Mordecai Micah Harvey. Doctor of Philosophy. Human Nutrition, Foods and Exercise. Matthew Hulver, Committee Chair. Kevin Davy.

Transcription

1 Characterization of an in vitro exercise model and the effects of metabolic endotoxemia on skeletal muscle adaptation to electric pulse stimulation Mordecai Micah Harvey Dissertation submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Human Nutrition, Foods and Exercise Matthew Hulver, Committee Chair Kevin Davy Madlyn Frisard Robert Grange Liwu Li May 17, 2017 Blacksburg, Virginia Keywords: (endotoxemia, skeletal, muscle, exercise, electric, stimulation)

2 Characterization of an in vitro exercise model and the effects of metabolic endotoxemia on skeletal muscle adaptation to electric pulse stimulation Mordecai Micah Harvey Abstract The prevalence of obesity and type II diabetes is increasing. Although exercise is widely accepted for prevention and treatment, evidence of resistance to exercise in patients with these diseases is also mounting. Muscle contraction during exercise stimulate cellular responses important for adaptation. These responses include the release of myokines and the subsequent increase in substrate metabolism. This study aimed to define a culture model for simulating exercise in human primary skeletal muscle cells. We hypothesized that chronic electric pulse stimulation (EPS) of human myotubes in vitro would emulate cellular and molecular responses to exercise observed in vivo. To define this model, we applied EPS to human myotubes for varied lengths of time and measured interleukin-6 (Il-6), peroxisome proliferator-activated receptor gamma coactivator 1-a (PGC1-a), superoxide dismutase 2 (SOD2), substrate metabolism, metabolic enzyme activity, heat stress markers, and ph. To recreate the inflammatory milieu observed in metabolic disease states we treated the myotubes with a low dose of 20 EU lipopolysaccharide (LPS). Following the 24-hour stimulation we observed significant increases in transcription of Il-6, PGC1-a, and SOD2. Basal glucose and fatty acid oxidation were also markedly increased in the cells after EPS. Cells treated with LPS elicited a blunted transcriptional, metabolic, and enzymatic response to EPS. These findings suggest that EPS is a viable model for simulating the effects of exercise. Our observations also indicate that an inflammatory environment could play a role in interfering with the adaptations to exercise.

3 Characterization of an in vitro exercise model and the effects of metabolic endotoxemia on skeletal muscle adaptation to electric pulse stimulation Mordecai Micah Harvey General Audience Abstract The prevalence of obesity and type II diabetes is increasing. Although exercise is widely accepted for prevention and treatment, evidence of resistance to exercise in patients with these diseases is also mounting. Muscle contraction during exercise stimulate cellular responses important for adaptation. These responses include the release of myokines and the subsequent increase in substrate metabolism. This study aimed to define a culture model for simulating the effects of exercise in human primary skeletal muscle cells. We hypothesized that stimulating these cells with electric pulses (EPS) cultured m in vitro would simulate cellular and molecular responses to exercise. To define this model, we applied EPS to human skeletal muscle cells for varied lengths of time and measured transcription of interleukin-6 (Il-6), peroxisome proliferator-activated receptor gamma coactivator 1-a (PGC1-a), superoxide dismutase 2 (SOD2), substrate metabolism, metabolic enzyme activity, heat stress markers, and ph. To recreate the inflammatory status observed in metabolic disease states we treated the cells with a low dose of 20 endotoxin units (EU) lipopolysaccharide (LPS). Following the 24-hour stimulation we observed significant increases in transcription of Il-6, PGC1-a, and SOD2. Basal glucose and fatty acid oxidation were also markedly increased in the cells after EPS. Cells treated with LPS elicited a blunted transcriptional, metabolic, and enzymatic response to EPS. These findings suggest that EPS is a viable model of an in vitro exercise model. Our observations also indicate that an inflammatory environment could play a role in interfering with the adaptations to exercise

4 Acknowledgements I would like to thank Dr. Matthew Hulver for accepting me as his student and granting me the opportunity to pursue a Ph.D. Your continued guidance and patience have been invaluable to me over the course of my graduate experience. I would like to thank my committee members, Dr. Kevin Davy, Dr. Madlyn Frisard, Dr. Robert Grange, and Dr. Liwu Li for your time and effort in giving me continued constructive feedback and assisting my progress throughout my academic career. I would also like to thank Dr. Deborah Good and my former mentor, Terry Mitchell for submitting letters of recommendation on my behalf prior to my admission to graduate school. I would be remiss in not thanking Dr. Ryan McMillan for his unwavering help and guidance throughout my time here. I want to thank Dr. Nabil Boutagy for collaborating with me in the early stages of my research project. His insight and effort was essential in getting the project off of the ground. I would also like to thank Dr. William Newton, my former biochemistry professor for providing a truly eyeopening experience in his course. His influence and teaching have heavily informed my research understanding. Dr. Angela Anderson, thank you for help and insight early on in my academic career. You influenced my approach as a teaching assistant for the better. I would also like to thank Dr. Michael Tarpey, Dr. Joey Stevens, Dr. Karen Strat, Dr. Kevin Voelker, Dr. Mostafa Ali, Dane Fausnacht, Jing Luo, Justin Sperringer, Will Moore, and Jasmine Hayes for your help at various stages of my academic career. To Dr. Lauren Kennedy, my wife, I am eternally grateful to you for your unwavering support and love. You are the best thing to happen to me and I fail to conceive who I would be as a person or student without you. To James, Mario, and Wes, thank you for your unconditional iv

5 acceptance. Your friendships have and will continue to fuel my life. To my brother, thank you for figuratively and at times literally making and showing me the path to follow. To my mom, dad, and step-dad, thank you for giving me a solid foundation in life without which none of my academic pursuits would have been possible. I love you dearly. v

6 Table of Contents Content Page ABSTRACT...ii ACKNOWLEDGEMENTS.iv TABLE OF CONTENTS.vi CHAPTER 1 INTRODUCTION...1 CHAPTER 2 REVIEW OF LITERATURE 5 REFERENCES 34 CHAPTER 3 SPECIFIC AIMS 47 SPECIFIC AIMS..48 RESEARCH DESIGN...49 EXTENDED METHODS...52 CHAPTER 4 Characterization of an in vitro exercise model and the effects of metabolic endotoxemia on skeletal muscle adaptation to exercise stimulus...58 ABSTRACT 59 INTRODUCTION 60 METHODS.62 RESULTS 67 DISCUSSION 78 REFERENCES.83 SUPPLEMENTAL FIGURES...87 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS..93 vi

7 Chapter 1 Introduction 1

8 In 2011, the prevalence of obesity in the United States reached a staggering high of 34.4% and as of 2013, an average of 37% of men and women were overweight globally. 1,2 The prevalence of cardiovascular disease and type II diabetes, which are strongly associated with obesity, is also increasing. Regular exercise is widely adopted as a strategy for treating and preventing these diseases apart from weight loss. 3 Skeletal muscle, which accounts for approximately 40% of body weight in lean individuals, is the primary affected organ during exercise. Other organs such as the brain, liver, lungs, and heart are also impacted. 4 Recently, researchers have uncovered the endocrine function of skeletal muscle and thus its crosstalk with the other organs during muscle contraction. The role of skeletal muscle as an endocrine organ acting on other tissues is integral in understanding how it mediates the physiological responses to exercise. During exercise, skeletal muscle secretes cytokines, which act locally on muscle and in an endocrine fashion affecting remote organs. In the context of muscle physiology these peptides are known as myokines. 5 Interleukin-6 (Il-6) was one of the first of these identified and is the prototypical myokine. Earlier research of Il-6 postulated that the cytokine was released by monocytes in response to muscle contraction. However, a decade of research has shown that muscle is primarily responsible for its secretion. 5 Dramatic increases in serum Il-6 occur at the onset of exercise and lead to increased glucose uptake, hepatic glucose production and lipolysis. 6-8 Other myokines influenced by muscle contraction are, Interleukin-1 Beta (Il-1b) 9, Il-8 10, Il-15 11, angiopoetin-like protein 4 (ANGPTL4) 12. fibroblast growth factor 21 (FGF-21) 13, secreted protein acidic and rich in cysteine (SPARC) 14, leukaemia inhibitory factor (LIF) 15, irisin 16, ciliary neurotrophic factor (CNTF) 17, 2

9 apelin 18, myostatin 19, and brain derived neurotrophic factor (BDNF). 20 Many of these are implicated in affecting metabolism in skeletal muscle and other tissues such as skin, bone, and adipose tissue. 7,20-22 In recent years, researchers have used electrical pulses to stimulate cultured skeletal muscle as an in vitro model for exercise. Findings from earlier studies using this model involve the culturing of C2C12 myotubes and demonstrate responses that mimic the effect of exercise observed in vivo. Significant increases in expression and secretion of interleukin-6 (Il-6), a notable myokine, is repeatedly shown in C2C12. 23,24 In vivo, Il-6 secretion due to exercise increases glucose uptake, hepatic glucose production, and lipolysis. 7 Findings from studies using the (EPS) model show rises in glucose consumption, GLUT4 translocation 25, and MAPK signaling 23,26 in C2C12 myotubes. Furthermore, C2C12 myotubes treated with EPS have significantly higher levels of reactive oxygen species (ROS) production 27 and gene expression of PGC-1a Taken together these findings suggest use of the EPS model may be a viable means of mimicking exercise in vitro. Throughout the past decade, the majority of researchers applying this model did so using C2C12 myotubes while only a few studies involved stimulation of human primary myotubes. Using EPS on human cells yields results that demonstrate a similar increase in creatine kinase activity, glucose uptake, expression of Il-6 and other myokines such as Il-8, LIF, and ANGPTL4. 10 Another group reported increased expression of Il-6, phosphorylated Akt, and insulin signaling. 30 Most recently a study stimulated cultured myotubes from lean, severely obese non-diabetic, and severely obese diabetic subjects, and observed increased glucose oxidation in all groups but only increased fatty acid oxidation for lean myotubes. 31 The data reported from studies using human subjects adds to the growing definition of the EPS model 3

10 and addresses the topic of chronic disease and the adaptation to exercise. We intend to fill the gaps in research concerning this model by measuring the effect of EPS on metabolic enzymatic activity, superoxide formation, and heat stress. The purpose of this study is to first further characterize the EPS model and explore how the response to EPS is affected by cell type, culture conditions, and the presence of endotoxin. We will test the hypothesis that the use of EPS on human primary myotubes will mimic the metabolic, transcriptional, and physical effects of exercise such as temperature and ph observed in contracting skeletal muscle. Recent studies have contributed to the evidence of a resistance to exercise observed in populations with obesity, type II diabetes, and CVD. 32,33 Chronic low-grade inflammation is associated with these diseases and is referred to as metabolic endotoxemia. 34 We will also investigate the effect of metabolic endotoxemia on the EPS response in human primary myotubes by treating with low levels of lipopolysaccharide (LPS or endotoxin) during stimulation. We hypothesize that the presence of endotoxin will blunt the response to EPS.. 4

11 Chapter 2 Review of Literature 5

12 The objective of the following review is to highlight key myokines and their corresponding roles in skeletal muscle and other tissues. The effect of exercise on cytokine response in skeletal muscle is also important in this review as our proposed model simulates it. This review will also address what is known regarding chronic inflammation and subsequent effects on metabolic function in skeletal muscle. We intend to identify the holes in the literature regarding the cytokine response to exercise and the effect of a proinflammatory environment on that response. Il-6 (Interleukin-6) Discovered in 1980, Il-6 is a member of the gp130 family of cytokines. It was first characterized by its role in inflammatory signaling. Over a decade after its discovery, exercise is shown to be a stimulant for Il-6. 8 Specifically Il-6 secretion is most responsive to prolonged running and has the highest plasma concentrations immediately following activity. 6 Although Il-6 has been characterized by an inflammatory response, the results of a study by Starkie et al show that skeletal muscle, not monocytes, is the cause of elevated Il- 6 after exercise. 35 The notion that Il-6 increases with muscle contraction were due to an immune response in muscle caused by tissue damage was debunked when the production of Il-6 was compared with concentric versus eccentric muscle contractions. There are no significant differences in secreted Il-6 based on the type of contraction, suggesting that muscular damage does not influence on this phenomenon. 36 Although the type of contraction does not appear to impact Il-6 activity, muscle fiber type appears to be important. However, there are conflicting research findings on this matter. Using immunohistochemistry in three studies, only one showed type II fibers to be 6

13 the predominant source of Il-6 during contractile activity Creating cause for further speculation another finding implicated type I fibers as the predominant source of Il Glucose, specifically muscle glycogen appears to have a potent effect on exerciseinduced Il-6 production. When muscle glycogen is low pre-exercise the Il-6 response is significantly augmented. 41,42 Intuitively, post-exercise glycogen levels are negatively correlated to the duration of exercise, and changes in Il-6 in skeletal muscle and plasma. 43 Heat shock protein 72 (HSP72) and phosphorylated p38 mitogen activated protein kinase (MAPK) are increased with carbohydrate deficits. 44,45 Il-6 is also secreted from adipose tissue in response to exercise and acts on skeletal muscle resulting in subsequent increases in mrna and plasma Il When carbohydrate supplementation is provided pre-exercise, Il-6 activity is blunted significantly. 47 In this context, Il-6 appears to have lipolytic effects by mobilizing free fatty acids (FFA s) in response to exercise. These effects were reconfirmed by a later study in which muscle and adipose tissue incubation with Il-6 yielded increased phosphorylation of AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase (ACC). AMPK and ACC are important metabolic markers because AMPK phosphorylates ACC and 6- phosphofructo-2-kinase, which leads to an increase in fat oxidation and glycolysis. When Il- 6 deficient mice are exercised the activity of these biomarkers is diminished indicating that Il-6 plays a role. 48 Chronic Il-6 exposure in human skeletal muscle also increases myotube fusion and formation, the expression of glycogen synthase, myocyte enhancer factor 2D (MEF2D) uncoupling protein 1 and 2 (UCP1, UCP2), and fatty acid transporter 4 (FAT4). These biomarkers are upregulated concomitantly with increased phosphorylation of AMPK, signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinases 1/2 (ERK1/2), and activity of phosphotidylinositol (PI) 3-kinase. 49,50 7

14 Il-6 treatment of L6 cells stimulates fatty acid oxidation and insulin stimulated glucose uptake and GLUT4 translocation. Another study done using Il-6 deficient mice demonstrates that acute exercise elicits an increase in GLUT1 and GLUT4 activity without a significant effect on fatty acid transporters. 51 This phenomenon appears to be mediated by AMPK because when cells infected with adenovirus containing control vector dominantnegative AMPK mutant the effects of the treatment are abrogated. 51 Only a relatively recent study done by O Neil et al yielded findings contrary to those previously described. Il-6 knockout mice were observed to have similar rates of glucose uptake and utilization and phosphorylation of AMPK as the wild type mice. 52 The exercised-induced Il-6 response is adaptable. Specifically endurance training is shown to increase the levels of resting muscle glycogen, capacity for fatty acid oxidation, and lessen the magnitude of Il-6 secretion from muscle in humans after 10 weeks. 53 Basal serum concentrations of Il-6 are lower in participants engaged in more physical activity than their sedentary counterparts. This trend is also observed for the levels of tumor necrosis factora (TNF-a) and C-reactive protein (CRP). 54 In contrast to Il-6, production of TNF-a is not responsive to exercise and this is likely due to TNF-a s down-regulation of glucose uptake. Presently, Il-6 is also known to inhibit TNF-a in this context. 5,55 The s relationship between Il-6 and TNF-a in the context of exercise is shown in Figure 1. Figure 1. The figure below taken from (Pederson et al 2008) further illustrates how signaling of Il-6 during exercise is different in that circulating TNF-a is only present during a pro-inflammatory insult such as sepsis. 8

15 The findings of other earlier inhibitor studies involving Il-6 are useful in differentiating how TNF-a and Il-6 signaling pathways are regulated. The results of one such study demonstrate how lipopolysacharride (LPS) exposure increases transcription of Il-6 and TNF-a. Exposure to the antioxidant dexamethasone blunted Il-6 transcription but not TNF-a and the proteasomal inhibitor MG-132 blunted TNF-a signaling without decreasing Il The implications of these findings suggest that Il-6 is activated by a different mechanism. A later discovery, resulting from the use of inhibitors shows that calcineurin is involved in the induction of Il-6. Human skeletal muscles stimulated with ionomycin were treated with the calcineurin inhibitor cycloporin A (CSA). CSA exposure knocked down Il-6 secretion while TNF-a secretion increased. 57 This phenomenon was mimicked in another study in which electric pulse stimulation (EPS) was used to increase Il-6 secretion and transcription. This response was blocked with CSA treatment. 24 Experimentation with EPS, which will be highlighted later in this review, has yielded findings that also uncover important details of Il-6 signaling. Induction of Il-6 via exogenous ATP and EPS elicited increases in Il-6 expression and subsequent janus kinase 2/ signal transducer and activator of transcription 3 (JAK2/STAT3) activation. When inositol 1,4,5 9

16 triphosphate (IP3)-dependent calcium channels are blocked STAT3 phosphorylation is abolished. 58 An earlier study in which K + depolarization was used as a means to stimulate Il- 6 transcription in human myotubes, yielded similar findings implicating IP3-mediated calcium signals. 59 Il-6 upregulates satellite cell mediated hypertrophic muscle growth via STAT3. This is shown in Wistar rats that undergo 10-week resistance training protocol that yielded increases in Il-6 and suppressor of cytokine signaling 3 (SOCS) and the phosphorylation of STAT1 and STAT3. 60 In summary, Il-6 is the prototypical myokine and one about which researchers have uncovered a wealth of knowledge. It is highly responsive to exercise, especially endurance running. It seems to be secreted primarily by type II muscle fibers. However, there are conflicting findings in this regard, which makes further investigation necessary. The exercise-induced effect on Il-6 secretion is heavily impacted by the availability of glucose and this secretion increases fatty acid and glucose metabolism via AMPK signaling. Ca++, IP3, and STAT3 also mediate il-6 activity. The Il-6 response brought on by muscle contraction is adaptable and lessens in magnitude with prolonged training. Il-1b (Interleukin-1 Beta) Il-1b is a cytokine that is released from blood monocytes in response to an infectious or inflammatory stimulus. It is also expressed in healthy and diseased skeletal muscle 61 and is also expressed in the heart, liver, kidney, and spleen. 62 Epinephrine and endotoxin are also stimuli for increased expression of Il-1b in rat skeletal muscle. 62,63 However, epinephrine levels induced here resembled those associated with septic shock

17 With regard to the effect of exercise, there are many conflicting results on the response of Il-1b. Earlier research implicates exercise as another stimulus for Il-1b secretion. Specifically, eccentric exercise elicits accumulation of Il-1b in the muscle and this increase is correlated to Z-band damage. 9 Increased intramuscular Il-b is sustained up 5 days post exercise. 64 According to other findings, plasma Il-1b also increases in response to exercise with no significant changes in mrna expression. 65 In contrast to eccentric exercise, prolonged running yields only modest increases in Il-1b expression. 66 Based on Ostrowski et al endurance exercise elicits increases in plasma within an hour but the same activity yielded in no change in Il-1b concentrations. 6,67 (See Table 1) There is a lack of conclusive data linking Il-1b to the exercise response in skeletal muscle. Although it is known that LPS induces increases in Il-1b, the association between its activity and chronic metabolic disease has not been made. To date, only one group investigating cytokine profile of type II diabetic patients showed no relationship between Il- 1b levels and chronic inflammation. 68 These unanswered questions enhance the need to further explore the effect of contraction and the impact of chronic inflammation on Il-1b. Il-8 (Interleukin-8) Il-8 is a chemokine of the CXC family that generally attracts neutrophils (5). Research findings throughout the past 15 years have revealed mixed conclusions on how exercise influences Il-8. According to Suzuki et al. plasma Il-8 is significantly increased in athletes following a marathon. 69 Other studies yielded similar findings after subjects completed an ultra-marathon, which may suggest that Il-8 is responsive to endurance training. 70,71 Il-8 has 11

18 also been shown to be responsive to exercise in horses following a km race as expression is increased. 72 Conversely exercise is shown to reduce serum Il-8 levels in lean and obese subjects. Basal plasma Il-8 is significantly elevated in overweight and obese participants. 73,74 (See Table 1) Investigation in to the effect of carbohydrate availability on Il-8 levels yielded findings similar to that of Il-6. Prolonged running in one study produced increases in Il-8 plasma with sharper rises in participants that did not receive carbohydrates. 75 Intervals of cycling elicited a similar response but only in transcription of Il-8, with serum levels unaffected. Il-8 expression was markedly increased in the low glycogen group aminoimidazole-4-carboxamide-1-b-D-ribonucleoside (AICAR), which has been shown to improve insulin sensitivity, inhibits Il-8 secretion and expression in skeletal muscle. 77 Alternatively, treatment with TNF-a in human myoblast increases expression of Il Myotubes cultured from type II diabetics have higher levels of Il-8 secretion and subsequent reduced capillary density. 79 This phenomenon is counterintuitive because of the long established role of Il-8 is the induction of de novo angiogenesis. 80 This however, is different in the context of chronic diseases such as diabetes. For example, when diabetes is induced by streptotocin in rats, Il-8 mediated angiogenesis is significantly blunted. 81 Taken together, these findings implicate the role of Il-8 in chronic metabolic disease. In summary, Il-8 is another important myokine that seems to be responsive to endurance exercise. Similar to Il-6, exercise induced Il-8 is significantly influenced by the availability of glucose. Il-8 is also associated with the presence of pro-inflammatory cytokines like TNF-a and with obese and diabetic status, which could suggest an anti- 12

19 inflammatory role. However, more data is needed to elucidate the role of Il-8 in disease and exercise adaptation. Il-10 (Interleukin-10) Il-10 is an anti-inflammatory cytokine found in skeletal muscle. It is a potent inhibitor of T-cell proliferative and pro-inflammatory cytokine responses. 82 There is a lack of evidence that training has a direct impact on Il-10. Plasma Il-10 increases substantially after prolonged running. 6,83 (See Table 1) However, exercise in post-myocardial infarction (MI) rats doesn t increase Il-10 levels but does increase the ratio of Il-10/TNF-a. Protein and mrna expression drop increasing the ratio and reverse the effects of MI. 84 This suggests that exercise decreases circulating TNF-a as an anti-inflammatory effect of exercise. Il-10 expression and activity has a strong positive relationship to insulin sensitivity. 85 Subjects with chronic diseases such as obesity and type II diabetes have lower capacity for Il-10 production. 86,87 When Il-10 is over-expressed specifically in muscle, mice on a high-fat diet develop the obese phenotype while retaining sensitivity to insulin. 88 Il-10 is also shown to protect against Il-6 and fatty acid induced insulin resistance in mice. 89 Il-10 knockout mice have more damaged mitochondria than similar aged counterparts and do not recover from muscle damage as muscle regeneration and growth are significantly blunted when Il-10 is ablated. 90,91 Il-10 is involved in producing the M2 macrophage phenotype in injured skeletal muscle. An application of this in mice with mdx muscular dystrophy demonstrates that ablating Il-10 increases muscle damage and that Il- 10 is important activating M2 macrophages and deactivating M1. 92 The absence of Il-10 is 13

20 also associated with a greater Il-6 response to endotoxin in skeletal and cardiac muscle of mice. 93 Il-10 is a characteristically anti-inflammatory cytokine that has a protective effect on skeletal muscle. Beyond the effect of training on plasma Il-10 and its relationship to TNF-a, more investigation is needed to identify the impact on exercise on Il-10 in muscle. Furthermore, understanding the role of chronic inflammation in the adaptation to exercise in skeletal muscle is of importance. Il-15 (Interleukin-15) Il-15 is an anti-inflammatory cytokine that has been detected in skeletal muscle, cardiac muscle, the lungs, kidneys, and placenta. 94 Its expression in skeletal muscle is highest in type II fibers. 11 Until recent years, the effect of exercise on Il-15 has not been clear. However, there is evidence that resistance training increases plasma and mrna of Il ,95 More recent findings indicate that exercise increases Il-15 activity. Specifically, endurance training in rats over 8 weeks is shown to significantly increase Il-15 production. 96 Similarly, prolonged cycling drives Il-15 protein up 40% but without increasing plasma or mrna levels. When cycling for a 3-hour bout circulating Il-15 is increased which suggests that Il- 15 is activated by exercise on a unique time-course. 97 Though limited, there is also evidence that resistance exercise in rats increases Il-15 protein content. 98 (See Table 1) Most notable of Il-15, is its endocrine action on white adipose tissue (WAT). Il-15 regulates WAT by decreasing its mass and inhibiting lipid accumulation and limiting plasma triacylglycerols. 99,100 Il-15 is not expressed in preadipocytes or differentiated adipocytes, 14

21 which implicates skeletal muscle as the source of this action. Adiponectin secretion from WAT is stimulated by Il-15, which promotes glucose uptake, fatty acid oxidation, and insulin sensitivity. 101,102 Il-15 inhibits muscle wasting in association with cancer relate cachexia. Over expression of Il-15 causes hypertrophic myotube morphology. 103 Findings by Li et al implicate a protective role of Il-15 in C2C12 myoblasts. Preincubation with Il-15 decreased peroxide-induced reactive oxygen species (ROS) production. 104 BDNF (Brain Derived Neurotrophic Factor) Brain-derived neurotrophic factor (BDNF), which is in the mammalian family of neurtrophins that include, nerve growth factor, neurotrophin-3 (NT-3), and neurotrophin- 4/5 (NT-4/5) is expressed in skeletal muscle satellite cells and also innervates motor neurons. 105 Collectively this family of neurtrophins are important in increasing the area of innervated muscle fibers, motor endplates per muscle fiber and the length of neurites according to findings of early research performed in rat embryo spinal chord explants. 106 BDNF binds the receptors tyrosine kinase B (TrkB) and or p75 NTR, which, initiates intracellular events important to neural development. The expression of BDNF is linked to a reduction myogenic differentiation and when it is silenced, myoblast differentiation is enhanced. 107,108 The majority of findings related to the effect of exercise on BDNF are similar in that its expression is enhanced by exercise. Wheel running, treadmill running, and electrical stimulation (ES) have shown to increase gene and protein expression of BDNF. Voluntary wheel running also enhances neuroplasticity, CREB, and synapsin 1 mrna, a receptor for BDNF. 109 Acute and endurance exercise via treadmill also enhance BDNF expression for as 15

22 long as 72 hours following activity The use of EPS in C2C12 cells shows increases in BDNF, ACC, and AMPK expression. 113 Only the findings of one study demonstrate no effect of exercise on BDNF in which fasting was shown to upregulate expression of BDNF. This is also the only study done in humans correlating BDNF with exercise to date. 113 (See Table 1) SPARC (Secreted Protein, Acidic and Rich in Cysteine) Secrete protein, acidic and rich in cysteine (SPARC) is a myokine, which is expressed in adipose tissue and skeletal muscle. Early findings link SPARC to myogenesis with observed increases in transcription during myoblast differentiation, which appears to be mediated by calcium. 114,115 SPARC expression is also linked to myopathies such as Duchenne and Beker dystrophy, congenital myopathy and inclusion body myositis. 116 In adipose tissue SPARC has a similar role and SPARC expression is increased during the differentiation of preadipocytes. 117 SPARC expression decreases with age and this drop is linked to decreases in muscle fiber diameter. 118 The relationship between exercise and SPARC is not clear. Resistance exercise suppresses SPARC expression and secretion. However, SPARC expression and secretion increases in response to single bout of aerobic exercise. 14,119 (See Table 1) The promotion of SPARC via exercise is linked to increased apoptosis of tumor cells. 119,120 Interestingly, AMPK activation without exercise increases SPARC expression in L6 cells. Glucose deprivation via GLUT4 is also a stimulus for SPARC. 121 Current findings regarding SPARC suggest that exercise is a stimulant for its activity. However, more investigation is necessary to fully address the role of SPARC in the response to contraction in skeletal muscle. 16

23 ANGPTL4 (Angiopoietin-like 4) ANGPTL4 is expressed in liver, adipose tissue, hypothalamus, and skeletal muscle and is an inhibitor of lipoprotein lipase (LPL)-mediated plasma triglyceride clearance. 12 Longterm fasting, endurance exercise, and serum free fatty acids (FFA) are stimulants for ANGPTL Recent findings demonstrate that FFA mediates ANGPTL4 via peroxisome proliferator-activated receptor-d (PPAR-d) to inhibit uptake of serum triglyceride derived fatty acids in exercising and non-exercising muscle. During exercise, however, ANGPTL4 blocked by AMPK and LPL activity is uninhibited. 12,122,123 (See Table 1) Exercise is also shown to stimulate expression and secretion of ANGPTL4 from adipose tissue and liver to a greater extent than muscle. 124 ANGPTL4 is expressed two-fold more in the hypothalamus and skeletal muscle of diabetic obese mice than wild types. Insulin treatment causes a decrease in serum ANGPTL4 and a subsequent in LPL activity. Interestingly, in ANGPTL4 deficient mice there is no observed effect on LPL or diabetic status. 125 Irisin Irisin is a novel myokine, which increases metabolic rate and mitochondrial content in skeletal muscle and adipose tissue. Irisin is activated by transcription of its precursor fibronectin type 3 domain containing 5 (FNDC5). A recent in vitro study yielded results that irisin stimulates oxidative metabolism and upregulates expression of PGC1-a, NRF1, TFAM, UCP3, and GLUT Exercise and the drug metformin are stimulants for irisin. FNDC5 and serum irisin are lower in diabetic mice. 127 While, metformin causes increased expression of FNDC5 and subsequent irisin release it does so independently of AMPK. 127 Resistance 17

24 exercise and treadmill running have been demonstrated to stimulate irisin. However, only treadmill running for mice has resulted in a rise in serum irisin. 16,128 Results of another recent study demonstrated no effect of exercise on FNDC5 or irisin in vivo but implicated plasma irisin with muscle mass, strength and increased metabolism. 129 (See Table 1) Myostatin Myostatin also known as growth and differentiation factor-8 (GDF-8) is a myokine and negative regulator of skeletal muscle mass that inhibits activation of muscle satellite cells. 130 The effects of myostatin are demonstrated in knockout mice, which have higher levels of hypertrophy and hyperplasia than wild types. Similarly when mice are treated with an inhibitory antibody, they exhibit increased muscle mass. 131,132 When muscular atrophy is induced with the glucocorticoid dexamethasone, myostatin mrna and protein expression is increased while blocking dexamethasone attenuates muscle atrophy. 133 Intuitively, muscle specific over-expression of myostatin induces atrophy and lower muscle mass. As a negative regulator of the Akt/mTor pathway myostatin elicits these effects independently of the ubiquitin-proteosome pathway. 134,135 Resistance and aerobic exercise downregulate myostatin mrna expression 6-fold and 3-fold, respectively. 19 (See Table 1) Also, inhibiting myostatin prior to exercise is shown to increase running time and sensitivity to insulin while mice treated with recombinant myostatin are less insulin sensitive. 136,137 Gene and protein expression of myostatin is elevated in necrotic fibers and lower in regenerating muscle fibers. 138 The present findings related to myostatin suggest that its regulation is important in treating muscle degenerative diseases. 18

25 LIF (Leukemia Inhibitory Factor) Conversely, leukemia inhibitory factor (LIF) has seemingly opposite effects on skeletal muscle. LIF is a part of the Il-6 superfamily and early research demonstrates its effect on muscular regeneration. Using the crush model, which involves the exposure by dissection of the vastus lateralis and the use of forceps to crush the muscle. With this methodology, in which skeletal muscle is severely damaged, LIF administration promotes regeneration hypertrophy without affecting the number of muscle fibers. LIF s action is most likely autocrine in nature because it is not detected in circulation. 139 LIF knockout mice have significantly less muscle regeneration than wild types and the infusion of LIF restores this function. 140 LIF is also a stimulator of muscle satellite cell proliferation and hypertrophy and glucose uptake in mice via Akt phosphorylation. 21,141 Exercise is shown to upregulate LIF mrna but not protein expression and treatment of myocytes with ionomycin upregulates both. 15 (See Table 1) Research exploring the effect of exercise on LIF is lacking to date. Additionally a study yielded contrary findings that showed LIF mrna decreased after two weeks of exercise in mice. 142 Studies have also drawn association between the upregulation of LIF and muscle disuse atrophy and muscle injury. 143,144 FGF-21 (Fibroblast Growth Factor-21) Fibroblast growth factor-21 (FGF-21) is a myokine that regulates glucose metabolism by enhancing clearance of plasma glucose. 145 It is expressed in and secreted by skeletal 19

26 muscle in response to insulin infusion in human males. 146 Diet induced obesity in mice is attenuated by FGF-21 in a dose-dependent manner. Mice undergo reduced body weight and whole body fat mass while total energy expenditure and physical activity levels increase. FGF-21 administration also reduced hepatic triglyceride levels and increased insulin sensitivity. 145 Mice overexpressing FGF-21 are resistant to diet induced obesity. 147 Additionally, mice that overexpress UCP1 were shown to have higher expression and circulation of FGF Research findings further demonstrate FGF-21 as a promising target for treatment of diabetes as lipid induced insulin resistance in human skeletal muscle cells is reversed by FGF-21 treatment allowing Akt phosphorylation and inhibition of NFkB activation. 149 Serum FGF-21 is elevated in patients with type II diabetes, which is counterintuitive given its demonstrated role in stimulating nutrient uptake. However, this could be due to a compensatory response given that type II diabetic patients have a compromised ability to clear nutrients from the bloodstream. 150 Findings related to the effect of exercise on FGF-21 are mixed and lacking. Aerobic exercise is shown to increase FGF-21 in plasma after two weeks and modestly decline after a 3-month training program. 13,151 (See Table 1) There is a need to uncover the effect of exercise acutely and chronically. CNTF (Ciliary Neurotrophic Factor) Ciliary neurotrophic factor is a myokine expressed in glial cells and skeletal muscle. Early findings show that its synthesis decreases with age and it is more highly expressed in denervated muscle and that it blunts denervation-induced atrophy. 17 In regenerating 20

27 muscle, CNTF is upregulated. 152 CNTF acts through the CNTFRa-Il-6R-gp130b receptor to increase fatty acid oxidation and lower insulin resistance by activating AMPK. It is also shown to induce weight loss and improve glucose tolerance in humans and rodents. 153 CNTFRa is highly conserved and mice lacking this component of the receptor die prenatally. 154 CNTF also promotes muscle progenitor cell viability in culture and does so through the PI3-Akt pathway. Inhibition of CNTF abrogates cell viability in mice lacking PI3 kinase. 155 Apelin Apelin is a myokine expressed in skeletal muscle and adipose tissue. Findings related to its function are relatively novel. A study done in 2008 revealed that apelin induces glucose utilization in skeletal muscle via AMPK and Akt activation. 156 Apelin is also shown to decrease adiposity and improve insulin resistance in mice. 157,158 Eight weeks of endurance exercise stimulates transcription of apelin but not secretion. Cylclic-AMP and calcium are also stimulants of apelin in vitro. 18 A recent similar study in which an eight week exercise program was used yielded findings that apelin plasma and gene expression are increased. 159 (See Table 1) To date there are only two investigations with published results linking exercise and apelin. PGC-1a upregulates apelin in adipocytes and overexpression of the transcription factor induces secretion and expression of apelin. 160 There is a need to explore the apelin and associated disease as only one study demonstrates that apelin is elevated in human obese subjects

28 Myonectin Another novel myokine is myonectin, which is in the C1qTNF-related protein (CTRP) family. It is induced in differentiating myotubes and is predominantly expressed in skeletal muscle. Feeding and exercise are stimulants for myonectin expression and secretion. Recombinant myonectin lowers FFA s without altering tissue lipolysis. Exercise is also shown to stimulate myonectin with no effect on irisin precursor FNDC. 162,163 (See Table 1) There is a lack of research investigating the effect of knocking down myonectin, which may offer clues to its mechanism of action. The study of cytokine signaling in skeletal muscle has evolved immensely over the past decade. Early discovered myokines are implicated in mediating glucose and fat metabolism, muscle growth and differentiation, and a responsiveness to muscle contraction. Identification of more recent myokines further demonstrates muscle s capacity for endocrine function by acting on itself, adipose and neural tissue, and tumor cells. The effect of exercise on the activity of these molecules is important to the context of the proposed study. The chart below provides a picture of what is known about the cytokine response in muscle to exercise. There are numerous areas of conflicting results and others in which more information is needed. 22

29 Table 1. The table below shows a list of myokines indicating expression in muscle and responsiveness to exercise. (+) denotes either yes or upregulation, (-) denotes downregulation, and (=) denotes no significant change. The reference number of the corresponding study is listed next to one of the three markers. Myokine Expression in Muscle Responsiveness to Exercise mrna protein mrna protein serum Il , 29, 34 +8, 29, , 29, 34 Il-1b = , 53 +6, , 53 Il , 60, , 63 No findings at present Il = 73 =73 + 6, 72 Il = , , 87 = , 85 BDNF = , 99, 100, = 102 SPARC No findings at present +108 ANGPTL , 112 No findings at present Irisin = , 117 Myostatin No findings No findings at present at present LIF = 15 No findings at present FGF No findings at present CNTF No findings at present No findings at present No findings at present + 13, 140 No findings at present Apelin No findings at present Myonectin No findings at present =

30 Skeletal muscle and electric pulse stimulation The means by which numerous myokines have been discovered and characterized is through an approach known as electric pulse stimulation (EPS). EPS is a method of simulating in vivo exercise related muscle contraction in vitro, by electrically stimulating myotubes with varying protocols for frequency (Hz), pulse duration (ms), and time interval. The earliest of studies in which EPS was used, yields findings that it promoted slow type I fiber phenotype at low frequencies and lowered ATP levels while increasing extracellular adenosine in contracted muscle. 164,165 Another important discovery is that EPS induces synchronous contractions of C2C12 myotubes. 166 At the turn of the century, Connor et al demonstrated that within an hour of eliciting acute contractile activity in C2C12 myotubes, mitochondrial factors such as early growth response (Egr-1) and chronic stimulation induced specificity protein 1 (Sp1) mediated expression of cytochrome C expression. 167 Another group reproduced similar findings, by showing that Egr-1 and serum response factor (SRF) were upregulated within an hour of EPS treatment. 168 Reactive oxygen and nitrogen species (ROS and NOS) are induced by low frequency electrical stimulation in H2k b cells. At higher frequencies, nitric oxide generation is also increased 169,170. EPS induced ROS s role in upregulating PGC-1a, UCP3, and hexokinase II (HKII) was revealed by Silveira et al in rat myotubes using antioxidant incubation. 171 At low frequencies EPS has been shown to increase repetitive calcium transients and the development of sarcomere structure in C2C12 myotubes. 172 These findings further demonstrate EPS as a simulation for in vivo exercise. Another study using this cell type demonstrated that EPS induces responses in skeletal muscle such as increased GLUT4 translocation, glucose uptake, and phosphorylation 24

31 of key metabolic targets, Akt, acetyl CoA carboxylase and AMP kinase. 25 GLUT4 and Akt activity responded to EPS in a fashion similar to insulin treatment. Il-6 another important target for exercise in vivo was upregulated by EPS. 25 Another group used EPS to induce contractile activity and showed that it induces mitochondrial adaptations in C2C12 myotubes via PGC1-a. The same group also showed that cells deficient of this transcription factor respond to EPS with increased phosphorylation of AMPK. 29 This finding could implicate a compensatory mechanism in stimulated myotubes. Other transcription factors such as estrogen related receptor alpha (ERR alpha), transcription factor A (TFAM), and GA binding protein A (GABPA) are upregulated in C2C12 myotubes treated with EPS. 28 These regulate transcription of nuclear genes encoding mitochondrial proteins (NUGEMPS). Chronic EPS elicits an increase in transcription of these genes, which include cytochrome C, ATP synthase and cytochrome C oxidase (COX), and COX4 and 5. 28,29 Applications of EPS in myotubes have shown activation of the mitogen-activated protein kinase (MAPK) pathways with increased phosphorylation of extracellular signalregulated kinases 1/2 (ERK1/2), c-jun NH2 terminal kinase (JNK) and p38 MAPK. 10,26 Interestingly, a study done yielded findings that similar to others with regard to EPS-induced MAPK and Akt phosphorylation and glucose uptake. However, ERK 1/2 was downregulated by EPS. Authors of this work also demonstrated that EPS decreased activation of nuclear factor kappa B (NFkB) and induced complete fatty acid oxidation and glucose uptake and oxidation. 30,173 EPS is shown to protect human skeletal muscle from induced impaired insulin signaling via chemerin, monocyte chemotactin protein 1(MCP1), and adipocyte conditioned media 30. These findings are nearly paralleled with the existing dynamic of exercise induced molecular events observed in vivo

32 Recent investigation reaffirms EPS as a stimulator of ROS, glucose uptake in skeletal muscle, and Il-6 expression and secretion. 24, One such group showed that JNK mediates the Il-6 response in C2C12 myotubes through inhibition. Additionally, JNK-deficient mice showed no changes in Il-6 from treadmill running. 23 Calcium and ROS are also important to the increase in p38, PGC-1a, and CaMKII activity brought on by EPS. The antioxidant NAC and reduces p38 phosphorylation and calcium chelation reduces CaMKII and PGC-1a promoter activity in C2C12 cells. 27 More recently it has be shown that EPS conditioned media can induce transcription in hepatocytes that have been treated with it. 178 This takes place in the absence of myotubes, which has compelling implications concerning the effects of using EPS as a model for exercise. The comparison of EPS responses in cultured myotubes from lean, severely obese non-diabetic, and severely obese type two diabetic subjects was also recently explored. Glucose oxidation is increased for all tissue types in response to a low frequency chronic stimulation. Fatty acid oxidation however, only increased significantly for stimulated lean myotubes. Il-6 expression only increased for lean and severely obese non-diabetic myotubes. When PPARd was activated, fatty acid oxidation increased for all groups without further increase with EPS. Severely obese type two diabetic tissue did not show increases in Akt phosphorylation without the administration of insulin. 31 These findings highlight the effect of chronic disease in vivo on adaptation to exercise and validate the use of EPS as a viable model for exercise in vitro. EPS is a proven in vitro model for exercise, producing many of the similar effects observed during muscle contraction in vivo. These responses range from increased generation of ROS and NOS, Il-6 upregulation, and fatty acid and glucose oxidation. Research 26

33 findings demonstrate that electrically stimulated muscle is protected from impaired insulin signaling which is a comparable response to exercise in vivo. The majority of researchers using EPS have done so with C2C12 myotubes. The aim of the proposed study is to use this model with human primary myotubes as there is a lack of published data characterizing the response to EPS in human muscle. The chart below shows a breakdown of the numerous studies in which this methodology was used. Table 2. The table above is a chronological list detailing studies in which electrical stimulation was used on skeletal muscle. (+) Denotes an increase and appears before the respective target measured. Reference Tissue/Cell Protocol Pulse Parameters Number Type Generator Measured 154 Wistar Rat myotubes 10 V at 2.5 Hz with a 4 ms pulse with a 250 ms pulse train for up to 13 days 155 C2C12 myotubes 3 V/cm 2 at 2 Hz with a 6 ms pulse for 48 hours 153 Primary Rat myotubes 50 V at 2.3 Hz and 1 Hz with a 1 ms pulse for 1,2,5,10,15, and 30 min 156 C2C12 myotubes 65 V at 5 Hz for 5, 15, 30, 60, 240 min or 3h/day for 4 days 167 C2C12 myotubes V at 5 Hz for 5, 15, 30, 60, 180, and 12 hours S88 Grass Instruments Custom built with 1 mm platinum electrodes Custom built with silver chloride electrodes S88 Grass Instruments Electrosonic Instruments +MHC1 +SERCA1 Adenosine formation +Sp1, +cytochrome c +ATP synthesis +Egr1, +SRF, +Sp1 27

34 158 H2k b myotubes 30 V/well at 1 or 50 Hz with a 2 ms pulse 159 H2k b myotubes 30 V/well at 50 Hz with a 2 ms pulse for 10 min 160 Primary rat myotubes 10 V at 1 Hz with a 1 ms pulse for 90 min 161 C2C12 myotubes 40 V/mm 2 at 1 Hz with a 24 ms pulse for 1, 2, and 6 hours 162 C2C12 myotubes 40 V/mm 2 at 1 Hz with a 2 ms pulse for 24 hours 164 C2C12 myotubes 14 V at 50 Hz with a 1 ms pulse for 90 min or 90 min/day for 4 days 169 C2C12 myotubes 4 ma at 1 Hz with a 20 ms pulse for 10 min 171 Primary rat myotubes 10 V at 50 Hz with a 1 ms pulse for 30 min, 500 ms pulse train and 500 ms pause 163 C2C12 myotubes 9 V at 5 Hz for 3 hours/day for 4 days Custom built with 1 mm platinum electrodes Custom built with 1 mm platinum electrodes Custom built with 1 mm platinum electrodes Ion Optix Ion Optix Ion Optix Porous alumina membrane based stimulator Custom built with silver chloride electrodes Ion Optix +ROS, +NOS +ROS +HK2, +PGC1-a, +UPC3 and +ROS Sarcomere architecture, +Ca ++ transients +Glucose uptake, +GLUT4 translocation +ERRa, +PGC1-a, +GABPA, +TFAM +Glucose uptake +Glucose uptake, +HK2, +PFK, +G6PDH +PGC1-a, +COX 166 Primary human 11.5 V at 1 Hz with a 2 Ion Optix +AMPK, +Akt, +Il-6, skeletal myotubes ms pulse for 2, 4, 8, 14, insulin signaling and 24 hours 170 C2C12 myotubes 45 V at 5 Hz with a 20 ms pulse for 45, 60, 75, 90, and 120 min TY-C type stimulator +Il-6, +ROS 28

35 167 Primary human 30 V at 100 Hz with a Custom built on site +Il-6, +CPT1b, +MHC1 skeletal myotubes 200 ms pulse every 5 th second for 5-60 min and 30 V at 1 Hz with a 2 ms pulse for 24 hours 165 C2C12 myotubes 50 V at 1 Hz with a 3 ms pulse on 997 ms intervals for 1, 2, and 3 hours 172 C2C12 myotubes 40 V/60 mm 2 at 1 Hz with a 10 ms pulse for 90 min, 4, 6, and 12 hours 46 C2C12 myotubes 40 V/60 mm 2 at 1 Hz with a 2 ms pulse for 24 hours Ion Optix Ion Optix Ion Optix +JNK, +p38, +glucose uptake +AP-1, +JNK, +Il-6 +Il-6 10 Primary human 14 V at 5 Hz with a 2 Ion Optix +Il-6, +Il-8, +LIF, skeletal myotubes ms +TNF-a, +ANGPTL4 47 Sprague-Dawley rat 400 at 1ms/pulse at S48 Grass Stimulator +Il-6, +STAT3 myotubes 45 Hz 173 C2C12 myotubes 9 V at 5 Hz for 3 hours/day for 4 days Ion Optix +Ca ++, +AMPK, +p38, +PGC1-a, +ROS 175 Primary human 30 V at 1 Hz with a 2 Porous membrane +MYH7, +Il-6, +Il-8 skeletal myotubes ms pulse for 48 hours based stimulator 174 C2C12 myotubes 11.5 V at 1 Hz with a 2 ms pulse for 24 hours Ion Optix +AMPK 29

36 Skeletal muscle, myokines, inflammation, and chronic disease There is a strong association between chronic low-grade inflammation and chronic diseases like diabetes and obesity. In this state overnutrition leads to an increasing supply of free fatty acids which exceeds the capacity for oxidation or storage by tissues. 179,180 This scenario causes the formation of diacylglycerols and ceramides, which leads to the phosphorylation of insulin receptor substrate 1 (IRS1) at inhibitory residues. Protein kinase C, which acts on IRS1 stops normal signaling that allows for GLUT4 translocation and glucose uptake. The mitochondria of muscle that are overloaded with fatty acids produce more ROS, which act on DNA, proteins, and lipids. The MAPK s JNK and p38 are activated in this state and can also target IRS In an insulin resistant state such as this AMPK, adiponectin, and PGC-1a activity are blunted. 181 Concurrently, NFkB, TNF-a, MCP1, Il-1b, and Il-6 are chronically elevated in plasma and mrna expression in skeletal muscle Pharmacological reduction of FFA has been shown to enhance insulin signaling and nutrient disposal in obese and type II diabetic subjects. Interestingly this was shown without a change in DAG or ceramide although these markers at baseline are higher in the obese and type II diabetic. 184 Acute increases of Il-6 have also been shown to promote the clearance and oxidation of fatty acids in muscle. 185,186 Other important markers for acute and chronic inflammation are adipocytes and monocytes in blood or macrophages in tissues such as adipose or skeletal muscle. In the context of chronic metabolic disease, M1 macrophage and adipocyte infiltration may occur in skeletal muscle and other tissues. 187,188 These macrophages secrete pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and Il-1Beta, which lead apoptosis in pancreatic islets and blunted insulin secretion. The immune cells residing in adipose tissue 30

37 in lean states recruit M2 macrophages that secrete anti-inflammatory cytokines like Il This process is facilitated by pattern recognition receptors called toll-like receptors (TLR s). Specifically, TLR4 signaling is involved in the development of obesity and diabetes. These protein receptors are expressed in immune cells, adipose tissue, and skeletal muscle. Ligands for TLR4 include resistin, free fatty acids, fetuin A, and lipopolysaccharide (LPS). 188 Upon activation of TLR4 an inflammatory response is initiated via nuclear factor kappa B (NFkB) and translation of Il-1b, TNF-a, and Il-6. TLR4 activity is increased in obese animals and infusion of LPS elicits similar metabolic outcomes to high fat feeding. 34 However, when TLR4 is knocked down the capacity for fatty acids to induce macrophage recruitment is blunted. Additionally, animals lacking TLR4 are protected from impaired insulin signaling induced by lipid infusion. 189,190 Myeloid differentiation primary response 88 (MyD88) an important downstream protein of TLR4 activation is required for the inflammatory environment marked by NFkB, JNK, Il-6, ceramide and impaired glucose uptake and brought on by physical inactivity. 191 Exercise provokes an acute inflammatory response in skeletal muscle that requires the recruitment of macrophages and is described with three phases. 192 The first is the mounting of an inflammatory response, which involves the infiltration of macrophages at the site of injury. This phase is followed by the resolution of inflammation in which the established pro-inflammatory transitions to an anti-inflammatory profile. Lastly, there is tissue repair and regeneration at the site of injury in muscle. This process is executed by Ly6C + macrophages expressing Il-1b and TNF-a. Phagocytosis of injured muscle debris by Ly6C + causes a shift in phenotype to Ly6C -, which express TGF-b and Il-10 and promote muscle regeneration. The magnitude of accumulating macrophages corresponds to the 31

38 degree to which the muscle is damaged. 192,193 The switch in macrophage phenotype is driven by AMPKa and is demonstrated when AMPKa is knocked down. Macrophages lacking this do not consume injured tissue and thus do not transition to an M2 phenotype during muscle regeneration. 194 Il-6 signaling in contracting muscle appears to be different from observations in macrophages. The Il-6 response brought on by exercise was previously thought to be mediated by immune cells in response to muscle damage. However, exercise does not result in increased expression of Il-6 in monocytes. 35 This finding implicates muscle as the source of Il-6 during exercise. Moreover, Il-6 expression in working skeletal muscle is not dependent on NFkB or increases in TNF-a. 5,55 (See Figure 1) As with septic conditions but to a lesser degree, significantly elevated circulation of TNF-a and Il-6 are observed in obese and insulin resistant subjects compared to lean. 68,195,196 It also known that these patients consume less oxygen during exercise and that skeletal muscle insulin resistance leads to decreased glucose uptake, glycogen stores, and capacity to produce ATP. 33 The molecular response in skeletal muscle to exercise stimuli in an inflammatory environment is not fully characterized. The use of EPS to understand cytokine signaling in skeletal muscle has proven useful in recent years. However, there is a lack of investigation in to human skeletal muscle using this model. Concluding Remarks and Rationale for Research Skeletal muscle is highly adaptable to exercise and serves as far more than the source of mechanical function. The secretory nature of muscle, combined with its constitution of nearly half of the human body s mass, prove that it is a very influential organ. The physiological impact of an inflammatory milieu is also large and seems to be associated with 32

39 a maladaptive response to exercise. 33 Using of EPS as a model for mimicking exercise related contraction in human myotubes is relatively novel and we are proposing to do so to determine the impact of chronic inflammation at the onset of exercise. Moreover, there is a great lack of published investigation into metabolic response in skeletal muscle using this model. Additionally, there are no findings at present that show how contracting muscle responds in the presence of chronic inflammation. The goal of the proposed study is to reaffirm EPS as a model for exercise and simulate the circulatory milieu present in obese and diabetic states. Using this model, we expect to understand how chronic inflammation affects contracting skeletal muscle. We also intend to uncover if and how TLR4 signaling mediates adaptation to EPS. To date there are conflicting findings on the effect of exercise on TLR4 signaling. 197 Our results will elucidate the cause of the dilemma of the maladaptive response to exercise observed in the presence of chronic disease. 33

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50 162. Seldin MM, Peterson JM, Byerly MS, Wei Z, Wong GW. Myonectin (CTRP15), a novel myokine that links skeletal muscle to systemic lipid homeostasis. J Biol Chem. 2012;287(15): Peterson JM, Mart R, Bond CE. Effect of obesity and exercise on the expression of the novel myokines, Myonectin and Fibronectin type III domain containing 5. PeerJ. 2014;2:e Hellsten Y, Frandsen U. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture. J Physiol. 1997;504 ( Pt 3): Wehrle U, Dusterhoft S, Pette D. Effects of chronic electrical stimulation on myosin heavy chain expression in satellite cell cultures derived from rat muscles of different fiber-type composition. Differentiation. 1994;58(1): Thelen MH, Simonides WS, van Hardeveld C. Electrical stimulation of C2C12 myotubes induces contractions and represses thyroid-hormone-dependent transcription of the fast-type sarcoplasmic-reticulum Ca2+-ATPase gene. Biochem J. 1997;321 ( Pt 3): Connor MK, Irrcher I, Hood DA. Contractile activity-induced transcriptional activation of cytochrome C involves Sp1 and is proportional to mitochondrial ATP synthesis in C2C12 muscle cells. J Biol Chem. 2001;276(19): Irrcher I, Hood DA. Regulation of Egr-1, SRF, and Sp1 mrna expression in contracting skeletal muscle cells. J Appl Physiol (1985). 2004;97(6): Pattwell DM, McArdle A, Morgan JE, Patridge TA, Jackson MJ. Release of reactive oxygen and nitrogen species from contracting skeletal muscle cells. Free Radic Biol Med. 2004;37(7): McArdle F, Pattwell DM, Vasilaki A, McArdle A, Jackson MJ. Intracellular generation of reactive oxygen species by contracting skeletal muscle cells. Free Radic Biol Med. 2005;39(5): Silveira LR, Pilegaard H, Kusuhara K, Curi R, Hellsten Y. The contraction induced increase in gene expression of peroxisome proliferator-activated receptor (PPAR)- gamma coactivator 1alpha (PGC-1alpha), mitochondrial uncoupling protein 3 (UCP3) and hexokinase II (HKII) in primary rat skeletal muscle cells is dependent on reactive oxygen species. Biochim Biophys Acta. 2006;1763(9): Fujita H, Nedachi T, Kanzaki M. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes. Exp Cell Res. 2007;313(9): Nikolic N, Bakke SS, Kase ET, et al. Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise. PLoS One. 2012;7(3):e Kramer HF, Goodyear LJ. Exercise, MAPK, and NF-kappaB signaling in skeletal muscle. J Appl Physiol (1985). 2007;103(1): Kaji H, Ishibashi T, Nagamine K, Kanzaki M, Nishizawa M. Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness. Biomaterials. 2010;31(27): Pan H, Xu X, Hao X, Chen Y. Changes of myogenic reactive oxygen species and interleukin-6 in contracting skeletal muscle cells. Oxid Med Cell Longev. 2012;2012: Pinheiro CH, Silveira LR, Nachbar RT, Vitzel KF, Curi R. Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells. Free Radic Biol Med. 2010;48(7):

51 178. Evers-van Gogh IJ, Alex S, Stienstra R, Brenkman AB, Kersten S, Kalkhoven E. Electric Pulse Stimulation of Myotubes as an In Vitro Exercise Model: Cell-Mediated and Non- Cell-Mediated Effects. Sci Rep. 2015;5: Pedersen BK. Exercise-induced myokines and their role in chronic diseases. Brain Behav Immun. 2011;25(5): Jialal I, Kaur H, Devaraj S. Toll-like receptor status in obesity and metabolic syndrome: a translational perspective. J Clin Endocrinol Metab. 2014;99(1): Eisele PS, Handschin C. Functional crosstalk of PGC-1 coactivators and inflammation in skeletal muscle pathophysiology. Semin Immunopathol. 2014;36(1): Liong S, Lappas M. Activation of AMPK improves inflammation and insulin resistance in adipose tissue and skeletal muscle from pregnant women. J Physiol Biochem. 2015;71(4): Marette A, Liu Y, Sweeney G. Skeletal muscle glucose metabolism and inflammation in the development of the metabolic syndrome. Rev Endocr Metab Disord. 2014;15(4): Liang H, Tantiwong P, Sriwijitkamol A, et al. Effect of a sustained reduction in plasma free fatty acid concentration on insulin signalling and inflammation in skeletal muscle from human subjects. J Physiol. 2013;591(11): Petersen EW, Carey AL, Sacchetti M, et al. Acute IL-6 treatment increases fatty acid turnover in elderly humans in vivo and in tissue culture in vitro. Am J Physiol Endocrinol Metab. 2005;288(1):E van Hall G, Steensberg A, Sacchetti M, et al. Interleukin-6 stimulates lipolysis and fat oxidation in humans. J Clin Endocrinol Metab. 2003;88(7): Hausman GJ, Basu U, Du M, Fernyhough-Culver M, Dodson MV. Intermuscular and intramuscular adipose tissues: Bad vs. good adipose tissues. Adipocyte. 2014;3(4): Watanabe Y, Nagai Y, Takatsu K. Activation and regulation of the pattern recognition receptors in obesity-induced adipose tissue inflammation and insulin resistance. Nutrients. 2013;5(9): Shi H, Kokoeva MV, Inouye K, Tzameli I, Yin H, Flier JS. TLR4 links innate immunity and fatty acid-induced insulin resistance. J Clin Invest. 2006;116(11): Tsukumo DM, Carvalho-Filho MA, Carvalheira JB, et al. Loss-of-function mutation in Toll-like receptor 4 prevents diet-induced obesity and insulin resistance. Diabetes. 2007;56(8): Kwon OS, Tanner RE, Barrows KM, et al. MyD88 regulates physical inactivity-induced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance. Am J Physiol Endocrinol Metab. 2015;309(1):E Chazaud B. Inflammation during skeletal muscle regeneration and tissue remodeling: application to exercise-induced muscle damage management. Immunol Cell Biol. 2016;94(2): Arnold L, Henry A, Poron F, et al. Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med. 2007;204(5): Mounier R, Theret M, Arnold L, et al. AMPKalpha1 regulates macrophage skewing at the time of resolution of inflammation during skeletal muscle regeneration. Cell Metab. 2013;18(2):

52 195. Roytblat L, Rachinsky M, Fisher A, et al. Raised interleukin-6 levels in obese patients. Obes Res. 2000;8(9): Winkler G, Lakatos P, Salamon F, et al. Elevated serum TNF-alpha level as a link between endothelial dysfunction and insulin resistance in normotensive obese patients. Diabet Med. 1999;16(3): Handschin C, Spiegelman BM. The role of exercise and PGC1alpha in inflammation and chronic disease. Nature. 2008;454(7203): Marinik EL, Frisard MI, Hulver MW, et al. Angiotensin II receptor blockade and insulin sensitivity in overweight and obese adults with elevated blood pressure. Ther Adv Cardiovasc Dis. 2013;7(1):

53 Chapter 3 Specific Aims, Research Design, and Extended Methods 47

54 Specific Aim 1: To further define the EPS model and its effect on cultured human primary myotubes. Hypothesis: The use of EPS on human primary myotubes will mimic the metabolic, transcriptional, translational, and physical effects of exercise observed in contracting skeletal muscle. Specific Aim 2: To determine the effect of metabolic endotoxemia on the adaptive response to EPS in cultured human primary myotubes. Hypothesis: The presence of LPS will attenuate the adaptive response to EPS in primary myotubes. 48

55 Research Design Specific Aim 1: To further define the EPS model and its effect on cultured human primary myotubes. Hypothesis: The use of EPS on human primary myotubes will mimic the metabolic, transcriptional, translational, and physical effects of exercise observed in contracting skeletal muscle. Research Model Cell Culture: Human primary skeletal muscle cells will be cultured, grown, and electrically stimulated after 7 days of differentiation. The stimulation protocol will be 14 volts at 5 hertz with a 2 milisecond pulse. To confirm transmission of the electrical pulse the Ion Optix C- Pace EP system will be connected to an oscilloscope and monitored prior to stimulation. Experimental Strategy: Human primary skeletal muscle cellswill be cultured and grown in media containing a 1:1 mixture of a-mem and Ham s F-12 supplemented with 20% FBS, 1% chicken embryo extract, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B until 80% confluency. The cells will be incubated for 7 days in differentiation media consisting of a-mem containing 5.5 mm glucose, 2 mm glutamine, 2% FBS, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B. After 7 days the resulting myotubes will be subjected to EPS. 49

56 Endpoint Measures To reaffirm some published findings and address the lack of data in muscle we will measure the following targets: Oxidation of radiolabled palmitate and glucose, gene expression of Il- 6, PGC1-alpha, SOD2, enzyme activity of MDH and citrate synthase, protein content of HSP70 in cells and media, creatine kinase released in media, and media temperature and ph. Specific Aim 2: To test the hypothesis chronic low-grade inflammation will abrogate the adaptive response to EPS. Hypothesis: The presence of LPS will attenuate the adaptive response to EPS in primary myotubes. Research Model Cell Culture: Human primary skeletal muscle cells will be cultured, grown, and electrically stimulated after 7 days of differentiation. The stimulation protocol will be 14 volts at 5 hertz with a 2 milisecond pulse. To confirm transmission of the electrical pulse the Ion Optix C- Pace EP system will be connected to an oscilloscope and monitored prior to stimulation. Experimental Strategy: Human primary skeletal muscle cellswill be cultured and grown in media containing a 1:1 mixture of a-mem and Ham s F-12 supplemented with 20% FBS, 1% chicken embryo extract, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B until 80% confluency. The cells will be incubated for 7 days in differentiation media consisting of a-mem containing 5.5 mm glucose, 2 mm glutamine, 2% FBS, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B. After 7 days the resulting 50

57 myotubes will be subjected to EPS. At the start of stimulation, media containing 20 EU (endotoxin units) of LPS will be added. Media containing LPS will remain in contact with the myotubes for the duration of the stimulation protocol and be removed immediately afterwards. Endpoint Measures To reaffirm some published findings and address the lack of data in muscle we will measure the following targets: Oxidation of radiolabled palmitate and glucose, gene expression of Il- 6, PGC1-alpha, SOD2, enzyme activity of MDH and citrate synthase, protein content of HSP70 in cells and media, and creatine kinase released in media. 51

58 Extended Methods Participants. Human primary skeletal muscle was obtained from the vastus lateralis of 19 healthy non-obese college aged (18-30) males via biopsy performed using a modified Bergstrom needle with suction as described. 198 Subjects provided written informed consent under approved protocols by Virginia Polytechnic Institute and State University Institutional Review Boards. Cell Culture. Muscle acquired from the biopsies was prepared using a trypsin cocktail containing 2.5% trypsin 10X, 2% EDTA, 1% collagenase type IV, and 1% BSA, and ultra-pure water. Human primary skeletal muscle cells were cultured and grown in either media containing a 1:1 mixture of a-mem and Ham s F-12 (Hyclone) supplemented with 20% FBS (Gibco), 1% chicken embryo extract (MP Biomedicals), 100 U/ml penicillin (Gibco), 100µg/ml streptomycin (Gibco), and 0.5µg/ml amphotericin B (Gibco) or Dulbecco's modified Eagle's medium (DMEM) low glucose (Hyclone) supplemented with 10% FBS (Gibco), 5.0 ml of BSA (Lonza), 0.5 ml of dexamethasone (Lonza), 5.0 ml of fetuin bovine (Lonza), 0.5 ml of gentamicin (Lonza), and 0.5 ml of rhegf (Lonza) until 80% confluency. The cells were differentiated for 7 days in either a-mem containing 5.5 mm glucose, 2 mm glutamine, 2% FBS, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B or DMEM low glucose supplemented with 2% horse serum (Gibco), 5.0 ml of fetuin bovine, 5.0 ml of BSA, and 0.5 ml of gentamicin. a-mem based media was used for all experiments unless otherwise indicated. C2C12 myotubes. C2C12 cells were purchased from the American Type Culture Collection (Manassas, VA) and grown to 80% confluence in DMEM containing 10% FBS, 50 U/ml 52

59 penicillin, and 50 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were grown to confluence and then differentiated into myotubes in DMEM containing 2% horse serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen). All experiments were performed after 7 days of differentiation. Murine Adipose-Derived Stem Cells. Murine mesenchymal stem cells were isolated from adipose tissue and grown in DMEM high glucose supplemented with 10 % FBS and 1% ABAM. Electrical Pulse Stimulation Protocol EPS Device. EPS was applied to differentiated myotubes in 6-well plates using the C-Pace EP system from Ion Optix (Dublin, Ireland). Oscilloscope. To confirm transmission of the electrical pulse the Ion Optix C-Pace EP system was connected to an oscilloscope prior to stimulation. Stimulation protocol. Human primary skeletal muscle was cultured, grown, and stimulated after 7 days of differentiation. The stimulation protocol was 14 volts at 5 hertz with a 2 milisecond pulse for 24 hours unless otherwise noted. Substrate Metabolism Fatty Acid Metabolism. Fatty acid oxidation was determined by measuring and summing 14 CO2 production and 14 C-labeled acid soluble metabolites from the oxidation of [1-14 C] palmitic acid (American Radiolabeled Chemicals, St. Louis, MO). Briefly, cells were incubated in media containing radiolabeled substrate for 3 hours at 37C, 5% CO2. Following incubation, media was removed and acidified with 70% perchloric acid to elute gaseous 14 CO2. 14 CO2 was 53

60 trapped in 1M NaOH over the course of 1 hour. The NaOH was then be placed in a liquid scintillation counter and counted. Data are expressed as means ± SEM and is normalized to total protein content. Glucose Metabolism. Glucose oxidation was assessed by measuring 14 CO2 production from the oxidation of [U- 14 C] glucose (American Radiolabeled Chemicals, St. Louis, MO) in a manner similar to fatty acid oxidation except for the substitution of glucose in place of palmitic acid. Real-time Quantitative PCR. Cells were collected in TRIzol and RNA was extracted. Total mrna was prepared using TRIzol reagent, according to the manufacturer s protocol (Life Technologies, Carlsbad, CA), and extracted using the RNeasy mini kit (Qiagen, Valencia, CA). Total mrna concentration was quantified using an Agilent Bioana- lyzer. Real-time quantitative PCR (RTQ-PCR) was performed using an ABI 7900 Fast HT RTQ-PCR instrument (PE Applied Biosystems, Foster City, CA). Il-6, PGC1-a, and SOD2 gene expression was determined with the TaqMan Universal PCR master mix, according to manufacturer s specifications (Applied Biosystems). Primer and probes sets for all target genes were purchased as pre-validated assays (Applied Biosystems). Relative quantification of target genes was calculated using the 2DCT method. Derivation of the 2DCT equation has been described in Applied Biosystems User Bulletin No. 2 (P/N ). Target gene expression was normalized to cyclophilin B and b-actin mrna levels. 54

61 Enzyme Activity. Malate Dehydrogenase activity was measured spectrophotometrically at 340nm at 37 C. Briefly, 10ul of sample will be pipetted in triplicate in wells. Then, 290ul of reaction media (0.1 M potassium phosphate buffer, PH 7.4 plus M oxaloacetic acid, prepared in potassium phosphate buffer plus M NADH, prepared in potassium phosphate buffer) was added to the wells and samples read for 5 minutes at 340nm. The rate of disappearance of NADH was analyzed and expressed relative to protein content. Citrate Synthase catalyzes the formation of citrate and CoASH from acetyl-coa and oxaloactetate. CoASH reduces DTNB and CS activity was determined from the reduction of DTNB over time. Briefly, ten microliters of a 1:5 diluted media was added, in triplicate, to 170μl of a solution containing Tris buffer (0.1M, ph 8.3), DNTB (1mM, in 0.1M in Tris buffer) and oxaloacetate (0.01M, in 0.1M Tris buffer). Following a 2min background reading, the spectrophotometer (SPECTRAmax ME, Molecular Devices Corporation, Sunnyvale California) was calibrated and 30μl of 3 mm acetyl CoA was added to initiate the reaction. Absorbance was measured at 405nm at 37C every 12 seconds for 7 minutes. Maximum CS activity was calculated and reported as μmol/min/mg. Heat Stress and Temperature, Cell Damage, and ph Human HSP70 ELISA. A Human HSP70 ELISA kit (ThermoScientific) was used to detect HSP70 content in cells and media after stimulation. Detection was measured according to the kit instructions. Temperature. Control and stimulation media temperature was measured after 24 hours of EPS in control and stimulated samples using a digital thermometer. 55

62 Creatine Kinase catalyzes the reversible conversion of creatine and ATP into ADP and phosphocreatine. Creatine kinase activity was measured using a colorimetric creatine kinase activity assay kit from Abnova. Creatine kinase detection was based on a curve representing the generation of NADH over time. ph. Control and stimulation media ph was measured after 24 hours of EPS in control and stimulated samples using an Accumet Basic ph meter from Fisher Scientific. LPS treatment with human primary myotubes. Cell culture experiments were performed with 20 EU/ml of LPS (E. coli 0111:B4, catalog no. L2630; Sigma-Aldrich). All LPS preparations were made using endotoxin free water. Plates were treated with differentiation media containing LPS at the start and throughout EPS. Immunocytochemistry and Immunofluorescence. To detect the presence of fast-type skeletal myosin, human primary myotubes were washed in PBS and fixed in PBS containing 4% paraformaldehyde (ph 7.4) for 15 minutes at room temperature. Cells were permeabilized with PBS containing 0.25% Triton X-100 for 10 minutes. Blocking was performed 1% BSA in PBS-T(0.1% Tween) for 30 minutes. Cells were incubated with a MyHCII antibody (M4276; Sigma-Aldrich) diluted 1:133 in blocking solution, washed three times with PBS and incubated with a Goat pab to Ms IgG (TRITC) secondary antibody (ab6786;abcam) diluted 1:200 in blocking solution for 1 hour and washed again. Cells were imaged using a X-Cite 120 Fluorescence Illumination System. 56

63 Statistics. Results were analyzed with Student s t-tests for two-condition comparisons and a one-way ANOVA for multiple-condition comparisons with Tukey s post hoc analysis. Data was expressed as means +/- SE. The level of significance was set a priori at P <

64 Chapter 4 Characterization of an in vitro model for exercise and the effects of metabolic endotoxemia on skeletal muscle response to electric pulse stimulation 58

65 Abstract The prevalence of obesity and type II diabetes is increasing. Although exercise is widely accepted for prevention and treatment, evidence of resistance to exercise in patients with these diseases is also mounting. Muscle contraction during exercise stimulate cellular responses important for adaptation. These responses include the release of myokines and the subsequent increase in substrate metabolism. This study aimed to define a culture model for simulating the effects of exercise in human primary skeletal muscle cells. We hypothesized that electric pulse stimulation (EPS) of human myotubes in vitro would simulate cellular and molecular responses to exercise observed in vivo. To define this model, we applied EPS to human myotubes for varied lengths of time and measured interleukin-6 (Il-6), peroxisome proliferator-activated receptor gamma coactivator 1-a (PGC1-a), superoxide dismutase 2 (SOD2), substrate metabolism, metabolic enzyme activity, heat stress markers, and ph. To recreate the inflammatory milieu observed in metabolic disease states we treated the myotubes with a low dose of 20 EU lipopolysaccharide (LPS). Following the 24-hour stimulation we observed significant increases in transcription of Il-6, PGC1-a, and SOD2. Basal glucose and fatty acid oxidation were also markedly increased in the myotubes after chronic EPS. Cells treated with LPS elicited a blunted transcriptional, metabolic, and enzymatic response to EPS. These findings suggest that EPS is a viable model for simulating the effects of exercise. Our observations also indicate that an inflammatory environment could play a role in interfering with the adaptations to exercise. 59

66 Introduction In 2011, the prevalence of obesity in the United States reached a staggering high of 34.4% and as of 2013, an average of 37% of men and women are overweight globally. 1,2 The global population of obese and overweight adults is projected to exceed 3 billion in The prevalence of cardiovascular disease and type II diabetes, which are strongly associated with obesity, is also rising. Regular exercise is widely adopted as a strategy for treating and preventing these diseases apart from weight loss. 4 Skeletal muscle, which accounts for up to 40% of body weight in lean individuals is the primary organ affected during exercise. In recent years, researchers have used electrical pulses to stimulate cultured skeletal muscle as an in vitro model for exercise. Findings from earlier studies using this model involve the culturing of C2C12 myotubes and demonstrate responses that mimic the effect of exercise observed in vivo. Significant increases in expression and secretion of interleukin-6 (Il-6), a notable myokine, is repeatedly shown in C2C12. 5,6 In vivo, Il-6 secretion due to exercise increases glucose uptake, hepatic glucose production, and lipolysis. 7 Findings from studies using the (EPS) model show rises in glucose consumption, GLUT4 translocation 8, and MAPK signaling 5,9 in C2C12 myotubes. Furthermore, C2C12 myotubes treated with EPS have significantly higher levels of reactive oxygen species (ROS) production 10 and gene expression of PGC-1a Taken together these findings suggest use of the EPS model may be a viable means of mimicking exercise in C2C12 cells. Throughout the past decade, the majority of researchers applying this model did so using C2C12 myotubes while only a few studies involved stimulation of human primary myotubes. Using EPS on human cells yields results that demonstrate a similar increase in creatine 60

67 kinase activity, glucose uptake, expression of Il-6 and other myokines such as Il-8, LIF, and ANGPTL4. 13 Another group reported increased expression of Il-6, phosphorylated Akt, and insulin signaling. 14 Most recently a study stimulated cultured myotubes from lean, severely obese non-diabetic, and severely obese diabetic subjects, and observed increased glucose oxidation in all groups but only increased fatty acid oxidation for lean myotubes. 15 The data reported from studies using human subjects adds to the growing definition of the EPS model and addresses the topic of chronic disease and the adaptation to exercise. We intend to fill the gaps in research concerning this model by measuring the effect of EPS on metabolic enzymatic activity, superoxide formation, and heat stress. The purpose of this study is to first further characterize the EPS model and explore how the response to EPS is affected by cell type, culture conditions, and the presence of endotoxin. We will test the hypothesis that the use of EPS on human primary myotubes will mimic the metabolic, transcriptional, and physical effects of exercise such as temperature and ph observed in contracting skeletal muscle. Recent studies have contributed to the evidence of a resistance to exercise observed in populations with obesity, type II diabetes, and CVD. 16,17 Chronic low-grade inflammation is associated with these diseases and is referred to as metabolic endotoxemia. 18 We will also investigate the effect of metabolic endotoxemia on the EPS response in human primary myotubes by treating with low levels of lipopolysaccharide (LPS or endotoxin) during stimulation. We hypothesize that the presence of endotoxin will blunt the response to EPS. 61

68 Methods and Materials Participants. Human primary skeletal muscle was obtained from the vastus lateralis of 19 healthy non-obese college aged (18-30) males via biopsy performed using a modified Bergstrom needle with suction as described. 19 Subjects provided written informed consent under approved protocols by Virginia Polytechnic Institute and State University Institutional Review Boards. Cell Culture. Muscle acquired from the biopsies was prepared using a trypsin cocktail containing 2.5% trypsin 10X, 2% EDTA, 1% collagenase type IV, and 1% BSA, and ultra-pure water. Human primary skeletal muscle was cultured and grown in either media containing a 1:1 mixture of a-mem and Ham s F-12 (Hyclone) supplemented with 20% FBS (Gibco), 1% chicken embryo extract (MP Biomedicals), 100 U/ml penicillin (Gibco), 100µg/ml streptomycin (Gibco), and 0.5µg/ml amphotericin B (Gibco) or Dulbecco's modified Eagle's medium (DMEM) low glucose (Hyclone) supplemented with 10% FBS (Gibco), 5.0 ml of BSA (Lonza), 0.5 ml of dexamethasone (Lonza), 5.0 ml of fetuin bovine (Lonza), 0.5 ml of gentamicin (Lonza), and 0.5 ml of rhegf (Lonza) until 80% confluency. The cells were differentiated for 7 days in either a-mem containing 5.5 mm glucose, 2 mm glutamine, 2% FBS, 100 U/ml penicillin, 100µg/ml streptomycin, and 0.5µg/ml amphotericin B or DMEM low glucose supplemented with 2% horse serum (Gibco), 5.0 ml of fetuin bovine, 5.0 ml of BSA, and 0.5 ml of gentamicin. a-mem based media was used for all experiments unless otherwise indicated. C2C12 myotubes. C2C12 were purchased from the American Type Culture Collection (Manassas, VA) and grown to 80% confluence in DMEM containing 10% FBS, 50 U/ml 62

69 penicillin, and 50 μg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were grown to confluence and then differentiated into myotubes in DMEM containing 2% horse serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen). All experiments were performed after 7 days of differentiation. Murine Adipose-Derived Stem Cells. Murine mesenchymal stem cells were isolated from adipose tissue and grown in DMEM high glucose supplemented with 10 % FBS and 1% ABAM. Electrical Pulse Stimulation Protocol EPS Device. EPS was applied to differentiated myotubes in 6-well plates using the C-Pace EP system from Ion Optix (Dublin, Ireland). Oscilloscope. In order to confirm transmission of the electrical pulse the Ion Optix C-Pace EP system was connected to an oscilloscope prior to stimulation. Stimulation protocol. Human primary skeletal muscle was cultured, grown, and stimulated after 7 days of differentiation. The stimulation protocol was 14 volts at 5 hertz with a 2 milisecond pulse for 24 hours unless otherwise noted. Substrate Metabolism Fatty Acid Metabolism. Fatty acid oxidation was determined by measuring and summing 14 CO2 production and 14 C-labeled acid soluble metabolites from the oxidation of [1-14 C] palmitic acid (American Radiolabeled Chemicals, St. Louis, MO). Briefly, cells were incubated in media containing radiolabeled substrate for 3 hours at 37C, 5% CO2. Following incubation, media was removed and acidified with 70% perchloric acid to elute gaseous 14 CO2. 14 CO2 was 63

70 trapped in 1M NaOH over the course of 1 hour. The NaOH was then be placed in a liquid scintillation counter and counted. Data are expressed as means ± SEM and is normalized to total protein content. Glucose Metabolism. Glucose oxidation was assessed by measuring 14 CO2 production from the oxidation of [U- 14 C] glucose (American Radiolabeled Chemicals, St. Louis, MO) in a manner similar to fatty acid oxidation except for the substitution of glucose in place of palmitic acid. Real-time Quantitative PCR. Cells were collected in TRIzol and RNA was extracted. Total mrna was prepared using TRIzol reagent, according to the manufacturer s protocol (Life Technologies, Carlsbad, CA), and extracted using the RNeasy mini kit (Qiagen, Valencia, CA). Total mrna concentration was quantified using an Agilent Bioana- lyzer. Real-time quantitative PCR (RTQ-PCR) was performed using an ABI 7900 Fast HT RTQ-PCR instrument (PE Applied Biosystems, Foster City, CA). Il-6, PGC1-a, and SOD2 gene expression was determined with the TaqMan Universal PCR master mix, according to manufacturer s specifications (Applied Biosystems). Primer and probes sets for all target genes were purchased as pre-validated assays (Applied Biosystems). Relative quantification of target genes was calculated using the 2DCT method. Derivation of the 2DCT equation has been described in Applied Biosystems User Bulletin No. 2 (P/N ). Target gene expression was normalized to cyclophilin B and b-actin mrna levels. Enzyme Activity. Malate Dehydrogenase (MDH) activity was measured spectrophotometrically at 340nm at 37 C. Briefly, 10ul of sample will be pipetted in triplicate in wells. Then, 290ul of reaction 64

71 media (0.1 M potassium phosphate buffer, PH 7.4 plus M oxaloacetic acid, prepared in potassium phosphate buffer plus M NADH, prepared in potassium phosphate buffer) was added to the wells and samples read for 5 minutes at 340nm. The rate of disappearance of NADH was analyzed and expressed relative to protein content. Citrate Synthase catalyzes the formation of citrate and CoASH from acetyl-coa and oxaloactetate. CoASH reduces DTNB and CS activity was determined from the reduction of DTNB over time. Briefly, ten microliters of a 1:5 diluted media was added, in triplicate, to 170μl of a solution containing Tris buffer (0.1M, ph 8.3), DNTB (1mM, in 0.1M in Tris buffer) and oxaloacetate (0.01M, in 0.1M Tris buffer). Following a 2min background reading, the spectrophotometer (SPECTRAmax ME, Molecular Devices Corporation, Sunnyvale California) was calibrated and 30μl of 3 mm acetyl CoA was added to initiate the reaction. Absorbance was measured at 405nm at 37C every 12 seconds for 7 minutes. Maximum CS activity was calculated and reported as μmol/min/mg. Heat Stress and Temperature, Cell Damage, and ph Human HSP70 ELISA. A Human HSP70 ELISA kit (ThermoScientific) was used to detect heat shock protein 70 (HSP70) content in cells and media after stimulation. Detection was measured according to the kit instructions. Temperature. Control and stimulation media temperature was measured after 24 hours of EPS in control and stimulated samples using a digital thermometer. Creatine Kinase catalyzes the reversible conversion of creatine and ATP into ADP and phosphocreatine. Creatine kinase activity was measured using a colorimetric creatine kinase activity assay kit from Abnova. Creatine kinase detection was based on a curve representing the generation of NADH over time. 65

72 ph. Control and stimulation media ph was measured after 24 hours of EPS in control and stimulated samples using an Accumet Basic ph meter from Fisher Scientific. LPS treatment with human primary myotubes. Cell culture experiments were performed with 20 EU/ml of LPS (E. coli 0111:B4, catalog no. L2630; Sigma-Aldrich). All LPS preparations were made using endotoxin free water. Plates were treated with differentiation media containing LPS at the start and throughout EPS. Immunocytochemistry and Immunofluorescence. In order to detect the presence of fasttype skeletal myosin, human primary myotubes were washed in PBS and fixed in PBS containing 4% paraformaldehyde (ph 7.4) for 15 minutes at room temperature. Cells were permeabilized with PBS containing 0.25% Triton X-100 for 10 minutes. Blocking was performed 1% BSA in PBS-T(0.1% Tween) for 30 minutes. Cells were incubated with a MyHCII antibody (M4276; Sigma-Aldrich) diluted 1:133 in blocking solution, washed three times with PBS and incubated with a Goat pab to Ms IgG (TRITC) secondary antibody (ab6786;abcam) diluted 1:200 in blocking solution for 1 hour and washed again. Cells were imaged using a X-Cite 120 Fluorescence Illumination System. Statistics. Results were analyzed with Student s t-tests for two-condition comparisons and a one-way ANOVA for multiple-condition comparisons with Tukey s post hoc analysis. Data was expressed as means +/- SE. The level of significance was set a priori at P <

73 Results Effects of cell type and media on responses to EPS Due to the variability between our preliminary findings and published results with regard to cell and media types used, we measured transcription in C2C12 and human primary myotubes grown and differentiated in different media. Based on previously published data EPS was administered for 24 hours. 12 Chronic (24 hours) electrical stimulation increased Il-6 with no effect on SOD2 and PGC1-a in C2C12 myotubes. (Figure 1). There were significant differences in transcriptional response to chronic stimulation between human myotubes grown and differentiated in DMEM-based versus alpha-mem based media used by Scheler et al Twenty-four hours of EPS in human myotubes fused in alpha-mem based media yielded significant increases in PGC1-a, SOD2, and Il-6 transcription (Figure 1). However, there were no significant transcriptional changes for myotubes differentiated in DMEM-based media. Detailed comparison of these media indicates remarkable differences in the components between DMEM-based and alpha-mem based media. Of note, are the presence of fatty acids, penicillin/streptomycin, and numerous inorganic salts in alpha-mem based media but not contained in the DMEM based media (Supplemental Figures 1-3). 67

74 Figure 1. Effects of chronic EPS on C2C12 cells and human primary myotubes. mrna expression of Il-6, PGC1-a, and SOD2 were determined in C2C12 and human primary myotubes after 24 hours of EPS. Values are normalized to levels of housekeeping gene b- actin and are shown as a fold increase relative to the control. Data are presented as mean +/- SEM. (* denotes P<0.05) 68

75 Effect of EPS duration on transcription Our findings indicated that the most robust effect on transcription occurred in human primary myotubes in a-mem based media after 24 hours of EPS. To determine the time course for the transcriptional response, mrna expression of PGC1-a, Il-6, and SOD2 was measured in human myotubes after 1, 2, 4, 6, 8, 12, and 24 hours of EPS. Analysis of mrna levels for PGC1-a, indicate a significant stimulation effect after 8 and 24 hours. Il-6 expression increased significantly after 1, 2, 4, 8, and 24 hours of EPS. SOD2 expression was significantly elevated at 2, 4, 8, and 24 hours of stimulation. A 6-fold increase in expression in comparison to the control was recorded for SOD2 while Il-6 mrna elevated 3-fold compared to the control at 24 hours Figure (2). Figure 2. Effects of EPS duration on transcription. PGC1-a, Il-6, and SOD2 mrna were measured in human primary myotubes after 1, 2, 4, 6, 8, 12, and 24 hours of EPS. Values are normalized to levels of housekeeping gene cyclophilin B (PPIB) and are shown as a fold increase relative to the control. Data are presented as mean +/- SEM. (* denotes P<0.05) 69

76 Effects of EPS duration on substrate metabolism and enzyme activity After determining that 24 hours of EPS yielded the greatest transcriptional response in human myotubes we measured substrate metabolism after 1, 2, 12, and 24 hours. Glucose oxidation in acutely stimulated myotubes was significantly greater after 1 hour of EPS while no increase was detected after 2 hours. Chronic EPS of 12 and 24 hours were associated with significant increases in glucose oxidation (Figure 3). These data indicate a demand for glucose at the onset and after prolonged stimulation. Acute EPS of 1 and 2 hours did not produce any significant changes in CO2, acid soluble metabolites (ASM), or total palmitate oxidation (Figure 3). However, 12 hours of stimulation yielded significant increases in CO2, ASM, and total palmitate oxidation. Analysis of the ratio of CO2 to ASM produced showed an increase in oxidative efficiency for stimulated myotubes. After 24 hours of EPS, CO2 production and oxidation efficiency levels were significantly increased with no changes in ASM or total oxidation (Figure 3). Similar to transcription the most significant increase in substrate metabolism occurred after chronic stimulation. We then used 24 hours of EPS to measure the activity of the metabolic enzymes citrate synthase and malate dehydrogenase. Analysis showed no significant changes in enzyme activity due to electrical stimulation (Figure 3). 70

77 71

78 Figure 3. Effects of EPS duration on substrate metabolism and enzyme activity. Glucose and palmitate oxidation were measured after 1, 2, 12, and 24 hours of EPS. Citrate synthase and malate dehydrogenase activities were measured after 24 hours of EPS. Data are presented as mean +/- SEM. (* denotes P<0.05) Effects of chronic EPS on heat stress markers, ph, and creatine kinase To further examine the EPS model as a mimetic for exercise, we measured HSP70 in cells and media, media temperature, ph and creatine kinase activity. Twenty-four hours of EPS applied to human primary myotubes yielded significant increases in HSP70 in cells but not in media. Media temperature was significantly increased 1.7 degrees (37.8 degrees Celsius) due to electrical stimulation compared to controls. There was a significant decrease in ph in the media of electrically stimulated myotubes (Figures 4). These data demonstrate a heat stress response in cells and a shift in proton concentration after electrical stimulation. To assess the effect of 24 hours of EPS as a mimetic of exercise-induced stress, creatine kinase content was measured in media. The level of creatine kinase in the media of stimulated myotubes was significantly higher than in control media (Figure 4). These data suggest increased muscle damage in associated with prolonged (24 hr) electrical stimulation. 72

79 73

80 Figure 4. Effects of EPS on heat stress markers, ph, and creatine kinase. Media temperature and ph, HSP70 in cells and media, and creatine kinase release were measured after 24 hours of EPS. Data are presented as mean +/- SEM. (* denotes P<0.05) Effect of LPS exposure on the response to EPS Chronic low grade inflammation is associated with metabolic disease. To simulate a similar environment in vitro, human myotubes were exposed to a low level (20EU) of endotoxin during chronic stimulation. Expression of Il-6, SOD2, and PGC1-a were measured after 24 hours of EPS and concurrent LPS exposure. After 24 hours of EPS and LPS treatment SOD2 and Il-6 expression increased significantly due to stimulation. However, the response to stimulation was significantly less than in myotubes not treated with LPS. For PGC1-a mrna exposure to LPS resulted in no significant stimulation effect. Il-6 and SOD expression were both significantly elevated in LPS treated myotubes independent of stimulation (Figure 5). Figure 5.EPS and LPS and mrna transcription. Il-6, SOD2, and PGC1-a gene expression was measured after 24 hours of EPS and concurrent LPS exposure. Data are presented as mean +/- SEM. (* or # denotes P<0.05) 74

81 LPS alone significantly increased glucose oxidation. The effect of stimulation was significantly lower in myotubes exposed to LPS. CO2 production after LPS exposure and a 24- hour stimulation was significantly increased. Marked increases in ASM, total palmitate oxidation, and oxidation efficiency in were lost in the presence of LPS (Figure 6). LPS treatment also significantly blunted malate dehydrogenase activity although there was no detectable effect of stimulation in absence of LPS. There was no significant change in citrate synthase for LPS-treated myotubes (Figure 6). LPS-treated myotubes released significantly lower levels of creatine kinase in response to EPS. Significant increases in HSP70 due to EPS were not present for cells exposed to LPS (Figure 7.) 75

82 Figure 6. Effect of chronic LPS exposure on the response to EPS. Glucose and palmitate oxidation, and citrate synthase and malate dehydrogenase activities were measured after 24 76

83 hours of EPS and concurrent LPS exposure. Data are presented as mean +/- SEM. (* or # denotes P<0.05) Figure 7. Chronic EPS and LPS treatment and creatine kinase and HSP70 content. Creatine kinase and HSP70 were measured after 24 hours of stimulation and LPS exposure. Data are presented as mean +/- SEM. (* or # denotes P<0.05) 77

84 Discussion The aim of the current study was to further define and determine the viability of EPS as an in vitro model for exercise. Given that myotubes in this study are fixed the type of exercise mimicked is comparable to a very low intensity isometric contraction. We also intended to use the model to determine the effect of metabolic endotoxemia on muscle adaptation to an exercise stimulus. The primary findings from this study indicate a significant transcriptional, metabolic, enzymatic, and physical response to electrical stimulation. The presence of low levels of endotoxin significantly alters the effect of stimulation on mrna transcription and metabolic enzyme activity. The findings of this study further support EPS as a viable means of mimicking exercise in vitro. Moreover, our results suggest that the EPS model can be used to understand the effect of a low-grade inflammatory milieu in muscle during physical stress. Previous work with this model in C2C12 cells demonstrated increases in the transcription of PGC1-a and Il-6 as well as ROS production. 5,6,10,11 Two of these studies used a similar protocol of 9 volts at 5 hertz 5,6 with the others using a higher voltage 40 volts at 1 hertz. 10,11 However, in our study in C2C12 cells, the use of EPS with 14 volts at 5 hertz only produced significant changes in Il-6 mrna. When we applied EPS to human primary myotubes grown and differentiated in DMEM based media, there were no changes in transcription of which had been previously shown by Scheler et al 13 and Lambernd et al. 14 After applying EPS in a- MEM based media, significant changes in transcription were detected. Detailed analysis of the contents of DMEM and a-mem based media revealed marked differences. a-mem based media contains lipoic and linoleic acid, penicillin/streptomycin, and numerous inorganic salts not contained in DMEM. Most notable, are the antibiotic supplements as previous 78

85 research implicates their role in altering membrane potential and thus, the electrical excitability of cultured cells. 20 We measured transcription of PGC1-a, Il-6, and SOD2 under various EPS durations and the results indicate that PGC1-a transcription is delayed with significant increases at 8 and 12 hours in comparison to Il-6 and SOD2 with increases at 1 and 2 hours respectively. The most robust response to stimulation occurred after 24 hours for all of the mrna targets. These data suggest SOD2 plays a role in neutralizing oxidative stress near the onset of an exercise stimulus. Il-6 s immediate role in the exercise adaptive response was also confirmed by our findings. To the authors knowledge, only one previous study has examined substrate metabolism in response to EPS. 15 However, to date, there are no published data demonstrating the metabolic response to EPS at varying durations. Using radiolabed glucose and palmitate, we measured substrate oxidation at 1, 2, 12, and 24 hours. Glucose oxidation, significantly increased after acute and chronic stimulation demonstrating a skeletal muscle metabolic response comparable to recorded observations in exercising humans. 21 Conversely, marked increases in fatty acid metabolism only occurred after chronic EPS. For this study, the data collected from applying EPS for varied durations was used to characterize the EPS model for heat stress, ph, enzyme activity, and substrate metabolism. The most robust metabolic response to stimulation was observed after 24 hours and this informed our approach toward further defining the EPS model. As previously demonstrated in human and animal muscle, HSP70 significantly increased in stimulated myotubes. 22,23 Media temperature increased similarly to observations made in in-vivo exercising muscle. 24 EPS effects on temperature 79

86 were modest and did not exceed 39 degrees Celsius. This outcome suggests our EPS protocol more resembles a model of exercise and not simply heat stress. However, to ensure that EPS is causing a heat stress response, pre-heated media could be used without any electrical stimulation. EPS yielded a marked decrease in media ph. However overall ph values were still significantly higher relative to values measured in exercising human subjects. 25 To determine the effect of EPS on muscle damage we also measured creatine kinase activity. Our findings indicate that chronic stimulation increases creatine kinase release which is comparable to previously published data on the effect of exercise in humans. 26 Taken together these data suggest that this model is sufficient for mimicking exercise. Recent studies have contributed to the growing evidence of exercise resistance in populations with chronic diseases such as type II diabetes, cardiovascular disease, and obesity. 16,17 The low grade inflammation associated with these diseases has been coined by the term metabolic endotoxemia. 18 These diseases are also linked to increased toll-like receptor 4 (TLR4) signaling and increased Il-6 and SOD2 expression. 27 Therefore, the secondary purpose of this study was to investigate the effect of low doses of endotoxin on the cellular responses to EPS. We hypothesized that chronic exposure to LPS, mimicking levels seen with metabolic endotoxemia, would attenuate the effects of electrical stimulation. This effect was confirmed because PGC1-a, Il-6, SOD2 transcription was blunted in the presence of 20 EU LPS during EPS. Additionally, the EPS-associated increases in glucose oxidation were absent when EPS was conducted in an environment simulating metabolic endotoxemia. LPS treatment alone also resulted in increased glucose oxidation and Il-6 and SOD2 expression independent of stimulation which is consistent with previous reports. 27,28 80

87 These data further implicate the role of chronic inflammation in the maladaptive response to exercise seen in obese and insulin-resistant populations. The presence of endotoxin altered the effect of electrical stimulation for creatine kinase and HSP70 which also suggests that LPS may disrupt the response to exercise in muscle. HSP70 is important to the protection against cellular stress. An earlier study demonstrated that its overexpression prevents LPS-induced increases in TNF-a and Il Recent findings also reveal that HSP70 and LPS have opposing regulatory effects on TLR4 in immune cells. 30 To understand the relationship between HSP70 content, LPS, and the EPS response, future studies should aim to block LPS binding during stimulation. To date, there are no published data revealing a relationship between LPS and creatine kinase released from skeletal muscle. Our findings suggest that LPS interrupts release of this indirect marker for muscle injury. To determine the interaction of LPS and creatine kinase similar future studies should also block LPS binding and measure creatine kinase release at varying intensities of electrical stimulation. The authors note a few limitations in this study. The use of a cell culture model inherently limited the authors ability to perfectly recreate the physiological environment present in working muscle. For this study, stimulated myotubes could not contract similarly to contracting muscle as they are fixed to plates. Using the human primary myotubes also precluded the authors from measuring less sensitive enzyme assays for targets such as b- hydroxacyl CoA dehydrogenase (b-oxidation) and cytochrome C oxidase (electron transport 81

88 chain) due to relatively lower cell content. The current study was also limited by the inherent variability associated with using tissue cultured from human subjects. In conclusion, EPS applied to human primary myotubes produces very low intensity exercise responses that are comparable to those demonstrated in vivo. Our findings demonstrate that reproducing the transcriptional effects of very low intensity exercise is dependent on media formulation used to culture and differentiate the cells. The time-course of electrical stimulation reveals that the response to EPS is also dependent on duration as shown herein and in a previous study. 13 Substrate metabolism, media temperature, ph, creatine kinase, and HSP70 are impacted by prolonged stimulation. Furthermore, simulating metabolic endotoxemia with LPS exposure results in markedly blunted responses to an exercise stimulus. Future studies should aim to further define this model for exercise by examining calcium transients, translational responses, and the possibility of rescuing the adverse effects of endotoxin. The findings of such studies have meaningful implications for understanding the physiological response to exercise in the presence of metabolic disease. This study contributes to the confirmation of an in vitro model for exercise. This model allows for future mechanistic studies and interventions that would be otherwise burdensome or not feasible in vivo. 82

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93 Supplemental Figures 87

94 Supplemental Figure 1. Detailed breakdown of components in high glucose DMEM based media. 88

95 Supplemental Figure 2. Detailed breakdown of components in low glucose DMEM based media. 89

96 Supplemental Figure 3. Detailed breakdown of components in alpha-mem based media. 90

97 Supplemental Figure 4. Confirmation of MyHCII in control and stimulated myotubes. Pictured on the left are control and on the right, are stimulated human primary myotubes. Immunofluorescence imaging show the expression of MyHCII in the cells and nuclei of control and stimulated tissue. Supplemental Figure 5. Cells isolated from stromal vascular fraction. As a negative control measure cells were incubated with an MyHCII antibody to detect its presence. Shown here are images confirming a lack of fluorescence indicating that MyHCII is not expressed in these cells. 91

98 Supplemental Figure 6. Effect of high voltage EPS. Pictured on the left are human primary myotubes not treated with EPS. On the right are myotubes treated with EPS for 24 hours with 40 volts, 5 hertz, and a 2-millisecond pulse. 92