University of Iowa

Iowa Research Online Theses and Dissertations

Summer 2012

Investigating the potential relationship between skeletal muscle atrophy and obesity Christopher John Elmore University of Iowa

Copyright 2012 Christopher John Elmore This thesis is available at Iowa Research Online: http://ir.uiowa.edu/etd/3289 Recommended Citation Elmore, Christopher John. "Investigating the potential relationship between skeletal muscle atrophy and obesity." MS (Master of Science) thesis, University of Iowa, 2012. http://ir.uiowa.edu/etd/3289.

Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Neuroscience and Neurobiology Commons

INVESTIGATING THE POTENTIAL RELATIONSHIP BETWEEN SKELETAL MUSCLE ATROPHY AND OBESITY

by Christopher John Elmore

A thesis submitted in partial fulfillment of the requirements for the Interdisciplinary StudiesMaster of Science degree in Molecular and Cellular Biology in the Graduate College of The University of Iowa July 2012 Thesis Supervisor: Associate Professor Christopher M. Adams

Graduate College The University of Iowa Iowa City, Iowa

CERTIFICATE OF APPROVAL _______________________ MASTER'S THESIS _______________ This is to certify that the Master's thesis of Christopher John Elmore has been approved by the Examining Committee for the thesis requirement for the Interdisciplinary StudiesMaster of Science degree in Molecular and Cellular Biology at the July 2012 graduation. Thesis Committee:__________________________________ Christopher M. Adams, Thesis Supervisor __________________________________ Christopher Benson __________________________________ David Motto

To my spouse, who makes my life better than I ever imagined. To my family, who have way more confidence in my abilities than I do. To my mentors and educators, for their commitment of time, and ability to inspire curiosity.

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Education is no substitute for intelligence. That elusive quality is defined only in part by puzzle-solving ability. It is in the creation of new puzzles reflecting what your senses report that you round out the definition. Frank Herbert Dune: Chapterhouse

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ACKNOWLEDGMENTS Thank you to Christopher Adams and all members of the Adams lab for their advice, support, and most of all laughter.

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ABSTRACT Skeletal muscle atrophy is the most common clinical disorder of skeletal muscle and typically occurs as a secondary consequence of fasting, disuse, acute and chronic illness, and aging. It can lead to prolonged recovery and loss of independent living. Of similar clinical significance, one third of Americans are obese and at risk for metabolic syndrome. Interestingly recent studies have demonstrated that both metabolic syndrome and obesity diminish skeletal muscle strength, power, and endurance. However, there are no effective pharmacological treatments for these debilitating effects on skeletal muscle. This is largely due to the fact that the molecular mechanisms underlying its pathogenesis remain uncharacterized. We have recently identified ursolic acid (UA) as a small molecule inhibitor of muscle atrophy. In the absence of atrophyinducing stress, UA-supplemented chow elicited muscle hypertrophy with little adiposity in mice. To further evaluate these data, mice were subjected to a high fat diet (HFD) with or without UA supplementation, or a standard chow (SC) control. Our data indicates that UA-supplemented HFD mitigates muscle atrophy and adiposity, while HFD significantly reduces muscle mass compared to SC. Furthermore, mice fed a HFD exhibited increased adiposity and reduced muscle mass, strength, and fiber diameter when compared to SC controls. Molecular analysis revealed diminished protein content and increased triglycerides. Gene expression analysis revealed a reduction in Pgc1!, a critical gene that regulates oxidative metabolism and mitochondrial biogenesis. Additionally, we found decreased expression of hormonal receptors AR, involved in signaling of testosterone, and Thr!, involved in signaling of thyroid hormones. Taken together, these data suggest that alterations in gene expression resulting from

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diet-induced obesity are an atrophy-inducing stress that may function by disrupting metabolic and hormonal signaling.

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TABLE OF CONTENTS LIST OF FIGURES .............................................................................................. viii LIST OF ABBREVIATIONS ................................................................................... x CHAPTER I.

INTRODUCTION .................................................................................. 1 Skeletal Muscle Atrophy ....................................................................... 1 Skeletal Muscle Physiology .................................................................. 1 Skeletal Muscle in Obesity ................................................................... 2 ATF4/Gadd45a/Cdkn1a Pathway......................................................... 4 Preliminary Results .............................................................................. 5

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MATERIALS AND METHODS ........................................................... 13 Animal Protocols ................................................................................ 13 Skeletal Muscle Histology .................................................................. 13 Skeletal Muscle Composition ............................................................. 14 Skeletal Muscle mRNA Analysis ........................................................ 14

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RESULTS ........................................................................................... 16 High Fat Diet-induced Obesity ........................................................... 16 Skeletal Muscle Weights .................................................................... 16 Skeletal Muscle Fibers ....................................................................... 17 Muscle Composition ........................................................................... 17 Gene Expression ................................................................................ 17

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DISCUSSION ..................................................................................... 41 Interpretation Of Results .................................................................... 41 Future Directions ................................................................................ 44

REFERENCES .................................................................................................... 46

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LIST OF FIGURES Figure 1.1 Novel atrophy pathway involving ATF4/Gadd45a/Cdkn1a. ........................... 7 1.2 UA inhibits weight gain from a HFD. ............................................................. 8 1.3 UA reduces fasting blood glucose on a HFD. ............................................... 9 1.4 UA inhibits hepatomegaly from a HFD. ....................................................... 10 1.5 UA inhibits loss of skeletal muscle mass from a HFD. ................................ 11 1.6 HFD increases expression of Cdkn1a. ........................................................ 12 2.1 HFD increases weight gain. ........................................................................ 19 2.2 HFD increases epididymal fat. .................................................................... 20 2.3 HFD increases retroperitoneal fat. .............................................................. 21 2.4 HFD induces hepatomegaly. ....................................................................... 22 2.5 HFD does not effect cardiac muscle weights. ............................................. 23 2.6 HFD has no effect on kidney weights. ......................................................... 24 2.7 HFD reduces weight of tibialis anterior. ....................................................... 25 2.8 HFD reduces weight of gastrocnemius. ...................................................... 26 2.9 HFD reduces weight of quadriceps. ............................................................ 27 2.10 HFD reduces weight of triceps. ................................................................... 28 2.11 HFD increases the weight of soleus. ........................................................... 29 2.12 HFD reduces weight of hindlimb muscles. .................................................. 30 2.13 HFD changes muscle histology. .................................................................. 31 2.14 HFD induces skeletal muscle atrophy. ........................................................ 32 2.15 HFD reduces fiber size distribution of skeletal muscle. ............................... 33 2.16 HFD reduces grip strength. ......................................................................... 34 2.17 Relationship between body weight and skeletal muscle atrophy. ............... 35 2.18 HFD decreases skeletal muscle protein content. ........................................ 36 2.19 HFD increases content of skeletal muscle triglycerides. ............................. 37 viii

2.20 HFD reduces the ratio of skeletal muscle protein to triglycerides. .............. 38 2.21 Relationship between body weight and skeletal muscle triglycerides. ........ 39 2.22 HFD suppresses gene expression of Pgc1!, AR, Thr!............................... 40

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LIST OF ABBREVIATIONS AA, amino acids AR, androgen receptor ATF4, activating transcription factor ANOVA, analysis of variance Bnip3, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 bZIP, basic leucine zipper domain Cdkn1a, cycline dependent kinase inhibitor 1a cDNA, complementary deoxyribonucleic acid Ctsl, cathepsin L DNA, deoxyribonucleic acid Fbox32, F-box only protein 32 Gadd45a, growth arrest and DNA damage inducible 45a H&E, hematoxylin and eosin IGF1, insulin-like growth factor 1 HFD, high fat diet mRNA, messenger ribonucleic acid Pgc1!, peroxisome proliferator-activated receptor gamma coactivator 1 alpha qPCR, quantitative real-time polymerase chain reaction SC, standard chow Thr!, thyroid receptor alpha Trim63, tripartite motif containing 63 UA, ursolic acid

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CHAPTER I INTRODUCTION

Skeletal Muscle Atrophy Skeletal muscle atrophy is a frequent consequence of prolonged disease and commonly attributed to illnesses such as cancer, kidney disease, congestive heart failure, lung disease, sepsis, and chronic infections like tuberculosis and HIV/AIDS. Atrophy can also be the result of situations fostering muscle disuse, such as spinal cord injury, prolonged bedrest, uncontrolled diabetes mellitus, and neurological disorders like multiple sclerosis. Characterized by a wasting of muscle, it can lead to complications and extended recovery times. Muscle atrophy also occurs during aging, making the consequences of atrophy something that may affect everyone at some point during their lifetime. Ultimately, skeletal muscle atrophy can leave an individual incapable of working, and result in loss of independent living. Consequently, skeletal muscle atrophy has an enormous impact on an individual’s quality of life for the patient, as well as their families. It also places an immense burden on the healthcare system and society in general. Currently, we lack effective medical treatments to rehabilitate or prevent skeletal muscle atrophy, largely due to an inadequate understanding of its molecular pathogenesis for targeted interventions. Skeletal Muscle Physiology During times of starvation or fasting, skeletal muscle serves as a reservoir for amino acids (AA), where proteins are catabolized and exported via atrophy. These AA serve in important physiological functions, including protein synthesis and gluconeogenesis. However, this increased catabolism results in an overall

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reduction of protein content and muscle mass. This transition is histologically seen as diminished size, rather than a quantitative reduction in myofibers (1). Normal adults prevent this effect primarily through proper nutrition, muscle use, and anaboilic signaling of insulin/IGF1. Starvation, disuse, and uncontrolled diabetes mellitus, are all circumstances resulting in skeletal muscle atrophy. In opposition to insulin/IGF1 signaling, other hormones can promote skeletal muscle atrophy, such as glucocorticoids, inflammatory cytokines, and autocrine/paracrine factors such as myostatin (1-3). Therefore, skeletal muscle mass is a balance between catabolic proteolysis and anabolic protein synthesis within the muscle. Skeletal Muscle In Obesity One-third of Americans are presently obese (4). A host of comorbidities are associated with obesity such as diabetes, heart disease, hypertension, and stroke. Obesity is one of the key diagnostic criteria for metabolic syndrome, a group of medical disorders including elevations in triglycerides, blood pressure, and fasting blood glucose (4). These factors in combination increase the likelihood of developing coronary artery disease, type 2 diabetes, and stroke (4). The risk factors for obesity and outcomes from metabolic syndrome display considerable similarity. Ultimately, metabolic syndrome defines the risk factors that result from chronic obesity. As obesity quickly becomes a worldwide epidemic, increasing demands have been placed on the healthcare system to treat obesity-related diseases. Studies conducted by the Centers for Disease Control estimate the annual medical spending for obesity, and obesity-related illnesses, at $147 billion in the United states alone, and may rise to $1.8 trillion by the year 2018 (5, 6). For the obese individual, the financial burden of long-term therapies, as well as a

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progressive decline in health and quality of life, makes prevention the best option. Obese individuals suffer from muscle weakness and reduced mobility. Poor diet and sedentary lifestyle are concomitant risk factors for skeletal muscle atrophy, obesity, and metabolic syndrome. Typically, dietary intervention and physical activity are initial therapies when treating metabolic syndrome and obesity. Research suggests that obesity impairs skeletal muscles ability to hypertrophy under conditions from either external loading or increased body weight (7). This makes exercise a poor therapy to rehabilitate muscles in the obese. Studies elucidating the connection between skeletal muscle and metabolic syndrome show diminished skeletal muscle strength, power, and endurance in human models (8, 9). Other data has demonstrated that obesity increases intramyocellular triglycerides in skeletal muscle, impairs insulin sensitivity, and decreases strength, in mice and human models (10, 11). Interestingly, studies of ob/ob mice using estimated cross-sectional areas from single fiber dissections, suggest an obesity-related reduction in myofiber size (12). Nevertheless, ob/ob mice are an extreme model of obesity. The use of single fiber dissection imparts mechanical strain on muscle fibers, making the estimate calculations an inaccurate technique to evaluate normal muscle histology. Currently, no evidence supports obesity-related muscle atrophy in a C57BL/6 mouse using less invasive techniques of histological analysis. This would allow us to determine the potential remodeling of skeletal muscle in a more applicable translational model of obesity. Taken together, these data lead us to hypothesize that obesity is an atrophy-inducing stress that impairs skeletal muscle function.

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ATF4/Gadd45a/Cdkn1a Pathway ATF4 (activating transcription factor 4) is a bZIP transcription factor with an evolutionarily ancient role in cellular stress signaling (13, 14). Diverse stresses, including fasting, disuse, insulin deficiency, and systemic disease, can all increase ATF4 expression in skeletal muscle (15-17). To examine the role of ATF4 in skeletal muscle, we overexpressed ATF4 by transfection in mouse tibialis anterior muscles and found it sufficient to reduce fiber size, indicating muscle fiber atrophy. To determine how ATF4 causes atrophy, we used an unbiased exon expression array and identify Gadd45a (growth arrest and DNA damage inducible 45a) as an atrophy-associated ATF4 target gene (18). Previous studies on Gadd45a elucidate its role in cell-type specific stress responses, such as growth arrest, differentiation, DNA damage repair, and DNA demethylation, however its function in skeletal muscle is uncharacterized (19-21) We used qPCR (quantitative real-time polymerase chain reaction) to confirm that ATF4 overexpression increases Gadd45a mRNA in mouse skeletal muscle. To determine the role of Gadd45a in atrophy, we reduced its expression by transfection in mouse tibialis anterior muscles with miRNAs targeting Gadd45a. This allowed us to identify Gadd45a as the mediator of a molecular pathway that is activated by skeletal muscle stress and drives skeletal muscle atrophy. Furthermore, we determined ATF4 induces Gadd45a expression, and Gadd45a was found to be required for skeletal muscle atrophy under immobilization, fasting, and denervation stresses (22). We next sought to determine the mechanism by which Gadd45a induces skeletal muscle atrophy. Using the exon expression arrays, we compared the effects of fasting, as well as Gadd45a overexpression, to identify atrophyassociated transcription targets. This identified Cdkn1a (cyclin-dependent kinase

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inhibitor 1a) as a Gadd45a target, and were consistent with previous findings that ATF4 increases Cdkn1a (18). Previous studies have shown Cdkn1a, which encodes the protein p21, is increased under atrophy stimuli such as aging, disuse, and denervation in muscle (21, 23-25). Further data, in preparation for publication, determined that Gadd45a increases the expression of Cdkn1a by demethylation at a specific 5-methylcytosine in the Cdkn1a promoter region (Figure 1A). Preliminary Results Previous studies using the exon expression arrays from fasting and denervated skeletal muscle in mice and humans identified conserved atrophyassociated mRNA signatures. These signatures were used to query the Connectivity Map, which characterizes the effects of more than 1,300 small molecules on global mRNA expression from several cultured human cell lines. We identified ursolic acid (UA) as having a strong negatively correlated mRNA signature to skeletal muscle atrophy (26). Experiments utilizing standard chow (SC) compared to UA supplemented (0.14% by weight) SC demonstrate induction of skeletal muscle hypertrophy, increased grip strength, as well as reductions in adiposity and fasting glucose, likely by increasing insulin/IGF1 signaling (26). These data suggests a potential utility for ursolic acid in the treatment of obesity and diabetes, leading us to investigate its use in combination with high fat diet (HFD). To further evaluate UA’s effects, experiments with mice fed HFD with or without UA supplementation demonstrate UA reduces obesity, glucose intolerance, and fatty liver disease, possibly by increases in metabolically active skeletal muscle and brown fat (27). We next sought to determine the efficacy of UA in obesity reduction with experiments utilizing HFD with or without UA (0.15%

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by weight) supplementation, as well as a SC control. We find that HFD (55% calories from fat) leads to increased total body weight, fasting blood glucose, and hepatomegaly. UA-supplemented HFD was able to attenuate these effects (Figure 1A-1C). Interestingly, HFD reduces skeletal muscle weights compared to SC, with UA supplementation normalizing this effect in mice subjected to HFD (Figure 1D). This suggests that UA may be working as an atrophy inhibitor under conditions of HFD-fed mice. Furthermore, preliminary data from HFD-fed mice show a significant increase in Cdkn1a compared to SC (Figure 1E). Taken together, these data leads us to hypothesize that obesity is an atrophy-inducing stress that is at least partly mediated by the ATF4/Gadd45a/Cdkn1a pathway.

7 Figure 1.1 Novel atrophy pathway involving ATF4/Gadd45a/Cdkn1a. Cellular stress activates translation of ATF4, which translocates to the nucleus. ATF4 gene target, Gadd45a, is transcribed and translated. In the nucleus, Gadd45a demethylates a specific 5-methylcytosine in the promoter region of Cdkn1a. This increases transcription of gene, resulting in an increase in its encoding protein p21. Increases in p21 promote skeletal muscle atrophy.

8 Figure 1.2 UA inhibits weight gain from a HFD. Effect of a HFD with or without 0.15% UA compared to SC after 5 weeks with C57BL/6 mice. Each data point represents one mouse and horizontal bars denote the means. *P2=/M8A/PC

9 Figure 1.3. UA reduces fasting blood glucose on a HFD. After 16 hours of fasting, blood glucose was measured. Each data point represents one mouse and horizontal bars denote the means. *P?

26 Figure 2.8. HFD reduces weight of gastrocmenius. Mice were provided ad libitum access to SC or HFD for 7 weeks. Each data point represents one mouse and the horizontal bars denote the means. P-value calculated by t-test.

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33 Figure 2.15. HFD reduces fiber size distribution of skeletal muscle. Mice were provided with ad libitum access to SC or HFD for 7 weeks. Each distribution represents the mean fiber diameter of one mouse determined from >500 quadricep muscle fibers from 10 mice. (>500 measurements / animal); p