 signaling mediated by REDD1 and REDD2 that is nonetheless responsive to a leucine stimulus. More importantly, they suggest that phosphorylation of p70S6K1 on
41 Thr389, which is required for full activation of the kinase and thus signaling to its downstream substrates, fails to respond appropriately to the leucine stimulus. This lack of response is apparently due to a defect in PDK1 signaling and would explain the phenomenon of “anabolic resistance” referred to in other models of disuse atrophy (50,
72, 172).
42
43 Table 3.1 Protein concentration of soleus muscle from immobilized and control hindlimbs after 1-3 days of hindlimb immobilization
Protein Concentration (µg/uL)
Days Immobilized Immobilized Hindlimb Control Hindlimb
Control 6.77 ± 0.48
1 day 6.65 ± 0.43 6.66 ± 0.44
2 days 6.51 ± 0.34 6.54 ± 0.50
3 days 6.73 ± 0.56 6.41 ± 0.44
Data are means ± SE, n = 19-20 muscles/group, and are expressed as the protein concentration (µg/µL). Rats had one hindlimb immobilized in plantarflexion (soleus fixed in a shortened position) for 1-3 days. No significant differences were observed between groups.
44
45
46 Table 3.2 End points not associated with immobilization-induced repression of mTORC1 signaling.
Compared to End point Non-Immobilized Limb (%) p53 protein 111.3 ± 12.8 Sestrin 1 mRNA 106.9 ± 14.9 Sestrin 1 protein 69.5 ± 15.9 Sestrin 2 mRNA 143.4 ± 31.2 AMPK Thr172 phosphorylation 100.6 ± 13.6 473 Akt Ser phosphorylation 94.7 ± 2.7 308 Akt Thr phosphorylation 106.1 ± 9.6 939 TSC2 Ser phosphorylation 61.7 ± 22.0 ERK Thr202Tyr204 phosphorylation 82.3 ± 6.0 eIF2α Ser51 phosphorylation 114.4 ± 13.6 ATF4 mRNA 120.3 ± 14.5 ATF4 protein 83.9 ± 12.6 HIF1-α protein 75.9 ± 17.7
Data are means ± SE, n = 5-6 rats/group, and are expressed as a percentage of measurements from immobilized hindlimbs relative to measurements from non- immobilized hindlimbs. No leucine effects were observed. Rats had one hindlimb immobilized for 1 day. * p < 0.05 versus non-immobilized limbs.
47
48
49 Figure 3.6
50 Chapter 4. CHANGES IN REDD1, REDD2, AND ATROGENE MRNA
EXPRESSION ARE PREVENTED IN SKELETAL MUSCLE FIXED
IN A STRETCHED POSITION DURING HINDLIMB
IMMOBILIZATION
Introduction
Disuse atrophy and depressed rates of protein synthesis are observed when skeletal muscle is immobilized in a shortened position, but not in a stretched position (75,
76, 137). Thus, shortened muscle length is likely a key physiological stimulus for disuse atrophy. The mechanism(s) through which immobilization in a stretched position acts to prevent disuse atrophy is currently unknown. The goal of this chapter was to gain an understanding of the role of fixed muscle length in the regulation of protein synthesis and degradation in the soleus muscle of an immobilized rat hindlimb. I hypothesized that the soleus placed in a stretched position would not exhibit disuse atrophy and would be protected from changes in gene expression known to be associated with disuse atrophy.
Methods
Experimental design
Rats (8-9 weeks of age; 250-350 g) were maintained on a standard 12:12-hr light- dark cycle (lights on at 0700 hours) for one week before the experiment began. They were anesthetized with isoflurane inhalation (2.5%) and subjected to unilateral hindlimb immobilization. One group of rats had the ankle joint of one hindlimb immobilized in full plantarflexion, which placed the soleus in a shortened position, as described in the
Methods section. Another group of rats had the ankle joint of one hindlimb immobilized
51 in full dorsiflexion, which placed the soleus in a stretched position (75). The hindlimbs were immobilized for 3 days prior to removal of the soleus muscle for subsequent analysis. An equal number of rats from the two experimental groups were processed on the days of tissue harvest. All rats were fasted overnight (18h), but allowed free access to water. On tissue harvest days, rats were anesthetized using isoflurane and remained anesthetized for the remainder of the procedure. Immobilization for 3 days was selected for all analysis based on the results of Chapter 3 showing that disuse atrophy was manifest at this time point in this experimental model. Results from Chapter 3 also showed that the hindlimb immobilization procedure did not affect the variables of interest in the contralateral, non-immobilized hindlimbs of immobilized rats. Therefore, the control hindlimb of immobilized rats was used as the control. Results from the immobilized hindlimb soleus are presented as a percentage of the non-immobilized hindlimb soleus ± SEM.
Results
Effect of immobilization on muscle mass and protein synthesis
As illustrated in Figure 4.1A, immobilization of soleus in a stretched position prevented the atrophy observed in the soleus muscle immobilized in a shortened position,
(i.e. muscle mass was significantly reduced (p < 0.05) in soleus immobilized in a shortened position when expressed relative to soleus muscle from the contralateral control hindlimb). To investigate the cause of the observed differences in muscle mass, rates of protein synthesis were measured in the soleus muscle after 3 days of immobilization. As illustrated in Figure 4.1B, immobilization of soleus muscle in a
52 stretched position prevented the reduction in rates of protein synthesis observed in the soleus muscle immobilized in a shortened position (i.e. rates of protein synthesis were reduced approximately 70% in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus muscle from the control hindlimb).
Effect of immobilization on regulation of mTORC1 signaling
To gain an understanding of potential molecular events contributing to the changes in protein synthesis during hindlimb immobilization, mTORC1 signaling was assessed after 3 days of immobilization. An analysis of the phosphorylation state of p70S6K1 at Thr389 (Figure 4.2) demonstrated that immobilization of the soleus muscle in a stretched position prevented the reduction in mTORC1 signaling observed in the soleus muscle immobilized in a shortened position (i.e. phosphorylation of p70S6K1 at
Thr389 was reduced approximately 85% in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus muscle from the control hindlimb).
Attenuation of mTORC1 signaling following hindlimb immobilization in plantarflexion is associated with induced expression of the genes encoding the mTORC1 repressors REDD1 and REDD2 (111). In the studies presented here, immobilization of the soleus muscle in a stretched position prevented the induction in both REDD1 (Figure
4.3A) and REDD2 (Figure 4.3B) mRNA expression that was observed in soleus muscle immobilized in a shortened position (i.e. mRNA expression of REDD1 and REDD2, both of which repress mTORC1 signaling through activation of the tuberous sclerosis complex
(152, 196), was enhanced approximately 250% and nearly 500%, respectively, in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus
53 muscle from the control hindlimb). Interestingly, soleus muscle immobilized in a stretched position displayed a 40% reduction in REDD2 expression compared to the contralateral control hindlimb (Figure 4.3B). Overall, these results are consistent with a model in which immobilization of soleus muscle in a stretched position prevents the induction of REDD1 and REDD2 expression that are responsible for attenuated mTORC1 signaling and protein synthesis in skeletal muscle immobilized in a shortened position.
Effect of immobilization on atrogene expression
Next, I assessed mRNA expression of the E3 ubiquitin ligases, MAFbx and
MuRF1, as markers of proteasome-mediated degradation of skeletal muscle proteins during atrophy (17). An analysis of mRNA expression of MAFbx (Figure 4.4A) and
MuRF1 (Figure 4.4B) demonstrated that immobilization of soleus muscle in a stretched position prevented the induction observed in soleus muscle immobilized in a shortened position (i.e. mRNA expression of MAFbx and MuRF1 increased over 150% and 100%, respectively, in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus muscle from the control hindlimb). These results suggest that fixed muscle length regulates atrogene expression whereby soleus muscle immobilized in a stretched position prevents induction of atrogene expression observed in soleus muscle immobilized in a shortened position.
54 Effect of immobilization on regulation of atrogene expression
To gain an understanding of potential molecular events contributing to the changes in atrogene expression during hindlimb immobilization, Akt and FoxO3a phosphorylation were assessed after 3 days of immobilization. An analysis of the phosphorylation state of Akt at Ser473 (Figure 4.5A) demonstrated that immobilization of the soleus muscle in a stretched position prevented the reduction in Akt phosphorylation observed in the soleus muscle immobilized in a shortened position (i.e. phosphorylation of Akt at Ser473 was reduced approximately 45% in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus muscle from the control hindlimb).
Akt suppresses atrogene expression through the phosphorylation of Forkhead box
O (FoxO) transcription factors (183). An analysis of the phosphorylation state of FoxO3a at Ser253 (Figure 4.5B) demonstrated that immobilization of the soleus muscle in a stretched position prevented the reduction in FoxO3a phosphorylation observed in the soleus muscle immobilized in a shortened position (i.e. phosphorylation of FoxO3a at
Ser253 was reduced approximately 40% in soleus muscle immobilized in a shortened position when expressed relative to that of the soleus muscle from the control hindlimb).
However, it should be noted that this reduction in phosphorylation of FoxO3a at Ser253 in the soleus muscle immobilized in a shortened position did not reach statistical significance due to a small number of samples used in this analysis (n = 2-4).
55 Discussion
The goal of the studies described in the second chapter was achieved by gaining an understanding of the role of fixed muscle length in the regulation of protein turnover in immobilized rat hindlimb skeletal muscle. In support of my hypothesis, soleus muscle placed in a stretched position did not exhibit disuse atrophy and was protected from changes in gene expression known to be associated with disuse atrophy. Though it has been shown that soleus muscle immobilized in a stretched position does not exhibit disuse atrophy or depressed rates of protein synthesis (21, 74, 75, 137, 184), the studies presented here investigated gene expression changes that might contribute to these observations. The results indicate that immobilization of soleus muscle in a stretched as compared to a shortened position prevents induction of gene expression for the mTORC1 repressors, REDD1 and REDD2, which modulate mTORC1 signaling (152, 196). In addition, immobilization of soleus muscle in a stretched as compared to a shortened position prevents induction of expression of atrogenes, which modulate rates of protein degradation (18). Together, these findings help to explain why soleus muscle fixed in a stretched position does not exhibit disuse atrophy.
In accordance with other reports (189, 195), the studies presented here confirm that induction of atrogene expression is dependent on fixed muscle length. Senf and colleagues (188, 189) observed induction of atrogene expression in rat soleus muscle immobilized in a shortened position for 3 days. In contrast, Soares and colleagues (195) observed a suppression of atrogene expression in rat soleus muscle immobilized for 1 and
2 days in a stretched position returning to non-stretched expression levels after 4 days. I did not observe a suppression of atrogene expression after 3 days of immobilization of
56 the soleus muscle in a stretched position. Perhaps atrogene expression was suppressed at earlier time points and returned to control levels by the third day. Still, the results of the present study agree with both Senf et al. (189) and Soares et al. (195) whereby muscle immobilized in a stretched position prevents the induction of atrogene expression while muscle immobilized in a shorted position leads to the induction of atrogene expression.
The induction in atrogene expression is associated with a reduction in the phosphorylation of Akt at Ser473 and FoxO3a at Ser253 in soleus muscle from a hindlimb immobilized in a shortened position. Atrogene expression is modulated by
FoxO transcription factors, such as FoxO3a (183). Akt regulates FoxO3a via phosphorylation on residues, such as Ser253. Phosphorylation prevents the translocation of FoxO3a from the cytoplasm to the nucleus. In my model, 3 days of hindlimb immobilization in a shortened position reduced Akt-mediated phosphorylation of
FoxO3a. FoxO3a in a less-phosphorylated state is likely responsible for induction of atrogenes at this time point. In contrast, no attenuation in the phosphorylation of Akt at
Ser473 and FoxO3a at Ser253 was observed in soleus muscle immobilized in a stretched position. Thus, the regulation of Akt and FoxO3a phosphorylation appears to be dependent on fixed muscle length.
Other studies have provided evidence to suggest that mTORC1 signaling is related to the extent to which a muscle is stretched. For example, acute passive stretch of skeletal muscle cells (185) and tissue (3, 95) enhances mTORC1 signaling. While some studies have shown an activation of Akt and signaling to the mTORC1 complex in response to stretch (3, 49, 185), Hornberger et al. (95) suggested that acute passive stretch of skeletal muscle ex vivo stimulates mTORC1 signaling even in the absence of PI3K/Akt
57 signaling. The latter suggestion would imply that muscle stretch stimulates mTORC1 signaling by an Akt-independent mechanism (95). Gene expression is also influenced by skeletal muscle stretch whereby muscle immobilized in a stretched position exhibits repression of fast type and activation of slow myosin genes (30, 31, 77). Stretch and other mechanical stimuli can influence muscle gene expression through mechanosensory proteins in the muscle sarcomere (132). Proteins that bind the Z-disk and M-line relay mechanical strain information to cellular systems that control gene expression in the nucleus (67). For example, transcription factors and their interacting proteins bind to the muscle scaffolding protein, titin, and are released in response to increases in sarcomere length (132). Like titin, obscurin also binds numerous sarcomeric proteins and contains a signaling domain near its C-terminus that can be coupled to serine/threonine protein kinase domains (113). Other proteins that link skeletal muscle strain with the regulation of gene expression include nebulin, myopalladin, muscle LIM protein, and nuclear factor of activated T-cells C (132, 207).
In conclusion, the results of this chapter support the hypothesis that immobilization of soleus muscle in a stretched position prevents changes associated with disuse atrophy such as induction of REDD1, REDD2 and atrogenes, consequently leading to the attenuation of mTORC1 signaling and rates of protein synthesis as well as accelerated rates of protein degradation. Thus, fixed muscle length plays an important role in the regulation of atrophic gene expression.
58
Figure 4.1. Mass and rates of protein synthesis in the soleus are reduced only when the muscle is immobilized in a shortened position. Rates of protein synthesis were measured by the amount of puromycin incorporated into protein as assessed by immunoblot analysis. Rats had one hindlimb immobilized for 3 days in a position to place the soleus muscle in either a stretched or shortened position (Imm: Immobilized; N Leg = Non-Immobilized (control) limb). Bars represent A) the mean mass of the soleus muscle, and B) the mean rates of protein synthesis in the soleus muscle from an immobilized hindlimb expressed as a percentage of the mass of the soleus muscle from the contralateral control hindlimb. Data are mean percentages ± SEM, n = 10 rats/group. * p = < 0.05 versus soleus muscle from the control hindlimb.
59
60
61
62
A
150 p = 0.0785 l Akt Leg) a
t o ed T z ili / 100 ob 473 * S mm t I k - 50 A
on N f pho o s 0 % ho (
P Shortened Stretched
B
150 l Protein Leg) a
t o ed T z / ili 100 ob 253 S
mm I - 3a 50 O on x N o f F
o 0 % ( pho Shortened Stretched s ho P
Figure 4.5 Phosphorylation of A) Akt at Ser473 and B) FoxO3a at Ser253 are reduced only when the soleus muscle is immobilized in a shortened position. Phosphorylation of Akt at Ser473 and FoxO3a at Ser253 were assessed by protein immunoblot analysis. Rats had one hindlimb immobilized for 3 days in a position to place the soleus in either a shortened or stretched position. Bars represent the mean phosphorylation of Akt at Ser473/total Akt protein or phosphorylation of FoxO3a at Ser253/total protein ratio in the soleus muscle from an immobilized hindlimb expressed as a percentage of that in the soleus muscle from the contralateral control hindlimb. No changes in total Akt expression were observed in any group across 3 days of hindlimb immobilization. Total FoxO3a pro- tein could not be accurately assessed by immunoblot. Data are mean percentages ± SEM, n = 10 rats/group. * p < 0.05 versus soleus muscle from the control hindlimb.
63 Chapter 5. AGE-RELATED RESPONSES OF MTORC1 SIGNALING
IN RAT HINDLIMB SKELETAL MUSCLE TO IMMOBILIZATION
AND REMOBILIZATION
Introduction
Loss of skeletal muscle mass and function in the elderly leads to physical frailty, increased risk of falls and fractures, and increased morbidity and mortality (35, 139, 200).
A period of inactivity (e.g. limb immobilization, bed rest, etc.) worsens the problem since inactivity causes skeletal muscle atrophy (199). Both age- and inactivity-induced muscle atrophy may, in part, be due to resistance to anabolic stimulation (e.g. exercise and amino acids) (55, 72, 170). In contrast, remobilization promotes skeletal muscle hypertrophy due, in part, to enhanced anabolic stimulation (98, 126). If the molecular mechanisms responsible for anabolic stimulation in muscle can be understood, then this will lead to the development of therapeutic interventions aimed at preventing age- and inactivity- induced skeletal muscle atrophy.
Based on the results of Chapters 3 and 4, I asked how mTORC1 signaling may be regulated by REDD1 and REDD2 in response to aging and remobilization. I tested the hypothesis that REDD1 and/or REDD2 mRNA expression is elevated in muscle from old compared to young rats in association with attenuated mTORC1 signaling in response to nutrient stimulation. Moreover, I hypothesized that the immobilization-induced stimulation of REDD1 and/or REDD2 expression is exacerbated in old compared to young rats. I also tested the hypothesis that remobilization reverses the immobilization- induced induction of REDD1 and/or REDD2 expression in conjunction with restoration of mTORC1 signaling.
64
Methods
Experimental Design
Rats 2 (juvenile), 9 (young adult), and 18 (old adult) months of age were anesthetized by isoflurane inhalation (2.5%) and subjected to unilateral hindlimb immobilization for 7 days prior to removal of the soleus muscle for subsequent analysis.
An additional group of rats aged 9 months was subjected to unilateral hindlimb immobilization for 7 days, and following removal of the cast, were allowed to remobilize for 7 days (remobilization group). All rats were fasted overnight (18h) but allowed free access to water. On tissue harvest days, rats were individually caged and provided with rodent chow (AIN-93M) for 10 min (rats consumed 2.1 – 7.0 grams). They were subsequently anesthetized using isoflurane and remained anesthetized for the rest of the experiment. The soleus muscle was chosen for analysis in the present study so that the results could be compared to those from Chapters 3 and 4, which investigated the effects of unilateral hindlimb immobilization on mTORC1 signaling in young rats. The soleus muscle was removed from the immobilized and the contralateral non-immobilized
(control) hindlimbs 45 min after the start of chow feeding. This time point was selected based on previous studies showing that the peak response of mTORC1 signaling to a nutrient-induced stimulus occurs between 45 and 60 min (6). Ribosome content was calculated by measuring the total RNA concentration in a portion of muscle as described in ‘Measurement of mRNA expression’ and applying this concentration to the whole muscle.
65 Statistical analysis
In order to assess the effects of remobilization following hindlimb immobilization, the soleus muscle from the remobilized limb of rats in the remobilization group was compared to the soleus muscle from 9-month old immobilized rats. Non- linear regression (curve fit) analysis was used to assess the relative relationship between p70S6K1 phosphorylation at Thr389 and REDD2 mRNA expression for all samples.
Results
Effect of age and remobilization on immobilization-induced loss of muscle mass
As illustrated in Figure 5.1A, the soleus muscle mass-to-body mass ratio declined with age, being significantly lower (p < 0.05) in 18-month old rats compared to both 2- and 9-month old rats. Absolute mass of the soleus (Figure 5.1B) was larger in 9- and 18- month old rats compared to 2-month old rats, but no significant differences in muscle mass were observed between 9- and 18-month old rats. In response to 7 days of hindlimb immobilization, muscle mass-to-body mass ratio was reduced in rats from all age groups.
Notably, the loss of muscle mass following hindlimb immobilization was inversely proportional to age, where the muscle mass-to-body mass ratio was reduced 40%, 15%, and 8% in the soleus muscle from the immobilized hindlimb of 2-, 9-, and 18-month old rats, respectively, when compared to the soleus muscle from the contralateral non- immobilized hindlimb (referred to hereafter as the control hindlimb). A similar relationship between age and disuse atrophy was observed when results were expressed as absolute muscle mass (Figure 5.1B) where muscle mass was reduced 40%, 15%, and
7% in the soleus muscle from the immobilized hindlimb of 2-, 9-, and 18-month old rats.
66 No difference in muscle mass-to-body mass ratio was observed between the soleus muscle from the control hindlimb of 9-month old immobilized and remobilized rats.
Following 7 days of remobilization, muscle mass-to-body mass ratio and absolute mass were further reduced (p < 0.05) in the soleus muscle from the remobilized hindlimb compared to either the immobilized or the control hindlimb of 9-month old rats.
Effect of age, immobilization, and remobilization on mTORC1 signaling
In order to gain an understanding of the potential molecular events responsible for age- and immobilization-induced skeletal muscle atrophy, rats were provided with a nutrient stimulus to produce an anabolic response. This was assessed by analysis of the phosphorylation state of residues on two proteins known to be direct targets of mTORC1, p70S6K1 Thr389 (Figure 5.2A) and ULK1 Ser757 (Figure 5.2B). The phosphorylation of ULK1 at Ser757 was included here as a direct readout of mTORC1 signaling that is not influenced by prior PDK1 phosphorylation. The analysis demonstrated that aging was associated with an 83% and 78% attenuation of the nutrient-induced stimulation of p70S6K1 phosphorylation at Thr389 in the soleus muscle from the control hindlimb of 9- and 18-month old rats, respectively, relative to the soleus muscle from the control hindlimb of 2-month old rats. In response to 7 days of hindlimb immobilization, phosphorylation of p70S6K1 at Thr389 was reduced 68% and 48% in the soleus muscle from the immobilized hindlimb relative to the control hindlimb of 2- and 9-month old rats, respectively, whereas it was statistically different from the control value in 18-month old rats. ULK1 phosphorylation at Ser757 also declined with age following the nutrient stimulus. In response to immobilization, it was reduced 48% relative to the control
67 hindlimb of 2-month old rats, but no difference was observed in its phosphorylation state between hindlimbs of 9- or 18-month old rats. Following 7 days of remobilization, mTORC1 signaling was augmented in the soleus muscle from the remobilized limb relative to the control hindlimb, i.e. phosphorylation of p70S6K1 at Thr389 and ULK1 at
Ser757 was elevated 160% and 42%, respectively, in the soleus muscle from the remobilized hindlimb.
Effect of age, immobilization, and remobilization on Akt activation
As a biomarker of an upstream signaling input to mTORC1, phosphorylation of
Akt at Ser473 was assessed as an index of the activation state of the kinase. The assessment demonstrated that aging was associated with attenuation of the nutrient- induced activation of Akt in soleus muscle from the control hindlimb of 9- and 18-month old rats relative to the soleus muscle from the control hindlimb of 2-month old rats
(Figure 5.3). In response to 7 days of hindlimb immobilization, the nutrient-induced phosphorylation of Akt at Ser473 was reduced 30% in the soleus muscle from the immobilized hindlimb relative to the soleus muscle from the control hindlimb of 2-month old rats. No difference in phosphorylation of Akt at Ser473 was observed between the soleus muscle from the immobilized and the control hindlimb of 9- and 18-month old rats. Following 7 days of remobilization, phosphorylation of Akt at Ser473 was augmented 90% in the soleus muscle from the remobilized limb relative to the soleus from the control hindlimb.
68 Effect of age, immobilization, and remobilization on REDD mRNA expression
In order to gain an understanding of the mechanism(s) responsible for the observed responses of mTORC1 signaling to aging, hindlimb immobilization, and remobilization, the mRNA expression was assessed of two repressors of the pathway, i.e.
REDD1 and REDD2. No change in REDD1 mRNA expression was observed in response to aging, hindlimb immobilization, or remobilization (Figure 5.4A). In contrast, aging was associated with increased expression of REDD2 mRNA, i.e. REDD2 mRNA expression was induced approximately 250% and 300% in the soleus muscle from the control hindlimb of 9- and 18-month old rats, respectively, relative to the control hindlimb of 2-month old rats (Figure 5.4B). In response to 7 days of hindlimb immobilization, REDD2 mRNA expression was induced by over 400% in the soleus muscle from the immobilized hindlimb relative to the soleus muscle from the control hindlimb of 2-month old rats. In 9-month old rats, REDD2 mRNA expression in the soleus muscle from the immobilized hindlimb relative to the soleus muscle from the control hindlimb was further increased by 77% above the aging-induced response.
However, this trend did not reach statistical significance (p = 0.16). Moreover, in 18- month old rats, immobilization did not enhance REDD2 mRNA expression above the aging-induced response. Following 7 days of remobilization, REDD2 mRNA expression was repressed 38% in the soleus muscle from the remobilized limb relative to the soleus muscle from the control hindlimb, and was significantly lower than the REDD2 mRNA expression in the soleus muscle from the immobilized hindlimb of 9-month old rats.
69 Relationship between p70S6K1 Thr389 phosphorylation and REDD2 mRNA expression
Across age groups and conditions, there appeared to be an association between mTORC1 signaling and REDD2 mRNA expression in the soleus from rat hindlimbs, i.e. the phosphorylation of p70S6K1 at Thr389 and ULK1 at Ser757 was inversely proportional to REDD2 mRNA expression. A scatter plot with the natural logarithm (ln) of REDD2 mRNA expression and the phosphorylation of p70S6K1 at Thr389 plotted on the x- and y-axes, respectively, revealed a linear relationship between these two variables. As illustrated in Figure 5.5, low REDD2 mRNA expression was associated with high phosphorylation of p70S6K1 at Thr389 and increases in REDD2 mRNA expression were associated with reductions in phosphorylation of p70S6K1 at Thr389.
Fitting a linear regression to these points reveals a negative correlation (r2 = 0.5372) between REDD2 mRNA expression and phosphorylation of p70S6K1 at Thr389 (p <
0.0001).
Effect of age and immobilization on PDK1 phosphorylation sites on Akt and p70S6K1
In order to gain an understanding of PDK1 signaling in response to aging and 7 days of hindlimb immobilization, I assessed the phosphorylation states of p70S6K1 at
Thr229 and Akt at Thr308. Aging was associated with an attenuation of the nutrient- induced stimulation of p70S6K1 phosphorylation at Thr229 in soleus muscle from the control hindlimb (Figure 5.6A). In response to 7 days of hindlimb immobilization, the phosphorylation of p70S6K1 at Thr229 was reduced in 2-month old rats, but not in 9- and
70 18-month old rats. I observed similar results for the phosphorylation of Akt at Thr308, whereby aging was associated with an attenuation of the nutrient-induced stimulation of
Akt phosphorylation at Thr308 in soleus muscle from the control hindlimb (Figure 5.6B).
In response to 7 days of hindlimb immobilization, the phosphorylation of Akt at Thr308 was reduced in 2-month old rats, but not in 9- and 18-month old rats.
Effect of age, immobilization, and remobilization on atrogene mRNA expression
I assessed the mRNA expression of the E3 ubiquitin ligases, MAFbx and MuRF1
(also known as 'atrogenes' (18), as biomarkers associated with the proteasome-mediated degradation of skeletal muscle proteins during atrophy. The mRNA expression of
MAFbx (Figure 5.7A) was elevated 63% and 47% in the non-immobilized hindlimb of 9- and 18-month old rats compared to the non-immobilized hindlimb of 2-month old rats.
Moreover, after seven days of immobilization, MAFbx mRNA expression was higher in the immobilized compared to the control hindlimb in each age group. Aging had no significant effect on the mRNA expression of MuRF1 (Figure 5.7B). Interestingly, although MuRF1 was elevated in the immobilized hindlimb of 2-month old rats, it was not elevated in response to immobilization in 9- and 18-month old rats. In response to 7 days of remobilization, MAFbx expression was lower in the remobilized hindlimb compared to the immobilized hindlimb of 9-month old rats. However, MAFbx mRNA expression was not statistically different between the remobilized and non-immobilized hindlimb of the same rat. In contrast, MuRF1 mRNA expression was reduced in the remobilized hindlimb compared to both the non-immobilized and immobilized hindlimbs of 9-month old rats.
71
Effect of age, immobilization, and remobilization on gastrocnemius muscle mass-to- body mass ratio and RNA content
In order to gain an understanding of the translational capacity of skeletal muscle in response to aging, hindlimb immobilization, and remobilization, ribosome biogenesis was assessed (32). Not enough soleus muscle tissue was available for this analysis, so gastrocnemius muscles from the same rats were used to measure ribosome content. Like the soleus muscle, gastrocnemius mass-to-body mass ratio declined with age, being significantly lower (p < 0.05) in 18-month old rats compared to both 2- and 9-month old rats (Figure 5.8A). Additionally, muscle mass-to-body mass ratio was reduced in rats from all age groups in response to 7 days of hindlimb immobilization. Total RNA content was measured as an indicator of ribosome biogenesis (Figure 5.8B). RNA content declined with age, being significantly lower (p < 0.05) in 9- and 18-month old rats compared to both 2-month old rats, but a statistically significant difference in RNA content was not observed between 9- and 18-month old rats. Although not significantly
(p < 0.05) different, RNA content declined with hindlimb immobilization and increased with remobilization.
Discussion
The goal of the studies presented in this third chapter was to assess REDD1 and
REDD2 mRNA as surrogates for protein expression in skeletal muscle in response to aging and remobilization. This goal was achieved and an association between mTORC1 signaling and REDD2, but not REDD1, mRNA expression in soleus muscle across a
72 number of variables including aging, immobilization and remobilization was observed.
Sarcopenia, defined as an age-related loss of muscle mass and function, is thought to be in large part a consequence of the development of resistance to nutrient (particularly amino acids)-induced stimulation of muscle protein synthesis (144, 170). In young adult humans (e.g. approximately 25 years old), muscle protein synthesis is stimulated in response to consumption of relatively small quantities (e.g. 7-10 g) of essential amino acids, whereas in older adults (e.g. approximately 65 years old) this amount of amino acids is ineffective (107, 108). However, ingesting larger quantities of amino acids (e.g.
25-30 g) effectively stimulates muscle protein synthesis in both young and older individuals (202, 203). Notably, the responsiveness of muscle protein synthesis in the elderly to small quantities of essential amino acids can be overcome by increasing the leucine content of the mixture (73, 108). Similar results have been reported for studies in rats in which the age-related resistance of skeletal muscle protein synthesis to stimulation by a protein diet was overcome by supplementation with leucine (39). Combined, the results from both human and rat studies suggest that the age-related loss of muscle responsiveness to amino acids is largely due to impaired leucine sensitivity.
In both animals (5) and humans (47), leucine induces stimulation of protein synthesis through activation of the mTORC1 signaling pathway. In agreement with the impaired responsiveness of muscle protein synthesis to stimulation by leucine, the sensitivity of mTORC1 signaling to activation by the amino acid is also attenuated in muscle from older individuals compared to young adults (83). In this study a similar phenomenon was observed in rats, i.e. aging was associated with a proportional reduction in nutrient-induced activation of mTORC1 signaling. Notably, the decline in sensitivity
73 of mTORC1 to activation by nutrients was inversely correlated with changes in expression of REDD2, which in addition to REDD1, is a dominant repressor of the pathway. Many studies have focused on the role of REDD1 and/or REDD2 in repressing mTORC1 signaling under conditions of cell stress (36, 57, 141), with many fewer assessing their role under physiological conditions. However, recent reports have demonstrated an inverse correlation between changes in mTORC1 signaling and REDD1 and/or REDD2 expression in skeletal muscle in response to fasting and re-feeding (145) as well as after a bout of exercise (151, 157). This study extends the observations to show that elevated REDD2 mRNA expression in 9- and 18-month old rats is associated with a reduction in mTORC1 signaling. The mechanism through which aging acts to induce REDD2 expression is unknown. However, it is notable that many of the stressors that have been shown to enhance REDD1 and/or REDD2 gene transcription, e.g. endoplasmic reticulum stress (214) or conditions that cause DNA damage (130) or increase production of reactive oxygen species (58), have been reported to be elevated in muscle from older compared to younger individuals (13, 133, 144, 158). Thus, it is tempting to speculate that one, or more, of these stressors is responsible for mediating the induction of REDD2 mRNA expression observed in the present study. It should be noted that, in a previous study in humans (53), no difference in expression of either REDD1 or
REDD2 RNA was observed in skeletal muscle from young compared to older individuals. However, the apparent discrepancy between the results of that study and this one is likely due to the age of the individuals assessed. In this study, a significant reduction in REDD2 mRNA was observed between 2 and 9 months of age, with a further slight, non-significant, reduction occurring between 9 and 18 months. Thus, much of the
74 decline occurred during the transition from young, rapidly growing to middle aged rats, thus emphasizing the importance of the age of the subject being studied.
Chapters 3 and 4 showed mTORC1 signaling to be inversely proportional to both
REDD1 and REDD2 mRNA expression in skeletal muscle of an immobilized rat hindlimb (110, 111). In those studies, a rapid induction of both REDD1 and REDD2 mRNA expression was observed following hindlimb immobilization of 1 to 3 days duration compared to the values observed in either the contralateral, non-immobilized limb or the limb of a control rat (111). In the present chapter, following 7 days of hindlimb immobilization, REDD1 mRNA expression had returned to the control value whereas REDD2 mRNA expression remained elevated. Thus, the rapid induction of
REDD1 mRNA expression observed in the prior studies likely resulted from a stimulus associated with the immobilization procedure. In contrast, based on the results presented here, REDD2 induction would seem to be the more relevant response to immobilization.
Such a possibility is consistent with the finding that of the tissues that have been examined REDD2 mRNA expression is highest in skeletal muscle (152). Again, it should be noted that the data presented herein are for mRNA and not protein expression for REDD2. Presently, assessing changes in REDD2 protein expression is not feasible due to the lack of anti-REDD2 antibodies.
Another key result of the studies presented in this chapter is that the nutrient- induced activation of mTORC1 signaling in response to remobilization is associated with repression of REDD2 mRNA expression. This result agrees with other studies that have also observed an activation of mTORC1 signaling in skeletal muscle following 1-7 days of remobilization (33, 126, 127, 155). Repression of REDD2 mRNA expression during
75 remobilization would provide a mechanism for relieving the resistance to nutrient- induced activation of mTORC1 signaling following immobilization. Notably, despite the activation of mTORC1 signaling, muscle mass continued to fall during the period of remobilization. A similar phenomenon has been previously reported (126, 140). The molecular basis for the delayed recovery of muscle mass with remobilization is unknown.
However, it is tempting to speculate that it may be related to a reduction in RNA content, and thus ribosome abundance. In young, growing animals ribosome number is elevated to allow for higher rates of protein synthesis after an anabolic stimulus, whereas fewer ribosomes would be needed to maintain the relatively static muscle mass in older animals. Thus, an increase in ribosome number in the older animals may be required for recovery of muscle mass following remobilization. Notably, ribosome biogenesis, which is under the control of mTORC1 signaling (99), is reduced during hindlimb suspension and immobilization (9), and enhanced during reloading following hindlimb suspension
(90). Due to the relatively slow turnover of ribosomes, it is not surprising to observe a delay in the response of skeletal muscle protein synthesis and growth (i.e. mass) to immobilization and/or remobilization.
Models of both aging- and inactivity-induced muscle atrophy are associated with insulin resistance in skeletal muscle (29, 35, 159, 205, 215, 220). The alterations in the nutrient-induced stimulation of phosphorylation of Akt at Ser473 observed in the studies described in this chapter in response to aging and hindlimb immobilization and re- mobilization are in agreement with other reports involving hindlimb immobilization and remobilization and functional overload of skeletal muscle (33, 98, 155, 220). It remains to be determined whether these changes in the phosphorylation of Akt at Ser473 are
76 linked with changes in REDD2 mRNA expression. Thus, future studies are needed to determine the contributions of induced REDD2 mRNA expression versus attenuated Akt phosphorylation on mTORC1 signaling during aging, hindlimb immobilization, and remobilization.
In conclusion, nutrient-induced activation of mTORC1 signaling was enhanced and REDD2 mRNA expression was reduced in skeletal muscle from young compared to older rats. Moreover, mTORC1 signaling was activated by 7 days of remobilization in association with reduced REDD2 mRNA expression. No differences in REDD1 mRNA expression were observed in skeletal muscle due to aging, hindlimb immobilization, or remobilization. Therefore, REDD2 expression appears to play a prominent role in the regulation of mTORC1 signaling in skeletal muscle during aging, and hindlimb immobilization and remobilization, and consequently in modulating skeletal muscle mass
(Figure 5.9).
77
78
79
80
81
82 A l
a + t o
T 2.0 # Immobilized Limb / ) s * Control Limb 229 1.5 ue T l
a 1 v
K y
6 1.0 r S a r t i b p70 r
0.5 a ( pho s 0.0
ho 2 9 18 P Age (months)
B l
a 1.0 t
o # Immobilized Limb ) T
s 0.8 Control Limb / ue l a 308
v 0.6
T y t r k a A r 0.4
t i b r pho a 0.2 ( s ho
P 0.0 2 9 18 Age (months)
Figure 5.6 Nutrient-induced activation of PDK1 signaling declines during 7 days of hindlimb immobilization and aging, but in older rats the pathway is not responsive to immobilization. Rats 2-, 9-, and 18-months of age had one hindlimb immobilized for 7 days. Relative phosphorylation of A) p70S6K1 at Thr229 and B) Akt at Thr308 were as- sessed by protein immunoblot analysis. Bars represent the mean phospho/total protein ratio in soleus muscle from control (gray) and immobilized (black) hindlimbs. No differ- ences were observed in total p70S6K1 or Akt protein expression. Data are mean ± SEM, n = 5-6 rats/group. * p < 0.05 compared to control limb; # p < 0.05 compared to equivalent limb in 9-month old rat; + p < 0.05 compared to equivalent limb in 18-month old rat.
83 A
300 Non-Immobilized Limb * # Immobilized Limb % Remobilized Limb A * 200 * RN m
+
x # b 100 # F A M
0 2 9 9R 18 Age (months)
B + # 250 * Non-Immobilized Limb
% Immobilized Limb
200
A Remobilized Limb
RN 150 m
# 1 100 F
R * u 50 M
0 2 9 9R 18 Age (months)
Figure 5.7 MAFbx mRNA increases with age and 7 days of immobilization, while MuRF1 increases with immobilization only in 2-month old animals and decreases in response to remobilization. MAFbx and MuRF1 mRNA expression were assessed by Taqman gene expression assay. Rats, 2-, 9-, and 18-months of age, had one hindlimb immobilized for 7 days, and an additional group of 9-month old rats had their casts removed after 7 days and were allowed to remobilize for an additional 7 days (9R). Bars represent the mean A) MAFbx mRNA-, or B) MuRF1 mRNA-to-Tbp mRNA ratio in soleus from control (dark gray), immobilized (black), and remobilized (light gray) hindlimbs expressed as a percent- age of the same ratio in soleus from control hindlimbs of 9-month old rats. Tbp gene ex- pression was verified against three common loading controls as a reliable housekeeping gene. Data are mean ± SEM, n = 4-6 rats/group. * p < 0.05 compared to control limb. # p < 0.05 compared to equivalent limb in 9-month old rat. + p < 0.05 compared to equivalent limb in 18-month old rat.
84 Figure 5.8
85 Figure 5.9 Regulation of mTORC1 signaling under conditions of aging and 7 days of hindlimb immobilization. Nutrient-induced stimulation of mTORC1 signaling is attenuated under such conditions in association with induction in REDD2 mRNA expression. It is postulated that an induction in REDD2 leads to repression of mTORC1 signaling by pro- moting TSC GAP activity towards Rheb. In addition, aging and 7 days of hindlimb immobi- lization were associated with impaired PDK1 signaling to p70S6K1 and Akt. Insulin resis- tance may play a role in this impairment, but the precise mechanism is unknown.
86 Chapter 6. CONCLUSIONS AND FUTURE DIRECTIONS
The overarching goal of the studies described in this dissertation was to understand the molecular mechanisms responsible for attenuated mTORC1 signaling in immobilized rat hindlimb skeletal muscle. Specifically, I set out to understand how mTORC1 signaling is repressed and becomes resistant to stimulation by nutrients in soleus muscle from hindlimb immobilized rats in order to understand the mechanisms responsible for depressions in the rates of protein synthesis, anabolic resistance and disuse atrophy. I hypothesized that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction of REDD1 and REDD2 expression. The studies conducted for my dissertation research project contribute three major findings to the fields of disuse atrophy and regulation of skeletal muscle mass. First, hindlimb immobilization induced REDD1 and REDD2 mRNA expression in association with attenuated mTORC1 signaling. Second, genes associated with disuse atrophy were induced in skeletal muscle when immobilized in a shortened position, and not in a stretched position. Third, aging was associated with attenuated mTORC1 signaling and an induction in the mRNA expression of REDD2, while remobilization was associated with augmented mTORC1 signaling and a repression in the mRNA expression of
REDD2. Collectively, these findings indicate that the mRNA expression of REDD1, and primarily REDD2 at time points longer than 3 days, is associated with changes in mTORC1 signaling under conditions of hindlimb immobilization, aging, and remobilization. Thus, the results support the overall hypothesis that mTORC1 signaling is repressed in immobilized rat hindlimb skeletal muscle due to induction of REDD1 and
REDD2 mRNA expression as surrogates for REDD1 and REDD2 protein.
87
mTORC1 Signaling and the Genesis of Disuse Atrophy
The studies conducted for my dissertation research project contribute to our understanding of mTORC1 signaling in disuse atrophy. Disuse atrophy is caused by a deficit in muscle protein (69, 72, 121). According to protein balance calculations (162,
171), the imbalance in human muscle protein turnover is the result of depressed rates of protein synthesis rather than augmented rates of protein degradation (61). As a master regulator of the rates of protein synthesis in skeletal muscle (51), much attention has focused on mTORC1 signaling during disuse (80, 143). Attenuated mTORC1 signaling has been observed during hindlimb immobilization (111, 126, 220) and unloading (10,
54, 94), but these studies assessed mTORC1 signaling after disuse atrophy was already manifest. Hindlimb immobilization depressed skeletal muscle protein synthesis after 6 hours (22) and 1 day (127) of immobilization, but mTORC1 signaling was not measured at these early time points. This led my colleagues and I to question whether attenuated mTORC1 signaling is an initial or adaptive response to hindlimb immobilization. I hypothesized that attenuated mTORC1 signaling is an initial response to hindlimb immobilization and plays a major role in causing disuse atrophy. If attenuated mTORC1 signaling causes depressed rates of protein synthesis, then attenuated mTORC1 signaling should be observed at the same time or prior to observing depressed rates of protein synthesis. Chapter 3 is the first to report attenuated mTORC1 signaling in muscle from an immobilized hindlimb after just 1 day of immobilization. The observance of attenuated mTORC1 signaling after 1 day and disuse atrophy after 3 days indicates that the attenuation of mTORC1 signaling is an initial response to hindlimb immobilization
88 that leads to disuse atrophy. Unfortunately, mTORC1 signaling and protein synthesis were not measured earlier than 1 day of immobilization because these experiments were designed to screen for repressors of mTORC1 signaling across the first few days of hindlimb immobilization. As a result, the sequence of initial responses within the first few hours of hindlimb immobilization (i.e. attenuated mTORC1 signaling followed by depressed protein synthesis) remains unknown. Future studies will need to assess both mTORC1 signaling and rates of protein synthesis in skeletal muscle from immobilized hindlimbs within 1 day of immobilization (particularly < 6 hours after immobilization).
Moreover, future studies will need to determine the definitive role of attenuated mTORC1 signaling in depressed rates of protein synthesis during hindlimb immobilization. Similar to what has been used in mouse denervation studies, this could be achieved by measuring rates of protein synthesis in muscle from an immobilized rodent hindlimb where mTORC1 signaling is increased using shRNA or genetic knockout of TSC1 (15).
The most important finding of the studies conducted for my dissertation research project is the identification of REDD1 and REDD2 mRNA induction in association with attenuated mTORC1 signaling during hindlimb immobilization. This is important because the induction of REDD1 and REDD2 mRNA as surrogates of REDD1 and
REDD2 protein may provide a mechanism responsible for repression of mTORC1 signaling during hindlimb immobilization. Before this thesis project, evidence supported an association between attenuated mTORC1 signaling and disuse atrophy (10, 94, 220), but the mechanism responsible for attenuated mTORC1 signaling during disuse was unknown. As described in Chapter 3, known repressors of mTORC1 signaling were
89 assessed in muscle samples from immobilized and non-immobilized hindlimbs and an induction in REDD1 and REDD2 mRNA expression was observed in soleus muscle from immobilized hindlimbs. Importantly, this induction was observed after 1 day of hindlimb immobilization in association with attenuated mTORC1 signaling. In the studies described in Chapter 4, I observed another link between induction of REDD1 and
REDD2 mRNA expression and attenuated mTORC1 signaling in soleus muscle that was immobilized in a shortened, but not a stretched position. Finally, I observed a link between REDD2 mRNA expression and mTORC1 signaling in soleus muscle with aging,
7 days of hindlimb immobilization and remobilization. The results of these studies indicate that hindlimb immobilization, particularly in a shortened position, induces
REDD1 and REDD2 mRNA expression in association with attenuated mTORC1 signaling. An induction in REDD1 and REDD2 mRNA likely leads to an induction in
REDD1 and REDD2 protein, which represses mTORC1 signaling. Specifically, the data indicate that both REDD1 and REDD2 modulate mTORC1 signaling in response to an acute stimulus (e.g. 1-3 days of hindlimb immobilization), while REDD2 modulates mTORC1 signaling in response to a chronic stimulus (e.g. 7 days of hindlimb immobilization/remobilization and aging). The reason for discrepancy between REDD1 versus REDD2 during aging and hindlimb immobilization is unclear because REDD1 and
REDD2 are believed to repress mTORC1 signaling by a similar mechanism (152, 196).
In addition, little is known about the regulation of REDD1 and REDD2 mRNA in response to aging and hindlimb immobilization. In order to understand the roles and discrepancies between REDD1 and REDD2 during aging and hindlimb immobilization, future studies should investigate the mechanisms responsible for regulation of REDD1
90 and REDD2 mRNA transcription, degradation, and translation in order to help us understand how REDD1 and REDD2 expression is induced under these conditions.
Anabolic Resistance
REDD1 and REDD2 are both repressors and governors of the capacity for mTORC1 signaling (57, 152). Reducing the maximum capacity for mTORC1 signaling, overexpression of REDD2 in muscle cells diminished mTORC1 signaling in response to leucine and mechanical stimulation (152). Moreover, elevated expression of REDD1 in diabetic rat skeletal muscle was associated with blunted stimulation of mTORC1 signaling in response to feeding (145). Blunting the stimulation of mTORC1 signaling,
REDD1 and REDD2 expression may act as a governor of mTORC1 signaling by modulating the capacity for mTORC1 stimulation. As repressors and governors of mTORC1 signaling, REDD1 and REDD2 are likely to repress mTORC1 signaling under non-stimulated conditions and make mTORC1 signaling resistant to stimulation by nutrients, growth factors and exercise. Therefore, if mTORC1 signaling modulates skeletal muscle protein synthesis, induction of REDD1 and REDD2 may provide a mechanism responsible for the depression in the rates of protein synthesis observed in a fasted state and anabolic resistance to nutrient stimulation of protein synthesis during hindlimb immobilization.
Induction of REDD1 and REDD2 during aging and hindlimb immobilization may provide a mechanism responsible for anabolic resistance to nutrient-induced stimulation of mTORC1 signaling. Nutrient and growth factor signaling are both necessary, but not sufficient, for full activation of mTORC1 signaling (46, 149). Nutrients, particularly
91 leucine, are important for stimulation of skeletal muscle protein synthesis (28, 37, 108,
173). Although this mechanism is still unclear, nutrients stimulate protein synthesis by signaling through the Rag-Ragulator complex and targeting mTORC1 to the lysosomal surface where mTORC1 is activated by Rheb•GTP (41, 179). However, amino acid- mediated stimulation of protein synthesis is blunted if plasma insulin levels are reduced or prevented from increasing in response to nutrients using somatostatin (6, 7). Insulin plays a permissive role in the stimulation of skeletal muscle protein synthesis by amino acids and exercise (117) and promotes stimulation of skeletal muscle protein synthesis through activation of mTORC1 signaling and eIF4F complex formation (5, 7, 117).
Insulin promotes mTORC1 activation by activating phosphatidylinositol-3-OH kinase
(PI(3)K)-Akt signaling, which leads to the phosphorylation of TSC2 and inhibition of
TSC2’s ability to regulate Rheb (46). In the absence or repression of PI(3)K-Akt signaling, the TSC complex maintains Rheb in its GDP-bound state and blocks the activation of mTORC1 by nutrients. If an induction in REDD1 and REDD2 expression impairs PI(3)K-Akt signaling and promotes TSC2 GAP activity towards Rheb, then this will block the activation of mTORC1. In response to a leucine bolus as described in
Chapter 3 and feeding as described in Chapter 5, respectively, the activation of mTORC1 was either blunted or lowered in association with an induction in REDD1 and/or REDD2 mRNA expression. The observations suggest that REDD1 and REDD2 may cause resistance to nutrient-induced activation of mTORC1 by promoting TSC2 GAP activity towards Rheb, allowing Rheb•GTP to be converted into Rheb•GDP. Since mTORC1 activation would be limited by the abundance of Rheb•GTP in this scenario, this could
92 provide a mechanism responsible for anabolic resistance to nutrient-induced stimulation of mTORC1 signaling.
In addition to repression by REDD1 and REDD2, insulin resistance likely impairs insulin signaling to mTORC1 during hindlimb immobilization. Physical activity plays a profound role in skeletal muscle Akt activation and insulin sensitivity. Stretching (3, 49,
185) and muscle contraction (52, 64, 104) increase Akt phosphorylation, mTORC1 signaling and skeletal muscle insulin sensitivity of glucose transport (91, 92, 175). A key regulator of muscle protein turnover, Akt promotes mTORC1 signaling through the phosphorylation of TSC2 (102) and the mTORC1-component, proline-rich Akt substrate of 40 kDa (181). Akt also suppresses atrogene expression through the phosphorylation of
FoxO transcription factors (183). In contrast to muscle contraction, physical inactivity is associated with insulin resistance (81). Attenuated phosphorylation of Akt at Ser473 has been observed in muscles during disuse (54, 87, 159, 220). This has led to speculation that attenuated Akt activation is responsible for attenuated mTORC1 signaling and induction of atrogene expression (220). While phosphorylation of Akt at Ser473 was attenuated in muscle fixed in a shortened position for 3 (Figure 4.5) and 7 (Figure 5.3) days, it was not attenuated after 1 day (Table 3.2). Since mTORC1 signaling was attenuated after 1 day of hindlimb immobilization, attenuated Akt signaling is not likely the cause of the initial attenuation of mTORC1 signaling. Rather, attenuated Akt phosphorylation is likely an adaptive response to hindlimb immobilization in a shortened position. Hindlimb immobilization in a shortened position increased the rates of protein degradation in soleus muscle after 2 days, but not 1 day (75). Perhaps the increase in rates of protein degradation is delayed 1 day because it takes longer for hindlimb
93 immobilization to reduce Akt signaling and induce FoxO-mediated atrogene expression.
Both the phosphorylation of Akt at Ser473 and FoxO3a at Ser253 (an Akt phosphorylation site) were attenuated after 3 days of immobilization in a shortened position (Figure 4.5) in association with induction of atrogene mRNA expression (Figure
4.4). This suggests that attenuated Akt signaling by at least 3 days of immobilization in a shortened position induces atrogene expression through FoxO3a. Interestingly, the phosphorylation of both Akt and FoxO3a at these sites was augmented when the soleus muscle was immobilized in a stretched position. This indicates that atrogene expression may be repressed in muscle immobilized in a stretched position through enhanced Akt signaling to FoxO3a. Future studies will need to determine the extent to which Akt signaling contributes to attenuated mTORC1 signaling and induction of atrogene expression in muscle from immobilized hindlimbs.
Insulin resistance and attenuated insulin signaling may contribute to anabolic resistance to nutrient-induced stimulation of mTORC1 signaling through impaired PDK1 signaling to p70S6K1. Insulin resistance is manifested in soleus muscle after 1 day of hindlimb immobilization (29, 187) and hindlimb unloading (159) measured by reduced uptake of 2-deoxyglucose. One day of hindlimb immobilization attenuated nutrient- induced phosphorylation of p70S6K1 at Thr229, a PDK1 phosphorylation site (112), in association with blunted stimulation of p70S6K1 phosphorylation at Thr389. Under fed conditions, an association between attenuated phosphorylation of p70S6K1 at Thr229 and
Thr389 was also observed in soleus muscle after 7 days of hindlimb immobilization
(Figure 5.6). As described in Chapter 3, phosphorylation of p70S6K1 at Thr229 by
PDK1 is required for phosphorylation of p70S6K1 at Thr389 by mTOR. If insulin
94 resistance lessens the activation of PDK1 in response to feeding, this could blunt PDK1- mediated phosphorylation of p70S6K1 at Thr229. This in turn would prevent phosphorylation of p70S6K1 at Thr389 by mTOR (112), which is necessary for full activation of p70S6K1 and stimulation of protein synthesis. In contrast to this theory, the phosphorylation of Akt at Thr308, a site phosphorylated by PDK1 (4), is not attenuated in soleus muscle after 1 day of hindlimb immobilization (Table 3.2). Therefore, blunted phosphorylation of p70S6K1 at Thr229 may be caused by a mechanism independent of insulin resistance or weakened activation of PDK1. Future studies will need to determine the role of phosphorylation of p70S6K1 at Thr229 in nutrient-induced stimulation of mTORC1 signaling and explore alternative causes for impaired stimulation of p70S6K1
Thr229 phosphorylation. Such causes might include impaired phosphorylation of other sites on p70S6K1 required for its full activation, localization of p70S6K1 or phosphatase activity towards Thr229 and Thr389.
Aging is also associated with insulin resistance and anabolic resistance to nutrient-induced stimulation of protein synthesis (for review see (35) and (170)).
Numerous studies have observed anabolic resistance to stimulation by nutrients in muscle from old compared to young subjects (38, 39, 83, 174). Although the mechanism responsible for age-induced anabolic resistance is unknown, insulin resistance may play a role. In addition to its permissive role in the stimulation of skeletal muscle protein synthesis at the cellular level (5, 7, 117), insulin also promotes nitric oxide-mediated, endothelial-dependent vasodilation, which improves amino acid delivery throughout muscle tissue by increased muscle blood flow and microvascular perfusion (35, 148,
205). Aging causes insulin resistance, which impairs insulin’s ability to augment skeletal
95 muscle blood flow (148). Interestingly, when the legs of insulin-resistant elderly subjects were infused with insulin plus a pharmacological vasodilator, sodium nitroprusside, this improved muscle microvascular perfusion, Akt phosphorylation and skeletal muscle protein synthesis (205). Therefore, insulin resistance (as determined by oral glucose tolerance test and muscle perfusion measurements) may contribute to anabolic resistance by impairing insulin-mediated delivery of amino acids and activation of the Akt- mTORC1 signaling pathway (215). In contrast, anabolic resistance was observed in muscle from elderly subjects despite no differences in interstitial amino acid concentrations or phenylalanine transport between muscle samples from young and elderly (55). As described in Chapter 5, aging and 7 days of hindlimb immobilization exhibited attenuated phosphorylation of Akt at Ser473 and nutrient-induced stimulation of mTORC1 signaling. In association with attenuated nutrient-induced stimulation of mTORC1 signaling, aging and 7 days of hindlimb immobilization attenuated phosphorylation of p70S6K1 at Thr229 (Figure 5.6A). Unlike what was observed after 1 day of hindlimb immobilization, aging and 7 days of hindlimb immobilization are associated with attenuated phosphorylation of Akt at Thr308 (Figure 5.6B), which indicates that PDK1 signaling is attenuated under these conditions. Since aging and hindlimb immobilization in humans and rodents are associated with insulin resistance, and insulin signaling activates PDK1 and Akt, the attenuated phosphorylation of Akt and p70S6K1 with aging and 7 days of hindlimb immobilization may result from insulin resistance. If so, this may provide further support for a mechanism of anabolic resistance where defective PDK1-mediated phosphorylation of p70S6K1 at Thr229 blunts full activation of p70S6K1.
96 Impaired growth factor signaling (e.g. impaired insulin signaling) may also contribute to anabolic resistance through induction of REDD1 and REDD2.
Gastrocnemius muscle from diabetic rats exhibited elevated REDD1 mRNA expression compared to non-diabetic controls (145). Moreover, fasting (low insulin signaling) caused an elevation in REDD1 mRNA and protein expression, while refeeding (higher insulin signaling) decreased REDD1 expression relative to fasted conditions. IGF-1 treatment decreased REDD2 mRNA expression in rat gastrocnemius muscle and myotubes (63), indicating that REDD2 expression is also repressed by growth factor signaling. Although the mechanism is still unclear, growth factor signaling may induce
REDD1 expression through a mechanism involving ER stress-mediated induction of
ATF4 (43). REDD1 mRNA expression in response to growth factor signaling seems to be regulated by mRNA transcription, as measured by nuclear run-on assay in Rat 2 fibroblasts in response to serum stimulation and deprivation. In contrast to this theory, soleus muscle in my model was resistant to stimulation of mTORC1 signaling (Figure 3.3 and Figure 3.5) and REDD1 and REDD2 mRNA expression (Figure 3.4) were induced after 1 day of hindlimb immobilization despite no apparent activation of ER stress (i.e. no increases in eIF2alpha Ser51 phosphorylation or ATF4 mRNA and protein expression,
Table 3.2). While impaired growth factor signaling may still contribute to the induction in REDD1 and REDD2 mRNA expression during hindlimb immobilization, it does not occur through ER stress and/or induction of ATF4. Future studies will need to elucidate the mechanism by which growth factor signaling and induction of REDD1 and REDD2 contribute to anabolic resistance.
97 Limitations
While this dissertation provides evidence of a link between induction of REDD1 and REDD2 mRNA expression and repression of mTORC1 signaling during aging and hindlimb immobilization, this link could be strengthened by additional experiments.
First, this link could be strengthened by demonstrating a temporal relationship between induction of REDD1 and REDD2 mRNA expression and repression of mTORC1 signaling at earlier time points following hindlimb immobilization. As demonstrated in
MEFs treated with cycloheximide, changes in REDD1 expression precede observed changes in mTORC1 signaling (116). In the genesis of disuse atrophy, I propose that hindlimb immobilization induces REDD1 and REDD2 mRNA expression as a cause for attenuated mTORC1 signaling and protein synthesis. If my proposal is correct, I would observe the following sequentially in skeletal muscle from an immobilized hindlimb: 1) induction of REDD1 and REDD2 mRNA expression; 2) an increase in REDD1 and
REDD2 protein expression; 3) attenuated mTORC1 signaling; 4) depressed rates of protein synthesis; and finally, 5) disuse atrophy. The earliest time point for which I have data (1 day of immobilization) already exhibits attenuated rates of protein synthesis, so I can only assume that the induction in REDD1 and REDD2 mRNA preceded the other events. I do not have data to support a temporal cause-and-effect relationship in which
REDD1 and REDD2 mRNA expression was induced before attenuated mTORC1 signaling. As mentioned earlier, future experiments will need to measure these variables at earlier time points (< 6 hours following hindlimb immobilization (22)) in order to understand the sequential progression of events leading to disuse atrophy.
98 A second limitation in these studies is the lack of data on REDD1 and REDD2 protein expression. REDD1 and REDD2 protein expression was not measured because presently there are no reliable, commercially available antibodies for measuring REDD1 and REDD2 protein in rat skeletal muscle. Though REDD1 mRNA and protein expression are both induced in response to glucocorticoids (211), starvation (145), ER stress (214), and phorbol 12-myristate 13-acetate treatment (221), cycloheximide treatment induced REDD1 protein but not REDD1 mRNA expression (116).
Consequently, while it is highly likely that the observed induction in REDD1 and
REDD2 mRNA expression increased REDD1 and REDD2 protein expression, respectively, REDD1 mRNA expression is not always synonymous with REDD1 protein expression. When reliable protein detection tools for REDD1 and REDD2 are available, future studies will need to measure these and confirm that REDD1 and REDD2 protein expression is increased in conjunction with induction of the mRNA.
Gene isolation-manipulation techniques are necessary to determine the precise roles of REDD1 and REDD2 in the modulation of mTORC1 signaling during hindlimb immobilization, aging, and remobilization. To determine the roles of REDD1 and
REDD2 in attenuated mTORC1 signaling under these conditions, REDD1 and/or REDD2 mRNA expression needs to be manipulated as an independent variable. Using techniques such as gene-transfection muscle-electroporation (189, 208), adenoviral gene delivery systems (59), si/shRNA injection (14), and genetically-modified animals (153), the gene expression of REDD1 and REDD2 can be increased, decreased, or removed.
Manipulating the gene expression of REDD1 and REDD2 using these techniques is necessary for two reasons. First, silencing or knocking-out REDD1 and REDD2 mRNA
99 expression during hindlimb immobilization will help us determine the extent to which these genes influence mTORC1 signaling. According to my overall hypothesis, silencing
REDD1 and REDD2 gene expression during hindlimb immobilization will prevent the attenuation of mTORC1 signaling and protein synthesis, and prevent disuse atrophy. In contrast, if silencing REDD1 and REDD2 gene expression has no effect on these variables during hindlimb immobilization, I would be forced to reject my hypothesis. A second reason for manipulating the gene expression of REDD1 and REDD2 is to understand the contributions of REDD1 versus REDD2 in the repression of mTORC1 signaling during hindlimb immobilization. Manipulating the gene expression of REDD1 and REDD2 individually and simultaneously is important to understand the extent to which these genes repress mTORC1 signaling in an independent versus cooperative manner.
In a pilot study, wild-type BL6/C129 and Bl6/C129 REDD1 knockout mice were procured from Dr. David Williamson (SUNY Oswego) and subjected to unilateral hindlimb immobilization for 5 days. The mice were subjected to unilateral hindlimb immobilization using a 1.5 mL Eppendorf tube as described previously (127). After 5 days of hindlimb immobilization in a shortened position, the soleus muscles were extracted from both the immobilized and contralateral control hindlimbs of wild-type and
REDD1 knockout mice. No differences in body mass, or soleus muscle-to-body mass ratio were observed between wild-type and REDD1 knockout mice (Figure 6.1). In response to 5 days of hindlimb immobilization, soleus muscle mass-to-body mass ratio was reduced in the immobilized hindlimb compared to the control hindlimb. However, no differences were observed between wild-type and REDD1 knockout mice. In contrast,
100 phosphorylation of p70S6K1 at Thr389 was significantly (p < 0.05) reduced in soleus muscle from the immobilized hindlimb of wild-type mice, but not in REDD1 knockout mice. This result supports my hypothesis that an induction in REDD1 expression is responsible for attenuated mTORC1 signaling during hindlimb immobilization. Based on this data, REDD1 is necessary for the repression of mTORC1 signaling in soleus muscle in response to 5 days of hindlimb immobilization, but not disuse atrophy. The reason for this discrepancy between muscle mass and mTORC1 signaling is unknown, but there are numerous possible explanations. First, the disuse atrophy after 5 days of hindlimb immobilization is mice may primarily be the result of augmented rates of protein degradation. Second, the hindlimb immobilization may depress rates of protein synthesis in skeletal muscle downstream of mTORC1 signaling. Third, there may be differences in the genesis of disuse atrophy between models (tube fitting versus casting) or species (mouse versus rat). Collectively, these results indicate that REDD1 plays an important role in the repression of mTORC1 signaling during hindlimb immobilization but not in disuse atrophy. Future studies will need to assess the regulation of mRNA translation downstream of mTORC1 signaling and determine the specific contributions of depressed rates of protein synthesis versus augmented rates of protein degradation to disuse atrophy in rodents subjected to hindlimb immobilization.
The mRNA expression of REDD2 and other genes associated with disuse atrophy were not measured in this study. It is possible that both REDD1 and REDD2 have overlapping functions in the repression of mTORC1 signaling across 5 days of hindlimb immobilization and perhaps mTORC1 signaling was repressed in the immobilized hindlimb during the first 4 days of hindlimb immobilization. Despite a lack of REDD1
101 induction, an induction in REDD2 mRNA during hindlimb immobilization may be enough to repress mTORC1 signaling. Future studies will need to assess mTORC1 signaling and muscle mass during hindlimb immobilization in REDD2 and REDD1/2 knockout mice in order to determine the individual versus overlapping roles of REDD1 and REDD2 in disuse atrophy.
At present, the role of Rheb GTP-state in the activation of mTORC1 during disuse is unknown. Although the mechanism is unclear, REDD1 and REDD2 repress mTORC1 signaling upstream of Rheb in a TSC2-dependent manner (152, 196). If an induction in
REDD1 and REDD2 expression is responsible for attenuated mTORC1 signaling during disuse, then the abundance of REDD1 and REDD2 should increase TSC2 GAP activity towards Rheb (101). This could be tested in two ways. First, future studies should confirm that Rheb-mediated activation of mTORC1 is in fact attenuated with disuse.
This could be accomplished by measuring the activation state of Rheb (muscle samples from immobilized hindlimbs should have a high proportion of Rheb in its inactive GDP state) and the phosphorylation state of Raptor sites such as Ser863 that are associated with Rheb•GTP (141). Second, Rheb-mediated activation of mTORC1 could be manipulated by transfecting normal or constitutively active Rheb (ca-Rheb•GTP) into skeletal muscles of immobilized hindlimbs. Overexpression of Rheb blocked the repressive effects of both REDD1 (196, 211) and REDD2 (152) on mTORC1 signaling.
If REDD1 and REDD2 repress mTORC1 signaling through TSC2 during hindlimb immobilization, then an increase in the expression and activation state of the downstream target, Rheb, will overcome this repression. Thus, no differences in mTORC1 signaling should be observed between immobilized and non-immobilized hindlimbs. Similarly, a
102 reduction in TSC2 expression by siTSC2 injection should prevent attenuated mTORC1 signaling during disuse, because REDD1 and REDD2 require TSC2 for repression of mTORC1 signaling (45, 152, 196). In this scenario, despite induction of REDD1 and
REDD2, a decrease in TSC2 expression would reduce the conversion of Rheb•GTP into
Rheb•GDP, allowing Rheb•GTP to activate mTORC1 signaling. Future experiments are needed to investigate the role of Rheb signaling in attenuated mTORC1 signaling during hindlimb immobilization.
Muscle Length and Disuse Atrophy
Identifying REDD1 and REDD2 as repressors of mTORC1 signaling with disuse, the studies described in Chapter 4 focused on understanding the physiological trigger responsible for induction of these genes and others associated with disuse atrophy.
Understanding the physiological trigger responsible for inducing these genes was important to elucidate the molecular mechanism responsible for regulation of REDD1 and REDD2 mRNA expression. It was known that muscle fixed in a stretched position does not exhibit a depression in the rates of protein synthesis and disuse atrophy during hindlimb immobilization (75, 76, 137). In addition, it was known that muscle fixed in a stretched position does not exhibit decreases in both functional and metabolic properties
(e.g. prevented decrease in oxidative capacity and maximum tension after 3 days of hindlimb suspension) (184), and stretching spared the expression of genes associated with slow muscle phenotype (147). The key contribution of the studies is that fixed muscle length also modulates mTORC1 signaling and the expression of key genes associated with disuse atrophy. This finding is important because it may explain the differences in
103 mTORC1 signaling observed during disuse between animal (10, 94, 220) and human studies (40, 72). Muscles are fixed in shortened versus neutral positions in animal versus human studies, respectively. Moreover, this finding is important because it helps us to understand how critical genes associated with disuse atrophy are induced with hindlimb immobilization.
In light of the results presented in Chapter 4, the next question becomes ‘how does fixed muscle length regulate the gene expression of key genes associated with disuse atrophy?’ Answering this question will require identification of transcription factors responsible for inducing the expression of these genes, and elucidation of the mechanisms by which muscle sarcomere proteins regulate gene expression. Future studies should investigate how muscle sarcomeric-binding proteins with mechanosensory functions are involved with the regulation of genes associated with disuse atrophy. For example, future studies could use chromatin immunoprecipitation (ChIP) assays and identify key transcription factors that bind promoter and other key binding regions in the
DNA for REDD1, REDD2 and atrogenes. Key transcription factors responsible for inducing the mRNA expression of genes associated with disuse atrophy should bind to the promoter region when muscle is placed in a shortened position and ‘pulled down’ during precipitation of the promoter DNA. These studies should also explore the role of proteins such as titin and other sarcomeric proteins that regulate muscle gene expression in response to mechanical strain on the sarcomere (124, 132). Titin is a particularly interesting protein to study in future investigations because it senses changes in muscle sarcomere length with stress and strain associated with muscle loading (67, 124, 210).
These changes in stress and strain lead to conformational changes in the titin protein
104 structure, which influence the binding properties of interacting proteins (67, 124, 128). It has been postulated that titin senses mechanical arrest in diaphragm muscle when patients are mechanically ventilated, leading to the changes in signaling and protein expression associated with muscle atrophy (161). However, no study to my knowledge has investigated the role of titin or its binding partners in attenuated mTORC1 signaling and protein synthesis during disuse. Other likely protein candidates with mechanosensory functions include obscurin, nebulin, myopalladin, muscle LIM protein, and nuclear factor of activated T-cells C (113, 132, 207). Therefore, future studies will need to elucidate the mechanism by which titin and other sarcomeric proteins regulate the transcriptional properties of disuse-specific transcription factors and other mechanisms that effect gene expression (e.g. expression of microRNAs, mRNA degradation, mRNA translation, etc.) in response to a shortened muscle length.
Elucidating the mechanism by which fixed muscle length regulates mTORC1 signaling and gene expression will require assessment of additional time points and fixed muscle lengths. One day of hindlimb immobilization in a stretched position was found to repress the expression of atrogenes (195) and genes associated with fast muscle phenotype (77), and activate genes associated with slow muscle phenotype and ribosome biogenesis in rats (76, 77). By 4 days of immobilization in a stretched position, atrogene expression returned to control values in association with an increase in the number of sarcomeres in series (195). Adding and subtracting the number of sarcomeres in series in response to hindlimb immobilization in either a stretched or shortened position, respectively, maintains optimal muscle strain and sarcomere length, while protecting the muscle from being overstretched (204, 216). Since skeletal muscle adds sarcomeres in
105 series in response to fixation in a stretched position, the muscle may experience a stronger stretch stimulus in the days immediately following hindlimb immobilization and a weaker stretch stimulus at later time points (184). If muscle atrogene expression is repressed by a stretch-sensitive mechanism, this may explain why atrogene expression was repressed in soleus muscle fixed in a stretched position after only 1 day, but not after the muscle has added sarcomeres in series by 4 days (195). If the transcriptional machinery responsible for induction of REDD1 mRNA is more sensitive to changes in muscle length than that of REDD2 mRNA, this may also explain why REDD1, but not
REDD2, mRNA returned to control values after 7 days of hindlimb immobilization
(Figure 5.4). Future studies should assess REDD1 and REDD2 mRNA expression after earlier (1 and 2 days) and later (>4 days) periods of hindlimb immobilization in a stretched position in order to understand the initial and adaptive responses of REDD1 and
REDD2 mRNA expression to hindlimb immobilization in a stretched position. In addition, more fixed muscle lengths should also be assessed in future studies. Comparing various shortened, neutral, and stretched muscle lengths during hindlimb immobilization will help us understand the extent to which REDD1 and REDD2 mRNA expression is either repressed by an increase in muscle length or induced by a decrease in muscle length (62). These experiments are also important to help us understand the role of muscle length in the regulation of other activators of mTORC1 signaling in response to mechanical stimuli such as phosphatidic acid and Akt (93, 95, 96, 185).
106 Conclusion
In conclusion, the mRNA expression of REDD1, and primarily REDD2 at times longer than 3 days, is associated with changes in mTORC1 signaling under conditions of hindlimb immobilization, aging, and remobilization (Figure 3.6 and Figure 5.9). My overall hypothesis was supported whereby mTORC1 signaling was repressed in immobilized rat hindlimb skeletal muscle in association with induction of REDD1 and
REDD2 mRNA expression. The studies conducted for this dissertation research project contributed three major findings to the fields of disuse atrophy and regulation of skeletal muscle mass including 1) hindlimb immobilization induced the mRNA expression of
REDD1 and REDD2 in association with attenuated mTORC1 signaling under both fasted and nutrient-stimulated conditions; 2) these genes were induced when skeletal muscle was immobilized in a shortened but not a stretched position; and 3) aging and 7 days of hindlimb immobilization induced REDD2 mRNA expression in association with attenuated mTORC1 signaling, while remobilization repressed REDD2 mRNA expression in association with augmented mTORC1 signaling. However, more studies are needed to elucidate the roles of REDD1 and REDD2, insulin signaling, and fixed muscle length in the regulation of mTORC1 signaling and anabolic resistance during hindlimb immobilization.
107 A o i t a
r #
0.0004 Control Limb ss a * Immobilized Limb m
0.0003 y * bod 0.0002 o t ss
a 0.0001 m
e l 0.0000 sc
u WT REDD1 KO M
B 9 38 r
h 0.8 ) T Control Limb t 1 a K Immobilized Limb 6 1 S K 0.6 6 S p70
l * a t p70 f
o 0.4 o
/ t ion t pho 0.2 s la y r pho (
pho 0.0 s WT REDD1 KO ho P
Figure 6.1. Phosphorylation of p70S6K1 at Thr389 was not attenuated despite reduced muscle mass-to-body mass ratio in soleus muscle from the immobilized hindlimb of REDD1 knockout mice. Mice had one hindlimb immobilized for 5 days in a position to place the soleus in a shortened position. Bars represent the mean A) muscle mass-to- body mass ratio or B) phosphorylation of p70S6K1 at Thr389/total p70S6K1 protein ratio. No changes in total p70S6K1 expression were observed in any group across 5 days of hindlimb immobilization. Data are mean percentages ± SEM, n = 5 mice/group. * p < 0.05 compared to the contralateral control hindlimb. # p < 0.05 compared to the equivalent limb in the wild type mouse.
108 REFERENCES
1. In: International Animal Research Regulations: Impact on Neuroscience
Research: Workshop Summary. Washington (DC): 2012.
2. Abadi A, Glover EI, Isfort RJ, Raha S, Safdar A, Yasuda N, Kaczor JJ,
Melov S, Hubbard A, Qu X, Phillips SM, and Tarnopolsky M. Limb immobilization induces a coordinate down-regulation of mitochondrial and other metabolic pathways in men and women. PLoS One 4: e6518, 2009.
3. Agata N, Sasai N, Inoue-Miyazu M, Kawakami K, Hayakawa K, Kobayashi
K, and Sokabe M. Repetitive stretch suppresses denervation-induced atrophy of soleus muscle in rats. Muscle Nerve 39: 456-462, 2009.
4. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, and
Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1.
Embo J 15: 6541-6551, 1996.
5. Anthony JC, Anthony TG, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J Nutr 130: 139-145, 2000.
6. Anthony JC, Lang CH, Crozier SJ, Anthony TG, MacLean DA, Kimball SR, and Jefferson LS. Contribution of insulin to the translational control of protein synthesis in skeletal muscle by leucine. Am J Physiol Endocrinol Metab 282: E1092-1101, 2002.
7. Anthony JC, Reiter AK, Anthony TG, Crozier SJ, Lang CH, MacLean DA,
Kimball SR, and Jefferson LS. Orally administered leucine enhances protein synthesis in skeletal muscle of diabetic rats in the absence of increases in 4E-BP1 or S6K1 phosphorylation. Diabetes 51: 928-936, 2002.
109 8. Ayuso MI, Hernandez-Jimenez M, Martin ME, Salinas M, and Alcazar A.
New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress. J Biol Chem 285: 34355-34363, 2010.
9. Babij P, and Booth FW. Alpha-actin and cytochrome c mRNAs in atrophied adult rat skeletal muscle. Am J Physiol 254: C651-656, 1988.
10. Bajotto G, Sato Y, Kitaura Y, and Shimomura Y. Effect of branched-chain amino acid supplementation during unloading on regulatory components of protein synthesis in atrophied soleus muscles. Eur J Appl Physiol 111: 1815-1828, 2011.
11. Baptista IL, Leal ML, Artioli GG, Aoki MS, Fiamoncini J, Turri AO, Curi
R, Miyabara EH, and Moriscot AS. Leucine attenuates skeletal muscle wasting via inhibition of ubiquitin ligases. Muscle Nerve 41: 800-808, 2010.
12. Bar-Shai M, Carmeli E, Ljubuncic P, and Reznick AZ. Exercise and immobilization in aging animals: the involvement of oxidative stress and NF-kappaB activation. Free Radic Biol Med 44: 202-214, 2008.
13. Bar-Shai M, Carmeli E, and Reznick AZ. The role of NF-kappaB in protein breakdown in immobilization, aging, and exercise: from basic processes to promotion of health. Ann N Y Acad Sci 1057: 431-447, 2005.
14. Ben Sahra I, Regazzetti C, Robert G, Laurent K, Le Marchand-Brustel Y,
Auberger P, Tanti JF, Giorgetti-Peraldi S, and Bost F. Metformin, independent of
AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer Res 71:
4366-4372, 2011.
15. Bentzinger CF, Lin S, Romanino K, Castets P, Guridi M, Summermatter S,
Handschin C, Tintignac LA, Hall MN, and Ruegg MA. Differential response of
110 skeletal muscles to mTORC1 signaling during atrophy and hypertrophy. Skelet Muscle 3:
6, 2013.
16. Biolo G, Ciocchi B, Lebenstedt M, Barazzoni R, Zanetti M, Platen P, Heer
M, and Guarnieri G. Short-term bed rest impairs amino acid-induced protein anabolism in humans. J Physiol 558: 381-388, 2004.
17. Bodine SC. Disuse-induced muscle wasting. Int J Biochem Cell Biol 45: 2200-
2208, 2013.
18. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA,
Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ, Valenzuela DM,
DeChiara TM, Stitt TN, Yancopoulos GD, and Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704-1708, 2001.
19. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R,
Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, and Yancopoulos GD.
Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol 3: 1014-1019, 2001.
20. Bolster DR, Crozier SJ, Kimball SR, and Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277: 23977-23980,
2002.
21. Booth FW. Time course of muscular atrophy during immobilization of hindlimbs in rats. J Appl Physiol Respir Environ Exerc Physiol 43: 656-661, 1977.
22. Booth FW, and Seider MJ. Early change in skeletal muscle protein synthesis after limb immobilization of rats. J Appl Physiol 47: 974-977, 1979.
111 23. Booth FW, and Seider MJ. Recovery of skeletal muscle after 3 mo of hindlimb immobilization in rats. J Appl Physiol 47: 435-439, 1979.
24. Brooks NE, and Myburgh KH. Skeletal muscle wasting with disuse atrophy is multi-dimensional: the response and interaction of myonuclei, satellite cells and signaling pathways. Front Physiol 5: 99, 2014.
25. Bruning A, Rahmeh M, and Friese K. Nelfinavir and bortezomib inhibit mTOR activity via ATF4-mediated sestrin-2 regulation. Molecular oncology 7: 1012-1018,
2013.
26. Budanov AV, and Karin M. p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134: 451-460, 2008.
27. Burnett PE, Barrow RK, Cohen NA, Snyder SH, and Sabatini DM. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl
Acad Sci U S A 95: 1432-1437, 1998.
28. Buse MG, and Reid SS. Leucine. A possible regulator of protein turnover in muscle. J Clin Invest 56: 1250-1261, 1975.
29. Butler DT, and Booth FW. Muscle atrophy by limb immobilization is not caused by insulin resistance. Horm Metab Res 16: 172-174, 1984.
30. Carson JA, and Booth FW. Effect of serum and mechanical stretch on skeletal alpha-actin gene regulation in cultured primary muscle cells. Am J Physiol 275: C1438-
1448, 1998.
31. Carson JA, and Booth FW. Myogenin mRNA is elevated during rapid, slow, and maintenance phases of stretch-induced hypertrophy in chicken slow-tonic muscle.
Pflugers Arch 435: 850-858, 1998.
112 32. Chaillou T, Kirby TJ, and McCarthy JJ. Ribosome Biogenesis: Emerging
Evidence for a Central Role in the Regulation of Skeletal Muscle Mass. J Cell Physiol
2014.
33. Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Temporal alterations in protein signaling cascades during recovery from muscle atrophy. Am J Physiol Cell
Physiol 285: C391-398, 2003.
34. Chopard A, Hillock S, and Jasmin BJ. Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of countermeasures. J Cell Mol Med 13: 3032-3050, 2009.
35. Churchward-Venne TA, Breen L, and Phillips SM. Alterations in human muscle protein metabolism with aging: Protein and exercise as countermeasures to offset sarcopenia. Biofactors 2013.
36. Corradetti MN, Inoki K, and Guan KL. The stress-inducted proteins RTP801 and RTP801L are negative regulators of the mammalian target of rapamycin pathway. J
Biol Chem 280: 9769-9772, 2005.
37. Crozier SJ, Kimball SR, Emmert SW, Anthony JC, and Jefferson LS. Oral leucine administration stimulates protein synthesis in rat skeletal muscle. J Nutr 135:
376-382, 2005.
38. Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, Atherton P,
Wackerhage H, Taylor PM, and Rennie MJ. Anabolic signaling deficits underlie amino acid resistance of wasting, aging muscle. Faseb J 19: 422-424, 2005.
113 39. Dardevet D, Sornet C, Bayle G, Prugnaud J, Pouyet C, and Grizard J.
Postprandial stimulation of muscle protein synthesis in old rats can be restored by a leucine-supplemented meal. J Nutr 132: 95-100, 2002.
40. de Boer MD, Maganaris CN, Seynnes OR, Rennie MJ, and Narici MV. Time course of muscular, neural and tendinous adaptations to 23 day unilateral lower-limb suspension in young men. J Physiol 583: 1079-1091, 2007.
41. Dennis MD, Baum JI, Kimball SR, and Jefferson LS. Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids. J Biol Chem 286:
8287-8296, 2011.
42. Dennis MD, Coleman CS, Berg A, Jefferson LS, and Kimball SR. REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling. Science Sig 2014.
43. Dennis MD, McGhee NK, Jefferson LS, and Kimball SR. Regulated in DNA damage and development 1 (REDD1) promotes cell survival during serum deprivation by sustaining repression of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1). Cell Signal 25: 2709-2716, 2013.
44. Desplanches D, Mayet MH, Sempore B, and Flandrois R. Structural and functional responses to prolonged hindlimb suspension in rat muscle. J Appl Physiol
(1985) 63: 558-563, 1987.
45. DeYoung MP, Horak P, Sofer A, Sgroi D, and Ellisen LW. Hypoxia regulates
TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev 22: 239-251, 2008.
114 46. Dibble CC, and Manning BD. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat Cell Biol 15: 555-564, 2013.
47. Dickinson JM, Fry CS, Drummond MJ, Gundermann DM, Walker DK,
Glynn EL, Timmerman KL, Dhanani S, Volpi E, and Rasmussen BB. Mammalian target of rapamycin complex 1 activation is required for the stimulation of human skeletal muscle protein synthesis by essential amino acids. J Nutr 141: 856-862, 2011.
48. Dodd SL, Gagnon BJ, Senf SM, Hain BA, and Judge AR. Ros-mediated activation of NF-kappaB and Foxo during muscle disuse. Muscle Nerve 41: 110-113,
2010.
49. Dogra C, Changotra H, Wergedal JE, and Kumar A. Regulation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-kappa B signaling pathways in dystrophin-deficient skeletal muscle in response to mechanical stretch. J Cell Physiol
208: 575-585, 2006.
50. Drummond MJ, Dickinson JM, Fry CS, Walker DK, Gundermann DM,
Reidy PT, Timmerman KL, Markofski MM, Paddon-Jones D, Rasmussen BB, and
Volpi E. Bed rest impairs skeletal muscle amino acid transporter expression, mTORC1 signaling, and protein synthesis in response to essential amino acids in older adults.
American Journal of Physiology Endocrinology and Metabolism 302: E1113-1122, 2012.
51. Drummond MJ, Dreyer HC, Fry CS, Glynn EL, and Rasmussen BB.
Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling. J Appl Physiol (1985) 106: 1374-1384, 2009.
52. Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman
KL, Volpi E, and Rasmussen BB. Rapamycin administration in humans blocks the
115 contraction-induced increase in skeletal muscle protein synthesis. The Journal of
Physiology 587: 1535-1546, 2009.
53. Drummond MJ, Miyazaki M, Dreyer HC, Pennings B, Dhanani S, Volpi E,
Esser KA, and Rasmussen BB. Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis. J Appl Physiol
(1985) 106: 1403-1411, 2009.
54. Dupont E, Cieniewski-Bernard C, Bastide B, and Stevens L.
Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK. Am J
Physiol Regul Integr Comp Physiol 300: R408-417, 2011.
55. Durham WJ, Casperson SL, Dillon EL, Keske MA, Paddon-Jones D, Sanford
AP, Hickner RC, Grady JJ, and Sheffield-Moore M. Age-related anabolic resistance after endurance-type exercise in healthy humans. Faseb J 24: 4117-4127, 2010.
56. Ebert SM, Dyle MC, Kunkel SD, Bullard SA, Bongers KS, Fox DK,
Dierdorff JM, Foster ED, and Adams CM. Stress-induced skeletal muscle Gadd45a expression reprograms myonuclei and causes muscle atrophy. J Biol Chem 287: 27290-
27301, 2012.
57. Ellisen LW. Growth control under stress: mTOR regulation through the REDD1-
TSC pathway. Cell Cycle 4: 1500-1502, 2005.
58. Ellisen LW, Ramsayer KD, Johannessen CM, Yang A, Beppu H, Minda K,
Oliner JD, McKeon F, and Haber DA. REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species.
Mol Cell 10: 995-1005, 2002.
116 59. Etgen GJ, Valasek KM, Broderick CL, and Miller AR. In vivo adenoviral delivery of recombinant human protein kinase C-zeta stimulates glucose transport activity in rat skeletal muscle. J Biol Chem 274: 22139-22142, 1999.
60. Favier FB, Benoit H, and Freyssenet D. Cellular and molecular events controlling skeletal muscle mass in response to altered use. Pflugers Arch 456: 587-600,
2008.
61. Ferrando AA, Lane HW, Stuart CA, Davis-Street J, and Wolfe RR.
Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. Am J
Physiol 270: E627-633, 1996.
62. Fournier M, Roy RR, Perham H, Simard CP, and Edgerton VR. Is limb immobilization a model of muscle disuse? Exp Neurol 80: 147-156, 1983.
63. Frost RA, Huber D, Pruznak A, and Lang CH. Regulation of REDD1 by insulin-like growth factor-I in skeletal muscle and myotubes. Journal of cellular biochemistry 108: 1192-1202, 2009.
64. Fry CS, Drummond MJ, Glynn EL, Dickinson JM, Gundermann DM,
Timmerman KL, Walker DK, Dhanani S, Volpi E, and Rasmussen BB. Aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. Skelet Muscle 1: 11, 2011.
65. Fujita S, Rasmussen BB, Cadenas JG, Drummond MJ, Glynn EL, Sattler
FR, and Volpi E. Aerobic exercise overcomes the age-related insulin resistance of muscle protein metabolism by improving endothelial function and Akt/mammalian target of rapamycin signaling. Diabetes 56: 1615-1622, 2007.
117 66. Gardetto PR, Schluter JM, and Fitts RH. Contractile function of single muscle fibers after hindlimb suspension. J Appl Physiol (1985) 66: 2739-2749, 1989.
67. Gautel M. Cytoskeletal protein kinases: titin and its relations in mechanosensing.
Pflugers Arch - Eur J Physiol 462: 119-134, 2011.
68. Gautsch TA, Anthony JC, Kimball SR, Paul GL, Layman DK, and Jefferson
LS. Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise. Am J Physiol 274: C406-414, 1998.
69. Gibson JN, Halliday D, Morrison WL, Stoward PJ, Hornsby GA, Watt PW,
Murdoch G, and Rennie MJ. Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization. Clin Sci (Lond) 72: 503-509, 1987.
70. Gibson JN, Smith K, and Rennie MJ. Prevention of disuse muscle atrophy by means of electrical stimulation: maintenance of protein synthesis. Lancet 2: 767-770,
1988.
71. Gingras AC, Raught B, Gygi SP, Niedzwiecka A, Miron M, Burley SK,
Polakiewicz RD, Wyslouch-Cieszynska A, Aebersold R, and Sonenberg N.
Hierarchical phosphorylation of the translation inhibitor 4E-BP1. Genes Dev 15: 2852-
2864, 2001.
72. Glover EI, Phillips SM, Oates BR, Tang JE, Tarnopolsky MA, Selby A,
Smith K, and Rennie MJ. Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion. J Physiol 586:
6049-6061, 2008.
118 73. Glynn EL, Fry CS, Drummond MJ, Timmerman KL, Dhanani S, Volpi E, and Rasmussen BB. Excess leucine intake enhances muscle anabolic signaling but not net protein anabolism in young men and women. J Nutr 140: 1970-1976, 2010.
74. Goldspink DF. A comparative study of the effects of denervation, immobilization, and denervation with immobilization on the protein turnover of the rat soleus muscle [proceedings]. J Physiol 280: 64P-65P, 1978.
75. Goldspink DF. The influence of immobilization and stretch on protein turnover of rat skeletal muscle. J Physiol 264: 267-282, 1977.
76. Goldspink DF. The influence of passive stretch on the growth and protein turnover of the denervated extensor digitorum longus muscle. Biochem J 174: 595-602,
1978.
77. Goldspink G, Scutt A, Loughna PT, Wells DJ, Jaenicke T, and Gerlach GF.
Gene expression in skeletal muscle in response to stretch and force generation. Am J
Physiol 262: R356-363, 1992.
78. Goodman CA, Mabrey DM, Frey JW, Miu MH, Schmidt EK, Pierre P, and
Hornberger TA. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB Journal: Official publication of the Federation of American Societies for Experimental Biology 25: 1028-
1039, 2011.
79. Gordon BS, Kazi AA, Coleman CS, Dennis MD, Chau V, Jefferson LS, and
Kimball SR. RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells. Cell Signal 26: 461-467, 2014.
119 80. Gordon BS, Kelleher AR, and Kimball SR. Regulation of muscle protein synthesis and the effects of catabolic states. Int J Biochem Cell Biol 45: 2147-2157, 2013.
81. Gratas-Delamarche A, Derbre F, Vincent S, and Cillard J. Physical inactivity, insulin resistance, and the oxidative-inflammatory loop. Free Radic Res 48: 93-108,
2014.
82. Greenhaff PL. The molecular physiology of human limb immobilization and rehabilitation. Exerc Sport Sci Rev 34: 159-163, 2006.
83. Guillet C, Prod'homme M, Balage M, Gachon P, Giraudet C, Morin L,
Grizard J, and Boirie Y. Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans. Faseb J 18: 1586-1587, 2004.
84. Gupta M, and Rath PC. Interferon regulatory factor-1 (IRF-1) interacts with regulated in development and DNA damage response 2 (REDD2) in the cytoplasm of mouse bone marrow cells. International journal of biological macromolecules 65: 41-50,
2014.
85. Hackney KJ, and Ploutz-Snyder LL. Unilateral lower limb suspension: integrative physiological knowledge from the past 20 years (1991-2011). Eur J Appl
Physiol 112: 9-22, 2012.
86. Haddad F, Adams GR, Bodell PW, and Baldwin KM. Isometric resistance exercise fails to counteract skeletal muscle atrophy processes during the initial stages of unloading. J Appl Physiol 100: 433-441, 2006.
87. Han B, Zhu MJ, Ma C, and Du M. Rat hindlimb unloading down-regulates insulin like growth factor-1 signaling and AMP-activated protein kinase, and leads to severe atrophy of the soleus muscle. Appl Physiol Nutr Metab 32: 1115-1123, 2007.
120 88. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, and Avruch J.
Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 273: 14484-14494, 1998.
89. Haystead TA, Haystead CM, Hu C, Lin TA, and Lawrence JC, Jr.
Phosphorylation of PHAS-I by mitogen-activated protein (MAP) kinase. Identification of a site phosphorylated by MAP kinase in vitro and in response to insulin in rat adipocytes.
J Biol Chem 269: 23185-23191, 1994.
90. Heinemeier KM, Olesen JL, Haddad F, Schjerling P, Baldwin KM, and
Kjaer M. Effect of unloading followed by reloading on expression of collagen and related growth factors in rat tendon and muscle. J Appl Physiol (1985) 106: 178-186,
2009.
91. Henriksen EJ. Invited review: Effects of acute exercise and exercise training on insulin resistance. J Appl Physiol (1985) 93: 788-796, 2002.
92. Henriksen EJ, Bourey RE, Rodnick KJ, Koranyi L, Permutt MA, and
Holloszy JO. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am J Physiol 259: E593-598, 1990.
93. Hornberger TA, Chu WK, Mak YW, Hsiung JW, Huang SA, and Chien S.
The role of phospholipase D and phosphatidic acid in the mechanical activation of mTOR signaling in skeletal muscle. Proc Natl Acad Sci USA 103: 4741-4746, 2006.
94. Hornberger TA, Hunter RB, Kandarian SC, and Esser KA. Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats.
Am J Physiol Cell Physiol 281: C179-187, 2001.
121 95. Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML, Chin ER, and Esser KA. Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism.
Biochem J 380: 795-804, 2004.
96. Hornberger TA, Sukhija KB, and Chien S. Regulation of mTOR by mechanically induced signaling events in skeletal muscle. Cell Cycle 5: 1391-1396, 2006.
97. Hunter RB, Mitchell-Felton H, Essig DA, and Kandarian SC. Expression of endoplasmic reticulum stress proteins during skeletal muscle disuse atrophy. Am J
Physiol Cell Physiol 281: C1285-1290, 2001.
98. Hwee DT, and Bodine SC. Age-related deficit in load-induced skeletal muscle growth. J Gerontol A Biol Sci Med Sci 64: 618-628, 2009.
99. Iadevaia V, Huo Y, Zhang Z, Foster LJ, and Proud CG. Roles of the mammalian target of rapamycin, mTOR, in controlling ribosome biogenesis and protein synthesis. Biochem Soc Trans 40: 168-172, 2012.
100. Imen JS, Billiet L, Cuaz-Perolin C, Michaud N, and Rouis M. The regulated in development and DNA damage response 2 (REDD2) gene mediates human monocyte cell death through a reduction in thioredoxin-1 expression. Free Radic Biol Med 46:
1404-1410, 2009.
101. Inoki K, Li Y, Xu T, and Guan KL. Rheb GTPase is a direct target of TSC2
GAP activity and regulates mTOR signaling. Genes Dev 17: 1829-1834, 2003.
102. Inoki K, Li Y, Zhu T, Wu J, and Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4: 648-657, 2002.
122 103. Inoki K, Zhu T, and Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115: 577-590, 2003.
104. Jacobs BL, You JS, Frey JW, Goodman CA, Gundermann DM, and
Hornberger TA. Eccentric Contractions Increase TSC2 Phosphorylation and Alter the
Targeting of TSC2 and mTOR to the Lysosome. The Journal of Physiology 2013.
105. Jin HO, An S, Lee HC, Woo SH, Seo SK, Choe TB, Yoo DH, Lee SB, Um
HD, Lee SJ, Park MJ, Kim JI, Hong SI, Rhee CH, and Park IC. Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia- inducible factor-1alpha and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway. Cell Signal 19: 1393-1403, 2007.
106. Kang SA, Pacold ME, Cervantes CL, Lim D, Lou HJ, Ottina K, Gray NS,
Turk BE, Yaffe MB, and Sabatini DM. mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin. Science 341: 1236566, 2013.
107. Katsanos CS, Kobayashi H, Sheffield-Moore M, Aarsland A, and Wolfe RR.
Aging is associated with diminished accretion of muscle proteins after the ingestion of a small bolus of essential amino acids. Am J Clin Nutr 82: 1065-1073, 2005.
108. Katsanos CS, Kobayashi H, Sheffield-Moore M, Aarsland A, and Wolfe RR.
A high proportion of leucine is required for optimal stimulation of the rate of muscle protein synthesis by essential amino acids in the elderly. Am J Physiol Endocrinol Metab
291: E381-387, 2006.
109. Kazi AA, Hong-Brown L, Lang SM, and Lang CH. Deptor knockdown enhances mTOR Activity and protein synthesis in myocytes and ameliorates disuse muscle atrophy. Mol Med 17: 925-936, 2011.
123 110. Kelleher AR, Gordon BS, Kimball SR, and Jefferson LS. Changes in REDD1,
REDD2, and atrogene mRNA expression are prevented in skeletal muscle fixed in a stretched position during hindlimb immobilization. Physiol Rep 2: e00246, 2014.
111. Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, and Jefferson LS. The mTORC1 signaling repressors REDD1/2 are rapidly induced and activation of p70S6K1 by leucine is defective in skeletal muscle of an immobilized rat hindlimb. American
Journal of Physiology Endocrinology and Metabolism 304: E229-236, 2013.
112. Keshwani MM, von Daake S, Newton AC, Harris TK, and Taylor SS.
Hydrophobic motif phosphorylation is not required for activation loop phosphorylation of p70 ribosomal protein S6 kinase 1 (S6K1). J Biol Chem 286: 23552-23558, 2011.
113. Kho AL, Perera S, Alexandrovich A, and Gautel M. The sarcomeric cytoskeleton as a target for pharmacological intervention. Current opinion in pharmacology 12: 347-354, 2012.
114. Kim J, Kundu M, Viollet B, and Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 13: 132-141, 2011.
115. Kimball SR. Integration of signals generated by nutrients, hormones, and exercise in skeletal muscle. Am J Clin Nutr 99: 237S-242S, 2014.
116. Kimball SR, Do AN, Kutzler L, Cavener DR, and Jefferson LS. Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis. J Biol Chem 283: 3465-
3475, 2008.
124 117. Kimball SR, Farrell PA, and Jefferson LS. Invited Review: Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise. J
Appl Physiol (1985) 93: 1168-1180, 2002.
118. Kimball SR, and Jefferson LS. Control of translation initiation through integration of signals generated by hormones, nutrients, and exercise. J Biol Chem 285:
29027-29032, 2010.
119. Kimball SR, and Jefferson LS. Induction of REDD1 gene expression in the liver in response to endoplasmic reticulum stress is mediated through a PERK, eIF2alpha phosphorylation, ATF4-dependent cascade. Biochem Biophys Res Commun 427: 485-
489, 2012.
120. Kimball SR, Jefferson LS, Fadden P, Haystead TA, and Lawrence JC, Jr.
Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle. Am J Physiol Cell Physiol 270: C705-709, 1996.
121. Kobayashi H, Kato H, Hirabayashi Y, Murakami H, and Suzuki H.
Modulations of muscle protein metabolism by branched-chain amino acids in normal and muscle-atrophying rats. J Nutr 136: 234S-236S, 2006.
122. Kojima E, Takeuchi A, Haneda M, Yagi A, Hasegawa T, Yamaki K, Takeda
K, Akira S, Shimokata K, and Isobe K. The function of GADD34 is a recovery from a shutoff of protein synthesis induced by ER stress: elucidation by GADD34-deficient mice. Faseb J 17: 1573-1575, 2003.
123. Krawiec BJ, Frost RA, Vary TC, Jefferson LS, and Lang CH. Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis. Am J Physiol Endocrinol Metab 289: E969-980, 2005.
125 124. Kruger M, and Linke WA. The giant protein titin: a regulatory node that integrates myocyte signaling pathways. J Biol Chem 286: 9905-9912, 2011.
125. Kumari R, Willing LB, Jefferson LS, Simpson IA, and Kimball SR. REDD1
(regulated in development and DNA damage response 1) expression in skeletal muscle as a surrogate biomarker of the efficiency of glucocorticoid receptor blockade. Biochem
Biophys Res Commun 412: 644-647, 2011.
126. Lang CH, Pruznak A, Navaratnarajah M, Rankine KA, Deiter G, Magne H,
Offord EA, and Breuille D. Chronic alpha-hydroxyisocaproic acid treatment improves muscle recovery after immobilization-induced atrophy. Am J Physiol Endocrinol Metab
305: E416-428, 2013.
127. Lang SM, Kazi AA, Hong-Brown L, and Lang CH. Delayed Recovery of
Skeletal Muscle Mass following Hindlimb Immobilization in mTOR Heterozygous Mice.
PLoS One 7: e38910, 2012.
128. Lange S, Xiang F, Yakovenko A, Vihola A, Hackman P, Rostkova E,
Kristensen J, Brandmeier B, Franzen G, Hedberg B, Gunnarsson LG, Hughes SM,
Marchand S, Sejersen T, Richard I, Edstrom L, Ehler E, Udd B, and Gautel M. The kinase domain of titin controls muscle gene expression and protein turnover. Science 308:
1599-1603, 2005.
129. Laplante M, and Sabatini DM. mTOR signaling in growth control and disease.
Cell 149: 274-293, 2012.
130. Lin L, Qian Y, Shi X, and Chen Y. Induction of a cell stress response gene
RTP801 by DNA damaging agent methyl methanesulfonate through CCAAT/enhancer binding protein. Biochemistry 44: 3909-3914, 2005.
126 131. Lin TA, Kong X, Haystead TA, Pause A, Belsham G, Sonenberg N, and
Lawrence JC, Jr. PHAS-I as a link between mitogen-activated protein kinase and translation initiation. Science 266: 653-656, 1994.
132. Linke WA, and Kruger M. The giant protein titin as an integrator of myocyte signaling pathways. Physiology (Bethesda) 25: 186-198, 2010.
133. Liu JP. Molecular mechanisms of aging and related diseases. Clin Exp
Pharmacol Physiol 2014.
134. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J,
Hawley SA, Udd L, Makela TP, Hardie DG, and Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. Embo J 23:
833-843, 2004.
135. Lloyd SA, Lang CH, Zhang Y, Paul EM, Laufenberg LJ, Lewis GS, and
Donahue HJ. Interdependence of muscle atrophy and bone loss induced by mechanical unloading. J Bone Miner Res 2013.
136. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, and Avruch J. Rheb binds and regulates the mTOR kinase. Curr Biol 15: 702-713, 2005.
137. Loughna P, Goldspink G, and Goldspink DF. Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles. J Appl Physiol (1985) 61:
173-179, 1986.
138. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, and Pandolfi PP.
Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 121: 179-193, 2005.
127 139. Machida S, and Booth FW. Regrowth of skeletal muscle atrophied from inactivity. Med Sci Sports Exerc 36: 52-59, 2004.
140. Magne H, Savary-Auzeloux I, Migne C, Peyron MA, Combaret L, Remond
D, and Dardevet D. Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing. The Journal of physiology 590: 2035-2049, 2012.
141. Maiese K, Chong ZZ, Shang YC, and Wang S. mTOR: on target for novel therapeutic strategies in the nervous system. Trends Mol Med 19: 51-60, 2013.
142. Manning BD, Tee AR, Logsdon MN, Blenis J, and Cantley LC. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 10: 151-162, 2002.
143. Marimuthu K, Murton AJ, and Greenhaff PL. Mechanisms regulating muscle mass during disuse atrophy and rehabilitation in humans. J Appl Physiol (1985) 110: 555-
560, 2011.
144. Marzetti E, Calvani R, Cesari M, Buford TW, Lorenzi M, Behnke BJ, and
Leeuwenburgh C. Mitochondrial dysfunction and sarcopenia of aging: from signaling pathways to clinical trials. Int J Biochem Cell Biol 45: 2288-2301, 2013.
145. McGhee NK, Jefferson LS, and Kimball SR. Elevated corticosterone associated with food deprivation upregulates expression in rat skeletal muscle of the mTORC1 repressor, REDD1. J Nutr 139: 828-834, 2009.
146. McGlory C, White A, Treins C, Drust B, Close GL, Maclaren DP, Campbell
IT, Philp A, Schenk S, Morton JP, and Hamilton DL. Application of the [gamma-32P]
128 ATP kinase assay to study anabolic signaling in human skeletal muscle. J Appl Physiol
(1985) 116: 504-513, 2014.
147. McKoy G, Hou Y, Yang SY, Vega Avelaira D, Degens H, Goldspink G, and
Coulton GR. Expression of Ankrd2 in fast and slow muscles and its response to stretch are consistent with a role in slow muscle function. J Appl Physiol (1985) 98: 2337-2343; discussion 2320, 2005.
148. Meneilly GS, Elliot T, Bryer-Ash M, and Floras JS. Insulin-mediated increase in blood flow is impaired in the elderly. J Clin Endocrinol Metab 80: 1899-1903, 1995.
149. Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H,
Cantley LC, and Manning BD. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156: 771-785, 2014.
150. Michel G, Matthes HW, Hachet-Haas M, El Baghdadi K, de Mey J,
Pepperkok R, Simpson JC, Galzi JL, and Lecat S. Plasma membrane translocation of
REDD1 governed by GPCRs contributes to mTORC1 activation. Journal of cell science
127: 773-787, 2014.
151. Miyazaki M, and Esser KA. Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals. J Appl Physiol (1985) 106: 1367-1373, 2009.
152. Miyazaki M, and Esser KA. REDD2 is enriched in skeletal muscle and inhibits mTOR signaling in response to leucine and stretch. Am J Physiol Cell Physiol 296: C583-
592, 2009.
153. Molitoris JK, McColl KS, Swerdlow S, Matsuyama M, Lam M, Finkel TH,
Matsuyama S, and Distelhorst CW. Glucocorticoid elevation of dexamethasone-
129 induced gene 2 (Dig2/RTP801/REDD1) protein mediates autophagy in lymphocytes. J
Biol Chem 286: 30181-30189, 2011.
154. Morey-Holton E, Globus RK, Kaplansky A, and Durnova G. The hindlimb unloading rat model: literature overview, technique update and comparison with space flight data. Adv Space Biol Med 10: 7-40, 2005.
155. Morris RT, Spangenburg EE, and Booth FW. Responsiveness of cell signaling pathways during the failed 15-day regrowth of aged skeletal muscle. J Appl Physiol 96:
398-404, 2004.
156. Moser BA, Dennis PB, Pullen N, Pearson RB, Williamson NA, Wettenhall
RE, Kozma SC, and Thomas G. Dual requirement for a newly identified phosphorylation site in p70s6k. Molecular and Cellular Biology 17: 5648-5655, 1997.
157. Murakami T, Hasegawa K, and Yoshinaga M. Rapid induction of REDD1 expression by endurance exercise in rat skeletal muscle. Biochem Biophys Res Commun
405: 615-619, 2011.
158. Nair KS. Aging muscle. Am J Clin Nutr 81: 953-963, 2005.
159. O'Keefe M P, Perez FR, Kinnick TR, Tischler ME, and Henriksen EJ.
Development of whole-body and skeletal muscle insulin resistance after one day of hindlimb suspension. Metabolism 53: 1215-1222, 2004.
160. Ortells MC, Morancho B, Drews-Elger K, Viollet B, Laderoute KR, Lopez-
Rodriguez C, and Aramburu J. Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin. Nucleic acids research
40: 4368-4384, 2012.
130 161. Ottenheijm CA, van Hees HW, Heunks LM, and Granzier H. Titin-based mechanosensing and signaling: role in diaphragm atrophy during unloading? Am J
Physiol Lung Cell Mol Physiol 300: L161-166, 2011.
162. Phillips SM, Glover EI, and Rennie MJ. Alterations of protein turnover underlying disuse atrophy in human skeletal muscle. J Appl Physiol 107: 645-654, 2009.
163. Pisani DF, Leclerc L, Jarretou G, Marini JF, and Dechesne CA. SMHS1 is involved in oxidative/glycolytic-energy metabolism balance of muscle fibers. Biochem
Biophys Res Commun 326: 788-793, 2005.
164. Powers SK, Smuder AJ, and Judge AR. Oxidative stress and disuse muscle atrophy: cause or consequence? Curr Opin Clin Nutr Metab Care 15: 240-245, 2012.
165. Powers SK, Wiggs MP, Duarte JA, Zergeroglu AM, and Demirel HA.
Mitochondrial signaling contributes to disuse muscle atrophy. American Journal of
Physiology Endocrinology and Metabolism 303: E31-39, 2012.
166. Prod'homme M, Balage M, Debras E, Farges MC, Kimball S, Jefferson L, and Grizard J. Differential effects of insulin and dietary amino acids on muscle protein synthesis in adult and old rats. The Journal of Physiology 563: 235-248, 2005.
167. Rasmussen BB, Fujita S, Wolfe RR, Mittendorfer B, Roy M, Rowe VL, and
Volpi E. Insulin resistance of muscle protein metabolism in aging. Faseb J 20: 768-769,
2006.
168. Reiling JH, and Hafen E. The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila. Genes
Dev 18: 2879-2892, 2004.
131 169. Rennie MJ. Anabolic resistance in critically ill patients. Crit Care Med 37: S398-
399, 2009.
170. Rennie MJ. Anabolic resistance: the effects of aging, sexual dimorphism, and immobilization on human muscle protein turnover. Appl Physiol Nutr Metab 34: 377-
381, 2009.
171. Rennie MJ, Selby A, Atherton P, Smith K, Kumar V, Glover EL, and Philips
SM. Facts, noise and wishful thinking: muscle protein turnover in aging and human disuse atrophy. Scand J Med Sci Sports 20: 5-9, 2010.
172. Rennie MJ, and Wilkes EA. Maintenance of the musculoskeletal mass by control of protein turnover: the concept of anabolic resistance and its relevance to the transplant recipient. Ann Transplant 10: 31-34, 2005.
173. Rieu I, Balage M, Sornet C, Debras E, Ripes S, Rochon-Bonhomme C,
Pouyet C, Grizard J, and Dardevet D. Increased availability of leucine with leucine- rich whey proteins improves postprandial muscle protein synthesis in aging rats.
Nutrition 23: 323-331, 2007.
174. Rieu I, Sornet C, Bayle G, Prugnaud J, Pouyet C, Balage M, Papet I, Grizard
J, and Dardevet D. Leucine-supplemented meal feeding for ten days beneficially affects postprandial muscle protein synthesis in old rats. J Nutr 133: 1198-1205, 2003.
175. Rodnick KJ, Holloszy JO, Mondon CE, and James DE. Effects of exercise training on insulin-regulatable glucose-transporter protein levels in rat skeletal muscle.
Diabetes 39: 1425-1429, 1990.
132 176. Rogers GW, Jr., Richter NJ, and Merrick WC. Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A. J Biol
Chem 274: 12236-12244, 1999.
177. Rosner M, Freilinger A, and Hengstschlager M. Akt regulates nuclear/cytoplasmic localization of tuberin. Oncogene 26: 521-531, 2007.
178. Sacheck JM, Hyatt JP, Raffaello A, Jagoe RT, Roy RR, Edgerton VR,
Lecker SH, and Goldberg AL. Rapid disuse and denervation atrophy involve transcriptional changes similar to those of muscle wasting during systemic diseases.
FASEB J 21: 140-155, 2007.
179. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, and Sabatini DM.
Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141: 290-303, 2010.
180. Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, and Sabatini DM. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496-1501, 2008.
181. Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E,
Carr SA, and Sabatini DM. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25: 903-915, 2007.
182. Sandri M. Protein breakdown in muscle wasting: Role of autophagy-lysosome and ubiquitin-proteasome. Int J Biochem Cell Biol 45: 2121-2129, 2013.
183. Sandri M, Lin J, Handschin C, Yang W, Arany ZP, Lecker SH, Goldberg
AL, and Spiegelman BM. PGC-1alpha protects skeletal muscle from atrophy by
133 suppressing FoxO3 action and atrophy-specific gene transcription. Proc Natl Acad Sci
USA 103: 16260-16265, 2006.
184. Sasa T, Sairyo K, Yoshida N, Fukunaga M, Koga K, Ishikawa M, and Yasui
N. Continuous muscle stretch prevents disuse muscle atrophy and deterioration of its oxidative capacity in rat tail-suspension models. Am J Phys Med Rehabil 83: 851-856,
2004.
185. Sasai N, Agata N, Inoue-Miyazu M, Kawakami K, Kobayashi K, Sokabe M, and Hayakawa K. Involvement of PI3K/Akt/TOR pathway in stretch-induced hypertrophy of myotubes. Muscle Nerve 41: 100-106, 2010.
186. Schwarzkopf M, Coletti D, Sassoon D, and Marazzi G. Muscle cachexia is regulated by a p53-PW1/Peg3-dependent pathway. Genes Dev 20: 3440-3452, 2006.
187. Seider MJ, Nicholson WF, and Booth FW. Insulin resistance for glucose metabolism in disused soleus muscle of mice. Am J Physiol 242: E12-18, 1982.
188. Senf SM, Dodd SL, and Judge AR. FOXO signaling is required for disuse muscle atrophy and is directly regulated by Hsp70. American Journal of Physiology Cell
Physiology 298: C38-45, 2010.
189. Senf SM, Dodd SL, McClung JM, and Judge AR. Hsp70 overexpression inhibits NF-kappaB and Foxo3a transcriptional activities and prevents skeletal muscle atrophy. FASEB J 22: 3836-3845, 2008.
190. Shah OJ, and Hunter T. Tuberous sclerosis and insulin resistance. Unlikely bedfellows reveal a TORrid affair. Cell Cycle 4: 46-51, 2005.
134 191. Shangraw RE, Stuart CA, Prince MJ, Peters EJ, and Wolfe RR. Insulin responsiveness of protein metabolism in vivo following bedrest in humans. Am J Physiol
255: E548-558, 1988.
192. Shaw RJ, Bardeesy N, Manning BD, Lopez L, Kosmatka M, DePinho RA, and Cantley LC. The LKB1 tumor suppressor negatively regulates mTOR signaling.
Cancer Cell 6: 91-99, 2004.
193. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, and Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 101:
3329-3335, 2004.
194. Siu PM, Pistilli EE, Murlasits Z, and Alway SE. Hindlimb unloading increases muscle content of cytosolic but not nuclear Id2 and p53 proteins in young adult and aged rats. J Appl Physiol 100: 907-916, 2006.
195. Soares AG, Aoki MS, Miyabara EH, Deluca CV, Ono HY, Gomes MD, and
Moriscot AS. Ubiquitin-ligase and deubiquitinating gene expression in stretched rat skeletal muscle. Muscle Nerve 36: 685-693, 2007.
196. Sofer A, Lei K, Johannessen CM, and Ellisen LW. Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol 25: 5834-5845, 2005.
197. Stein TP, and Schluter MD. Excretion of amino acids by humans during space flight. Acta Astronaut 42: 205-214, 1998.
198. Stein TP, and Schluter MD. Human skeletal muscle protein breakdown during spaceflight. Am J Physiol 272: E688-695, 1997.
135 199. Stein TP, and Wade CE. Metabolic consequences of muscle disuse atrophy. J
Nutr 135: 1824S-1828S, 2005.
200. Strawbridge WJ, Shema SJ, Balfour JL, Higby HR, and Kaplan GA.
Antecedents of frailty over three decades in an older cohort. J Gerontol B Psychol Sci Soc
Sci 53: S9-16, 1998.
201. Suetta C, Frandsen U, Jensen L, Jensen MM, Jespersen JG, Hvid LG, Bayer
M, Petersson SJ, Schroder HD, Andersen JL, Heinemeier KM, Aagaard P,
Schjerling P, and Kjaer M. Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy. PLoS One 7: e51238, 2012.
202. Symons TB, Schutzler SE, Cocke TL, Chinkes DL, Wolfe RR, and Paddon-
Jones D. Aging does not impair the anabolic response to a protein-rich meal. Am J Clin
Nutr 86: 451-456, 2007.
203. Symons TB, Sheffield-Moore M, Wolfe RR, and Paddon-Jones D. A moderate serving of high-quality protein maximally stimulates skeletal muscle protein synthesis in young and elderly subjects. J Am Diet Assoc 109: 1582-1586, 2009.
204. Tabary JC, Tabary C, Tardieu C, Tardieu G, and Goldspink G. Physiological and structural changes in the cat's soleus muscle due to immobilization at different lengths by plaster casts. The Journal of Physiology 224: 231-244, 1972.
205. Timmerman KL, Lee JL, Fujita S, Dhanani S, Dreyer HC, Fry CS,
Drummond MJ, Sheffield-Moore M, Rasmussen BB, and Volpi E. Pharmacological vasodilation improves insulin-stimulated muscle protein anabolism but not glucose utilization in older adults. Diabetes 59: 2764-2771, 2010.
136 206. Toth MJ, Matthews DE, Tracy RP, and Previs MJ. Age-related differences in skeletal muscle protein synthesis: relation to markers of immune activation. Am J Physiol
Endocrinol Metab 288: E883-891, 2005.
207. Tsukamoto Y, Hijiya N, Yano S, Yokoyama S, Nakada C, Uchida T,
Matsuura K, and Moriyama M. Arpp/Ankrd2, a member of the muscle ankyrin repeat proteins (MARPs), translocates from the I-band to the nucleus after muscle injury.
Histochemistry and cell biology 129: 55-64, 2008.
208. Tuckow AP, Vary TC, Kimball SR, and Jefferson LS. Ectopic expression of eIF2Bepsilon in rat skeletal muscle rescues the sepsis-induced reduction in guanine nucleotide exchange activity and protein synthesis. American Journal of Physiology
Endocrinology and Metabolism 299: E241-248, 2010.
209. Vega-Rubin-de-Celis S, Abdallah Z, Kinch L, Grishin NV, Brugarolas J, and
Zhang X. Structural analysis and functional implications of the negative mTORC1 regulator REDD1. Biochemistry 49: 2491-2501, 2010.
210. Voelkel T, and Linke WA. Conformation-regulated mechanosensory control via titin domains in cardiac muscle. Pflugers Arch 462: 143-154, 2011.
211. Wang H, Kubica N, Ellisen LW, Jefferson LS, and Kimball SR.
Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. J Biol Chem 281: 39128-39134, 2006.
212. Wang X, Li W, Williams M, Terada N, Alessi DR, and Proud CG. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. Embo J 20: 4370-4379,
2001.
137 213. Welle S, Thornton C, Jozefowicz R, and Statt M. Myofibrillar protein synthesis in young and old men. Am J Physiol 264: E693-698, 1993.
214. Whitney ML, Jefferson LS, and Kimball SR. ATF4 is necessary and sufficient for ER stress-induced upregulation of REDD1 expression. Biochem Biophys Res
Commun 379: 451-455, 2009.
215. Wilkes EA, Selby AL, Atherton PJ, Patel R, Rankin D, Smith K, and Rennie
MJ. Blunting of insulin inhibition of proteolysis in legs of older subjects may contribute to age-related sarcopenia. Am J Clin Nutr 90: 1343-1350, 2009.
216. Williams PE, and Goldspink G. Changes in sarcomere length and physiological properties in immobilized muscle. J Anat 127: 459-468, 1978.
217. Williamson DL, Li Z, Tuder RM, Feinstein E, Kimball SR, and Dungan CM.
Altered nutrient response of mTORC1 as a result of changes in REDD1 expression:
Effect of obesity versus REDD1 deficiency. J Appl Physiol (1985) 2014.
218. Yang HS, Knies JL, Stark C, and Colburn NH. Pdcd4 suppresses tumor phenotype in JB6 cells by inhibiting AP-1 transactivation. Oncogene 22: 3712-3720,
2003.
219. Yarasheski KE, Zachwieja JJ, and Bier DM. Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women. Am J
Physiol 265: E210-214, 1993.
220. You JS, Park MN, Song W, and Lee YS. Dietary fish oil alleviates soleus atrophy during immobilization in association with Akt signaling to p70s6k and E3 ubiquitin ligases in rats. Appl Physiol Nutr Metab 35: 310-318, 2010.
138 221. Zhou Y, Wang Q, Guo Z, Weiss HL, and Evers BM. Nuclear factor of activated T-cell c3 inhibition of mammalian target of rapamycin signaling through induction of regulated in development and DNA damage response 1 in human intestinal cells. Mol Biol Cell 23: 2963-2972, 2012.
139 VITA Andrew R. Kelleher Education: 2009 M.S. Exercise Science, Syracuse University, Syracuse, NY 2007 B.S. Biology, Muhlenberg College, Allentown, PA
Professional Experience: 2007 - 2008 Manager; Personal Fitness Inc., Syracuse, NY 2005 - 2007 Owner-Operator; ARK Fitness, Greater Lehigh Valley, PA Official Personal Trainer & Fitness Class Instructor, Muhlenberg College, Allentown, PA 2005 - 2007 Rehab Assistant; Rehabilitation Management Specialists, Bethlehem, PA
Professional Organizations 2009 - 2014 American Physiological Society 2009 - 2014 Mid-Atlantic Regional Chapter of the American College of Sports Medicine
Honors and Awards TechCelerator@Hershey Start-up Award, Ben Franklin Technology Partners (2013) College of Medicine Endowed Scholarship, Penn State College of Medicine (2013) Graduate Alumni Endowed Scholarship, Penn State College of Medicine (2013) Class of 1971 Scholarship, Penn State College of Medicine (2012) Doctoral Student Investigator Award, Mid Atlantic Regional Chapter of the American College of Sports Medicine (2011) IGDP Professional Research Presentation Award, Penn State University (2011) School of Education Summer Research Grant Award, Syracuse University (2008) Graduate Research Grant, National Strength & Conditioning Association (2008) Doctoral Assistantship Award, Syracuse University (2008)
Selected Publications Kelleher AR, Gordon BS, Kimball SR, Jefferson LS. Changes in REDD1, REDD2, and atrogene mRNA expression are prevented in skeletal muscle fixed in a stretched position during hindlimb immobilization. Physiol Rep. 2(2):e00246, 2014. Gordon BS1, Kelleher AR1, Kimball SR. Protein synthesis in catabolic states. Int J Biochem Cell Biol. 45(10):2147-2157, 2013. 1 These authors contributed equally to the manuscript. Hackney KJ, Kelleher AR, Ploutz-Snyder LL. Resting energy expenditure is increased following multiple bouts of resistance exercise with amino acid-carbohydrate intake. ISRN Nutrition. Vol 2013 Article ID 948695, 2013. Kelleher AR, Kimball SR, Dennis MD, Schilder RJ, Jefferson LS. The repressors of mTORC1 signaling, REDD1 and REDD2, are rapidly induced in skeletal muscle of an immobilized rat hindlimb. Am J Physiol: Endo Metab. 304(2):E229-36, 2013. Kelleher AR, Fairchild TJ, Keslacy S. STZ-induced skeletal muscle atrophy is associated with increased p65 content and downregulation of insulin pathway without NF-kappaB canonical cascade activation. Acta Diabetol. 47(4):315-323, 2010. Kelleher AR, Hackney KJ, Fairchild TJ, Keslacy S, Ploutz-Snyder LL. The metabolic costs of reciprocal supersets vs. traditional resistance exercise in young recreationally active adults. J Strength Cond Res. 24(4):1043-1051, 2010.